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176 Posters / Journal of Reproduc

Results: In comparison to the control healthy popu-lation, in prepubertal boys with cryptorchidism a trendfor lower representation HLA-DRB1*08 and HLA-DQB1*04was observed. Prepubertal boys suffering from bilateralcryptorchidism differed from controls in HLA-DRB1*11 fre-quency (p = 0.0338). Boys with history of infertility in afamily exhibited highly significant difference (p = 0.0005,odds ratio 8.167) from control in HLA-DRB1*11 frequency.No significant differences were seen between healthyprepubertal boys and prepubertal boys with antispermantibodies. However statistically significant differenceswere observed in HLA-DRB1*04 (p = 0.042) and HLA-DQB1*06 (p = 0.0329) when allelic frequencies of boysunder study with and without antisperm antibodies werecompared.

Conclusions: Some prepubertal boys with (differencesin allelic class II differences in respect to unilateraland bilateral) cryptorchidism demonstrate detectable andstatistically significant circulating antisperm antibodies.There was found a strongly significantly higher frequencyof DRB1*11 allele in patients with cryptorchidism infamilies with history of infertility. It is plausible that cryp-torchidism associated with infertility may present distinctyet unidentified genetic background.

doi:10.1016/j.jri.2011.06.083

P25

Analysis of granulosa cells by single cellPCR—establishment of an in vitro model system

J. Pastuschek a,∗, S. Hoelters a, S. Neubeck a, J.S. Fitzgerald a,E. Schleussner a, C. Holzhauer b, G. Barthel b, R. Kirchner b,M. Alunni b, U.R. Markert a

a Placenta-Lab, Department of Obstetrics, University HospitalJena, Germanyb Beckman Coulter Biomedical GmbH, Munich, Germany

Background: Granulosa cells compose a layer betweentheca cells and the oocyte within the ovarian follicle.They support the development of follicles through “bi-directional communication” between oocyte/granulosacells and granulosa/theca cells. This communication ismoderated by regulatory proteins, mostly members of theTGF-ß and the IGF family. Understanding these complexcommunicatory signals may serve for a better under-standing of folliculargenesis. Furthermore, these moleculesmight serve as developmental markers for assessing ovar-ian follicles or oocyte “quality”.

Methods: Single granulosa cells, either human primarycells from follicular fluid (obtained during IVF treat-ments) or from the immortalised cell line COV434, weremicromanipulated on the AmpliGrid platform® (BeckmanCoulter Biomedical GmbH). Subsequent single cell mul-tiplex OneStep RT-PCR analysis of 5 specific genes ofthe TGFß and IGF mediator families, and addition of oneleukocyte (contamination control) and one housekeeping

gene (technical control) were performed. The differentialgene expression pattern of single cells was analysed byPAGE and silver staining and compared according to var-

unology 90 (2011) 164–183

ious culturing circumstances prior to micromanipulation(alone vs. pooled, varying culture periods). The method hasbeen established on granulosa cell RNA using the OneStepRT-PCR kit (Qiagen) and is being optimized to allow stan-dardization for future clinical use.

Results: Preliminary analyses of single human primarygranulosa cells obtained during IVF treatments suggesteddiffering inhomogeneous gene expression patterns forInhibin B� and Bß, anti-Müllerian hormone receptor 2 andthe IGF-binding-protein-1/5, while pooled cells showedidentical homogenous and more complex patterns. Cul-ture of these primary granulosa cells altered the expressionof the above-mentioned genes. Further preliminary mRNAanalyses of an immortalised COV434 granulosa cell line(pooled cells), which were used to established an in vitromodel system for granulosa cell studies, suggested cleardiscriminative expression patterns between the COV434cell line and follicular fluid derived granulosa cells. How-ever experiments with RNA templates of both sources onthe AmpliGrid system demonstrated differences within theset of replicates. To overcome this technical problem ahousekeeping gene as an internal control was integratedto optimize the multiplex PCR system and further geneexpression profile comparisons on the basis of single cells– either primary or cell line derived – were performed.

Conclusions: Follicular fluid derived single human pri-mary granulosa cells show a heterogeneous expressionpattern. This information suggests that single primarygranulosa cells act differentially. For future studies, an arti-ficial system with the COV434 cell line will be developedin which the granulosa cell marker panel will be associatedwith oocyte quality.

doi:10.1016/j.jri.2011.06.084

FETAL SEX-SPECIFIC IMMUNITY IN EARLY PREGNANCY

P26

Immunological features of male and female fetusespregnancies

Y. Goncharova a,∗, I. Sudoma a,c, B. Donskoy b

a Clinic of reproductive medicine NADIYA, Kyiv, Ukraineb Laboratory of Immunology, Institute of Pediatrics, Obstet-rics and Gynecology, National Academy of Medical Sciences ofUkraine, Kiev, Ukrainec National Medical Academy for Postgraduate EducationNamed After P. Shupyk, Kiev, Ukraine

Introduction: The differences in male (M) and female(F) fetus and newborn growth are well known and arethought to be connected to the distinction of androgenand other sex-related products level. At the same time, thevariations in M and F embryos development at preimplan-tation and implantation stages have been shown. M and Fembryos have different metabolism. On the other hand, itwas found that M fetuses are more likely to survive than

F in allo-immune recurrent spontaneous abortion (RSA)due to allo-immune reproductive wastage of chromosoma-lly normal F concepti in early human pregnancy, and that

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llo-immune RSA makes up the highest proportion of unex-lained RSA. So, it can be supposed that immunologicalechanisms of fetus sex recognition exist.Material and methods: Immunological parameters of

0 pregnant women (30 women with IVF ICSI pregnanciesnd 50 women with natural conception) were retrospec-ively analyzed in 2 groups depending on fetus sex, with

ale fetus pregnancies (MP; 37 cases) and female fetusregnancies (FP; 43 cases). All examined pregnancies wereingletons with unremarkable course and healthy termelivery.

Blood collection for immunological evaluation waserformed at 7–12 weeks of gestation. Lymphocyte sub-ets, Treg cells (CD4+CD25 high CD127 low), intracellularytokines in CD4+ cells (IFN�, IL-4, TNF, IL-10), expres-ion of activating molecules (CD69, HLA-DR, CD95) andhemokine receptors (CXCR3, CCR4) were quantified byow cytometry.

Results: The mean age of women with IVF pregnanciesas 35.1 years; the mean age of women with natural preg-ancies was 33.7 years (p < 0.07). The M to F ratio in the

VF group was 1.5 (18 M, 12 F), mean delivery term in Mnd F was 38.7 and 39.4 weeks, mean newborn weightas 2915 g and 3263 g (р < 0.1), mean baby length was

0.5 and 51.0 cm respectively; in the natural pregnancyroup the M to F ratio was 0.61 (19 M, 31 F), mean deliv-ry term was 39.9 and 39.1 weeks, mean newborn weightas 3770 g and 3233 g (р < 0.01), mean baby length was

3.0 and 51.0 cm (р < 0.01). The F babies in IVF and natu-al pregnancies did not differ by weight, but the M babiesn the IVF group had a significantly lower weight than Mabies in natural pregnancies. We did not find any signif-

cant difference between MP and FP in T cells, B cells, NKells, CD4+ cells or CD8+ lymphocytes or in expression ofctivating markers (CD69, CD25, and HLA-DR), chemokineeceptors (CXCR3 and CCR4), intracellular cytokines (IFN�,L-4, TNF, and IL-10) or NK cell cytotoxicity. A significantifference between the MP and FP was observed in the

evels of KIR receptors (CD158a) on CD3+CD56+ cells. Theean percentage of CD3+CD56+CD158a+ cells was 16.8% inP and 9.57% in FP (p < 0.05). Qualitative analysis of CD158a

xpression on CD3+CD56+ cells shows that 11/37 (29.7%)P and only 4/47 (9.3%) FP had a pronounced (more than

5%) increase in CD3+CD56+CD158a+ cells. The differencen CD158a expression between M and F fetus pregnanciesemained significant in both IVF and natural pregnancies.

Conclusions: It may be assumed that the detectedncrease in CD3+CD158a+ cells could be the result of mater-al immune system response to the foreign (to the female

mmune system) products encoded by the fetal Y chromo-ome. This might contribute to a physiological mechanismf reproduction and gender-population balance control ormmunological sex control.

Keywords: Sex; Pregnancy; Immunity; KIR receptor; CD58a

oi:10.1016/j.jri.2011.06.085

unology 90 (2011) 164–183 177

INFLAMMATION AND EARLY PREGNANCY LOSS

P27

Individual dynamic immune parameters before preg-nancy and in first trimester of non-complicated andcomplicated IVF pregnancy

V. Chernyshov a,∗, I. Sudoma b,c, B. Dons’koi a, A. Chernov a,A. Kostyuchyk a, Y. Goncharova b

a Laboratory of Immunology, Institute of Pediatrics, Obstet-rics and Gynecology, National Academy of Medical Sciences ofUkraine, Kiev, Ukraineb Reproductive Medicine Clinic “Nadiya”, Kiev, Ukrainec National Medical Academy for Postgraduate Education, Kiev,Ukraine

Introduction: Previous studies from our laboratoryhave shown some prognostic changes in immune param-eters observed in the first trimester of complicated IVFpregnancy. Our research question is whether the changesare related to pregnancy or inherent to the individualfemale irrespective of pregnancy. Individual immune vari-ability before and during pregnancy is one of the possibleway to understand immune mechanisms of human preg-nancy.

Materials and methods: Blood samples for analysiswere taken from 91 women before pregnancy, in earlyphase (5–7 gestational weeks) and late phase (9–12 weeks)of the first trimester of IVF pregnancy. Retrospectivelywomen were divided into two groups: 62 women withnon-complicated pregnancy (NCP) and 29 women withobstetric complications (OC) including threatened abor-tion before 22 weeks (n = 17), missed abortion (n = 5),or preeclampsia (n = 7). Lymphocyte subsets, Treg cells(CD4+CD25 high CD127 low), intracellular cytokines inCD4+ cells (IFN�, IL-4, TNF, IL-10) and expression of acti-vating molecules (CD69, HLA-DR, CD95) and chemokinereceptors (CXCR3, CCR4) were quantified by flow cytom-etry.

Results: High initial variability in values of immuneparameters before pregnancy and individual tendency tonormalization in 1st trimester was observed in both groupsof women. Percentage levels of CD56+CD8+CD3− cellshad a strong tendency to decrease in NCP women andto increase in OC women in 1st trimester of pregnancy.Absolute values of leucocytes, granulocytes, CD3+DR+ andCD3+CD69+ cells were elevated in both groups, but moreactivation markers were seen in OC women (CD69+CD4+;CD69+CD8+; IFN�+CD4+; CD25+CD4+; CD8+CD3+ cells).The percentage levels of granulocytes were elevated andlymphocytes were accordingly decreased in both groups. InNCP TNF+CD4+ and CD19+CD5+ cells were decreased and inthe OC group, CD3+CD69+ and CD4+CD69+ cells were ele-vated. Absolute values of lymphocytes were higher in NCPbefore pregnancy compared with the OC group. When wecompared the percentage values of the two groups, higherlevels of CCR4+CD4+ and CD4+CD25+ (but not CD127−)

cells were observed in OC women before pregnancy. Adecrease in NCP and an elevation in OC, with greater dif-ferences in the latter parameter between the two groups ofwomen in the 1st trimester were detected.