Conclusion: With sufficient numbers of seminiferous tubule profilesexamined (at least 200 in this study), histology can be considered the goldstandard for the detection of spermatozoa in testicular biopsy samples.The inability to detect spermatozoa by examining wet preparations in 2cases in which spermatozoa could be detected in histological prepara-tions suggests that the current methods used for sperm recovery fromtesticular biopsy samples for ICSI may benefit from further technicalimprovements.

P-400

Sperm Aneuploidy and ART Cycles Failure: Mosaic Germ Cellsor Preferential Non Disjunction? 1M. Ben Khalifa, 1N. Frydman,2R.Franchin, 1S. Hamamah,2R. Frydman,1G. Tachdjian. 1ReproductiveGenetics Unit. BDR Laboratory and2Obstetrics and Gynecology Depart-ment A Beclere Hospital, 157 rue de la Porte de trivaux. 92140 Clamart,France.

Objective and Design: Since thirty years a major aria of cytogeneticsinvestigation has been focused into the nature of genes defect and chromo-somes abnormalities on association with subfertility and reproductive pa-thology. This study was conducted to assess the rate of chromosome aneu-ploidy in semen sample from a male how had 7 ART cycles with very badembryo quality. During the last tentative the sequential culture was termi-nated without transfer. Multicolor Fluorescent In Situ Hybridization (MFISH) was used for chromosomes 8, 9, 13, 18, 21, X and Y to understandthe possible male contribution in embryos development and ART failure.

Materials and Methods: The semen was collected in the laboratory. Afterpure sperm preparation the sample presented 6.4 millions spz, 3.2 millionsof round cells and 20% of mobility. After centrifugation, 2 times washingin distilled water, hypotonic treatment was done in natrium citrate solution,the sperm was fixed in carnoy and spread on slides. For DNA deconden-sation slides were treated with NaOH before probes hybridization. Foraneuploidy assessment, specific probes for chromosomes 8, X and 13 werelabeled directly using FITC, parallely for chromosomes 9, Y, 21 probeswere labeled by Texas Red. For chromosome 18 a centromeric probelabeled with aqua was used. This combination of probe mix and haptenlabeling allowed us to practice double and triple FISH permitting in eachhybridization the use of one chromosome for the internal control. For FISHfour different probes mix were used (8,9), (13,21), (X,Y) and (X, Y,18). Theprotocol of hybridization was done using the hybrite and completed by rapidwashing and staining. Each hybridization was repeated twice and twodifferent observers screened the slides.

Results and Discussion: The results revealed severe unbalanced sex ratiousing X and Y probes. The data from 3000 nuclei showed around 75% ofX sperm and 20% of Y bearing. Triple color FISH, using X, Y and 18probes confirmed this percentage. The disomy rate for chromosomes X andY it was 1.4 and 1.3 respectively. After this preliminary results, the data ofautosomes analysis demonstrated: 3.6% of disomy 13, 2.8% of disomy 21,3% of disomy 8, 2.4% of disomy 9 and 3.7% of disomy 18. These disomyrates confirm an abnormal disjunction of the analyzed chromosomes com-pared to normal sperm. These aneuploidies or the total non-disjunction canexplain the ART cycles failure by producing aneuploid or triploid embryoswith severe disorders incompatible with embryo development and implan-tation. The question is, there are any mosaic germ cells (diploid/triploid) inthe testis and if the answer is yes, what’s the ratio of this mosaicism? Theanalysis of testicular biopsy will be helpful to understand the behavior ofchromosomes segregation during the spermatogenesis. This study illustratesthe interest of sperm aneuploidy analysis in ART failure.

P-401

Clinical Pregnancy Resulting from ICSI with Frozen Testicular Spermin Non Mosaic 47, XXY Patients. R. Wainer,1 M. Bergere,2 M. Albert,2

M. Bailly,1 Y. Ville 1 J. Selva2. Departments of1Gynecology and Obstetricsand 2Reproductive Biology and Cytogenetics, CHI Poissy-St Germain,78303 POISSY, Paris-Ouest University, France.

Objectives: To our knowledge, only seven pregnancies have been re-ported in azoospermic non mosaic 47,XXY patients. The aim of our studywas to evaluate the efficiency of delayed microinjection using frozentesticular sperm from non mosaic 47,XXY patients.

Design: Testis biopsies have been performed in 3 azoospermic 47,XXYpatients and testicular sperm were frozen before ICSI.

Materials and Methods: The following table summarizes the biologicalinvestigations, the biopsy results and ICSI outcome.

Patient 1 Patient 2 Patient 3

Height/weight 176 cm/85 kg 170 cm/65 kg 184 cm/80 kgGynecomasty Mild Absent AbsentTestis volume R:6 ml L:6 ml R:4 ml L:5 ml R:6 ml L:6 mlSperm parameters Azoospermia Azoospermia AzoospermiaKaryotype (blood) 47, XXY 47, XXY 47, XXYXXY FISH (blood cells) 241 216 225DAZ deletion Not deleted Not deleted Not deletedFSH (N,12 ui/l) 30.6 ui/l 33.7 ui/l 26 ui/lTesto (2.8–8 ng/ml) 3.2 ng/ml 4.7 ng/ml 4.18 ng/mlInhibine B 22 pg/ml ,5 pg/ml 15 pg/mlSeminal markers Low Low Low

Results:

Testis biopsy (TB) 6 TB: few motilesperm

4 TB: few motilesperm

8 TB: very few im-motile sperm

ICSI 1 embryo type I trans-ferred

2 embryos type IItransferred

Planned

Pregnancy Evolutive pregnancy(ET :31/10/99)

(2)

Following events Genetic counsellingbut no chromosomalprenatal diagnosis

Next ICSI plannedwith stored frozentesticular sperm

No clinical nor biological parameters could make decision for the oppor-tunity of biopsy which was productive for these 3 patients.

Conclusion: Biopsy followed by delayed microinjection of frozen testic-ular sperm was efficient in 47,XXY non mosaic patients. In cases of nonproductive testis biopsy, it will avoid a useless oocyte retrieval. Moreover,freezing of testicular sperm allows to practice iterative microinjectionattempts instead of a new testis biopsy.

P-402

Conventional In Vitro Fertilization (IVF) and Intracytoplasmic SpermInjection (ICSI) Using Sibling Oocytes: A Strategy for Avoiding Com-plete Fertilization Failure. M. Plachot, J. M. Mayenga, J. Belaisch Allart.CHI Jean Rostand, 141 Grande Rue, 92310 Se`vres, France.

Objectives: In order to avoid complete fertilization failure after IVF orunnecessary use of ICSI, we conducted a study in which couples withmoderate OATS, and those with previous fertilization failure after conven-tional IVF, had half their oocytes inseminated (IVF) and the other halfmicroinjected (ICSI).

Materials and Methods: Fifty nine couples were included in the study: 37had male infertility, 14 had male and female infertility and 8 had had anunexplained fertilization failure or a low rate of fertilization (,20%) inprevious cycles. Sibling oocytes were randomly allocated to IVF or ICSI.

Results: Two of the 59 IVF/ICSI attempts resulted in fertilization failurewith both techniques (3.4%), 16 in fertilization after ICSI only (27.1%) and41 in fertilization with IVF and ICSI (69.5%).Fertilization failure in IVFand ICSI:A low number of oocytes and a high rate of immature and atreticoocytes seem to be the reason of the failure.Fertilization after ICSI only:The characteristics of the semen sample used for IVF and ICSI were asfollows: 26.56 39; 13 106 sperm/ml; 26.96 14.5% motile sperm; 70.8618.1% abnormal forms; 75 oocytes were inseminated, no fertilization oc-curred; 83 sibling oocytes were microinjected: 63.0% were fertilized. Amean of 2.2 embryos per patient were transferred, leading to 8 clinicalpregnancies (50%). The implantation rate was 24.3%.Fertilization in IVFand ICSI:All semen characteristics were slightly better in this group. Of the210 mature microinjected oocytes, 130 were fertilized (61.9% of the mi-croinjected oocytes, 55.8% of the oocytes allocated to ICSI). This rate offertilization was similar to that of the oocytes allocated to IVF (59.7%).About 80% of the embryos had normal morphology in both groups. Re-garding the rate of development, 50.8% of embryos resulting from ICSI and42.9% of embryos resulting from IVF had at least 4 cells on day 2 (notsignificant). For the 41 couples as a whole, the mean number of embryos

S220 Abstracts Vol. 74, No. 3, Suppl. 1, September 2000