Anti mullerian hormone (AMH) level and expression in mural and cumulus cells in relation to age

DESIGN: A prospective controlled study. MATERIALS AND METHODS: Sperm was induced for capacitation with Krebs-Ringer solution containing calcium and BSA or in normal saline. The sperm was assessed for routine markers and for the expression of a2V-ATPase (a2 isoform of vacuolar ATPase). Assessments were made by confocal microscopy and PCR. Immunolocalization to evaluate a2V protein expression, realtime PCR to evaluate gene expression of a2V and different cytokines, sandwich ELISA to evaluate the secretion of a2NTD, which is the secreted portion of a2V. RESULTS: The capacitation of sperm induced a2V transcription and upregulated a2V protein expression. The expression of a2V was only detected in the appropriate capacitation buffer. No transcription or translation products were detected in sperm incubated in normal saline or PBS. Minimal expression of a2V was measured in Krebs-Ringer buffer without calcium or BSA. Capacitated sperm released a2NTD which can induce IL-1b, TNFa and MCP-1 (Monocyte chemotactic protein 1) expression. The results are compared between fertile couples and those being seen for treatment for recurrent pregnancy loss. CONCLUSION: We have previously reported that sperm capacitation induces the expression of the immune regulatory molecule a2V, which in turn causes the induction of inflammatory cytokines in the uterus. Capacitated sperm initiates the immunity of pregnancy by inducing a2V that in turn induces inflammatory cytokine expression during onset of pregnancy. We demonstrate that the maternal-fetal immune dialogue begins in the male with the capacitation of sperm and could be an immune predictor of fertility in women. O-324 Wednesday, October 19, 2011 05:30 PM GENOME-WIDE IDENTIFICATION OF CHLAMYDIA TRACHOMATIS ANTIGENS ASSOCIATED WITH TUBAL FACTOR INFERTILITY. N. M. Budrys, A. K. Rodgers, S. Gong, A. Holden, R. S. Schenken, G. Zhong. Obstetrics and Gynecology, University of Texas Health Science Center, San Antonio, TX; Microbiology and Immunology, University of Texas Health Science Center, San Antonio, TX. OBJECTIVE: To identify Chlamydia trachomatis (CT) antigens associated with tubal factor infertility using a genome-wide proteome array. DESIGN: Subjects were assigned to the tubal factor infertility (TFI) or infertile control (IC)group based on results of pelvic laparoscopy. CT antibody response profiles were assessed to identify antigens associated with tubal pathology. MATERIALS AND METHODS: 31 TFI and 23 IFC patients from a university-based fertility clinic were enrolled after IRB approval. Patients with a history of appendicitis or endometriosis were excluded. Serum samples were analyzed for CT titre utilizing immunofluorescence assay. The antibody recognition of each of 933 CT antigens was independently confirmed using enzyme-linked immunosorbent assay. Log transformation of data was used for normalization prior to analysis with either Student T-test or Fisher’s Exact test. RESULTS: A greater percentage of patients in the TFI group had high antibody titers to CT (>1:10,000) compared to the IC group (61% vs 4.4%, p < 0.001). A total of 30 antigens were preferentially recognized by antibodies from the TFI group with a detection sensitivity and specificity of 80.6% and 56.5%, respectively, with 10 antigens showing 100% specificity. A combination of CT443 and CT381 yielded the highest sensitivity (67.7%) while maintaining 100% specificity, when compared with single antigens alone or in combination. CONCLUSION: Our findings show that antibodies to CT443 and CT381, when used in combination, have higher sensitivity and specificity in prediction of TFI than other indicators for TFI such as HSP60 (35.5%, 100%) or hysterosalpingogram (65%, 83%). In the future, utilization of a serum antibody panel may lead to a more reliable diagnosis of TFI which does not require expensive, invasive methods for diagnosis. Supported by: This work was supported in part by NIH grant R01AI64537 (to G. Zhong).

REPRODUCTIVE ENDOCRINOLOGY: RESEARCH O-325 Wednesday, October 19, 2011 03:45 PM WHAT IS THE MAJOR ESTROGEN BINDING PROTEIN IN THE FOLLICULAR FLUID? Y. Bentov, N. Esfandiari, R. F. Casper. Toronto Centre for Advanced Reproductive Technology, Torornto, ON, Canada; Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON, Canada; University of Toronto, Division of Reproductive Sciences, Department of Obstetrics and Gynecology, Toronto, ON, Canada. OBJECTIVE: Estrogens orchestrate multiple changes in gonadal and extragonadal tissues directly and indirectly important for reproduction. The main site of estrogen production is the granulosa cells of the ovarian follicle, with concentration of estrogen in follicular fluid (FF) 1000-fold higher than in serum. In se-

FERTILITY & STERILITYÒ

rum, almost all estrogen is bound to sex hormone binding globulin (SHBG) and albumin. We have previously shown that estrogen binding in the FF is maintained by the action of a yet unknown binding protein other than albumin or SHBG. The objective of this study was to determine if other estrogen binding proteins are responsible for maintaining the high level of estrogen in FF. DESIGN: Proteomic analysis of FF MATERIALS AND METHODS: Samples of FF were collected from women undergoing IVF treatment. FF was stripped using charcoal coated dextran and then incubated with biotinylated 17-b-estradiol. The FF was then passed through a Softlink Avidin column. We collected the flow-through (FT), and then washed the column with buffer to remove nonspecifically bound proteins, followed by a biotin solution wash (BW) to remove specifically bound proteins. The native TT, FT and BW were then analyzed with Mass-spectrometry. RESULTS: The protein with the highest rate of enhancement with biotin was basement membrane-specific heparan sulfate proteoglycan core protein (Perlecan). The concentrations in the native FF, FT and BW were 11, 0, and 325 nmol/L, respectively, representing a 30 fold enhancement. CONCLUSION: Perlecan was shown to be present in FF and is known to be synthesized by granulosa and cumulus cells. Its synthesis is stimulated by FSH and correlates with follicular maturation. It was previously speculated to have a physiological role in folliculogenesis and oocyte maturation. Our current finding suggests another role for Perlecan as the main estrogen binding protein in the follicle. The physiologic significance of enhanced estrogen binding in the FF is unknown but may protect the oocyte from excessive free estrogen. O-326 Wednesday, October 19, 2011 04:00 PM PROGESTERONE STIMULATES PITUITARY ADENYLATE CYCLASE-ACTIVATING POLYPEPTIDE (PACAP) EXPRESSION IN PITUITARY GONADOTROPES. J. L. Morgan, C. M. Grafer, C. Wang, L. M. Halvorson. Obstetrics and Gyencology, University of Texas Southwestern Medical Center, Dallas, TX. OBJECTIVE: PACAP has been shown to have widespread distribution and varying roles throughout the reproductive axis. In the anterior pituitary, PACAP secretion is well known to influence gonadotropin synthesis and release. Our objective was to investigate a potential role for progesterone in the regulation of pituitary PACAP expression. DESIGN: Progesterone-stimulated expression of the PACAP gene was analyzed in vitro. MATERIALS AND METHODS: 1) Gonadotrope-derived LbT2 cells were co-transfected with rat PACAP promoter-luciferase vectors and a progesterone receptor B (PR-B) expression construct and then treated with progesterone (100nM x 24h). A Renilla reporter vector controlled for transfection efficiency. 2) LbT2 cells were treated with progesterone (100nM x 6 or 24h) or vehicle followed by qPCR measurement of PACAP mRNA levels. RESULTS: Progesterone increased the full length -1906/+906 PACAP promoter activity in a dose-dependent manner with a peak of 14-fold at 300 nM. Progesterone-stimulated PACAP promoter activity was blunted with deletion to position -402 (52% of full length, P<0.001) and eliminated with further truncation to position -77 relative to the transcriptional start site (7% of full length, P<0.001). Further dissection of region -402 to -77 showed a significant decrease in PACAP responsiveness with deletion from position -308 to -242 (64% of the -402 construct, P<0.001) with further loss following deletion to position -171 (16% of the -402 construct, P<0.001). In addition to transcriptional activation, progesterone treatment increased endogenous PACAP mRNA levels by 3- to 5-fold at 6 and 24 hrs. CONCLUSION: Our results show that progesterone increases pituitary PACAP promoter activity and mRNA expression. These findings suggest a novel mechanism for ovarian steroid modulation of pituitary function, which ultimately influences gonadotopin gene expression and reproductive homeostasis. Supported by: R01HD054782

O-327 Wednesday, October 19, 2011 04:15 PM ANTI MULLERIAN HORMONE (AMH) LEVEL AND EXPRESSION IN MURAL AND CUMULUS CELLS IN RELATION TO AGE. A. Kedem, Y. Yung, H. Kanety, M. Hanochi, J. Dor, A. Hourvitz. Department of Obstetrics and Gynecology, In Vitro Fertilization Unit and Human Embryonic Stem Cell and Reproduction Lab, Sheba Medical Center, Israel. Sackler School of Medicine, Tel Aviv University, Tel Aviv Israel, Ranat-Gan, Israel; Endocrinology Institute, Ranat-Gan, Israel. OBJECTIVE: Anti-Mullerian hormone (AMH) has recently been shown to be one of the most important markers of ovarian reserve.

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Serum AMH is declining with age and is highly associated with ovarian follicular development. However, the precise role of AMH in the process of human follicular aging still has to be determined.This study investigates AMH level in the follicular fluid (FF) and mRNA expression pattern in cumulus and mural cells of human ovarian follicles in relation to age. DESIGN: Prospective study. MATERIALS AND METHODS: Sixty nine women undergoing In vitro fertilization (IVF) treatment in our unit were enrolled in the study. FF from large preovulatory follicles (16-24mm) of 21-36 years old women (n ¼ 40) and 40-45 years old women (n ¼ 29) were aspirated during oocyte pickup. FF AMH levels were determined using ELISA method and AMH expression pattern in human mural and cumulus granulosa cells (Gcs) were analyzed by Real Time PCR, normalized to b-actin, and expressed in arbitrary units. RESULTS: AMH mRNA expression in cumulus cells was significantly higher then in mural granulosa cells in both age groups. Interestingly, higher AMH mRNA expression in cumulus cells was observed in the older age group then in the younger group (22.3  5.1vs 5.6  2.2, P <0.01 respectively). AMH mRNA expression in mural cells was not significantly different among both age groups. In addition FF AMH levels were significantly higher in the older group than in the younger group (4.7  1.1 ng\ml and 2.3  0.2 ng\ml respectively, P<0.002). CONCLUSION: To our knowledge, this is the first report which compares AMH level and expression pattern in human cumulus and mural cells at different age groups. This study shows a sharp increase in cumulus expression with increasing age and higher FF AMH level in the older age group then in the younger age group. This remarkable correlation between AMH mRNA levels in cumulus cells in respect to age suggests that AMH may be involved in follicular aging process.

O-328 Wednesday, October 19, 2011 04:30 PM ALTERATIONS IN RETINOID SIGNALING IN ENDOMETRIOSIS MAY LEAD TO DIFFERENCES IN DECIDUALIZATION. M. E. Pavone, M. Dyson, E. Pearson, T. Kakinuma, S. E. Bulun. Obstetrics and Gynecology, Northwestern University, Chicago, IL. OBJECTIVE: Decidualization alters molecular pathways in endometrial stromal cells to permit successful embryo implantation. We have previously reported significant differences in retinoid uptake, metabolism and action in endometriosis compared to normal endometrium. Here we characterize retinoid signaling during decidualization in human stromal cells from normal endometrium (HESC) and endometriosis (OSIS). DESIGN: Basic Science MATERIALS AND METHODS: Primary HESC and OSIS were isolated, cultured and treated with MPA, E2, and 8-Br-cAMP over 2 weeks to induce decidualization. Protein and mRNA expression of genes responsible for retinoid metabolism and trafficking including STRA6, CRBP1, ALD1A2, CRABP2, FABP5, RARa, RXRa, PPARb/d and CYP26B1 were examined using RT-PCR and western blotting. Decidualization markers including prolactin and IGFBP1 were examined using RT-PCR and ELISA. Knockdown of CRABP2 prior to inducing decidualization was also performed. RESULTS: ESC and OSIS expressed all intracellular proteins involved in retinoid uptake and metabolism all time points (n ¼ 5). However, decidualization significantly reduced expression of the genes responsible for RA uptake and shuttling to the nucleus (P<0.05). Furthermore, CYP26B1, which inactivates RA, was strongly induced during the same period (P<0.05). Expression of RAR family members did not change. Knockdown of CRABP2 significantly decreased decidualization markers. In OSIS, there was an overall decreased expression of genes involved in RA uptake and shuttling, as well as markers of decidualization (P<0.05). However, CYP26B1 expression was still induced. CONCLUSION: Progesterone alters RA trafficking in ESC during decidualization to favor paracrine rather than intacrine signaling, which may enhance signaling to the adjacent epithelium. This action is blunted in endometriosis. These alteration in retinoid signaling may help explain the decidualization differences seen in endometriosis. Supported by: K12HD050121, ASRM Career Development Award (to MEP), R37HD038691 (to SEB)

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Abstracts

O-329 Wednesday, October 19, 2011 04:45 PM MITOCHONDRIAL PROGESTERONE RECEPTOR (PR-M) DISTRIBUTION IN UTERINE LEIOMYOMA AND MYOMETRIUM AND IMPACT ON MITOCHONDRIAL MEMBRANE POTENTIAL (MMP) IN IMMORTILIZED HUMAN MYOMETRIAL CELLS (HTERT-HM). J. R. Crochet, Q. Feng, Q. Dai, P. C. Leppert, T. M. Price. Obstetrics and Gynecology, Duke University Medical Center, Durham, NC; Obstetrics and Gynecology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China. OBJECTIVE: A mitochondrial progesterone receptor (PR-M) increases cellular respiration and MMP. We define the distribution of PR-M in uterine fibroid and myometrial tissue and study its impact on cellular respiration in hTert-HM cells. DESIGN: Basic science MATERIALS AND METHODS: Western blots were performed with total protein from the edge of a human fibroid and adjacent myometrium in 10 subjects with polyclonal PR (C-19) and monoclonal mitochondrial porin and GAPDH antibodies. Transcript levels of PR-M, nPR A+B and GAPDH were compared in 5 subjects with real-time RT-PCR. hTert-HM cells were treated with PR agonist R5020, and MMP was determined by JC-1 assay. Specificity and non-genomic action were determined by treatment with PR antagonist RTI 6413-049b, dexamethasone (DEX) and the translation inhibitor cycloheximide (CHX). Additional MMP studies were performed with pGFP-N1-PRM transfected cells. RESULTS: PR-M protein was greater in fibroid tissue compared to adjacent myometrium in 9 of 10 subjects and significantly greater overall (P<0.001). Porin expression showed the same pattern (P<0.05). There was no difference in nPR A+B expression. PR-M transcript levels were greater in the myometrium in 3 of 5 subjects and significantly greater overall (P<0.05). There was no difference in nPR A+B transcript levels. A dose-dependent increase in MMP was seen in hTert-HM cells treated with R5020 compared to control (P<0.0001). R5020+RTI showed no increase in MMP. There was no change in MMP following DEX treatment. CHX did not block the R5020-induced increase in MMP. Cells overexpressing PR-M had a greater R5020-induced MMP compared to empty vector (P<0.05). CONCLUSION: 1) Fibroid edge has greater expression of PR-M, and a greater number of mitochondria compared to adjacent myometrium. 2) There is a non-genomic, progestin-induced increase in cellular respiration in hTert-HM cells. 3) Overall, an increase in cellular respiration via PR-M may be a major factor in the progestin-dependent growth of fibroids. Supported by: Susan Fiery Hughes Foundation

O-330 Wednesday, October 19, 2011 05:00 PM IN VIVO IMPACT OF DOXORUBICIN ON OVARIAN GERM CELL AND NON-GERM CELL POPULATION: DOUBLE STRAND DNA BREAKS AND MICROVASCULAR DAMAGE. R. Soleimani, E. Heytens, K. Oktay. Department of Obstetrics & Gynecology, Laboratory of Molecular Reproduction, Institute for Fertility Preservation, New York Medical College, Valhalla, NY; Department of Cell Biology & Anatomy, New York Medical College, Valhalla, NY. OBJECTIVE: Doxorubicin (doxo) is frequently used in the treatment of cancers common to young females but its impact on ovarian function is not fully understood. We investigated the in vivo impact of doxo on primordial follicle (PF) reserve and the molecular mechanisms of this damage. DESIGN: Experimental MATERIALS AND METHODS: Ten days after xenografting human ovarian cortical strips (OCS) (n ¼ 12), doxo 10 mg/kg was administered iv. Grafts were recovered 24h later and immunostained for g-H2AX (double strand DNA break-DSB-marker), cleaved caspase-3 (apoptosis), and CD31 (vascular marker). Total and apoptotic PF densities were also compared in ovaries of doxo-treated SCID mice vs. vehicle-treated controls (n ¼ 6). The effect of doxo was further studied by confocal microscopy in GVoocytes and preantral follicles isolated from doxo vs vehicle-treated mice. RESULTS: The % of gH2AX-positive (58.1  1.0 vs. 15.6  0.9 %, P<0.001) and apoptotic PFs were significantly higher in doxo-treated xenografts compared to controls (55.4  5.7 vs. 11.8  5.7 %, P<0.001). Doxo resulted in reduced vascular density compared to controls (6.4  1.9 vs. 13.9  2.1/mm2, P<0.001). In addition, doxo treatment significantly reduced PF density in mice (4.31  2.1 vs. control 10.57  6.7/mm2, P<0.001), and similar to its effect in human ovarian xenografts in the same mice, resulted in

Vol. 96., No. 3, Supplement, Sepetmber 2011