Study funding/competing interest(s): This trial is supported by a grant of theNetherlands Organisation for Health Research and Development (ZonMw Clinic-al fellow grant 90700154).Trial registration number: ISRCTN 48210491

Embryology

P-075 ACCU-VIT: a new strategy for managing poor responders

G. Gandhi1, G. Allahbadia2, S. Kagalwala1, A. Allahbadia2, S. Ramesh2, K. Patel2,R. Hinduja2, V. Chipkar1, M. Madne1, and R. Ramani1

1Rotunda - Centre for Human Reproduction, Assisted Reproduction Laboratory,Mumbai, India, 2Rotunda - Centre for Human Reproduction, AssistedReproduction, Mumbai, India

Study question: To evaluate the efficacy of serial minimal stimulation IVF cycleswith vitrification of embryos for treatment of poor responders as compared to con-ventional IVF protocols.Summary answer: Accumulating vitrified embryos in serial minimal stimulationcycles (ACCU-VIT) followed by a frozen embryo transfer is a better treatmentoption for poor ovarian responders as compared to conventional IVF.What is known already: Previous trials have shown that neither conventionalIVF nor natural cycle IVF is an effective treatment option for poor ovarian respon-ders. However, none of the trials have examined the efficacy of accumulatingembryos with serial minimal stimulation cycles and vitrifying the resultingembryos. Women with poor ovarian reserves, who commonly do not respond toconventional stimulation protocols, are left with few options when planning afamily.Study design, size, duration: This is a retrospective data analysis of poor respon-ders from February 2011 to March 2012. A total of 140 patients were included inthe study. 55 patients were offered minimal stimulation cycles with vitrificationand embryo banking (ACCU - VIT Group) and 85 patients underwent convention-al controlled ovarian stimulation for IVF.Participants/materials, setting, methods: The inclusion criteria for ACCU-VITgroup were patients with at least one previous conventional IVF cycle with poorresponse (defined as ≤ 4 MII oocytes). Embryos were vitrified using Cryotec Vitr-fication protocol on Day 3. Once six embryos were banked with us, a frozenembryo transfer was planned. A maximum of 3 embryos were transferred. Mainoutcomemeasurewas the clinical pregnancy rate defined as positive fetal heartbeatat 12 weeks of pregnancy.Main results and the role of chance: The mean age was 38.5 years in theACCU-VIT group and 35.7 years in the conventional IVF group. In theACC-VIT group, each patient underwent an average of 2.7 cycles of embryo ac-cumulation before planning a frozen embryo transfer. An average of 6.2embryos were vitrified for each patient. The cycle cancellation rate was 16.6%in the ACCU-VIT group and was significantly higher in the conventional IVFgroup (22.2%). The clinical pregnancy rate was higher in the ACCU-VIT group(28.5%) than the conventional IVF group (18.7%). The cumulative pregnancyrate was statistically higher in the ACCU – VIT group (41.5%) than the conven-tional IVF group (22.3%).Limitations, reason for caution: A limitation of our analysis is a small samplesize. However, we are very encouraged by the better results observed in theACCU-VIT group and are continuing to apply this approach to more patients.Wider implications of the findings: ACCU-VIT using minimal stimulation andreliable vitrification methods is a successful approach to treat poor responders cre-ating a situation similar to normal responders. This approach allows the poor re-sponder women to have consecutive cycles of embryo accumulation before thefollicular reserve is depleted. It will maximize the ovaries’ already limited lifespan, allowing patients the opportunity to store embryos while oocyte productionis still active.Study funding/competing interest(s): No funding was used. There are no com-peting interests to declare.

Trial registration number: Not Applicable

P-076 Sucrose needs not to be added in vitrification solution for freezingthe artificial shrunken mouse blastocysts

J.K. Joo1, J.E. Jeung1, K.R. Go2, and K.S. Lee1

1Pusan National University Hospital, OB/GY, Busan, Korea South, 2PusanNational University Hospital, infertility clinic, Busan, Korea South

Study question: Does sucrose need to be added in vitrification solution for freez-ing the artificial shrunke mouse blastocysts?Summary answer: Sucrose needs not to be added in vitrification solution forfreezing the artificial shrunken mouse blastocysts.What is known already: Sucrose included in freezing medium causes embryos toshrink by losing intracellular water, so that intracellular ice formation is reducedand embryos can be frozen rapidly. Blastocyst consists of trophoblast and innercell mass. It may be considered that the artificial shrunken blastocyst is notembryo but tightly packed somatic cells. Sucrose is not added to somatic cell freez-ing medium.Study design, size, duration: Mouse morulaewere collected from superovulated,mated mice(C57BL/CBA), cultured in G2.2 to develop into expanded blastocysts.Vitrification and thawing were prepared. Two vitrification solutions with andwithout sucrose were prepared. Control(G25E25) and treatment(G25E25S0.5)were composed of 25%glycerol + 25%ethyleneglycol and 25%glycerol +25%ethyleneglycero +0.5 mol/l sucrose, respectively.Participants/materials, setting, methods: Blastocoel fluid was aspirated in theexpanded mouse blastocysts and shrunken blastocysts were equilibrated inEBS1, EBS2. After loading capped pulled-straw blastocysts were vitrified or rehy-drated. Assisted hatching was performed using assisted hatching pipette afterthawing procedure or rehydration. Rates of re-expanding and hatching were exam-ined after culture for 6h.Main results and the role of chance: Re-expanding rate of mouse blastocystexposed to VS(without and with 0.5 mol sucrose) were not different inG25E25(98%) and G25E25S0.5(92%) (P . 0.05) and hatching rate was higherin G25E25(95%) than in G25E25S0.5(88%) but did not differ in two treatments(P . 0.05). Re-expanding rate of mouse blastocyst vitrified in G25E25 andG25E25S0.5 was 95% and 90%, respectively (P . 0.05), and hatching rate washigher in G25E25(90%) than in G25E25S0.5(76%) (P . 0.05).Limitations, reason for caution: This study was done with mouse blastocysts.So, we are not sure about the effect in human blastocyst. Before applying thismethod to human blastocyst, we have to confirm the negative effect of sucrose-freevitrification solution to human blastocyst.Wider implications of the findings: It can be applied to human embryo freezingprocedure. This technique make freezing process easy, convenient, so we canreduce laboratory mistakes in freezing precess.Study funding/competing interest(s): NoneTrial registration number: None

P-077 ATP contents in immature oocytes obtained from graafian folliclesdecreased compared with those from small follicles

H. Goto1, S. Hashimoto1, A. Amo1, T. Yamochi1, H. Iwata2, and Y. Morimoto3

1IVF Namba Clinic, Reserch division, Osaka, Japan, 2Tokyo University ofAgriculture, Department of Animal Science, Atsugi, Japan, 3IVF Namba Clinic,Medical office, Osaka, Japan

Study question: To assess the relationships among ATP contents in oocytes,ovarian stimulation procedures, donor age, oocyte cell cycle, and oocyte diameter,we measured ATP contents in immature oocytes obtained from graafian and smallfollicles.Summary answer: ATP contents in immature oocytes obtained from small folli-cles (diameter: approx. 10-mm) followed by maturation culture was significantlyhigher than that in immature oocytes obtained from graafian follicles after ovarianstimulation and natural cycles (diameter: approx. 19-mm).What is known already: ATP contents in oocytes have been suggested to be amarker of oocyte quality including maturity and developmental competence.On the other hand, it has been also reported that oocytes containing extremelyhigh ATP content had low developmental competence.

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Study design, size, duration: This was an experimental study using 97 immatureoocytes obtained from graafian follicles (diameter: approx. 19-mm) after ovarianstimulation or natural cycles and 79 immature oocytes obtained from small folli-cles (diameter: approx. 10-mm) followed by maturation culture between April2012 and October 2012. The local IRB approved this study.Participants/materials, setting, methods: Donated oocytes were used afterinformed consent. ATP contents in oocytes were measured individually afterthe measurement of their diameter and the removal of their cumulus cells. TheATP assay was performed individually based on the luminescence reaction. Lumi-nescence was measured using a luminometer. Data were compared using studentt-test.Main results and the role of chance: There were no differences in the ATP con-tents between GV (6.7 pM, n: 110) and MI stage oocytes (6.2 pM, n: 66), andbetween stimulation (5.0 pM, n: 66) and natural cycles (4.4 pM, n: 32). Moreover,there were no relationships between the ATP contents and oocyte diameter (diam-eter: 107.5-135.0 mm, 120.5 mm, n: 147, r2 ¼ 0.01, P ¼ 0.2), and between theATP contents and donor age (age: 25-45 years old, n: 176, r2 ¼ 0.02, P ¼0.06). However, the ATP content in oocytes obtained from small follicles (8.6pM, n: 79) was significantly higher (P , 0.05) than that in oocytes obtainedfrom graafian follicles (4.8 pM, n: 97).Limitations, reason for caution: Further studies are required to clarify the linkbetween a decrease of ATP contents in immature oocytes obtained from large fol-licles compared with oocytes obtained from small follicles.Wider implications of the findings: This study provided new insights on theimplications of a decrease of ATP contents during follicle growth.Study funding/competing interest(s): Part of this work was supported by a grantfrom the Japan Society for the Promotion of Science (JPS-RFTF 23580397 toS.H.). No other competing interests are declared.Trial registration number: None.

P-078 Is ICSI yielding better clinical outcome compared to IVF in poorresponders with normal sperm analysis?

M. Koifman, S. Lahav-Baratz, E. Blais, Z. Megnazi-Wiener, D. Ishai,R. Auslender, and M. DirnfeldCarmel Medical Center, Gyn/Obs IVF unit, Haifa, Israel

Study question: To compare the effect of insemination using standard In vitro fer-tilization (IVF) Vs Intra Cytoplasmic Sperm Injection (ICSI) on clinical preg-nancy and delivery rates in poor responders with 4 or less available oocytes anda normal sperm count.Summaryanswer: Clinical pregnancy and delivery rates were higher but not stat-istically significant in IVF Vs ICSI in poor responders with normospermia.

Significantly lower fertilization rates and more "NO embryo transfer " eventsoccurred with ICSI Vs IVF.

In poor responders with normal sperm the use of ICSI was not beneficial.What is known already: The introduction of ICSI has led to dramatic improve-ment in pregnancy rates in severe male factor and cases with failed fertilization. Inview of high pregnancy rates and take home baby rates and reports on the relativesafety of the ICSI technique, centers have considered using ICSI for other indica-tions than Male factor.

With only few available oocytes retrieved, embryologists and clinicians face areal dilemma regarding the choice of the insemination technique.Study design, size, duration: The study reviewed 195 treatment cycles of knownpoor responders, with 4 or less retrieved oocytes.

Only cases with normal sperm count were included.The study group included 148 cycles in which standard IVF was used and 47

cycles with ICSI.Participants/materials, setting, methods: The parameters studied were age,number of retrieved oocytes, basal day 3-5 serum FSH, number of MII oocytes,fertilization rates, cleavage rate, number of embryos transferred, the percentageof cycles with embryo transfer, clinical pregnancy and delivery rates, analyzedin the group of IVF and ICSI cycles.Main results and the role of chance: There was no difference between the groupsof IVFand ICSI cycles in the parameters of age, numberof oocytes retrieved, FSH,number of MII oocytes per cycle and cleavage rates.

A significantly higher fertilization rate was observed in the IVF group as com-pared with ICSI group (81.51% and 67.73% respectively, p ¼ 0.004).

The mean numberof transferred embryos after IVF was 1.54+0.83 Vs 1.21+1.10 with ICSI (p ¼ 0.019). A "No embryo transfer" event was observed in 32%of the ICSI group Vs 11.50% in the IVF group (p ¼ 0.004).

Clinical pregnancy rates and delivery rates were higher but not statistically sig-nificant in IVF Vs ICSI (24.2% Vs 15.6%, 8.1% Vs 6.4% respectively).Limitations, reason for caution: This study is retrospective. As all casesincluded were treatments with normal sperm count, the decision to choose theuse of IVF or ICSI was based on patients’ and clinicians’ request and thereforethe IVF group was larger as compared to the ICSI group.Wider implications of the findings: In view of the above results and recentreports on increased malformation rates in ICSI, it is reasonably to advise thatin the presence of normal sperm counts, the technique of choice for inseminationin poor responders should be IVF rather than ICSI.Study funding/competing interest(s): The researchers declare that this study isfree of any financial support or other interests.Trial registration number: N/A

P-079 A modified cryotop vitrification protocol with polimer cryopro-tectant agent ficoll in the cryopreservation of human oocytes

V. Zaletova1, E. Zakharova2, I. Krivokharchenko2, and S. Zaletov1

1MAMA Fertility Center, Medical Department, Moscow, Russia C.I.S, 2MAMAFertility Center, Embryology and Genetic Laboratory, Moscow, Russia C.I.S

Study question: We examined a modified cryotop vitrification protocol in thecryopreservation of human oocytes. Suggested modification had an additionalpolimer cryoprotectant agent ficoll in vitrification medium with standard cryopro-tectants compound.Summary answer: Applying of modified cryotop vitrification protocol withficoll enables a high survival rate of thawed oocytes, a high rate of blastocyst for-mation and pregnancy rate.What is known already: Until now all standard human oocytes vitrification pro-tocols used two- and tree-component vitrification mediums with different sets ofcryoprotectants, excluding polimers. Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide. Cryoprotectant effect of ficoll is that thispolimer increases viscocity of vitrification medium, and therefore hinders forma-tion of crystals. A number of published articles on cryopreservation of animalsoocytes demonstrated efficiency of protocols with polimer cryoprotectants, in-cluding ficoll.Study design, size, duration: 388 oocytes were obtaned from 54 anonymousdonors and vitrified using modified cryotop vitrification protocol with ficoll. Vit-rified oocytes were stored in Cryobank for 1 to 6 months. Thawed oocytes of goodquality were donated for IVF treatment, in 30 cases. Then oocytes were fertilizedby husband’s sperm. Embryos were transferred on Day 5 after fertilization (blasto-cyst stage, 1-2 embryos), in 28 cycles.Participants/materials, setting, methods: During ccryopreservation oocyteswere successively exposed to three vitrification mediums with increased concen-trations of ethylen glycol (3,75 - 15%); DMSO (3,75 - 15%); ficoll (2,5 - 10%);sucrose (0,125 - 0,5 M) at 370 C. Oocyte exposure time to each solution variedfrom 1 to 6 min. Oocytes were vitrified by cryotop method. Thawing oocyteswere successively exposed to four sucrose solutions with decreased concentration(1M - 0,125 M). Then oocytes were fertilized 3 hours after thawing by ICSImethod.Main results and the role of chance: In 30 IVF cycles 263 donor’s oocyteswere thawed. After thawing 19 out of 263 oocytes degenerated (7,2% – 19/263), 244 oocytes showed no signes of degeneration (92,8% – 244/263).Normal fertilization occured in 207 oocytes (84,8% – 207/224), all zygotesstarted to cleave at 48 hours after fertilization. By Day 5 of their culture, wehad 98 embryos of good quality (47,3% - 98/207). Embryos were transferredinto the uterus in 28 IVF cycles. 10 out of 28 patients had a positive beta-hCGblood test. All 10 pregnancies were clinically recognized (35,7% for anembryo transfer - 10/28), 8 out of which resulted in the birth of healthy chil-dren an one - of healthy twins.Limitations, reason for caution: Four-component vitrification medium contain-ing ficoll was used only for human oocytes vitrification and not for cryopreseva-tion of embryos/sperm.Wider implications of the findings: Applying modified cryotop vitrificationprotocol with ficoll resulted in laboratory and clinical data comparable to those

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published in regard to early known vitrification protocols. We demonstrated itsstable efficiency, which enables us to create and successfully use Cryobank ofdonor’s oocytes.Study funding/competing interest(s): None.Trial registration number: None.

P-080 Cryopreservation of cleavage stage embryo or blastocyst: whichoption is better

L. Zhu, Y. Li, H. Zhang, J. Ai, and L. JinTongji Hospital Affiliated to Tongji Medical College, Huazhong University ofScience and technology, Wuhan, China

Study question: Surplus embryos available for cryopreservation in fresh cyclehave been considered as a good resource for future use. However, the optimalstage of embryo cryopreservation remains unclear.Summary answer: Blastocyst cryopreservation is more time-effective and cost-effective as compared to cleavage stage embryo cryopreservation though blasto-cyst culturewill not improve the embryo developmental potential. It is a more pref-erable option to patients with surplus embryos destined for future use.What is known already: Blastocyst culture is an effective tool to aid embryo se-lection. But the benefits of blastocyst transfer versus cleavage stage embryo trans-fer are usually evaluated in fresh cycles. Few studied have compared the results infrozen-thawed embryo transfer (FET) cycles in a prospective way.Study design, size, duration: A prospective cohort study was performed. Patientsin a university-based IVF center with failed pregnancy in fresh cycle and surplusembryos available on Day 3 were enrolled during March 2011 and September2011. Each patient was followed up at least one year.Participants/materials, setting, methods: A total of 627 patients were enrolledin the study. Patients were divided into two groups: cleavage stage embryo cryo-preservation group (309 patients) and blastocyst cryopreservation group (318patients). Clinical outcomes of the FET cycles in each patient were evaluated.Main results and the role of chance: Cycle characteristics in terms of female age,duration of infertility, basal FSH, mean numberof retrieved oocytes, mean numberof available embryos/top quality embryos were comparable between the twogroups. Although a decreased cryopreservation rate (74.5%, 237/318), the blasto-cyst group achieved significantly higher rates of pregnancy/cycle (42.8% vs31.7%, PLimitations, reason for caution: This study was not designed for randomizedfashion. Patients made the assignment decision mainly by themselves.However, the two groups had similar cycle characteristics, supporting theabsence of any selection bias.Wider implications of the findings: The conclusion will be helpful in counselingpatients regarding the optimal stage of cryopreservation of surplus embryos infresh cycles and utilization of cryopreserved embryos in future FET cycles.Study funding/competing interest(s): The authors have no connection to anycompanies or products mentioned in this article.Trial registration number: None

P-081 Quantitative analysis of operator-dependent variability in blasto-cyst vitrification with a High Security Vessel (HSV) vitrification system

X. Zhang, N. Rajan, A. Kovacs, C. Foley, J. Flanagan, J. O’Callaghan,J. Waterstone, and T. DineenCork Fertility Centre, Embryology, Cork, Ireland

Study question: This study was performed as part of process validation whenembryo vitrification was introduced at the fertility center. The purpose of thisstudy was to quantify variability in the performance of blastocyst vitrificationwith a closed HSV system by a number of operators with a wide-spectrum of ex-perience.Summary answer: Survival rates after blastocyst vitrification were high and didnot vary significantly between operators both with and without previous embryovitrification experience. Quantitative analysis of operator-dependent variabilityprovides a practical approach for training when implementing a successful vitrifi-cation program.What is known already: A successful outcome in vitrification is highly operatordependent, and a quite different skill set is necessary compared to that required for

slow rate freezing. The embryologist needs to be aware of several critical proced-ural details that can impact negatively on results. Skill variations for all operatorsmust be monitored and compared in order to achieve consistent and predictableresults.Study design, size, duration: 347 Blastocysts (day5/6) derived from IVF treat-ment were vitrified and 130 were warmed between Dec 2010 and Dec 2012. Sixembryologists, with experiences of vitrification ranging from 0 to 5 years, allunderwent vitrification training, practicing with silicone beads and mock proce-dures before participating in the study.Participants/materials, setting, methods: Blastocyst survival rates postwarming were used to compare for different operators. The performance of vitri-fication was also quantified by recording a number of key parameters: loadingtime, loading volume, warming speed, and speed of handling the HSV straws.Main results and the role of chance: Operators with less experience showedhigher coefficients of variation (CVs) in comparison with experienced operatorswith regard to loading volume (28.5% vs. 18.1%) and loading time (10.4% vs.6.7%), but not warming speed (13.7% vs. 12.8%) nor straw handling (13.4%vs. 13.1%). However, all operator-dependent parameters remained within proto-col limits, even though they varied among individuals. 130 vitrified blastocystswere warmed. The overall survival rate post warming was 93.8% (122/130)with 93.3% (84/90) for inexperienced and 95.0% (38/40) for experienced opera-tors. These rates were not significantly different.Limitations, reason forcaution:This audit of operator performance is being con-tinued as vitrification is applied clinically and will be supplemented by successdata.Wider implications of the findings: Quantitative analysis of operator-dependentvariability is both practical and essential when implementing a successful vitrifi-cation program.Study funding/competing interest(s): N/ATrial registration number: N/A

P-082 For now, I would rather have the transfer of one fresh blastocyst.The surplus, please, vitrify them!

E.M. Dahdouh1, P. St-Michel2, L. Granger1, B. Carranza-Mamane3, F. Faruqi2,T.V. Kattygnarath2, and F.L.A. Ferreira Gomes2

1PROCREA Clinics & University of Montreal, ART Centre, Montreal, Canada,2PROCREA Clinics, ART Centre, Montreal, Canada, 3PROCREA Clinics &University of Sherbrooke, ART Centre, Montreal, Canada

Study question: Is a frozen elective single-embryo transfer (eSET) at the blasto-cyst stage effective for good prognosis patients?Summary answer: Though less effective than fresh single blastocyst transfer,frozen SET (FET) at the blastocyst stage yields adequate clinical outcomes.This will further contribute in decreasing multiple pregnancy rates, and improvingthe cumulative pregnancy rate in the setting of an eSET policy.What is known already: Vitrified blastocysts retained good developmental com-petency after warming. High pregnancy and implantation rates can be expectedonce the blastocysts go through vitrification and warming because they have ad-equate developmental potential as fresh blastocysts.Study design, size, duration: This is a prospective non-randomized control studyincluding 244 women who underwent a SET at the blastocyst stage (fresh - eSETand frozen- FET) in a 11 months period between January 9th 2012 and December15th 2012.Participants/materials, setting, methods: Women aged 34 years or youngerundergoing a fresh elective single blastocyst transfer cycle (eSET) (n ¼ 152) ora frozen-thawed single blastocyst transfer (FET) (n ¼ 92). The study was per-formed in a tertiary private infertility clinic.Main results and the role of chance: Patients in both groups (fresh and frozen)were homogeneous for age, duration and cause of infertility, and for levels ofday 3 FSH. In the Fresh group, the biochemical pregnancy rate was 52.6%while in the Frozen group it was 33.7% (p ¼ 0.0052). Implantation rate washigher in the Fresh group compared to the Frozen group (47.4% and 31.5% re-spectively, p ¼ 0.0161). Ongoing pregnancy rates (41.4% and 28.3% respective-ly, p ¼ 0.0406) were also significantly higher in the Fresh group. Patients whounderwent a fresh eSET had a median of two surplus blastocysts cryopreservedafter transfer. Multiple pregnancy rates were very low, 0.7% and 1.1% for theFresh and the Frozen group, respectively.

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Limitations, reason for caution: This is a non-randomized study and thereforesubject to bias. Primary clinical outcomes do not include live birth rates, soresults must be interpreted with caution.Wider implications of the findings: Improvement in cryopreservation techni-ques and better synchronization between the endometrium and the embryo devel-opment, will further contribute to increase FET efficiency. Blastocysts can bevitrified in patients for whom fresh blastocyst transfer is unsuitable, such aspatients at risk of OHSS, or those in need for preimplantation genetic diagnosis.Cumulative pregnancy rate might offset the lower pregnancy rate for fresh blasto-cyst transfer observed in this study.Study funding/competing interest(s): This study received no funding and thereare no conflicts of interests to be declared.Trial registration number: None.

P-083 Effectiveness of vitrification in women undergoing electivefreezing of oocytes and/or embryos when embryo transfer conditions aresuboptimal

N. Christoforidis, C. Ioakimidou, C. Papas, M. Moisidou, and A. ChatziparasidouEmbryolab Assisted Reproduction Unit, IVF Unit, Thessaloniki, Greece

Study question: Certain conditions that are associated with reduced ARToutcomefollowing controlled ovarian stimulation, such as raised progesterone levels on dayofhCG triggering,ovarian hyperstimulation syndrome, endometrialpolypsand thinendometrium. Elective vitirification of all oocytes/embryos and frozen embryotransfer in subsequent cycles may provide a valid alternative approach.Summary answer: Elective vitrification of oocytes/embryos after controlledovarian stimulation in suboptimal conditions for embryo transfer, is associatedwith high survival rates of oocytes/embryos and high pregnancy rates in frozenembryo replacement cycles.What is known already: Successful implantation relies on both embryologicalfeatures and endometrial receptivity. It is known that certain conditions have anegative impact on endometrial receptivity, in particular, raised progesteronelevels on the day of hCG triggering, hyperoestrogenemia associated withovarian hyperstimulation syndrome, as well as endometrial pathology, such asendometrial polyps and presence of endometrial fluid. Vitrification is an effectiveprocedure that allows preservation of oocytes/embryos and transfer of embryos incycles with optimal endometrial recpetivity.Study design, size, duration: Retrospective analysis of 88 cases undergoingelective freeze-all with vitrification of oocytes/embryos following controlledovarian stimulation, when endometrial conditions were deemed suboptimal,from March 2011 to March 2012.Participants/materials, setting, methods: Cases were categorized according toindication of elective freeze-all procedure as follows: repeated implantationfailure, endometrial polyp, raised progesterone on day of hCG triggering,OHSS, embryo collection for PGD, endometrial fluid, thin endometrium. Sur-vival rate of oocytes/embryos post thawing and pregnancy rates in subsequentembryo transfers were evaluated.Main results and the role of chance: Survival rate of oocytes/embryos and CPRper category were recorded as follows: repeated implantation failure: 95% oocytesurvival, 50% CPR, endometrial polyp: 96% oocyte survival, 98% embryo sur-vival, 85% CPR, raised progesterone: 98% oocyte survival, 54.8% CPR,embryo collection for PGD: 98% embryo survival, 48% CPR, OHSS: 97%oocyte survival, 64.5% CPR, endometrial fluid: 99% embryo survival, 25%CPR, thin endometrium: 96% embryo survival, 46% CPR.Limitations, reason for caution: Number of cases that underwent elective cry-preservation for endometrial receptivity reasons may not be large enough.Wider implications of the findings: Oocyte and embryo survival rates after vitir-ification are quite high and pregnancy rates are very satisfactory following embryotransfer in a subsequent endometrial preparation cycle. It is suggested that when-ever endometrial conditions are deemed suboptimal oocyte and/or embryo vitri-fication may be a valid option to allow optimal endometrial preparation andhence,high pregnancyrates. Elective vitirification may minimize the riskof devel-oping OHSS, augment the number of available embryos for PGD.Study funding/competing interest(s): NoneTrial registration number: None

P-084 Cleavage stage embryo versus blastocyst transfer in patients with3 failed IVF/ ICSI cycles, a retrospective cohort study

M. Klaver, K. Tilleman, and P. De SutterUniversity Hospital Gent, Assisted reproduction, Gent, Belgium

Study question: Does day 5 embryo transfer (ET) in patients with repeated im-plantation failure (3 failed IVF/ICSI cycles) increase pregnancy rates in thefourth cycle if necessary after correction of thrombophilic or autoimmune path-ology?Summary answer: Day 5 ET in patients with 3 failed IVF/ICSI cycles and inwhom thrombophilia or autoimmune disorders are treated if present, increasesthe pregnancy rate per embryo transfer significantly until 67% when comparedto 37% after day 3 ET.What is known already: Pregnancy rate in the fourthcycle after 3 failed IVF/ICSIcycles is usually rather low, making this a therapeutically challenging group ofpatients. There is a lot of controversy about testing for auto-immunity or thrombo-philia and about which is the best therapeutic strategy for this group.Study design, size, duration: This is a monocentric retrospective study. Weincluded 72 patients with 4 consecutive IVF or ICSI treatment cycles in ourcentre between 2007 and 2012. The first 3 cycles had ET at cleavage stage (day2 or 3) and negative HCG as outcome. Patients aged above 36 years wereexcluded.Participants/materials, setting, methods: In the fourth cycle 51 patients wereplanned for ETat cleavage stage and 21 for blastocyst transfer.A repeated implant-ation failure protocol (RIF), including testing for thrombofilia, auto-immunity,karyotype disorders and hysteroscopy was performed. Depending on the testresults treatment consisted of operative hysteroscopy, prednisolon, heparin oraspirinaccordingly.Main results and the role of chance: In the group scheduled for day 5 ET 81%(17 of 21 patients) were tested and 28.5% of the tests (6 patients) were abnormaland treated. In one patient (4.7%) the embryos did not reach the blastocyst stageand no transfer was performed. In the cleavage stage group 63% (32 of 51 patients)were tested and 37.5% (12 patients) were abnormal and treated. Pregnancy rate pertransfer in cleavage stage transfer was 37% (19 out of 51 patients). Pregnancy rateper transfer in the blastocyst group (day 5) was 67% ( 14 out of 21 patients). Thisdifference is statistically significant (x2-test, P ¼ 0.023.).Limitations, reason forcaution: The patient group is rather small, but still resultsare significant. These are preliminary results and the cohort will be expanded toinclude patients with 3 IVF/ICSI cycles of which the first 2 have failed. Resultsfrom frozen embryo’s and subsequent cryo cycles have not been included inthis study.Wider implications of the findings: With blastocyst transfer chances for patientswith repeated implantation failure increase significantly. Besides correcting forimplantation hindering pathology, performing day 5 embryo culture also allowsto discriminate between real implantation and embryo quality problems. This pro-vides useful information, allowing better counselling of patients regarding furthersteps of treatment.Study funding/competing interest(s): This study was not funded and there wereno competing interests.Trial registration number: not applicable

P-085 Effect of female underweight on embryo morphokinetic usingtime-lapse

J. Lammers, T. Freour, C. Splingart, and P. BarriereCHU Nantes, 38 Bd Jean Monnet, Nantes Cedex 01, France

Study question: The aim of our study was to compare early embryo morphoki-netic parameters with a time-lapse monitoring system (Embryoscope) accordingto female BMI (Body Mass Index) category, in order to identify an eventual det-rimental effect of underweight on in vitro embryo development.Summary answer: We showed that morphokinetics differs according to femaleBMI category, with almost all cellular events delayed in underweight women.We also found that Zona Pellucida (ZP) thickness was different according tofemale BMI. However, IVF cycle outcome does not appear to be affected in under-weight women.What is known already: Consequences of underweight (BMI , 18.5 kg/m2) onreproductive age women’s health are numerous, including fertility. Indeed, these

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women often face with infertility, anovulation, high miscarriage rate and obstetric-al complications, leading to lower live birth rate after ARTcycles. Whether under-weight affects preimplantatory embryo development in underweight womenundergoing IVF remains to be explored.Study design, size, duration: This retrospective study was conducted on all ICSIcycles performed between 2011 and 2012 with the Embryoscopew. All clinical,ovarian reserve, ovarian stimulation and embryonic parameters (conventionalmorphology and kinetic events in hours post injection) were recorded.Participants/materials, setting, methods: This study was conducted at the Uni-versity Hospital of Nantes, France, on all ICSI cycles. After oocyte retrieval andICSI, all oocytes were incubated in the Embryoscopew until transfer. Clinical,ovarian reserve, ovarian stimulation and embryo morphokinetic parameterswere compared according to female BMI category.Main results and the role of chance: Among the 366 couples included (2270oocytes collected), 34 women had low BMI (,18.5 kg/m2), 224 had a normalBMI (18.5-25), 65 were overweight (25-30) and 43 were obese (.30). Patients’clinical characteristics were not significantly different between the 4 groups(age, smoking status, infertility, AMH, semen characteristics and stimulation para-meters). Day 3 FSH, E2 and LH and peak E2 were significantly higher, whereastotal FSH dose was lower in underweight group than in others. Number ofoocytes retrieved, fertilization rate, number and quality of cleaved embryos (con-ventional morphology) were not significantly different between underweight andothers groups. Most embryo development events (4 cell, 5 cell, 8 cell stages) oc-curred significantly later in underweight than in other groups. ZP was also thickerin underweight women.Limitations, reason for caution: This retrospective study was conducted in alimited cohort of patients.Wider implications of the findings: Time-lapse analysis is a relevant method forthe evaluation of female clinical characteristics’ influence on early embryo devel-opment. Not only obesity, but also female underweight, should be studied as a co-factor potentially leading to modifications in early embryo development.Study funding/competing interest(s): NoneTrial registration number: Local IRB approved study

P-086 Effect of oocyte activation by calcium ionophore A23187 orstrontium chloride in patients with low fertilization rates

T. Ikeno1, Y. Nakajyo2, Y. Sato1, K. Hirata1, T. Kyoya1, and K. Kyono1

1Kyono ART Clinic, Department of Gynechology, Sendai, Japan, 2Kyono ARTClinic Takanawa, Department of Gynechology, Tokyo, Japan

Study question: To evaluate the effectiveness of oocyte activation (OA) bycalcium ionophore A23187 or strontium chloride in patients with low fertilizationrates.Summary answer: Artificial OA using A23187 or SrCl2 is beneficial to patientswith low or no fertility.

Further studies are needed to confirm the safety of oocyte activation.What is known already: Oocytes unfertilised after ICSI have been subsequentlyactivated using chemical, mechanical, and electrical stimulation, and theseoocytes have been able to form pronuclei.Study design, size, duration: We obtained informed consent from 144 coupleswho had had unsuccessful ICSI treatment at our clinic between April 2004 andAugust 2012. These were then divided into two groups.Participants/materials, setting, methods: Ninety two couples who had unsuc-cessful ICSI treatments sequentially received A23187 OA after ICSI. We com-pared the clinical results with and without OA.

Fifty two couples who had unsuccessful previous ICSI treatments sequen-tially received SrCl2 OA after ICSI. We compared the clinical results with andwithout OA.Main results and the role of chance: Comparing the results without and withA23187, the results were as follows: two pronucleus (2PN) zygote rates, 24.3%(85/350) vs. 61.2.% (316/516): P , 0.01; top quality embryo rates, 26.1%(23/88) vs. 43.4%(115/265): P , 0.01; expanded blastocyst rates, 13.8% (9/65)vs. 15.6% (30/192): NS; clinical pregnancy rates, 2.7% (1/36) vs. 14.9% (26/174): NS; and miscarriage rates, 100% (1/1) vs. 38.5% (10/26): NS.

Comparing the results without and with SrCl2, the results were as follows: 2PNzygote rates, 24.5% (81/330) vs. 57.5% (192/334): P , 0.01; top quality embryorates, 27.0% (24/89) vs. 25.3% (42/166): NS; expanded blastocyst rates, 14.1%

(9/64) vs. 8.4% (11/131): NS; clinical pregnancy rates, 0%(0/52) vs. 19.2% (14/73) :P , 0.05; and miscarriage rates, 0% (0/0) vs. 21.4% (3/14): NS.Limitations, reason for caution: None.Wider implications of the findings: In conclusion, AOA using A23187 or SrCl2is beneficial for patients with low or no fertilization by ICSI.

Comparing the results for babies with A23187 and SrCl2 OA showed no signifi-cant difference between babies born after OA and naturally conceived babies.

As with all assisted reproductive technology treatment, we must inform patientsof the risks and benefits of undergoing fertility treatment and possible implicationsfor the future health of their children.Study funding/competing interest(s): None.Trial registration number: This study is not RCT.

P-087 Are there any differences in terms of kinetic parameters betweenmale or female embryos - a time lapse analysis

F. Bronet Campos1, M. Meseguer2, M. Nogales3, E. Martinez3, M. Ariza3,D. Agudo1, L. Rodrigo3, and J.A. Garcia-Velasco4

1IVI Madrid, IVF, Madrid, Spain, 2IVI Valencia, IVF, Valencia, Spain, 3IVIMadrid, PGD, Madrid, Spain, 4IVI Madrid, Gynaecology, Madrid, Spain

Study question: Is it possible to estimate embryo gender according to the embryocleavage timing?Summary answer: There is no correlation between sex ratio and developingembryo rate.What is known already: In some species male and female embryos develop atdifferent rate during preimplantational period. It has been suggested that XYhuman embryos could have faster growing rate and also they could reach blasto-cyst stage earlier. However, no time lapse studies have been reported so far.Study design, size, duration: Retrospective, observational study including 365embryos from our PGS (preimplantation genetic screening) program fromJanuary 2012 to December 2012. Embryos were grouped according to their sex:male (166 embryos) and female (161 embryos). Additionally another group wasincluded with 38 embryos with Turner Syndrome (TS).Participants/materials, setting, methods: All embryos were cultured in an incu-bator with time lapse technology, Embryoscope. Cleavage timing from insemin-ation to day 3 was studied and all kinetic parameters that have been described inprevious studies by our group have been taken into account and comparedamong the three groupsMain results and the role of chance: Chromosomal abnormal rate was similar inboth groups (male: 61.4%; female: 57.7%, p ¼ 0.4955). We did not find any stat-istical differences between male or female embryos according to the growing ratein any specific cleavage check point.

Surprisingly, we found statistical differences in cleavage time from one cell totwo cells (T2) in TS embryos compared with male or female embryos (TS: 27.6+3.7; male: 26.3+3.0; female: 26.3+2.8; p ¼ 0.032).Limitations, reason for caution: Sample size may limit our findingsWider implications of the findings: Embryo development is not affected byembryo gender and sex ratio is not affected by the embryo selected to transferbased on kinetic parameters.Study funding/competing interest(s): The authors declare no conflicts of inter-est. The study did not receive any external grant.Trial registration number: None

P-088 Blastocyst survival, re-expansion, and % live cells following vitri-fication and warming using two commercially-available vitrification systems

A.S. Lopes, V. Frederickx, G. Vankerkhoven, A. Serneels, P. Roziers,P. Puttermans, R. Campo, and S. GordtsLIFE (Leuven Institute for Fertility and Embryology), Unit for ReproductiveMedicine, Leuven, Belgium

Study question: The aim of this study was to evaluate and compare survival,re-expansion, and % live cells of individual Day 5-6 human blastocysts, vitrifiedand warmed with the Vit Kit Freeze/Thaw (Irvine Scientific, CA), or with two pro-tocols using the Global Fast Freeze/Thaw Kits (LifeGlobal, Canada).Summary answer: Survival, re-expansion, and % live cells were not different forblastocysts vitrified and warmed between the two vitrification/warming kits, orbetween the two protocols for the Global Fast Freeze/Thaw Kits.

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What is known already: Vitrification is a modern method for cryopreservation ofhuman embryos, currently available in most IVF centers. Several cryoprotectantsand devices are available in the market but some require excellent training skillsand others led to inconsistent survival rates following thawing.Study design, size, duration: Group 1 blastocysts were vitrified with the Vit Kit(n ¼ 41) and Hemi-Straw devices, Group 2 (n ¼ 50) and Group 3 (n ¼ 57) blas-tocysts were cryopreserved with the Global Fast Freeze Kit and 0.25 ml straws,using a direct plunge or a -1008C holding step, respectively. Group 4 (Controls,n ¼ 31) were not vitrified.Participants/materials, setting, methods: Frozen/thawed Day 2-3 or discardedembryos were cultured to blastocyst (culture day 5-6). After vitrification/warming, blastocysts were cultured for 24h. All embryos were stained individu-ally with propidium iodide and Hoechst. Live and total cell number was assessedwith ImageJ (NIH), and the percentage of live cells calculated for each blastocyst.Main results and the role of chance: Survival immediately following thawingwas 90%, 94%, and 83% for Groups 1, 2 and 3, respectively, and not significantlydifferent (P . 0.05). Following 24h culture, survival was 72%, 74% and 77%,and not significantly different. Re-expansion at 24h after thawing was 55%,68% and 68%, and not significantly different. Mean (+ SD) % live cells were90.1+2.5, 92.7+2.5, 90.4+2.2, and 95.8+1.1, for Groups 1, 2, 3, and 4, re-spectively. There was no significant effect of treatment, culture day (Day 5 or 6), orinteraction between treatment and culture day.Limitations, reason for caution: The power of the comparisons of proportions islow (, 0.40) due to the relatively small sample sizes.Wider implications of the findings: The simplicity of blastocyst vitrificationusing the Global Fast Freeze/Thaw Kits using freezing straws may provideincreased safety, and efficiency with lower cost, compared with vitrificationusing specialized embryo vitrification devices.Study funding/competing interest(s): Global Fast Freeze/Thaw Kits were pro-vided by IVFonline. A.S. Lopes is a paid technical consultant for IVFonline.Trial registration number: Not applicable

P-089 Analysis of mitochondrial DNA copy number provides an insightinto the ploidy and implantation potential of human blastocysts

E. Fragouli1, S. Alfarawati2, K. Spath3, and D. Wells1

1Reprogenetics UK/ University of Oxford, Institute of Reproductive Sciences,Oxford, United Kingdom, 2Reprogenetics UK, Institute of Reproductive Sciences,Oxford, United Kingdom, 3University of Oxford, Institute of ReproductiveSciences, Oxford, United Kingdom

Abstract withdrawn by the author.

P-090 Serum anti-Mullerian hormone measurements and humanblastocyst development after IVF

J. Liss, K. Lukaszuk, J. Glowacka, and A. BruszczynskaInvicta, Fertility and Reproductive Center, Gdansk, Poland

Studyquestion: To report on relationships among baselineAMH level, blastocystdevelopment and other selected embryology parameters observed in non-donoroocyte IVF cycles.Summary answer: The current investigation present that baseline serum AMHlevel can be a useful parameter to estimate blastocyst developmental potentialduring IVF.What is known already: AMH was a predictive correlate for ovarian reserve andthe number of oocytes retrieved, with a high positive value compared with age,early follicular FSH level and pregnancy after IVF. Its role in forecasting invitro embryo morphology and developmental potential to the blastocyst stage isstill emerging.Study design, size, duration: Between January and December 2011, 830 patientswithout endometriosis, who had their AMH levels analyzed were incorporatedinto this prospective study.Participants/materials, setting, methods: Mean age, AMH level, antral folliclecount (AFC), number of follicles, oocyte retrieved, MII oocytes, early cleavageembryos, blastocyst for transfer were evaluated.

Main results and the role of chance: Three groups were formed according to theAMH level: 1: ,0.6 ng/ml (n ¼ 55), 2: 0.6-1.4 ng/ml (n ¼ 134) and 3: .1.4 ng/ml (n ¼ 641). Blastocyst formation was significantly lower (p ¼ 0.001) in group1 and 2 compared to group 3 (23.6%, 43.3% and 67% respectively). There werestatistical differences in first group of AMH with age (p ¼ 0.001) and early cleav-age embryos (p ¼ 0.02). By multivariate analysis, only age (OR ¼ 0.81; 95% CI,0.66-0.98) showed significant association with the blastocyst formation in thisgroup. All parameters were found to be associated with blastocyst formation inAMH group 2 and 3. By multivariate analysis, only early cleavage embryos(OR ¼ 2.56; 95% CI, 1.58-4.13 and OR ¼ 1.71; 95% CI, 1.52-1.93 respectively)showed significant association with the blastocyst formation in these two groups.Limitations, reason for caution: Different types of reproductive pathologymight impact serum AMH in different ways (endometriosis, PCOS). Culture tothe blastocyst stage require strict environmental. This can affect the resultsobtained in other laboratories.Wider implications of the findings: Our study confirmed correlation betweenAMH level and age, AFC, total number of retrieved oocytes as well as thenumber of MII oocytes and early cleavage embryos. The correlation betweenearly cleavage embryos and blastocyst formation suggests that early cleavage ob-servation (e.g. time laps procedure) will be a good prognostic factor for patientwith different AMH level. Decision about embryo transfer with smaller amountof good quality embryos could be taken earlier, without long culture.Study funding/competing interest(s): There are not any commercial associationof the author of any coauthors that might pose a

P-091 Effect of sperm selection using hyaluronic acid binding on repro-ductive outcome with donated oocyte cycles

S. Cortes Gallego, L. Ortega Lopez, E. Olaya Vila, M. Gago Garcıa, C. Luna Canas,A. Garcıa Segovia, A. Guijarro Ponce, R. Nunez Calonge, and P. Caballero PeregrınClinica Quirurgica Tambre, Laboratorio FIV / Andrologia, Madrid, Spain

Study question: Compare the efficiency of routine sperm selection method poly-vinylpyrrolidone (PVP) with HA-selection method (Sperm-Slow) for fertilizationrate (FR), cleavage rate (CR), embryo quality (EQ) and pregnancy rate (PR) aswell as evaluating the relationship between HA-binding ability with sperm pro-tamine deficiency and DNA fragmentation using donated oocyte cycles.Summary answer: Contrary to our expectation, selection of HA-boundspermato-zoa had no benefits in terms of fertilization, em-bryo cleavage,embryo quality and pregnancy rate in ICSI cycles.

In conclusion, our prospective and randomized trial suggests that both,PVP and Sperm-Slow allow a comparable clinical efficiency in selectingspermatozoa.What is known already: Intracytoplasmic sperm injection (ICSI) using hyalur-onic acid (HA) containing media has been suggested to have higher specificityand lower biological risk than other selection methods. HA-binding ability ofspermatozoa is related to sperm membrane maturity and fertilizing potential,thus it has been suggested that sperm selection using HA before ICSI mighthelp to optimize the treatment. However, there are conflicting data regardingsubsequent improvement of FR, CR and PR after ICSI using HA-boundspermatozoa.Study design, size, duration: This was a single-center, prospective, randomizedstudy of 144 ICSI cycles (2012) with donated oocytes to avoid female infertility asa bias factor, randomly carried out with PVP (1119 ovocites microinjectated) orwith Sperm-Slow (1104 ovocites microinjectated) for sperm selection.Participants/materials, setting, methods: Our primaryoutcome was to compareFR, CR, EQ and PR between two groups.

A secondary outcome was to better de?ne the role of HA for selection of sperm-atozoawith normal chromatincontent to optimize ICSI outcome. To determine thevalue of Sperm DNA fragmentation (SDF) levels, the Sperm Chromatin Disper-sion test (SCD) was measured.

Between groups differences of normally distributed continuous variables wereassessed with a parametric statistic (Student’s t test).Between-group differences inno continuous variables were assessed using the c2 method. Differences were con-sidered significant when a P value was ,.05.Main results and the role of chance: There were no significant differences inregard the total number of injected MII oocytes, semen quality or SDF.

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No statistical difference was found regarding FR p: 0,961), CR (p: 0,884), EQ(0,848) and PR (0,329). Oocytes injected with HA-bound spermatozoa had notdifferent FR (82, 33) than the conventional group (82, 46) neither CR (96, 71 vs97, 03), or EQ (74, 23 vs 75, 6). Lower pregnancy rate was observed in oocytesinjected with HA-bound spermatozoa than the conventional group, but the differ-ence was not significant (61, 0 vs 53, 2).

When the study population was divided by the SDF level (cutoff 30 %), therewere no statistical differences regarding FR, CR, EQ and PR when HA-selectedspermatozoa were used.Limitations, reason for caution: We havent limitations in our studyWider implications of the findings: The majority of studies have consistentlyreported that HA-binding method is effective in selection of spermatozoawithout DNA fragmentation and with normal nucleus.

This is the first report with HA-bound spermatozoa using oocytes obtained fromyoung and fertile donors in previous cycles and this way to isolate the real influ-ence of HA in clinical results.Study funding/competing interest(s): No study funding or competing interestTrial registration number: No trial registration numbre

P-092 Does growth retardation in human blastocysts decrease implant-ation potential after embryo transfer through an increase of the abnormalspindles

S. Hashimoto1, A. Amo1, K. Ito2, Y. Nakaoka2, and Y. Morimoto2

1IVF Namba Clinic, Research division, Osaka, Japan, 2IVF Namba Clinic,Medical office, Osaka, Japan

Study question: To assess the potential of growth-retarded embryos, the implant-ation potential and the spindle shape of vitrified–warmed blastocysts wereassessed among normally developing and growth-retarded blastocysts.Summaryanswer: The incidence of abnormal spindle morphology increased andthe implantation competence decreased following vitrification in growth-retardedembryos compared with normally developing embryos, but there were no signifi-cant differences in the chromosomal abnormalities of abortuses and the incidencesof peromelus of babies between 2 groups.What is known already: There are conflicting data on whether the human embryogrowth rate affects the outcome of vitrified–warmed blastocyst transfer. Varioustypes of spindle abnormality occur in human cleavage- and blastocyst-stageembryos. A recent systematic review and meta-analysis concluded thatgrowth-retarded embryos that develop to the blastocyst stage by day 6 have thesame implantation potential as their day 5 counterparts if the morphology ofday 6 blastocyst is similar to that of day 5 blastocyst.Study design, size, duration: This was a retrospective cohort study including 878single vitrified–warmed blastocyst transfers between January 2010 andJuly 2012,and an experimental study using 108 vitrified–warmed blastocysts donated to re-search. The local IRB of IVF Namba clinic approved this study. Data were com-pared using the Mann–Whitney nonparametric U-test.Participants/materials, setting, methods: In a clinical study, we compared theimplantation rates of vitrified–warmed embryos that developed to the blastocyststage on day 5 after insemination with those that required culture to day 6. In anexperimental study, vitrified blastocysts were immunostained with an anti-a-tubulin antibody, an anti-g-tubulin antibody and DAPI.Main results and the role of chance: In the clinical study, the implantation rateof growth-retarded embryos (47%, n: 270) was significantly lower (P n:608). However, there were no differences in the chromosomal abnormalitiesof abortuses and the incidences of peromelus of babies between 2 groups. Inthe experimental study, a total of 533 spindles were analyzed in both day 5and day 6 blastocysts. Confocal image analysis was accomplished by captur-ing a z-series stack of 0.5-mm-thick optical sections encompassing the entireblastocyst. Only spindles with fusiform poles and with chromosomes alignedat the equator were classified as normal. The incidence of abnormal spindlesin growth-retarded embryos (47%, n: 274) was significantly higher(P n: 259).Limitations, reason for caution: Further studies are required to clarify the linkbetween an increase in abnormal spindle formation and a decrease in embryonicimplantation potential.

Wider implications of the findings: This study provided new insights on theimplications of an increase in abnormal spindle formation in growth-retardedhuman blastocysts.Study funding/competing interest(s): Part of this work was supported by a grantfrom the Japan Society for the Promotion of Science (JPS-RFTF 23580397 toS.H.). No other competing interests are declared.Trial registration number: None.

P-093 In vitro maturation of human oocytes selected by Brilliant CresylBlue staining

D.D. Alcoba1, E.G. Valerio2, M. Conzatti1, J. Tornquist1, A.P. Kussler3,A.M. Pimentel3, H.E. Corleta3, and I.S. Brum1

1Universidade Federal do Rio Grande do Sul, Departamento de Fisiologia, PortoAlegre, Brazil, 2Hospital de Clınicas de Porto Alegre, Servico de Ginecologia eObstetrıcia, Porto Alegre, Brazil, 3Hospital Moinhos de Vento, Nucleo deReproducao Humana Gerar, Porto Alegre, Brazil

Study question: Evaluate the efficiency and safety of Brilliant Cresyl Blue (BCB)staining as a selection of developmentally competent immature human oocytesbefore in vitro maturation (IVM).Summary answer: BCB test can be a good marker in pre-selection procedures ofdevelopmentally competent human oocytes and, apparently, it is not toxic to thegamete.What is known already: Growing oocytes synthesize glucose-6-phosphate de-hydrogenase (G6PDH), but this enzyme is inactivated in oocytes that have com-pleted their growth phase. Once BCB can be reduced by G6PDH, the selectionof developmentally competent oocytes before IVM by BCB staining is widelyused for many animal species (bovine, porcine, mice, etc). Using animalmodels, it has been already demonstrated that this selection can improve notonly nuclear and cytoplasmic oocyte maturation, but also fertilization and blasto-cyst rates.Study design, size, duration: Cross sectional experiment – control versus treat-ment groups. Three kinds of patients were included in the study: 6 hormonal sti-mulated patients with immature oocytes at retrieval (17 oocytes); 3ooforectomized patients (20 oocytes recovered); and 32 pregnant patientsduring cesarean section (92 oocytes recovered).Participants/materials, setting, methods: Immature oocytes were divided intogroups: control (disposed directly to IVM) and treated - exposed to BCB26 mM during 60 minutes. After staining, treated group was classified as cyto-plasm coloration, BCB positive or negative, and then disposed to IVM. After 24and 48 hours of IVM nuclear status was checked.Main results and the role of chance: The IVM rate of immature oocytes recov-ered from stimulated and ooforectomized patients was equal among groups(control, BCB positive and BCB negative) either after 24 or 48 hours of IVM.Nevertheless, IVM was higher in BCB positive compared to BCB negative after24 and 48 hours of IVM analyzing oocytes recovered from all patients (P ¼0.024 and P ¼ 0.015) and from cesarean patients (P ¼ 0.004 and P ¼ 0.032).The control group was equal to BCB positive. The meiosis resumption rate(absence of germinal vesicle) after 24 hours of IVM was equal among groups,but higher in BCB positive compared to other groups after 48 hours when allgametes were analyzed jointly (P ¼ 0.035). Degeneration rate was lower inBCB positive compared to their counterparts when oocytes were analyzedjointly (P ¼ 0.002).Limitations, reason for caution: Human oocytes that can be destined to researchare limited. We have to look for effects of ovarian puncture during cesarean sectionafter some years. As the eggs were not fertilized, embryo characteristics were notaccessed; so the effects of BCB on human embryo development remain unknown.Wider implications of the findings: In human oocytes the IVM rate of BCB posi-tive was higher than that observed for BCB negative, but equal to control group.The degeneration rate was lower in BCB positive oocytes, compared to othergroups (control and BCB negative). These results were similar to that describedin animals.Study funding/competing interest(s): The experiment was supported by univer-sity hospital and it has not any vested conflict.Trial registration number: Not applicable.

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P-094 Local evaluation and validation of open systemoocyte vitrificationmethod

P. Boyer, D. Montjean, P. Tourame, and M. Gervoise-BoyerHopital Saint Joseph, Reproductive Medicine and Embryology Department,Marseille, France

Study question: Is there a really need to validate oocyte vitrification technique inan IVF laboratory before establishing it in daily practice?Summary answer: Validation of micromanipulation-based technique, in thiscase oocyte vitrification, is essential to evaluate its efficiency prior to enlargingthe use of the system to routine practice.What is known already: Oocyte vitrification is a new worldwide used techniqueand legal recently in France. This micromanipulation needs to be performed by aconfirmed embryologist and requires an internal assessment in each IVF unitbefore any wide use.Study design, size, duration: We designed a prospective study, from September2011 to July 2012, using sibling oocytes from women who recovered more than 12Metaphase II oocytes. A part of freshly recovered oocytes underwent immediateICSI while the remaining oocytes were vitrified.Participants/materials, setting, methods: 62 couples (872 Metaphase IIoocytes) undergoing ICSI were selected based on number of oocytes availableat recovery. 405 Metaphase II oocytes were microinjected after retrieval and467 were vitrified using an open system (Cryotopw).Main results and the role of chance: To date, 145 oocytes have been thawed for24 couples. This allowed us to estimate both the mean survival rate (84.9 %+17.9) and fertilisation rate after oocytes thawing at 66.9 %+ 32.9 comparedto 56.4 % +27.8 in freshly recovered oocyte sub-group No statistical differencewas found.

The number of embryos transferred/number of Metaphase II ratio is 38/157 forfresh oocytes and 39/145 for warmed oocytes (p . 0.05). Among the 62 includedcouples, 21 clinical pregnancies were recorded in the fresh ICSI oocytes and 6were registered in the vitrified/warmed oocyte cohort. Interestingly, the presentevaluation led to a satisfactory cumulative pregnancy rate: 43.5%. One additionalbenefit of such practice is the limitation of embryo freezing.Limitations, reason for caution: The study design delays oocytes warmingcycles, due to pregnancies triggered by the transfer of fresh derived oocyteembryos and to the priority to transfer all the frozen embryos before startingoocytes warming. Furthermore, no data is available about children’ health.Wider implications of the findings: Oocyte vitrification represents a dramaticchange in our daily practice. Indeed, it enables us to be in line with the will oflocal authorities to decrease the number of cryopreserved embryos while ensuringsatisfactory pregnancy chances. The encouraging results suggest that oocyte vit-rification may also be used for a larger panel of purposes including oocyte storingin cases of sperm retrieval failure, fertility preservation or egg donation.Study funding/competing interest(s): Kitazato BioPharma provided Cryotopsafety Kits.Trial registration number: Clinical Trials Registration number: 209 R02

P-095 Oocyte ERM proteins play a crucial role in gamete adhesion/fusion

J. Cohen, B. Lefevre, C. Ialy Radio, J.P. Wolf, and A. ZiyyatUniversite Paris Descartes Institut Cochin, Inserm U1016, Paris, France

Study question: Using siRNA intracytoplasmic injection in mouse oocytes, thepurpose of the study was to determine the role of oocyte Ezrin, Radixin andMoesin (ERM) proteins in gamete adhesion/fusion.Summary answer: We found that fertilization index (mean number of spermfused by zona pellucida free egg) was 57% decreased, and that oocyte microvilliwere thicker after oocyte ERM inhibition.What is known already: On the mammalian oocyte, Cd9 is the only moleculeknown to be essential for gamete membrane adhesion/fusion. In the absence ofCd9, oocyte microvilli are thicker and adhesion exists but remains abortive.Cd9 generates adhesion sites, named pro-fusional, with strong adhesion. This ad-hesion is strong because of the connection between the membrane and the cyto-skeleton, which is realized indirectly through other molecules, Cd9 partners,such as ERM that connect actin cytoskeleton to plasma membrane.

Study design, size, duration: We performed a cross sectional (control versustreatment) study. Number of oocytes: 149 (siRNA control injected¼ 82 /siRNA ERM injected ¼ 67). The oocytes where injected at the germinal-vesiclestage oocytes (GV).Participants/materials, setting, methods: Animals were 6 weeks-old femalesB6CBAF1 mice.

We realized RNA silencing by oocyte intracytoplasmic siRNA microinjectionunder microscopic control at GV stage oocytes.

Zona pellucida was removed before IVF. Efficiency of the protein silencing andFertilization Index (FI) were assessed by immunofluorescence (Dapi). Oocytemicrovilli morphology was studied by electronic microscopy.Main results and the role of chance: Efficiency of the silencing was assessed bythe decrease or the disappearance of oocyte membrane ERM. The fluorescenceintensity was quantified with specialized software.

After ERM combined siRNAs injection (N ¼ 67 oocytes), Fertilization Indexwas 1.2 sperm per egg and 2.8 after control si RNA injection (N ¼ 82 oocytes),which means a 57% decrease (p , 0,001).

After ERM siRNAs injection (N microvilli ¼ 521; N oocytes ¼ 5), oocytemicrovilli radius of curvature was 37.8% increased (34.6 nm versus 47.7 nm;p , 0,001) compared to control injected siRNA (N microvilli ¼ 350;N oocytes ¼ 5).

Fertilization Index was not reduced when silencing separately Ezrin, Radixin orMoesin.Limitations, reason for caution: The main limitation of this study is the partialinhibition provided by RNA silencing. It was impossible to use a triple knock-outmodel because of the premature lethality of Ezrin2/- mice.Wider implications of the findings: This work provides new elements in theunderstanding of Cd9 mechanisms in gamete adhesion/fusion. These observa-tions are a strong argument in favor of the cis-action of Cd9, which is the mostlikely mechanism: gamete fusion could succeed thanks to a strong link betweenplasma membrane and its underlying cytoskeleton mediated by Cd9 and ERMproteins.Study funding/competing interest(s): We declare no competing interest.Trial registration number: No registration number.

P-096 Clinical outcome of vitrified single blastocyst transfer as related toblastocyst morphology before vitrification

I. De Croo, A. Tolpe, S. Degheselle, A. Van de Velde, K. Tilleman, P. De Sutter,and E. Van den AbbeelGhent University Hospital, Centre for Reproductive Medicine, Ghent, Belgium

Study question: How does the clinical outcome of vitrified single blastocysttransfer compare between poor-quality and good-quality blastocysts before vitri-fication?Summary answer: Vitrification and warming of poor-quality blastocysts hasclinical significance since in our hands acceptable clinical outcome is achievedas compared to vitrification and warming of good-quality blastocysts.What is known already: Results with blastocyst transfer allow the use of singleembryo transfer, thereby reducing the incidence of multiple pregnancies. Withsuccessful vitrification techniques, cryopreserved embryo transfer cycles havebecome an integral part of IVF treatment. In fresh cycles, several reports have indi-cated the importance of the blastocyst morphology on clinical outcome after trans-fer. In most IVF centers poor-quality blastocysts are not cryopreserved andtherefore little is known about their clinical outcome after vitrification.Study design, size, duration: Between February 1st 2012 to October 10th 2012,462 vitrified blastocysts were warmed in 366 warming cycles. After warming andovernight culture, we retrospectively investigated clinical pregnancy rates (CPR)as related to blastocyst morphology before vitrification in 223 single vitrifiedblastocyst transfer (SVBT).Participants/materials, setting, methods: Blastocysts were graded according toSchoolcraft&Gardner (1999). Early, full, expanded and hatching blastocysts werevitrified. On day 5, early, full and expanded/hatching blastocysts with an ICM Cand/or a TE C were considered poor-quality blastocysts. Full and expanded/hatch-ing blastocysts with ICM A/B and/or TE A/B were considered good-quality blas-tocysts.Main results and the role of chance: Morphological survival rate immediatelyafter warming was 85.7% (396/462). After overnight culture, we performed

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278 vitrified blastocyst transfers. Overall pregnancy rate (% positive HCG)was 40.3%/ET and 30.6% per warming cycle. CPR was 29.1%/ET with animplantation rate of 21.4% (99/462) per warmed blastocyst. More specifical-ly and independent of ICM and TE quality, CPR per SVBT was 19.1% (14/73), 27.9% (17/61) and 27.0% (24/89) for early, full and expanded blasto-cysts respectively (p ¼ 0.3). In the expanded blastocyst group with ICM orTE grade C, CPR was 25.9% (7/27) compared to 28.4% (35/123) in thegroup with ICM and TE grade A and B. Taken together the CPR perSVTB was 21% (21/100) for poor-quality blastocysts versus 28.4% 35/123) for good-quality blastocysts (p ¼ 0.2).Limitations, reason for caution: Much larger study groups are required to evalu-ate clinical pregnancy and miscarriage rates by the specific blastocyst morpho-logical scores especially as related to the different types of ICM and TE in fulland expanded/expanding blastocysts.Wider implications of the findings: Our findings could change cryopreservationpolicies in IVF centres performing blastocyst vitrification because so far in mostcentres only good-quality blastocysts are considered eligible for vitrification.Study funding/competing interest(s): This study was not funded and there wereno competing interests.Trial registration number: Inapplicable.

P-097 A comparison of two different vitrification methods for cryo-preservation of mature human oocytes

S. Kagalwala1, G. Gandhi1, G. Allahbadia2, M. Kuwayama3, A. Allahbadia2,V. Chipkar1, A. Khatoon1, R. Ramani1, M. Madne1, and S. Alsule1

1Rotunda CHR, Assisted Reproduction Laboratory, Mumbai, India, 2RotundaCHR, Assisted Reproduction, Mumbai, India, 3Repro-Support Medical ResearchCenter, Assisted Reproduction, Tokyo, Japan

Study question: To compare the efficacy of two different methods of vitrificationfor cryopreservation of human oocytes: The Cryotop method and the Cryotechmethod.Summary answer: Cryotech vitrification method for oocyte cryopreservationgives slightly higher survival and fertilization rates and significantly higher cleav-age rate compared to the Cryotop method.What is known already: Vitrification is a highly effective method for successfulcryopreservation of oocytes. Various media, methods and carrier devices are beingused for optimizing the vitrification technique. Cryotop vitrification method isknown to give excellent results. The Cryotech method is different from theCryotop method in terms of the constituents of its solutions as well as thedesign of the plates and the carrier device.Study design, size, duration: This is a retrospective data analysis of donor egg-IVF cycles using vitrified oocytes from October 2010 to August 2012. Theoocytes were vitrified using either the cryotop or cryotech vitrification method.A total of 611 mature oocytes were vitrified and 131 embryo transfer cycleswere performed using the embryos created after fertilizing the warmed oocyteswith ICSI.Participants/materials, setting, methods: Donor oocytes were vitrified usingeither cryotech or cryotop vitrification method. The frozen oocytes were thenwarmed using the respective warming media. The surviving oocytes were ferti-lized by ICSI. Embryo transfer was performed in the recipients using theseembryos. The survival, fertilization, cleavage and the clinical pregnancy rateswere compared in the two groups.Main results and the role of chance: A total of 275 mature oocytes were vitrifiedusing cryotech method and 336 mature oocytes using cryotop method. The meanage of the egg donors was 25.3+2.8 years and 24.8+2.4 years for Cryotech andCryotop methods respectively. The survival rate of warmed oocytes with Cryotechmedia was 97.1% (n ¼ 267/275) while for Cryotop media it was 95.1% (n ¼ 319/336; p ¼ 0.19). The fertilization rate of Cryotech group was 90.7% (n ¼ 240/267)and Cryotop group was 86.1% (n ¼ 274/319; p ¼ 0.05). The cleavage rate forCryotech group was 96.8% (n ¼ 232/240) and Cryotop group was 91.9% (n ¼251/274; p ¼ 0.01). The clinical pregnancy rate for Cryotech group was 54.8%(n ¼ 34/62) and Cryotop group was 40.6% (n¼ 28/69).Limitations, reason for caution: A limitation of this study is that it is not doneusing sibling oocytes. However, there was no statistically significant differencein the age and fertility parameters of the donors of both the groups.

Wider implications of the findings: Even though the survival rate of oocytes inboth the groups did not show a statistically significant difference, the embryo de-velopment was better in the Cryotech group, as reflected by higher cleavage rate.This may be attributed to less trauma to the oocytes vitrified and warmed usingCryotech method. The Cryotech method would be very useful in building donoroocyte banks and fertility preservation of cancer patients.Study funding/competing interest(s): No funding was used.Trial registration number: Not Applicable

P-098 Influenceof storageperiod of vitrified embryos on clinical outcome

M. Inaba1, A. Ohgaki1, A. Ohtani1, H. Matsumoto1, S. Mizuno1, R. Mori2,A. Fukuda2, and Y. Morimoto3

1IVF osaka clinic, Division of reproductive technology, Osaka, Japan, 2IVF osakaclinic, Doctor, Osaka, Japan, 3IVF namba clinic, Doctor, Osaka, Japan

Study question: The present study was conducted to investigate the influence ofvitrification on laboratory and clinical outcomes depending on the period ofstorage in the stage of 2PN, cleaved embryo and blastocyst.Summary answer: The present study suggested that cryopreservation of embryosat any stages by vitrification had no detrimental effect on cryosurvival and clinicaloutcome.What is known already: Frozen-thawed or vitrified-warmed embryo transfer(FET) in ART has been recently increasing with not only an advancement of cryo-preservation techniquesuch as vitrification, but also a trend of single embryo trans-fer (SET) to prevent multiple pregnancy. Cryopreservation of embryos by slowfreezing method has been used more than 30 years and the influence of thismethod has been evaluated. However, vittrificaion is a relatively new techniqueand influence of vitrification on clinical outcome has not been elucidated.Study design, size, duration: In 3392 FET cycles of SET (1674 patients), 8796embryos (6069 2PNs, 1573 cleaved embryos and 1154 blastocysts) werewarmed for transfer from January 2010 to December 2012. Data obtained fromthese embryos were analyzed retrospectively.Participants/materials, setting, methods: Those embryos were divided into 4groups based on the storage period, A: ,1 year, B: 1-2 years, C: 2-3 years, D:.3 years. Survival after warming in each period was compared in each stage ofembryos. Moreover, the influence of storage period on the rates pregnancy andlive birth were compared.Main results and the role of chance: 1. Survival rates in group A, B, C, D andaverage were as follows. 2PN: 98.0%, 98.7%, 100.0%, 100.0% and 98.1%.Cleaved embryo: 96.3%, 95.1%, 94.9%, 100.0% and 96.1%, respectively. Blasto-cyst: 97.9%, 97.8%, 100.0%, 100.0% and 97.9%, respectively. There were no sig-nificant differences among these 4 groups in each stage.

2. Pregnancy rates in group A, B, C, D and average were as follows. Cleavedembryos: 32.6%, 42.9%, 26.7 % and 25.0% and 33.0%. Blastocyst: 42.9%,45.7%, 56.3% and 41.7%, and 43.7%, respectively. Live birth rates were asfollows. Cleaved embryos: 76.5%, 83.3 %, 100.0%, 50.0%, and 77.1%. Blasto-cyst: 80.5%, 79.3%, 77.8%, 60.0%, and 80.0%, respectively. There were no sig-nificant differences among 4 groups either in cleaved embryo or blastocyst.Limitations, reason for caution: NoneWider implications of the findings: The present study suggested that cryopreser-vation of embryos at any stages by vitrification had no detrimental effect on cryo-survival and clinical outcome regardless of storage period for at least 3 years. Thelongest period was 5 years and 6 months with healthy baby born. Vitrification isvery effective, laboratory friendly and safe cryopreservation method.Study funding/competing interest(s): NoneTrial registration number: None

P-099 Outcome of blastocyst vitrification with cryoloops: clinicaloutcome of 2,924 cycles comparing with 2,003 fresh blastocyst transfer cycles

Y. Umekawa, A. Yoshida, S. Tanigiwa, K. Seida, H. Suzuki, and M. TanakaKiba Park Clinic, Tokyo, Japan

Study question: Is ultra rapid vitrification method of blastocysts using a cryoloopsafe and effective?

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Summary answer: Ultra rapid vitrification method of blastocysts using a cryo-loop is a safe and valid method as a result of comparison between vitrified-thawedblastocyst ET and fresh blastocyst ET cycles.What is known already: Blastocyst transfer has become a promising option to getthe higher pregnancy rate, comparing with cleavage stage embryo transfer. Vitri-fication is expected to achieve a high rate of survival because of the absence of ice.Vitrification has become the method to cryopreserve blastocysts.Study design, size, duration: This is a retrospective analysis of clinical outcomein 2,924 vitrified-thawed blastocyst ET cycles and 2,003 fresh blastocyst ETcycles conducted for women under the age of 40 from 2002 to 2011.Participants/materials, setting, methods: IVFand ICSI procedures were carriedout with sequential media. The protocol for the cryoloop vitrification of blasto-cysts from IVF/ICSI patients was adopted. Embryo transfers were performedunder ultrasound examination using a soft catheter.Main results and the role of chance: The average embryo transfer number ofvitrified-thawed blastocyst transfer was significantly lower than that of freshblastocyst transfer (1.4+0.5 vs 1.5+0.6: PLimitations, reason for caution: Long term follow-up of children from ultrarapid vitrification method of blastocysts using a cryoloop, including motor andpsychological development, is recommended.Wider implications of the findings: Ultra rapid cryoloop vitrification methodof blastocysts is easy-to use, safe and effective. Outcome of ultra rapid cryoloopvitrification method of blastocysts is comparable with that of fresh blastocysttransfer.Studyfunding/competing interest(s): This study was not funded and there are noconflicts of interest.Trial registration number: n/a

P-100 Anthocyanin improves viability and maturation of sheep oocytescultured In the presence of cAMP modulators

Z. Vahabi1, P. Eftekhari Yazdi1, A. Dalman1, B. Ebrahimi1, F. Mostafaei2, andM. Rajabpour Niknam1

1Department of Embryology Reproductive Biomedicine Research Center, RoyanInstitute for Reproductive Biomedicine ACECR, Tehran, Iran, 2Animal CoreFacility Reproductive Biomedicine Research Center, Royan Institute for AnimalBiotechnology ACECR, Tehran, Iran

Study question: Can high levels of cAMP alone support sheep cultured oocytesfrom apoptosis?Summary answer: Our findings showed that, cAMP modulators couldn’tsupport maturation and viability of sheep oocytes. While the Pre-IVM and IVMmediums supplemented with delphinidin chloride could significantly Decreaseapoptosis and increase maturation of sheep oocytes.What is known already: To our knowledge, no studies to data have focused onapoptosis repression in sheep oocytes cultured at high levels of cAMP.Study design, size, duration: Cumulus-oocyte complexes (COCs) were culturedin two biphasic mediums: 1) Pre-IVM and IVM mediums supplemented with del-phinidin chloride and 2) Pre-IVM and IVM mediums without delphinidin chlor-ide. Differences in maturation and viability rates between COCs cultured in twoexperimental groups were determined by the Chi-square test.Participants/materials, setting, methods: COCs were recovered from ovineovaries collected at a local slaughterhouse. Good-quality COCs were randomlytransferred into two biphasic IVM mediums. After maturation period, oocyteswere denuded from cumulus cells and MII oocytes with first polar body werecounted. Oocyte viability was determined by means of the TUNEL technique.Main results and the role of chance: The percentage of oocytes that reachedmetaphase II was significantly higher for group 1 (93.5% and 73.0%, respectivelyP , 0.0001) than group 2 oocytes. The percentage of viable oocytes at group 1was higher ( 98.9% and 85.1%, respectively P , 0.001) than group 2 oocytes.

Maturation rate and viability detection by TUNEL kit in two experimentalgroups in sheep oocytes.Limitations, reason for caution: We have any limitation.Wider implications of the findings: Some studies reported that, High levels ofcAMP suppress apoptosis by inhibition of Caspase 3 activation. In additionArnault and Colleagues (2008) showed that caspases may be dispensable forfinal oocyte death in mouse. In this study we used delphinidin chloride, the acti-vator of GSH synthesis. Based on our findings, it seems that GSH synthesis

activators more effective than inhibitors of Caspase-3 activators in oocyte deathcontrol.Study funding/competing interest(s): This work was supported by EmbryologyDepartment of Royan Institute.Trial registration number: Our study wasn’t clinical trial

P-101 Correlation of the duration of the first cleavage cycle of embryoswith good quality blastocyst and implantation rates in single frozen-thawedblastocyst transfer

S. Watanabe1, M. Kamihata1, T. Tanaka1, R. Matsunaga1, N. Yamanaka1, C. Kani1,T. Ishikawa1, T. Wada1, H. Morita1, H. Miyamura2, E. Nishio2, M. Ito3, A. Kuwahata3,M. Ochi3, and T. Horiuchi41Ochi Yume Clinic Nagoya, ART lab., Nagoya, Japan, 2Fujita Health UniversityHospital, Obstetrics and Gynecology, Toyoake, Japan, 3Ochi Yume ClinicNagoya, Reproductive Medicine, Nagoya, Japan, 4Prefectural University ofHiroshima, Graduate School of Comprehensive Scientific Research, Shobara,Japan

Study question: Is there any correlation of the first cleavage duration of embryoswith the success rate of obtaining good quality blastocysts and the implantationrates in single frozen-thawed blastocyst transfer cycles?Summary answer: There is a correlation between the duration of the first cleav-age cycle of embryos and good quality blastocyst and implantation rates.What is known already: It has been reported that there is a correlation betweenthe duration of the first cleavage cycle of an embryo and the formation of a viableblastocyst.Study design, size, duration: A retrospective study on 357 embryos from 182cycles which were cultured to the blastocyst stage by using a time-lapse incubatorbetween July and October 2012.Participants/materials, setting, methods: We recorded and analyzed the dur-ation of the first cleavage of embryos by using a time-lapse incubator (Embryo-Scope, Unisense Fertilitech, Denmark), to examine the correlation of the firstcleavage duration with the success rate of obtaining good quality blastocystsand with the pregnancy rate in single frozen-thawed blastocyst transfer cycles.Main results and the role of chance: Among the 357 normal fertilized embryoscultured by using the ES, 124 (34.7%) were good quality blastocysts. The durationof the first cleavage in the good blastocyst group and the non-good-quality blasto-cyst group (mean+SD) was 25.4+2.8 and 28.7+4.6 hours, respectively,which showed a significant difference (P , 0.01). Seventy-three singlefrozen-thawed blastocyst transfers were performed in the good blastocystgroup. The pregnancy rate of the group with a first cleavage duration of 24–25hours (69.2%, 9 of 13) tended to be greater than that of the 20–24 hours group(44.4%, 12 of 27) (P ¼ 0.186), and significantly greater than that of the 25–35hours group (27.3%, 9 of 33) (P , 0.05).Limitations, reason for caution: None.Wider implications of the findings: We confirmed that the duration of the firstcleavage cycle of embryos might be an effective parameter for ensuring viablehigh- quality blastocysts for transfer.Study funding/competing interest(s): No external funding was obtained forthis study.Trial registration number: None.

.........................................................................................

Experimentalgroups

Oocytes Maturedoocytes

OocytesforTUNEL

Viableoocytes

0.1 mg/mldelphinidinchloride (group 1)

217 202 (93.5%) 90 89

withoutdelphinidinchloride (group 2)

215 175 (73.0%) 87 74

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P-102 In vitro maturation does not affect the morphology of the meta-phase II spindle in oocytes collected from antral follicles in IVM cycles

M. Dal Canto1, M.C. Guglielmo1, R. Fadini1, M. Mignini Renzini1, D.F. Albertini2,P. Novara1, M. Lain1, F. Brambillasca1, D. Turchi1, M. Sottocornola1, andG. Coticchio1

1Biogenesi Reproductive Medicine Centre, Istituti Clinici Zucchi, Monza, Italy,2Department of Molecular and Integrative Physiology, University of KansasMedical Center, Kansas City, U.S.A

Study question: In vitro maturation (IVM) might affect the organization and func-tion of the metaphase II (MII) spindle, compromising oocyte quality. The studyobject was to assess comparatively the morphology of the MII spindle inoocytes matured in vitro from IVM cycles and oocytes matured in vivo in stimu-lated cycles (IVO).Summary answer: IVM does not alter the geometry of the MII spindle in humanoocytes recovered in IVM cycles matured in vitro. In addition, distribution of cor-tical actin appears to be polarized showing an accumulation in proximity of thespindle, perhaps to ensure localization and anchorage of this cytoskeletal compo-nent.What is known already: In the mouse, it is widely recognized that in vitro mat-uration can alter the structure of the oocyte MII spindle. This could have implica-tions for chromatid segregation and embryo chromosome status. In the human, ithas been repeatedly reported that IVM oocytes may be affected by spindle anom-alies, but such findings are questionable because were generated using a rather in-appropriate model, i.e. leftover denuded germinal vesicle (GV) oocytes obtainedfrom conventional ovarian stimulation cycles.Study design, size, duration: Fifty-nine IVO and 10 IVM oocytes were analysed.No leftovers GV oocytes from stimulated cycles were included. Chromosomeswere classified as displaced or aligned at MII plate. Their internal and external ar-rangement was classified as two perfect rings or partially/totally disarranged. Cor-tical and cytoplasmic actin was also compared.Participants/materials, setting, methods: IVO oocytes derived from womenundergoing conventional ovarian stimulation. IVM oocytes matured in vitrofrom cumulus-oocyte complexes in the presence of FSH/HCG. Chromatin,tubulin and actin were detected by fluorescence confocal microscopy. Spindleswere reconstructed as 3D images. P ,0.05 was adopted as criterion to considerdifferences statistically significant.Main results and the role of chance: The MII spindles of the two groups weremorphologically equivalent as shown by comparing pole-to-pole axis (11.8+2.6 mm in IVO vs. 12.0+2.3 mm in IVM oocytes), equatorial axis (8.9+1.7 mm in IVO vs. 8.6+1.5 mm in IVM oocytes), and area of maximum projection(88.8+29.5 mm2 in IVO vs. 94.7+18.1 mm2 in IVM oocytes). Chromosomealignment and internal/external disposition were not related to spindle characteris-tics. Proportions of normal chromosome arrangement were 64.5% and 60.0% inIVO and IVM oocytes respectively. Signal intensity of cortical actin was highernear the spindle, inbothoocyte types. Conversely, the mean intensityofcytoplasmicactin in IVM oocytes was higher than in IVO oocytes. (21.1 vs. 35.0).Limitations, reason for caution: Data regarding IVM oocytes are numericallylimited and require further confirmation.Wider implications of the findings: This is thefirst studyfocusingonspindlechar-acteristics in oocytes obtained from normo-ovulatory patients undergoing IVM.Data suggest that IVM conditions do not affect the structure of the MII spindle, dis-puting a widespread preconception. Moreover, this study introduces new criteria forthe analysis of chromosome arrangement and reveals a polarization of cortical actin.Accumulation of cytoplasmic actin in immature oocytes suggests the basis forfutures studies aimed at understanding spindle morphogenesis during IVM.Study funding/competing interest(s): This study was partly funded by the ItalianMinistry of Health. The authors declare to have no interests conflicting with the study.Trial registration number: Not applicable

P-103 Effects of sperm concentration after swim-up on conventional invitro fertilization rates: Analysis of sperm motility using a sperm motilityanalysis system

M. Kato1, N. Fukunaga2, R. Nagai1, H. Kitasaka2, T. Yoshimura1, F. Tamura2,N. Hasegawa1, K. Nakayama2, M. Takeuchi2, H. Ohno2, N. Aoyagi1, E. Kojima1,F. Itoi3, Y. Hashiba4, and Y. Asada5

1Asada Ladies Kachigawa Clinic, Laboratory, Aichi, Japan, 2Asada LadiesNagoya Clinic, Laboratory, Aichi, Japan, 3The Asada Institute for ReproductiveMedicine, Laboratory, Aichi, Japan, 4Asada Ladies Kachigawa Clinic, Doctor,Aichi, Japan, 5Asada Ladies Nagoya Clinic, Doctor, Aichi, Japan

Study question: Does sperm concentration after swim-up affect fertilization ratesin conventional in vitro fertilization (C-IVF)?Summary answer: It is thought that using a high concentration of spermatozoawith motility after swim-up and high curve speed and head amplitude improvethe normal fertilization rate of C-IVF.What is known already: In conventional in vitro fertilization, it is important toimprove the rate of normal fertilization. Our C-IVF procedure entails adjustingthe count to 200,000 spermatozoa after swim-up. However, there may be differ-ences in some cases regarding the normal fertilization rate with our procedure,and the reason for this is currently not clear. To address this problem, we used asperm motility analysis system (SMAS) for the first time in our clinic.Study design, size, duration: Concentration of spermatozoa with motility afterswim-up was divided into two groups: not less than 1.0 × 106/ml and not morethan 2.0 × 106/ml (Group A); and not less than 2.1 × 106/ml and not more than14.9 × 106/ml (Group B). Fertilization rates were compared in this retrospectivestudy.Participants/materials, setting, methods: From January through May 2011, weconducted a review of 524 cycles in 483 patients. In addition, we conducted areview of 42 cycles in 42 patients between May 2012 and September 2012. Themotility rates of Group A and group B were analyzed via SMAS.Main results and the role of chance: The normal fertilization rate of Group Awas48%; that of Group B was 63.0%. Thus, the normal fertilization rate was signifi-cantly higher in Group B compared to Group A (P , /SSF . , 0.01). The rateof abnormal fertilization (1PN, ≥3PN) of Group A was 6.1%; that of Group Bwas 5.8%. With SMAS, the straight line speed, straight advancement, curvespeed, and head amplitude were 31.5 mm/s, 0.36, 96.1 mm/s, and 2.04 mm, re-spectively for Group A and 34.8 mm/s, 0.34, 112.5 mm/s, 2.68 mm, respectivelyfor Group B. Group B had a higher curve speed and head amplitude than Group A.Limitations, reason for caution: These are preliminary data from a retrospectiveanalysis with a limited sample size.Wider implications of the findings: Not determinable until further studies usingSMAS with a larger sample have been performed.Study funding/competing interest(s): None.Trial registration number: None.

P-104 Study of the most effective embryo transfer with blastocyst grade

H. Kikuchi, Y. Iwasa, T. Kamono, A. Suzuki, K. Yamada, H. Kanno, K. Sasaki,H. Murakawa, M. Matsubara, and H. YoshidaYoshida Ladies Clinic, Center for Reproductive Medicine, Sendai, Japan

Study question: It has reported that blastocyst transfer is able to obtain betterresult by synchronization with endometrium. However, depends on the situation,such as priming egg retrieval or patient’s offer, it is necessary to screen the blasto-cyst for fresh transfer.Summaryanswer:Regarding to blastocyst ICM andTE grade, grade A blastocystresulted in significantly high pregnancy rate in frozen-thawed cycle. It is provedthat frozen-thawed cycle is susceptible to improve the pregnancy rate by classifi-cation of grade. It is suggested that not only good embryos but inferior ones areworth cryopreservation.What is known already: Recently, the viability after thawing is getting remark-ably high through the improvement of cryopreservation vitrification. It hasreported that blastocyst transfer is able to obtain better result by synchronizationwith endometrium.Study design, size, duration: We classified 141 cases (138 patients) in fresh cycleand 673 cases (473 patients) in frozen-thawed cycle, which underwent singleblastocyst transfer from January 2010 through October 2012 in our clinic.Participants/materials, setting, methods: We compared the clinical offspringand abortion rate in fresh cycle and frozen-thawed cycle with Gardner’s classifica-tion which were Inner Cell Mass (ICM) and Trophectoderm (TE) cell numbers.Main results and the role of chance: The patient mean age was 33.8+3.8 yearsold in fresh cycle, 35.6+4.0 years old in frozen-thawed, so there was a significantdifference (P , 0.05). In the view of ICM grade, the pregnancy rate infrozen-thawed cycle is significantly high in ICM grade A (P , 0.05) although

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no significant difference in fresh pregnancy cycle (P , 0.005). In the view of TEgrade, the pregnancy rate in frozen-thawed cycle is significantly high in TE gradeA (P , 0.025). As compared with each grade in same group, TE grade A has sig-nificantly high rate, and grade B was the following in frozen-thawed cycle thoughno significant difference was observed in fresh cycle.Limitations, reason for caution: None.Wider implications of the findings: We suggested that the best way to improvetotal pregnancy rate per oocyte pick-up is need to cryopreservation for effectivefrozen-thawed cycle in case of good quality embryo was obtained, and thenperform fresh second grade embryo transfer.Study funding/competing interest(s): No competing interest is declared.Trial registration number: None.

P-105 Comparison of four techniques for artificial shrinkage of theblastocoele cavity pre-vitrification

C. Valdespin1, M. Elhelaly2, P. Chen3, M. Pangestu3, and S. Catt3

1Nascere, Reproductive Medicine, Mexico City, Mexico, 2Al-ahram FertilityCenter, Embryology, Mansoura, Egypt, 3Monash University, Obstetrics &Gynecology, Melbourne, Australia

Study question: Pre-vitrification artificial shrinkage: are hyperosmotic solutionsas effective as laser pulse or micro-puncture techniques?.Summary answer: Collapsing expanded mouse blastocysts prior to vitrificationby either laser, micro-puncture/suction or the hyperosmotic solutions: sucrose andtrehalose is associated with greater survival, expansion and hatching rates whencompared to non-collapsed controls. The novel use of trehalose provides an excel-lent low cost, low skill option with proven implantation.What is known already: Improvements in survival and implantation rates ofexpanded blastocysts have been demonstrated with laser pulse and micro-puncture collapse, but these techniques require expensive equipment or advancedskill sets. A simple hyperosmotic pre-vitrification collapsing technique usingsucrose has been tested successfully as an alternative method. We have postulatedthat trehalose may be a superior sugar to use due to its membrane stabilizationproperties.Study design, size, duration: 367 expanded blastocysts were assigned to 5groups: laser-pulse [LP], micro-puncture/suction [MS], use of trehalose pre-vitrification [TPV], and use of sucrose pre-vitrification [SPV] and compared toa control group (CG). 30 embryos/group were assigned for transfer (at 4h post-warm) to pseudo-pregnant female mice while the remaining blastocysts were cul-tured overnight.Participants/materials, setting, methods: Blastocysts were vitrified, using ashort protocol on Cryotops. Blastocysts were assessed prior to transfer for survivaland re-expansion (classed as .70% of blastocoel re-expanded) and then, for im-plantation on day 12 post-transfer. The remaining cultured blastocysts were eval-uated for re-expansion, and hatching rates at 24h post-warm.Main results and the role of chance: Overall 98.7% of all warmed blastocystssurvived as assessed at 4h, but ,50% were classed as re-expanded at transfer.At 24h, re-expansion rates were higher for the combined collapsed groups(82%, P , 0.05, Chi Square) compared to CG (66%), with TPVand LP exhibitingthe highest rates of expansion (87% and 82% respectively), and TSV and MSslightly lower (80% and 76.3% respectively). Hatching rates were also higherfor the combined collapsed groups (40%, P , 0.05) compared to the control(22%), with TVP and SVP having the highest rates (56.5% and 40% respectively),compared to CG (P, 0.01 and p , 0.05 respectively). All five groups had at leastone blastocyst implant and the highest rate was 6/30 for TPV.Limitations, reason forcaution: Although we have demonstrated that collapsinggives betteroutcomes andhyperosmotic treatmentswas successful, further studiesare required to examine these findings. Implantation was demonstrated but futuretransfers would benefit from improved synchronization as, in this study, we trans-ferred day 4.5 blastocysts into surrogate female mice 2.5 days after mating.Wider implications of the findings: Vitrification has become the popular choicefor blastocyst preservation, and in general, studies have shown that the expandedblastocyst benefits from collapse prior to vitrification. We have demonstrated thatpassing through two hyperosmotic solutions, just prior to the equilibration step inthe vitrification process, can improve post-warm outcomes, particularly when tre-halose is used. Thus, collapse can be performed cheaply and efficiently withoutthe need for specialized equipment or advanced technical skill.

Study funding/competing interest(s): No direct funding or competing interests.Trial registration number: NA

P-106 Morphokinetics of embryos acquired from poor responders aged40 years and above: comparison between natural and stimulated cycles

N. Hojnik, B. Kovacic, P. Roglic, M. Taborin, M. Zafosnik, J. Knez, andV. VlaisavljevicUniversity Medical Centre Maribor, Department of Reproductive Medicine andGynaecologic Endocrinology, Maribor, Slovenia

Study question: Is there a difference in morphokinetics of embryos derived fromnatural cycles compared to stimulated cycles within the group of poor respondersaged ≥40 years?Summary answer: There is no difference in morphokinetic parameters in groupof poor responders aged ≥40 years whether embryos derive from natural or stimu-lated cycles.What is known already: In patients with previous unsuccessful IVF attemptswith hormonal stimulation of high dosages and low number of cells retrieved,natural cycle is used as an alternative. Although the success of natural cycles inthis group of patients is relatively low, controlled ovarian hyperstimulation result-ing in small number of oocytes might not be better choice. Our interest is to definethe quality of the embryos with the morphokinetic parameters.Study design, size, duration: Prospective study included 42 poor-responderpatients, aged ≥40 years, treated in our center in year 2012. Natural cyclesgroup: 28 patients, 27 embryos for analysis. Stimulated cycles group: 14 patients,23 embryos for analysis

Participants/materials, setting, methodsDuring 3-day cultivation pictures of embryos were taken in 5-minute intervals.

Timing of the appearance and disappearance of the pronuclei, the formation of thecleavage furrow of the first and second mitosis and the beginning of the 2,3,4-cellstage was monitored. Time intervals (TI) were compared with Mann-Whitney test.Main results and the role of chance: TI from fertilization to disappearance of thepronuclei (p ¼ 0,42), TI from fertilization to 1st cleavage furrow (p ¼ 0,9), TIfrom fertilization to 2-cell stage (p ¼ 0,88), TI from 2-cell stage to 2nd cleavagefurrow (p ¼ 0,33), TI from 3-cell stage to 4-cell stage (p ¼ 0,14), TI from fertil-ization to 4-cell stage (p ¼ 0,19). We also compared the development (TI from3-cell stage to 4-cell stage) of the subset of embryos without the occurence of ab-errant or reverse divisions (14 embryo from the natural cycle group compared tothe 7 embryos from the stimulated group; p ¼ 0,09).

There seems to be no significant difference in morphokinetics of embryos fromthe two groups studied.Limitations, reason for caution: Selection of the treatment (natural/stimulated)was done according to previous attempts and patient preferences. Neverthelessboth approaches seemed equally suitable for all patients. Small sample sizeposes limitations of our study.Wider implications of the findings: Our findings are in agreement with the litera-ture that find the two approaches for treating poor responders comparable.Study funding/competing interest(s): .Trial registration number: .

P-107 Hydroxypropyl Cellulose as a novel cryoprotectant for oocyte/embryo vitrification

C. Mori, A. Yabuuchi, K. Ezoe, Y. Takayama, F. Aono, and K. KatoKato Ladies Clinic, Advancedmedical Research Institute of Fertility, Tokyo,Japan

Study question: To determine hydroxypropyl cellulose (HPC) serves as a substi-tute for serum substitute solution (SSS) as a novel cryoprotectant for oocyte/embryo vitrification.Summary answer: The use of HPC as a novel cryoprotectant is advantageouswith the higher embryo survival rate after vitrification.What is known already: Despite the proposal toward designing chemicallydefined media in human ART, SSS still has been supplemented widely in the vit-rification solution.

HPC is an inert viscoelastic polymer, harmless and non-ionic water-soluble cel-lulose known as a material for food and medical-supply additive agent and also a

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free-water retentive polymer which may acts as cryoprotectant of the vitrificationsolution.Study design, size, duration: Inbred and hybrid mouse blastocysts with/withoutzona pellucid (ZP) with different cryoresistance (inbred: C57BL/6J, n ¼ 80,hybrid: C57BL/6JxDBA/2, n ¼ 30) were selected. Generally, hybrid embryosshow better freezing tolerance than inbred. Embryos were vitrified/thawed inHPC or SSS supplemented solution with Cryotop and there survival rates werecompared using a cell viability assay kit.Participants/materials, setting, methods: 2-cell embryos were collected fromsuperovulated mice and cultured in KSOM media (37C, 5%CO2, 95% Air) for 4days to obtain blastocyts. ZP-free blastocysts were obtained by the treatmentwith acid tyrode’s solution. The survival rate of these blastocysts vitrified/thawed in HPC (1%/vol) or SSS (1%/vol) solutions was assessed.Main results and the role of chance: The rate of blastocyst development in inbred(C57BL/6) and hybrid (C57BL/6JxDBA/2: B6D2F1) 2-cell embryos was 85.3%and 86.8%, respectively. The survival rate of inbred blastocysts with ZP was sig-nificantly higher in HPC 83.4% (121/145) than in SSS 50% (70/140), whereas inhybrid blastocysts, the survival rate in both HPC and SSS was 100% (32/32 inHPC, 33/33 in SSS). The survival rate of ZP free inbred blastocysts in HPC andSSS was 67.9% (38/56) and 19.6% (9/46), respectively. The survival rate was sig-nificantly higher in HPS than SSS when embryos with less freezing tolerance(inbred blastocysts, ZP blastocysts) were selected. In addition, we observed thestickiness of different blastocysts to the Cryotop using HPC and SSS. Whenusing SSS, the blastocyst stuck to the Cryotop, but when using HPC it didn’tstick at all.Limitations, reason forcaution: The limitation of this work was the use of animalmodel only. Monitoring of outcomes with human embryos vitrified/thawed inHPC supplemented solution is imperative to convince the safety and efficacyfor transferring in the clinical settings.Wider implications of the findings: We demonstrated that HPC is advantageouswith higher survival rates of blastocysts even without ZP revealing that HPC is aneffective cyroprotectant as a substitute for SSS. We also observed that HPC over-came the stickiness of embryos during the thawing procedure suggesting that HPCmay minimize technical complications. Our study shows that the use of HPC as acryoprotectant will be applicable and would be a standard agent in human ART.Study funding/competing interest(s): The authors declare that they have no con-flicts of interest in the research.Trial registration number: N/A

P-108 Morphokinetics and time-lapse techniqueas a predictorof embryodevelopment to the blastocyst stage

P. Radwan, R. Krasinski, K. Chorobik, and M. RadwanGameta Szpital, Department of Repoduction, Rzgow, Poland

Study question: Does the dynamic parameters of cell division assessed by time-lapse analysis predicts selection of most viable embryos for embryotransfer ?Summary answer: Adapted for embryology, the time lapse analysis enables thecontinous monitoring of embryo development and observation of cell division ab-normalities and can optimize the selection of most viable embryos for embryo-transfer.What is known already: The standard assessment of embryo morphology atgiven time points not always allows to transfer the embryo with highest im-plantation potential. The effect of transfer of the improper embryo is the lackof pregnancy or miscarriage and, as a consequence, exposure of patient tounnecessary emotional stress and necessity to perform the frozen embryotransferStudy design, size, duration: Prospective cohort study. Development of50 embryos was continuously monitored with the use of Primo Visio system(Cryo Innovation, Hungary), with image acquisition every 10 minutes. January2012- May 2012.Participants/materials, setting, methods: The following parameters wereanalysed: t2 – time after ICSI to the division to two cells; t3 - time after ICSIto the division to three cells; t4 - time after ICSI to the division to four cells; t3– t2 – time from division to two cells until division to three blastomeres; t4 –t3 – time from division to three cells until division to four blastomeres.Main results and the role of chance: The embryos developing to the blastocyststage reached the two cell stage earlier (25,94 +2,17 h vs 28,89 +3,75 h). The

time between division to 2 cells and subsequent division to three cells and thetime between three- and four cell stage were shorter comparing to the arrestedembryos (t3: 37,36 +2,55 h vs 42,09 +5,23 h; t4: 38,49 +2,63 h vs 42,93+5,01 h). The time interwal between the two- and three cell stage (t3 – t2) incase of embryos developing to the blastocyst stage was shorter. (11,42 +0,80 hvs 13,20 +2,25 h). The direct transition of zygote to the three blastomere stageor too short second cell cycle (t3 – t2), ,5h, was the negative predictive factor.No difference in the duration of third cell cycle (t4 – t3) was observed (0,88+0,80 h vs 0,84 +1,28 h).Limitations, reason for caution: The study was not powered to test differences inpregnancy ratesWider implications of the findings: Adapted for embryology, the time-lapse ana-lysis can optimize the selection of most viable embryos for embryotransfer.Study funding/competing interest(s): No specific funding was obtained for thisstudy; it was solely funded by Gameta Ferility Center. None of the authors haveany economic affiliation.Trial registration number: Not applicable.

P-109 Effect of early blastomere multinucleation on pre-implantationdevelopmental timings of human embryos

M. Stoppa1, R. Maggiulli1, A. Capalbo1, E. Ievoli1, L. Dovere1, C. Scarica1,L. Albricci1, S. Romano1, F. Sanges1, N. Barnocchi2, L. Papini2, A. Vivarelli1,F.M. Ubaldi1, and L. Rienzi1

1Genera Center for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy,2Genera, Center for Reproductive Medicine, Umbertide (PG), Italy

Study question: The aim of the study was to evaluate whether or not the occur-rence of blastomeres multinucleation may disturb the early developmentalevents of preimplantation embryos.Summary answer: We observed a different behavior in the early events of devel-opment of multinucleated embryos with respect to the sibling mononucleatedcounterpart.What is known already: The occurrence of blastomeres multinucleation may bedue to nuclear replication without cytokinesis, nuclear fragmentation or defectivemigration at mitotic anaphase leading to errors in DNA packaging. Retrospectiveanalyses of human embryo development have previously showed an association ofmultinucleation with impaired cleavage, increased incidence of aneuploidy andlow implantation rates in IVF cycles. Time-lapse cinematography has been recent-ly applied in human IVF, as useful tool to optimize embryo assessment withoutinterfering with embryo growth.Study design, size, duration: The influence of early blastomere multinucleationon cleavage timing was retrospectively evaluated by comparing the developmen-tal behavior of 114 sibling embryos. To avoid confounding factors, each embryoshowing multinucleation at 2-cell stage (MNBs group ¼ 38) was compared withtwo randomly selected sibling embryos from the same cohort with mononucleatedblastomeres (NBs group ¼ 76).Participants/materials, setting, methods: Microinjected oocytes were culturedin a time-lapse incubation system (Embryoscope, Unisense). Early events of de-velopment (timing of pronuclear appearance, syngamy, 1st to 7th cell divisionand cell cycle duration) were monitored and recorded. Multinucleation wasassessed as the presence of two or more nuclei in both blastomeres, at 2-cells stage.Main results and the role of chance: Embryonic cohorts of 32 patients (meanfemale age ¼ 35.8+3.5) containing at least one MNB embryo and two randomlyselected sibling NB embryos were evaluated. We observed a different behavior ofmultinucleated embryos with respect to the sibling mononucleated counterpart.The median time value of third (T3) cell cycle was found to be significantly differ-ent between NBs (T3 ¼ 37.5+4.7, CI:36.4-38.7) and MNBs group (T3 ¼40.4+4.2, CI:39.0-41.9) (p ¼ 0.003). No significant difference in the mediantime value of first (T2), third (T4), fourth (T5), seventh (T8) cell cycle was high-lighted, although the MNBs group showed consistently slower cleavage timings(T2 ¼ 28.3+3.3, CI:27.2-29.3, T4 ¼ 41.3+4.4, CI ¼ 39.8-42.8; T5 ¼51.4+8.5,CI:48.4-54.4, T8 ¼ 61.1+7.4,CI:58.2-63.9) compared to the NBsgroup (T2 ¼ 27.7+3.8, CI:26.8-28.6, T4 ¼ 40.0+4.9, CI ¼ 38.8-41.2; T5 ¼49.8+6.9,CI:48.1-51.5, T8 ¼ 59.2+10.7,CI:56.6-61.9).Limitations, reason forcaution: The main limitation of the study is the rather lownumber of embryos enrolled (N ¼ 114).

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Wider implications of the findings: We identified distinctive early cleavagetiming ranges for mononucleated and multinucleated embryos. When continuousmonitoring of embryo development cannot be performed, the choice of the bestembryo to transfer usually relies on the static evaluation of morphologicalaspect of the embryo. According to confidence intervals observed in ourdataset, the persistence of 3-cell stage later than 39.0 hours post insemination sug-gests the occurrence of blastomere multinucleation at 2-cell stage.Study funding/competing interest(s): noneTrial registration number: none

P-110 Human embryo aneuploidies cannot be predicted by morphoki-netic assessment during the first phases of in vitro culture

L. Rienzi1, S. Bono2, A. Capalbo1, L. Spizzichino2, C. Rubio3, F.M. Ubaldi1, andF. Fiorentino2

1Genera c/o Clinica Valle Giulia, Reproductive Medicine, Roma, Italy, 2Genoma,Molecular biology, Roma, Italy, 3IVIOMICS, Molecular Cytogenetics Lab,Valencia, Spain

Study question: The aim of our study was to evaluate the reliability of morpho-kinetic assessments in predicting embryo aneuploidies as assessed by array com-parative genomic hybridization (aCGH).Summary answer: No correlation has been found between morphokinetics vari-ables and embryo aneuploidies.What is known already: Preimplantation genetic screening (PGS) has beenintroduced in IVF to avoid the transfer of aneuploid embryos. However, PGS isan invasive procedure, technically challenging that requires embryo extra-manipulation and thus potentially detrimental. For this reason there is an impera-tive search for new non-invasive techniques able to identify healthy embryos in areliable way. Time lapse cinematography and embryo morphokinetic assessmentsis a promising approach. This technologyhas been recently correlatedwith signifi-cant improvements in pregnancy rate.Study design, size, duration: Embryos were individually monitored by timelapse cinematography (Embryoscope, Unisense). Subsequently, biopsy andchromosomal screening by aCGH (Bluegnome, UK) were performed either atday 3 (N ¼ 137) or at day 5 (N ¼ 158). Finally, embryos were categorized aseuploid (group1), single aneuploid (group 2) or complex aneuploid (≥errors,group 3).Participants/materials, setting, methods: 295 embryos from 64 PGS patientswere enrolled. Morphokinetic variables assessed were: timing of pronuclear for-mation (PN), 2-cells (T2), 3-cells (T3), 4-cells (T4), 5-cells (T5) and 8-cells (T8)divisions, length of the second cell cycle (cc2) and the synchrony in the divisionfrom two to four cells (s2).Main results and the role of chance: 83/295 (28.1%), 69/295 (23.4%), and 143/295 (48.5%) embryos were euploid or had single or multiple aneuploidies, re-spectively. No statistically significant differences were observed for all the vari-ables analyzed. The timings of PN formation were: 12.09 (CI 11,56-12,63)11,50 (CI 11,00-12,01) and 11,97 (CI11,56-12,39), of T2: 26,37 (CI25,73-27,02), 26.69 (CI 25,89-27,49) and 27,00 (CI 26,40-27,61), of T3: 37,24(CI 35,98-38,49), 36,60 (CI 34,96-38,25) and 37,46 (CI 36,45-38,47), of T4:39,07 (CI 38,22-39,91), 39,04 (CI 37,80-40,28) and 39,99 (CI 38,94-41,04), ofT5: 49,97 (CI 47,81-52,13), 48,94 (CI 46,61-51,28) and 51,72 (50,31-52,12),of T8: 53,05 (CI 48,92-57,18), 54,68 (CI 49,67-59,69) and 56,52 (CI53,23-59,81) in group 1, 2, and 3, respectively (NS). No significant differenceswere also found for cc2 and s2 variables. Irregular cell divisions (transitionfrom 1- to 3- and 2- to 5- cell stage) were observed in 2/83 euploid embryos(2,4%), 0/69 (0%) single aneuploid embryos and 5/143 (3,4%) multiple aneuploidambryos (NS). Finally, direct cleavage (second cell division in less than 4 hours)was found in 3/83 (3,6%), 9/69 (13,0%) and 9/143 (6,3%) embryos in group 1, 2and 3, respectively (NS).Limitations, reason for caution: A relatively low number of euploid embryoswere transferred (52). For this reason it has been impossible to compare the behav-ior of implanted (22) vs non implanted euploid embryos (30). It cannot beexcluded that extending the observations up to day 5 some correlations betweenmorphokinetics behavior and aneuploidies could be found.Wider implications of the findings: Embryo behavior in the first 3 days of invitro culture is not useful to select embryos for chromosomal aneuploidies.What remains to be understood is if morphokinetics assessment may provide

an effective tool in determining embryo developmental competence andthus be helpful in optimizing euploid embryo selection in combination toPGS approach.Study funding/competing interest(s): noneTrial registration number: none

P-111 Cleavage and blastocyst development rate is diminished and sexratio is shifted by oocyte exposure to bisphenol A in Bos taurus

J. Ferris, L.A. Favetta, N. MacLusky, and W.A. KingUniversity of Guelph, Biomedical Sciences, Guelph, Canada

Study question: The primary objective of the current study is to evaluate theeffects of bovine oocyte exposure to bisphenol A (BPA) on embryo cleavageand blastocyst development rates, as well as the sex ratio of resulting embryos sur-viving to blastocyst.Summary answer: In comparison to various controls, oocyte exposure to BPAduring maturation resulted in a decrease in cleavage and blastocyst rates, and ahigher proportion of female embryos in comparison to multiple controls.What is known already: Optimal hormone levels are important for the occur-rence and maintenance of a successful pregnancy. BPA, an endocrine disruptingchemical, can have detrimental effects on fertility and reproductive success,having been linked to reproductive issues such as reduced in vitro fertilization(IVF) success in humans and recurrent miscarriage. Feminization of males anda sex skew towards females have been found in fish and amphibia in vivo as aresult of early exposure to BPA.Study design, size, duration: Oocytes were matured in in vitro maturation mediaunder various conditions. Controls included no-treatment, vehicle and estradiolcontrols. Treatment group media were supplemented with BPA (3 and 30 mg/mL). Blastocysts were collected at day 8 post fertilization, treated briefly withpronase to remove the zona pellucida and frozen individually.Participants/materials, setting, methods: Standard IVF was completed follow-ing maturation. Cleavage was calculated at 2 days post-fertilization (dpf) andblastocyst rate at 8dpf. PCR was used to lyse blastocysts and positive controls,and amplify TSPY and GAPDH. Embryos displaying GAPDH and TSPY bandswere deemed male; GAPDH without TSPY band were deemed female.Main results and the role of chance: Cleavage rates in the 30 mg/mL BPA treat-ment group were significantly lower compared to both the no-treatment andvehicle controls (p ¼ 0.0297 and p ¼ 0.0049 respectively, Fisher’s exact, two-tailed). The number of cultured embryos to reach blastocyst were significantlylower in the 30 mg/mL BPA treatment group compared to the no-treatment andvehicle controls (p ¼ 0.0087 and p ¼ 0.0035 respectively, Fisher’s exact, two-tailed). There is a trend showing that oocyte treatment with high BPA results ina higher number of females surviving to blastocyst (80% females compared to54% in the no-treatment control, p ¼ 0.0872, Fisher’s exact, two-tailed). Allresults are dose-dependent with the high BPA, but not low BPA, treatmentshowing significant decreases in cleavage and blastocyst rates and a trendtowards increased number of female embryos.Limitations, reason for caution: The sexing analysis is limited by the number ofembryos analyzed; between 25 to 45 embryos per group. Additional analyses areunderway to increase sample size. Analyses were conducted solely in vitro usingBos taurus. Fertility and mortality rates are underway using Onchorynchus mykissunder the same treatment conditions.Wider implications of the findings: BPA supplementation to maturation mediaresulted in a significant and dose-dependent decrease in cleavage and blastocystrates, and a trend towards a female sex skew. These results suggest that bovineoocyte exposure to BPA can compromise the resulting embryo’s early develop-mental competence, and influence sex ratio of surviving blastocysts. Consideringthe high prevalence of human exposure, these results may provide insightinto oocyte viability of reproductive aged women who are regularly exposedto BPA.Study funding/competing interest(s): This research was funded by the NaturalSciences and Engineering Research Council (Canada), with no competinginterests.Trial registration number: N/A

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P-112 The impact of laser-assisted hatching (LAH) on clinical outcome ofassisted reproduction technology and the role of maternal age

T. Madani, N. Jahangiri, and R. AflatoonianDepartment of Endocrinology and Female InfertilityatReproductive BiomedicineResearch Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran,Iran

Study question: This study was considered for further assessment of the LAH forpromoting implantation and evaluating impact of maternal age on the embryonichatching.Summary answer: Zona thickness appears to be impacted by a woman’s age, andLAH procedure seems to be effective in improving pregnancy and implantationrates in patients with advanced female age.What is known already: Despite great effort, there is still debate among scientistsabout the reported benefits of AH.Study design, size, duration: This retrospective cross-sectional study was con-ducted from December 2010 until March 2012 at the Reproductive BiomedicineResearch Center. The total number of patients entered in this study was 273 in thestudy group (LAH was performed) and 434 in the control group (LAH was not per-formed).Participants/materials, setting, methods: The participants were women aged20-45 years undergoing IVF or ICSI using their own gametes with history of ≤3 ART cycles. LAH was performed on cleavage stage fresh embryos and the out-comes were compared with the intact control group. These two groups were furthersubdivided into women of advanced age (.36 y) and younger (≤ 36).Main results and the role of chance: The developmental rate of embryos at dif-ferent stagesandalso rate of womenwith male factor infertility were comparable inboth groups. With respect to age, our data showed that LAH is of no benefit inwomen ≤ 36 years of age. As the chemical pregnancy and clinical pregnancyrates after LAH were insignificantly lower among women aged ≤ 36 years inthe study group compared to control group (32.5% vs. 39.2%, P ¼ 0.131;31.3% vs. 36.4%, P ¼ 0.249). However the only statistically significant differenceobserved in implantation rate (16.5% vs. 23.2%, P ¼ 0.005). In women aged over36 years, the chemical pregnancy, clinical pregnancy and implantation rates werehigher in the study group than controls, but these differences were not statisticallysignificant (29% vs. 6.3%, P ¼ 0.053, 21.5% vs. 6.3%, P ¼ 0.193 and 9.9% vs.2.8%, P¼ 0.162 respectively).Limitations, reason for caution: -Wider implications of the findings: Some investigators introduce this method asa routine plan in ART, whilst others do not. No published studies recommend AHfor younger women. Some researchers also claim that LAH of embryos is effectivein improving the pregnancy and implantation rates of women with advanced ma-ternal age. These disputes may be related to the type of AH and quality and stage ofembryos selected for AH technique.Study funding/competing interest(s): This study has .Trial registration number: This study is a retrospective study.

P-113 Retrospective analysis of clinical outcome of vitrified blastocystpost thaw using time-lapse imaging and standard incubation

E. Cater1, D. Hulme1, K. Berrisford1, L. Jenner1, A. Campbell2, and S. Fishel1

1CARE Fertility, Embryology, Nottingham, United Kingdom, 2CARE Fertility,Embryology, Manchester, United Kingdom

Study question: Does a continuous incubation (ES), Embryoscope, (UnisenseFertilitech) environment and time-lapse imaging improve the clinical outcomeof blastocyst transfer post thaw compared to standard incubation (SI)?Summary answer: Blastocysts incubated in a continuous environment have animproved chance of pregnancy and significantly improved clinical outcome com-pared to SI. Time-lapse imaging may give more information but as yet does notappear to contribute to the increased outcome.What is known already: Many papers such as Kirkegaard K. et al (2012) andMeseguer, M. et al (2012) report a significant increase in clinical outcome in com-parison to SI.

Time-lapse imaging has also been used to investigate embryo morphokinetics(I. Rubio, 2012), prognostic markers of viability (J. Herrero, 2012) and theimpact of ploidy on embryo development (A Campbell 2012,2013).

However, little evidence is documented regarding the use of continuous incu-bation and time-lapse imaging for embryos post thaw.Study design, size, duration: 96 vitrified/ warmed day 5/ 6 blastocyst cycles fromJuly to October 2012 were included in the data analysis. Group A (n ¼ 52, averagematernal age 36.3 + /-3.99) were cultured in a standard incubator (HeraCell) andGroup B (n ¼ 44, average maternal age 32.2 ¼ /-5.58) in a continuous time-lapseincubator.Participants/materials, setting, methods: Embryos were warmed as per proto-col. Retrospective analysis examined the achievement of an embryo transfer(ET), positive bhCG, presence of a foetal heart (FH) per embryo(s) transferred,clinical pregnancy (CP), time of re-expansion and time of full recovery. Twotailed Fishers exact test will be used to assess significance.Main results and the role of chance: 88.5% (46/52) of Group A achieved an ET.39.1% had a positive bhCG/ET (18/46) and 21.7% (10/46) a CP/ET. Of the 56embryos transferred, 12 (21.4%) achieved FH, with an average of 1.22embryos/ET.

88.6% (39/44) of Group B achieved an ET. 59.0% (23/39) achieved a positivebhCG/ETand 46.2% (18/39) a CP/ET. FH/embryo transferred was 34.0% (16/47)with an average of 1.02 embryos/ET.

CP rate was found to be significantly greater in Group B (p ¼ 0.0215). All othercomparisons were non significant.

In Group B, embryos that implanted were found to begin re-expansion (average0.95hrs) and be fully recovered (average 1.35hrs) faster than those that didn’t(re-expansion begins, average 2.82 hrs, fully recovered 3.35hrs. Time lapseimaging played no role in embryo selection for transfer.Limitations, reason forcaution: The decision to culture in the ES or SI was basedon availability of the ES rather than randomisation. Culture dishes varied betweenGroup A and B and there is a difference in age between the groups. These factorscannot be dismissed as contributory.Wider implications of the findings: The use of uninterrupted culture is consid-ered likely to increase implantation in most fresh culture cycles but the data pre-sented suggests that it also has a role in increasing the outcome of frozenembryo replacements, allowing the thaw and transfer of a single blastocyst tomaximise positive outcome and minimise multiple births.

To assess this further, we propose a randomised study to eliminate the limita-tions above.Study funding/competing interest(s): NoneTrial registration number: None

P-114 Minimal waste of healthy human embryos due to chromosomalmosaicism

X.Y. Zhang, A. Yilmaz, H. Hananel, and A. AoMcGill University Health Center, Obstetrics and Gynecology, Montreal QC,Canada

Study question: What is the extent and clinical relevance of chromosomal mosai-cism in the ’spare’ preimplantation embryos (i.e., embryos that are found to be un-suitable for freezing or transfer based on the results of chromosome screeningperformed on a single blastomere).Summary answer: Only 3.5% of the 454 embryos were mosaic which had suffi-cient quality grade, not arrested, and thus, expected to implant if transferred.What is known already: Previous studies have shown that the presence of over38% abnormal cells in mosaic embryos is detrimental and prevent implantation.However, the extent of mosaicism that may result in healthy births in spare preim-plantation human embryos in the literature is subject to controversy.Study design, size, duration: The embryos were collected from 122 patients in148 preimplantation genetic screening cycles performed at a hospital-based repro-ductive center from January 2006 to March 2011. The embryos were analyzed onday 4 or 5 post-insemination.Participants/materials, setting, methods: Fluorescent in situ hybridization(FISH) procedure for chromosomes 13, 15, 16, 17, 18, 21, 22, X and Y wasused to determine chromosomal complement in each of the 5,455 nuclei obtainedfrom 454 spare embryos.Main results and the role of chance: 165 of the 454 spare embryos (36.3%) weredevelopmentally arrested. 26.1% (43/165) of the arrested and 24.6% of the nonar-rested (70/ 284) embryos had over 38% diploid cells. However, only 16 of these454 (3.5%) embryos were mosaic, not arrested and were of good quality,

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suggesting that they had the potential to implant. Percentage of diploid cells in thenon-arrested spare embryos were not associated with changes in maternal age,number of cumulus oocyte complexes (COC) collected, or the number of chromo-somally normal embryos obtained per cycle. Pearson’s coefficients for the correla-tions of percent diploid cells with the maternal age (r ¼ -0.10), number of COCcollected (r ¼ 0.10), and number of normal embryos obtained per cycle (r ¼0.07) were all small.Limitations, reason for caution: Only nine pairs of chromosomes were analyzedin this study. Screening of a larger number of chromosomes may detect mosaicismrates higher than those found in our study. FISH has an error rate of 5 to 7% result-ing from inaccuracy of probe hybridization.Wider implications of the findings: Only 3.5% of the non-arrested mosaic spareembryos analyzed in this study had over 38% diploid cells, were of sufficientquality, and thus, had the potential to implant if transferred. These results are inagreement with some of the previous publications reporting less than 5% of mis-diagnosis due to mosaicism in spare embryos using FISH. These results suggestthat waste of healthy human embryos due to mosaicism after chromosome screen-ing is minimal.Study funding/competing interest(s): N/ATrial registration number N/A

P-115 Near-equilibrium rapid freezing versus cryotop vitrification of2-cell mouse embryos

T. Vutyavanich, W. Piromlertamorn, U. Saenganan, and S. SamchimchomChiang Mai university, Obstetrics and Gynecology Faculty of medicine, ChiangMai, Thailand

Study question: We aimed to develop a novel method of freezing that combinesthe advantages of both the non-equilibrium vitrification and slow equilibriumfreezing.Summary answer: Our method gave comparable results to vitrification in termsof embryo survival and developmental competence. The method is user friendly,and allows fast and simple cryopreservation of embryos in a large volume of cryo-protectants in a closed container.What is known already: Slow cooling and vitrification are the most commonlyused methods for cryopreservation. Vitrification can be done rapidly and achievesa high survival rate of embryos, without the need of a programmable freezing.However, it is time and operator dependent. Slow freezing, on the other hand,can be done in a closed container, and the volume and time of exposure to cryo-protectants, as well as the thawing speed, are not as critical as the vitrificationmethod.Study design, size, duration: Mouse 2-cell embryos (n ¼ 723) were divided intocontrols (n ¼ 211), vitrification (n ¼ 192) and near-equilibrium rapid freezing(n ¼ 320) groups in a ratio of 1:1:1.5. After one week of storage in liquid nitrogen,embryos were warm/thawed and culture up to the blastocyst stage.Participants/materials, setting, methods: Embryos were exposed to 10% EG,5% PROH, 0.2M trehalose in PBS for five minutes, loaded into sealed straws,and inserted into holes inside an aluminum cylinder, pre-cooled in liquid nitrogen.They were thawed at 37oC in serial dilutions of Trehalose. In another group,embryos were vitrified/warmed using Cryotop kits.Main results and the role of chance: The survival, further cleavage, and blasto-cyst formation rate of 2-cell mouse embryos in both groups was comparable byChi-squared tests (98.8% vs. 96.4%, P ¼ 0.06; 94.9 vs. 95.1%, P ¼ 1.00; and86.7% vs. 88.1%, P ¼ 0.67 in the rapid freezing and vitrification groups, respect-ively, but further cleavage and blastocyst formation was lower than those in thecontrols (99.1% and 98.1%, P , 0.05). There was no significant difference(ANOVA tests) in the average number of inner cell mass (35.5+7.2, 36.2+6.4 and 35.8+5.6: P ¼ 0.856), trophectoderm cells (50.9+9.3, 52.1+9.9and 53.2+6.4: P ¼ 0.293), and total cells (86.4+15.2, 88.3+14.8 and88.9+10.4: P ¼ 0.228) in blastocysts in the rapid freezing, vitrification andcontrol groups, respectively.Limitations, reason for caution: Another study is now underway to transferfrozen/thawed embryos into pseudo-pregnant mice to evaluate the implantationand pregnancy rates. We also plan to employ this freezing protocol on discardedhuman embryos, as data obtained from the mouse might not be directly applicableto the human.Wider implications of the findings: We postulated that this novel method createda “hybrid” condition, i.e. the co-existence of small ice crystals interspersed among

the vitrified extracellular fluid, in contrast to the presence of large ice crystals inslow freezing or the total absence of any ice crystal in vitrification. The methodrequires lower cooling and warming rates, and the volume is not critical. It isequally effective, but less demanding and easier to perform than the current vitri-fication techniques.Study funding/competing interest(s): The funding was provided by the Facultyof Medicine Endowment Fund for Medical Research, Chiang Mai University. Theauthors declared s that could influence the study results.Trial registration number: (Not applicable)

P-116 Aseptic oocyte vitrification to circumvent oocyte aging as strategywhen semen sample production is delayed

B. Wirleitner1, B. Lejeune2, N.H. Zech1, and P. Vanderzwalmen2

1IVF-Centers Prof. Zech-Bregenz GmbH, Bregenz, Austria, 2Centre HospitalierInter Regional Cavell, (CHIREC), Braine-l’Alleud, Belgium

Study question: When production of the semen sample on the day of oocytepick-up (OPU) is delayed or impossible, e.g. due to erectile dysfunction or azoo-spermia, aging of oocytes becomes a matter. The efficiency of a rescue strategywhere oocytes are vitrified and warmed when sperm collection is possible wastested.Summary answer: Our results show that in all centers with a good, standardizedprotocol, aseptic oocyte cryopreservation can safely be applied in cases of unex-pended failure of sperm production.What is known already: Although several articles deal with the efficiency ofoocyte vitrification using open vitrification devices in donor cycles, still little isknown about vitrification in closed aseptic conditions and its application in stand-ard IVF-patients. To apply the oocyte vitrification technique on a larger scale andlift its experimental label, it is important also to test its efficiency, especially of theaseptic closed devices, on our daily clientele in routine IVF-work.Study design, size, duration: This retrospective study included 56 IVF-cycleswhere aseptic oocyte vitrification was performed due to absence of sperm produc-tion at least 3 hours after OPU during January - December 2011.Participants/materials, setting, methods: In all cycles no sperm was retrieveduntil 3 hours after the OPU. Oocytes were vitrified in a closed vitrificationdevice (VitriSafe) ensuring aseptic conditions during vitrification and storage.After sperm retrieval in a subsequent cryo-cylce, oocytes were warmed and ferti-lized. As main outcome live-birth rate was evaluated.Main results and the role of chance: For 56 patients 455 oocytes were vitrified ofwhich 416 were fertilized after warming and ICSI/IMSI. The cleavage rate on day3 was 95.3%. Embryo transfer was performed on day 5 (blastocyst selection). Anongoing pregnancy rate of 43.8% and a birth rate of 35.7% was obtained and 26healthy babies were born. These results are encouraging, especially as meanage of women in this study was 34.5 years and sperm parameter were verypoor. The additionally safety and efficiency of aseptic vitrification (i.e. reducingthe risks of contamination and increasing the viability after warming) duringcooling and storage turn this strategy in to the focus not only when its applicationis inevitable but also when sperm parameters are very poor (e.g. after recent infec-tions).Limitations, reason for caution: Although baby-take-home rates and health ofbabies born do not give any reason for concern, a long-time follow-up will be ne-cessary to exclude any later appearing health problems originating from theapplied techniques.Wider implications of the findings: In contrast to earlier findings, we report veryhigh efficiency of aseptic vitrification in closed carriers. The reduced cooling ratesin closed carriers as compared to open devices can be overcome bya slightlymodi-fied vitrification protocol. Aseptic vitrification achieves the same warming rates asopen devices and thereby ensures high survival rates.Study funding/competing interest(s): NoneTrial registration number: None

P-117 Sequential versus single step media in a time lapse system: is therea difference in pregnancy rate?

E. Albani, V. Parini, A. Smeraldi, F. Menduni, R. Antonacci, A. Marras, S. Levi,G. Morreale, B. Pisano, A. Di Biase, A. Di Rosa, and P.E. Levi Setti

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IRCCS Istituto Clinico Humanitas, Department of Gynecology Division ofGynecology and Reproductive Medicine, Rozzano, Italy

Study question: Is the pregnancy rate, in infertile patients, influenced by the typeof culture media used in association with time lapse analysis?Summary answer: According to our experience, the use of universal mediainstead of sequential media could improve the pregnancy rate.What is known already: Recent studies of different culture media on humanembryo development show that there are no differences in implantation rate andclinical pregnancy rate. Furthermore embryo evaluation is often based on astatic observation.Study design, size, duration: In a retrospective study, from June 2012 to Decem-ber 2012, 141 infertile patients/cycles underwent Intracytoplasmic Sperm Injec-tion (ICSI) cycles with embryo transfer. After which ICSI oocytes wereincubated in a time lapse system (Unisense FertiliTech A/S Embryoscope).Participants/materials, setting, methods: The 141 patients were divided in twogroups, group A and B. In group A (n ¼ 90) we used a sequential media (Sagew)and B (n ¼ 51) a single step media (Irvine Scientificw). The variables studied werethe fertilisation, implantation, pregnancy, and blastocyst cryopreservation rate.Main results and the role of chance: In Group A 1217 oocytes were retrieved,722 injected (8.02+1.42), 511 fertilised (FR ¼ 70.8) and 201 embryos weretransferred; in B 618 retrieved, 383 injected (7.5+1.5), 269 fertilised (FR ¼70.2) and 120 transferred.No differences in mean age (A¼ 36.6+3.8; B ¼36.6+3.8), fertilisation rate (A ¼ 70.8%; B ¼ 70.2%) and transferred embryos(A ¼ 2.2+0.54; B ¼ 2.3+0.5). Furthermore there are no differences in timelapse analysis at two, four, eight, morula and blastocycsts development betweentwo groups. A pregnancy rate (A ¼ 34.4%; B ¼ 47%) and percentage of cryopre-served sovrannumerary blastocysts (A ¼ 25.5%; B ¼ 33.3%) positive trend wasobserved, although not statistically significantly different.Limitations, reason for caution: The use of time lapse systems without compari-son with 3 gas incubators normally used in clinical practice. The sample consid-ered had not sufficient statistical power to detect the difference observed in thisstudy.Wider implications of the findings: Although not statistically different, ourresults show a clinical interesting increase in pregnancy rate between the twostudied groups, to be confirmed in a larger sample comparing 3 gas incubatorsand time lapse systems.Study funding/competing interest(s): NoneTrial registration number: Not applicable

P-118 Reverse phase protein array (RPPA): a new approach for theidentification of cumulus cells biomarkers of oocyte quality

V. Puard1, V. Cadoret2, T. Tranchant2, C. Gauthier2, E. Reiter2, F. Guerif1, andD. Royere1

1CHRU Tours - Hopital Bretonneau, GYN-OBS 1 REPRODUCTION, ToursCedex 1, France, 2INRA, UMR85 Physiologie de la Reproduction et desComportements, Nouzilly, France

Study question: We proposed to evaluate the Reverse Phase Protein Array(RPPA) to detect and analyze potential biomarkers related to oocyte developmen-tal competence through a non-invasive procedure involving each individualcumulus (CCs) in human.Summary answer: Reverse Phase Protein Array allowed us to detect specific pro-teins at a level as low as the equivalent of one cell of one human cumulus.What is known already: Morphological criteria are mostly used in ART laborator-ies but remain poorly predictive of embryo potential to develop. Various approachesinvolving transcriptomics, proteomics, and metabolomics on the embryo or its cel-lular or non cellular environment have been proposed. RPPA is a sensitive and quan-titative technique which allowed the detection of specific proteins in very lowquantities of biological samples to help clinical management of cancer.Study design, size, duration: Six patients undergoing intracytoplasmic sperm in-jection (ICSI) for male infertility from January to May 2012 were included in thisstudy. Thirteen human individual cumulus from 4 patients and a pool of 16cumulus from 2 patients were collected during ICSI procedure.Participants/materials, setting, methods: The expression of the targeted pro-teins was suppressed by siRNA in HEK293 and the antibodies used for RPPAwere validated by correlating the signal observed by Western Blot (WB) and

RPPA.Proteins weredetected in a poolof cells of 16 cumulus, then in 13 individualcumulus.Main results and the role of chance: Specificity of antibodies targeting the pro-teins of interest was assessed by WB. The expression of 4 proteins was partly sup-pressed in HEK293 cells by siRNA. The correlation of knockdown efficiencies ofthe VCL and SRC proteins between WB and RPPA validated the antibodies forRPPA used. We detected in cells of a pool of 16 cumulus the proteins VCL,SRC and ERK2 at the level of less than one equivalent cell of one cumulus.Then, these proteins were detected at the same sensibility in individualcumulus. No correlation was observed for the antibody targeting RGS2 and ledus to reject it for RPPA used. Therefore, validation the antibodies is a crucialstep for RPPA used and its application for biomarkers identification.Limitations, reason for caution: Antibody validation remains a challengingproblem for RPPA. While suppressing the expression of the proteins by siRNA ap-proach is efficiency, this technique required heavy development. Overexpressionthe proteins in HEK293 cells might be an interesting alternative for high through-put validation.Wider implications of the findings: Once validated antibodies targeting potentialbiomarkers of oocyte quality at protein level and assessed their robustness bytaking into account the patient variability, RPPA might be used in ART laboratory.Indeed RPPA is a sensitive, high throughput, cost and material effective techniquewhich might be used to predict the developmental ability and implantation of theembryos in ART laboratory to increase single embryo transfer and limit multiplepregnancies.Study funding/competing interest(s): This work was supported by MerckSerono Grant for Fertility Innovation Award 2010.Trial registration number: -

P-119 Abnormal distribution of type 1 Inositol 1,4,5 tri-phosphate recep-tor in fertilization failed human eggs

S.Y. Yoon, J.H. Eum, E.A. Park, T.Y. Kim, T.K. Yoon, D.R. Lee, and W.S. LeeCHA University, Department of Biomedical Science, Seoul, Korea South

Study question: IP3R1 is a key protein to induce intracellular Ca2+-oscillationduring fertilization. Does fertilization failed human eggs have normal IP3R1distribution?Summary answer: Fertilization failed human eggs showed abnormal IP3R1 dis-tribution including inconsistent clusters in cytoplasm instead of cortical clusters.What is known already: The capacity of [Ca2+]i oscillation is acquired duringegg maturation and coincides with an increase in the sensitivity of the IP3R1and their localization in cytoplasm. Cluster formation of IP3R1 in the eggcortex is important to initiation of [Ca2+]I oscillations during egg and spermfusion.Study design, size, duration: Immunofluorescence analysis of IP3R1 in in vitromatured egg, fertilization failed egg and tripronuclear human eggs.Participants/materials, setting, methods: Patients enrolled in the assisted repro-duction program at the Fertility Center of CHA Gangnam Medical Center wereparticipated with written consent in this study. Total 62 eggs (GV, 8 eggs; NF 42eggs, 3PN; 12 eggs) were immunostained with anti IP3R1 antibody, and examinedwith a confocal laser-scanning microscope.Main results and the role of chance: Distribution of IP3R1 in human eggschange gradually from GV stage to MII stage. In GV stage, most of the eggsshows progressively disperse from GV to cortex. In MII stage, 6 out of 8 eggshave that IP3R1 distributed in whole cytoplasm including clusters which ismore 3 � 6um in diameter on the cortex. After fertilization, in PN stage with3PN, most of the eggs have IP3R1distribution with gradually disperse from PNto cortex, but they do not have cortical clusters. In fertilization failed MII eggson the next morning post fertilization, more than 50% of eggs have severalIP3R1 aggregates in the middle of the cytoplasm, which is more than 10um indiameter instead of cortical clusters as in MII.Limitations, reason for caution: The low numbers of human eggs analysed maynot be ideal for conclusive statistical analysis. Also, evaluation of other IP3R1 bio-chemical changes would be need to conclusion.Wider implications of the findings: The present findings provide new under-standing the reason of the fertilization failure during ART program, and suggestfurther evidence for the clinical application as maker of egg quality.

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Study funding/competing interest(s): This work was supported by a grant fromthe Korea Healthcare Technology R&D Project, Ministry for Health, Welfare &Family affairs, Republic of Korea (A084923).Trial registration number: Basic research

P-120 The shipment of oocytes or embryos vitrified in minimum volumeby means of dry shipper containers does not impair the clinical outcome

A. Cobo Cabal1, B. Vallejo1, P. Campos1, E. Sanchez1, J. Serrano1, and J. Remohi2

1Instituto Valenciano de Infertilidad, IVF laboratory, Valencia, Spain, 2InstitutoValenciano de Infertilidad, Ob. Gyn., Valencia, Spain

Study question: Is shipping impairing survival or clinical outcome of vitrifiedoocytes or embryos?Summary answer: Oocytes or embryos vitrified in minimum volume with an opensystem can be safely transported by means of dryshippers equipped with temperaturemonitoring devices without impairment of survival or clinical outcome.What is known already: Vitrification has revealed as most efficient method foroocyte cryopreservation, especially by means of open systems using minimumvolume for loading samples. Shipping vitrified material could be of great utilityfor donor egg-banks as well as for patients needing the transportation of theiroocytes or embryos due to change of residence or clinic. Usually cryopreservedsamples are transported in vapor dry shippers. Serious concerns have beenraised about the shipping of micro-drop- vitrified samples in this type of contain-ers, due to possible deleterious changes in temperature caused by the manipulationneeded to load the containers or during transportation.Study design, size, duration: Retrospective cohorts study, January 2007- April2011.Participants/materials, setting, methods: University affiliated infertility center.674 oocytes (N¼ 72 cycles) and 304 embryos (N ¼ 132 cycles) were shipped todifferent Spanish cities. Matched controls consisted of IVF and warming cyclesconducted in situ including 1436 non-shipped vitrified oocytes (N ¼ 144cycles) and 524 non-shipped vitrified embryos (N ¼ 272 cycles). All sampleswere vitrified using Cryotop method. MEV vapor dry shipperw equipped with atemperature tracking system (ShipslogTM) was employed for monitoring the tem-perature of the device throughout the trip. Any air-contact was avoided whenloading the dry shipper. Full history of temperature throughout the journey wasdownloaded. Survival and ongoing pregnancy rate (OPR) were analyzed asmain outcome measurements. x2 test was used for statistical analysis and oddsratios (OR) with CI 95% were calculated with significance under 0.05.Mainresultsand the role of chance:Theshipmentofoocytesorembryosdoesnotimpair survival and clinical outcome. 89.8 % vs. 91% (NS) and 94.9% vs. 97.8%(NS) embryo transfers were performed when oocytes and embryos were shippedcompared to controls respectively. The odds ratio (OR) for survival and OPR/cycle in the case of shipped oocytes was 1.134 (CI95% 0.838-1.534; P¼ 0.433)and 0.912 (CI95% 0.498-1.670; P ¼ 0.877). Similarly, no statistical differenceswere found for shipped embryos vs. controls: OR ¼ 0.751 (CI95% 0.325-1.738;P ¼ 0.548)and1.051(CI95%0.682-1.622;P ¼ 0.912) for survivalandOPR/cycle.Limitations, reason for caution: Heterogeneity of study sample including ovumdonation and autologous oocytes cycles and natural cycles or hormonal replace-ment therapy for vitrified embryo transfers, although matched controls have beenincluded.Wider implications of the findings: The safe shipment of vitrified oocytesand embryos has great implications for the flexibility of ovum donation andIVF programs.Study funding/competing interest(s): None of the authors are affiliated to thebrands that commercialize the devices employed in the study.Trial registration number: N/A

P-121 Cleavage timing of euploid embryo development is age-related

V. Nagornyy1, P. Mazur1, D. Mykytenko2, L. Semeniuk1, and V. Zukin1

1Clinic of Reproductive Medicine ’NADIYA’, Embryology department, Kiev,Ukraine, 2Clinic of Reproductive Medicine ’NADIYA’, Department of moleculardiagnostics, Kiev, Ukraine

Study question: Can age-related differences in timing of embryo development beindentified and do they correlate with embryo ploidy?

Summary answer: According to our investigation, transfer quality embryos ofwomen of advanced age show delayed cleavage times, especially those withcorrect chromosomal constitution confirmed by array comparative genome hy-bridization (aCGH) analysis.What is known already: Time-lapse analysis of human embryo morphokineticshad already produced sufficient amount of data to help improve selection ofembryos with higher implantation potential. However, there is little evidencefor connection of morphokinetic parameters with embryo ploidy status andwomens ageStudy design, size, duration:This cross-sectional study included 878 embryos of133 patients, cultured in time-lapse imaging system during one year period. Fromthese, 113 embryos of 24 patients were analysed for ploidy by aCGH. Of aCGHembryos 45 originated from 10 women over 34 years, other 68 embryos werefrom 14 younger women.Participants/materials, setting, methods: Embryos were cultured in Embry-oScopeTM (UnisenseFertiliTech A/S, Denmark). Array CGH, if applied, was per-formed after trophectoderm biopsy, using 24surew kit (BlueGnome, UnitedKingdom). After 5 or 6 days of embryo culture, time-lapse image data were ana-lysed and event times determined.Main results and the role of chance: In general group, embryos were comparedby timing of first divisions. No differences between mean values for young andadvanced age women were found.

Embryos analysed by aCGH have developed to blastocyst at day 5 or day 6,allowing trophectoderm biopsy. Thus, timing was also established for laterembryo development events, e.g. morula formation, cavitation and expansionstart. Morphokinetic data were compared by women’s age and embryo ploidy.Euploid embryos of women aged 35 years or more, showed significantly slowertimes of early development when compared to euploid embryos of youngerpatients. First cleavage time was 29.10h (CI95% 26.97 to 31.33h) compared to24.57h (CI95% 23.92 to 25.22h), second cleavage was at 41.93h (CI95% 39.60to 44.26) compared to 35.91h (CI95% 34.94 to 36.88h)Limitations, reason forcaution: Presented data shows only limited possibilityofnoninvasive embryo ploidy assessment.Wider implications of the findings: Our findings suggest that currently usedtime-lapse imaging analysis criteria should be adjusted when applied toembryos of women of advanced age. In this group of patients optimal timepoints of cytokinesis are delayed.Study funding/competing interest(s): Authors have nothing to disclose.Nofunding was received for this research.Trial registration number: None

P-122 Does the type of culture media or oxygen tension affect embryofertilization and development - analysis of sibling o ocytes

P. Guilherme1, C. Madaschi1, T.C.S. Bonetti2, G. Fassolas3, and C.R. Izzo4

1Originare - Centro de Reproducao Humana, IVF Laboratory, Sao Paulo SP,Brazil, 2Universidade Federal de Sao Paulo, Laboratory of MolecularGynecology and Proteomics, Sao Paulo SP, Brazil, 3Originare - Centro deReproducao Humana, Laboratory director, Sao Paulo SP, Brazil, 4Originare -Centro de Reproducao Humana, Clinical director, Sao Paulo SP, Brazil

Study question: Are the morphokinetics of growing embryos affected by the typeof culture media utilized and does the oxygen tension of the incubator may influ-ence sequencial or single step media?Summary answer: The oxygen tension does not influence the fertilization, butthe GlobalTM (single-step) media presents better fertilization rate. On the otherhand, embryo development was not affected by the type of culture media, butfor the oxygen tension. Lower oxygen tension gives rise to better qualityembryo, and subsequently better blastocysts.What is known already: Optimal culture conditions and media composition arecritical for the development of embryos in vitro. It is commercially available singlestep and sequential media for embryo culture; however the best type of media forembryo development is not defined yet. Besides the type of media, the atmosphereculture conditions are equally important. It is known that laboratory culture ofembryos with oxygen at atmospheric tension impairs embryo metabolism andblastocyst development in several speciesStudy design, size, duration: Retrospective cohort study carried out inprivate infertility clinic. We analyzed 253 ICSI cycles with ejaculated

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sperm between January,2011 and December,2012. Embryos were culturedwith two different types of media, single-step ( GlobalTM) or sequential(VitrolifeTM), in tri-gas (5% CO2, 378C, 6% O2) or conventional incubator(5% CO2, 378C, 20% O2).Participants/materials, setting, methods: Oocytes were split according to mediaand incubator: A: atmospheric O2 tension/single-step media; B: atmospheric O2

tension/sequential media; C: 6% O2 tension/single-step media; D: 6% O2

tension/sequential media. Variables studied included normal fertilization,embryo grade on day 3, and blastocyst expansion on day 5. p ≤ 0.05 was consid-ered statistically significant.Main results and the role of chance: When normal fertilization rate was evaluated,it seems the single step media result in higher rates as groupA (77.6%) versus groupB(68.5%) (p¼ 0.286) and groupC (80.9%) versus groupD (67.5%)(p , 0.001)showed numerical and statistical significant differences, respectively. No differenceswere observed in relation to the oxygen tension. When we analyzed the embryomorphology on day 3, the low oxygen tension was relevant, as we observed statistic-ally significant difference between groupA (67.8%) and groupC (80.3%), p¼ 0.020.No difference was seen between groupB and groupD, showing that for sequencialmedia, the oxygen tension does not matter. In spite of seem that group A hadlower blastocyst rate (39.1%) than other groups (B: 51.6%, C:49.3% and D:44.2%), there is no statistical difference among them.Limitations, reason forcaution: The study was not powered to test differences inpregnancy rates between the two culture media, as embryos from different groupswere transferred for the most of patients.Wider implications of the findings: The absence of differences in the develop-ment of blastocyst between two different media concepts validates the algorithmfor embryo selection in diverse culture conditions. On the other hand, the lowoxygen tension seems to lead to improved embryo quality on day 3, and shouldbe adopted specially in short time cultures.Study funding/competing interest(s): No specific funding was obtained for thisstudy; it was solely funded by ORIGINARE. None of the authors have any eco-nomic affiliation any culture media company.Trial registration number: Not applicable.

P-123 Is intrafollicular retinoic acid associate with nuclear oocytematuration

M.J. De Los Santos1, D. Beltran1, V. Garcıa-Laez1, M.J. Escriba1, N. Grau1,L. Escrich1, C. Albert1, J.L. Zuzuarregui2, and A. Pellicer1

1IVI Valencia, Clinical embriology, Valencia, Spain, 2IVI Valencia, Clinicalanalysis, Valencia, Spain

Study question: Does intrafollicular concentration of retinoic acid (RA) correlatewith nuclear maturity of human oocytes?Summary answer: Despite the presence of RA in follicular fluids (FF), it seemsthat the concentration of RA is not directly associated with the resume of meoisis.What is known already: RA synthesis is a requirement to sustain meiosis inhuman ovary. ALDH1A2 gene expression which converts Vitamine A to retinoicacid have been found in human cumulus cells (CC) from both unstimulated andstimulated cycles and it seems that, at least in the bovine, is associated with the ac-quisition of oocyte cytoplasmic competence through the promoting growthfactors, COX2 expression suppression.Study design, size, duration: This sampling procedure studywas performed from2007 to 2009. A total of 39 FF were analysed; 13 FF from immature oocytes and 26FF from mature oocytes. Oocyte retrieval was schedule 36 hours after hCG admin-istration.Participants/materials, setting, methods: All FF were measured and centri-fuged, aliquoted and stored at -80C for subsequent analysis. All the oocytes asso-ciated to the aspirated follicles were kept in culture individually. FF were analyzedfor RA by means of liquid chromatography. Student T-test was used for statisticalcomparisons.Main results and the role of chance: We observed no differences between thegroups of immature and mature oocytes in terms of age of the patients (35.5 +3.6 vs. 34.5+6.5 years), size of the follicle (4.1 + 2.1 ml vs. 4.2 + 1.6 ml )and RA concentration (4.3 + 2.3 ug/ml vs. 4.1+1.2 ug/ml) respectively.Limitations, reason for caution: Sample size may be a limitation of the study,however since the RA concentration frame was very narrow among samples webelieve results will not vary that much.

Wider implications of the findings: Despite meiosis initiation in the humanovary relies partially on RA, it seem that is not important in later steps ofmeiosis since no differences were found in FF from preovulatory follicles contain-ing metaphase II oocytes an immature ones.Study funding/competing interest(s): The present project was supported by theR + D programme from the Generalitat Valenciana (Regional Valencian Gover-ment).Project identification number: IMPIVA IMDTG/2008/26 and IMIDTF/2009/142Trial registration number: This is not a clinical trial

P-124 Evaluation of calcium machinery in a meiosis defect mouse model

Y. LU1, D. Nikiforaki1, F. Vanden Meerschaut1, J. Neupane1, W.H. De Vos2,S. Lierman1, T. Deroo1, B. Heindryckx1, and P. De Sutter1

1University Hospital Ghent, Department for Reproductive Medicine, Gent,Belgium, 2Ghent University, Department of Molecular Biotechnology, Gent,Belgium

Study question: Is nuclear and cytoplasmic maturation impaired in in vivo andin vitro matured oocytes from LT/Sv mice, a mouse model showing oocytemeiotic arrest?Summary answer: In addition to abnormal spindle-chromosome complexes, de-fective intracellular calcium (Ca2+) signalling during in vitro maturation (IVM)points to both a nuclear and cytoplasmic maturation defect in LT/Sv oocytes.What is known already: Acquisitionofoocytemeiotic competencecoincides withspontaneous Ca2+- oscillations during germinal vesicle breakdown (GVBD)mediated by the type 1 inositol 1, 4, 5-triphosphate receptor (IP3R1). In addition,the occurrence of Ca2+-oscillations during fertilization serves as a marker of effi-cient cytoplasmic maturation. LT/Sv mice show a significant proportion of arrestedmetaphase I (MI) oocytes. Furthermore, LT/Sv GVoocytes that are in vitro maturedto the MI and MII stage show aberrant Ca2+-responses at fertilization.Study design, size, duration: Spontaneous Ca2+-oscillations were analyzed in GVoocytes following 16h IVM from LT/Sv (n¼ 38) and B6D2F1 mice(n¼ 40). Strontium-induced Ca2+-oscillations were measured for 2h in in vivo andIVM LT/Sv (n¼ 91) and B6D2F1 oocytes (n¼ 94). IP3R1 localization and spindle-chromosome complexes were assessed in in vivo and IVM LT/Sv oocytes (n¼ 72).Participants/materials, setting, methods: Oocytes were collected from 6- to 10-week-old LT/Sv and control B6D2F1 mice 48h after FSH (GV) and 14h after hCGinjection (MI and MII). Spontaneous and strontium-induced Ca2+-responses weremeasured by fluorescence time lapse imaging. IP3R1 localization and spindle-chromosome complexes were analysed by immunostaining and confocal micros-copy.Main results and the role of chance: During maturation none of the IVM MI and25% of the IVM MII oocytes from LT/Sv mice showed spontaneous Ca2+-oscillations compared to 60% (P , 0.01) and 64% of B6D2F1 oocytes, respect-ively. After strontium activation, the number of Ca2+-rises was significantlydecreased in IVM LT/Sv-MI (5.50+3.73) and IVM LT/Sv-MII oocytes(4.59+2.34) compared to in vivo LT/Sv-MI (14.28+5.83) and in vivo LT/Sv-MII oocytes (11.25+4.23) (P , 0.01) respectively. Furthermore, in vivoLT/Sv-MI oocytes showed more Ca2+-oscillations than in vivo LT/Sv-MII(P , 0.05) and B6D2F1 in vivo MII oocytes (P , 0.05). IP3R1 localization wassimilar in both in vivo and IVM LT/Sv-MI and LT/Sv-MII oocytes. However, a sig-nificantly lower number of normal spindle-chromosome complexes was observedin IVM LT/Sv-MI and LT/Sv-MII oocytes compared to in vivo matured LT/Sv-MIand LT/Sv-MII oocytes (P , 0.05).Limitations, reason forcaution: LT/Sv mice are a good model for human clinicalmixed MI arrest cases, yet the findings are species-specific and cannot be fullyextended to the human. The mechanism and pattern of strontium-induced Ca2+-oscillations may differ from those of fertilization.Wider implications of the findings: Defective Ca2+ signalling during oocytematuration might underlie certain types of oocyte maturation arrest. Nuclear trans-fer may be a way to overcome this, but given the high rate of spindle abnormalitiesin in vitro matured oocytes, this should be applied to collected in vivo maturedoocytes. Further studies are needed to determine the contribution of the IP3R1and other signalling pathways to impaired cytoplasmic maturation.Study funding/competing interest(s): This study was supported by the ChinaScholarship Council and Special Research Fund from Ghent University (Bijzon-der Onderzoeksfonds, BOF). No competing interest declared.

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Trial registration number: No trial registration number.

P-125 Embryonic humanchorionic gonadotropin (hCG) in spent culturemedium may tell embryo viability in IVF-ET program: a multi-center study

J. Li1, X.Y. Chen1, G. Lin2, G.N. Huang3, Z.Y. Sun4, Y. Zhong5, B. Zhang6, T. Li1,S.P. Zhang2, H. Ye3, S.B. Han3, S.Y. Liu5, J. Zhou4, G.X. Lu2, and G.L. Zhuang1

1The First Affiliated Hospital Sun Yat-Sen University, IVF Center-Dept. of Ob/Gyn, Guang Zhou, China, 2Central South University, Institute of Reproductionand Stem Cell, Changsha, China, 3Chongqing Obstetrics and GynecologyHospital, Chongqing Reproductive and Genetics Institute, Chongqing, China,4Peking Union Medical College Hospital, Department of Obstetrics andGynecology, Beijing, China, 5Chengdu Xinan Gynecological Hospital,Reproductive Center, Chengdu, China, 6Maternal and Child Health Hospital ofGuangxi Zhuang Autonomous Region, Obstetrics and Gynecology, Nanning,China

Study question: Could embryonic beta-hCG be as a biomarker for embryo selec-tion in IVF-ET procedure?Summary answer: A higher embryonic beta-hCG level was found in embryoswith higher morphologic grading or implantation potential and beta-hCG mightbe a predictor for embryo viability, especially in blastocyst transfer program.What is known already: hCG was one of the first foundembryonic secretions anddetected at variable levels by different methods in spent embryo culture mediumsince 1984. A highly sensitive electrochemiluminescence immunoassay (ECLIA)with strong repeatability, efficiency and stability was performed to detectbeta-hCG in culture medium in our previous study.Study design, size, duration: It’s a cross sectional experiment study. Total 719samples were individually collected at day3 (n ¼ 300) and day5 (n ¼ 419)which included fresh (n ¼ 161), frozen-thawed (n ¼ 55) and further culture toblastocyst after thawing (n ¼ 203) from 382 women in 6 IVF centers from Nov.2011 to Oct. 2012 in China.Participants/materials, setting, methods: A sequential culture system was per-formed from 2PN to blastocyst stage. Samples were collected individually andstored at -808C until beta-hCG detection by ECLIA. Clinical data of these parti-cipants were collected at the same time.Main results and the role of chance: 1) Beta-hCG was found in culture media atfresh day3 (87.7%, 263/300), fresh day5 (98.1%, 158/161), further culture to day5after thawing (96.6%, 196/203) and thawed day5 (100%, 55/55). 2) There was nodifference among conventional IVF group, ICSI group and PGD group (p ¼0.067). 3) A higher beta-hCG concentration appeared in subgroups of freshblastocysts with expanded cavity or high trophectoderm grading (A OR B)(p ¼ 0.042) and day5 (fresh/thawed and single/double embryos transferred). 5)The concentration of embryonic beta-hCG correlatedpositively with implantationrate (r ¼ 0.56, p , 0.001).Limitations, reason for caution: 1) Embryo transferred was still dependent onthe morphology grading rather than beta-hCG concentration in spent culturemedium in this study. 2) The sample size of single embryo transfer (n ¼ 88)was relatively small to describe the predictive power of beta-hCG for embryo via-bility assessment.Wider implications of the findings: Selecting the embryo with highest compe-tence by embryonic beta-hCG detection in spent culture media with ECLIAalone and/or combined with morphology grading may reduce the number ofembryos transferred, resulting in a decrease of multiple pregnancies rate in clinicalapplication.Study funding/competing interest(s): This study was supported by NatureScience Foundation of China (2008; no.30872762) and the Science Foundationof Guangdong Province (2009B030801022).Trial registration number: None.

P-126 Viability markers: optimal dynamic range for implantation incompetent blastocyst

L. Muela1, M. Roldan1, B. Gadea1, M. Martinez1, I. Perez1, M. Meseguer2, andM. Munoz3

1IVI Alicante, IVF Laboratory, Alicante, Spain, 2IVI Valencia, IVF Laboratory,Alicante, Spain, 3IVI Alicante, Medical Director, Alicante, Spain

Study question: Is there an optimal range in cleavage kinetics that increases im-plantation potential?Summary answer: For five of the morphokinetic parameters studied, we havefound a higher significant implantation probability for those embryos within anoptimal timing range.What is known already: Evaluation of embryos outside the incubator enables theassessment of timing of events, but also exposes embryos to undesirable changesin temperature, humidity and gas composition (Zhang et al., 2010). Culture ofembryos in a time-lapse monitoring system improves pregnancy outcome com-pared with a standard incubator. (Meseguer et al 2012). Addition of kinetics toconventional morphological assessment can be used as predictor of embryo im-plantation. (Meseguer et al 2011)Study design, size, duration: In a retrospective cohort study, we analyzed 236embryos with known implantation from 240 cycles included in our egg donationprogram. Embryos were culture in a time-lapse incubator after intracytoplasmicinjection or conventional in vitro fertilization and monitored until transfer onday5. It was conducted from November-2009 till December-2012.Participants/materials, setting, methods: The current study took place in aprivate IVF clinic, on embryos with Known Implantation Data (KID). Weincluded in the study embryos with 100% implantation (KIDpositive) andembryos failing to implant 0% (KIDnegative).

For each embryo, the timing of each cellular event was annotated: pronuclearformation and fading, cleavage to 2 cells (t2), t3,t4,t5,t6,t7,t8,t9, Morula(tM),early blastocyst(tB), and expanded blastocyst. We also evaluated the durationof the second cycle cc2 (t3-t2), time between 2 and 5 cells(t5-t2), and the blasto-mere synchrony s2 (t4-t3)

Selection of embryos for transfer was exclusively based on morphologicalcriteria.

The optimum timing range is established according to embryo distribution foreach parameter studied depending on implantation status.

Pearson’s Chi-square was performed, p values , 0.05 were consideredsignificant.

Main results and the role of chanceThe implantation rate (IR) of the study was 40.4%, KID IR was 39.0%, preg-

nancy rate was 64.8%. These results showed how the probability to implantrises when a blastocyst is selected inside the optimal timing range.

Limitations, reason for cautionThese are data from a retrospective analysis, although sample size is consider-

ably high. Results are based on observations with embryos from oocyte donorsand need to be repeated with embryos from infertile patients of different ages.Wider implications of the findings: Inclusion of kinetics parameters withinembryo morphology classification may increase pregnancy rates. The finding ofthe optimal kinetic range is the first step in this study. From now on, we willinclude this optimal kinetic range in our embryo selection criteria to demonstratean improvement on pregnancy rates, although previous studies already did it. Thepresent analysis will be evaluated by logistic regression in order to develop an al-gorithm for blastocyst selection.Study funding/competing interest(s): Not applicableTrial registration number: Not applicable

..............................................................

.........................................................................................

Timing ofcellular event

Probability to implant.

Outside optimumkinetic range

Inside optimumkinetic range

t5 (47- 60 h) 28.8 % (n ¼ 73) 42.9% (n ¼ 92)*

t5t2 (22-33h) 26.4% (n ¼ 14) 42.6% (n ¼ 78)*

t8 (49-62h) 32.5 % (n ¼ 38) 45.4% (n ¼ 54)*

tm (82-92h) 32.2% (n ¼ 58) 60.7% (n ¼ 34)*

tb (92-105h ) 30.3% (n ¼ 46) 54.8% (n ¼ 92)*

* p values , 0.05.

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P-127 Impact of exposure to music during in vitro culture on embryodevelopment

C. Castello1, M. Asensio1, P. Fernandez1, A. Farreras1, S. Rovira1, J.M. Capdevila1, E. Velilla1, and M. Lopez-Teijon2

1Instituto Marques, Reproductive Biology, Barcelona, Spain, 2Instituto Marques,Reproduction Medicine Service, Barcelona, Spain

Study question: The aim of this work is to determine whether or not exposure tomusic during in vitro culture conditions could improve outcomes in terms ofoocyte fertilization and embryo quality.Summary answer: Our preliminary results show a statistically significant in-crease in fertilization rate in oocytes exposed to music, but no statistically signifi-cant differences were found regarding embryo quality in terms of cleavage stageand multinucleation.What is known already: Mechanical micro-vibration can alter both the expres-sion of some molecules and neurogenesis itself (Alladi 2005). It has been reportedthat exposure of in vitro cultured human embryos to 5s intervals 44hz/h of micro-vibrations can improve embryo development and quality (Heo 2010; Matsura2010). Nothing is known about music as a source of mechanical vibrations andits effect on human embryos whilst undergoing in vitro development prior to im-plantation.Study design, size, duration: 985 oocytes from 114 patients were analyzed(01-08/2012). Inseminated oocytes from each patient were divided in groups:A (culture with music (n ¼ 497)) and B (culture without music (n ¼ 488)). Fertil-ization rates and embryo quality were compared. We tested 3 types of music(A1:Pop,A2:Heavymetal,A3:Classical) by placing speakers inside standard incu-bators (Labotec C200).Participants/materials, setting, methods: Sibling embryos were randomlyassigned to groups A/B. Morphological quality was established (scale 1-10,quality 7-10 ¼ first-choice embryos). Generalised Linear-Mixed model. Oocytefertilization rates, embryo quality (10-7 score), cleavage and multinucleationwere analysed. Two (patient/cycle) or three levels (patient/cycle/day of transfer)were considered. Bayesian inferences were made using Integrated Nested Lapla-ce(INLA).Main results and the role of chance: Fertilisation rates in group A (music) were4.82% higher than in Group B (no music). Regarding the parameters used to assessembryo quality, no statistically significant difference was found in embryo cleav-age rates or the percentage of multinucleated embryos created. In group A therewas no statistical difference in the percentage of first choice transfer embryos(score 10-7) created, compared with those in group B. However, no statisticallysignificant difference was observed between the different types of music used(pop, heavy metal and classical).Limitations, reasonforcaution:Bayesian inferences weremadeusing integratedNested Laplace (INLA).Wider implications of the findings: Limitednumberof Publications . The routineuse of music inside the incubators during in vitro culture would appear to be auseful tool to improve fertilisation rates. It is important to evaluate the effect ofmusic on embryo development to day 5. We would recommend confirmingthese results in a larger series of cases.Study funding/competing interest(s): Not applicable.Trial registration number: Not applicable.

P-128 Can a composite score based on time lapse observation aid embryoselection for single embryo transfer n an interim report

P. Kovacs1, S.Z. Matyas1, V. Forgacs2, A. Reichart2, F. Rarosi3, A. Bernard1,A. Torok4, S.G. Kaali1, A. Sajgo1, and C.S. Pribenszky5

1Kaali Institute, IVF Center, Budapest, Hungary, 2Forgacs Intezet, IVF Center,Budapest, Hungary, 3Department of Medical Physics and Informatics BolyaiInstitute, University of Szeged Hungary, Szeged, Hungary, 4Pannon ReprodukciosIntezet, IVF Center, Tapolca, Hungary, 5St. Istvan University Faculty of VeterinaryScience, Department of Animal Breeding and Genetics, Budapest, Hungary

Study question: The aim of this study is to compare pregnancy rates in humanin-vitro fertilization treatments when embryos are monitored and evaluatedusing Primo Vision time-lapse system (Vitrolife Ltd., Hungary) (TL) or culturedand evaluated in the traditional way to support the selection of a single blastocystfor transfer (ET).

Summary answer: Based on the interim results of this ongoing study TL monitor-ing may assist embryo selection for single blastocyst transfer. The 32% increase inpregnancy rate (PR) in the TL group is encouraging.What is known already: A multiple pregnancy is an undesired outcome ofassisted reproduction. Current embryo classification is inefficient in identifyingthe embryo with the highest implantation potential. Time-lapse embryo monitor-ing provides additional information about embryo development and therefore mayaid embryo selection. It appears that the kinetics and dynamics (fragmentation,blastocyst formation) of embryo development predict embryo viability.Study design, size, duration: Ongoing, prospective, randomized, multicentertrial started in Jan/2012. All patients use the long agonist protocol withrecombinant-FSH stimulation. The single blastocyst for ET is selected based onday-5 morphology (Control) or on composite score based on TL observations.Fifty patients are planned to be included per protocol.Participants/materials, setting, methods: Eligible patients are randomized toTL vs. standard daily embryo monitoring. For the TL monitoring a scoringsystem was developed including: timing of 1st division, duration of 2-cell cytokon-esis, timing of 2-3 and 3-4 cell divisions, time to reach the 5-cell stage, fragmen-tation, blastocyst morphology). Patient/ cycle, parameters were compared.Main results and the role of chance: This report is based on the first 59 rando-mized patients (28 TL vs 31 control). 12 patients dropped out (5 TL, 7 control)for various reasons. Patient and stimulation parameters are comparable betweenthe groups.

The per randomization pregnancy rates (PR): 14/28 (50%, TL) vs 13/31 (41.9%control) and ongoing PR: 13/28 (46.4% TL) vs 12/31 (38.7% control) were notsignificantly different (p ¼ 0.5).

The per protocol PR: 14/23 (60.8% TL) vs 11/24 (45.8% control) and ongoingPR: 13/23 (56.52% TL) vs 10/24 (41.6% control) were not significantly different(p ¼ 0.3). The mean time lapse score was higher among those who achieved preg-nancy in the TL group: (14.5 + /2 1.8 vs 13.1 + /2 2.0; p ¼ 0.09).Limitations, reason for caution: The sample size is relatively small to allow firmconclusions but we observed a favorable trend with TL monitoring. The scoringsystem that was developed based on currently available time-lapse findings andown experience also needs to be validated on a larger dataset.Wider implications of the findings: The development of effective embryo selec-tion tools could lead to further reductions in the number of embryos transferred foran even wider patient population without affecting pregnancy rates in the freshcycle. TL monitoring provides immediate information, is non-invasive andallows us to keep the embryos under ideal culturing conditions throughout the ob-servation.Study funding/competing interest(s): NoneTrial registration number: NCT01694641

P-129 p38 MAPK signal activity is critical for GLUT1 and GLUT4expressions, maintaining pluripotency and survival of early mouse embryos

B. Sozen1, S. Ozturk1, A. Yaba-Ucar2, and N. Demir1

1Akdeniz University Faculty of Medicine, Histology and Embryology, Antalya,Turkey, 2Istanbul Bilim University Faculty of Medicine, Histology andEmbryology, Istanbul, Turkey

Study question: Does p38 MAPK signaling pathway has any role on the regula-tion of GLUT1 (glucose transporter 1) and GLUT4 (glucose transporter 4) expres-sions, cell death, pluripotency and, thus cell fate in the preimplantation embryodevelopment?Summary answer: The suppression of p38 MAPK activity affected the expres-sion of GLUT1 and GLUT4 proteins and mRNA levels; increased cell deathand altered expression of the pluripotency markers in preimplantation embryos.What is known already: Cleavage divisions after 8-cell stage generate differen-tiation within the preimplantation embryos, namely the segregation of primitiveendoderm, epiblast, trophectoderm lineages. Nanog, Sox2, Oct4/Pou5f areknown as cell fate and pluripotency regulators during this period. On the otherhand, to accomplish proper preimplantation development, glucose transport inearly embryo achieved by facilitative glucose transporters, GLUTs. However, al-though some signaling pathways associated with these processes have been iden-tified, the role of p38 MAPK signaling is remained elusive.Study design, size, duration: Two cell stage embryos were cultured up to 8-cell,compact morula and blastocyst stages in three microdrop culture treatments;

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control (n ¼ 195), vehicle (%0.1 DMSO-treated, n ¼ 195), SB203580-treated(p38 MAPK specific inhibitor, CSAIDTM, n ¼ 223). Statistical analysis were per-formed by one-way ANOVA test.Participants/materials, setting, methods: 6-week-old Balb/C female micesuperovulated with 5 IU/mouse PMSG/hCG. Cell death was assayed byTUNEL and whole mount immunofuorescence staining used for p-MK2,p-hsp27, GLUT1, GLUT4. The relative Glut1, Glut4 mRNA levels were deter-mined by quantitative RT-PCR. Expression of Oct4/Pou5f, Nanog, Sox2 deter-mined by RT-PCR , /SSF . and analysed by Image-J software.Main results and the role of chance: With the inhibition of p38 MAPK activity,embryos displayedmorphological abnormalities, developmental blockade at8-16cells onwards (P , 0.05). In the presence of SB203580, blastocysts containedfewer cells (mean 62+2.35) and an increased percentage of TUNEL positivenuclei (8.25+1.32%) compared to control (1.11+0.33%) and vehicle groups(1.08+0,34%) (P , 0.05). Expectedly, SB-treated embryos displayed a com-plete loss of p-MK2 and p-hsp27 expressions in all groups confirming the effect-iveness of inhibitor. Besides, dramatically decreased both GLUT1 and GLUT4protein expressions and increased mRNA levels in SB-treated blastocysts wereobserved, indicating an altered glucose metabolism. Pluripotency related genesNanog, Sox2, and Oct4/Pou5f showed altered expressions in SB-treatedembryos. Nanog, a transcription factor key for epiblast differentiation, mRNAlevels significantly decreased at 8-cell stage onwards in the presence ofSB203580 (P , 0.05).Limitations, reason forcaution: This study was performed invitro conditions andshown only in mice. In vivo applications of CSAIDTM molecules is not appropriateto determine the effects of p38 MAPK inhibition on preimplantation embryos.Wider implications of the findings: These results demonstrated how the inhib-ition of p38 MAPK activity could affect the preimplantation development. Asclearly shown in the present study, the expression of glucose transporters and plur-ipotency genes to ensure lineage allocation are most likely regulated by p38MAPK activity in early mouse embryos. Therefore, our study might providenew insights into the mechanism of regulation of preimplantation developmentand new approaches for idiopatic infertility problems.Study funding/competing interest(s): This study was supported by AkdenizUniversity Research Project Coordination Unit (Project no: 2011.02.0122.005).Trial registration number: None.

P-130 A prospective study evaluating the effect of high (21%) and low(5%) oxygen levels on the human embryo development

N. Gelo, P. Stanic, V. Hlavati, S. ogoric, D. Pavicic-Baldani, M. prem-Goldtajn,B. Radakovic, M. Kasum, M. Strelec, T. Canic, V. imunic, and H. VrcicUniversity Hospital Centre Zagreb, Department of Gynecology and Obstetrics,Zagreb, Croatia

Study question: The purpose of this study was to examine effect of low O2 (5%)levels in order to obtain more embryos with excellent morphology (gradeZ1,A,AA) as well as higher clinical pregnancy rates (CPR).Summary answer: Cultivation of embryos in incubators with low oxygen (5%)levels contributes their better development on day 2, enhances the rate of blasto-cysts and those blastocysts results with higher number of CPR.What is known already: Oxygen is the major factor in embryo development invitro along with cultivation medium. Excess of reactive oxygen species (ROS)results with damage known as oxidative stress (OS) which can cause DNA frag-mentation, apoptosis, slowing or even stopping of embryo development. Excessof ROS is more common in cultures with high (21%) oxygen levels.Study design, size, duration: Between March 2012 and May 2012, 70 IVF andICSI stimulated cycles were prospectively randomized for cultivation with high(21%) or low (5%) oxygen level. Until the time of fertilization all oocytes werecultivated with high (21%) oxygen level and after that cultivated according tothe prior randomization.Participants/materials, setting, methods: Embryo development was monitoredevery 24 hours (except on day 4) for 3 - 5 days according to the day of transfer.Statistical analysis was performed with StatSoft program and results were com-pared with chi-square test with Yates correction with df ¼ 1. A P value ≤0. 05was considered statistically significant.Main results and the role of chance: Statistically significant difference wasobserved with embryos on day 2 – 52.2% (47/90) grade A embryos developed

with 5% O2 and 37.4% (34/91) with 21% O2, number of embryos that stoppedin development – 5.6% (5/90) with low oxygen and 15.4% (14/91) with highoxygen levels and number of embryos that reached blastocyst stadium – 21.1%(19/90) with 5% O2 versus 6.6% (6/91) with 21% O2.Number of transfers onday 3 showed statistically significant difference for cultivation with high (21%)oxygen levels while number of transfers on day 5 showed statistically significantdifference for cultivation with low (5%) oxygen levels-88% versus 71.2% and26% versus 8.9%.Number of CPR after transfer of blastocyst was statistically sig-nificant after cultivation in incubator with 5% O2–46.6% versus 10%.Limitations, reason for caution: Results are specific and limited due to our re-strictive law in that period - maximum of 3 oocytes per couple were fertilized.Wider implications of the findings: Themost interesting result is statistically sig-nificant difference observed with more grade A embryos on day 2 in favor of lowoxygen cultivation. These results are in contrast with those of some previousstudies that found no benefit of culturing human embryos at lower O2 concentra-tions at this stage of development (Dumoulin et al.,1999; Bahceci et al., 2005).Other results are in correlation with previous findings (Kovacic et al.,2010;Meintjes et al.,2009; Waldenstrom et al.,2009).Study funding/competing interest(s): We have no relevant interests to declare.Trial registration number: None

P-131 Growth factors status in follicular fluid of patients undergoingICSI

M. Ajina1, D. Negra1, H. Ben-Ali2, S. Jallad1, I. Zidi1, S. Meddeb3, M. Bibi3,H. Khairi3, and A. Saad3

1Unit of Reproductive Medicine, University hospital F.Hached, Sousse, Tunisia,2Laboratories of Cytogenetic Molecular Biology and Human Biology ofReproduction, University hospital F.Hached, Sousse, Tunisia, 3Department ofObstetrics and Gynaecology, University hospital F.Hached, Sousse, Tunisia

Study question: we try to find any association between follicular fluid (FF) levelsof TGFb1, IGF 1 and EGF from individual follicles and the subsequent fertiliza-tion results after ICSI of the oocytes derived from the same follicles.Summary answer: The intrafollicular concentrations of growth factors were notcorrelated with ICSI outcomes but TGF b1 expression in the ovary seems to beregulated differentially by FSH and LH since recombinant- FSH group producedhigher levels of TGF b1 compared to HMG one.TGF b1 production appears todecrease with age.What is known already: Although the exact role of each growth factor is not en-tirely known, a large body of evidence suggests that their harmonic cooperation isof crucial importance for the development of mature and competent oocyte. Byusing ICSI as the fertilization technique, the fertilization or even pregnancyoutcome seems to be mainly dependant on the quality of the oocytes, and conse-quently, the relationship between oocyte and its environment and fertilizationoutcome could be investigate.Study design, size, duration: A prospective study during oneyear , including 100women aged under than 38 years old undergoing ovarian stimulation followingstandard long agonist protocol, divided in two groups for analysis according tothe treatment modalities, HMG(n¼ 35) or recombinant FSH(n¼ 65) for ICSItreatment with an indication of male factor infertility.Participants/materials, setting, methods: Follicular Fluid and its matchedoocyte from each single follicle were collected individually during oocyte re-trieval and stored at -208c until subsequent assays for TGF b1, IGF1 and EGFby ELISA using commercially available kits. After ICSI the levels of thesefactors and the subsequent fertilization results were analyzed.Main results and the role of chance: total retrieve and metaphase II oocytenumbers were significantly higher in the r-FSH group (p ¼ 0.0001), cleavageand pregnancy rates were higher in this group (p ¼ 0.0001,), in contrast withlower rates of fertilization and top embryos. r-FSH group produced higherlevels of TGF b1 (p , 0.05) and IGF 1(p . 0.05). The intrafollicular concentra-tions of the above factors were not significantly associated with the ICSI out-comes. However, a strong positive correlation was found between TGF b1levels and IGF1 (r ¼ 0.467; p ¼ 0.0001), but a negative one between TGF b1or IGF1 and EGF(r ¼ -0.222, p ¼ 0.029; r ¼ -0.237, p ¼ 0.028. TGF b1 levelsappeared significantly higher in patients aged less than 30 years old (p ¼ 0.04)in whose pregnancy rate was higher and correlated negatively with age after 30years (r ¼ -0.262, p ¼ 0.03).

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Limitations, reason for caution: IGF 1 tented to be higher in r-FSH group al-though the difference was not significant (p . 0.05) and that was correlated posi-tively with TGF b1, so this result could be explained by our small sample size.EGF levels were very low and slightly detected coming probably through bloodpassive diffusion.Wider implications of the findings: The intrafollicular concentrations of growthfactors can not predict ICSI outcomes but TGFb1 expression in the ovaryseems tobe regulated differentially by FSH and LH; it is stimulated by FSH and inhibited byLH. The lower IGFI levels in HMG group may suggest that LH inhibits IGF1 and/or stimulates IGF binding protein 1. We suggest that TGFb1 together with IGF I,may regulate ovarian follicle growth in response to gonadotropin stimulation.Study funding/competing interest(s): TGFb1 production is stimulated par FSHand inhibited by LH and appears to decrease with age; So Studies on the follicularfeatures of the aging ovary are much needed.Trial registration number: BASIC SCIENCE

P-132 Detailed kinetics and morphology analyis of human triplonu-cleated embryos: a comparison with correctly fertilized transferred embryos

L. Escrich, N. Grau, M. Meseguer, P. Gamiz, T. Viloria, and M.J. EscribaIVI Valencia, FIV, Valencia, Spain

Study question: Is the in vitro dynamics of tripronucleated embryos (TPN) com-parable to the morphokinetics of correctly fertilized transferred embryos (BPN)?Summary answer: TPN and BPN embryos showed different dynamic featureswhich became reflected into a different embryo morphokinetic quality distribution.What is known already: Classical assessment have shown that ICSI-TPN cleaveinto two cells as BPN embryos do, but on day 3, the number of cells and ability toprogress in vitro to the blastocyst stage are inferior to that observed in TPNembryos.

Using time lapse technology, six discriminative morphokinetic parameters(t2-t3-t4-t5-cc2-s2) were defined for implantation of BPN. Using these parametersand a hierarchical classification procedure (A-E) we identified embryos with highimplantation potential (Meseguer et al 2011).Study design, size, duration: Retrospective study of the morphokinetic para-meters of 378 BPN transferred embryos and 163 TPN embryos.Participants/materials, setting, methods: Embryos were evaluated by detailedtime-lapse analysis, which measured the exact timing of t2, t3, t4, t5, cc2, s2 (inhours post-ICSI). They were then classified according to a hierarchy based onthese parameters. These parameters were compared in the two embryo groups(BPN and TPN).Main results and the role of chance: We observed significant differencesbetween BPN and TPN embryos in t2(25.9hrs vs 31.1hrs), t3(36.9hrs vs39.7hrs), t4(38.4 vs 44.2), cc2(11.0 vs 9.4hrs), cc3(14.7hrs vs 13.1hrs),s2(1.5hrs vs 4.6hrs), s3(8.2hrs vs 10.4hr) and t5-t2(25.6hrs vs 23.4hrs). Concern-ing t5, timings were comparable in both groups.

Differences in the hierarchical embryoclassification were evident. Significantlymore BPN embryos were classified as “A” and “B” than TPN embryos (49.5% vs22.2%), whereas more embryos were classified as “E” in the TPN than in the BPNgroup (44.4% vs 6.9%, respectively). Comparable percentages of BPN and TPNembryos were classified as “D” (16.1% vs 11.1%, respectively).Limitations, reason for caution: Although none of our TPN embryos were even-tually transferred, the results reported here suggest that embryo quality is impairedin these abnormally fertilized embryos.Wider implications of the findings: Normallyand abnormally fertilized embryosseem to be distinguished by the morphokinetics of in vitro embryo development.The present results reinforce the relevance of morphokinetics as a reliable markerof embryo quality.Study funding/competing interest(s): NoneTrial registration number: Not Applicable

P-133 Embryo morphokinetic after artificial oocyte activation by usingcalcium ionophore

E. Taboas Lima1, M. Perez Fernandez1, J.A. Aguilar Prieto1, M. Ojeda Varela1,D. Kassa1, and E. Munoz Munoz2

1IVI Vigo, FIV laboratory, Vigo, Spain, 2IVI Vigo, Gynelogy, Vigo, Spain

Study question: Is embryo morphokinetic affected after artificial oocyte activa-tion (AOA) with Calcium ionophore?Summary answer: Pronucleus fading(PN fading), The time from 3 to 4 cells (T4)and the time from 4 to 5 cells (T5) occurred earlier in the Calcium ionophore (iCa)group than in the control group, although this maynot affect the pregnancyand livebirth rates.What is known already: Total fertilization failure occurs in almost 10% of ICSIcycles. Artificial oocyte activation (AOA) with calcium ionophore (iCa) showedacceptable fertilization rates and successful pregnancies and deliveries of a healthyinfant however, previous animal studies have demostrated that the use of AOAmay influence the embryonic development to blastocyst stage.Study design, size, duration: Between January 2011 and January 2013, 13couples who had experienced previous total failed fertilization after ICSI cycleswith oligoasthenozoospermic sperm characteristics (iCa group) were comparedwith 12 couples with similar sperm quality (control group). Written informedconsent to share the outcomes for research purposes was obtained from all them.Participants/materials, setting, methods: The AOA consisted of sperm injectionwith buffered medium containing iCa (Ionomicym from Streptomyces congloba-tus). After sperm injection, oocytes were incubated for 20 minutes in culturemedium with iCa. In the control group conventional ICSI was performed.

To evaluate the embryo morphokinetic, oocytes were cultured in a tri-gas time-lapse incubator.Main results and the role of chance: No significant differences were observedbetween groups for age, fertilization rate, proportion of TQE and number ofembryos transferred.Significant differences were shown in embryo morphokinetics.

The implantation rate was 29.41% in control versus 30% in ICa group andongoing pregnancy rate was 80% and 83.33%, respectively. To date 4 live birthswere achieved in the control group and 5 in iCa group.Limitations, reason for caution: Total fertilization failure occurs in almost 10%of ICSI cycles. The AOAwith Ionomicym from Streptomyces conglobatus is anexperimental technique, at this moment.Wider implications of the findings: AOA induced by microinjecting sperm andcalcium ionophore is an acceptable technique in couples who faced previous failedfertilization cycles.

Pronucleus fading, T4 and T5 occurred earlier in the studied group than incontrol group, although this not affect the pregnancy and live birth rates.Study funding/competing interest(s): NoneTrial registration number: no RCT trial

P-134 Improvement of blastocyst culture by a time-lapse incubator

H. Morita1, S. Watanabe1, M. Kamihata1, R. Matsunaga1, T. Wada1, K. Kani1,T. Ishikawa1, H. Miyamura2, M. Ito2, A. Kuwahata3, M. Ochi3, and T. Horiuchi4

1Ochi Yume Clinic Nagoya, ART laboratory, Nagoya, Japan, 2Fujita HealthUniversity Hospital, Obstetrics and Gynecology, Nagoya, Japan, 3Ochi YumeClinicNagoya, Reproductive medicine, Nagoya, Japan, 4Prefectural UniversityofHiroshima Graduate School, Comprehensive Scientific Research, Hiroshima,Japan

Studyquestion: We compared the results of embryos incubated in the convention-al incubator and those cultured in the time-lapse incubator.

.........................................................................................Control iCa Sig

Age 39.35+2.7 38+2.9 .0.05

Fertilization rate 66.28+19.63 58.88+21.77 .0.05

% TQE 41.01+29.84 47.18+33.66 .0.05

Number ET 1.74+0.53 1.80+0.52 .0.05

Pn Fading 26.15+3.40 23.50 +3.65 0.03

T4 41.70+5.57 38.24+3.47 ,0.01

T5 54.92 +5.60 50.17+7.38 ,0.01

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Summaryanswer: Culture using the time-lapse incubator largely reduced the fre-quency of removal and the time required to remove embryos from the culturechamber, and improved the development rate of blastocysts. Furthermore,frozen-thawed embryo transfer had high success rates.What is known already: A time-lapse imaging incubator for embryos facilitatesthe observation of embryos within the culture chamber. This can minimize stressfactors such as decrease in temperature, change in culture medium pH, and lightexposure, and may allow ’gentle culturing of ovumStudy design, size, duration: The time-lapse imaging incubator culture groupcomprised 428 procedures (350 individuals; average age, 40.1 years) fromwhom oocytes were collected from August to December 2012. The conventionalculture group comprised 1273 procedures (757 individuals; average age, 38.5years) from whom oocytes were collected in 2011.Participants/materials, setting, methods: Embryos were placed in the time-lapse imaging incubators (TLI) or the conventional culture incubators (CCI) forup to 7 days. We removed embryos form culture chamber twice in TLI groupand up to 17 times in CCI group for inspection and changing the medium.Good quality blastocysts were cryopreserved.Main results and the role of chance: The rate of obtaining a good quality blasto-cyst was 43.5% (486 of 1116) in the ES culture group and 33.1% (1032 of 3120) inthe conventional culture group, and the difference was significant (P , 0.05). Thepregnancy rate from frozen-thawed blastocyst transfer in the the time-lapseimaging incubators culture group and the conventional culture group was48.9% (65 of 133) and 52.7% (371 of 704), respectively, and there was no signifi-cant difference.Limitations, reason for caution: noneWider implications of the findings: The improvement of the culture environ-ment by using the time-lapse imaging incubators increased the success rate ofobtaining good quality blastocysts.Study funding/competing interest(s): No ezternal funding was obtained for thisstudy.Trial registration number: Nothing

P-135 The effect of piezo-assisted biopsy on preimplantation mouseembryo development and gene expression

M.N.K. Nor-Ashikin1, J.M.Y. Norhazlin1, S. Norita1, W.J. Wan-Hafizah1,M. Mohd-Fazirul1, D. Razif2, and B.P. Hoh1

1Institute of Medical Molecular Biotechnology, Faculty of Medicine UniversitiTeknologi MARA, Sungai Buloh Selangor, Malaysia, 2Faculty of Health Science,Universiti Teknologi MARA, Puncak Alam Selangor, Malaysia

Study question: Are the development and genes expressed by piezo-biopsiedembryos significantly different from non-biopsied embryos, at morula and blasto-cyst stages?Summaryanswer: A significantdifference (p , 0.05)was observed between thedevelopment of piezo-biopsied and non-biopsied embryos at the blastocyst stage,and a total of 75 and 66 genes of piezo-biopsied embryos were found significantlydifferent in expression (p , 0.05) compared to non-biopsied controls, at morulaand blastocyst stages respectively.What is known already: Zona breaching by chemical and laser drilling are impli-cated in delayed development and abnormal hatching of blastocysts. Although ithas been reported that acid Tyrodes biopsy did not significantly affect develop-ment and gene expression of blastocysts, the effect of piezo-assisted biopsy ongene expression has not been examined.Study design, size, duration: A 2 x 2 factorial study design was used to comparedevelopment and gene expression profiles of non-biopsied with piezo-biopsiedembryos at morula and blastocyst stages. The development of 291 embryoswere observed. Thirty embryos were pooled from each treatment group forRNA extraction, with three replicates per group.Participants/materials, setting, methods: Piezo-assisted (Eppendorf Piezo-Xpertw) single-blastomere biopsies were performed on 8-cell ICR mouseembryos (68 h post-human Chorionic Gonadotropin injection) in Ca2+/Mg2+-free M2 medium. Biopsied embryos were cultured in M16 medium untilRNA extraction. cDNAwas amplified, labelled and hybridized on the Affymetrix-GeneChipw. Microarray was analysed using Gene Spring GX 12.Main results and the role of chance: Significant difference (p , 0.05) betweenthe development of non-biopsied and piezo-biopsied embryos was observed only

at the blastocyst stage (96.8% versus 89.0%, respectively). Gene expression ofnon-biopsied and piezo-biopsied mouse embryos were compared at morula andblastocyst stages, using the unpaired t-test (p , 0.05), including fold changeof ≥ 1.5 for each gene. At the morula stage, 32 genes were found upregulatedand 43 genes downregulated. At the blastocyst stage, 43 genes were upregulatedand 23 genes downregulated. DAVID pathway analysis software was used for an-notation and visualization of statistically significant genes. KEGG database anno-tated 14 genes for morula and 11 genes for blastocyst into different categories ofpathways. Both stages showed regulation of genes involved in oxidative phos-phorylation and fatty acid metabolism.Limitations, reason for caution: The use of in vivo instead of in vitro fertilizedembryos may have reduced the impact on gene expression profiles. Criticalchanges in gene expression profiles may be diluted because of the pooling ofembryos for RNA extraction.Wider implications of the findings: Elucidation of gene expression of preim-plantation embryos after biopsy will add to a better understanding of the effectof piezo-assisted blastomere biopsy on early development. This knowledge cansubsequently contribute towards the improvement of Assisted ReproductiveTechnology (ART) outcomes.Study funding/competing interest(s): This research was supported by our insti-tutional grant (600-RMI/ST/DANA5/3/Dst(337/2011) and national FundamentalResearch Grant Scheme (600-RMI/ST/FRGS5/3/FST(71/210). Animal proce-dures were approved by institutional Animal Care and Use Committee(ACUC-7/11).Trial registration number: Not applicable

P-136 Retrospective analysis of sperm incubation time pre IVFinsemination

S. Dale, E. Cater, G. Woodhead, L. Jenner, and S. FishelCARE Fertility, Embryology, Nottingham, United Kingdom

Study question: Does sperm incubation pre IVFeffect fertilisation, embryo trans-fer and clinical outcome?Summary answer: From the data presented in the abstract, clinical outcome wasnot significantly affected by duration of sperm incubation pre IVF fertilisation.What is known already: Hyperactivation and capacitation of sperm is known tobe induced by warming to body temperature, as would occur naturally in thefemale genital tract, and this is a prerequisite for fertilisation. Seminal plasma inhi-bits this hyperactivation (Mortimer et al 1998).

Ragaa et al (2008) reported the rate of acrosomally reacted sperm was greatestafter incubation for 5 hours but that a better fertilisation rate was achieved with 3hours incubation pre-ICSI.Study design, size, duration: Retrospective analysis, non randomised analysis ofIVF cycles from a single independent UK clinic. 153 continuous IVF cyclesincluded in analysis from January to October 2012, regardless of age, previoushistory, sperm or oocyte source.

Results to be statistically analysed using unpaired t-test and Fisher’s exact test.Participants/materials, setting, methods: Cycle preparation and stimulationvaried as per consultant recommendation based on previous history. Oocyte re-covery, insemination, culture and embryo transfer were performed as per protocol.Transfer day varied between day 3 and day 5 based on embryo development, andnumber of embryos transferred varied based on age and previous history.Main results and the role of chance: 222 patients were included in the study. Ofthese 201 achieved embryo transfer. These patients were divided into 2 groups de-pending on pre IVF sperm incubation times (Group 1 60-89 mins and Group 290-119 mins). Fertilisation rates were comparable (71.9%, 72.9%, p ¼ 0.8308 re-spectively) as were the abnormal fertilisation rates (3pn) (4.3%, 4.4%, p ¼ 0.9222respectively). Both were analysed using unpaired t tests. Patients that didn’tachieve embryo transfer due to failed fertilisation, embryo development orfreeze all due to OHSS risk were analysed but showed no correlation. PositivebhCG (55.4%, 60.6%), clinical pregnancy (47.5%, 47.9%) and biochemicalloss rate (14.3%, 20.9%) were analysed using Fisher’s exact test but showed nosignificance (p ¼ 0.5339, p ¼ 1.000 & p ¼ 0.4286 respectively).Limitations, reason forcaution: The study was limited due to a change in labora-tory protocol in January 2012. Sperm was incubated for 1-2 hours prior to insem-ination, therefore data pre 1 hour and post 2 hours is limited. The majority of casesperformed at the clinic are ICSI, giving low numbers for analysis.

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Wider implications of the findings: There is little literature available regardinglength of time sperm should be incubated for IVF inseminations. There aremany studies discussing sperm activity and capacitation upon which laboratoriesmust base their protocols for incubation pre insemination. Many of these studiesassess acrosome activity but do not relate to IVF outcome. This study, althoughlimited, provides evidence that for the specific times of incubation includedthere is no statistical difference in outcome.Study funding/competing interest(s): NoneTrial registration number: None

P-137 The effect of CO2 levels in embryo culture on clinical outcome

S. Andronikou, G. Francis, S. Tailor, M. Vourliotis, and P.A. AlmeidaChelsea & Westminster Hospital, ACU, London, United Kingdom

Study question: We sought to determine whether a change in CO2 level suppliedto IVF incubators from 6.0% to 5.5% had an effect on fertilization rate (FR) andclinical pregnancy rate (CPR). This retrospective analysis of women ≤34 and≥35 years of age was conducted in the context of the recommendations of theculture media manufacturer.Summaryanswer: Although not statistically significant, overall FR was higher witha CO2 supplyof 5.5% compared to 6.0%. In contrast to younger women, a statisticallysignificant decrease in CPR was observed in the older group of patients whenembryos were cultured under 5.5% rather than 6% CO2 level. There was no statisticaldifference in FR for either of the groups under the different CO2 conditions.What is known already: Most culture media utilize a bicarbonate/CO2 buffersystem to keep pH in the range of 7.2-7.4.Media manufacturing companies recom-mend a value of CO2, having taken into consideration the Henderson-Hasselbachequation, under which their media will achieve the desired pH. In theory 1% dif-ference in CO2 should only alter the pH by 0.1. However, as pH is a fundamentalfactor impacting oocyte viability and subsequent embryo development, any slightfluctuation may affect the resulting embryo’s developmental potential.Study design, size, duration: A retrospective analysis to examine the effects ofchanges in CO2 levels on FR and CPR in 384 ART cycles over a 12 monthperiod from January -December 2012. Results were compared among two differ-ent age groups (≤34 and ≥35 years old). A proprietary commercially availablemedium was used for gamete/embryo culture.Participants/materials, setting, methods: In 209 cycles the gametes/embryoswere cultured under 6.0% CO2 supply (Condition A) and in the remaining 175cycles under 5.5% CO2 supply (Condition B). These conditions were comparedamong two different age groups. 72 cycles of Condition A were compared to 54cycles of Condition B in patients ≤34 years (Group I). 137 cycles of ConditionA were compared to 121 cycles of Condition B in patients ≥35 years (GroupII). T-test was employed to examine the difference in the mean maternal ageamong the groups. One sample Z-test with significance level of 0.05 was usedfor statistical analysis of FR and CPR.Main results and the role of chance: The mean maternal age of Group I underCondition Awas comparable to the mean maternal age of Group I under ConditionB (30.6 years+3.0 vs. 31.4 years+2.5, NS). Similar results were observed forGroup II (Condition A: 38.7 years+2.5 vs. Condition B: 38.4 years+2.3, NS).

FR in Group I was slightly higher under Condition B than in cycles under Con-dition A (63.2% vs. 58.4%, NS). Almost identical results were observed when FRof Group II was compared under different CO2 conditions (Condition B: 60.8% vs.Condition A: 60.0%; NS).

CPR in Group I under Condition Awas comparable to CPR under Condition B(41.7% vs. 40.7%, NS). However, a statistically significant decrease in CPRwas observed in Group II under the different CO2 conditions (ConditionA: 28.5 % vs. Condition B: 17.4%; p ≤ 0.05).Limitations, reason for caution: Though the findings are intriguing, more data isrequired to determine whether these findings are valid. Ideally, the pH of the mediashould be measured as well as the CO2 levels.Wider implications of the findings: Thedatademonstrated that slight difference inCO2 levels in the IVF culturehadsignificant impactonclinical outcomeamongolderwomen. One possible explanation is that when handling gametes/embryos outsidetheir normal environment, replacing them into an incubator with a higher CO2

level allows for a faster recovery to physiological pH levels; older patients’embryos due to poorer quality may be more prone to pH fluctuation induced

damage.A larger sample inaprospective studywillbe required toconfirmthesefind-ings are robust, and whether there is an effect on embryos of younger women.Study funding/competing interest(s): NoneTrial registration number: N/A

P-138 Cyclin E1 as a new ICM marker identifies a discrete lineage in epi-blast cells of the human blastocyst

M. Krivega1 and H. Van de Velde2

1Vrije Universiteit Brussel, Reproduction and Genetics - REGE, Brussels,Belgium, 2Vrije Universiteit Brussel and UZ Brussel, Reproduction and Genetics(REGE) and Centre for Reproductive Medicine (CRG), Brussels, Belgium

Study question: This study was aimed to describe new molecular players inhuman embryonic cells. In particular, we investigated Cyclin E1 (CCNE1) as a po-tential regulator of inner cell mass (ICM) in human embryos.Summary answer: The new marker of ICM in expanding blastocysts, CCNE1, isrestricted to a defined cell type in human blastocysts at the moment of implant-ation.What is known already: Cyclin E1 is one of the major regulators of the cell cycle.It forms a complex with CDK2 (cyclin-dependent kinase 2) and together theypromote transition from G1 to S phases of the cell cycle. CCNE1 protein isknown to be sustained to high levels in pluripotent cells. CCNE1 is necessaryfor re-entry of G0 cells into the cell cycle and for oncogenic transformation.Knock-out studies in mice proved redundancy for CCNE1 and CDK2.Study design, size, duration: We analyzed the expression pattern of CCNE1 inhuman preimplantation embryos and hESC in combination with pluripotencyand early differentiation markers. Day 3 frozen-thawed embryos were culturedin presence of Roscovitine and harvested on day 6 for gene expression analysis.HESC were cultured on matrigel and transfected with FUGENE.Participants/materials, setting, methods: The study was approved by the Localand Federal Ethical Committees for research on human embryos. Human embryoswere obtained from patients treated for infertility at our IVF Centre. Human em-bryonic stem cell (hESC) lines had been derived by our group. Samples were ana-lyzed by qRT-PCR and immunocytochemistry.Main results and the role of chance: CCNE1 protein is exclusively expressed innuclei of all ICM cells in expanded blastocysts. Later at the moment of implant-ation (day 6-7) CCNE1-positive cells only mark NANOG-negative cells of theepiblast. Inhibition of CCNE1 function during the cell cycle using Roscovitinedid not affect preimplantation embryo development and expression of general em-bryonic markers for pluripotency and differentiation. Moreover, CCNE1 wasfound in hESC, particularly in VIMENTIN-positive cells acquiring epithelial-mesenchymal transition and VIMENTIN-negative cells, but not in NANOGexpressing cells. Overexpression of CCNE1 in hESC strongly upregulatedSOX17 and mildly induced GATA4 and GATA6. This effect implies specificationinto the endodermal lineage, although SOX17 marks both endoderm progenitorsand PGCs. We currently investigate this hypothesis to find out the nature ofCCNE1-positive epiblast cells.Limitations, reason for caution: There are limited numbers of good qualityhuman embryos donated for research.Wider implications of the findings: The improved knowledge on human preim-plantation development will contribute to refine artificial reproductive techniquesand consequently increase live birth rates.Study funding/competing interest(s): Our research is supported by grants fromthe Fund for Scientific Research - Flanders (FWO-Vlaanderen) and the Methusa-lem (METH) of the VUB.Trial registration number: none

P-139 Correlation of PRSS35 and SERPINE2 gene expression levels incumulus cells with oocyte maturation and the potential as a biomarker topredict embryo quality

R.K. Lee1, Y.M. Hwu1, C.H. Lu2, and S.H. Li2

1Mackay Memorial Hospital, Department of Obstetrics and Gyneocology, Taipei,Taiwan R.O.C, 2Mackay Memorial Hospital, Department of Medical Research,New Taipei, Taiwan R.O.C

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Study question: Are PRSS35 and SERPINE2 gene expression levels in cumuluscells correlated with oocyte maturation, fertilization, and embryo quality?Summary answer: PRSS35 and SERPINE2 gene expression levels in cumuluscells were correlated with oocyte maturation and fertilization as well as goodembryo quality.What is known already: The oocyte is surrounded by several layers of cumuluscells and that bi-directional communication between the oocyte and thecumulus cells is crucial for oocyte developmental competence and embryo de-velopment. PRSS35, SERPINE2, PTX3, and GJA1 are expressed in humancumulus cells. PTX3 and GJA1 mRNA levels are known to be associatedwith embryo quality. However, the correlation of PRSS35 and SERPINE2 ex-pression levels with oocyte maturation and embryo developmental potentialremains to be clarified.Study design, size, duration: Total 211 cumulus cell samples of individualoocytes were obtained from 30 patients in the IVF laboratory. Patients under-going intracytoplasmic sperm injection treatment program were recruited intothe study.Participants/materials, setting, methods: PRSS35, SERPINE2, PTX3, andGJA1 mRNA levels were assessed in cumulus cells of individual oocytesusing quantitative RT-PCR. PTX3 and GJA1 expression levels were alsoassayed for reference. The housekeeping gene RPL19, ribosomal proteinL19, was used as the internal loading control to normalize the relative geneexpression levels. Differences were analyzed by one-way analysis of variancefollowed by the Bonferroni post hoc test using GraphPad software. P , 0.05was considered significant.Main results and the role of chance: Cumulus cells from mature oocytesexpressed significantly lower PRSS35 and SERPINE2 mRNA levels than thosefrom immature oocytes (P , 0.05 and P , 0.0001, respectively). Conversely,Cumulus cells surrounding mature oocytes expressed significantly higher PTX3mRNA levels than those encircling immature oocytes (P , 0.05). GJA1 mRNAlevels also showed the tendency, although there was no significant differences.PRSS35 mRNA levels were 5-fold lower in cumulus cells from mature and ferti-lized oocytes than those from mature but non-fertilized oocytes. GJA1, PRSS35,and SERPINE2 mRNA levels in cumulus cells from mature oocytes that devel-oped to high quality embryos (grades 1 and 2) were also significantly lowerthan those developed to poor quality embryos (grades 3 and 4) (P , 0.05).However, PTX3 mRNA levels exhibited the reverse tendency, which is similarto the previously published results.Limitations, reason for caution: A small sample size may cause insufficientpower. Thus, further large cohort studies are required.Wider implications of the findings: PRSS35 and SERPINE2 mRNA levels incumulus cells correlate with oocyte maturation, fertilization, and good-embryoquality. This may provide novel biomarkers to predict oocyte developmental com-petence and embryo development.Study funding/competing interest(s): This work was funded by a grant, NSC101-2314-B-195-009-MY3, from the National Science Council of Taiwan. isdeclared.Trial registration number: none

P-140 Biologic efficiency of fresh oocytes in a large Italian ART programin relation to International benchmarks

A. Vaiarelli, R. Antonacci, A. Smeraldi, M. Desgro, E. Albani, A. Baggiani,E. Zannoni, and P.E. Levi SettiIRCCS Istituto Clinico Humanitas, Department of Gynecology Division ofGynecology and Reproductive Medicine, Rozzano, Italy

Study question: Is the ratio of babies delivered in relation to number of utilizedoocytes a constant element in different infertile populations and countries? Aimof the present study was to compare retrospectively the biological efficiency ofour ART program in relation to USA and European benchmark published data.Summary answer: Although great differences in term of delivery rates have beenreported between European and USA results, representing still a matter of debate.The mean number of oocytes utilized to obtain a single baby delivered seems notsignificantly different among our results and European and USA benchmarks.What is known already: A large and constant number of oocytes is needed toreach a delivered baby. This number seems a constant biological limitation, al-though improvements in ART technology during the last 30 years have been

implemented. According to the American data “live birth” per oocyte retrievedwas 4.6 %. European data shows that 4.4% to 3.8% (according to the age ofpatients) oocytes are needed to deliver a baby.Studydesign, size, duration:Retrospective analysis of clinical and embryologic-al data of cycles performed at the Humanitas Clinical and Research Institute (Italy)in the period from 1 January 2011 – to 31 December 2011.Participants/materials, setting, methods: 1739 patient’s cycles with 9194oocytes inseminated (7989 ICSI and 1205 IVF) were analyzed. Mean femaleage was 37+3.9 years. Data were divided by age into two groups (≤38 A and≥39 B). Live born rate was calculated from the total number of oocytes includingonly fresh embryo transfer.Main results and the role of chance: The live baby born rate per inseminatedoocytes was 3.5%. In group A and B the live baby born rate was respectively 4.4% and 2.1% per oocyte used. The number of eggs needed to have a baby was 22.6in group A while 48.4 in group B. American and European data show no increasein babies born if . 15-20 oocytes were collected. This range of oocytes could bethe optimal number to maximise delivery rate, minimizing the risk of OHSS infresh ART cycles. Our results even without considering pregnancies obtainedfrom oocytes and embryos cryopreservation are not so far from benchmark results.Limitations, reason for caution: Our results do not consider cumulative deliveryrate, because most of patients have still cryopreserved oocytes and embryos. Ourdata consider only used oocytes and not all mature retrieved oocytes.Wider implications of the findings: A strict correlation exists between thenumber of the eggs collected and babies born in ART cycles. This enormous bio-logical wastage is probably due to the great number of intrinsically abnormaloocytes. This knowledge could be important in couple’s counseling. Our effortsshould be addressed to modify earlier stages of folliculogenesis, to improveoocyte intrinsic quality, before ovulation induction, were little could be done.Study funding/competing interest(s): NoneTrial registration number: Not applicable

P-141 Factors affecting the twin-delivery rate in sperm donationprogramme

L. Bacer Kermavner1, I. Virant Klun1, B. Pinter2, and E. Vrtacnik-Bokal2

1University Medical Centre Ljubljana, Dep. of Obstetrics and Gynaecology - IVFLaboratory, Ljubljana, Slovenia, 2University Medical Centre Ljubljana, Dep. ofObstetrics and Gynaecology Reproductive unit, Ljubljana, Slovenia

Study question: In our sperm donation programme there is still twin-pregnancyrate 20%, therefore the aim of this study was to elucidate if the female age and thenumber of retrieved or transferred blastocysts affect the twin-delivery rate and thepotential role of elective single embryo transfer (eSET) in these couples.Summaryanswer: eSETis recommended in sperm donation programme when atleast 4 good quality blastocysts are developed after prolonged embryo culture re-gardless the female age.What is known already: eSET has already became a practice in the in vitro fer-tilization programme using autologous fresh partner’s semen to prevent the twin-pregnancy and to avoid the risk for both the woman and the baby. It is less knownabout the factors affecting the twin-pregnancy in the sperm donation programmeusing frozen-thawed sperm.Study design, size, duration: The data on 497 in vitro fertilization cycles usingfrozen-thawed donated sperm were performed at our department during the timeperiod from 2001 to 2011 and were retrospectively analyzed to elucidate the effectof female age and number of retrieved or transferred blastocysts on twin-deliveryrate in these couples.Participants/materials, setting, methods: One or two blastocysts were trans-ferred. The couples were divided into three groups: 1.) non-pregnant, 2.) withsingle-delivery or 3.) with twin-delivery. All groups were divided according tothe female age (≤36 or .36 years) and compared in terms of the number ofretrieved and transferred blastocysts by the Chi-Square test. Statistical signifi-cance was set up at P , 0.05.Main results and the role of chance: In couples with transferred one blastocystthe mean number of blastocysts per cycle was lower than in couples with a transferof two blastocysts; after the transfer of two blastocyts there was a higher numberofblastocyts per cycle in couples with pregnancy/delivery than in couples with nopregnancy regardless the female age. In couples with twin-delivery there was ahigher number of blastocysts per cycle (younger: 3.8, older: 4.0) than in

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couples with single delivery (younger: 2.9, older: 2.1). This indicated that thenumber of at least 4 blastocysts retrieved per cycle is an important tool to introducethe eSETinto the embryo transfer strategy regardless the female age. In all coupleswith twin-delivery number of blastocyst per cycle was significantly higher than incouples with single delivery.Limitations, reason for caution: The number of cycles was realtively low.Wider implications of the findings: These results indicated that developmentalpotential of embryos and a number of blastocysts per cycle may be a good prog-nostic factor for twin-pregnancy/delivery and an important tool to introduce theeSET in the sperm donation programme regardless the female age.Study funding/competing interest(s): The study was performed in the frame ofour clinical programme. There are no competing interests to be declared.Trial registration number: 0

P-142 Differentiation during early human embryogenesis

C. De Paepe1, G. Cauffman2, G. Verheyen2, D. Stoop2, I. Liebaers3, andH. Van de Velde2

1Vrije Universiteit Brussel, Department of Reproduction and Genetics (REGE),Jette, Belgium, 2Universitair Ziekenhuis Brussel, Centre for ReproductiveMedicine (CRM), Jette, Belgium, 3Universitair Ziekenhuis Brussel, Centre forMedical Genetics (CMG), Jette, Belgium

Study question: How are the trophectoderm (TE) key regulators CDX2, TEAD4and YAP expressed during early human embryogenesis?Summary answer: In human embryos, the restriction of TE-specific componentsto the TE occurs after expansion of the embryo, when the two cell lineages (TEversus inner cells mass (ICM)) are morphologically distinguishable, suggesting arole for other molecules in the initial segregation between the ICM and TE lineages.What is known already: In mice, the Hippo Signaling pathway has been describedin thefirst lineagesegregation (Nishiokaetal. 2009).Differences incell-cell contactsthrough differences in cell position lead to activation or inactivation of YAP, whichsubsequently can activate or inactivate TEAD4 in the nucleus and consequentlyregulate CDX2 expression in the inner versus outer cells. In the human it is notknown how these Hippo signaling pathway components regulate TE specification.Study design, size, duration: In this study, human preimplantation embryos wereanalysed for the expression of the transcription factors CDX2 and TEAD4, and theco-activator protein YAP. Both localization within the embryo and the time-specific expression were analysed.Participants/materials, setting, methods: The study was approved by the Localand Federal Ethical Committees for research on human embryos. Good qualityembryos were obtained at our IVF Laboratory after informed consent of thepatients. They were fixed in 4% paraformaldehyde, permeabilized with 0,1%triton and stained for the respective proteins using immunocytochemistry.Main results and the role of chance: In cleavage-stage embryos, compaction andearly blastocyst stages CDX2was found weakly cytoplasmic. From the full blastocyststage onwards, some cells showed nuclear CDX2 expression. In expanded blasto-cysts CDX2 was found either in the TE nuclei or in the ICM and TE nuclei. In hatch-ing/hatched blastocysts CDX2 was found in the ICM and TE nuclei except forone bighatched blastocyst that started to downregulate CDX2 expression in ICM nuclei. YAPand TEAD4 started to be expressed in the nuclei of some cells of late cleavage-stageand compacted embryos. In expanded, hatching and hatched blastocysts TEAD4 andYAP were expressed in ICM and TE nuclei, except for one big hatched blastocyst inwhich YAP started to become downregulated in the nuclei of ICM cells.Limitations, reason for caution: Due to ethical concerns the amount of materialis limited. This work is rather descriptive, but functional studies will be performedin the future.Wider implications of the findings: Information about the key players leading tothe first lineage segregation in the human embryo and the mechanisms underlyingdifferentiation will contribute to our basic knowledge on human embryogenesis.This fundamental research is unique and crucial to the field of reproductive medi-cine. Even though a lot is known about this subject in mice, results obtained in thehuman are different. This study shows that data obtained in mice cannot always beextrapolated to humans.Study funding/competing interest(s): This research is supported by grantsfrom the Scientific Research Foundation- Flanders (FWO-Vlaanderen) and theResearch Council (OZR) of the VUB.Trial registration number: None

P-143 Oocyte activation increases fertilization rate resulting in a higherfinal number of blastocysts without compromising implantation potentialper transferred embryo: a sibling prospective study

A. Stecher, B. Wirleitner, P. Vanderzwalmen, M. Zintz, A. Neyer, M. Bach,B. Baramsai, D. Schwerda, and N.H. ZechIVF-Centers Prof. Zech Bregenz GmbH, Bregenz, Austria

Study question: Low fertilization rates (FR) -predominantly observed in severemale factor patients- negatively affects IVF outcome. We evaluated whether acti-vation with calcium ionophore after sperm injection increases FR in these patientsand studied the impact on blastocyst development, implantation rates (IR) andpregnancy rates (PR) in a sibling study.Summary answer: Oocyte activation significantly increased FR in male factor in-fertility (e.g. severe OAT, testicular sperm extraction) and unexplained fertilizationfailure. Activation did not compromise blastocyst development but led to a highernumberof fertilized embryos. Thus, overall moreblastocysts with similar potentialto implant are achieved per cycle.What is known already: To release the meiotic arrest in the oocyte and initiate thefertilization process, repeated calcium oscillations are essential. One main triggerfor this intracellular signal was identified as the sperm-specific phospholipase Czeta I. Therefore failed fertilization is thought to be mainly a sperm factor. FRwas shown to be increased with oocyte activation by calcium ionophore in patientswith previous implantation failure and case series described no impact on health ofbabies born.Study design, size, duration: This prospective study included 62 IVF-cyclesbetween November 2009 and December 2012. Inclusion criteria were severe malefactor infertility and/or unexplained fertilization failure (,30%) in previous cycles.Sibling analysis was performed. Injected oocytes were randomly grouped in halfand allocated either to the active or non-activated group for direct comparison.Participants/materials, setting, methods: ICSI or IMSI was performed 2-3hours post-OPU. Half of the injected oocytes of each patient were activatedwith calcium ionophore. FR was checked 17 hours post injection, embryoquality on day 3 and blastocyst rate on day 5 before embryo transfer. IR was cal-culated by embryonic heart beat/embryo transferred.Main results and the role of chance: In 62 OPUs 966 oocytes were retrieved and798 MII oocytes were injected. Of 397 non-activated oocytes 256 (64.5%) werefertilized on day 1 as compared to 302/401 in activated (75.3%; p , 0.001). Inthe further development the blastocyst rate/ MII oocyte was 31.8% without activa-tion as compared to 36.6% with calcium ionophore. On day 5 the best blastocystswere chosen for transfer by morphological criteria. In the male-factor group thenumber of transferred blastocysts was the same in both groups, but in thenon-male factor patients more embryos from the non-activated group werechosen. Similar pregnancy rates (54.5% vs. 52.9%) were obtained with non-activated and activated, the IR was 35.3% vs. 29.6% and the ongoing PR 45.5%vs. 41.2%.Limitations, reason for caution: A higher number of cases as well as a long-termfollow-up of the children’s health will be necessary to prove the safety of this pro-cedure.Wider implications of the findings: Our results are in line with the literature.Additionally, we show for the first time, that oocyte activation does not negativelyimpact on blastocyst development. Similar IR and ongoing PR for activated andnon-activated sibling embryos were observed. By applying this technique thetotal number of developing blastocysts/ cycle is increased. This may lead to ahigher cumulative PR.Study funding/competing interest(s): NoneTrial registration number: Informed consent was signed by all patients.

P-144 Synchronized blastomere cleavage at cryopreservation: Effect onsubsequent embryo survival, pregnancy and live birth rates

Z. Wiener-Megnazi, M. Fridman, M. Koifman, S. Lahav-Baratz, I. Blais,R. Auslender, and M. DirnfeldCarmel Medical Hospital, IVF Unit Obstetrics and Gynecology, Haifa, Israel

Study question: To evaluate the effect of blastomere synchronicity of frozenembryos on post - thaw survival, morphological grading and outcome parametersin frozen embryo transfer (FET) cycles.

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Summary answer: Embryos with both synchronous and asynchronous blasto-mere cleavage can be safely cryopreserved. Embryos for cryopreservationshould be selected by classical embryo grading and not by the criteria of synchron-icity of blastomere cleavage.What is known already: While morphologic scoring of embryos in IVF isestimated subjectively, number of blastomeres and the distinction betweensynchronous and asynchronous cleavage are objective parameters. Embryos withsynchronized blastomere cleavage are usually preferentially selected for cryopreser-vation. Time-lapse analysis techniques implicated synchronicity of blastomere

cleavage as a possible predictor of treatment success.Study design, size, duration: Retrospective analysis of 1060 FET cycles per-formed from 2004 - 2006. Cycles were divided into 3 groups: 1: cycles withonly embryos with synchronous blastomere cleavage were frozen; 2: cycleswith only embryos with asynchronous blastomere cleavage were frozen; 3:cycles with both, synchronous and asynchronous blastomere cleavage embryosfrozen.Participants/materials, setting, methods: One thousand and sixty FET cycleswere analyzed. Clinical and laboratory data was recorded and analyzed. Mainoutcome parameters were: Post - thaw embryo survival, morphologic grading,pregnancy and live birth rates.Main results and the role of chance: Out of 1863 embryos, embryo survival ratewas 68%; in 90%, ≥1 embryo survived. This was similar among all groups. Fullblastomere survival rate was higher among the synchronous group (62.7%,43.3%, 38.3% respectively, P ¼ 0.0001). Mean and maximal embryo grading atcryopreservation and at thawing were higher among the synchronous group(P ¼ 0.0001, P ¼ 0.037, P ¼ 0.004, P ¼ 0.03, respectively). When controlledfor number of thawed embryos, differences remained significant for 2 embryos(P ¼ 0.028). Mean and maximal thawing parameters were in association withconception. (P ¼ 0.056, P ¼ 0.023, respectively). Groups were similar regardingpregnancy and live birth rates. In a multivariate regression analysis, number oftransferred embryos and maximal embryo grading at thawing affected the occur-rence of conception (P ¼ 0.021, P ¼ 0.027, respectively).Limitations, reason for caution: Validity of results may be limited due to theretrospective nature of the study.Wider implications of the findings: Our study suggests that synchronicity ofblastomere cleavage at cryopreservation should not serve as a criteria to selectembryos for freezing. Although some of the embryo survival parameters werebetter among synchronous embryos, this was not associated with the chances ofimplantation. Therefore, the implantation potential of asynchronous and syn-chronous thawed embryos should be regarded as equal, in the same manner asfresh embryos, and not be discarded from being candidates for cryopreservationas such.Study funding/competing interest(s): The study was not funded by any com-mercial company. There were no competing interests in the study.Trial registration number:Thestudy is not an RCT, so there is no need fora studyregistration number.

P-145 Histidine-rich glycoprotein improves embryo development

H. Akerud, K. Lindgren, K. Karehed, K. Wanggren, and J. HreinssonDepartment of Women’s and Children’s Health, Uppsala University, Uppsala,Sweden

Study question: Does a Histidine-rich glycoprotein (HRG) peptide improvehuman embryo development and is the Embryoscope useful for evaluation ofembryo development?Summary answer: A 35 amino-acid long HRG peptide seems to improve humanembryo development and maturation when added to culture media. An Embryo-scope is useful for monitoring embryo development.What is known already: Histidine-rich glycoprotein (HRG) is a protein known tointeract with a number of different biological pathways. We have previouslyshown that a single nucleotide polymorphism (SNP) in the HRG gene (HRGC633T) is associated with recurrent miscarriages and pregnancy success rateafter IVF treatment. In addition, infertility seems to associate with the HRG geno-type. The different pathways through which HRG affects embryo development,implantation and placentation are not yet well established.Study design, size, duration: The study was designed as a case-control study andwas approved by the regional ethics committe. Fourty-eight couples donated their

surplus embryos which were cultured for four days and continoulsy monitored bya time-lapse methodology using an Embryoscope (UniSense Fertilitech, Denmark).Participants/materials, setting, methods: The study was carried out at the Re-productive Center, Akademiska sjukhuset, Uppsala, Sweden. Embryos frozenon day two after fertilization were thawed and cultured with or without HRGpeptide added to culture media. Time-lapse sequences were used for timing of de-velopmental events and to set final blastocyst scores.Main results and the role of chance: After four days of culture 71% of theembryos in the peptide group (n ¼ 24) and 58% of the embryos in the controlgroup (n ¼ 24) had developed to blastocysts. 46% of the embryos in thepeptide group and 25% of the embryos in the control group were given thehighest final blastocyst scores. The time needed from first cleavage to develop-ment of morula differed significantly between the groups (43.1+9.8 h peptidegroup vs 34.6+14.3 h control group, p ¼ 0.049). In conclusion, the embryoscultured with the HRG peptide added developed slower and the difference intime needed between first cleavage and development of morula might reflect abetter timing of events of relevance for adequate maturation of an embryo.Limitations, reason for caution: This is a relatively small study and the resultsneed to be confirmed in a larger study population. Also, the protocol for scoringthe embryos is new and needs to be evaluated further. The results are although sig-nificant and the protocol was easy to use and reproducible.Wider implications of the findings: The results indicate that HRG is a protein ofimportance in human embryo development and that the HRG peptide might be ofinterest to add to culture media to optimize maturation of embryos and also toimprove IVF treatment results. The Embryoscope is useful for evaluation ofhuman embryo development.Study funding/competing interest(s): This study was funded by SwedishSociety of Medicine and the Family Planning Foundation in Uppsala, Sweden.The authors declare no competing interests.Trial registration number: Not applicable

P-146 The efficacy of hyaluronate (HA): a comparative study to evaluatethe selection of mature sperm in ICSI-IMSI cycles

S. Rovira1, J.M. Capdevila1, B. Freijomil1, C. Castello1, A. Farreras1,P. Fernandez1, M. Asensio1, M. Lopez-Teijon2, and E. Velilla1

1Instituto Marques, Reproductive Biology, Barcelona, Spain, 2Instituto Marques,Reproduction Medicine Service, Barcelona, Spain

Study question: The objective of the study is to determine whether or not spermselection by means of hyaluronate selection by SpermSlow is better than poly-vinylpyrrolidone (PVP) in terms of the subsequent embryo fertilisation rates, via-bility, and pregnancy rates, in patients with teratozoospermiawho undergo ICSI orICSI-IMSI.Summary answer: There is a statistically significant increase in the number ofviable embryos created when hyaluronate rather than PVP is used to selectsperm prior to ICSI. This difference was not noted if IMSI is used. No statisticallysignificant difference was noted in fertilisation rates or pregnancy rates with hya-luronate.What is known already: Mature sperm have receptors for hyaluronic acid, aprotein found on the outer membrane of the oocyte. These sperm can thenremodel the protein on the oocyte. Hyaluronidate in the microinjection mediaimproves the selection of sperm with greater DNA integrity (Gabor Huszar,2003), improves fertilisation rates (Nasr-Esfahani, 2008) and improves embryoquality, development and implantation (Parmegiani, 2009; 2010). However,these improvements have not been noted in all groups (Van der Bergh, 2009).Study design, size, duration: A prospective year-long study was set up in 2012 tocompare hyaluronate (HA) by SpermSlow media (Medicult) and polyvinylpyr-rolidone (PVP) (Vitrolife). The comparison was made both in patients whoused ICSI and those who used IMSI-ICSI, resulting in the creation of 4groups:G1: PVP-ICSI, G2: HA-ICSI, G3: PVP-IMSI-ICSI; G4: HA-IMSI-ICSI.Participants/materials, setting, methods: Inclusion criteria included allpacients who required egg donation, and whose partner’s sperm showed asevere teratozoospermia (normal morphology ,4%, Kruger et al, 1988).Main results and the role of chance: 537 oocytes were fertilised, 323 usingPVP-ICSI (G1) and 214 using HA-ICSI (G2). No significant differences werenoted between the two groups in terms of fertilisation rates (76,47% vs 77,1%p ¼ 0,8652), nor pregnancy rates (41,46% vs 46,43 p ¼ 0,6829). There was

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howevera significantdifference in the numberof embryos that continued to evolve– i.e. the number of viable embryos created (G1: 112: 45,34% - vs G2: 102:61,82%, p ¼ 0,001).

516 oocytes were fertilised, 361 using PVP-IMSI-ICSI (G3) and 155 usingHA-IMSI-ICSI (G4). No significant differences were noted between the twogroups in terms of fertilisation rates (70,91% vs 69,03% p ¼ 0,6679), pregnancyrates (47,73% vs 35,29 p ¼ 0,3807), or in the number of viable embryos created(G3: 179: 69,92% - vs G4: 69: 64,49%, p ¼ 0,3101).Limitations, reason for caution: The study was not randomised.Wider implications of the findings: The use of hyaluronate in order to selectmature sperm prior to ICSI would appear to be a good technique to improveembryo viability in patients with teratozoospermia, and therefore could be usedas an alternative to IMSI.Study funding/competing interest(s): Not applicable.Trial registration number: Not applicable.

P-147 Oocyte maturity rate as function of time from ovulation trigger tooocyte aspiration in IVF-ICSI cycles

A. Weiss1, R. Neril2, J. Geslevich1, R. Beck-Fruchter1, M. Lavee1, J. Golan1,A. Ermoshkin1, and E. Shalev2

1Emek Medical Center, Obstetrics and Gynecology, Afula, Israel, 2Technion-Israel Institute of Technology, Rappaport Faculty of Medicine, Haifa, Israel

Study question: Is the rate of oocyte maturity dependent on the time from ovula-tion trigger to oocyte aspiration within a four hour time frame during which thepickup procedures take place and does variation in this time interval warrant indi-vidualized scheduling of ovulation induction injection and oocyte aspiration?Summaryanswer: Thirtyfourhours is theminimumtimerequired fromoocytemat-uration injection to ovum pickup. Variation in time between 34 and 37 hours did notaffect the maturity rate of oocytes. Within this time frame there is no need for indivua-lized scheduling of ovulation induction injection and oocyte aspiration.What is known already: In spite of the prevalence of IVF, few studies haveaddressed the exact timing of oocyte aspiration after ovulation trigger. While 36hours is an accepted time interval, the outpatient nature of the procedure maymake meticulous timing of oocyte pickup difficult. Furthermore, advances inIVF, such as the emergence of antagonist protocols and the introduction ofGnRH agonist for ovulation trigger in high responders, may make older studiesobsolete.Study design, size, duration: A retrospective study analyzing 526 IVF/ICSIcycles from January 2010 to May 2012.Participants/materials, setting, methods: Patients were instructed to inject HCG(or GNRH agonist for high responders) at 23:00 two days prior to ovum pickup.Patients were aspirated in the order they arrived at the clinic. The time of ovumpickup, laboratory and patient records were analyzed retrospectively.Main results and the role of chance: A total of 526 ICSI cycles were analyzed.On average 8.38+6.22 oocytes were aspirated per cycle. Overall, 64+28% ofaspirated oocytes were MII oocytes. A one-way ANOVA demonstrated thatoocyte maturity differed significantly among four different time groups: 33.45to 34 hours (n ¼ 94), 34 to 35 hours (n ¼ 256), 35 to 36 hours (n ¼ 125) and36 to 37 hours (n ¼ 34); [F (3,506) ¼ 5.244, P , .001]. Tukey post hoc compari-son of the four groups, showed that only the first group differed significantly fromeach of the other three groups (54+31%, 66+27%, 66+27% and 72+25%respectively). The time groups did not differ regarding other clinical and treatmentparameters.Limitations, reason for caution: The study is limited by its retrospective nature.The sample size was sufficient to reach significance, though the fourth time groupis smaller than the other three time groups.Wider implications of the findings: Simply changing the time patients areinstructed to administer the ovulation triggering agent from 23:00 to 22:00should increase the rate of MII oocytes in our unit. There is no need to individualizeand meticulously schedule injections and ovum pickups as long as between 34 and37 hours transpire. Further studies should reveal if an even longer delay will in-crease or decrease MII oocyte maturity rates.Study funding/competing interest(s): We declare no outside funding or compet-ing interest.

Trial registration number: The study was approved by the local ethics commit-tee. Due to its retrospective non-interventional nature, trial registration is notrequired.

P-148 Factors related to clinical pregnancy of vitrified-warmed embryotransfer: a retrospective and multivariate logistic regression analysis of2313 transfer cycles

W. Shi, S. Zhang, W. Zhao, X.I.A. Xue, M.I.N. Wang, H. Bai, and J. ShiMaternal & Child Health Care Hospital, Assisted Reproduction Center,Xi’anShaan’xi, China

Study question: what factors affect clinical pregnancy in vitrified-warmedembryo transfer (VET)?Summary answer: Assisted hatching (AH) had a positive effect on clinicalpregnancy and the reason of freezing was one of the most significant factorsrelated to the clinical pregnancy of VET.No. of embryos transferred showedthe lowest relative importance in 8 variables on the effect of successful clinicalpregnancy.What is known already: Most previous studies have analyzed some of the factorsrelated to the outcome of VET using one-factor analysis. To take account of con-founding factors such as patient age, embryo quality, number of embryos to trans-fer, different laboratory procedure and clinical alternations, it is important toperform a multivariate logistic regression analysis to determine which one or agroup of factors are most related to clinical pregnancy of VET.Study design, size, duration: The study was a retrospective analysis of 2313 VETcycles from 1481 patients performed between January 2008 and April 2012.A multivariate logistic regression analysis was performed to identify the factorsto affect clinical pregnancy outcome of VET.Participants/materials, setting, methods: There were 22 candidate variablesidentified from clinical experiences and the literature. Eleven variables werechosen by a bootstrapping stepwise variable selection algorithm (n ¼ 1000)with the thresholds of aentry ¼ aremoval ¼ 0.05 for both variable entry and variableremoval.3 factors were excluded and 8 factors were chosen to contribute themodel.Main results and the role of chance: For the reason of freezing, OHSS groupshowed a high OR than the other groups (OHSS vs Other, OR: 2.145 CI:1.4-3.286; Supplement vs Other, OR: 1.152 CI: 0.761-1.743). Assisted hatchingalso showed a high OR (OR: 2.105, CI: 1.554-2.85). The 3 variables (age of COH,damaged blastomere, and presence of blood on catheter) had an adverse effect onthe outcome of VET (b , 0).

The number of good-quality embryos showed the highest marginal PEV andpartial PEV (marginal PEV 3.91%, partial PEV 2.28%). The reason of freezingshowed the second highest marginal PEV (2.77%). However, No. of embryostransferred showed the lowest marginal PEV and partial PEV (marginal PEV0.21%, partial PEV 0.46%).Limitations, reason forcaution: This was a retrospective multivariate analysis ofthe data obtained in 5 years from a single IVF center. Prospective analysis of largedata sites from a multicentre study is necessary to confirm this finding .Wider implications of the findings: Except for the quality of embryos and thenumber of good embryos, assisted hatching and the reason for freezing weretwo important variables on clcinical pregnancyof VET.No. of embryos transferredshowed less relative importance on the effect of successful clinical pregnancyStudy funding/competing interest(s): This study was financially supported bythe Scientific and Technological Research Projects of Shaanxi Province (projectnumber: 2011k15-02-01). All the authors have to declare.Trial registration number: N/A

P-149 Understanding the impact of Assisted Reproductive Technologies(ART) on embryo health, child health and disease and longevity in later life

H.L. Smith1, L. Shaw1, S. Kimber1, and D. Brison2

1University of Manchester, reproductive medicine, Manchester, United Kingdom,2St. Mary’s Hospital, reproductive medicine, Manchester, United Kingdom

Study question: To use existing microarray data and amplified cDNAs from dif-ferent stages of human embryo development to investigate the expression of

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metabolic and epigenetic pathways identified as important in embryonic health,disease and longevity, e.g. mTOR, IGF, AMPK and zfp57.Summary answer: Genetic variability between embryos results in inconsistentexpression of manyof the key genes. However, several alternative genetic and epi-genetic pathway members appear to be differentially expressed between differentstages of development, indicating potential points of control in embryo develop-ment.What is known already: Many studies have shown the relationship between thematernal environment and offspring health, using animal models and epidemio-logical data. Children with diabetic mothers/ pre-blastocyst stage embryosremoved from a diabetic environment have an increased risk of developingobesity, diabetes and metabolic syndrome in later life. Mouse studies demonstratethe importance of epigenetic methylation stability during embryonic develop-ment. However few studies have attempted to clarify the link between the envir-onment/ART technology and human embryo health.Study design, size, duration: To use existing microarray data and amplifiedcDNAs from different stages of human embryo development. Initial analysis con-ducted on oocytes,4cell andblastocyst stageembryos, 3 samples per developmen-tal stage.Participants/materials, setting, methods: Embryos were donated by patientsundergoing IVF at St. Mary’s hospital, Manchester, UK. cDNA was amplifiedvia polyAPCR and analysed using the Affymetrix microarray HG U133 plus 2chip, at the Paterson cancer Institute, Manchester. CEL. files are downloadedand statistical analysis performed within Partek and Metacore.Main results and the role of chance:The epigenetic regulator Trim28 was shownto be expressed during early human embryonic development. Although Trim28was not differentially expressed between stages, its co-repressing bindingpartner MM-1 was 98 fold over-expressed in oocytes/4cell stage, but not at theblastocyst stage. MM-1 over-expression may lead to increased methylatedregions, transcription factor inhibition and a point of gene silencing withinoocyte/embryo development. IGF-related genes are also suggested as keypoints of metabolic control, relating the maternal nutrient availability to down-stream pathways in the embryo. IGFBP1 was heavily (373 fold) over-expressedin oocytes compared to blastocysts. IGFBPs bind and inhibit IGF’s thereforethe differential regulation is an apparent point of control in oocyte and embryo de-velopment. A number of other genes were upregulated by the blastocyst stage.Limitations, reason forcaution: The number of samples was kept low in order tofocus on genetic variability, however; utilisation of other microarray sampleswithin the database will help clarify/verify findings. Embryos are inherently dif-ferent to one another and therefore less stringent settings need to be applied toidentify trends in gene expression.Wider implications of the findings: Understanding developmentally importantpathways which may be perturbed by ARTaids our understanding of potential riskfactors to which human embryos are subject in vitro. Assessing the perturbation ofsuch genes in response to ART is an essential part of a fully-informed risk assess-ment. This will ultimately lead to increased understanding of long term health out-comes for ART children and will help to improve ART conditions to avoidunwanted impacts.Study funding/competing interest(s): This research is funded by the EuropeanCommission under the FP7 Health programme, as part of the EpiHealth consor-tium (co-ordinator Professor Andras Dinnyes)Trial registration number: no trial registration number

P-150 Developmental regulation of apoptosis related-genes during earlyembryonic development: a comparison between human and mouse species

I. Boumela1, S. Assou1, D. Haouzi1, O. Ait Ahmed1, H. Dechaud2, andS. Hamamah3

1CHU Montpellier Institute for Research in Biotherapy Hopital Saint-Eloi,INSERM U1040, Montpellier Cedex5, France, 2CHU Montpellier Institute forResearch in Biotherapy Hopital Saint-Eloi,Hopital Arnaud de Villeneuve Dept. ofOb/Gyn, Montpellier Cedex5, France, 3CHU Montpellier Institute for Researchin Biotherapy Hopital Saint-Eloi, INSERM U1040 Hopital Arnaud de VilleneuveDept. of ART/PGD, Montpellier Cedex5, France

Study question: What are the apoptotic-regulatory genes expressed during earlyembryonic development in humans and mice and are theycomparable between thetwo species?

Summary answer: The apoptotic machinery is expressed during human andmouse early embryonic development. Although some differences may exist,most apoptosis-related genes exhibit similar expression patterns in both species.What is known already: Apoptotic cell death has been reported in human andmouse oocytes and pre-implantation embryos in vivo and in vitro, but its regula-tion and the conservation of the molecular factors between human and mice arenotwell known.Study design, size, duration: This experimental study included 15 patients re-ferred to our ART department for IVF treatment and 25 mice (B6CBAF1)between 2010 and 2012. Both patients and female mice underwent controlledovarian stimulation.Participants/materials, setting, methods: Human mature MII oocytes, 8-cellstage embryos and blastocystswere includedafter informedconsentof the patientsand IRB approval. Mouse MII oocytes, 2-cell stage embryos and balstocysts werecollected from mice in vivo. Using DNA microarray, the expression profiles ofapoptosis-related genes were determined before and after embryonic genome ac-tivation (EGA) in human and mouse.Main results and the role of chance: In human, genes involved in the extrinsicpathway of apoptosis are overexpressed in MII oocytes (Edar, ×11, p ¼ 0.008)and day5 blastocysts (Tnfrsf10b, ×10, p ¼ 0.03; Tnfrsf21, ×115, p ¼ 0.04)while in mouse Tnfrsf25 and Tnfsf12 are constitutively expressed. Severalmembers of the Bcl2 family, that regulate the intrinsic pathway, show similarexpression patterns in human and mouse such as for Bcl2l10 which followed a ma-ternal profile (×10, p ¼ 0.01; ×3.5, p ¼ 0.03) and Mcl1 which was overex-pressed after EGA at the 8- and 2-cell stages in human and mouse respectively(×8, p ¼ 0.008; ×6, p ¼ 0.002). Among the numerous caspase genes expressedduring preimplantation development, Caspase6 showed differential expressionpatterns between the two species with abundant levels in human MII oocytes(×7, p ¼ 0.01) and a specific increase in mouse blastocysts (×12, p ¼ 0.008).Limitations, reason for caution: Human and mouse oocytes and embryos wereobtained after controlled ovarian stimulation, and thus the gene expression pro-files of apoptosis-related genes could have been influenced. In addition, the invitro culture conditions of human embryos may also have an impact.Wider implications of the findings: This study opens new perspectives forunderstanding the molecular regulation of oocyte and pre-implantation embryosurvival and death.Study funding/competing interest(s): This work was partially supported by agrant from Ferring Pharmaceuticals company. The authors of the study have nocompeting interests to report.Trial registration number: Not applicable

P-151 Ultrastructural alterations in different developmental stages ofin vivo and in vitro preimplantation murine embryos exposed tocryopreservation

R. Dasiman1, A.R. Nor-Shahida2, W.J. Wan-Hafizah2, J.M.Y. Norhazlin2,M. Mohd-Fazirul2, O. Salina2, R.A.F. Gabriele2, and M.N.K. Nor-Ashikin2

1Universiti Teknologi Mara, Faculty Of Health Science, Puncak Alam Selangor,Malaysia, 2Universiti Teknologi Mara, Faculty Of Medicine, Sungai BulohSelangor, Malaysia

Study question: This study was designed to address the issues listed below:1) Which stage of development is best for cryopreservation in vivo and in vitro

embryos?2) What are the effects of cryopreservation using vitrification and slow

freezing methods on ultrastructural organizations of the in vivo and in vitroembryos?Summary answer: The findings indicated that 8-cell stage of in vivo and in vitroembryos is more suitable for cryopreservation in terms of embryonic cryotoler-ance and ultrastructural damages. Highly significant changes were observed inthe ultrastructural organizations of both in vivo and in vitro cryopreservedembryos (p , 0.001).What is known already: Ultrastructural organizations of the preimplantationembryos are highly unique and consist of a fragile intracellular network of micro-filaments and microtubules that is essential for embryonic development. The orga-nizations undergo dramatic architectural changes which show variablephysiological necessities and requirements in order to survive. Cryopreservationreduced embryonic survivability and caused deleterious changes on the

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ultrastructural elements. However, to date, limited information is available aboutthe ultracellular alterations in in vivo and in vitro cryopreserved embryos.Study design, size, duration: This study is carried out using a 2 x 3 x 2 factorialdesign. The sample size is 50 embryos for each treatment arm. The cryopreservedin vivo and in vitro produces embryos at 2-, 4-, and 8-cell stages were used inthis study.Participants/materials, setting, methods: The ultrastructural elements of theembryos namely tubulin, actin and nucleus were labelled by immunofluorescentstaining, after being cryopreserved by either vitrification or slow freezingmethod. The embryos were then viewed under Confocal Laser Scanning Micro-scope and finally the intensities of the labelled elements were analyzed usingQWin Software V.3.Main results and the role of chance: The results clearly showed that both cryo-preservation methods caused highly significant changes in ultrastructural ele-ments of in vivo and in vitro embryos as compared to non-cryopreservedembryos (p , 0.001). In all cryopreserved group, as the numberof cells increased,fluorescent intensities of tubulin, actin and nucleus were decreased slightly ascompared to non-cryopreserved groups. Vitrification and slow freezing causedhighly significant changes in tubulin intensities (p , 0.001), but the intensitiesof actin and nucleus were not significant between 4- and 8-cell in in vivoembryos. In the in vitro embryos, the differences between intensities of tubulin,actin and nucleus were highly significantly between 2-, 4- and 8-cell (p , 0.001).Limitations, reason for caution: The use laser with high energy and wavelengthsduring confocal microscopy observations is absorbed by the pigment granules thatcan cause intense local heating and contribute to ultrastructural alterations. Otherthan that, the fluorescent signals may be overlapped during the immunofluorescentstaining and confocal analysis.Wider implications of the findings: Information obtained from this study willprovide vital information needed in the selection of the best stage and cryotolerantembryo candidates for cryopreservation and directly reduce the cost of long termstorage by eliminating unnecessary storage of non-viable embryos.Study funding/competing interest(s): This research was funded by Dana Kece-merlangan Universiti Teknologi MARA (DANA-600-RMI/ST/DANA5/3/Dst/37/2009) and Fundamental Research Grant Scheme (FRGS-600-RMI/ST/FRGS/5/3/SFT/71/2010), Ministry of Science, Technology and Innovation(MOSTI), Malaysia.Trial registration number: None

P-152 Time-lapse microscopic analysis for exploring the dynamics ofembryonic cell cycles following blastomere biopsy

D. Ben-Yosef, T. Shwartz, T. Cohen, A. Carmon, N. Mey Raz, M. Malcov,T. Frumkin, B. Almog, I. Vagman, R. Kapustiansky, A. Reches, F. Azem, andA. AmitTel Aviv Sourasky Medical Center, Racine IVF Unit, Tel Aviv, Israel

Study question: Preimplantation genetic diagnosis (PGD) involves the aspirationof single blastomeres from a 6-8 cell embryo at day 3 of development. This studyexplored the dynamics of the developmental events following blastomere biopsyby determining the timing of subsequent cleavages and compaction followingculture in the EmbryoScopeTM concomitantly with time-lapse analysis.Summary answer: Analysis of morphokinetic parameters enables unraveling ofdevelopmental events associated with blastomere biopsy. Synchronized cleavingembryos biopsied at the 8-cell stage enter the next cell cycle later than non-synchronized ones. The latter are biopsied during the M phase of the cycle, thusresuming cleavage or entering compaction shortly after biopsy.What is known already: Morphokenetics evaluated by time-lapse analysis wasrecently added to the embryo scoring system to assist in embryo selection. Todate, selection of healthy embryos following PGD was based mainly on theirdynamic development following biopsy as observed by static evaluation, i.e., add-ition of cells following cleavage and/or compaction. There is only one publication(Kirkegaard et al., 2012) in which time-lapse analysis was performed followingblastomere biopsy in order to study its effect on embryo development.Study design, size, duration: Embryos from all PGD treatments from September,2012 to date were cultured in a time-lapse incubator (EmbryoScopeTM) until days4-5 after fertilization. Images of 178 embryos from 24 couples carriers of geneticdisorders were acquired every 20 min. Time-points of key embryonic eventsbefore and after embryo biopsy were registered.

Participants/materials, setting, methods: Participants included 13 couple car-riers of monogenic disorders and 11 couple carriers of chromosomal transloca-tions. The acquired images of each embryo were retrospectively analyzed withthe EmbryoViewer by image analysis software. All the selected embryo develop-mental events were annotated together with their corresponding time points (inhours) after insemination.Main results and the role of chance: Blastomere biopsy was performed at71.2+2.9h post ICSI on embryos with 8.6+1.4 cells. A dynamic in embryo de-velopment was observed within 7.8+4.7h following biopsy: most embryos(77.5%) initially cleaved, as evidenced by the addition of blastomeres, followedby compaction. Interestingly, embryos biopsied at the 8-cell stage took longer toprogress into subsequent developmental stages compared to embryos whichunderwent biopsy at a stage 8 cells (median 8.8h vs. 4.4h until cleavage and22h vs. 10h until compaction, respectively). Fifty of the 178 cultured embryoswere diagnosed as inheriting the normal allele (41 embryos transferred; 9frozen). In 21 cycles 41 healthy embryos were transferred resulting in 8 pregnan-cies and 9 beating hearts (38% ongoing pregnancy rate; 22% implantation rate).Limitations, reason for caution: These new findings warrant validation in alarger cohort of embryos.Wider implications of the findings: Blastomere biopsy at �70 h post-fertilization probably does not interfere with the embryonic cell cycle. This sup-ports our previous observations that non-synchronized cleaving embryos with agood cleavage pattern probably have the same potential for further developmentas synchronized cleaving embryos. These findings make it possible to explorethe timing of developmental events characterizing human preimplantationembryos, and they may have broader implications in choosing the optimaltiming for blastomere biopsy for PGD.Study funding/competing interest(s): No funding,Trial registration number: 06/214

P-153 An additive model using morphokinetics to predict embryos ofhighest implantation potential

M. Cetinkaya, C. Pirkevi, H. Yelke, Y. Kumtepe, Z. Atayurt, and S. KahramanIstanbul Memorial Hospital, Assisted Reproductive Technologies andReproductive Genetics Center, Istanbul, Turkey

Study question: Can we predict embryos with highest implantation potential frommorphokinetic parameters obtained until day3?Summary answer: The five formulas cited below were used to identify cleavagesynchronicity: (F1): (t8-t2)/((t3-t2) + (t5-t4)); (F2): (t8-t5)/(t8-t4); (F3): (t4-t3)/(t4-t2); (F4): (t4-t2)/(t8-t4); (F5): (t3-t2), additionally two criteria (irregular divi-sions and evenness of blastomeres at 2 and 4-cell stages) were applied to build anadditive embryo scoring model predicting implantation.What is known already: A hierarchical model, predicting embryo implantation,was built through the retrospective morphokinetic analysis of 247 embryos (Mese-gueret al., 2011). Themost predictive parameters weredescribedas being; first, thetime of division to 5 cells (t5), followed by, the time between division to 3 cells andsubsequent division to 4 cells (s2) and the time between division to 2 cells and div-ision to 3 cells (cc2).Study design, size, duration: This retrospective cohort study was conductedbetween October 2011 and October 2012 in a private ART Center and approvedby the institutional review board. The study included 454 embryo having a (t8)with known implantation data (KID ¼ 0 or1) from 257 infertile patients(Female age: 33.1+5.1; BMI: 24.4+6.8; MII: 5.3+4.1).Participants/materials, setting,methods: Embryos werecultured ina single stepculture medium, which was refreshed on day3. Incubation was performed underoil at 378C, 5%O2 and 6%CO2. Each time of cleavage was recorded. Clinical preg-nancy was defined as the presence of a gestational sac with fetal heartbeat detectedon ultrasound on week7.Main results and the role of chance: Embryos should mainly stay in even cellstages, which should be balanced when compared to each other, ideally reflectingcleavage synchronicity. Each formula was applied to KID embryos, which wereclassified in 5%groups and implantation rates (IR) were computed for the 20inter-vals. In order to follow mathematically the natural trends obtained in IR, theaverage of ≥3consecutive 5%groups was subtracted from the average of thetwo following groups, and divided to the initial IR until the theoretical thresholdset at 0.3 was reached. Each class had a mean IR, which was subtracted from

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the initial IR, giving a positive or negative score. Obtained scores were added forthe final prediction of the implantation potential. The logistic regression modelbuilt gave an AUC ¼ 0.684 (95% CI 0.628-0.740).Limitations, reason for caution: Embryos having a (t8) were exclusivelyincluded in the additive model, leaving out evaluation of day2 embryo transfers.Thecohort studied involved only infertile patients (female,male orcombined)andis in this respect a heterogeneous population.Wider implications of the findings: Time points defining precise embryo cleav-age events may not be generalized to infertile patients with different etiologies.However, using ratios based on selected cleavage cycles defining synchronicityof embryos, allows in this retrospective cohort study an individualized analysisgiving high predictivity of implantation. The proposed additive model has to befurther tested in a randomized prospective trial to evaluate its efficacy, and mayin the future improve embryo selection in IVF treatments.Study funding/competing interest(s): Authors have nothing to disclose.Trial registration number: The study was approved by the institutional reviewboard.

P-154 Safespeed technology - the myth of ultrahigh cooling rates: a closedevice and a serum-free media for the vitrification human oocytes/embryoswith the highest recovery rates

R. Risco1, M. Hebles2, A.M. Saa3, M.A. Vilches-Ferron4, P. Sanchez-Martin5,E. Lucena3, and M. Lucena6

1Engineering School, University of Seville, Seville, Spain, 2Clinica Ginemed, IVFLaboratory, Seville, Spain, 3Cecolfes, Fertility Laboratory, Bogota, Colombia,4Hospital Torrecardenas, Ginecology Department, Almeria, Spain, 5ClinicaGinemed, IVF Laboratory, Sevilla, Spain, 6Nidacon International, ProductDevelopment, Molndal, Sweden

Study question: While no studies have demonstrated unintentional uptake by ahuman or mammalian oocyte or embryo of any pathogen during vitrification orstorage, under experimental conditions such contamination may occur (Bielanskiet al., 2000); Also the presence in the vitrificationsolutions of substances of humanor animal origin is a drawbackSummary answer: SafeSpeed is brand new technology that accomplishes theoptimal requirements unveiled in recent studies for oocyte vitrification (Mazuret al., 2011), although with evidences since 1990 (Steponkus et. al, 1990). Wehave reached the highest recovery rates of human oocytes by optimizing thecooling and warming ratesWhat is known already: Various techniques have been developed in the past toovercome this difficulty by improving the heat transfer, such as Open PulledStraws, ‘‘solid-surface vitrification’’, the use of electron microscope coppergrids, cryoloops, and more recently cryotop and cryotip (Kuwayama, 2007).However, in each of those systems, the philosophy underneath is increasing,but not optimizing, the cooling/warming rate (Mazur, 2011). SafeSpeed hasborn as the best optimization of those parameters in a close system.Study design, size, duration: Experimental trials have been performed withmouse and human oocytes. Out of 1700 samples, ninety five percent of vitrifiedmouse oocytes survived after warming, and ninety three percent of humanoocytes (out of 94) were recovered (Ginemed, Torrecardenas Hospital, Spain) .Fertilization rate observed after ICSI was 70 %.Participants/materials, setting, methods: Currently transfer trials are carriedout in Cecolfes (Colombia). Images of fluorescence immunochemistry withtubulin-specific antibodies to visualize microtubular structures of humanoocytes vitrified with SafePreservation technology were excellent, showing thegood behavior of the cryoprotectant as well (Torrecardenas).Main results and the role of chance: This work has been possible thanks to aninternational collaboration of several partners: Nidacon, SafePreservation,Cecolfes, Clinica Ginemed, Hospital Torrecardenas and the Engineering Schoolof the University of Seville.Limitations, reason for caution: By optimizing the cooling/warming rates, wehave been able to maintain the use of conventional liquid nitrogen for our closedevice.Wider implications of the findings: In SafeSpeed, the container is heat-sealed atboth ends, and consequently the solution containing the oocyte or embryo and theliquid nitrogen is hermetical isolated during cooling and storage. The technique issimple and robust, without sophisticated devices and easy to learn. Our system

does not require special cryogenic agents, like slush nitrogen or other special mix-tures (Risco et al., 2007), what would make the everyday practice very cumber-some, and would elevate the costsStudy funding/competing interest(s): This study is relevant in the field of oocyteand ambryo vitrificationTrial registration number: 1

P-155 Vitrification: a useful tool in IVF lab when fresh embryo transfercan not be performed

M. De Las Heras1, J.A. Agirregoikoa2, E. Martinez1, G. Barrenetxea2, andJ.L. De Pablo1

1Quiron Bilbao, IVF Lab, Bilbao, Spain, 2Quiron Bilbao, Gynaecology, Bilbao,Spain

Study question: The aim of this retrospective observational study is to assess theeffectiveness of embryo vitrification on day 3 of embryo development comparingpregnancy rate, implantation rate and ongoing pregnancy rate between freshembryo transfer and warmed embryo transfer.Summary answer: Vitrification could be not only a good cryopreservationmethod for good quality embryos that are not transferred in the fresh cycle butit is also a good strategy when the fresh embryo transfer must be postponedWhat is known already: Cryopreservation allows improving the cost-effectiveness of IVF and expanding the options available to infertile couples.Many studies have shown that vitrification in contrast to slow freezing offers ahigher survival rate, minimal deleterious effects on post-warming embryo morph-ology and it can improve clinical outcomes. Other studies show a betterembryo-endometrial synchrony in frozen-thawed embryo transfer cycles thanfresh embryo transfer with higher pregnancy and implantation rates after the trans-fer of warmed embryos.Study design, size, duration: This retrospective observational study includes887 fresh and 690 warmed embryo transfers performed on day 3 between 2009and 2012. Only transfers in which the two embryos transferred had the samescore or single embryo transfers were included. A total number of 1456 freshand 1153 warmed embryos were transferred.Participants/materials, setting, methods: Patients were ≤ 39 years old and theirown oocytes were used. Embryos were scored according to the Spanish Associ-ation for Reproductive Biology. Vitrification and warming was performed usingcryotop method. Clinical pregnancy was determined 7 weeks after embryo trans-fer. Statistical analysis was done using the Fisher test (p , 0,05).Main results and the role of chance: The pregnancy rate after warmed embryotransfer of two very good quality embryos was 43,7%, 29,9% when only one verygood quality embryo was transferred, 32,3% when the transfer included two goodquality embryos and 26,6% when two medium quality embryos were transferred.The pregnancy rates after fresh cycles were 42,1%, 22,9%, 24,1% and 20,2% re-spectively not showing significant differences with those from warmed cycles(p . 0,05). The implantation rates did not show significant differences betweenthe two groups either (29,4% vs. 27,7%; 29,9% vs. 22,9%; 18,2% vs. 24,1%and 14,4% vs. 10,6%) (p . 0,05). There were not significant differencesbetween ongoing pregnancy rate (12 weeks after embryo transfer) (20,7% vs.23,6%; 22,4% vs. 18,3%; 14,6% vs. 18,2% and 13,3% vs. 9,6%) (p . 0,05).Limitations, reason for caution: Although the study included many patients, aprospective randomized study should be carried out in order to confirm our data.Wider implications of the findings: It is neccesary an efficient cryopreservationmethod that allows the storage of the remaining embryos maximizing the cumu-lative effectiveness of an in vitro fertilization cycle. Our data show that vitrifica-tion offers the patient the same pregnancy rate as fresh cycles so it could be a goodstrategy when transfer must be postponed for personal or medical reasons such asin the ovarian hyperstimulation syndrome or when the endometrium is not pre-pared.Study funding/competing interest(s): noTrial registration number: no

P-156 Embryo density in group culture of human embryos mayinfluenceembryo quality

A. Lehner1, C. Pribenszky2, A. Murber1, J. Rigo Jr.1, J. Urbancsek1, andP. Fancsovits1

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1Semmelweis University, First Department of Obstetrics and GynaecologyDivision of Assisted Reproduction, Budapest, Hungary, 2Szent Istvan University,Department of Animal Breeding and Genetics Faculty of Veterinary Sciences,Budapest, Hungary

Study question: The aim of this study was to analyze the effect of embryo densityon embryo quality and development when culturing in Well-of-the-Well (WOW)Petri dishes (Primo Dish, Vitrolife Hungary) in human IVF treatments.Summary answer: According to our results 3.5-5 ml/embryo density seems tohave a beneficial effect on the embryo quality compared to lower and higher dens-ities. Embryo density can affect embryo development when culturing embryos ingroups.What is known already: Group culture of human embryos is a widely usedculture method, which may have a beneficial effect via autocrine and paracrinemechanisms. However an optimal embryo density (volume of culture mediadivided by the number of embryos ¼ ml/embryo) in human in vitro culture hasnot been defined yet. The WOW dish, which contains 9 + 1 microwells in thecenter of the dish, allows us to identify individual embryos cultured together inthe same microdrop.Study design, size, duration: Data of 675 normally fertilized oocytes from 119IVF cycles were retrospectively analyzed. Embryos were classified into fourgroups: Group A (n ¼ 336): individual culture, Group B (n ¼ 62): 2-4embryos, Group C (n ¼ 152): 5-7 embryos, Group D (n ¼ 125): 8-10 embryoscultured in a single droplet of 25 ml media in WOW dishes.Participants/materials, setting, methods: Embryo densities were the following:Group A: 25 ml/embryo, Group B: 6-12.5 ml/embryo, Group C: 3.5-5 ml/embryo,Group D: 2.5-3 ml/embryo. Number of blastomeres, fragmentation and morph-ology score on Day 2 and Day 3 were analyzed with one-way ANOVA andTukey HSD test. Frequency of top quality embryos were compared withChi-squared test.Main results and the role of chance: Number of blastomeres was similar in thefour groups (P ¼ 0.36) on Day 2. However cell number in Group C was signifi-cantly higher than in Group A (7.5+2.1 vs. 6.4+2.2; P , 0.001) and inGroup D (7.5+2.1 vs. 6.6+2.0; P ¼ 0.007) on Day 3. Fragmentation ratewas significantly lower in Group C compared to Group A (11.5+8.3% vs.15.1+11.9%; P ¼ 0.01) on Day 2. The ratio of top quality embryos werehigher in Group C compared to Group A (29.3% vs. 17.2%; P ¼ 0.004) and toGroup B (29.3% vs. 8.3%; P ¼ 0.003), as well as in Group D compared toGroup B (22.6% vs. 8.3%; P ¼ 0.03) on Day 3.Limitations, reason for caution: This study presents preliminary data. A largerstudy is needed to have more exact information about the effect of embryodensity on embryo quality.Wider implications of the findings:Embryo densityof 3.5-5 ml/embryo seems tohave the most beneficial effect on embryo development. Culturing embryos in aWOW group culture dish may increase embryo quality and allows assessmentand tracing of individual embryos.Study funding/competing interest(s): -

Trial registration number: This investigation is a retrospective analysis of a sub-group of data obtained from a larger prospective randomized study (Clinical-Trials.gov: NCT01774006).

P-157 Effect of oil density in donor oocyte IVF cycles

D. Gumbao Bano1, A. Sanchez-Leon1, J. Marcos1, M. Molla1, B. Amorocho1,M. Nicolas2, L. Fernandez2, and J. Landeras2

1IVI Murcia, FIV Laboratory, Murcia, Spain, 2IVI Murcia, Gynecologist, Murcia,Spain

Study question: The aim of this study was to evaluate the effect of two differentculture oils with different densities on the donor oocyte clinical outcome rates atday five of embryo development using a time-lapse culture system.Summary answer: The density of the oils used in a time-lapse culturesystem at day five of embryo development may have some impact on ARToutcomes.What is known already: The quality of oil used in an embryo culture is one of themost critical issues for successful IVF treatment. In fact, different quality oils mayaffect embryonic development in vitro by containing inappropriate or toxicorganic substances.Study design, size, duration: To evaluate the oil density influence in the clinicaloutcomes on our embryo culture system, two different densities of the same oilwere evaluated retrospectively. 91 FIV-ICSI treatments were carried outbetween 2011 and 2012 with patients who were included in our oocyte donorprogram.Participants/materials, setting, methods: Embryos were divided into twogroups and cultured to day 5 in a time-lapse system using culture plates with 12wells including 24 mL of medium and overlaid with 1400 mL of the two differentculture oils. The statistical t-student software analysis significance was definedwhen P , 0.05.Main results and the role of chance: No significant differences were foundexcept in clinical miscarriage rate which was significantly lower in the group ofembryos cultured in light oil. The results are shown in Table 1.

Table 1. Clinical outcome rates. * statistical significance p ,0.05.Limitations, reason for caution: Although culture oil density did not affect preg-nancy and implantation rates, statistic differences in the miscarriage rates wereobserved, so further prospective and randomised studies are underway in orderto discover something else related with the oil density including fertilization today 5 time-lapse division parameters analysis.Wider implications of the findings: The results appear to point towards the im-portance of the oil using in culture. These results would be in agreement with theliterature and the results obtained by other groups that also defend the role playedby oil and the idea that further studies will be necessary in order to provide newproofs.Study funding/competing interest(s): The present study was supported by IVIMurcia SL. Murcia. SpainTrial registration number: No trial registration number.

...............................................................................................................................................................................................High-density oil Low-density oil p

Nº of treatments 34 57

Nº of transfers 32 55

Age 40.6 39.6

Average nº embryo transfers 1.5 1.7

Fertilisation rate 76.8 %(298/388) 77.9 %(514/660) 0.69

Clinical pregnancy rate 65.6 %(21/32) 69.1 %(38/55) 0.74

Implantation rate 50 %(24/48) 55.3 %(52/94) 0.55

Biochemical pregnancy rate 4.5 %(1/22) 11.6 %(5/43) 0.27

Clinical miscarriage rate 33 %(7/21)* 5.3 %(2/38)* 0.02

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P-158 A comparative gene expression analysis of slow frozen and vitri-fied metaphase II human oocytes

O.A. Adeniyi, S.M. Ehbish, and D.R. BrisonCentral Manchester University Hospitals, NHS Foundation Trust, ManchesterAcademic Health Science Centre

Study question: To investigate the impact of metaphase II (MII) oocyte cryo-preservation by slow freezing and by vitrification on the expression of genesinvolved in oocyte cryo-survival and in-vitro developmental competence.Summary answer: Oocytes showed a higher survival rate (86.7%) from vitrifica-tion compared to slowfreezing (76.2%). This was not significantlyassociated withmaternal age. Gene expression data for BRCA 1 and 2, OCT4, TUBB4Q and anumber of other candidate genes show differences between the vitrified andslow frozen oocytes.What is known already: Routine application of oocyte cryopreservation proce-dures for both long and short-term fertility preservation and treatment has beenwidely accepted. Several modifications in the processes of vitrification andslow oocyte freezing, have significantly improved the survival rates, pregnancyand live-birth rates obtained from both procedures. However, vitrificationseems to produce better results than slow oocyte freezing. Unfortunately, thesafety of these procedures and their impact on oocytes at the molecular level isnot completely understood.Study design, size, duration: A prospective study of 15 consenting patients in thevitrification group had controlled ovarian stimulation between May and October2012. Oocytes were vitrified using a closed system. Two patients in the slow freez-ing group had oocytes cryopreserved between 2002 and 2003. A minimum of 10oocytes were analysed per group.Participants/materials, setting, methods: Fifteen pairs of unfertilised MIIoocytes were obtained from fresh ICSI cycles in a standard NHS public sector hos-pital, of which 15 were vitrified and 15 sham treated as paired controls. Fertilitypreservation patients donated 21 slow frozen MII oocytes. Survival rates werecompared and cDNAwas analysed by polyAPCR and qPCRMain results and the role of chance: The average age of patients and oocyte sur-vival rates from slow freezing, vitrification and control groups were 31.4yrs and76.2%, 32.8yrs and 85.7% and 32.8yrs respectively. The survival rate in the vit-rification group was not statistically significant, when compared with the slowfreezing group (p ¼ 0.6897). Ongoing gene expression analysis of genes includ-ing TUBB4Q, OCT 4, BRCA 1, BRCA 2 and some specific genes associated withoocyte cryo-survival, show different gene expression patterns in both groups com-pared with the study control group.Limitations, reason for caution: The use of unfertilised MII oocytes from ICSIcycles in the vitrification and control groups can be argued to be a sub-optimalsource of MII oocytes, pre-exposed to micromanipulation and overnightculture. However, this did not appear to compromise the higher survival rateobtained, compared with slow freezing group. Moreover we have made genetic-ally normal embryonic stem cell lines from this source of oocytes demonstratingthat they retain developmental capacity.Wider implications of the findings: Our preliminary results show that MIIoocyte vitrification using a closed system produces better survival outcome com-pared with slow freezing. Vitrification can be argued to be essential in optimisingoutcomes involving fertility preservation and treatment. However, further inves-tigations involving sibling MII oocytes to compare cryopreservation techniques iswarranted. In particular, gene expression patterns in pre and post-cryopreservedMII oocytes can allow us to fully evaluate the association between oocytes cryo-preservation techniques and molecular gene expression.Study funding/competing interest(s): Adeniyi, OA and Brison, DR are fundedby Central Manchester NHS Trust; Ehbish, SM is funded by the Libyan Govern-ment.Trial registration number: None

P-159 Comparison of two different cryo-devices for vitrified-warmedsingle blastocyst transfer using chemically defined vitrification/warmingsolutions supplemented with recombinant human albumin

A. Egashira1, M. Murakami2, E. Nagafuchi1, K. Tanaka1, A. Tomohara1, C. Mine1, H. Otsubo1, A. Nakashima3, M. Otsuka3, N. Yoshioka3, and T. Kuramoto4

1Kuramoto Women’s Clinic, IVF Laboratory, Hakata-ku Fukuoka City, Japan,2Kuramoto Women’s Clinic, Reseach Laboratory, Hakata-ku Fukuoka City,Japan, 3Kuramoto Women’s Clinic, Reproductive Medicine, Hakata-ku FukuokaCity, Japan, 4Kuramoto Women’s Clinic, Clinical Department, Hakata-kuFukuoka City, Japan

Study question: To investigate whether a closed devise (Rapid-i) is useful forhuman blastocyst vitrification compared with an open vitrification devises(Cryotop)Summary answer: Closed vitrification system using recHAwere effective for thecryopreservation of human blastocysts to obtain good clinical outcome and toavoid the possibility of disease transmission and potential risk of cross-contamination.What is known already: The vitrification/warning solutions supplemented withrecHA used for human blastocysts vitrification with cryotop were first validated ina preclinical study (Murakami et al. 2012, abstract O-183 ESHRE). However,there are no proven viral transmissions between embryos or to the patient atembryo transfer, the use of an open system for vitrification potentially introducesa risk of cross-contamination during liquid nitrogen storage.Study design, size, duration: From August 2012 to December 2012, 79 patientsforvitrified-warmed single blastocyst transfer undera hormone-replacement cyclewere divided into two groups (group A: Cryotop, n ¼ 41; group B: Rapid-i,n ¼ 38). On day 5 and day 6, blastocysts with ICM and trophectoderm type Aor B (Gardner’s scoring system) were vitrified.Participants/materials, setting, methods: The base solution was PBS with2.5 mg/ml recHA. Blastocysts were exposed to two solutions: equilibration(10% EG) and vitrification (15% EG + 15% DMSO + 0.5 mol/l sucrose). Forthawing, embryos were placed into four sucrose solutions (0.5M, 0.25M, 0.1M,and 0M respectively). Data were analysed by x2 test.Main results and the role of chance: There were 41 warmed cycles in group Aand 38 warmed cycles in group B. Mean female age was 35.8+3.9 years in groupA and 35.1+ 2.5 years in group B.There were no significant different in embryosurvival rates (100% [41/41] vs. 100% [38/38]) and the implantation rates (53.7%[22/41] vs. 63.2% [24/38]) between groups A and B. The ongoing pregnancy ratesper embryo transfer were no significant different between group A and group B(43.9% [18/41] vs. 52.6% [20/38]).Limitations, reason for caution: In our study, study population and number ofvitrified blastocysts were small. Therefore, large-scale investigation includingfollow-up of children born after embryos vitrification using a Rapid-i are required.Wider implications of the findings: Closed vitrification method used in thisstudy useable each blastocyst to be vitrified in super-cooled air and loaded in aclosed straw. As a result, the blastocyst has supposed to have low risk of diseasetransmission and cross-contamination from liquid nitrogen, without impairing de-velopmental competence. This method will be useful vitrified-warmed singleblastocyst transfer in the future.Study funding/competing interest(s): NoneTrial registration number: None

P-160 Fresh embryo transfer versus frozen embryo transfer withGnRH-antagonist protocol in ART

D. Choi, H. Yang, J.H. Park, J.H. Jung, H.G. Hwang, J.H. Lee, J.E. Lee, A.S. Kang, J.H. Yoo, H.C. Kwon, and S.J. LeeMirae and Heemang Ob/Gyn Clinic, Obstetrics and Gynecology, Seoul, KoreaSouth

Study question: In ART of high ovarian responders undergoing aGnRH-antagonist protocol, which has benefit for patients between freshembryo transfer (FT) and frozen-thawed embryo transfer (FTT)?Summary answer: If fresh embryo transfer(FT) could be replace byfrozen-thawed embryo transfer(FTT) in high ovarian responder undergoingART with GnRH-antagonist protocol, the patients will have some advantages inboth higher pregnancy rate and avoiding OHSSWhat is known already: Some studies reported that in ART, the pregnancy rateof FTTare higher than FT. Also several meta-analyses mentioned similar results.The endometrial receptivity during ART is very important in implantation of thetransferred embryo. The endometrial receptivity can be controlled more preciselyin FTT than in FT.

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Study design, size, duration: All ART cycles were performed from January 1,2012 to December 31, 2012. FT were divided into three sub-groups: FT-LR:low-ovarian-responder (344 cycles), FT-NR : normal-ovarian-responder(227-cycles), FT-HR : high-ovarian-responder (104 cycles). Also, FTT weredivided into three sub-groups: FTT-LR: low-ovarian-responder (16 cycles),FTT-NR; normal-ovarian-responder (78 cycles), FTT-HR: high-ovarian-re-sponder (104 cycles).Participants/materials, setting, methods: All patients had GnRH-antagonistprotocol. The embryos used in FTT were cryopreserved by vitrification method.We classified the patients in low, normal and high ovarian responder, accordingto the number of retrieval oocytes: low-ovarian-responder: from 1 oocyte to 9oocyte, normal-ovarian-responder: from 10 to 19 oocytes, high-ovarian-respond-er: over 20. Main results and the role of chance By analyzing the results, weinvestigated the clinical-pregnancy rate, the implantation rate and the ongoing-pregnancy of all groups and compared them. However, we failed to find statistic-ally significant difference in comparison of FT-LR and FTT-LR [the clinical preg-nancy rate: 26.8% vs. 25%, the implantation rate: 9.8% vs. 13.5%, the on-goingpregnancy rate: 16.8% vs. 18.8%] or in FT-NR and FTT-NR [the clinical preg-nancy rate: 39.6% vs. 38.5%; the implantation rate: 12.4% vs. 15.4%; theon-going pregnancy rate: 28.2% vs. 29.5%.]. However, in comparison withFT-HR and FTT-HR, we could finally find significant difference (FT-HRvs. FTT-HR: the clinical pregnancy rate; 42.3% vs. 50.9%, p < 0.01; the im-plantation rate: 10.5% vs. 18.9%, p < 0.01, ongoing-pregnancy rate: 25.9%vs. 30.7%, p < 0.05) Limitations, reason for caution There were some variationin this study, including GnRH dosage, the day of antagonist treatment, and classi-fication of patients.Wider implications of the findings: The High ovarian responder undergoingGnRH-antigonist treatment can choose the strategy of cryopreservation of allembryos and transferring them subsequently in optimal conditions and this proto-col increases the synchrony between embryo and endometrial development, andtherefore, improves the ongoing pregnancy rate and outcome of ART cyclesStudy funding/competing interest(s): noneTrial registration number: none

P-161 Induction of autophagy in the vitrified-warmed mouse oocytes

S. Bang, H. Shin, and H.J. LimKonkuk University, Biomedical Science & Technology, Seoul, Korea South

Study question: Is autophagy induced during vitrification-warming process inmouse oocytes?Summary answer: Autophagy is induced in mouse oocytes during vitrification-warming process.What is known already: Vitrification uses cryoprotectants and liquid nitrogen,which may cause osmotic stress and cryodamage to oocytes. Autophagy iswidely considered as a survival or responsive mechanism to various environmen-tal and cellular stresses. However, the status of autophagy in vitrified-warmedoocytes has not been studied. In this work, we investigated if vitrification-warmingprocess induces autophagy in mouse oocytes.Study design, size, duration: Oocytes obtained from several mice were pooledand divided into three groups. Group1: fresh oocytes. Group2: oocytes treatedwith vitification solutions (1.3M EG + 1.1M DMSO and 2.7M EG + 2.1MDMSO + 0.5M sucrose) and warming solutions. Group3: vitrified-warmedoocytes (loaded onto an EM copper grid, and were stored in LN2 for 2weeks).Participants/materials, setting, methods: Four-week-old female ICR mice andGFP-LC3 transgenic mice were used. The mice were superovulated with 5IUPMSG and 5IU hCG and ovulated MII oocytes were collected from oviducts.RT-PCR and confocal live imaging of GFP-LC3 were performed to examine theeffects of vitrification-warming process on autophagy in oocytes.Main results and the role of chance: In RT-PCR analyses, the expression ofautophagy related (Atg) genes, such as Atg5, Atg7, Atg12, LC3a, LC3b, andBeclin1 were examined. In Group 2, the expression of Beclin1 was increased incomparison to fresh oocytes (Group 1), and it was recovered in Group 3. Thus, in-duction of Beclin1 mRNA is associated with the exposure to the vitrification so-lution. The expression of other Atg genes did not change. Confocal liveimaging analysis using oocytes from GFP-LC3 transgenic mice revealed thatsome vitrified-warmed oocytes showed green puncta which indicate autophagic

activation. All oocytes of Group 1 and Group 2 show no puncta formation. Eachexperiment was performed three times.Limitations, reason for caution: Increased autophagy was only confirmed bylive imaging of GFP-LC3 as a qualitative analysis, thus quantitative analysissuch as Western blotting needs to be performed. Further investigation is warrantedto address the role for autophagic activation in vitrified-warmed oocytes.Wider implications of the findings: Induction of autophagy may serve as an in-dicator of conditions of vitrification-warming process. Moreover, it offers the pos-sibility that development of methods to modulate autophagic response duringcryopreservation could improve efficacy of oocyte cryopreservation.Study funding/competing interest(s): This study was supported by a grant of theKorea Health technology R&D Project, Ministry of Health & Welfare, Republic ofKorea (A120080).Trial registration number: N/A

P-162 Forced blastocoele collapse of the blastocoel improves develop-mental potential in cryopreserved bovine blastocysts by vitrification andslow-rate freezing methods

S.H. Min, J.Y. Yeon, and D.B. KooDaegu University, Department of Biotechnology, Daegu, Korea South

Study question: The purpose of this study was to evaluate the effectiveness offorced blastocoele collapse of the blastocoel before vitrification and slow-ratefreezing of bovine blastocysts.Summary answer: Cryopreservation of bovine blastocysts has been proposed asa tool to improve the feasibility of cattle production by using embryo transfer tech-nique.What is known already: In general, the slow-rate freezing using a programmedcryo-machine are traditionallyemployed for the cryopreservation of embryos. Vit-rification with higher concentrations of cryoprotectants and a fast cooling rate, ittransforms cells into a glassy state without ice crystal formation.Study design, size, duration: Bovine in vivo and in vitro blastocysts were slow-rate frozen and vitrified after forced blastocoele collapse of the blastocoel by punc-turing the blastocoele with a pulled pasteur pipet.Participants/materials, setting, methods: The differences in survival and preg-nancy rates of blastocysts derived from forced blastocoele collapse and non-forcedblastocoele collapse groups were found in both slow-rate freezing and vitrifica-tion. Furthermore, we examined the total cell numbers and apoptosis index of blas-tocysts.Main results and the role of chance: We found that the total cell numbers of blas-tocysts in forced blastocoele collapse groups were increased and the numbers ofblastomeres undergoing apoptosis in forced blastocoele collapse groups weredecreased. Consistent with the results, qPCR data showed that the expression ofanti-apoptotic gene (Bcl-xL) was significantly increased by forced blastocoelecollapse groups, whereas the expression of pro-apoptotic gene (Bax) was signifi-cantly decreased. Furthermore, pregnancy rates in the both slow-rate frozen andvitrified bovine in vivo blastocysts could be improved by reducing the fluidcontent after forced blastocoele collapse of the blastocoel.Limitations, reason for caution: However, the low efficiency of frozen-thawedembryos survival and further development is a crucial problem.Wider implications of the findings: The forced blastocoele collapse of the blas-tocoel method suggesting that it could be improving the developmental compe-tence and qualities of frozen-thowed bovine embryos that may be used formammalian embryo preservation.Study funding/competing interest(s): We have no conpeting interest.Trial registration number: We have no trial registration number.

P-163 Effect of Hydroxypropyl cellulose on survival of bovine andhuman oocytes after vitrification

M. Kuwayama1, S. Higo1, and L. Ruvalcaba2

1Repro-Support Medical Research Center, Research & Development, Tokyo,Japan, 2Infertility Institute of Mexico, Research & Development, Guadalajara,Mexico

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Study question: Is Hydroxypropyl cellulose in vitrification solution effective onpost-warm survival of bovine oocytes and blastocysts, and human oocytes aftervitrification ?Summary answer: Hydroxypropyl cellulose was very effective as a macromol-ecule supplement in vitrification solution on post-warm survival of bovineoocytes and blastocysts, and human oocytes after vitrification. It is more effectivethan conventional synthetic serm substitute which has been normaly used inhuman IVF .What is known already: Supplementation of synthetic macromolecule to thecryopreservation solution is necessary to obtain stable and high post-warm sur-vival for human and animal oocytes and embryos vitrification.Study design, size, duration: Perspective study. Comparison of in vitro survivaland growth of oocytes after treatment. Total number of oocytes and embryos was874, the duration is 20 months from 04/2011.Participants/materials, setting, methods: Four experiments were conducted toassess the efficacy of HPC for use in vitrification solutions for bovine oocytes andblastocysts and for human oocytes. Four-hundred and twenty bovine oocytes and300 blastocysts and 154 human oocytes were vitrified by the Cryotop method(Kuwayama, 2005). The solutions for vitrification were supplemented with0.06 mg/ml HPC or with 10% commercial Synthetic Serum Substitute (SSS) orwith no added macromolecule (NA). Some HPC solutions and SSS solutionswere stored at 48C or -808C for 12months before vitrification.Main results and the role of chance: The survival rates of bovine oocytes vitri-fied in HPC, SSS and NAwere 100%, 100% and 86% and the respective blastocystrates were 22%, 18% and 8%, respectively. The survival rates of bovine blasto-cysts vitrified in HPC, SSS and NA were 99%, 93% and 88%, respectively.Thesurvival rate of bovine oocytes vitrified in HPC solutions stored for 12 monthsat 48C or -808C was 100%; the rates for those vitrified in SSS solutions were some-what lower than HPC. The survival rates of human oocytes vitrified in HPC, SSSand NAwere 100%, 98% and 92%, respectively.Limitations, reason for caution: This study is in vitro only.Wider implications of the findings: Found macromolecule hydroxypropyl cel-lulose may be used effectively for embryo culture media and freezing solutions forsperm.Study funding/competing interest(s): The study was performed by an ordinaryresearch expenses in the clinicTrial registration number: The study does’t contain clinical trials.

P-164 Restoration of developmental competence of oxidatively damagedoocytes by meiotic spindle transfer

M. Kobayashi1, T. Takeuchi2, and A. Yoshida1

1Kiba Park Clinic, Tokyo, Japan, 2Kyono ART Clinic Takanaw, Tokyo, Japan

Study question: Whether induced oxidative damage compromise the maturation-al and developmental capabilities of maturing oocytes as well as such insult can bereversed by spindle transfer (ST).Summaryanswer: Oxidative stress impaired meiotic and mitotic development ofmammalian oocytes with reduced mitochondrial activity and aberrant spindle for-mation. Ooplasmic replenishment by STwas able to conquer such injuries on ma-turing oocytes.What is known already: Oxidative stress during the in vitro maturation (IVM)process compromises the oocyte maturation. In addition, it is suggested that oxi-dative stress confronted during oocyte maturation might contribute to theage-related oocyte aneuploidy and consequent infertility in the human.However, the ability of ooplasmic replenishment by ST at the metaphase I (MI)stage to treat oxidatively damaged oocytes has not yet been assessed.Study design, size, duration: The spindle morphology, mitochondrial membranepotential (MMP), maturation rate and developmental ability were comparedbetween intact MI oocytes and those treated with H2O2. In order to assess theability of ST to treat oxidatively damaged oocytes, spindles isolated fromoocytes exposed to H2O2 were transferred to intact ooplasts.Participants/materials, setting, methods: Mouse MI oocytes were exposed to50 mM H2O2 for 15 min. H2O2 treated MI spindle was transferred into an enu-cleated intact oocyte with inactivated Sendai virus. After IVM, morphology ofMII spindle was examined immunohistochemically, MMP was evaluated withJC-1 dye, and matured oocytes were fertilized by ICSI.

Main results and the role of chance: Maturation rate of the H2O2 treated MIoocytes was significantly lower than the control (51.7% vs. 79.4%, p , 0.05).In oocytes reached MII stage after H2O2 treatment, length of the spindle was re-markably shorter, and MMP was lower than that of simply in vitro maturedoocytes. After ICSI, only 34.1% of the H2O2 treated oocytes developed to the2-cell stage, and none reached to the blastocyst stage. However, following ST,73.5% of the H2O2 treated oocytes matured normally exhibiting MII spindlewith normal morphology. MMP in the ST oocytes was higher than the H2O2

treated oocytes. In addition, all of the ST oocytes were fertilized normally afterICSI, and 20.5% developed to the blastocyst stage.Limitations, reason forcaution: This study was conducted using a mouse modelwith artificially induced oxidative stress. This finding does not directly representhuman infertility.Wider implications of the findings: This study shows clearly that the oxidativedamage induced in cytoplasm/mitochondria of mammalian oocytes impairs theirdevelopmental competence. Such ooplasmic damage can be reversed by replen-ishment of ooplasm, indicating that STcan playan important role in understandingage-related oocyte pathology.Study funding/competing interest(s): There are no conflicts of interest todeclare.Trial registration number: n/a

P-165 Avoiding the occurrence of smooth endoplasmic reticulum clus-ters in oocytes improves ART outcomes

A. Miwa, Y. Nagai, Y. Momma, K. Takahashi, M. Chuko, A. Nagai, and J. OtsukiNagai Clinic, Obstetrics and Gynecology, Saitama, Japan

Study question: Is it possible to prevent the presence of smooth endoplasmic re-ticulum clusters (sERC) in human oocytes and improve the take home baby rate?Summary answer: In this study, it was found that presence of sERC in oocytescould be avoided in most patients. About 30% of patients who previously hadsERC positive oocytes and had failed to become pregnant were able to havehealthy babies in their sERC negative cycles.What is known already: The presence of sERC in MII human oocytes indicatescytoplasmic damage. We have previously reported that the presence of sERCdecreased pregnancy rates and increased the miscarriage rate (ESHRE 2002,Hum Reprod 2004) and that elevated oestradiol and progesterone concentrationsand larger follicles induced sERC formation (ESHRE 2003, Hum Reprod 2004).Study design, size, duration: Consecutive ICSI and combined IVF and ICSIattempts from April 2002 to December 2012 in our clinic are included in thisstudy. Based on our previous findings three procedures described below werecarried out for patients previously positive for sERC.Participants/materials, setting, methods: The following three procedures, i)earlier administration of hCG than in previous sERC positive cycles, ii) alternativeovarian stimulation methods, iii) exclusion of even smaller sized sERC, werecarried out and pregnancy outcomes were subsequently investigated. sERC posi-tive oocytes rates were also compared in seven different stimulation procedures.Main results and the role of chance: Unfavorable sERCs were present in 12.1%(68/564) of patients representing 7.3% (86/1174) of cycles. Among the 68 patientswho had sERC positive cycles, 14 (20.6%) patients delivered healthy babies.Among the 54 coupleswho failed to deliver, 46 coupleshad further ART treatmentusing the three procedures described above and 41 out of 46 (89.1%) patients werefound to have sERC negative cycles. In their sERC negative cycles 12 (29.3%)patients delivered healthy babies. The following are the sERC positive rates foreach stimulation protocol: long, 8.9% (31/349); short, 25.0% (2/8);FSH-antagonist, 6.3% (32/512); CC-antagonist, 6.7% (11/164); CC, 6.9% (2/29); FSH, 9.1% (1/11) and letrozole-antagonist, 0% (0/29). The short protocolhad statistically lower sERC rates than the letrozole-antagonist (p , 0.01) andthe FSH-antagonist protocols (p , 0.05).Limitations, reason for caution: Since we previously proposed that the transferof embryos derived from sERC positive oocytes should be avoided (Hum Reprod2004), sERC positive oocytes in this study were not fertilized in all sERC positivecycles. Thus, the pregnancyoutcomes derived from sERC positive oocytes cannotbe investigated in this study.Wider implications of the findings: As reports show that babies derived fromsERC positive oocytes had multiple abnormalities, it is important to avoid the oc-currence of sERC formation. Since there were no sERC positive oocytes in the

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letrozole protocol, it could be used as a stimulation method for patients who haverecurrent sERC positive cycles.Study funding/competing interest(s): The authors received no funding for thisstudy and there is .Trial registration number: 0022654

P-166 Thawing in the day of embryo transfer had improved pregnancyrate in HRT cycles, but not in the natural cycles

S.G. Kim1, J.H. Lee1, Y.Y. Kim1, H.J. Kim1, I.H. Park1, H.G. Sun1, K.H. Lee1, andH.J. Song2

1Mamapapa&baby Obstetrics Gynecology Clinic, Infertility Lab., Ulsan city,Korea South 2Bucheon Seoul Women’s Hospital, BuCheon City, Korea South

Study question: What is the optimal thawing time in day 3 frozen-thawed embryotransfer(FET) during natural and hormone replacement therapy(HRT) cycles?Summary answer: Immediate embryo transfer after thawing in the morning hadimproved pregnancy rate compared with thawing in the evening 1 day beforeembryo transfer in HRT cycles.What is known already: Embryo-endometrial synchronization seems to beimproved by control of thawing day in FET cycles. However, the optimaltiming for thawing still remains uncertain.Study design, size, duration: In a retrospective study, the relationship betweenthawing time and pregnancy rate has been studied in 678 FET cycles betweenJan 2008 and Dec 2011. FET cycles were divided into four groups according tothawing time and endometrium preparation methods.Participants/materials, setting, methods: Embryos which were thawed evening1day before ET were cultured overnight and transferred the next day morning(group A: in natural cycle(n ¼ 160), group B: in HRT cycle (n ¼ 277)), whileembryos from thawed in the morning were transferred within 3h (group C:natural cycle (n ¼ 138), group D: in HRT cycle (n ¼ 103)).Main results and the role of chance: There were no differences among fourgroups in patient age(group A: 34.7+4.0, group B: 34.7+4.5, group C:33.9+3.5 and group D: 34.2+4.2), the mean number of transferredembryos(2.2+0.4, 2.1+0.4, 2.5+0.5 and 2.1+0.3), the mean number ofcryopreservedembryos(9.4+4.8, 10.9+6.2, 10.9+5.9 and 11.3+5.6), endo-metrial thickness(10.4+1.9, 10.3+2.0, 9.9+1.5 and 10.1+1.6) and embryoquality. The mean embryo survival index before transfer was comparable in thefour groups(72.4%, 71.7%, 70.6% and 71.4% p ¼ 0.30). The clinical pregnancyrates in natural cycles were no significant difference between group A(28.1%) andgroup C(37.7%, p ¼ 0.19). However, the clinical pregnancy rates in HRT cycleswere significantly higher in group D(50.5%) than group B(31.0%, p , 0.01).Limitations, reason for caution: The number of cycles between each group wasdifferent. In particular, the number of HRT cycles was small.Wider implications of the findings: In our results, the overall pregnancy rate inFET cycles had improved in immediate embryo transfer after thawing in themorning. Especially, pregnancy rate was significantly higher in the HRT cycles.We do not know the exact reason for the high pregnancy rate in the HRT cycles.Probably, it seems that better embryo-endometrial synchronization was achievedin HRT cycles.Study funding/competing interest(s): NoneTrial registration number: None

P-167 Single medium culture in a time-lapse incubator until the blasto-cyst stage with or without medium renewal on Day-3: a prospective rando-mised study with donor oocytes

N. Costa-Borges1, M. Belles1, J. Herreros1, J. Teruel1, A. Ballesteros2, A. Pellicer3,and G. Calderon1

1IVI Barcelona, IVF Laboratory, Barcelona, Spain, 2IVI Barcelona, ClinicalDepartment, Barcelona, Spain, 3IVI, Clinical Department, Valencia, Spain

Study question: Is it really necessary to renew the medium on Day-3 when using asingle medium for embryo culture to the blastocyst stage?Summary answer: Our preliminary data suggest that embryo quality, morphoki-netics, and clinical performance are not affected by single-step (uninterrupted)embryo culture in a single medium. The same medium can be used throughout

the 5 days of culture with no replacement on Day-3. Moreover, this strategydoes not affect embryo kinetics.What is known already: Embryo culture to blastocyst stage can be accomplishedusing either a single medium or sequential media. Traditionally, culture in a singlemedium includes medium renewal on Day-3. There are insufficient data to con-clude that this step can be avoided without affecting embryo quality and clinicalperformance.

In this study, we wanted to clarify this question and evaluate whether embryoculture until blastocyst stage in single medium can be performed safely withoutmedium replacement on Day-3.Study design, size, duration: We compared embryo development and quality,morphokinetics, and clinical outcomes (implantation and clinical and ongoingpregnancy rates) between single-step culture (without renewal) and two-stepculture (Day 3 renewal) culture. This prospective, randomised study comprised302 embryos (from donor oocytes) from 29 patients. ICSI was performed on alloocytes.Participants/materials, setting, methods: Injected oocytes were evenly distrib-uted in EmbryoSlidesw of a time-lapse incubator (EmbryoScopeTM, Unisense,FertiliTech). Slides were prepared with Global Totalw medium (LifeGlobal),equilibrated overnight, and randomised for medium renewal or no renewal onDay-3. Results were compared by Student’s t-test or X-test, P , 0.05 was consid-ered statistically significant.

Main results and the role of chance Blastocyst rates (67.5 vs 64.9%, P ¼0.751), percentages of frozen (49.0 vs 58.2%, p ¼ 0.248) and discardedembryos (22.5 vs 25.5%, P ¼ 0.745) were similar between two-step and single-step culture groups. The proportion of good-quality blastocysts (transferred plusfrozen) was also identical for both groups (77.5 vs 74.5%, P ¼ 0.745). Morphoki-netic variables from early cleavage until late blastocyst stage were not differentbetween culture treatment groups. No differences (P . 0.05) were found in clin-ical and ongoing pregnancy rates between transfers performed with embryos fromtwo-step culture (69.2 and 77.8%), single-step culture (75.0 and 100%) and mixedtransfers (42.9 and 66.7%). A total of 24 of 45 transferred embryos implanted andno significant differences were found among pure and mixed transfers.Limitations, reason for caution: These are preliminary results of an ongoingstudy and a larger sample size is required to increase statistical power. Resultsare based on observations with embryos from oocyte donors and need to be con-firmed with embryos from infertile patients of different ages.Wider implications of the findings: Simplified laboratory protocols, lower costsand fewer human errors are associated with the use of a single step culture systemwithout medium renewal on Day-3. The absence of differences in morphokineticsbetween both groups demonstrates that medium renewal does not affect timings ofembryo development events.Study funding/competing interest(s): This study was funded by IVI-Barcelona.The authors do not have any

P-168 Regulation of inositol 1,4,5-trisphosphate receptor during humanoocyte maturation and fertilization

D. Nikiforaki1, L. Vossaert2, F. Vanden Meerschaut1, C. Qian1, Y. Lu1, J.B. Parys3,W.H. De Vos4, D. Deforce2, T. Deroo1, E. Van den Abbeel1, L. Leybaert5,B. Heindryckx1, and P. De Sutter1

1University Hospital Ghent, Department for Reproductive Medicine, Ghent,Belgium, 2Ghent University, Laboratory for Pharmaceutical Biotechnology,Ghent, Belgium, 3KU Leuven, Department of Cellular and Molecular Medicine,Leuven, Belgium, 4Ghent University, Department of Molecular Biotechnology,Ghent, Belgium, 5Ghent University, Department of Basic Medical Sciences,Ghent, Belgium

Study question: What are the modifications occurring to the inositol1,4,5-trisphosphate receptor type 1 (IP3R1) during human oocyte maturationthat enhance its sensitivity for intracellular Ca2+ release during fertilization?Summary answer: During human oocyte maturation, the IP3R1 level increaseswith a concomitant rise of its phosphorylation. Simultaneously, the IP3R1,which is located in the endoplasmic reticulum (ER), relocalizes and formsdense clusters that decrease after fertilization. The amount of Ca2+ stored in theER ([Ca2+]ER) increases significantly during in vitro maturation (IVM).What is known already: Intracellular [Ca2+] oscillations mediated by the IP3R1regulate oocyte activation and embryo development. Prior to fertilization, the

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oocyte’s Ca2+ machinery is optimized. In animal models, multiple IP3R1 modi-fications occur during maturation from the germinal vesicle (GV) to metaphaseII (MII) stage, affecting its sensitivity and ensuring that the optimum Ca2+ re-sponse concurs with fertilization. These IP3R1 modifications include its redistri-bution, elevated level, enhanced Ca2+-releasing ability and relative IP3R1phosphorylation (p-IP3R1) potentially by M-phase kinases.Study design, size, duration: Immature, in vivo-matured and failed-fertilizedoocytes were obtained with informed consent (2010/182) from 98 women(32.2+4.6 years old) undergoing infertility treatment. Fifty oocytes were usedfor immunoblotting to assess the p-IP3R1 status, 35 for [Ca2+]ER evaluationand 37 for IP3R1 immunostaining and confocal microscopy.Participants/materials, setting, methods: GVand MI oocytes were matured inthe presence of inhibitors of Polo-like kinase 1 (Plk1) (BI 2536) and cyclin-dependent kinase 1(CDK1) (roscovitine, RO-3306) respectively. MPM-2 andRbt03-IP3R1 antibodies were used for immunoblotting and CT1-IP3R1 for immu-nostaining. [Ca2+]ER was assessed after thapsigargin treatment in Ca2+-freemedium. Results are expressed as median (interquartile range).Main results and the role of chance: Immunoblotting analysis indicated a2.2-fold maturation-associated increase in IP3R1 level between the GV and MIIstage. The p-IP3R1 increased by 75% from the GV to GV breakdown (GVBD)stage and reached a maximum level in MII oocytes. Specific inhibition of Plk1(until GVBD stage) and CDK1 (until MII stage) decreased the p-IP3R1 by 30%and 50% respectively. During maturation, IP3R1 redistributed from a diffusepattern to an increasingly localized pattern in both in vitro- and in vivo-maturedoocytes with prominent clusters that decreased in size after fertilization and invitro aging. [Ca2+]ER increased from 0.4 (0.3-0.5) AU at the GV stage to 1(0.8-1.4) AU at the GVBD, 1.4 (1.2-1.7) AU at the MI and 1.8 (1.7-2.1) AU atthe MII stage (P , 0.05).Limitations, reason forcaution: It is currently unclear which is the critical IP3R1modification underlying its increased sensitivity. Further analysis is required toclarify the effect of M-phase kinases on IP3R1 phosphorylation and IP3R1 Ca2+-releasing ability in human oocytes.Wider implications of the findings: This is the first report showing that variouscomponents of cytoplasmic maturation can render human oocytes competent toinitiate [Ca2+] oscillations at fertilization. These not only include the increasein [Ca2+]ER and IP3R1 redistribution but also the rise in IP3R1 level and phosphor-ylation, which in animal models lead to increased sensitivity for Ca2+ release. Anyperturbations of these processes could potentially hinder normal human fertiliza-tion and embryo development.Study funding/competing interest(s): This work was supported by a Ghent Uni-versity grant to B.H. The authors have no competing interests to declare.Trial registration number: Not applicable.

P-169 Exosomes as bioactive messengers in a critical time for preimplan-tation embryos n an ultrastructural study

L. Surlan1, V. Otasevic2, K. Velickovic3, I. Golic3, M. Vucetic3, V. Stankovic1,J. Stojnic4, N. Radunovic4, I. Tulic4, B. Korac2, and A. Korac3

1Clinic for Obstetrics and Gynaecology Clinical Centre of Serbia, ARTDepartment, Belgrade, Serbia, 2University of Belgrade Institute for BiologicalResearch “Sinisa Stankovic”, Physiology Department, Belgrade, Serbia,3University of Belgrade Faculty of Biology, Cell Biology, Belgrade, Serbia,4Clinic for Obstetrics and Gynaecology Clinical Centre of Serbia, Faculty ofMedicine, Belgrade, Serbia

Study question: Among the embryo fragments and blastomeres, various popula-tions of smaller but distinctive membranous extracellular organelles: exosomes,shedding microvesicles and apoptotic blebs deserve particular attention. Thegoal of this study was to provide an ultrastructural characterization of blastomere-derived exosomes in a critical period of embryo blastulation and fragmentation.Summary answer: Based on morphology, size and origin we defined the releaseof exosomes from both intact and fragmented blastomeres, their uptake by frag-ments and other blastomeres, and we provide their first extensive characterizationin human IVF embryos.What is known already: IVFembryos showrather craftydevelopmental capacityand various phenotype markers. Recent works confirmed exosomes as importantintercellular communication mediators – they carry proteins, miRNAs and menuof other molecules. They are formed by the inward budding and releasing of

multivesicular bodies, and are frequently fused with the neighboring cells.Various cell types are carrying exosomes. To date, their characterization, basedon morphology and, more importantly, biochemical and functional activity hasnot been provided.Study design, size, duration: Total of 35 embryos, having 6C to 8C, with variousdegrees of fragmentation, are stopped at Day 3 and examined. Overall cohort ofembryos were developed under standardized IVF clinical programme anddonated for science with written informed consent provided from couples.Participants/materials, setting, methods: Using transmission electron micros-copy (TEM) on human preimplantation embryos, we observe presence, distribu-tion and ultrastructural details of various membranous extracellular structures.A prospective observational ongoingstudywas initiated atObGinClinic -ClinicalCentre of Serbia and Faculty of Biology – Belgrade University.Main results and the role of chance: The blastomeres certainly do shed exo-somes and show their agglomerations in sub-plasmalemal area. Releasedexosomes are observed in inter-blastomeric space and not clearly confirmed inperivitelline space. Release zone was characterized with sER vesicles and mito-chondria presence. TEM shows characteristic cup-shaped exosomes with theirmean size of 165 nm+0.5 nm. Standard bell-shaped curve indicates homoge-neous population. We observed exosome production in A-class embryos andslightly negative correlation between their number and embryo quality. Presenceof various multivesicular bodies and structures resembling apoptotic bodiesincreases with lower embryo score. There are clear signs of exosome uptake byblastomeres, and it correlates with fragmentation degree. Fragments showedretained capability to endocytose exosomes as well. Rather excessive exosomeuptake was found in embryos showing apoptotic blastomeres.Limitations, reason for caution: Embryo-derived exosomes are mostly locatedin the central inter-blastomere environment. Their position, small quantity andhighly dynamic uptake make their isolation, purification and content depictionvery challenging, when compared with other cell systems. Revealing theirspatial dynamics is the first step for their future molecular and physiologicalmapping.Wider implications of the findings: To our knowledge, this is the first report thatembryos release exosomes. Studies on various cell types showed immunomodu-latory, antigen and transcriptomic activity. Excessive exosome uptake by dyingblastomeres suggests their involvement in activation-induced cell death. Theirpresence in good embryos implies their role in normogenesis and deserves greatattention. In future, improved purification and nomenclature strategies will helpto illuminate their place in complex and fragile biological system - early humanembryo.Study funding/competing interest(s): Study has institutional review board ap-proval and no conflicts of interest exist.Trial registration number: This work was supported by Serbian Ministry ofEducation, Science and Technological Development, Grant #173054.

P-170 Prospective randomized study comparing human embryo culturein group in the Well-of-the-Well dish or individually in droplets. Results froman intermediate analysis

P. Fancsovits1, C. Pribenszky2, A. Lehner1, A. Murber1, J. Rigo Jr.1, andJ. Urbancsek1

1Semmelweis University, Division of Assisted Reproduction First Department ofObstetrics and Gynaecology, Budapest, Hungary, 2Szent Istvan University,Department of Animal Breeding and Genetics Faculty of Veterinary Sciences,Budapest, Hungary

Study question: The aim of this study is to compare embryo development andpregnancy rate in human in vitro fertilization treatments when embryos are cul-tured individually or in group culture in Well-of-the-Well (WOW) Petri dish(Primo Dish, Vitrolife Hungary).Summary answer: Group culture in WOW Petri dish results in a higher cellnumber and better embryo morphology than individual culture on Day 3 ofembryo development. A non-significant increased clinical pregnancy rate isobserved after group culture.What is known already: Embryos in human IVF treatment are frequently cul-tured individually in microdrops allowing individual assessment and tracing ofembryo quality. Culturing more than one embryo in the same microdrop mayresult in accumulation of autocrine and paracrine factors in culture media

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having a beneficial effect on embryo development. The WOW dish contains 9 + 1microwells in the center of the dish allowing identification of individual embryoscultured together in the same microdrop.Study design, size, duration: One hundred nineteen consecutive IVF-ET cycleswere enrolled in this prospective randomized study between September and De-cember 2012. Cycles were randomized using a computer generated table. Fivehundred thirty-five embryos from 57 cycles were cultured in WOW dish and581 embryos from 62 cycles were cultured individually (Control group).Participants/materials, setting, methods: IVF cycles were randomized intoWOW group or individual culture. In WOW group up to 10 embryos were culturedtogether in 25 ml culture media. In Control group embryos were placed into a 25 mldroplet individually. Pregnancy rate and embryo morphology were compared.Main results and the role of chance: Fertilization rate in ICSI cycles -whereoocytes were placed into culture dish immediately after sperm injection- were sig-nificantly higher in WOW than in Control group (74.5% vs. 66.7 %, P ¼ 0.022).Number of blastomeres was similar in the two groups on Day 2. However, degreeof fragmentation was significantly lower in WOW than in Control group (12.7+8.4% vs. 15.0+11.8; P ¼ 0.005). We observed a higher number of blastomeres(7.0+2.0 vs. 6.5+2.1; P ¼ 0.002) and a lower degree of fragmentation (13.5+10.6% vs. 15.5+12.8; P ¼ 0.045) in WOW group on day 3. A higher proportionof embryos were available for cryopreservation in WOW group (41.3% vs. 32.9%;P ¼ 0.024). Clinical pregnancy rate was 50.9% in WOW and 38.7% in Controlgroup but the difference did not reach significance (P ¼ 0.182).Limitations, reason for caution: These are preliminary data of a larger study.There is more than 10% difference in clinical pregnancy rate which is not signifi-cant due to limited case numbers.Wider implications of the findings: Our intermediate findings suggest that usingWOW group culture dish may have a beneficial effect on IVF outcome. This is inagreement with previous findings on the superiority of group culture, however,WOW system allows for individual assessment and tracing of individualembryo quality. A larger study is currently ongoing.Study funding/competing interest(s): -Trial registration number: ClinicalTrials.gov: NCT01774006

P-171 Origin and role of transient DNA strand breaks duringspermiogenesis

R. Elias1, Q.V. Neri1, T. Fields1, P.N. Schlegel2, Z. Rosenwaks1, and G.D. Palermo1

1Weill Cornell Medical College, Reproductive Medicine, New York, U.S.A, 2WeillCornell Medical College, Urology Department, New York, U.S.A

Study question: To assess the etiology, localization, and meaning of DNA strandbreaks occurring during sperm production versus those generated in the malegenital tract.Summary answer: From this preliminary data, it appears that the origin of DNAfragmentation in these infertile men is caused by a post-spermatogenic insult.Therefore, the utilization of testicular specimen may enhance pregnancy outcome.What is known already: Tests for sperm DNA integrity have been proposed toassess male gamete competence. They are currently gaining popularity and aremore often used as a supplemental assay to traditional semen analysis. SpermDNA nicks and breaks are considered the culprits for the impaired reproductivecapacity in natural and assisted reproduction, however, it remains to be elucidatedwhen and at what level during sperm production the mishap occurs.Study design, size, duration: In patients with recurrent ICSI failure in ejaculatedspermatozoa, DNA fragmentation was assessed. To determine whether a testicularbiopsy would yield spermatozoa with a healthier chromatin, men underwent ICSIwith this specimen. Embryo developmental capacityand pregnancy outcomewerecompared among the two semen source.Participants/materials, setting, methods: We identified infertile men (n ¼ 9)with high DNA fragmentation in their ejaculates that agreed to undergo testicularbiopsy for this indication. Chromatin fragmentation index was assessed by SCSAor TUNEL on both types of sperm sources. Reproductive outcomes of ICSI cyclesutilizing the two sperm sources were analyzed and compared.Main results and the role of chance: In 9 men with recurrent ICSI failure (3.2cycles) their sperm yielded 60.9+29% fragmentation (up to 96%). After counsel-ing, eight underwent TESE with 6.5+5% DFI.

In a paired analysis the ejaculated cycle closest to the TESE cycle, gave a fertil-ization of 55.9% and 50.0%, respectively. The embryo cleavage was lower in theejaculate at 63.6% with a pregnancy rate of 12.5%, while in the TESE cohort allembryos cleaved resulting in a 25.0% clinical pregnancy.

When the overall ejaculated cycles (n ¼ 28) and TESE (n ¼ 13) were analyzed,fertilization was higher in the former (60.0 vs 46.9%;P , 0.01) with a comparableembryo cleavage rate. Following the replacement of an average of 2.8 concep-tuses, the clinical pregnancy was 10.7% (3/28) in the ejaculate and 30.8%(4/13) for the TESE.Limitations, reason for caution: Patients need to be informed of the risks withregards to the surgery, anesthesia, and the possibility that even with TESE a preg-nancy may not occur. Therefore, appropriate counseling should be conductedsince these men have spermatozoa in their ejaculate.Wider implications of the findings: When couples have recurrent pregnancy fail-ures after ICSI, associated with a high sperm DNA fragmentation, they should beoffered the option to undergo testicular biopsy.Study funding/competing interest(s): InstitutionalTrial registration number: Not applicable

P-172 Clinical outcomes following the transfer of vitrified blastocystsderived from a single or sequential culture medium

A. Gilson, N. Piront, B. Heens, C. Vastersaegher, A. Vansteenbrugge, andP.C.P. PauwelsCentre Hospitalier Regional de Namur, Procreation Medicalement Assistee,Namur, Belgium

Study question: The purpose of this study was to examine the effect of singleversus sequential culture medium on the survival of post-thawed blastocystsand their related chances of implantation and pregnancy rates.Summary answer: No statistical differences in terms of survival, implantationand pregnancy rates of post-thawed blastocysts were observed between the twoculture media studied.What is known already: Both single and sequential culture media contribute tothe development of zygote to the blastocyst stage. A large number of clinicalstudies have shown that blastocyst transfer results in higher implantation and preg-nancy rates than cleavage-stage embryo transfer. Obtaining good quality blasto-cysts before a vitrification procedure is a crucial step in predicting successfulpregnancy rates. However, the impact of culture medium on blastocyst cryotoler-ance remains poorly studied.Study design, size, duration: The current study includes a total of 177 warmingcycles of blastocysts from infertile women with a mean age of 32. Among these,101 were derived from culture in the single medium and 76 from culture in the se-quential medium. Data were retrospectively analysed for blastocyst survival, im-plantation and pregnancy rates. Chi-square test was used for statistical analysis.Participants/materials, setting, methods: Embryos arising from IVF cycleswere randomly cultured in four-well dishes containing either Global (single) orSage (sequential) medium covered with oil. Surplus embryos meeting our labora-tory standards for cryopreservation were vitrified in closed condition. Followingwarming, blastocysts were incubated for 3 hours prior to their transfer. Morpho-logical assessment was used to evaluate embryo viability/survival before andafter vitrification. Only blastocysts that show sign of reexpansion and with a cellviability above 50% were considered for transfer.Main results and the role of chance: There were 142 thawed embryos vitrifiedafter a 5 day culture in Global medium and 96 thawed embryos vitrified after a5 days culture in Sage medium.

The survival of post-warmed blastocysts was similar for both Global and Sageculture medium (78.9% vs. 82.3% respectively). Similarly, no significant differ-ences were observed between the two culture medium studied with respect to im-plantation rate (35.7% vs. 43.0%), clinical pregnancy rate (38.4% vs. 46.6%) andongoing pregnancy rate (32.6% vs. 34.6%).Limitations, reason for caution: N/AWider implications of the findings: This study shows that the use of single or se-quential culture medium does not affect the recovery of vitrified blastocysts andthat both media ensure similar chances of implantation and pregnancy followingfrozen blastocyst transfer.Study funding/competing interest(s): N/ATrial registration number: N/A

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P-173 Embryo culture under two different incubator temperature:a double blind randomized clinical trial

M.F. Abdel-Raheem1, M.Y. Abdel-Rahman2, H.M. Abdel-Gaffar2, M. Sabry2,H. Kasem3, S.M. Rasheed2, M. Amin2, A. Abdelmonem2, and A.S. Ait-Allah2

1IBN-SINA IVF/ICSI Center, IVF Lab, Sohag, Egypt, 2Sohag University, OB/GYN, Sohag, Egypt, 3Akhmim General Hospital, OB/GYN, Sohag, Egypt

Study question: Does embryo culture at 36.5 degree Celsius result in better preg-nancy rate compared with embryo culture at the traditional 37 degree Celsius in-cubator temperature?Summary answer: There is a statistically significant increase (*P ¼ 0.0196) inthe pregnancy rate at the group cultured at incubators with lower temperature36.5 degree Celsius (65.79%) compared to the pregnancy rate at the group cul-tured at incubators with traditional 37 degree Celsius (44.44%).What is known already: To date all IVF/ICSI procedures are conducted at 37degree Celsius, yet little studies had discussed the effect of temperature on devel-opment of embryos. It was principally accepted that 37 degree Celsius is the besttemperature for all aspects of human in vitro procedure. The ‘quiet embryo hy-pothesis’ proposed that viable embryos have a ‘quiet’ metabolism. Others pro-posed that gametes and early embryos function in vivo at a lower temperaturethan core body temperature.Study design, size, duration: This is a double blind randomized controlled clin-ical trial conducted at a private IVF/ICSI center comprising 130 participant at theperiod between October 2012 to January 2012.Participants/materials, setting, methods: One hundred thirty women undergo-ing fresh IVF/ICSI cycles in a private center were randomlyassigned either to 36.5degree Celsius incubator temperature (Group I, N¼ 76) or 37 degree Celsius in-cubator temperature (Group II, N¼ 54).Main results and the role of chance: There was a clear statistical significant in-crease (*P ¼ 0.0196) in the pregnancy rate at (group I) which cultured at incuba-tors with lower temperature 36.5 degree Celsius 50/76 (65.79%) compared to thepregnancy rate at (group II) which cultured at incubators with traditional 37 degreeCelsius 30/54 (44.44%).Limitations, reason for caution: Lack of clear molecular background that canexplain the interesting increase of the pregnancy rate in the group cultured atlower temperature.Wider implications of the findings: To change the traditional incubator tempera-ture to 36.5 degree Celsius.Study funding/competing interest(s): There was no study funding or competinginterests.Trial registration number: ClinicalTrials.gov identifier: NCT01706900

P-174 An uninterrupted culture medium protocol in conjunction with anon-humidified incubation environment can provide more patient treatmentoptions for IVF programs

M. VerMilyea, J. Anthony, J. Bucci, S. Croly, and C. CoutifarisUniversity of Pennsylvania, Penn Fertility Care, Philadelphia, U.S.A

Study question: The purpose of this study was to investigate the effects of embryodevelopment and alternative patient treatment options when using a renewedtwo-step sequential media protocol and traditional “big-box” incubators com-pared to a continuous uninterrupted single-medium protocol and benchtop incu-bators with no humidity.Summary answer: Embryos grown in a non-invasive single culture mediumunder oil and retained in humidity-free benchtop incubators are three-timesmore likely to become usable blastocysts thereby doubling the number of day-5embryo transfers and providing supernumerary blastocysts for vitrification foruse in subsequent frozen embryo transfer cycles.What is known already: Culture media is based on either a sequential (two/three-step) media protocol, which requires the use of one media followed by a differentmedia, or a single step culture medium. Previous reports have often suggested thata sequential series of media are necessary to accommodate changes in the physi-ology and metabolism of embryos but recent studies have shown comparablepregnancyoutcomesbut improved blastocyst development when embryos arecul-tured in an uninterrupted media and humid environment.Study design, size, duration: This retrospective study consisted of data from 956zygotes, which resulted in 179 embryo transfers over the course of nine months.

304 embryos were cultured in a sequential culture media system in standard sizeincubators while 652 embryos were place in a single-step medium in non-humidified benchtop incubators.Participants/materials, setting, methods: Irvine Scientific’s Continuous SingleCulture (CSC) was compared with LifeGlobal’s Fertilization and Global sequen-tial renewed-medium with respect to human embryo development, blastocyst util-ization rates and patient treatment options. Embryos cultured in CSC andK-Systems G-185 incubator were compared to GF and G media in Forma 3130incubators.Main results and the role of chance: Overall, significantly more embryos cul-tured in CSC and non-humidified benchtop incubators were transferred onday-5, compared to those cultured in Global sequential medium in humidity con-trolled “big-box” incubators (57% vs. 29%, P ¼ 0.0013). Autologous patients indifferent age groups (,35, 35-37, 38-40, .40) had more blastocyst transfers as aresult of CSC compared to Global (74%vs. 47%,60% vs. 28%, 27%vs. 21%,33%vs. 0%, respectively). In addition, surplus blastocysts available for cryopreserva-tion, which were cultured in CSC compared to LifeGlobal medium, were signifi-cantly greater (30% vs. 6%, P ¼ 0.0001). Ultimately, patients across all agegroups had a significantly higher blastocyst utilization rate when cultured in a non-invasive one-step embryo culture protocol (45% vs. 16%, P ¼ 0.0001 respective-ly). Data were analyzed by Chi-square analysis or Fisher’s exact test with P ≤0.005 regarded as statically significant.Limitations, reason forcaution: Although the data set is not extremely large, thisobservation clearly shows an upward trend supporting the benefit of a non-invasive culture system, which incorporates an uninterrupted single culturemedium (from day-0 to -6) and benchtop incubators. Further investigation intopregnancy outcomes will also soon be reviewed.Wider implications of the findings: The interest in single step culture mediumhas grown due to several practical and theoretical advantages over a sequentialmedia protocol. A non-invasive static approach to embryo culture, where unwar-ranted embryonic stresses from varying composition of media, potential osmoticshock and deprivation of any paracrine or autocrine factors has recently beenshown to be beneficial for embryo development. Practical advantages of using asingle step protocol include a substantial reduction in labor, error and cost.Study funding/competing interest(s): NoneTrial registration number: None

P-175 Efficiency of biopsy strategy and vitrification procedure in PGScycles

R. Maggiulli1, L. Rienzi1, D. Cimadomo1, A. Capalbo1, L. Dusi2, S. Colamaria1,E. Baroni1, M. Giuliani1, A. Vaiarelli1, F. Sapienza1, L. Buffo2, and F.M. Ubaldi1

1Genera Center for Reproductive Medicine, Clinica Valle Giulia, Rome, Italy,2Genera Center for Reproductive Medicine, Clinica Salus, Marostica, Italy

Study question: We aim to evaluate the efficiency of biopsy strategy and vitrifi-cation procedure, assessedas impact on implantationpotential of euploid embryosand on laboratory workload, in PGS cycles.Summary answer: Embryo implantation potential of euploid embryos seems notto be affected by the biopsy day and/or the vitrification procedure at blastocyststage. Laboratory workload is higher for day 3 biopsy, without any advantageon the clinical outcome.What is known already: The prevalence of aneuploidy in human embryos pro-vides a likely explanation for the relatively low success observed during IVFcycles, especially in advanced maternal age patients. Unfortunately, a poor correl-ation between conventional embryo morphological evaluation and chromosomalcomplement has been shown. This observation led to the introduction of PGS toavoid the transfer of aneuploid embryos. However, there is not yet a clear consen-sus about the impact of embryo biopsy strategy on implantation potential.Study design, size, duration: We compared day 3 biopsy (of one blastomere) andtransfer on day 5 (strategy 1) (36 cycles); day 5 biopsy (of 5-10 trofoectodermcells) and transfer on day 6 (strategy 2) (11 cycles) and day 5 biopsy, vitrificationand transfer in a subsequent prepared cycle (strategy 3) (10 cycles).Participants/materials, setting, methods: The patients enrolled in this studyunderwent ICSI/PGS cycles due to advanced maternal age (N ¼ 57). The meanfemale age was 38.1+2,9, 37,5+3,4 and 37,9+3,5 years for strategy 1, 2,and 3 respectively. No differences were observed in the basic patients character-istics between the 3 groups. Aneuploidies were assessed by whole genome

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amplification and array CGH (Bluegnome, UK). The primary outcome measurewas ongoing implantation rate (.12 weeks of gestation), while secondary out-comes measures were related to number of biopsied embryos and euploid blasto-cyts obtained.Main results and the role of chance: A significant higher mean number ofembryos per cycle were biopsied on day 3 vs day 5 (8,3+2,5 vs 4,9+2,2 and4,8+1,3 respectively, p , 0.001). A similar percentage of embryos were diag-nosed as euploid when strategy 1 was applied: 81/300 (27,0%), vs 20/54(37,0%) strategy 2 and 18/48 (37,5%) strategy 3 (NS). Moreover, the meannumber of euploid embryos that reached the blastocyst stage (thus available fortransfer and/or vitrification) per cycle was also comparable (1,9+0,7 (70/36),1,8+0.9 (20/11), 1,8+0,6 (18/10), for strategy 1,2,3 respectively). Inwarming cycles, blastocyst survival rate was 100% (16/16). Ongoing implantationrate was similar in the 3 groups [40,0% (28/70), 44,4% (8/18) and 43,8% (7/16)]for strategy 1,2,3 respectively).Limitations, reason forcaution: The main limitation of this study is the relativelylow number of embryos included (402 biopsied, 104 transferred) and the retro-spective approach.Wider implications of the findings: Since embryo biopsy strategy and/or cryo-preservation procedure do not seem to have any impact on embryo developmentalpotential, the most appropriate PGS strategy (day 3 or day 5 biopsy) can be adoptedaccording to the required genetic information and the laboratory workload.Study funding/competing interest(s): noneTrial registration number: none

P-176 Use of culture media amino acid mass spectrometry/liquid chro-motography (LCMS) measurments in identifing the embryo with the best im-plantation potential

E. Zivi1, E. Aizenman1, D. Barash2, D. Gibson2, and Y. Shufaro1

1Hadassah Ein Kerem, IVF unit, Jerusalem, Israel, 2Hebrew university, Institutefor Drug Research School of Pharmacy, Jerusalem, Israel

Study question: Is there an assocoation between culture media amino acids’(serine, proline, taurine, aspartic acid) alterations, as detected in mass spectrom-etry (MS), with reproductive outcome?Summary answer: Serine and proline excretion into the fertilization media wasfound to be in association with pregnancy.What is known already: Several studies investigated the correlation betweenpyruvate and glucose uptake with embryo viability and pregnancy. Othersfound that an increased uptake is correlative with embryo development and im-plantation. Amino-acids metabolism has also been investigated. Studies havefound an association between low concentrations of glycine and leucine andhigh concentration of aspargine in the culture media, with high pregnancy rate.High glutamate levels also correlate with pregnancy and live birth.Study design, size, duration: The study is observational.45 IVF patients ofvarious ages, infertility etiologies and different embryo qualities undergoing treat-ment, are included. Exclusion criteria: patients with a significant male factor ab-normality, structural anomaly of the uterus, endometriosis and pregestationaldiagnosis embryos. Spent culture media samples are collected and analyzedon MS.Participants/materials, setting, methods: Day 1 and day 3 samples of spentmedia are collected from each embryo, and analyzed on MS, in comparison tocontrol empty medium droplets incubated identically. The samples preparationincludes adding distilled water and hexane in order to remove the oil phase thatcoats the incubated embryos.Main results and the role of chance: Spent medium samples from each oocyte/embryo were obtained in 45 IVF cycles, performed in different patients, were ana-lyzed by LCMS. 22 patients conceived after fresh embryo transfer (all singletons).Day 1 spent fertilization media of 219 oocytes was analyzed, of which 84 embryoswere transferred fresh.

On day 1, excretion of serine and / or proline were found highly predictive forpregnancy; for proline excretion the PR’s were 40% compared to 18.5% (OR2.9,95% CI 1.1-8, p ¼ 0.04). For serine excretion the PR’s were 50% compared to17% (OR 5, 95% CI 1.75-14.28, p ¼ 0.003).

No significant differences in PRs were found in regard to aspartic acid andtaurine excretion or uptake.

The results of day 3 embryo culture media are currently analyzed.

Limitations, reason for caution: Thechnical limitations due to difficulties insample preparations regarded to the oil phase in the incubation.Wider implications of the findings: Serine and proline excretion into the fertil-ization media was found to be in association with pregnancy. The biological sig-nificance of this observation is that an embryo’s quality and implantation potentialcan be determined even immediately after fertilization. Accurate measurements ofmedia minute amino acid concentration changes are feasible even at this earlystage.Study funding/competing interest(s): research fund of the hospital. there is nocompeting interests.Trial registration number: the study is observational - not a clinical trial

P-177 Pronuclear score does not predict embryo implantaion.Retrospective study on different checkpoint times

M. Perez1, J. Aguilar1, E. Taboas1, M. Ojeda1, L. Suarez1, and E. Munoz2

1IVI VIGO, IVF Lab, Vigo (Pontevedra), Spain, 2IVI VIGO, IVF Unit, Vigo(Pontevedra), Spain

Study question: Can pronuclear score predict the embryo implantation?Summary answer: The aim of this study is to compare the pronuclear score(Z-score) at 17 hours post-insemination and at the frame before both pronucleus(PN) disappear to correlate themwith implantation rate byusinga time-lapsesystem.What is known already: A major objective of IVF laboratories is the identifica-tion and selection of embryos with the highest implantation potential after transfer,avoiding multiple pregnancies.

Pronuclearevaluation under light microscopyhas beenemployed as a determin-ing method for embryo fertilization assessment, and also for embryo selection.Previous studies endorse the prognostic effect of pronuclear evaluation forembryo viability and chromosomal abnormalities, while other authors have notbeen able to find any correlation.Study design, size, duration: Retrospective study of 2697 microinyected oocytesfrom 420 ICSI patients between January 2011 and November 2012, all of themincubated in an incubator with time-lapse technology.

Ax2-test was performed as the statistical analysis to assess the influence of themultinucleation in the implantation rate, and Yates correction when applicable.Participants/materials, setting, methods: The embryos which either failed toimplant or fully implanted, with full implantation meaning that all transferredembryos in a treatment implanted, were defined as KID (Known ImplantationData) positive (+) and negative (-) respectively. Only they were included in thestudy.

The distribution of nuclear precursor bodies (NPB) is classified into four groupsgiven Z-scores 1, 2, 3 and 4. Score group 1 includes zygotes with three to sevenpolarized NPB; score group 2 consists of zygotes with homogeneously dispersedNPB; score group 3 covers all the zygotes with a NPB distribution not included inthe other three groups, i.e., more than seven polarized NPB, one PN polarized andthe other dispersed or both dispersed but not homogeneously; and score group 4 isformed of zygotes with only one or two NPB as described by Gamiz et al (2003).

Z-score was registered at 17 hours post-insemination and the frame before thepronuclear disappearance (BPD).

Embryo transfer was performed on day 2-3 of development and embryo im-plantation was confirmed at an ultrasound scanning for gestational sacs.Main results and the role of chance: 268 KID embryos wereanalyzed. 33.5%(n ¼ 90) KID + , and 66.5% (n ¼ 178) KID-6. At 17h post-insemination,theZ-score was Z1 ¼ 10% (n ¼ 27); Z2 ¼ 7.8 (n ¼ 21); Z3 ¼ 15,6% (n ¼ 42) andZ4 ¼ 0% for theKID+ embryos, and Z1 ¼ 17.1% (n ¼ 46); Z2 ¼ 14.2% (n ¼38); Z3 ¼ 32.8% (n ¼ 88); andZ4 ¼ 2.2% (n ¼ 6) for the KID- embryos. AtBPF, the Z-score was Z1 ¼ 14.9% (n ¼ 40);Z2 ¼ 3.7% (n ¼ 10); Z3 ¼ 13.8%(n ¼ 37) and Z4 ¼ 1,1% (n ¼ 3) for the KID + , and Z1 ¼ 23.13%(n ¼ 62);Z2 ¼ 7.5% (n ¼ 20); Z3 ¼ 30.9% (n ¼ 83) and Z4 ¼ 4.8% (n ¼ 13) for KID-.

Statistical analysisshowed no significant differences in implantation rate andZ-score between groups,(p ¼ 0.30) and p ¼ (0.32) respectively. It showed an in-crease in the percentage ofZ1, Z3 and Z4 and a decrease in Z2 which could beobserved comparing 17h vs.BPD (p , 0,05).Limitations, reason for caution: Limitations due to the number of embryos ana-lysed.Wider implications of the findings: The two PN show a dynamic pattern andZ-score depends on when it is performed. As closer to BPD, NPB tend to be

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polarized. According to our data, there is not any prognostic effect of pronuclearevaluation at 17h or at BPD on implantation rate.

Further studies with a greater number of embryos analyzed are necessary toconfirm these preliminary results.Study funding/competing interest(s): No commercial interestTrial registration number: Trial none

P-178 Time laps monitoring of embryo development in patients with tes-ticular or ejaculated sperm: a comparative study

V. Casciani1, M.G. Minasi1, F. Scarselli1, M. Terribile1, D. Zavaglia1,A. Colasante1, G. Franco2, and E. Greco1

1European Hospital, Centre for Reproductive Medicine, Rome, Italy, 2SapienzaUniversity, Gynaecological-Obstetrical and Urological Sciences, Rome, Italy

Study question: Our purpose was to compare, with the use of time-lapse technol-ogy, the developmental stages of embryos obtained with intracytoplasmic injec-tion of spermatozoa retrieved either by ejaculation or by the testicles, in order tofind out possible differences in the developmental kinetics between the twogroups.Summaryanswer: No difference appeared in the fertilization timing between tes-ticular and normal ejaculated sperm, except a veryslight anticipation of the secondpolar body (IIPB) extrusion. In the following cellular divisions, it was possible toobserve a significant retardation in the 4-cells stage in embryos deriving from tes-ticular sperm.What is known already: It is well known that ejaculated and testicular spermdiffer in the degree of nuclear maturation. During spermiogenesis, the transit ofspermatozoa in the epididymal tract favors DNA packaging by stabilizing thechromatin structure through protamine dephosphorilation and the formation ofmolecular bridges. Additional difference in sperm maturity may involve the cen-triols as structures implicated in embryo cleavage. Based on these observations,sperm maturity can affect the timing of fertilization and later cellular divisions.Study design, size, duration: In this retrospective observational study (Septem-ber 2012-January 2013), we analyzed the developmental kinetics of embryosobtained either with testicular sperm (N ¼ 40 embryos; TS-group) or withnormal (WHO, 2010) ejaculated sperm (N ¼ 101 embryos; ES-group). The de-velopmental markers observed were: IIPB extrusion, 2PN appearance, 2PN dis-appearance, divisions at 2 to 8-cells.Participants/materials, setting, methods: Development timings were recordedfor all embryos (TS-group: 9 ICSI cycles, female mean age ¼ 33.78+4.58;ES-group: 25 cycles, mean age ¼ 35.57+4.09). Only correctly fertilizedoocytes were observed (40/50 ¼ 80% in TS; 101/117 ¼ 86.3% in ES, NS). Stu-dent’s t-test was used for statistical analyses. Average hour+SD from ICSI in-semination are reported for all stages.Main results and the role of chance: In TS versus ES groups respectively, IIPBwas extruded at 3.86+1.48h (N ¼ 38) and 4.03+1.39h (N ¼ 88) (p ¼ 0.035);2PN appeared at 10.05+1.77h (N ¼ 40) and 10.33+2.78h (N¼ 101); 2PN dis-appeared at 24.93+3.59h (N ¼ 32) and 23.91+2.48h (N ¼ 100); cleavage at2-cells was at 27.94+3.75h (N ¼ 25) and 26.66+3.00h (N¼ 92); 3-cells at39.07+6.27h (N ¼ 18) and 38.65+4.68h (N ¼ 53); 4-cells at 42.08+4.62h(N ¼ 23) and 39.70+3.65h N ¼ 88) (p ¼ 0.019); 5-cells at 52.23+9.45h(N ¼ 16) and 51.08+7.87h (N ¼ 70); 6-cells at 54.09+10.08h (N ¼ 17) and53.18+5.05h (N ¼ 53); 7-cells 52.65+9.21h (N ¼ 16) and 52.26+3.95h(N ¼ 58); 8-cells 54.99+9.73h (N ¼ 12) and 57.56+6.63h (N ¼ 60). Inbrackets are reported numbers of embryos at a specific developmental stage.Data on embryos are relative to cultures up to day-2 or day-3.Limitations, reason forcaution: A limitation of the present study is the small sizeof our population due to the fact that we have started using the time-lapse technol-ogy only in September 2012. An additional limitation may relates to the imagingsystem of such technology that allows the visualization of only 7 focal layers.Wider implications of the findings: Differences in maturity of ejaculated and tes-ticular sperm seem not to interfere with fertilization when ICSI is performed.Sperm factors involved in oocyte activation are likely present in testicular sperm-atozoa in similar amount and with similar biological activity as in normal ejacu-lated sperm. The retardation in the 4-cell stage observed with testicular spermcould be due to immaturity at different levels which may involve the spermhead as well as other structures participating in cellular divisions.

Study funding/competing interest(s): No external funding was obtained for thepresent study; has to be declared with any financial organization regarding the ma-terial discussed in the manuscript.Trial registration number: Not applicable

P-179 Time affects time: increased maternal age delays embryo morpho-kinetics

C. Hickman, C. Cook, D. Gwinnett, G. Trew, A. Carby, and S. LaveryBoston Place Clinic, Embryology, London, United Kingdom

Study question: To determine whether maternal age affects embryo morphoki-netic parameters.Summary answer: Embryos derived from older mothers have a slower develop-ment to the morula and blastocyst stage compared to embryos derived fromyounger mothers.What is known already: Maternal age is the most significant factor to affectembryo implantation potential due to the increased incidence of aneuploidy inembryos derived from older patients. Aneuploid embryos have been shown tohave delayed embryo morphokinetics. Therefore, it was hypothesized thatincreased maternal age delays embryo development.Study design, size, duration: This blind retrospective study in an IVF clinicassessed morphokinetic parameters of 348 human embryos cultured using a time-lapse imaging system (EmbryoscopeTM, Unisense-Fertilitech, Denmark) fromApril to October 2012. Embryos were categorized as derived from older (agegreater or equal to 37 years) versus younger (36 years and under) patients.Participants/materials, setting, methods: Morphokinetic data was collectedfrom 24 ICSI patients cultured in the EmbryoScopeTM (sensitivity: 15 minutes).Following ICSI, hours for embryos to reach the 2-cell, 3-cell, 4-cell, 5-cell,6-cell, 7-cell, 8-cell, 9 + cell, morula, expanded and hatched blastocyst wererecorded and compared between 10 older and 14 younger patients.Main results and the role of chance: Embryo morphokinetic parameters werecompared between embryos derived from older and younger patients and differ-ences tested for significance using a two-tailed two sample t-test. Differenceswere found to be significant at p , 0.05. Data are presented as mean+ standarddeviation. Patient age did not significantly affect the time for embryos to reach the2-cell, 3-cell, 4-cell, 5-cell, 6-cell, 7-cell, 8-cell or 9 + cell stages. Youngerpatients reached the morula (92.9+11 vs 98.8+14 hours post insemination,hpi, p ¼ 0.03), expanded blastocyst (111.7+8.4 vs 118+8.8hpi, p ¼ 0.008)and hatched blastocyst (115.1+7.5 vs 129+10.7hpi, p , 0.001) stages fasterthan older patients (younger vs older patients respectively).Limitations, reason for caution: As expected, the mean FSH dose for olderpatients (3069+1531,n ¼ 144) was significantly greater (p , 0.001) than foryounger patients (2148+1052, n ¼ 204). Therefore, further studies are requiredto determine whether the differences between the two treatments were due to ma-ternal age or differences in dose of FSH.Wider implications of the findings: Time-lapse technology has the potential toimprove embryo selection if parameters are identified as accurate predictors of im-plantation potential. Improving our understanding of how these parameters inter-act with current established predictors of implantation, such as age, may aid in theformation of models to predict implantation. Such models would have consider-able clinical value, with the potential to improve IVF success rates.Study funding/competing interest(s): NoneTrial registration number: None

P-180 The effect of Sildenafil Citrate (Viagraw) in vivo on endometrialreceptivity, ovulation and embryo development in mice

L. Asgari, D. Paouneskou, K. Jayaprakasan, W. Maalouf, and B.K. CampbellUniversity of Nottingham, School of Clinical Sciences, Nottingham, UnitedKingdom

Study question: What are the effects of pre-conception sildenafil treatment on:subsequent embryo development through culturing of embryos until the blasto-cyst stage, endometrial receptivity through assessment of the thickness of the epi-thelial cell layer and oocyte yield through the estimation of the number of corporalutea in mice.

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Summary answer: Pre-conception administration of sildenafil at both the lowerand higher doses tested impaired embryonic development relative to vehicletreated controls and the median dose of sildenafil. Further, the higher doseincreased endometrial thickness but none of the doses had any effect on ovulationrate or follicle number.What is known already: Sildenafil has been suggested as a useful adjuvanttherapy in women undergoing ART to increase the blood flow to both the devel-oping endometrium and the ovary. Previous studies from our laboratory haveshown that while addition of sildenafil to mouse embryos cultured in vitro hadno effect on embryo development at doses in the normal physiological range,higher concentrations had a detrimental effect. This study therefore testedwhether preconception sildenafil administration had a similar negative effect.Study design, size, duration: Mice were randomly assigned to either vehicle con-trols (n ¼ 16) or three different doses of sildenafil (S1:1.25 mg/kg n ¼ 16,S2:2.5 mg/kg n ¼ 16, S3:5 mg/kg n ¼ 16) administered by oral gavage twicedaily over 2 days before mating on the morning of Day 3 before sacrifice fortissue recovery on Day 4.Participants/materials, setting, methods: Day 1 zygotes were collected and cul-tured in vitro to the blastocyst stage before being scored and the total cell number(TCN) counted. Ovaries were analysed histologically for number of corpora lutea(CL) and follicles whereas in the uterus endometrial thickness and number ofblood vessels (neo-vascularisation) were counted.Main results and the role of chance: Relative to vehicle treated controls, cleav-age rates, speed of cleavage and blastocyst rates were significantly lower (P ,

0.05) in embryos from mice treated with the lowest and highest doses of sildenafil(S1 and S3, P , 0.05),while therewas no significantdifferencewith the middleS2dose. No difference was detected in the TCN of blastocysts formed (P . 0.05).Sildenafil at the highest concentration (S3: 5 mg/kg) also caused a significantincrease in the uterine thickness (P , 0.05), while ovulation rate and follicu-lar development remained unaffected (P . 0.05). These results suggest there-fore that sildenafil may be acting via different mechanisms at differentconcentrations.Limitations, reason for caution: This work has been carried out in mice socaution is needed when translating to the human situation as it is known thatthe pharmacokinetics of sildenafil differ between species.

Wider implications of the findings Although our study supports the possibleuse of sildenafil as adjuvant therapy to improve endometrial receptivity it alsoraises concernsover the possible safetyof this drug in terms of possible detrimentaleffects on embryo development. Further animal studies on the potentially deleteri-ous effects of pre-conception sildenafil treatment of females on subsequentembryo and fetal development are required before the introduction of sildenafilas an adjuvant therapy could be adopted.Study funding/competing interest(s): Study funding came from my belovedfather, Mohandes Ehsan Asgari and the University of Nottingham.Trial registration number: NA

P-181 Binucleation versus multinucleation in 2-cells embryos how dothey affect to implantation rate?

J. Aguilar1, E. Taboas1, M. Perez1, E. Munoz2, M. Ojeda1, and J. Remohi3

1IVI Vigo, FIV, Vigo, Spain, 2IVI Vigo, Gynecology, Vigo, Spain, 3IVI Valencia,Gynecology, Valencia, Spain

Study question: How do binucleation and multinucleation in 2-cells embryoaffect the implantation rateSummary answer: Embryos with one blastomere multinucleated (more than twonuclei per cell) have a lower implantation rate than those with one binucleatedblastomere and those with both blastomeres multinucleated (binucleated or multi-nucleated)What is known already: Multinucleation has been related with an increase in theaneuplody rate, in chromosomal anomalies and a lower blastocyst rate, althoughthe impact of multinucleation on live birth rate remains elusive. Nucleationerrors are relatively frequent and, can be transitory phenomenon. They are classi-fied either as binucleation (2 nuclei per cell), multinucleation (3 or more nuclei percell).Study design, size, duration: Cross-sectional study of 2697 microinjectedoocytes from 420 ICSI patients between January 2011 and November 2012, allof them incubated in an incubator with time-lapse technology.

Participants/materials, setting, methods: Only KID (Known ImplantationData) embryos were analized.They were classified in 6 groups according to im-plantation (positive/negative),and the number of multinucleated blastomeres on2-cells stage, (group 0¼ none; group 1 ¼ one; group 2 ¼ two)

Multinucleation on 4-cells embryo was employed as exclusion criteria. x2-testand a Fisher exact t-test were performedMain results and the role of chance: 245 out 475 embryos transferred providedknown implantation information and were analyzed, whereas 32.24% (n ¼ 79)fully implanted and 67.75% (n ¼ 166) failed to implant. Multinucleation waspresent in 46.17% of the embryos.

17.1% (n ¼ 42) fully implanted embryos were group 0, 8.97% embryos (n ¼22) were group 1, and 6.1% (n ¼ 15) group 2. The distribution for thenot-implanted embryos was (n ¼ 90) 36.7 % of embryos group 0, 17.5%, onecell multinucleated (group 1) (n ¼ 43), and 13.46% both cells multinucleated(group 2) (n ¼ 33).

In group 1, those embryos binucleated (n ¼ 17) showed greater implantationrate than those multinucleated (n ¼ 5), 26.15% v.s 7.69% (p ¼ 0.02)

In group 2, three subcategories were analyzed, 2 binucleated cells, 2 multinu-cleated cells, 1 binucleated + 1 multinucleated. No significant differences werefound among them.Limitations, reason for caution: Studies with a greater number of embryos ana-lyzed are necessary to confirm these preliminary resultsWider implications of the findings: Multinucleation in 2 cells embryos is rela-tively frequent during the embryo development, even in those transferredembryos selected by morphology and could disappear when embryos developinto 4-cells.The early cleavage checkpoint helps to improve embryo selectioncriteria.Study funding/competing interest(s): This work has not received any financialsupport from any commercial entity.

None of the authors have any particular benefit to run this project.Trial registration number: none

P-182 Improvement in blastocysts formation byselectinghyaluronic acidbinding sperm

E. Rega, A. Alteri, R.P. Cotarelo, P. Rubino, A. Colicchia, and P. GianniniFerticlinic-Villa Margherita, Laboratorio PMA, Roma, Italy

Study question: To evaluate the ability of hyaluronic acid (HA) binding sperm toincrease the efficiency of intracytoplamic sperm injection (ICSI) in terms of blas-tocysts formation.Summary answer: The selection of normal sperm for ICSI with hyaluronic acidbinding assay (HA-ICSI) led to increase of the blastocyst formation rate, in com-parison with the conventionally selected spermatozoa.What is known already: The majority of studies are focused on the use ofHA-binding as a method to select spermatozoa with normal nucleus and intactDNA. These parameters play a critical role in the paternal contribution to success-ful preimplantation embryogenesis; however, there are conflicting data about thepositive correlation between HA-ICSI and improvement of fertilization rate andembryo development.Study design, size, duration: In this cohort study, we retrospectively analyzed thepercentage of blastocysts obtained, between July and December 2012, from 67sibling oocytes injected with either HA-bound (n ¼ 33) or conventionallyselected spermatozoa (n ¼ 34) in a randomized way.Participants/materials, setting, methods: Patients using testicular or criopre-served sperm were excluded from the study. Half of the oocytes of women retriev-ing more than 4 metaphase II oocytes were injected by the HA procedures. Semensamples were treated via swim-u and placed in two different ICSI dishes with HAand PVP drops.Main results and the role of chance: A clear trend towards a higher blastocystformation rate in oocytes injected with HA-bound spermatozoa has beenobserved: the frequency of blastocyst formation in HA-ICSI was 62.0%, whilefor PVP-ICSI was 40.0% (p ¼ 0,0598). These data suggest that the embryosdeveloped from HA-ICSI seem to have a greater chance of developing to theblastocyst stage.Limitations, reason for caution: Low number of oocytes available for the study.

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Wider implications of the findings: This study suggest that the selection ofnormal sperm with HA might overcome potential embryo arresting due to patho-genesis inherent in the paternal genome.Study funding/competing interest(s): This study received no funding and thereare no conflicts of interests to be declared.Trial registration number: This was a coohort study and a trial registration doesnot apply

P-183 Embryo quality predictive models based on cumulus cells geneexpression for clinical application

R. Devjak1, T. Burnik Papler1, K. Fon Tacer2, and I. Verdenik1

1University Medical Centre Ljubljana, Departmen of Obstetrics and Gynecology,Ljubljana, Slovenia, 2University of Ljubljana Veterinary Faculty, Institute forHygiene and Pathology of Animal Nutrition, Ljubljana, Slovenia

Study question: The aim of the study was to use cumulus cells (CC) gene expres-sion differences of leukemia inhibitory factor (LIF), anti – Mullerian horomonereceptor, type II (AMHR2), follicle stimulating hormone receptor (FSHR), vascu-lar endothelial growth factor C (VEGFC) and serpin E2 (SERPINE2) to differen-tiate high grade and low grade embryos.Summary answer: As significant resulted AMHR2 (p ¼ 0.03) and LIF showed atendency to significance (p ¼ 0.11) between high grade and low grade embryoswhere their combination resulted in the area under the curve (AUC) 0.72+0.08 and 0.73+0.03 for binary logistic or decision tree prediction model,respectively.What is known already: Cumulus cells (CC) have a specific gene expressionprofile according to the developmental potential of the oocyte they are surround-ing and therefore specific gene expression could be used as a biomarkers. So far nosingle uniform biomarker has been found to be accurate enough for clinical appli-cation. The combination of several biomarkers and various statistical modelsmight improve the usefulness of CC biomarkers in the clinics.Study design, size, duration: The CC samples from oocytes developed in highgrade embryos (n ¼ 26) and low grade embryos (n ¼ 32) from 21 patients wereanalyzed. The CC expressions of LIF, AMHR2, FSHR, VEGFC and SERPINE2were compared between high grade and low grade embryos and two modelswere used to test their prediction value.Participants/materials, setting, methods: Infertile women with tubal, unex-plained infertility, aged less than 35 years and normal partners’ spermiogramswere included in the study. Quantitative PCR was used to analyze CC expression.A Mann – Whitney test was used for testing differences and a binary logisticmodel and data a decision tree model were used.Main results and the role of chance: The CC gene expression between highgrade and low grade embryos was significantly different at AMHR2 (p ¼ 0.03)and among other genes only LIF showed a tendency to significance (p ¼ 0.11).The CC expression values of AMHR2 and LIF were used in two models for pre-diction of high grade embryos. The first, binary logistic model yielded in AUC0.69+0.08 for AMHR2 and AUC 0.63+0.08 for LIF alone. Combining bothgenes the binary logistic model yielded in AUC 0.72+0.08. The second, deci-sion tree model yielded in AUC 0.67+0.01 for AMHR2 and AUC 0.57+0.02 for LIF alone. Combining both genes in the decision tree model yielded anAUC 0.73+0.03.Limitations, reason for caution: Only two genes were significant enough for im-plementation in models. With more genes in the model the predictive power wouldimprove.Wider implications of the findings: The present study indicates that prediction ofhigh grade embryos with CC expression is dependent on a type and number of CCbiomarkers used. Even though LIF is not significantly different between highgrade and low grade embryos it improves prediction value of AMHR2 in bothtested models. In term of eventual use in clinical practice the decision treemodel has easy rules which can be applicable when making clinical decisions.Study funding/competing interest(s): This work was supported by SlovenianResearch Agency (www.arrs.gov.si) grants P1-0104 and L3-4162.Trial registration number: Not a clinical trial.

P-184 Pronuclear behavior and timing of embryo development:a time-lapse point of view

C. Scarica1, F.M. Ubaldi1, M. Stoppa1, R. Maggiulli1, A. Capalbo1, E. Ievoli1,L. Dovere1, L. Albricci1, S. Romano1, F. Sanges1, A. Vaiarelli1, and B. Iussig2

1GENERA, Center for Reproductive Medicine Clinica Valle Giulia, Rome, Italy,2GENERA, Center for Reproductive Medicine Clinica Salus, Marostica (VI), Italy

Study question: Evaluate whether specific morphodynamic events during pro-nuclear (PN) formation are correlated with the early stages of embryo develop-ment, investigated with time-lapse cinematography.Summary answer: Through the use of time-lapse cinematography is possible tohighlight that a delay in the PN appearance is predictive for abnormal PN forma-tion. Moreover, PN eccentricity, asymmetry in size and failure of juxtaposition arerelated to a delay in timing of cleavage.What is known already: It was hypothesized that the PN evaluation at 16-18hpost-ICSI is correlated with embryo development and chromosomal abnormal-ities and proposed as an important embryo selection parameter. However, conflict-ing data are available in literature. Parameters analyzed are normally related to thenumber and distribution of nuclear precursors bodies in PNs, the size and the pos-ition of the PN. Due to the dynamicity of PN formation events, static evaluation isprobably insufficient evaluating this stage of embryo development.Study design, size, duration: We retrospectively evaluated the impact of specificPN abnormal characteristics (eccentricity, asymmetry in size and failure of juxta-position) on timing of embryo development by comparing 86 sibling embryos,cultured in a time-lapse incubator. To avoid confounding factor, each embryowith abnormal PN dynamics was compared with two sibling embryos withnormal PN dynamics of the same cohort.Participants/materials, setting, methods: All the embryos analyzed derivedfrom oocytes microinjected and cultured in a time-lapse incubator system(Embryoscope, Unisense). Events of development, such as timing of PN appear-ance, syngamy, timing of 1st to 7th cell division and cell cycle duration were eval-uated by time-lapse cinematography.Main results and the role of chance: The morphokinetic parameters analyzedshow that the timing of appearance of the PNs is significantly lower in zygoteswith normal PN dynamics (10,6+1,75 hours, CI 10,10-11,12), than those withabnormal PN dynamics (12,01+1,96 hours, CI 11,27-12,45) p ¼ 0,002. Thisdelay is reflected in the subsequent development and, in particular, in the time div-ision into 4 cells (39,56+5,01 hours CI38,15-40,57, vs 42,83+9,7 hoursCI38,43-46,52 respectively) p ¼ 0,05Limitations, reason for caution: Further analysis is necessary to confirm thisdata on a larger population and to understand the impact of abnormal PN behavioron implantation potential and chromosomal abnormality of the deriving embryos.Wider implications of the findings: Due to the high dynamicity of pronuclearformation, different events can be missed during static evaluation normally per-formed at 16-18 hours post ICSI procedure. Our results suggest the predictivevalue of pronuclear appearance on successive abnormal PN behavior. An evalu-ation around half past 11 hours could help to avoid the selection of embryosthat have the risk to display PN eccentricity, abnormal size and/or failure of juxta-position during early formation.Study funding/competing interest(s): noneTrial registration number: none

P-185 Artificial collapse of blastocoelic cavity improves clinical outcomeof closed blastocyst vitrification: a randomized controlled trial

A. Gala1, A. Ferrieres1, S. Assou1, C. Vincens2, S. Bringer-Deutsch2, C. Brunet2,and S. Hamamah1

1Universite Montpellier 1 Inserm U1040 CHU Montpellier, ART/PGDDepartment Institut de Recherche en Biotherapie, Montpellier Cedex 5, France,2CHU Montpellier, ART/PGD Department, Montpellier Cedex 5, France

Study question: Does blastocyst artificial shrinkage (AS) prior to vitrification in aclosed carrier device improve clinical outcomes?Summary answer: Artificial shrinkage of blastocoelic cavity significantlyimproves clinical pregnancy rate after closed vitrified blastocysts transfer.What is known already: Expanded blastocysts on day 5/6 are more sensitive tovitrification than early blastocysts because of an insufficient dehydration of theblastocoelic cavity. Based on this hypothesis, several authors have described tech-niques to reduce the blastocoele and prevent the formation of damaging ice crys-tals. However, all those studies have been realized with opened devices.

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Concerning the income of artificial shrinkage (AS) prior to vitrification in closedsystems, data remain unclear.Study design, size, duration: Prior to vitrification with closed device, blastocystswere either collapsed by laser pulse or non treated at all according to a randomizedprocedure. Clinical pregnancy rate was assessed in 147 warming cycles from April2011 to December 2012.Participants/materials, setting, methods: On day 5/6, full (3), expanded (4) orhatching (5-6) blastocysts with at least a trophectoderm quality type B accordingto Gardner classification were cryopreserved. Clinical pregnancy rate was com-pared between blastocysts with AS (n¼ 35 cycles) and blastocysts without AS(n ¼ 112 cycles). The mean women age was 33.2+4.9 years.Main results and the role of chance: The overall clinical pregnancy rate aftertransfer was 41.8% (59/141). The clinical pregnancy rate was significantlyhigher in the AS group (19/35; 54.2%) compared with the control group (40/106; 37.7%) (1 ¼2.414, p , 0.02). The survival rate was higher in the ASgroup but no significantly (52/53, 98.1% Vs. 157/170, 92.3%; 1 ¼1.468, p .

0.09). The two groups were similar concerning women age, endometrial prepar-ation and vitrification dayLimitations, reason forcaution: This results need to be completed with the livingbirth rates to ensure the efficiency of our treatment.Wider implications of the findings: To our knowledge, this is the first rando-mized controlled trial assessing the impact of AS prior to vitrification in aclosed device. This study reveals that blastocyst AS with laser pulse is a successfultechnique to improve clinical pregnancy rate after closed vitrification. Our resultsare in line with previous publications on the benefit of AS.Study funding/competing interest(s): This work was partially supported by agrant from Ferring Pharmaceuticals company. The authors declare that there is .Trial registration number: Not applicable

P-186 Dynamic assessment of early embryo fragmentation by time-lapseanalysis may improve cell cycle timing-based embryo selection

J. Conaghan1, L. Tan2, M. Gvakharia3, K. Ivani4, A. Chen2, and R. Reijo Pera5

1Pacific Fertility Center, IVF Lab, San Fransisco CA, U.S.A, 2Auxogyn Inc.,Clinical R&D, Menlo Park CA, U.S.A, 3Fertility and Reproductive Health,Reproductive Laboratories, San Jose CA, U.S.A, 4RSC of the SF Bay Area, IVFLab, San Ramon CA, U.S.A, 5Stanford University, Stem Cell Biology andRegenerative Medicine, Stanford CA, U.S.A

Study question: What are the fragmentation dynamics in human embryos from 1-to -4-cell stages, and could time-lapse assessment of fragmentation improveembryo selection when used together with early cell cycle timings?Summary answer: For most embryos, fragmentation occurs between the 2- and4-cell stages; time-lapse analysis of early embryo fragmentation can augment thepredictive power of cell cycle timing parameters for selecting the embryo with thehighest chance of implantation.What is known already: Fragmentation is commonly associated with decreasedembryo viability, and most clinics assess fragmentation on Day 2 and/or 3 (4- to8-cell stage). However, a recent paper by Chavez et al. (Nat Comm, 2012) suggeststhat dynamic assessment of embryo fragmentation, together with cell cycle timingparameters, is indicative of human embryo ploidy at the 4-cell stage. The currentstudy examined the incidence and outcomes for embryos with early fragmentationin a clinical IVF setting.Study design, size, duration: Retrospective analysis of 850 embryos from 95patients (6/2011-10/2012)with embryos imaged using the EevaTM Test, a platformthat makes blastocyst predictions by integrating time-lapse imaging and auto-mated analysis of cell cycle timing parameters P2 (time between cytokinesis 1and 2) and P3 (time between cytokinesis 2 and 3).Participants/materials, setting, methods: Embryo videos were analyzedbetween the 1- to 4-cell stages for fragmentation appearance, volume andpattern over time. This data was used to determine if fragmentation analysiscould augment P2 and P3 to better predict implantation. Statistical significancewas determined by Fischer’s Exact or Chi-Square test.Main results and the role of chance:Dynamicassessmentoffragmentationbytime-lapse imaging revealed that fragmentation was present in 94% (795/850) of embryos,and a majority of fragments first appeared at the 2-cell stage (90%, 720/795). At Day3, conventional fragmentation reported that only 77% (653/850) of embryos had frag-mentation, suggesting that time-lapse may be more sensitive in detecting fragments.

Time-lapse assessment also showed that the percentage of fragmentation was persistentto the 4-cell stage for 89% (707/795) of embryos. Without the fragmentation assess-ment, specific ‘in-window’ cell cycle timings (P2, P3) were predictive of embryo im-plantation (implantation 39% vs. 6%, in window vs. out of window, p , 0.0001).The addition of the early fragmentation assessment to ‘in-window’ cell cycle timings(P2, P3) resulted in a further improvement to implantation (46%).Limitations, reason for caution: The dynamic fragmentation assessment wasdone manually and thus subject to inter-observer variances. Automated andmore quantitative fragmentation assessment based on state-of-the-art computervision technologies is currently underway to address this limitation.Wider implications of the findings: Our results supportprevious findings suggest-ing that early fragmentation assessment from 1 to 4-cell stages, together with cellcycle timings P2 and P3, is predictive of embryo developmental competence, includ-ing aneuploidy status. Furthermore, our findings provided two additional insights: 1)time-lapse assessment of fragmentation is more sensitive than traditional Day 3evaluation; 2) adding early fragmentation assessment may further augment cellcycle timing-based embryo selection and improve implantation rates.Study funding/competing interest(s): This work was supported by Auxogyn, Inc.Trial registration number: ClinicalTrials.gov # NCT01369446 and NCT01617993

P-187 Comparisonof clinical results, between standard and uninterrupt-ed embryo culture, when there are no embryos to select between

N. Bowman1, S. Montgomery1, L. Best1, A. Campbell2, S. Duffy1, and S. Fishel2

1CARE Manchester, Embryology, Manchester, United Kingdom, 2CARE Fertility,CARE Fertility Group, Nottingham, United Kingdom

Study question: Is there a benefit of uninterrupted (time-lapse) incubation overstandard incubation for ICSI patients who only have the number of embryos avail-able that they require for transfer?Summary answer: When there is no selection of embryos required, as all createdare transferred and where female age is ≥38 years, there appears to be a significantbenefit of uninterrupted embryo culture compared to standard (interrupted)methods.What is known already: Meseguer et al (2012) reported a significant improve-ment in the relative probability of clinical pregnancy (CP) when the Embry-oScopeTM (ES)(Unisense Fertilitech, Denmark) was used compared to standardincubation methods for ICSI patients. This uplift was reported likely to be dueto a combination of uninterrupted culture and the enhanced embryo selection,made possible by the time-lapse imaging.Study design, size, duration: Retrospective analysis of CP and implantation ratesin a private IVF centre between May 2011 and October 2012. 85 embryos wereincubated in the ES’s uninterrupted conditions and 591 in Galaxy incubators(SI), both 5%O2, 5.5%CO2, 89.5%N2.Participants/materials, setting, methods: All patients undergoing ICSI treat-ment under either incubation condition were included, when they only had thenumber of embryos they required for transfer. IVF patients and oocyte recipientswere excluded. 53 patients underwent ES culture and 391 SI.Main results and the role of chance: There were no significant differencesbetween the CP rates and implantation rates when embryos were cultured in SIand ES culture when patients were aged ≤35 (n ¼ 244 patients SI, n ¼ 19ES).When two embryos were transferred, the CP rate was 27.3% SI vs 33.3% ES(p ¼ 0.7381 not significant (NS)) and the implantation rate, per embryo was16.4% SI vs 20.8% ES (p ¼ 0.5703 NS). For patients aged ≥36 there was atrend towards an increased CP and implantation rate in ES. For patients ≥38,there was a significant increase in implantation rate under ES incubation whentwo embryos were transferred (n ¼ 51 patients SI, n ¼ 16 ES, 3.9% SI vs15.6% ES p ¼ 0.035) and a trend towards an increased CP rate (7.8% SI vs25% ES p ¼ 0.085).Limitations, reason for caution: Embryos were cultured under different incuba-tion conditions at patient request. Therefore, other than age, patient history, has notbeen taken into account. The number of ‘interruptions’ during SI was variablewhen door openings of the 48L incubators and dish removal for developmentalprogress checks are included.Wider implications of the findings: This relatively new technology is not widelyavailable. Clinics commonly run both ES and SI and may prioritise which embryocohorts are placed into ES culture. Our previous data showed overall increased CPrates for patients over 36(Best 2013, ACE) but these findings suggest an

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implantation rate benefit to ES incubation for patients ≥38 even without embryoselection. Therefore, this may encourage units to culture embryos for this patientgroup in ES culture.Study funding/competing interest(s): None.Trial registration number: None.

P-188 The zona pellucida thickness of a human embryo is associatedwith implantation potential in fresh blastocyst transfer but not in day3 transfer

R. Hirata1,2, Y. Aoi1, T. Habara1, and N. Hayashi1

1Okayama Couples Clinic, Gynecology, Okayama, Japan, 2Laboratory ofReproductive Physiology, Graduate School of Environmental and Life Science,Okayama University

Study question: Is the zona pellucida thickness (ZPT) of cleavage embryo asso-ciated with implantation potential in a fresh cycle?Summaryanswer: The day 5 embryos with thickened zona pellucida (ZP) on day3 had a lower implantation rate compared with normal embryos. Assisted hatching(AH) could be beneficial for fresh day 5 transfer. However, the day 3 embryo hadno difference in implantation rate regardless of ZPT.What is known already: The ZPTis considered to be a good indicator for embryopotential. However, several studies have reported that the notion of a relationshipbetween ZPT and implantation has not been supported.Study design, size, duration: This study was a retrospective analysis of implant-ation rate after fresh elective single-embryo transfer cycles. A total of 376 embryoswere included between 2010 and 2012.Participants/materials, setting, methods: The AH procedure was performed onpatients who had ≥2 failed IVF cycles, or were ≥38 years of age, by laser, regard-less of ZPT. The measurement of ZPT was performed by Zilos imaging softwareon day 3 embryo images.Main results and the role of chance: The overall mean of ZPT was 18.44+2.4 mm. In day 3 transfer, ZPT did not correlate with implantation rate whetherAH was performed or not. In day 5 transfer, unhatched embryos whose zonawere thicker than 20 mm on day 3 had a significantly lower implantation rate com-pared with the embryos with a normal ZP of 15-19 mm (30.8% vs. 54.8% respect-ively, P , 0.05). However, in day 5 transfer with AH treatment, no significantdifferences were observed between the thick ZP group and normal ZP group inthe rates of implantation (42.9% vs. 36.7% respectively).Limitations, reason for caution: The characteristics of the two groups; theAH-treated group and non-treated group, are not comparable because the AHwas performed on patients with a poor prognosis; those with ≥2 failed IVFcycles and older women (≥38 years of age).Wider implications of the findings: Our results suggest that the AH procedure isuseful for embryos with a thick ZP in day 5 transfer but not in day 3 transfer.Extended embryo culture may affect the hatching of embryos, especially inembryos that have a thick ZP.Study funding/competing interest(s): This study received no funding and thereare no conflicts of interests to be declared.Trial registration number: Not applicabe.

P-189 A novel biological role of embryo derived oxytocin in earlystages of embryo development in mice

V. Dinopoulou, G.A. Partsinevelos, R. Bletsa, D. Mavrogianni, E. Anagnostou,K. Stefanidis, P. Drakakis, and D. LoutradisAthens University Medical School, Division of Human Reproduction IVF Unit 1stDepartment of Obstetrics and Gynaecology Alexandra Hospital, Athens, Greece

Study question: What is questioned in this study is whether oxytocin levels mea-sured in the culture medium post-fertilization are correlated with the stage ofembryo development (2-cell, 4-cell, morula/blastocyst and blastocyst stage)and whether these levels reflect embryo implantation potential in mice.Summary answer: Oxytocin was secreted at all stages of early embryo develop-ment, although a gradual decrease until morula-early blastocyst stage was found,which was followed byan increase at the blastocyst stage. Thesefindings suggest arole of oxytocin in early embryo development as well as in the implantationprocess in mice.

What is known already: Oxytocin receptor (OTR) is downregulated at the endo-metrial areas surrounding the attaching embryo, but is upregulated at the peri-implantation areas, throughout the later stages of implantation. OTR mRNA isdetected in mouse oocytes and embryos up to the blastocyst stage. The increasein OTR expression immediately after fertilization suggests a possible role of oxy-tocin in this process, whereas the gradual decrease after the 4-cell stage implies apossible role in implantation.Study design, size, duration: This prospective observational animal study wasperformed on a study population consisted of 10 female mice and 10 male mice(C57BL/6 x CBA) F1 hybrids, which were allocated in two sequential experi-ments of 5 female and 5 male mice each.Participants/materials, setting, methods: Female mice, superovulated andpaired with male, were sacrificed and zygotes were flushed from the fallopiantubes. Two-cell embryos were cultured in groups of 20. The culture mediumwas sampled and stored at -20o C at 2-cell, 8-to-16-cell, morula-blastocyst andblastocyst stage for oxytocin determination using an enzyme immunoassay kit.Main results and the role of chance: Baseline oxytocin levels in the mediumused to culture mouse embryos was 0.03+0.000 ng/ml (mean+SEM) onDay 0. On day 2, 56 hours post-hCG injection, at the 4- to 8-cell embryo stage,oxytocin levels were 0.070+0.009 ng/ml (mean+SEM). On day 3, 76 hourspost-hCG injection, at the 8- to 16-cell embryo stage, oxytocin levels were0.068+0.014 ng/ml (mean+SEM). On day 4, 109 hours post-hCG injection,at the morula-blastocyst embryo stage, oxytocin levels were 0.054+0.055 ng/ml (mean+SEM). On day 5, 131 hours post-hCG injection, at the blastocystembryo stage, oxytocin levels were 0.079+0.007 ng/ml (mean+SEM).Limitations, reason for caution: A possible limitation of our study is the use of arestricted number (20) of mouse embryos for 5-day culture. We believe that agreater difference in oxytocin levels would have probably been seen betweenstages of embryo development in case a higher number of mouse embryos hadbeen utilized.Wider implications of the findings: Taking into account that oxytocin mighthave some biological role at the early stages of development of fertilizedoocytes and the fact that embryos and their secreted oxytocin may exert localeffects, we could implement measurement of oxytocin levels in the culturemedium as a biological marker in the selection of the best embryos, in terms ofimplantation potential, for transfer in IVF protocols.Study funding/competing interest(s): No funding supported this study and nocompeting interest is declared.Trial registration number: The study was registered to Local University Hos-pital Ethics Committee (registration number: 15/26-01-2009).

P-190 Incidence of live birth using hyaluronic acid (HA) for spermselection

J. Hernandez1, C. Lorenzo Leon1, M. Puopolo2, and A. Palumbo1

1Centro de Asistencia a la Reproduccion Humana de Canarias, La Laguna,Spain, 2Istituto Superiore di Sanita, Department of Cell Biology andNeurosciences, Rome, Italy

Study question: PICSI has been proposed as a sperm selection technique capableof improving pregnancy rates in coupleswith male factor infertility. Since 2009 wehave introduced this technique in our IVF laboratory for selected couples to test itseffect on live birth rates.Summary answer: Our data suggest that use of hyaluronic acid for sperm selec-tion by PICSI may have a beneficial effect in patients with male factor by reducingthe miscarriage rate and improving the live birth rate.What is known already: PICSI is a technique of sperm selection based on theability of mature sperm to bind to hyaluronic acid in vitro. Although somereports suggest a beneficial effect of PISCI in male factor cases, current literatureis controversial.Study design, size, duration: This is a retrospective observational study on 44patients recruited from 11/2009 to 12/2012 who underwent a treatment cycleusing HA for sperm selection (PICSI). We selected an historical control grouprecruited from 1/2008 to 10/2009 composed of 104 ICSI cycles in couples withsperm parameters comparable to the study population.Participants/materials, setting, methods: Patients in the study group had asperm count in the range 5.000.000-20.000.000/ml. Variables analyzed includedpatients and partners age, fertilization rate, viable, transferred and top quality

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embryos, clinical pregnancy and miscarriage rate. Statistical analysis was per-formed using t-test for continuous variables and Fisher’s exact probability testfor categorical variables.

[Maria1]era nei parametri fissati per la selezione dei controlli?Main results and the role of chance: The study group and the control group didnot differ in terms of mean patients age and partners’ age (35.8+1 vs 36.4+0.4and 40.76+0.9 vs 39.1+0.6 respectively); fertilization rate (64.4% vs 65.9%),and pregnancy rate [50% (22/44) vs 40% (42/104)]. There was a trend towards alower miscarriage rate in the study group [9% (2/22)] vs 21% (9/42) in controls,however this difference did not reach statistical significance, which may be dueto the small sample analyzed.Limitations, reason for caution: The main limitation is the retrospective designand the small number of cases in the PICSI group. A further weakness is the use ofan historical control group which might be biased due to improvements in clinicalpractice, however no major changes occurred in our center during this time frame.Wider implications of the findings: The possibility that the PICSI techniquereduces the miscarriage rate increasing live birth rates is attractive and further pro-spective randomized studies are warranted.Study funding/competing interest(s): NoneTrial registration number: Not applicable

P-191 Impact of seminal trace elements and glutathione levels on spermDNA fragmentation and assisted reproductive techniques outcome

F. Atig1, A. Kerkeni2, A. Saad3, and M. Ajina3

1University Farhat Hached Hospital, Unit of reproductive medicine, Soussa,Tunisia, 2University of Monastir, Research Laboratory of “Trace elements freeradicals and antioxidants” Biophysical Department Faculty of Medicine,Monastir, Tunisia, 3University Farhat Hached Hospital, Unit of reproductivemedicine, Monastir, Tunisia

Study question: Can seminal non-enzymatic antioxidants and sperm DNA frag-mentation help to predict the in vitro fertilization (IVF) outcome of Tunisian infer-tile couples?Summary answer: Standard semen parameters were poor predictors of the IVFoutcome. Besides; sperm genome quality, seminal trace elements “Zinc and Sel-enium” and glutathione levels were strongly inter-related and demonstrated sig-nificant and close relationships with the in vitro early embryogenesis, thus withthe success of IVF attempt.What is known already: Valuable evidence has emerged about the crucial role ofseminal trace elements and glutathione as non-enzymatic antioxidants that protectspermatozoa against lipid peroxidation and oxidativedamages.Nevertheless, con-trary to the seminal reactive oxygen species, there is no clear consensus about theinvolvement of these antioxidants to protect sperm from oxidative DNA fragmen-tation and to predict the success of the early embryonic development after IVF.Study design, size, duration: This is a prospective controlled study carried out onTunisian infertile men participating in the conventional IVF program at our Unit ofReproductive Biology, Farhat Hached University Hospital (Tunisia). A total of200 consecutive males (24-50 years) were recruited in our investigationbetween June 2011 and July 2012.Participants/materials, setting, methods: Obtained semen samples were ana-lyzed according to WHO criteria (1999) and divided into four groups: nomozoos-permics (controls, n ¼ 70), oligozoospermics (n ¼ 40), asthenozoospemics (n ¼45) and teratozoospermics (n ¼ 45). Seminal zinc and selenium concentrationswere determined using flame and furnace atomic absorption. The differentforms of glutathione andsperm DNA fragmentation levels weremeasured spectro-photometrically and by TUNEL assay, respectively. IVF outcome was presentedas follows: fertilization, cleavage and embryo quality (Grade I, II and III).Main results and the role of chance: The key results in this study were: (1)increased seminal trace element amounts were observed in normozoospermicswhen compared with abnormal groups mainly with Asthenozoodpermics and Ter-atozoospermics. Total and reduced forms of glutathione (GSHt and GSHr) werealso significantly elevated in seminal plasma of controls than asthenozoospermics(P ¼ 0.002, P ¼ 0.003; respectively). (2) Controls established highly significantdecline of sperm DNA fragmentation levels compared to the three abnormalgroups (P , 0.001). (3) Negative correlations were found between enhancedseminal antioxidant profile and sperm DNA fragmentation (zinc [r ¼ -0.71**,

P , 0.001], selenium [r ¼ 0.63**, P , 0.001], GSHt [r ¼ -0.21*, P ¼ 0.01],GSHr [r ¼ -0.72**, P , 0.001]). (4) Inversely to the non-enzymatic antioxidants,sperm DNA fragmentation was strongly correlated to poor fertilization rate (r ¼-0.53*, P ¼ 0.02), cleavage (r ¼ -0.71**, P , 0.001) rate and embryo quality(Grade III) (r ¼ 0.62*, P ¼ 0.01).Limitations, reason for caution: The results may be biased by the determinationof sperm DNA fragmentation on the same day as the IVF procedure. It would beinteresting to measure the sperm DNA fragmentation and antioxidant levels beforeIVF, in order to predict their role in the procedure.Wider implications of the findings: We identified that the routine determinationof non-enzymatic antioxidants and sperm DNA fragmentation levels is a more re-liable prognostic tool for male infertility assessment which can help selection ofsemen samples with the least amount of sperm DNA fragmentation and thusreduce the risks associated with the use of DNA-fragmented sperm for fertilizationand avoid financial, social and emotional problems associated with failed IVFattempts.Study funding/competing interest(s): None of the authors have any competinginterest. This work is part of a scientific project (Thesis).Trial registration number: None.

P-192 Matured human oocytes with cumulus cells and assisted reproduc-tion techniques

G. D’Ommar, A.K. Herrera, L. Lozano, and M. MajerfeldCentro Medico Docente La Trinidad, Fertility Clinic, Caracas, Venezuela

Study question: Determine effects in fertilization rate and quality of embryosobtained from oocytes remnants of assisted reproduction techniques (ART),matured with or without their cumulus cells (CC).Summary answer: Mature oocytes with CC, equates the fertilization rate andembryo quality to levels similar to those obtained with mature oocytes collected.What is known already: The oocyte plays an important role regulating its owndevelopment, by the production of paracrine growth factors that affect the sur-rounding granulosa cells (Gilchrist et al., 2008). These cells represent an essentialcomponent in the maturation and fertilization of oocytes. Because oocytesundergo intracytoplasmic sperm injection (ICSI) are denuded, the potential posi-tive effects of cumulus cells in future development cannot be observed (Ebneret al., 2006).Study design, size, duration: Retrospective analysis of the effect of CC on com-pletion of oocyte maturation. Population: 124 patients (n ¼ 1239 oocytes), age:33.0 + 3.9 years, performing ART and ICSI, is compared fertilization andembryo quality.Participants/materials, setting, methods: Oocytes classification:Denuded: Appeared to be mature, were immature when denuded. (n¼ 198)Not denuded: Appeared to be immature, were not denuded. (n¼ 360)Control group: Weremature by crown classification, resulting MII when denuded(n ¼ 681).

Results were analyzed by the chi-square test.Main results and the role of chance: In collected mature oocyte, fertilization ratewas 69.6 %; in Denuded group 35.2 %; in Not denuded group 68.3 %. With valuesof P¼ 0.000001 between mature oocytes and Denuded group, P¼ 0.000002between Denuded group and Not denuded group, but with P¼ 0.8426 betweenmature oocytes and Not denuded group.

In relation to embryos quality, in mature oocytes 43.7 % corresponds to goodquality embryos; 24 % of good quality embryos in Denuded group; in Notdenuded group 43.3 %. With values of P¼ 0.003271 between mature oocytesand Denuded group, P¼ 0.003874 between Denuded group and Not denudedgroup, but with P¼ 0.9586 between mature oocytes and Not denuded group.For values ??of P ,0.05 there is no association between the variables.Limitations, reason for caution: The main limitation in this study because it ishuman oocytes was obtaining informed consent, which was reflected in thesample size.Wider implications of the findings: Was observed that coculture with cumuluscells promotes maturation and meiotic progression, which is reflected in thehigh rates of fertilization in vitro matured oocyteswith cumuluscells, which is con-sistent with other studies.Study funding/competing interest(s): Clınica de Fertilidad, Centro MedicoDocente La Trinidad, Caracas, Venezuela.

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Trial registration number: No

P-193 Direct unequal cleavages: Cell stage onsets, embryo developmen-tal potential and chromosomal abnormalities

Z. Ye, N. Zaninovic, R. Clarke, R. Bodine, and Z. RosenwaksCenter for Reproductive Medicine, Reproductive Medicine, New York, U.S.A

Study question: To investigate cell stage occurrence, developmental potential,and chromosomal abnormalities in embryos displaying direct unequal cleavages(DUC).Summary answer: When identified correctly, direct unequal cleavages at earlycell stages have a greater negative impact on subsequent embryo developmentthan later stage onsets, and are correlated with a high incidence of chromosomalabnormalities, but not triploidy.What is known already: The occurrence of DUC (1 to 3 cells) has been reportedin many IVF labs, a phenomenon unrelated to specific patient population, culturemedia or fertilization technique. A recent multicenter study showed limited im-plantation potential of embryos with direct unequal cleavages at first mitosis. Trip-loidy has been suggested as a cause; however developmental potential andchromosomal composition of these embryos in relation to the cell stage onsetwas not reported.Study design, size, duration: Fertilized oocytes (n ¼ 5604 from 408 patients)were cultured in time-lapse EmbryoScope incubators (Unisense Fertilitech) andannotated for patterns and times of cleavage. PGS analysis was performed onthe subset of patients (n ¼ 26, avg. age 37.7) that demonstrated DUC.Participants/materials, setting, methods: Direct unequal cleavages weredefined as cleavage of one cell directly to three or more cells which were annotatedduring the first, second or third cleavage division. PGS analysis (biopsy day 3 or 5)was performed on 26 patients exhibiting DUC using FISH, CGH or SNP array.Main results and the role of chance: DUC were observed in 12% of the embryos:58% at 1st cleavage, 25% at 2nd cleavage and 6.7% at 3rd cleavage division. DUCembryos were developmentally compromised, and only 9% reached blastocyststage and were frozen. The developmental potential of DUC embryos was asso-ciated with cell stage onset, where DUC occurrence at first cleavage is more crit-ical to development than DUC at second or third division. 255 PGS embryos wereanalyzed; 74 (29%) annotated as DUC. Chromosome abnormalities were detectedin 89% of DUC embryos, which included different patterns of chromosomal aber-rations (but not triploidy), and were not of specific maternal or paternal origin. Thedetailed analysis of PGS normal embryos, originally annotated as DUC, revealedambiguous cell or fragment identification, and/or cell fusion.Limitations, reason for caution: N/AWider implications of the findings: When identified correctly with time-lapsemicroscopy, DUC at various cleavage stages can be a useful tool for selectingembryos for transfer. Embryos demonstrating DUC have limited developmentalpotential and are associated with high level of chromosomal abnormalities; there-fore they should be avoided for ET or freezing.Study funding/competing interest(s): InstitutionalTrial registration number: N/A

P-194 Can timing of post-cleavage embryo development stages revealploidy or implantation potential?

P. Mazur1, V. Nagorny1, D. Mykytenko2, L. Semeniuk1, and V. Zukin1

1Clinic Of Reproductive Medicine "Nadiya", Embryology, Kyiv, Ukraine, 2ClinicOf Reproductive Medicine "Nadiya", Department of Molecular Diagnostics,Kyiv, Ukraine

Study question: Can detailed time-lapse observation of embryo post-cleavagedevelopment stages suggest embryo ploidy or its enhanced implantation?Summary answer: Although ploidy is not related with timing of compaction,cavitation and expansion, thorough analysis of blastocyst expansion may helpin assessment of embryo implantation potential.What is known already: Limited data connects timing of embryo developmentwith its ploidy. Human embryo genome activation is known to start at day 3 of cul-tivation in-vitro, so analysis of late stages of in-vitro culture seems promising.Study design, size, duration: This cross-sectional study includes 84 patientswhose embryos were cultured in time-lapse imaging system. Of those, 106

blastocysts of 60 patients were transferred in fresh cycles and have known preg-nancy outcome. Second group consisted of 24 infertility treatment patientswhere embryo ploidy was established by array comparative genome hybridization(aCGH).Participants/materials, setting, methods: Embryos were cultured in Embry-oScopeTM (Unisense FertiliTech, Denmark). After 5 or 6 days of embryoculture, time-lapse image data were analysed. Array CGH, if applied, was per-formed after trophectoderm biopsy, using 24surew kit (BlueGnome, UnitedKingdom). In aCGH group of 113 analysed embryos 58 were euploid and 56were aneuploid.Main results and the role of chance: Using time-lapse observation, developmenttime points like morula formation, cavity appearance and expansion time weredefined. Efficient expansion was presumed when expanding blastocyst startedto stretch zona pellucida (ZP). To locate this moment, inner ZP diameter was cal-culated from image data with 1h interval and subsequent linear regression analysisperformed. In aCGH group assisted hatching performed at day 3 of culture causedexpansion time to match with moment of trophectoderm protrusion trough ZPopening.

No correlation was seen between timing of late in-vitro culture events whenembryos were compared by ploidy or implantation fate. However graphic inter-pretation of blastocyst volume expansion speed in group of embryos withknown implantation showed that implanted embryos usually had uniform mid-speed expansion without deep collapsing or oversized growth.Limitations, reason for caution: Comparison of timing of late in-vitro develop-ment events is complicated by lags often observed in pre compaction and morulastages. It is possible that in future investigations these difficulties will be over-comed.Wider implications of the findings: To the date, assuming that each embryo isunique, we cannot recommend to select embryos for transfer relying on theirtiming of post-cleavage stages. Blastulation dynamics, after development oflabour-saving software, can be applied for embryo scoring.Study funding/competing interest(s): The authors have nothing to disclose. Noexternal funding was received for this work.Trial registration number: This study was not an RCT.

P-195 Prospective trial of vitrification of all embryos in cycles of ART:implantation and pregnancy rate

A. Zabala1, T. Pessino2, S. Outeda2, L. Blanco2, F. Leocata2, and R. Asch3

1Hospital Interzonal de Agudos Jose de San Martin, Ginecology, La Plata,Argentina, 2PROCREARTE, Ginecology, C.A.B.A, Argentina, 3Ministerio deSalud de la Provincia de Buenos Aires, Ginecology, La Plata, Argentina

Study question: To determine the pregnancy outcome after vitrification of allfresh embryos produced in stimulated assisted reproduction technique cycles(ART) and replacing them in subseuqemt non-gonadotropin stimulated cycleSummary answer: Vitrification of all fresh embryos produced in stimulatedART cycles and replacing them in subsequent non-gonadotropin stimulatedcycles resulted in highly successful implantation rate and cumulative pregnancyrates.What is known already: It has bee propoced that supraphysiological hormonelevels during ovarian stimulation may adversely affect embryo implantation inassisted reproductive treatments. Very few studies have advocated the electivecryopreservation of all embryos as a methos to avert the undesirable effect of go-nadotropin ovarian stimulation on implantation and pregnancy rate, as well as toprevent the occurrence of iatrogenic events such as ovarian hyperstimulation syn-drome (OHSS).Study design, size, duration: A prospective trial of series of cases in a private fer-tility center-ART program. 122 patients (average age 35 years) with risk todevelop OHSS or with uterin factor (poor endometrium or difficult transfer)who underment vitrificaion of all fresh embryos from Junuary 2011 to December2012Participants/materials, setting, methods: 1000 embryos were vitrified at 8 blas-tomeres or blastocyst stage. Embryo survival rate (at last 80% of blastomeres intactpost-thawing) was 97%. After thawing, 527 embryos were transferred intohormone replacement cycles in a total of 215 cycles. the average number ofembryos transferred per cycle was 2,4.

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Main results and the role of chance: 103 pregnancies were achieved for a cumu-lative clinical Pregnancy Rate (CCPR) of 84% per patient and 48% per cycle.These results are higher than fresh embryos transfer cycles for the center andstudy period (48% vs 31%). Implantation Rate (IR) was 22%. Pregnancies wereachieved 57% in the first, 29% in the second, 11% in the third and 3% in thefourth cycle of thawing per patient, respectively. Of those patients that did notachieved successful clinical pregnancies, 57 % sill have embryos vitrified (4,5embryos/ patient).Limitations, reason for caution: Descriptive measure of prospective trial.Wider implications of the findings: These results reassure the role of embryo vit-rification in an IVF program, and could also be a possible approach to prevent thealleged adverse effects of ovarian hyperstimulacion on the implantation process,and it is tempting to propose its use routinely in ART cycles in the future.Study funding/competing interest(s): noTrial registration number: no

P-196 Early cleaving mouse embryos are better candidates forvitrification

W.J. Wan-Hafizah1, M.H. Rajikin2, A.S. Nuraliza2, M. Mohd-Fazirul3,J.M.Y. Norhazlin3, D. Razif4, and M.N.K. Nor-Ashikin3

1Faculty of Medicine, Universiti Kuala Lumpur-Royal College of Medicine Perak,Ipoh, Malaysia, 2Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh,Malaysia, 3Institute of Medical Molecular Biotechnology Faculty of Medicine,Universiti Teknologi MARA, Sungai Buloh, Malaysia, 4Faculty of Health SciencesPuncak Alam, Universiti Teknologi MARA, Sungai Buloh, Malaysia

Study question: Do early cleaving (EC) embryos make better candidates forvitrification compared to late-cleaving (LC) embryos?Summary answer: EC embryos make better vitrification candidates compared toLC embryos, because they exhibit significantly higher post-vitrification survivalrate and viability.What is known already: The use of EC embryos has been suggested for embryotransfer in Assisted Reproductive Technology (ART) because of theirgood qualityand high viability. However, no study has been conducted to investigate whetherthese EC embryos also make better vitrification candidatesStudy design, size, duration: In a prospective study, post vitrification survivabil-ity and viability were compared between EC embryos and LC embryos. A total of124 EC embryos and 156 LC embryos were vitrified in liquid nitrogen for 1 hour.Participants/materials, setting, methods: Embryos were retrieved from in vivo-fertilized ICR mice, 28 hours after human Chorionic Gonadotrophin injection.Two-cell embryos were categorized as EC embryos and two-pronuclear zygotes asLC embryos. Vitrification using EFS20/40 method was performed at 2-cell stage.Post-vitrificationviabilityofECandLCembryos was comparedusingchi-square test.Main results and the role of chance: The survival rate was significantly higher invitrified EC embryos (96.8%) compared to vitrified LC embryos (89.2%) (p ,

0.05). A significantly higher proportions of vitrified EC embryos developed tothe blastocyst stage, compared with vitrified LC embryos (90% versus 57.1%,p , 0.0001). This may be related to the ability of early cleaving embryos tosurvive the high cooling rates, high osmotic changes and toxicity of cryoprotectantduring the vitrification process.Limitations, reason for caution: The use of in vivo- instead of in vitro-derivedembryos may have resulted in greater cryotolerance, as in vivo-derived embryosexhibit reduced sensitivity to chilling and freezing due to their lower lipid toprotein ratio compared with in vitro-derived embryos.Wider implications of the findings: The selection of early cleaving embryos asvitrification candidates could provide better cryopreservation outcomes. Thisfinding can subsequently contribute towards the improvement of ART outcomesand reduce the cost related to procedures in ART.Study funding/competing interest(s): This research was supported by institu-tional grant [600-RMI/ST/DANA5/3/Dst(335/2011)] and national grant[600-RMI/ST/FRGS5/3/FST(71/2010)]. There were no competing interests.Trial registration number: Not applicable

P-197 A novel method for human oocyte vitrification with a closed deviceusing super-cooled air

S. Machac1, V. Hubinka2, M. Larman3, and M. Koudelka1

1Reprofit International s.r.o., Gyneacology, Brno, Czech Republic, 2ReprofitInternational s.r.o., Embryology, Brno, Czech Republic, 3Vitrolife, ResearchDepartment, Englewood, U.S.A

Abstract withdrawn by the author

P-198 Is embryo quality affected by different approaches to stimulationin IVF?

T. Pehlivan Budak1, O. Oliana Membrado1, E. Sevillano Martinez1, P. Wilson1,A. McClure1, and G. Nargund2

1Create Health Clinics, IVF Laboratory, London, United Kingdom, 2CreateHealth Clinics, Obstetrics and Gynecology, London, United Kingdom

Study question: Does stimulation protocol affect embryo quality on day 2 in dif-ferent age groups?Summary answer: In patients ,40 years and ≥40 years of age, higher percent-age of Grade A and B embryos were obtained from patients submitted to naturalcycle IVF compared to stimulated cycles. The differences were statistically signifi-cant in the group ≥40 years of age.What is known already: In previous studies, natural cycle approaches to stimu-lation in IVF have been associated with better embryo quality and IVF outcome.Studydesign, size, duration:This retrospective observational data included3044embryos assessed on day 2 of embryo development during 2011 and 2012.Embryos scored based on ASEBIR criteria were compared in patients ,40years and ≥40 years of age whom underwent following treatment protocols:Natural cycles(NC), Modified Natural cycles(MN) and stimulated cycles (SC).Participants/materials, setting, methods: Three main groups were made: NC,MN and SC (Group 1, 2 and 3 respectively).

Group 1a(,40 years, 73 embryos) and Group 1b(≥40 years, 104 embryos).Group 2a(,40 years, 167 embryos) and Group 2b(≥40 years, 392 embryos).Group 3a(,40 years, 1644 embryos ) and Group 3b (≥40 years, 743 embryos).

Main results and the role of chance: In Group 1a, 65.8% were grade A and B,34.2% were grade C and D.

In Group 1b, 73.1% were grade A and B, 26.9% were grade C and D.In Group 2a, 64.1% were grade A and B, 35.9 % were grade C and D.In Group 2b, 65% were grade A and B, 35% were grade C and D.In Group 3a, 58.9% were grade A and B, 41.1% were grade C and D.In Group 3b, 62.2% were grade A and B, 37.8 were grade C and D.In general, independent of age, there was a tendency for better embryo quality in

NC, and MN compared to SC.The difference was statistically significant between groups 1b and 3b (p¼ 0.04).

Limitations, reason for caution: Due to the study design, selection bias and con-founding factors may have affected the results.Wider implications of the findings: In women undergoing IVF, natural cycle ap-proach to IVF seems to yield better quality embryos on Day 2 compared to stimulatedcycles. This finding is more significant in women with advanced maternal age .Study funding/competing interest(s): NoneTrial registration number: None

P-199 Accumulation of vitrified oocyte: a successful strategy to achievean elective embryo transfer in young low responders patients

D. Raso1, M.F. Insua2, B. Lotti1, S. Giordana2, C. Baldi2, J. Barattini1, M. Cogorno1, N. Fernandez Peri1, and F. Neuspiller1

1Instituto Valenciano de Infertilidad, Ginecology, C.A.B.A., Argentina, 2InstitutoValenciano de Infertilidad, FIV Laboratory, C.A.B.A., Argentina

Study question: To describe the results of vitrified oocytes accumulation (VOA)in low responder (LR) patients as a way of achieving an elective embryo transfer.Summary answer: The pregnancy rate in low responder patients is usually sub-optimal due to the low number of embryos. Vitrification techniques allow oocytesto accumulate several cycles of ovarian stimulation, increasing the possibilities ofelective embryo transfer.What is known already: The low response represents a difficulty to the manage-ment within women who must undergo assisted reproductive treatment. Reduced

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number of oocytes obtained in these patients is usually associated to poor eggquality, low number of embryos and high rate of cancellation of their cycles.

The pregnancy rate in this group of patients is usually suboptimal due to the in-ability to perform an elective embryo transfer.Study design, size, duration: We carried out an observational, retrospective,comparative and descriptive study. During the period from January 1st 2011 toJuly 31st 2012, 705 assisted reproduction cycles were performed, 20% (140)out of which behaved as low responders. LR was defined as those patients whoobtained ≤4 oocytes after puncture.Participants/materials, setting, methods: VOA was offered to 87 patients (22accepted). Patients were divided in two groups: ≤39 years and ≥40 years.Each group was also divided in those that did not accumulate oocytes (A), andthose who did (B).

Stimulation was performed with clomiphene citrate and hMG. Oocytes werecryopreserved (Cryotop method).Main results and the role of chance: Average age was 36.4 [31-43]. Averagecycles/ patient were 2.2+0.4. Main retrieved oocytes/aspiration were 4.1+2.1; average MII was 2.9+1.3; main retrieved oocytes/patient were 9.1+4.3and main retrieved MII 6.4+2.1. Oocytes survival rate was 81.8%. Global fertil-ization rate (FR) was 62.1%. Pregnancy rate (PR)/transfer was 34.7% and PR/patient was 36.3%.

Women ,40 years of GroupB exhibit significantly better results than theirpeers in GroupA. Elective embryo transfer was 50% vs. 25%, PR 33.3% vs15% and the implantation rate (IR) 25% vs 8.3%, respectively. The main agewas 37.2+1.74 vs 35.81+2.46.

In patients ≥40 years, results for GroupA and GroupB elective embryo transferwere 28.6% vs 23.5%, PR 14.3% vs 18.8%, IR 14.3% vs 11.4%. The main age forthese sub groups was 41.9+1.5 vs 41.9+1.4.Limitations, reason for caution: NoneWider implications of the findings: Oocyte vitrification is a useful technique toaccumulate oocytes of different stimulation cycles, increasing the initial pool.According to our results, in women ,40 years, this procedure significantlyimproves the possibility of an elective transfer, impacting positively on pregnancyrates. For ≥40 years, this strategy would not provide significant benefits whencompared with fresh cycles. This could be due to oocyte ageing, which in turnimpacts negatively on reproductive outcomes.Study funding/competing interest(s): NoneTrial registration number: None

P-200 Cryopreserved versus fresh blastocyst transfer: a way to improveimplantation rate

S. Resta, A. Filannino, E. Maggi, G. Cafueri, A.P. Ferraretti, M.C. Magli, andL. GianaroliS.I.S.M.E.R. s.r.l., Reproductive Medicine Unit, Bologna, Italy

Study question: Do blastocysts belonging to the same cohort have differentchances of implantation when transferred in fresh or a frozen cycles?Summary answer: Blastocysts transferred in frozen cycles seem to have higher im-plantation rates compared to the sibling blastocysts transferred in the fresh cycle.What is known already: Traditionally, the tendency in ART is to give priority tothe fresh cycle by selecting the best quality embryos for transfer. Several reportssupport the culture to the blastocyst stage with the aim of improving the selectioncriteria.

More recently, in view of the high efficiency of vitrification, the advantages oftransferring warmed blastocysts have been underlined. Actually, the rates of preg-nancyand implantationseem to be increasedcompared with fresh blastocyst trans-fer cycles.Study design, size, duration: Between 2008 and 2012, 437 blastocysts transferwere performed (309 fresh, 128 frozen). A subset of 36 patients who did notachieve term pregnancy in fresh cycles and had spare cryopreserved blastocystswere included in the study for a total of 36 fresh transfers and 38 cryopreservedtransfers.Participants/materials, setting, methods: Private center. Blastocyst develop-ment and quality, and implantation were evaluated in 36 patients that transferredat the blastocyst stage in the fresh cycle and cryopreserved one or more spare blas-tocysts for further attempts. The studied parameters were compared between freshand cryopreserved cycles.

Main results and the role of chance: In all, 136 blastocysts were obtained (73%blastocyst rate over 185 fertilized oocytes). As priority was given to fresh trans-fers, 35 patients (97%) transferred their best quality blastocysts (all day-5 blasto-cysts) in the fresh cycle (1.7+0.5 mean embryos transferred) with a pregnancyrate of 14% (5/36) and an implantation rate of 6% (4/62).

After the transfer of the cryopreserved blastocysts in subsequent 38 cycles(1.3+0.4 mean embryos transferred), the pregnancy rate was 40% per transfer(15/38, P , 0.05) and 42% per patient (15/36, P , 0.025). The implantationrate of 31% (15/48) was significantly higher when compared to fresh cycles(P , 0.005). In the group of frozen cycles, 29 transferred day-5 blastocysts and9 day-6 blastocysts with implantation rates of 29.7% and 36.4% respectively.Limitations, reason for caution: An extremely efficient cryopreservationprogram is necessary to minimize the possible damage to blastocysts during theprocedure. The results reported here regard a special group of patients that didnot achieve a term pregnancy in the fresh cycle, and should be verified on alarger set of data.Wider implications of the findings: The high implantation rate after the transferof spare blastocysts in frozen cycles suggests an improved endometrial receptivity,especially when considering that the best quality blastocysts were selected forfresh transfers. Transfers in frozen cycles have the additional advantages of 1) re-ducing the risk of OHSS, 2) increasing the cumulative pregnancy rate due to thehigher implantation, and 3) providing the better obstetric and perinatal outcomethat are associated with the transfer of thawed embryos.Study funding/competing interest(s): None.Trial registration number: Not applicable.

P-201 Vitrification of cleavage and morula stage mouse embryos withDMSO/ EG and PROH/ EG: effects on blastomere viability, cytoskeletonand development

A. Sioga1, Z. Oikonomou1, K. Chatzimeletiou2, L. Oikonomou1, E. Kolibianakis2,and B.C. Tarlatzis2

1Aristotle University Medical School, Lab. of Histology - Embryology,Thessaloniki, Greece, 2Aristotle University Medical School, 1st Department ofObstetrics and Gynaecology Papageorgiou Hospital, Thessaloniki, Greece

Study question: Is there a difference in blastomere viability, cytoskeletal abnor-malities and development of cleavage and morula stage mouse embryos vitrifiedwith a DMSO/EG kit compared with those vitrified with a PROH/EG kit and freshembryos?Summary answer: There was no significant difference in blastomere viability,cytoskeletal abnormalities and development between the DMSO/EG and thePROH/EG groups but fresh embryos showed a lower incidence of spindle abnor-malities compared to the vitrified groups.What is known already: Morula and blastocyst stage vitrification has successful-ly been applied in human and mouse embryos. Most studies assess feasibility ofthe methodology based on survival rates and development and only limitedstudies in human blastocysts report on the incidence of cytoskeletal abnormalities.Here, we investigate in the mouse immediate effects on blastomere viability fol-lowing staining with viability markers and any potential effects on the cytoskel-eton, by analysing spindle and chromosome configurations by confocalscanning microscopy.Study design, size, duration: This study was performed between January2011-January 2013 in an Embryology Laboratory. 280 embryos were flushedout of BalbC female mice oviducts. 215 were vitrified with DMSO/EG (n¼110) and PROH/EG (n¼ 105) and 65 were used fresh for comparison of Immedi-ate effects (n ¼ 15(4-6cell),n ¼ 15(8-cell),n ¼ 15morulae) and cytoskeleta ana-lysis, n ¼ 20 blastocysts).Participants/materials, setting, methods: 50 DMSO/EG (n ¼ 15(4-6 cell), n ¼18(8-cell), n ¼ 17morulae) and 45 PROH/EG (n ¼ 15(4-6 cell), n ¼ 15(8-cell),n ¼ 15morulae) vitrified embryos were subjected to viability CFSE/ PI staining.60 DMSO/EG (n ¼ 20(4-6 cell), n ¼ 20(8-cell), n ¼ 20morulae) and 60 PROH/EG (n ¼ 20(4-6 cell), n ¼ 20(8-cell), and n ¼ 20morulae) vitrified embryos werecultured to the blastocyst stage and stained with a-tubulin/DAPI.Main results and the role of chance: This is the first study to examine in detailusing viability markers the immediate effects of DMSO/EG and PROH/EG vitri-fication on blastomere survival and compares the type and incidence of cytoskel-etal abnormalities in vitrified cleavage and morula stage embryos with fresh

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embryos. There was no significant difference in the survival rates followingwarming between the two vitrified groups at all stages (p . 0.05). Cytoskeletalanalysis revealed that fresh embryos that had developed to the blastocyst stagehad a significantly higher incidence of normal spindles (72.2%) and lower inci-dence of abnormally shaped (22.2%) or multipolar spindles (2.8%) compared toboth DMSO/EG and PROH/EG vitrified embryos (p,0.05). The level of abnor-mal spindle/chromosome configurations including abnormal shape, and multipo-larity, was similar between the two vitrified groups (p.0.05).Limitations, reason for caution: Mouse embryos may differ from humanembryos in the way they survive post-warming.Wider implications of the findings: The results presented in this study documenthigh survival rates post warming and no major immediate effects following stain-ing with viabilitymarkers. This suggests that vitrification with both the DMSO/EGand PROH/EG kits at cleavage and morula stages is a safe procedure that can beapplied in routine clinical IVF practice.Study funding/competing interest(s): No external funding was raised for this study.Trial registration number: Not applicable

P-202 Impact of oxygen concentration on early human embryodevelopment in vitro

M. Roychoudhury Sarkar1, D. Ray1, and J. Bhattacharya2

1A.H IVF & Infertility Research Centre Pvt Ltd, Embryology, Kolkata, India, 2A.HIVF & Infertility Research Centre Pvt Ltd, IVF Specialist, Kolkata, India

Study question: How do the oxygen concentration affect early human embryo de-velopment in - vitro from fertilized oocytes to blastocyst?Summary answer: Result in this study demonstrated that embryo culture inhigher oxygen concentration reduce the human embryo developmental ratein vitro.What is known already: Early embryo development in-vitro is not only dependenton the culture media but also upon the physical environment of incubator such as con-centration of CO2 and O2 gas.Studies on mammalian embryo culture have demon-strated that culture at reduced O2 results in better embryo development. However,it has also been observed that embryo can also grow in elevated O2 level, this hasled to some confusion regarding the role of oxygen in human embryo culture.Study design, size, duration: Retrospective study. Embryos cultured in 3 differ-ent groups:- Group-I: Fertilisation upto blastocyst in 20% Oxygen, Group-II:Fertilisation(day O) in 20% Oxygen, followed by (day 1 to day 5)of embryoculture in 5% Oxygen and Group-III: Fertilisation upto blastocyst in 5%Oxygen. Only (PN2) oocytes were scored from (day 1-5).Participants/materials, setting, methods: Patients selected with a criteria of ≥35 years of age, devoid of endometriosis and PID and ≥ 8 oocytes after IVF / ICSI.Group-I: 60 patients with 421 normally fertilized oocytes. Group-II: 40 patientswith 289 normally fertilized oocytes and Group-III: 45 patients with 311 normal-ly fertilized oocytes.Main results and the role of chance: Result in this study shows that proportion ofdevelopment of 2-celled, 4-celled embryo are almost similar in the three groupsand from this stage, embryo development is significantly reduced in group-I com-pared to that of group-II and group-III. Therefore, percentages of compact embryoand blastocyst development in the 3 groups are as follows:Group-I: 45.1% and 39.9% Group-II: 63.4% and 57.09% and Group-III:65.8% and 59.16% respectively.We observed that there was no significant differ-ences in any parameters in group-II and group-III.Limitations, reason for caution: Care was taken to ensure that other parameterssuch as culture time and CO2 (6%) were kept constant.Wider implications of the findings: Significant dip in the growth rate is observedafter day-3. The delay in the late cleavage stage is probably due to maturation arrestof embryos cultured at 20% O2 before reaching the blastocyst stage.Study funding/competing interest(s): NATrial registration number: NA

P-203 Randomised assisted localised hatching in cryopreservedblastocyst transfers

J. Marcos Alises1, D. Gumbao1, A. Sanchez-Leon1, B. Amorocho1, M. Molla1,M. Nicolas2, L. Fernandez2, and J. Landeras2

1IVI Murcia, Infertility, Murcia, Spain, 2IVI Murcia, Gynecologist, Murcia, Spain

Study question: The aim of this work was to evaluate if there was any differencewhen the assisted hatching (AH) was carried out at the level of the inner cell mass(ICM) or the trophoectoderm (TRP)Summary answer: The results appear to point towards a favourable trend in ap-plyingassisted hatching techniques at the level of the ICM andmayexplain the roleplayed by the ICM in the embryo hatching processWhat is known already: In order for implantation to take place, the blastocystmust be hatched from the zona pellucida (ZP). In fact, different ART techniquestry to simulate this process in vitro, a process which is known as ‘Assisted Hatch-ing’ (AH). Despite the reported results, the effectiveness of AH dose not seem en-tirely clearStudy design, size, duration: Prospectively, 148 treatments from frozen-thawedday-five blastocysts were randomly selected in the period since 2010 to undergoAH treatment in the area corresponding to the ICM or TRPParticipants/materials, setting, methods: Blastocysts were vitrified-devitrifiedusing Kuwayama method and the laser-zona-thining technique was carried outrandomly in the area corresponding to the ICM or TRP after their re–expansion.Clinical outcomes were analysed computationally and t-student significance wasdefined when p , 0.05Main results and the role of chance: In the clinical outcomes no statistic differ-ences were observed when either MCI or TRP assisted hatching was performed .However a small trend in the pregnancy rate was shown (table 1)Table 1Limitations, reason for caution: This study could be limited by the number ortreatments and our vitrification system so further prospective and randomisedstudies must be necessary in order to provide new evidenceWider implications of the findings: Although no statistically significant differ-ences were observed, the pregnancy rate results appear to point towards a favour-able trend in applying assisted hatching techniques at the level of the inner cellmass. These results would be in agreement with the results obtained by othergroups that also defend the role played by the ICM in the embryo hatchingprocess, but further studies will be necessary in order to provide proof.Study funding/competing interest(s): This study was supported by IVI MurciaSL. Murcia. SpainTrial registration number: No trial registration number

P-204 Does gender affect pre-implantation embryonic developmentalrates?- looking back at the baby pictures

S. Duffy1, A. Campbell2, S. Montgomery1, C.F.L. Hickman3, and S. Fishel2

1CARE Fertility, Embryology, Manchester, United Kingdom, 2CARE Fertility,Embryology, Nottingham, United Kingdom, 3Mouwasat CARE Fertility,Embryology, Damamm, Saudi Arabia

Study question: The study objective was to establish whether time-lapse derivedmorphokinetic variables differ between male and female embryos.Summary answer: Only 1 out of 11 morphokinetic variables studied showed asignificant difference according to embryo gender. A trend for male embryos todevelop at a faster rate up to 8 cells, take less time to achieve full compaction,but to blastulate and expand at a slower rate was observed.

........................................................................................AH ICM AH TRP p

N 72 76

Nº transfer 68 76

Age 37 38

Average nº embryo transfers 1.6 1.5

Pregnancy % 51.4 (35/68) 42.1 (32/76) 0.26

Implantation % 36.4 (39/107) 34.5 (39/113) 0.77

Biochemical miscarriage % 16.7 (7/42) 17.9 (7/39) 0.88

Clinical miscarriage % 17.1 (6/35) 15.6 (5/32) 0.87

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What is known already: No studies published to-date using time lapse cite a re-lationship between embryo morphokinetics and offspring gender. Alfarawati et al(2011) discussed a possible connection between blastocyst morphology andembryo gender when considering a correlation between embryo morphologyand chromosomal status. Results of their study demonstrated that male embryosachieved full blastulation at a faster rate than female embryos and a significantlyincreased number of male embryos were of a higher grade than the female cohort.Study design, size, duration: This observational study included the retrospectiveanalysis of the morphokinetic parameters of 29 ICSI embryos following embryotransfer and live birth. These embryos had undergone time-lapse imaging in theEmbryoscopeTM (Unisense Fertilitech, Denmark) for between 3 and 5 daysbefore being selected for embryo transfer.Participants/materials, setting, methods: Patient data was obtained from 58fresh ICSI cycles that had utilized EmbryoScopeTM culture in a private IVFclinic setting and had achieved a live birth. Only embryo transfers that resultedin 100% implantation were included in the data analysis to ensure complete accur-acy of embryo gender.Main results and the role of chance: The time-points in hours post insemination(hpi) of the first cleavage (t2) up to the 9+ cell stage (t9+) and the time to reachmorula (tM), full blastocyst (tB) and expanded blastocyst (tEB) were recordedfor embryos of known gender. The mean time-points for male and femaleembryos respectively were; (t2) 23.01 vs 24.2, (t3) 33.54 vs 35.14, (t4) 35.41vs 36.77, (t5) 45.44 vs 47.61, (t6) 47.34 vs 49.94, (t7) 50.72 vs 54.02, (t8)53.47 vs 55.07, (t9+) 62.17 vs 59.71, (tM) 73.96 vs 77.42, (tB) 102.08 vs101.37, (tEB) 110.65 vs 107.71. A two sample t-test was performed to statisticallycompare the mean time-points of known gender embryos using a p value ,0.05.Statistical significance was observed only at t7( p ¼ 0.037).Limitations, reason for caution: Data set size is a limiting factor, making reliablestatistical analyses difficult. Morphokinetic analyses of known gender embryos isongoing as more live births occur, which, in addition to the continual availability oftime lapse in this clinic setting, will allow analysis of a larger data set to be presented.Wider implications of the findings: The concept of differing morphokineticvalues according to embryo gender is of scientific interest. The results of our pre-liminary analysis, disagree with recent publications, but this may alter as the dataset increases. The morphokinetic differences between the known genderembryos,although too subtle to be observed in standard incubation, may be observed inembryos cultured in other time lapse systems.Study funding/competing interest(s): N/ATrial registration number: N/A

P-205 Bovine embryo development in vitro: a sensitive assay to evaluateplga peg nanoparticle toxicity

I. Fiorentino1, R. Gualtieri1, V. Barbato1, S. Braun1, V. Mollo2, P. Netti2, andR. Talevi1

1University of Naples Federico II, structural and functional biology, Naples, Italy,2University of Naples Federico II, Materials and Production Engineering,Naples, Italy

Study question: The heterogeneityof nanoparticles (NPs) developed for biomed-ical use emphasizes the need of sensitive toxicity tests. Data on somatic cells werecontroversial and only a few studies were done on extremely sensitive processessuch as embryo development.Summary answer: Biodegradable 65nm rhodaminated poly(d,l-lactic-co-gly-colic acid)-block-polyethylene glycol (PLGA-PEG) NPs exert a cytotoxiceffect on embryo development, directly affecting 8 cell embryo (day 3) and blasto-cyst (day 8) rates, in a dose-dependent manner.What is known already: Most studies were performed on somatic cells. NPs, de-pending on their chemical and physical features, may promote sperm apoptosisand ROS production in a dose and size-dependent manner. NPs may alsoimpair folliculogenesis in mouse. Toxicity assays on zebrafish indicate that NPsmay damage embryo development, but no data are available in mammals.Study design, size, duration:The study was performed on 594oocytes. Cleavageand 8 cell embryo rates were scored at day 3, blastocyst rates at day 8. Moreover,NP internalization, blastocyst mean cell numbers (MCN) and percentage of nucleiwith fragmented DNA were assessed by confocal and transmission electron mi-croscopy (TEM).

Participants/materials, setting, methods: Fertilized oocytes treated with NPs(10 or 50 mg/mL) or vehicle for 8 days, were assessed for cleavage and 8 cellembryos (day +3) and blastocyst rates (day +8). Blastocyst MCN was analyzedthrough labelling with Hoechst 33342; DNA fragmentation was investigatedthrough TUNEL assay and NPs internalization by TEM.Main results and the role of chance: NPs treatment did not affect cleavage com-petence at day 3 after insemination. NPs at 50ug/ml reduced 8 cell embryo andblastocyst rates (treated vs control: 8 cell, 40 vs 60%, P , 0,05; blastocyst, 34vs 46,6%, P , 0,05) but MCN and DNA fragmentation were not influenced(treated vs control: MCN, 134+ 40 vs 132+65; DNA fragmentation,7,06%+ 3,44 vs 7,09%+4,14). The confocal z-sectioning showed thatPLGA/PEG-NPs were efficiently internalized by embryos and at higher magnifi-cation their localization was cytoplasmic and particulated. In agreement with con-focal data, TEM analysis showed NPs localized in cytoplasmic vacuoles andvesicles.Limitations, reason for caution: NPs behavior toward biological systems andtheir toxic effects are not fully understood. Contrasting finding could depend onthe chemical-physical nature of NPs, the variety of cell types and experimentalconditions used in different studies.Wider implications of the findings: In conclusion, PLGA-PEG-NPs exert acytotoxic effect on embryo development in a dose-dependent manner. In vitroembryo development in animal models may represent a sensitive and predictiveassay of NP toxicity before their application in biomedicine.Study funding/competing interest(s): Department of Biology, Department ofMaterials and Production EngineeringTrial registration number: none

P-206 The relationship of morphokinetic events and euploidy in humancleavage-stage embryos

A. Bayram1, N. Findikli2, M. Serdarogullari1, O. Sahin1, U. Ulug3, S.B. Tosun3,and M. Bahceci4

1Bahceci Umut IVF Center, Embryology, Istanbul, Turkey, 2Bahceci Fulya IVFCenter, Embryology, Istanbul, Turkey, 3Bahceci Umut IVF Center, ClinicalDepartment, Istanbul, Turkey, 4Bahceci Fulya IVF Center, Clinical Department,Istanbul, Turkey

Study question: This study questions whether there is a link between early mor-phokinetic parameters and aneuploidy/euploidy status of human embryos in pre-implantation genetic screening (PGS) cycles.Summary answer: Our results show that embryos that are eligible forembryo biopsy in PGS cycles reveal similar cleavage profiles irrespective of theeuploidy status. The possible use of time lapse technology in PGS cycles inorder to pinpoint euploidy status of embryos may therefore require largersample size and a different study design that includes post compaction follow-upand analysis until blastocyst stage.What is known already: Recent studies indicate that embryos carryingchromosomal abnormalities can show a variety of cleavage disturbances duringpreimplantation development. However, current literature correlating the devel-opmental competence and euploidy status of human embryos are mostly per-formed on static embryo culture hence data regarding early cleavage divisioncharacteristics of such embryos are very limited.Study design, size, duration: Embryos obtained from 43 PGS cycles in whichchromosomes 13, 15, 16, 17, 18, 21, 22, X and Y were analyzed by fluorescentin situ hybridization have been monitored in a special tri-gas incubator withbuilt-in time-lapse monitoring system. The morphokinetic characteristics ofeach embryo were traced until day 3 of embryo development. All embryobiopsy procedures were performed on day 3. The data consisted of embryos andPGS results of patients that were admitted in our clinics between March2011-December 2012.Participants/materials, setting, methods: Morphokinetic parameters of 254 bi-opsied day 3 embryos were retrospectively analyzed and grouped according to thechromosomal status after PGS as euploid (normal) or aneuploid (including mono-somies, trisomies and complex abnormalities). Grouped data were than comparedfor each time points (T2 through T8) as well as the duration between these clea-vages such as S2, S3 and cc2. Data has been analyzed with Student’s t test anda P-value less than 0.05 has been considered statistically significant.

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Main results and the role of chance: Morphokinetic parameters of 45 euploidand 209 aneuploid embryos carrying single or complex chromosomal abnormal-ities shows similar cleavage time characteristics on each time points and para-meters analyzed as shown in the Table. This may indicate that although theyhave different chromosome constitutions, embryos that are selected for embryobiopsy with a certain selection criteria are indifferent in their cell division kineticsuntil day 3 embryo development.

p . 0.05 for all the parameters compared among groups.Limitations, reason for caution: Our study includes only embryos that are suit-able for biopsy hence the possible correlation for embryos with developmental ab-normalities could not be assessed in this setting. Data including extended embryoculture parameters as well as reanalysis of PGS results on day 5 could also contrib-ute more to our study.Wider implications of the findings: Our results can indicate that morphokineticparameters on early developmental stages fail to indicate any distinct chromosom-al abnormality pattern hence cannot be used as an additional indicator for aneu-ploidy in good quality cleavage stage embryos.Study funding/competing interest(s): This study received no funding and thereare no conflicts of interests to be declared.Trial registration number: This study was not an RCT and therefore there is noregistration number.

P-207 Vitrification effect on the oocyte morphometry and the hardeningof zona pellucida

A. Sanchez Leon1, D. Gumbao1, J. Marcos1, M. Molla1, B. Amorocho1,M. Nicolas2, L. Fernandez2, and J. Landeras2

1IVI Murcia, IVF Laboratory, Murcia, Spain, 2IVI Murcia, Gynecologist, Murcia,Spain

Study question: The aim of this study was to focus on the possible morphologicaland developmental alterations in our oocyte vitrification system, paying particularattention to the oocyte zona pellucida (ZP) and its morphological peculiarities,whilst studying different oocyte morphometric measurements and the possiblehardening effect associated with the cryopreservation processSummary answer: We may conclude that in our oocyte vitrification system nonegative effect was evidenced on the embryo clinical outcomes when eitherfresh or vitrified oocytes were used and neither morphometric nor ZP hardeningchanges after vitrification were causedWhat is known already: The ability to cryopreserve human oocytes confers sig-nificant advantages in IVF-ICSI cycles, not only for the improvement of clinicalresults but also when considering ethical and legal aspects. Since the firsthuman vitrification studies different alterations have been reported and it isclear that all oocytes and embryos could suffer considerable morphological andfunctional damage during the cryopreservation process that may affect in vitroembryo development and IVF results.

Study design, size, duration: Three experiments were designed between 2010and 2012. In the first experiment 86 treatments from our oocyte donationprogram were analyzed. Furthermore, 28 metaphase II matured oocytes remainigfrom donor treatments were studied morphometrically. In the third experimentoocyte hardening was assessed in 47 fresh vs 37 vitrified oocytesParticipants/materials, setting, methods: The metaphase II matured oocyteswere vitrified-devitrified using the Kuwayama method and the clinical outcomestreatments were studied depending on oocyte origin.The morphometric measure-ments were performed using an image capture software and to study the ZP hard-ening the time of digestion mediated by an acidific tyrodes solution was evaluatedMain results and the role of chance: No clinical otucomes differences wereobserved when different oocytes were used (Table 1). Focusing on the morphom-etry, no differences were found (Table 2). when oocyte hardening was studied nodifferences were observed in fresh vs vitrified oocytes (23.1 sec vs 19.8 sec; p ¼0.18 ). Statistical differences were analysed computationally and t-student signifi-cance was defined when P , 0.05.Table 1 Table 2

...............................................................................................................................................................................................Euploid Monosomy Trisomy Complex Abnormality

T2 27,03+3,58 (45) 27,41+3,42 (56) 26,82+4,069 (46) 28,044+4,038 (89)

T3 37,21+5,085 (45) 37,22+5,07 (56) 37,41+4,767 (46) 35,73+5,59 (89)

T4 39,99+4,50 (45) 40,23+6,468 (56) 39,77+5,479 (46) 39,22+5,95 (89)

T5 50,8+7,44 (44) 49,24+7,28 (54) 49,07+6,78 (43) 46,251+8,138 (86)

T6 53,6+6,058 (43) 51,48+7,40 (53) 52,16+6,393 (42) 50,128+7,467 (81)

T7 55,48+5,45 (40) 54,54+6,30 (46) 54,92+5,427 (38) 53,343+7,296 (74)

T8 56,75+5,89 (35) 56,11+6,31 (39) 56,94+5,531 (35) 55,149+6,685 (63)

cc2 (t3-t2) 10,17+4,024 (45) 9,59+4,98 (56) 10,593+3,46 (46) 7,928+5,218 (88)

S2 (t4-t3) 2,78+3,764 (45) 3,001+4,37 (56) 2,356+3,717 (46) 3,537+5,096 (88)

S3 (t8-t5) 6,93+5,81 (35) 8,423+6,28 (39) 8,448+6,28 (35) 9,628+6,502 (63)

.........................................................................................

Vitrifiedoocytes

Freshoocytes

p

Age 40.4 39.8

Fertilization % 80.5 77.9 0.34

Clinical pregnancy % 77.8 66.0 0.27

Implantation % 56.5 55.8 0.94

Biochemical % 16 5.4 0.21

Clinicalmiscarriage %

19 11.4 0.24

.........................................................................................

Vitrifiedoocytes

Freshoocytes

p

ZP diameter (mm) 19.4 19.1 0.91

Oocyte diameter (mm) 163 162 0.74

Ooleme diameter (mm) 113 112 0.89

Polar body size (mm2) 332.2 355.1 0.57

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Limitations, reason for caution: These studies could be limited to our vitrifica-tion system and because of the results obtained further prospective and rando-mised studies must be necessary in order to provide new evidenceWider implications of the findings: The results show that, in our oocyte vitrifi-cation system, no negative effect was seen in embryo development when eitherfresh or vitrified oocytes were used. So we may conclude that the vitrificationmethod works well adn also no morphometric changes after vitrification werefound. On the other hand, the hardening associated process was discarted inorder to know if any hatching assisted method would be necessary to useStudy funding/competing interest(s): This study was supported by IVI MurciaSL. Murcia. SpainTrial registration number: No trial registration number

P-208 The influenceof sequential scoring in determining the best embryoclassification for transfer when not using time lapse technology

M.C.A. Cardoso1, A.P.S. Aguiar1, C. Sartorio2, A. Evangelista2, P. Gallo-Sa2, andM.C. Erthal-Martins2

1Vida Centro de Fertilidade da Rede D’Or, IVF Laboratory, Rio de Janeiro,Brazil, 2Vida Centro de Fertilidade da Rede D’Or, Clinic, Rio de Janeiro, Brazil

Study question: What are the benefits of removing the embryos out of the incu-bator on day 2 (D2) to fulfill the classification scores for the before transfer?Summary answer: The ASEBIR morphological classification assesses embryoevolution from D2 to day 3 (D3): similar embryos can be graded differently onD3 depending on classification they had on D2. On the other hand, taking theembryos out of the incubator increases stress on them, probably decreasingtheir chance in implanting.What is known already: Embryo morphology classification is still an importanttool of selection for transfer, no matter if the transfer is on day 2, day 3 or blasto-cyst. The ASEBIR embryo morphological classification takes in account theembryo evolution from day 2 to day 3. This is the method for embryo classificationwhich best correlated with the pregnancy outcomes in our clinic and it was thechosen method, since 2010.Study design, size, duration: Cases were randomlyselected to be either classifiedor not on D2. PGD cases, D2 transfers, oocyte donation and when all the embryoswere vitrified (no fresh transfer) were not included. A total of 112 IVF cycles wereanalyzed. The study was conducted from April to December of 2012.Participants/materials, setting, methods: The study groups were divided intwo: Group 1 ¼ D2 + D3 scoring and Group 2 ¼ D3 only scoring. Clinical preg-nancy and implantation rates were compared between groups. In Group 1, a blindreclassification was made as if the D2 assessment had not been done.Main results and the role of chance: Group 1 had 59 cycles and Group 2, 53cycles. The groups were similar among patients age (34,3 and 35 years). The preg-nancy rate and implantation rate were 22.0% and 15.5% for Group 1, and 34.0%and 24.5% for Group 2 (p ¼ 0.15 and p ¼ 0.09 respectively). Group 1 had 116embryos transferred in which 40.5% were grade A, 26.7% grade B, 19.0%grade C and 13.8% grade D. Group 2 had 101 embryos transferred in which inwhich 69.6% were grade A, 17.7% grade B, 7.8% grade C and 4.9% gradeD. In Group 1, if the classification had been done only in D3, the score wouldbe different (higher) in 11.2% of embryos transferred.Limitations, reason for caution: When assessments were made only on Day 3, itwas presumed that the embryos had the best score on Day 2, which falselyincreases the scores of some embryos.Wider implications of the findings: There was no significant difference betweenGroups 1 and 2 in pregnancyand implantation rates, but the validityof consideringthe embryo development profile until the day of transfer, while not having a time-lapse incubator, is still questionable. Subtracting one day of observationdiminishes exposure of the embryos. The loss in accuracy on embryo gradingaffects only a few percentage of them, not impairing their implantation potential.Study funding/competing interest(s): This study was conducted in a privateclinic without third party sponsorship.Trial registration number: None

P-209 Factors affecting the transcriptome of human preimplantationembryos

E. Mantikou1, M.J. Jonker2, M. de Jong2, K.M. Wong1, A.P.A. van Montfoort3,T.M. Breit2, S. Repping1, and S. Mastenbroek1

1Center for Reproductive Medicine, Academic Medical Center University ofAmsterdam, Amsterdam, The Netherlands, 2Swammerdam Institute for LifeSciences Faculty of Science (FNWI) University of Amsterdam, MicroArrayDepartment and Integrative Bioinformatics Unit (MAD-IBU), Amsterdam, TheNetherlands, 3Maastricht University Medical Center, Obstetrics andGynaecology, Maastricht, The Netherlands

Study question: What is the relative effect of common environmental and bio-logical factors on the transcriptome changes during human preimplantation devel-opment?Summary answer: Developmental stage and maternal age have a larger effect onthe global gene expression profile of human preimplantation embryos than theculture medium or oxygen concentration used in in-vitro culture.What is known already: Studies on mouse and bovine embryos have shown thatdifferent conditions in the in-vitro culture of embryos can lead to changes in tran-scriptome profiles. For humans this is not yet known. An effect of developmentalstage on the transcriptome profile of embryos has been demonstrated, but studieson the effect of maternal age or various culture conditions are lacking.Study design, size, duration: Human preimplantation embryos were randomisedto two culture-media (G5-medium or HTF-medium) and to two oxygen concen-trations (5% or 20%), with stratification for maternal age. Next to these variables,developmental stage after culture was also taken into account in the transcriptomeanalysis.Participants/materials, setting, methods: After thawing, donated humanembryos that were cryopreserved on day four, were cultured for two days underthe randomized conditions (N¼ 89). Only embryos developing to morula orblastocyst stage (N ¼ 39) were assessed for genome-wide gene expressionusing microarrays and a regression model was built to select the contributingfactors.Main results and the role of chance: Based on the number of differentiallyexpressed genes (DEGs), developmental stage (3,519 DEGs) and maternal age(1,258 DEGs) had a larger effect on the global gene expression profile ofhuman preimplantation embryos than either tested culture medium (596 DEGs)or oxygen concentration (492 DEGs) used during in-vitro culture. Interactionsbetween the factors were found, indicating that culture conditions might have adifferent effect depending on the developmental stage or the maternal age of theembryos. Affected pathways included metabolism, cell cycle processes and oxi-dative phosphorylation.Limitations, reason for caution: Culture of embryos for only two days mighthave limited the effect on global gene expression by the investigated culture con-ditions. Earlier stages of development (day 0 until day 4) were not analyzed andmight respond differently to the experimental conditions.Wider implications of the findings: Our results show that when studying geneexpression in single human preimplantation embryos under various experimentalconditions, one should take into account the confounding effect of biological vari-ables, such as developmental stage and maternal age. This makes these experi-ments different from gene expression experiments where these variables can betightly controlled, for example when using cell lines.Study funding/competing interest(s): This study received no external fundingand there were no competing interests.Trial registration number: None

P-210 Can completion of compaction predict implantation outcome?

E. Power, S. Montgomery, S. Duffy, K. Jordan, A. Campbell, and S. FishelCARE Fertility, Manchester embryology, Manchester, United Kingdom

Study question: If cellular material is excluded from the embryo at the stage ofcompaction (defined as incomplete compaction), does this affect the ability ofthe embryo to implant successfully?Summary answer: Significantly more embryos where compaction was in-complete failed to implant, compared to embryos with complete compaction(p ¼ 0.033).What is known already: There is evidence with conventional microscopyshowing snap shots of embryo development, that incomplete compaction maybe related to the ability to form a blastocyst (Ivec et. al.). Time lapse technology

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has allowed us to study this in greater detail and retrospectively compare this toimplantation outcome.Study design, size, duration: The use of time lapse technology in the IVF labora-tory has facilitated the ability to study embryo development as a dynamic processand to retrospectivelyassess how compaction mayaffect embryo development andultimately implantation. Using the EmbryoScopeTM (Unisense Fertilitech,Denmark) it has been possible to compare the process and degree of compaction,whether cellular material is excluded, in those embryos that are known to haveimplanted to those that are known to have not.Participants/materials, setting, methods: Embryos cultured in the Embryo-Scope (ES) with a minimum of 5% fragmentation (by volume) at the cleavagestage (up to 8 cell) that had developed to full blastocyst stage were included.This group was further restricted to embryos of known implantation outcome(KID) where a single embryo was replaced and implanted, or 2 embryos werereplaced and both implanted (KID +) and those where no implantation occurred(KID -). A database of the embryo development videos was set up in the ES viewerand all were viewed and annotated according to whether or not any cellular mater-ial was excluded at compaction. The end point of compaction was taken as the startof blastulation. The data was then exported to excel and compared to implantationoutcome.Main results and the role of chance: In the 75 embryos studied, there was a sig-nificant difference (p ¼ 0.033) in the number of embryos that completely com-pacted and went on to implant (48% 29/61), compared to the number withincomplete compaction that went on to implant (14% 2/14).Limitations, reason for caution: The embryo numbers are restricted by the factthat the embryos from many double embryo transfers where a single implantationoccurred have been excluded. Data from the KID embryos will continue to be col-lected.Wider implications of the findings:This may be a useful clinical tool, in conjunc-tion with time lapse technology, in de-selecting embryos with a reduced potentialto implant.Study funding/competing interest(s): N/ATrial registration number: N/A

P-211 Vitrification/warming before embryo biopsy can positivelycontribute to clinical outcome in Preimplantation Genetic Screeningcases

N. Findikli1, T. Aksoy1, M. Gultomruk1, A. Aktan2, C. Goktas1, U. Ulug3, andM. Bahceci2

1Bahceci Fulya IVF Centre, Embryology Laboratory, Istanbul, Turkey, 2BahceciFulya IVF Centre, Clinical Department, Istanbul, Turkey, 3Bahceci Umut IVFCentre, Clinical Department, Istanbul, Turkey

Study question: Does vitrification/warming before embryo biopsy have anyeffect or additional role in cases where Preimplantation Genetic Screening(PGS) is performed?Summary answer: The use of vitrified/warmed cleavage stage embryos beforebiopsy can positively contribute to cycle outcome compared to cases in whichonly embryos obtained in fresh cycles are biopsied.What is known already: Numerous studies report that cryopreserving embryosfollowing biopsy for PGS may affect the clinical outcome. However, thedata about the efficiency of vitrification/warming of cleavage stage embryosbefore biopsy for PGS and subsequent embryo transfer are scarce and containslimited data.Study design, size, duration: This retrospective cohort study includes 222patients undergoing a total of 231 PGS cycles performed for numerical andstructural chromosomal abnormalities between August 2010 – December 2012in Fulya and Umut Bahceci Assisted Reproductive Technology Centers.Participants/materials, setting, methods: PGS cycles that were included in thisstudy were grouped into three according to the nature of biopsied embryos: Group1 includes110 PGS cycles in which 792 fresh embryos werebiopsied, group 2 con-sists of 38 PGS cycles in which a total of 350 fresh and vitrified/warmed embryoswere biopsied and group 3 includes 83 PGS cycles that utilized only vitrified/warmed embryos (803 embryos biopsied). Upon availability of chromosomallycompetent embryos, cycles in group 1 and group 2 proceeded with freshembryo transfers, embryos in 3 were transferred in frozen embryo transfersetting with proper endometrial preparation.

Main results and the role of chance: A total of 1945 embryos (fresh and vitrified/warmed) were biopsied and an informativePGS result were obtained in 1903/1945(97.8%). Depending on the availability of chromosomally normal embryos, 71cycles in group 1 (65%), 25 cycles in group 2 (66%) and 65 cycles in group 3(79%) reached embryo transfer stage. Average number of embryos transferredin each group was 1.36, 1.52 and 1.46 respectively. Likewise, clinical pregnancyrates in group 1, 2 and 3 were 38%, 48%, 53.8% and implantation rates were foundto be 29%, 34% and 41%, respectively. In group 2 and 3, a total of 1060 embryoswere warmed in which 952 were utilized for embryo biopsy on day 3 of embryodevelopment (90.8%).Limitations, reason for caution: This study includes cases in which fluorescent insitu hybridisation (FISH)-based PGS were performed for 5, 7 or 9 chromosomes, re-spectively. Clinical outcome can be improved if array comparative genomic hybrid-ization (aCGH) or similar technologies can be utilized in the same approach.Wider implications of the findings: This study may be generalized in PGS casesin which the number of embryos in a fresh cycle is very limited and there is a highprobability of not finding a chromosomally normal embryo after analysis. In suchcases, embryos of consecutive IVF cycles can be vitrifiedand pooled. Also,PGS incombination with frozen embryo transfer programmes can produce better clinicaloutcome with more physiological endometrial environment.Study funding/competing interest(s): This study received no funding and thereare no conflicts of interests to be declared.Trial registration number: This study was not an RCT and therefore there is noregistration number.

P-212 Impact of HIV infection in women submitted to ICSI cycles

R. Petracco, L. Okada, R. Azambuja, F. Badalotti, J. Michelon, V. Reig, D. Kvitko,A. Tagliani-Ribeiro, M. Badalotti, and A. PetraccoFertilitat, Centro de Medicina Reprodutiva, Porto Alegre, Brazil

Study question: Analyze the human immunodeficiency virus (HIV) infection inwomen submitted to assisted reproduction technology on the oocyte quality,embryo quality and overall pregnancy rate.Summary answer: Our data showed that HIV patients have no difference whensubmitted to intracytoplasmic sperm injection (ICSI) cycles regarding to stimula-tion response, laboratory and pregnancy outcome.What is known already: There are 34.2 million people infected by HIVaroundthe world. Half of this population is women and three quarters are in their repro-ductive years. Considering best quality of life and higher survival rates usinganti retroviral therapies, the patients are considering pregnancy planning usingassisted reproductive technologies. Cumulative evidence suggests that ART issafe and effective for avoiding horizontal and vertical transmission in HIV serodis-cordant couples. The literature is controversial about the HIV patient results, butsome of the publications relates worse IVF/ICSI outcome in this population.Study design, size, duration: Retrospective cohort study with 11 HIV serodiscor-dant couples, whose women were infected, submitted to ICSI between 2002 and2012.Participants/materials, setting, methods: The HIV patients were matched totwo different control patients according to infertility cause, age, semen qualityand ovarian stimulation protocol. In the HIV patients group, mean age was 38.7years old and 22 cycles were performed, in the control groups was 39 years oldand were performed 44 cycles. The results were compared using t test (P , 0.05).Main results and the role of chance: The number of oocytes retrieved, mature(MII) oocytes, oocytes fertilized, number of embryos transferred and embryoquality were compared. In the serodiscordant group, there were 18 cycles withembryo transfer. Out of those, there were 9 pregnancies (50%), from those 1 bio-chemical and 8 clinical. Out of the clinical pregnancy there were 2 miscarriages, 2ongoing pregnancy and 4 newborn. In the control group there were 41 cycles withembryo transfer. Out of those, there were 18 pregnancies (43%), from these 4 bio-chemical and 14 clinical. Out of the clinical pregnancies 3 miscarriage, 2 areongoing and 9 newborn. There were no statistical differences between the groups.Limitations, reason for caution: The study was conducted with a small numberof patients.Wider implications of the findings: Our data showed that HIV patients presentedsimilar results to the control group regarding to stimulation, laboratory and preg-nancy outcome, however the number of embryos transferred had a trend to signifi-cance in the control group (p ¼ 0,054).

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Study funding/competing interest(s): There were no competing interests in thisstudyTrial registration number: Not available

P-213 Synchronicity of cleavage cycles predicts blastocyst formationand quality

C. Pirkevi, M. Cetinkaya, H. Yelke, Y. Kumtepe, Z. Atayurt, and S. KahramanIstanbul Memorial Hospital, Assisted Reproductive Technologies andReproductive Genetics Center, Istanbul, Turkey

Study question: Can we predict high quality blastocyst formation potential fromday3 embryos using morphokinetics?Summary answer: The five formulas cited below were used to identify cleavagesynchronicity: (F1): (t8-t2)/((t3-t2) + (t5-t4)); (F2): (t8-t5)/(t8-t4); (F3): (t4-t3)/(t4-t2); (F4): (t4-t2)/(t8-t4); (F5): (t3-t2), additionally two criteria (irregular divi-sions and evenness of blastomeres at 2 and 4-cell stages) were applied to build anadditive embryo scoring model predicting high quality blastocyst formation.What is known already: The blastocyst formation was found to correlate withcc2 (time between division to 2 cells and division to 3 cells), s2 (time between div-ision to 3 cells and subsequent division to 4 cells) and with the duration of the firstcytokinesis (Wong, 2010). Cruz et al. have added t4 and t5 and Dal Canto et al.used different subtractions giving the duration of cell cycles (t3-t2; t4-t3; t4-t2;t8-t4; t8-t5) to predict blastocyst quality.Study design, size, duration: This retrospective cohort study was conductedbetween October 2011 and October 2012 in a private ART Center and approvedby the institutional review board. The study included 1199 embryo having a(t8) from 190 infertile patients transferred on day5 (Female age: 31.7+4.4; Pre-vious ART cycles: 1.6+1.8; BMI: 24.4+6.2; MII: 8.2+3.4).Participants/materials, setting, methods: Embryos were cultured in a singlestep culture medium, which was refreshed on day3. Incubation was performedunder oil at 378C, 5%O2 and 6%CO2. Each time of cleavage was recorded. Blas-tocysts were scored before transfer according to Gardner’s classification(115-120h post ICSI).Main results and the role of chance: Embryos should mainly stay in even cellstages, which should be balanced when compared to each other, ideally reflectingcleavage synchronicity. Each formula was applied and embryos were classified in5%groups and good/ top quality blastocyst rate (BR) was computed for the20intervals. In order to follow mathematically the natural trends obtained inBR, the average of ≥3consecutive 5%groups was subtracted from the averageof the 2following groups, and divided to the initial BR until the theoretical thresh-old set at 0.15 was reached. Each class had a mean BR, which was subtracted fromthe initial BR, giving a positive or negative score. Obtained scores were added forthe final prediction of the blastocyst formation potential. The logistic regressionmodel built gave an AUC ¼ 0.841 (95%CI 0.819-0.861).Limitations, reason for caution: The model described may depend on cultureconditions applied in this particular IVF laboratory (low O2, single stepmedium, mainly antagonist ovarian stimulation protocol). The cohort studiedinvolved only infertile patients (female, male or combined) and is in thisrespect a heterogeneous population.Wider implications of the findings: Time points defining precise embryo cleav-age events may not be generalized to infertile patients with different etiologies.However, using ratios based on selected cleavage cycles defining synchronicityof embryos, allows in this retrospective cohort study an individualized analysisgiving high predictivity of blastocyst formation and quality. The proposedmodel has to be further tested in a randomized prospective trial to evaluate itsef?cacy, and may in the future improve embryo selection in IVF treatments.Study funding/competing interest(s): Authors have nothing to disclose.Trial registration number: Not applicable

P-214 The relationship between homocysteine, embryo quality andpregnancy in embryo culture medium in assisted reproductive techniques

B. Aydin and I. CepniCerrahpasa Tip Fakultesi (Medical Faculty), Obstetric and Gynecology, FatihIstanbul, Turkey

Study question: Can homocysteine in embryo culture medium affect the embryoquality?Summary answer: Homocysteine in embryo culture medium affect the embryoquality.What is known already: Homocysteine is an oxidant aminoacid product thataffect the reproductive process.Study design, size, duration: Our study is acase control study. The study has beencompleted in 1 year.40 cases are included.23 cases are non pregnant and 16 casesare pregnant.Participants/materials, setting, methods: 40 cases which admitted to our clinicwith fertility desire were included into study. In each case; the level of homocyst-eine in culture medium was measured by spectrophotometry. Also the correlationbetween Hcy levels in culture medium and embryo quality were investigated.Main results and the role of chance: Age, the duration of infertility, infertilitytype, VKI, FSH levels in 3 th day of the menstruation, serum AMH levels werenot statistically significant between pregnant and non-pregnant group in whichHcy levels were detected in embryo culture medium. And also total dosage of go-nadotropin, the number of total oocyte and oocyte which ICSI was performed, thedaytime in which the stimulation performed were not significant between twogroups. . Positive correlation was detected between Hcy in embryo culturemedium and embryo grade (r:0,376,p , 0.034). 24 of40 patients were not pregnantand 16 of 40 were pregnant. Also significant correlation was found between beingnot able to conceive and Hcy levels in culture medium (r:0,5131, p , 0.0004).Limitations, reason for caution: Our case numbers are so low. We can redoublethe case numbers and also the validity of the study.Wider implications of the findings: In other studies, homocysteine in semen andfollicular fluid has been also found as an oxidant product that affect inversly thereproductive parameters.Study funding/competing interest(s): Homocysteine is an important marker forembryo quality.Trial registration number: There is no registration number.

P-215 Comparison of gender-specific human embryo developmentcharacteristics by time lapse technology

M. Serdarogullari1, N. Findikli2, A. Bayram1, C. Goktas2, O. Sahin1, U. Ulug3,and M. Bahceci4

1Bahceci Umut IVF Centre, Embryology Laboratory, Istanbul, Turkey, 2BahceciFulya IVF Centre, Embryology Laboratory, Istanbul, Turkey, 3Bahceci Umut IVFCentre, Clinical Department, Istanbul, Turkey, 4Bahceci Fulya IVF Centre,Clinical Department, Istanbul, Turkey

Study question: This study asks whether there exist gender-specific embryo de-velopment kinetics or parameters between human male and female embryos thatcan be observed and distinguished by time lapse technology.Summary answer: Our results indicate that the cavitation time of a femaleembryo was slightly earlier compared to the male embryos. However, other mor-phokinetic parameters used, such as early cleavage time points, duration betweeneach cleavages, early signs of compaction, cavitation and blastocoel expansion donot show any variation.What is known already: Numerous studies in laboratory animals and mammalsindicate that there might be differences in embryo growth dynamics between maleand female embryos. However, current data and literature that had analyzedgender-specific preimplantation growth parameters in humans so far are scarceand the results are inconclusive or conflicting.Study design, size, duration: Embryos were cultured in special tri-gas incubatorswith built-in time-lapse monitoring system in 116 cycles. In these cycles, genderof the off-springs was also assessed through ultrasonography performed at 14-16weeks of gestation. Study included data from cycles performed between March2011-December 2012 in Bahceci Assisted Reproductive Technology Centers.Participants/materials, setting, methods: Embryos of 18 cycles were excludedfrom analysis since they were twin pregnancies of different genders. Remaining114 embryos were analysed for parameters including cleavage time points andduration in each cleavage from two cells - until hatching blastocyst stages (t2 totHB), time interval between cleavages (cc2, S2 andS3).Main results and the role of chance: Morphokinetic parameters 62 female and52 male embryos from a total of 98 cycles processed for data analysis. Table showsthe time kinetics of each observed parameter and the number of embryos scored.

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Limitations, reason for caution: The number of embryos and cycles included inthe study. To use of time lapse technology in predicting the gender-specific devel-opment of the human embryos may require further research and larger sample sizewith the current parameters and technical setting.Wider implications of the findings: Results of this study may indicate that al-though there is a trend of early cavitation in females, there are no distinct gender-specific differences in embryo development kinetics in human preimplantationembryos. Therefore, either novel dynamic embryo culture and selection or con-ventional embryo culture and scoring systems are not expected to change sex-ratioof the resulting pregnancies irrespective of the day of embryo transfer.Study funding/competing interest(s): This study received no funding and thereare no conflicts of interests to be declared.Trial registration number: This study was not an RCT and therefore there is noregistration number.

P-216 Final outcome of embryo transfers on day 3, 4 and 5 using own anddonated oocytes

D. Rodrıguez-Arnedo1, J. Ten1, J. Guerrero1, I. Ochando1, M. Perez2, andR. Bernabeu3

1Instituto Bernabeu, Embryology Dept., Alicante, Spain, 2Instituto Bernabeu,Embryology Dept., Cartagena, Spain, 3Instituto Bernabeu, ReproductiveMedicine Dept., Alicante, Spain

Study question: Is transfer on day 4 a reasonable alternative in ART (assisted re-production treatments) and are there differences between own or donated oocytesin term of clinical pregnancy and implantation rates?Summary answer: Transfer on day 4 is a reasonable alternative to day 5 in treat-ments using own and donated oocytes considering that at this time of developmentthe embryo has full genomic activation.What is known already: The day 4 human embryo appears as a mass of cellscomposed of 16-32 blastomeres; the morula. The blastomere adherence processis mediated by changes in distribution of E-cadherin proteins from the cyto-plasm to the cell membrane. This process has been linked to activation ofthe embryonic genome (Desai et al., 2000). Transfer on day 4 occurs post-genome activation and allows us to select the highest developmental potentialembryo from a cohort.Study design, size, duration: A cross-sectional study was performed in a retro-spective analysis of 2,685 IVF/ICSI treatments with own oocytes (670) anddonated oocytes (2015) between January 2008 and December 2012. Embryotransfers were performed on day 3, 4 or 5.Participants/materials, setting, methods: A multivariant analysis was per-formed including confusing factors like maternal age, number of oocytes col-lected, embryo quality on day 3, number of transferred embryos and theirquality. A multiple lineal regression for quantitative dependent variables andbinary logistic regression for categorical dependent variables were applied. Wecompared clinical pregnancy rates (CPR) and implantation rates (IR) in 3groups: day 3, 4 and 5 transfers.Main results and the role of chance: In case of own oocytes, there were no stat-istically significant differences between CPR and transfers on day 3, 4 and 5(43.3%; 49.4%; 50.5%, respectively). In terms of IR, we found a significant differ-ence between day 3 and day 5 (28.00 vs 38.86, respectively, p ¼ 0.004). Withdonated eggs, there were statistically significant differences between day 3 vs.day 4 and 5 with respect to CPR (44.1%, 51.0%, 56.4% respectively), p ¼0.044(day 3 with day 4), p ¼ 0.001 (day 3 with day 5), IR (28.71%, 37.26%,42.57% respectively), p ¼ 0.002 (day 3 with day 4), p ¼ 0.001 (day 3 with day5) and number of useful embryos (transferred plus frozen) (3.26, 3.68, 3.82, re-spectively), p¼ 0.003(day 3 with day 4), p ¼ 0.001 (day 3 with day 5).Limitations, reason for caution: According to our results, day 4 is a reasonablealternative for transfer in treatments of own and donated oocytes, but this study isretrospective and has some limitations. A prospective randomized study should bedone to confirm our findings.Wider implications of the findings: These results confirm the contributions ofother authors on the subject, considering the transfer on day 4 as an alternativeto transfer on day 5 without reducing success rates. In this case we have analyzeda total of 2,685 cycles, coming from day 3 transfers (901), day 4 (430) and day 5(1354); probably the greatest number of cases analyzed.

Study funding/competing interest(s): Conflicts of interest and source of fundingnot declared.Trial registration number: There is not trial registration number

P-217 Pregnancy rates comparison between fresh versus frozen blasto-cyst transfers

L. Okada, R. Petracco, R. Azambuja, F. Badalotti, J. Michelon, V. Reig,A. Tagliani-Ribeiro, D. Kvitko, M. Badalotti, and A. PetraccoFertilitat-Centro de Medicina Reprodutiva, Center for Medicine Reproductive,Porto Alegre, Brazil

Study question: To compare the pregnancy rates of transferred blastocystsderived from fresh or frozen cycles.Summary answer: Our data suggest that frozen blastocyst transfer does notreduce the chances of pregnancyWhat is known already: The embryo cryopreservation has become a fundamen-tal technique in assisted reproduction to store indefinitely and viably the surplusembryos. The culture of embryos to the blastocyst stage has shown to be a moreefficient way of selection and with better chances of implantation.Study design, size, duration: Retrospective cohort study with only cycles trans-ferring blastocysts embryos (fresh ¼ 116 or frozen ¼ 58), during the year 2012.Participants/materials, setting, methods: The results of blastocyst transfercycles were evaluated. The mean age of patients in the fresh cycle and FET(frozen embryo transfer) were 33.8 and 34.8, respectively. The technique of cryo-preservation was vitrification (Kuwayama et al. 1998). Statistical analysis was per-formed using Fisher’s exact test (p ,0.05).Main results and the role of chance: The number of embryos transferred in bothgroups (fresh and FET) were 238 and 109, respectively, with an average of 2.1 and1.9 per patient. The rates of pregnancies and clinical pregnancies were 45.7% and40.5% for fresh embryos and 50.0% and 43.1% transferring frozen/thawed blas-tocysts. No statistically significant difference (p. 0.05) was observed in theresults obtained.Limitations, reason for caution: The study was conducted with a small numberof patients in a short period of time.Wider implications of the findings: The results suggest that frozen blastocysttransfer does not reduce the chances of pregnancy, according to published data.Study funding/competing interest(s): There were no competing interests in thisstudy

.........................................................................................Female Male P

T2 25,5 +2,7(62) 25,6+3,51(52) 0.86

T3 36,3+4,1 (62) 36,4+3,93 (52) 0.89

T4 37,5+3,5 (62) 37,96+3,52 (51) 0.48

T5 48,3+6,5 (61) 48,83+6,14 (48) 0.66

T6 51,1+4,8 (61) 51,21+4,95 (48) 0.90

T7 53,2+5,2 (61) 53,1+5,5(47) 0.92

T8 55,9+5,6 (61) 56,98+8,2 (46) 0.42

T9 65,07+7,6 (54) 66,91+8,22 (45) 0.25

Tcomp 70,4+8,2 (48) 71,14+9,84 (41) 0.69

Tm 81,25+8,6 (46) 81,4+12,4 (41) 0.94

Tcav 90,9+ 8,1 (42) 94,4+9,65 (37) 0.08

Teb 103,1 +6,7 (40) 104,7+ 6,1 (33) 0.29

Thb 112,2+6,05( 14) 112,2+2,06 (4) 1.0

cc2 (t3-t2) 10,8+2,2 (62) 10,72+3,08 (52) 0.87

S2 (t4-t3) 1,2 +2,1 (62) 1,58+2,61 (51) 0.67

S3 (t8-t5) 7,5 +5,9 (61) 8,35+7,63 (46) 0.51

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Trial registration number: Not available

P-218 Transfer of frozen/thawed embryos: pregnancy comparisonbetween blastocysts and cleavage stage embryos

V. Reig, D. Kvitko, A. Tagliani-Ribeiro, L. Okada, R. Azambuja, R. Petracco,J. Michelon, F. Badalotti, A. Petracco, and M. BadalottiFertilitat, Centro de Med. Reprodutiva, Porto Alegre, Brazil

Study question: To compare the pregnancy rate in patients submitted to FET(Frozen Embryos Transfer) of cleavage stage embryos (Group 1), or at the blasto-cyst stage (Group 2).Summary answer: Our data showed that the transfer of frozen blastocysts has abetter pregnancy rate than cleavage embryos.What is known already: The first birth of a baby arising from a cryopreservedembryo occurred in 1983. Since then, improvements in embryo cryopreservationhave occurred. However since early 90′s a vitrification technique has also beendeveloped to cryopreserve embryos in any stage of development.Study design, size, duration: Retrospective cohort study during 2012, with atotal of 132 cycles.Participants/materials, setting, methods: In the group 1 the embryos had beenfrozen on the 2nd, 3rd or 4th day after fertilization, while in the group 2 the embryoswere frozen on 5th or 6th day at blastocyst stage. All embryos were cryopreservedby vitrification followed Kuwayama et al, 1998 protocol. The pregnancy rateswere compared through the chi-square test (p , 0.05).Main results and the role of chance: The Group 1 consisted of 74 patients, whichpregnancy and clinical pregnancy rate were 27.0% (20/74) and 21.6% (16/74), re-spectively. The Group 2 had 58 patients, and pregnancy and clinical pregnancyrate were 50.0% (29/58) and 43.1% (25/58) respectively. There was statistical dif-ference between the groups (P¼ 0.0107). The mean age were 34.8 and years oldfor groups 1 and 2 respectively.Limitations, reason for caution: The study was conducted during a short periodof time.Wider implications of the findings: This result suggests that the transfer ofembryos at the blastocyst stage would be the best choice for IVF clinics.Study funding/competing interest(s): There were no competing interests in thisstudy.Trial registration number: Not Available

Endometriosis, endometrium, implantation andfallopian tube

P-219 Down-regulation of dipeptidyl peptidase 4 under hypoxia couldenhance endometrial stromal cell migration in endometriosis

C.W. Tan1, Y.H. Lee1, M. Choolani2, H.H. Tan3, L. Griffith4, and J. Chan5

1Singapore-MITAlliance for Research and Technology (SMART), BioSystemsand Micromechanics, Singapore, Singapore Rep. of, 2National University ofSingapore (NUS), Yong Loo Lin School of Medicine, Singapore, Singapore Rep.of, 3KK Women & Children Hospital, Department of Reproductive Medicine,Singapore, Singapore Rep. of, 4Massachusetts Institute of Technology (MIT),Department of Biological & Mechanical Engineering, Boston, U.S.A, 5DukeNUS Graduate Medical School, Cancer & Stem Cell Biology Program,Singapore, Singapore Rep. of

Study question: To determine whether hypoxic microenvironment might be afactor in influencing cell migration of endometrial stromal cells in endometriosisSummaryanswer: Endometrial stromal cells (ESCs) derived from endometrioticpatients could migrate and invade more aggressively than control ESCs underhypoxia and this phenomenon may act through a downregulation of dipeptidylpeptidase 4 (DPPIV/CD26)- involved pathway and enhanced expression ofangiogenesis- related genes.

What is known already: Previous reports showed that hypoxia-inducible-factor-1(HIF-1) was significantly higher in endometriotic women. Besides,increased expression of focal adhesion kinase (FAK) in endometriotic tissuealso alluded to altered cell motility in endometriosis, yet there is little knownabout the relationship between hypoxia and cellular migration in the pathogenesisof such lesions.Study design, size, duration: ESCs were isolated from endometrial curettagesamples obtained from women with endometriosis (Stage IV AFS, n ¼ 3) orother benign gynaecological disease (Stage 0 AFS, n ¼ 3) undergoing laparo-scopic surgery.Participants/materials, setting, methods: Cells were exposed to hypoxia (2%O2) and assayed for motility with Oris Pro Cell migration/ invasion assay. TotalRNA was extracted for PCR Array Human Cell Motility (SA Biosciences).DPPIV/CD26 expression was determined by flowcytometry whereas conditionedmedium was applied to Human Angiogenesis Antibody Array (R&D Systems).Main results and the role of chance: Endometriotic ESCs could migrate andinvade through collagen gel (p , 0.005) more under hypoxic condition as comparedtoESCsderived fromhealthypatients. PCRarray revealeddown-regulationofmigra-tion inhibitors (Fibroblast activation protein (FAP), DPPIV/CD26) in patient ESCsunder hypoxia and was confirmed via flow cytometry (normoxia: 30.62% vshypoxia: 12.37%; p¼ 0.004) Protein array studies showed that angiogenesis-related genes such as TIMP-1, angiogenin and IGFBP-3 was aberrantly upregulatedin endometriotic cells whenbeingexposed tohypoxicenvironment. This could implythat ESCs from endometriosis patients may be enhanced their cell motility underhypoxic microenvironment, leading to up- regulation of migration/ angiogenesis-related genes while keeping the migratory inhibitors at low concentration.Limitations, reason for caution: One limitation is the ability to test downstreamgene expression of DPPIV/CD26 pathway. Since DPPIV/CD26 is proven tomodulate Stromal Cell Derived Factor-1 (SDF-1 / CXCL12) and its receptor,CXCR4, inhibiting DPPIV/CD26 may be possible to test whether DPPIV/CD26 down- regulation of in endometriosis is SDF- 1/ CXCL12-CXCR4 axisdependent.Wider implications of the findings: This is the first study to show a statisticallysignificant difference in DPPIV/CD26 expression associated with this disease.DPPIV/CD26, a membrane-bound extracellular peptidase, is found to be ableto cleave CXCL12 and thereby immobilize hematopoietic stem and progenitorcell (HSCs/HPCs) populations. Overexpressing DPPIV/CD26 to modulateSDF-1 / CXCL12 and their subsequent chemotactic activity may represent newtargets for novel therapies in women with endometriosis.Study funding/competing interest(s): The authors declare no competing finan-cial interests.Trial registration number: N.A.

P-220 Inhibition of annexin A2 by prostaglandin E2 results in reducedphagocytic ability of peritoneal macrophages and contributes to the develop-ment of endometriosis

P.C. Chuang1, M.H. Wu2, Y.J. Lin3, and S.J. Tsai4

1Chang-Gung Memorial Hospital Kaohsiung, Department of Medical Research,Kaohsiung City, Taiwan R.O.C, 2National Cheng Kung University MedicalCollege, Department of Obstetrics and Gynecology, Tainan City, Taiwan R.O.C,3Chung Hwa University of Medical Technology, Department of Nursing, TainanCity, Taiwan R.O.C, 4National Cheng Kung University Medical College,Department of Physiology, Tainan City, Taiwan R.O.C

Study question: Is annexin A2 involved in the reduced phagocytic ability ofmacrophages in endometriosis?Summary answer: Data from women with endometriosis and a murine modelof the disease show that expression of annexin A2 in peritoneal macrophages isinhibited by prostaglandin E2 (PGE2) and this impairs the phagocytic ability ofmacrophages.What is known already: Endometriosis is a chronic inflammatory disease thatrecruits many immune cells, especially macrophages, to the peritoneal cavity.The phagocytic ability of peritoneal macrophages isolated from women withendometriosis is reduced.

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