Dimeric Inhibin Research Articles

Early pregnancy prediction of gestational diabetes mellitus risk using prenatal screening biomarkers in nulliparous women

AimsTo evaluate the clinical utility of first and second trimester prenatal screening biomarkers for early pregnancy prediction of gestational diabetes mellitus (GDM) risk in nulliparous women. MethodsWe conducted a population-based cohort study of nulliparous women participating in the California Prenatal Screening Program from 2009 to 2011 (n = 105,379). GDM was ascertained from hospital discharge records or birth certificates. Models including maternal characteristics and prenatal screening biomarkers were developed and validated. Risk stratification and reclassification were performed to assess clinical utility of the biomarkers. ResultsDecreased levels of first trimester pregnancy-associated plasma protein A (PAPP-A) and increased levels of second trimester unconjugated estriol (uE3) and dimeric inhibin A (INH) were associated with GDM. The addition of PAPP-A only and PAPP-A, uE3, and INH to maternal characteristics resulted in small, yet significant, increases in area under the receiver operating characteristic curve (AUC) (maternal characteristics only: AUC 0.714 (95% CI 0.703–0.724), maternal characteristics + PAPP-A: AUC 0.718 (95% CI 0.707–0.728), maternal characteristics + PAPP-A, uE3, and INH: AUC 0.722 (0.712–0.733)); however, no net improvement in classification was observed. ConclusionsPAPP-A, uE3, and INH have limited clinical utility for prediction of GDM risk in nulliparous women. Utility of other readily accessible clinical biomarkers in predicting GDM risk warrants further investigation.

Plasma concentrations of dimeric inhibin and oestradiol in heifers undergoing superovulation with eCG or FSH

Plasma concentrations of dimeric inhibin and oestradiol in heifers undergoing superovulation with eCG or FSH

Quad Screen Test, A Multiplexed Biomarker Assay for Prenatal Screening to Assess Birth Defects: The Columbia University Experience Using the Beckman Access2 Immunoassay Analyzer and Benetech PRA.

In the prenatal quad screen, the levels of four analytes in maternal serum are used to calculate the risk of serious birth defects. The Beckman Access2 Immunoassay System is an automated analyzer that enables rapid measurement of alpha-fetoprotein, unconjugated estriol, human chorionic gonadotropin, and dimeric inhibin A. The Benetech PRA software package is used to convert maternal serum analyte concentrations to multiples of the median (MoM) and calculates the risks of particular birth defects. The results from this simple and minimally invasive screen determine the need for more sensitive, specific, and usually riskier diagnostic procedures. We present herein some recent data from our experience at Columbia University Medical Center in New York, NY, using the Beckman Access2 immunoassay analyzer and Benetech PRA software package.

Inhibin Is a Novel Paracrine Factor for Tumor Angiogenesis and Metastasis.

Inhibin is a heterodimeric TGFβ family ligand that is expressed in many cancers and is a selective biomarker for ovarian cancers; however, its tumor-specific functions remain unknown. Here, we demonstrate that the α subunit of inhibin (INHA), which is critical for the functionality of dimeric inhibin A/B, correlates with microvessel density in human ovarian tissues and is predictive of poor clinical outcomes in multiple cancers. We demonstrate that inhibin-regulated angiogenesis is necessary for metastasis. Although inhibin had no direct impact on tumor cell signaling, both tumor cell-derived and recombinant inhibin elicit a strong paracrine response from endothelial cells by triggering SMAD1/5 activation and angiogenesis in vitro and in vivo Inhibin-induced angiogenesis was abrogated via anti-inhibin α antibodies. The endothelial-specific TGFβ receptor complex comprising ALK1 and endoglin was a crucial mediator of inhibin signaling, offering a molecular mechanism for inhibin-mediated angiogenesis. These results are the first to define a role for inhibin in tumor metastasis and vascularization and offer an antibody-based approach for targeting inhibin therapeutically.Significance: Inhibin is a predictor of poor patient survival in multiple cancers and is a potential target for antiangiogenic therapies. Cancer Res; 78(11); 2978-89. ©2018 AACR.

Role of FSH glycan structure in the regulation of Sertoli cell inhibin production.

Variations in follicle-stimulating hormone (FSH) carbohydrate composition and structure are associated with important structural and functional changes in Sertoli cells (SCs) during sexual maturation. The aim of the present study was to investigate the impact of FSH oligosaccharide structure and its interaction with gonadal factors on the regulation of monomeric and dimeric inhibin production at different maturation stages of the SC. Recombinant human FSH (rhFSH) glycosylation variants were isolated according to their sialylation degree (AC and BA) and complexity of oligosaccharides (CO and HY). Native rhFSH stimulated inhibin α-subunit (Pro-αC) but did not show any effect on inhibin B (INHB) production in immature SCs isolated from 8-day-old rats. Activin A stimulated INHB and had a synergistic effect on FSH to stimulate Pro-αC. The less acidic/sialylated rhFSH charge analogues, BA, were the only charge analogue mix that stimulated INHB as well as the most potent stimulus for Pro-αC production. Native rhFSH stimulated both Pro-αC and INHB in SCs at a more advanced maturation stage, isolated from 20-day-old rats. In these cells, all rhFSH glycosylation variants increased INHB and Pro-αC production, even in the presence of growth factors. The BA preparation exerted a more marked stimulatory effect on INHB and Pro-αC than the AC. Glycoforms bearing high mannose and hybrid-type oligosaccharides, HY, stimulated INHB and Pro-αC more effectively than those bearing complex oligosaccharides, CO, even in the presence of gonadal growth factors. These findings demonstrate the modulatory effect of FSH oligosaccharide structure on the regulation of inhibin production in the male gonad.

Expanded conventional first trimester screening.

ObjectiveThe study aims to determine the performance of a five (5) serum marker plus ultrasound screening protocol for T21, T18 and T13.MethodSpecimens from 331 unaffected, 34 T21, 19 T18 and 8 T13 cases were analyzed for free Beta human chorionic gonadotropin, pregnancy‐associated plasma protein A, alpha‐fetoprotein, placental growth factor and dimeric inhibin A. Gaussian distributions of multiples of the median values were used to estimate modeled false positive and detection rates (DR).ResultsFor T21, at a 1/300 risk cut‐off, DR of screening with all five serum markers along with nuchal translucency and nasal bone was 98% at a 1.2% false positive rate (FPR). Using a 1/1000 cut‐off, the DR was 99% with a 2.6% FPR. For T18/13 with free Beta human chorionic gonadotropin, pregnancy‐associated plasma protein A, placental growth factor and nuchal translucency at a 1/150 cut‐off, DR was 95% at a 0.5% FPR while at a 1/500 risk cut‐off, DR was 97% at a 1.2% FPR.ConclusionAn expanded conventional screening test can achieve very high DRs with low FPRs. Such screening fits well with proposed contingency protocols utilizing cell‐free DNA as a secondary or reflex but also provides the advantages of identification of pregnancies at risk for other adverse outcomes such as early‐onset preeclampsia. © 2017 Eurofins NTD, LLC. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Autoimmune Disorders during Pregnancy: Effects of Biomarkers on Maternal Serum Screening and Course of Disease

Pre-existing autoimmune disease during pregnancy is infrequent occurring in 1.0-2.0% of pregnant women. Autoimmune diseases (ADs) are not lethal and can co-exist with other autoimmune types. In the present report, ADs were found to produce false positive maternal serum screens for both neural tube defects and Down syndrome. The course of ADs is highly influenced by maternal and placental proteins, hormonal factors, and cytokines. Such agents appear to serve as protective factors at the induction and effector stages of the immune response during pregnancy. During second and third trimester pregnancies and in postpartum, autoimmune-afflicted patients experience stages of remissions and relapses. Most remissions occur in the second and third trimesters when soluble immunoprotective factors attain peak levels. Some plausible immunosuppressive factors include quad biomarkers and pregnancy associated glycoproteins such as alpha-fetoprotein, human chorionic gonadotrophin, dimeric inhibin-A, and unconjugated Estriol. The mechanism of remission of ADs is not fully understood but is associated with induction of an immunosuppressive and/or immunotolerant state coincident with the presence of known and unknown soluble factors. Keywords: Alpha-fetoprotein, autoimmunity, dimeric inhibin, human chorionic gonadotrophin, pregnancy, unconjugated Estriol.

Maternal characteristics and mid-pregnancy serum biomarkers as risk factors for subtypes of preterm birth.

To examine the relationship between maternal characteristics, serum biomarkers and preterm birth (PTB) by spontaneous and medically indicated subtypes. Population-based cohort. California, United States of America. From a total population of 1004039 live singleton births in 2009 and 2010, 841665 pregnancies with linked birth certificate and hospital discharge records were included. Characteristics were compared for term and preterm deliveries by PTB subtype using logistic regression and odds ratios adjusted for maternal characteristics and obstetric factors present in final stepwise models and 95% confidence intervals. First-trimester and second-trimester serum marker levels were analysed in a subset of 125202 pregnancies with available first-trimester and second-trimester serum biomarker results. PTB by subtype. In fully adjusted models, ten characteristics and three serum biomarkers were associated with increased risk in each PTB subtype (Black race/ethnicity, pre-existing hypertension with and without pre-eclampsia, gestational hypertension with pre-eclampsia, pre-existing diabetes, anaemia, previous PTB, one or two or more previous caesarean section(s), interpregnancy interval ≥60months, low first-trimester pregnancy-associated plasma protein A, high second-trimester α-fetoprotein, and high second-trimester dimeric inhibin A). These risks occurred in 51.6-86.2% of all pregnancies ending in PTB depending on subtype. The highest risk observed was for medically indicated PTB <32weeks in women with pre-existing hypertension and pre-eclampsia (adjusted odds ratio 89.7, 95% CI 27.3-111.2). Our findings suggest a shared aetiology across PTB subtypes. These commonalities point to targets for further study and exploration of risk reduction strategies. Findings suggest a shared aetiology across preterm birth subtypes. Patterns may inform risk reduction efforts.

First and second trimester maternal serum markers in pregnancies with a vanishing twin.

The aim of this study was to assess the concentration of the first and second trimester maternal serum markers in pregnancies with a vanishing twin. This is a retrospective case-control study of pregnancies screened for Down syndrome in one Ontario center. Singleton pregnancies with ultrasound evidence of a vanishing twin were identified, and each was matched with five normal singleton controls for ethnicity, maternal age, gestational age, and blood sampling date. The median MoM of the first and second trimester serum markers was compared between cases and controls. The differences were assessed using the Mann-Whitney U-test. The study included 174 pregnancies that had a vanishing twin. Compared with control pregnancies, pregnancy associated plasma protein A increased by 21% (p = 0.0026), alpha-fetoprotein (AFP) increased by 10% (p < 0.0001), and dimeric inhibin A (DIA) increased by 13% (p = 0.0470) in pregnancies with a vanishing twin. Unconjugated oestriol and total human chorionic gonadotrophin were not significantly changed in these pregnancies. Pregnancy associated plasma protein A is not an adequate marker for pregnancies with a vanishing twin. The impact of elevated AFP on risk estimation is offset by that of DIA to certain extent. Further studies are needed to establish an adequate adjustment method for AFP and DIA to improve the accuracy of screening results for these pregnancies.

Effect of sialylation and complexity of FSH oligosaccharides on inhibin production by granulosa cells

Granulosa cell (GC) inhibin A and B production is regulated by FSH and gonadal factors. This gonadotrophin is released as a mixture of glycoforms, which induce different biological responses in vivo and in vitro. Our aim was to determine the effect of recombinant human FSH (rhFSH) glycosylation variants on inhibin A and B production by rat GCs. Preparative isoelectro focusing was used to isolate more acidic/sialylated (pH <4.00) and less acidic/sialylated (pH >5.00) rhFSH charge analogues. Concanavalin A was used to isolate unbound and firmly bound rhFSH glycoforms on the basis of their oligosaccharide complexity. GCs, obtained from oestrogen-primed immature rats, were cultured with either native rhFSH or its glycosylation variants. Inhibin A and B were determined using specific ELISAs. Results were expressed as mean±s.e.m. Under basal conditions, inhibin A was the predominant dimer produced (inhibin A: 673±55; inhibin B: 80±4 pg/ml). More acidic/sialylated charge analogues stimulated inhibin B production when compared to inhibin A at all doses studied; by contrast, less acidic/sialylated charge analogues stimulated inhibin A production and elicited no effect on inhibin B. Glycoforms bearing complex oligosaccharides showed a potent stimulatory effect on inhibin B when compared to inhibin A production (i.e. dose 1 ng/ml: 4.9±0.5 vs 0.9±0.1-fold stimulation, P<0.001). Glycoforms bearing hybrid-type oligosaccharides favoured inhibin A production (i.e. dose 4 ng/ml 2.9±0.1 vs 1.6±0.1-fold stimulation, P<0.05). These results show that the sialylation degree as well as the complexity of oligosaccharides present in the rhFSH molecule may be considered additional factors that differentially regulate dimeric inhibin production by rat GCs.

Expression of Inhibin/Activin Proteins and Receptors in the Human Hypothalamus and Basal Forebrain

The inhibin/activin family of proteins is known to have a broad distribution of synthesis and expression in many species, as well as a variety of functions in reproductive and other physiological systems. Yet, our knowledge regarding the production and function of inhibin and activin in the central nervous system is relatively limited, especially in humans. The present study aimed to explore the distribution of inhibin/activin protein subunits and receptors in the adult human brain. The human hypothalamus and surrounding basal forebrain was examined using post-mortem tissues from 29 adults. Immunocytochemical studies were conducted with antibodies directed against the inhibin/activin α, βA, and βB subunits, betaglycan and the activin type IIA and IIB receptors. An immunoassay was also utilised to measure dimeric inhibin A and B levels in tissue homogenates of the infundibulum of the hypothalamus. Robust βA subunit immunoreactivity was present in the paraventricular, supraoptic, lateral hypothalamic, infundibular, dorsomedial and suprachiasmatic nuclei of the hypothalamus, in the basal ganglia, and in the nucleus basalis of Meynert. A similar staining distribution was noted for the βB subunit, betaglycan and the type II receptor antibodies, whereas α subunit staining was not detected in any of the major anatomical regions of the human brain. Inhibin B immunoreactivity was present in all tissues, whereas inhibin A levels were below detectable limits. These studies show for the first time that the inhibin/activin protein subunits and receptors can be co-localised in the human brain, implicating potential, diverse neural functions.

Mechanisms of FSH synthesis: what we know, what we don't, and why you should care

The pituitary gonadotropin hormones, FSH and LH, are key regulators of reproductive physiology. Though the two hormones are produced by the same cell type, often in response to the same endocrine and paracrine regulators, they sub-serve different biological functions and their synthesis and secretion are differentially regulated. This stems largely from differences in transcriptional, post-transcriptional, and post-translational regulation of their unique beta subunits. That is, both hormones are dimeric glycoproteins and share a common alpha subunit. Their unique beta subunits, however, derive from different genes encoding distinct proteins. Past and recent research indicates synthesis and release of the two hormones are subject to extensive and independent regulation. LH appears to be secreted predominantly via the regulated secretory pathway, whereas FSH release is largely constitutive. As such, investigations of FSH-beta subunit synthesis may lend direct insight into mechanisms underlying patterns of secreted FSH, more so than investigations of the LHbeta subunit. Here, we review recent investigations of transcriptional regulation of the FSH-beta subunit gene from different mammalian species, including humans. The results reveal both conserved and species-specific regulatory mechanisms that might contribute to inter-species variation in FSH release.

Performance Characteristics of the UniCel DxI 800 Immunoassay for the Maternal Serum Quadruple Test, Including Median Values for Each Week of Gestation, in Korean Women

Maternal serum prenatal quadruple screening includes testing for alpha-fetoprotein (AFP), human chorionic gonadotrophin (hCG), unconjugated estriol (uE3), and dimeric inhibin A (DIA). We evaluated quadruple screening using an automated platform and looked for any ethnic differences in the median values of each marker. We measured the concentrations of each quadruple test analyte using the UniCel DxI 800 system (Beckman Coulter, USA) in 788 Korean mid-trimester maternal serum samples and calculated their median values using Benetech software (Benetech, Canada). We also compared the results with those obtained using the Immulite 2000 assay (Siemens Healthcare Diagnostics, USA) or ELISA (DSL, USA) in 442 samples. We obtained mid-trimester median values for each marker. The following are the comparative results for each test using the Immulite 2000 assay or ELISA (x) and the UniCel DxI 800 immunoassay (y): AFP, y=1.10x+0.01, r=0.925; uE3, y=0.28x+0.24, r=0.885; hCG, y=1.22x-3047.8, r=0.944; and DIA, y=0.86x+15.31, r=0.833. Assay results for each of the four markers showed good correlations. However, significant biases necessitated new median calculations of prenatal risk estimates in all four tests. We established gestational age-specific second-trimester median values for four markers in Korean samples using the UniCel DxI 800 immunoassay system. Despite significant bias, there were good correlations between the results obtained using the UniCel DxI 800 immunoassay and those obtained using the Immulite 2000 assay.

Ovarian Expression of Inhibin-Subunits, 3.BETA.-Hydroxysteroid Dehydrogenase, and Cytochrome P450 Aromatase during the Estrous Cycle and Pregnancy of Shiba Goats (Capra hircus)

The cellular localization of the inhibin subunits (α, β(A), and β (B)), steroidogenic enzymes (3β-hydroxysteroid dehydrogenase (3βHSD) and cytochrome P450 aromatase (P450arom) were evaluated in the ovaries of cyclic (n=6) and pregnant (n=2) Shiba goats (Capra Hircus). The immunointensity of inhibin α and β(A) subunits showed an increase in the granulosa cells (GC) of developing follicles. Inhibin β(B) subunit and P450arom showed high expression in GC of antral follicles. 3βHSD immunoreactivity was uniform in preantral and antral follicles. In follicular phase and late pregnancy, there was a strong expression of inhibin α subunit in GC of antral follicles. Although in mid pregnancy, antral follicles GC showed moderate immunostaining of inhibin β subunits, the immunoreactivity of inhibin β(A) and β(B) subunits was high during the follicular and luteal stages, respectively. While, immunoreactivity of GC to P450arom was moderate during all studied stages, and 3βHSD immunoreactivity was plentiful in antral follicles during the luteal phase. The immunoreactivity to inhibin α subunit and P450arom was abundant during mid pregnancy in the luteal tissues. Immunoreaction to inhibin β subunits was faint-to-moderate in cyclic and pregnancy corpora lutea. Immunoexpression of 3βHSD was maximal in late pregnancy corpora lutea. The present results suggest that, in goats, the GC of antral follicles are the main source of dimeric inhibins and that corpora lutea may partially participate in the secretion of inhibin. Changes in ovarian hormonal levels might depend on the synthesizing capacity of hormones in the follicles and corpora lutea to regulate the goat's reproductive stages.

Inhibin and premature ovarian failure

Elucidation of the causes of premature ovarian failure (POF) is difficult due to the heterogeneity of the condition. Inhibin is a potential candidate gene for POF based on its dual actions on FSH secretion by the pituitary and gametogenesis in the gonads. A missense mutation in the inhibin alpha subunit gene (INHA G769A) is associated with POF in several populations. However, there is phenotypic heterogeneity in INHA G769A mutation carriers. Relevant studies were identified by searching PubMed and mutational frequencies combined for meta-analysis. Meta-analysis of published studies revealed a risk difference of 0.04 (-0.030 to 0.11). The occurrence of asymptomatic carriers in populations suggests incomplete penetrance and/or a multi-genetic cause of POF. We propose that a decline in inhibin bioactivity caused by the mutation could increase FSH levels; and in a susceptible individual, the heightened sensitivity to gonadotrophins causes POF. Impaired paracrine effects of inhibin could impact folliculogenesis due to reduced antagonism of activin, bone morphogenetic protein 15 and growth differentiation factor 9. Functional studies of this mutation indicate normal production of dimeric inhibin A and B and impaired bioactivity of inhibin B. The identification of an autosomal mutation in the inhibin alpha subunit gene that is significantly linked to POF in certain ethnic populations highlights the role of inhibin in the regulation of ovarian biology and fertility. Although the reduction of inhibin B bioactivity by the INHA G769A mutation is clearly not the only cause, evidence suggests that this change may serve as a susceptibility factor, increasing the likelihood of POF.

Growth Differentiation Factor 9 Is a Germ Cell Regulator of Sertoli Cell Function

Oocyte-secreted growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 are critical regulatory factors in female reproduction. Together, they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study we show that the expression and stage-specific localization of both factors are limited to the germ cells of the rat seminiferous epithelium, with GDF9 being principally localized in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of signaling receptors [activin-like kinase (ALK) 5, ALK6, and BMP receptor, type II)]. Individual receptor types were expressed throughout the seminiferous epithelium, but coexpression of ALK5 and BMP receptor, type II was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFbeta ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction function and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localization of the junctional proteins claudin-11, occludin, and zonula occludens-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 up-regulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together, these results demonstrate that GDF9 and BMP15 are germ cell-specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.

Combined test + inhibin A at week 13 in contingent sequential testing: an interesting alternative for first‐trimester prenatal screening for Down Syndrome

To analyze various possibilities of using dimeric inhibin A (DIA) as a first-trimester marker, during week 13, for diverse sequential strategies together with the combined test (CT) [which uses pregnancy-associated plasma protein A, free beta-human chorionic gonadotrophin and nuchal translucency]. We analyzed three sequential strategies (nondisclosure, stepwise and contingent) for measuring DIA in week 13. Multivariate Gaussian modelling was used to estimate second-trimester, false-positive and detection rates. Model parameters were taken from the Serum, Urine and Ultrasound Screening Study (SURUSS) assay. The three sequential strategies provided high and comparable levels of effectiveness, with false-positive rate for fixed detection rate of 85% and detection rate for fixed false-positive rate of 5% values of about 91 and 2%, respectively. The contingent strategy (with two CT risk cutoffs, of 1 in 50 and 1 in 2000) produced a screening-positive rate of 0.8% with CT, while only 23% of the women needed subsequent DIA measurement. First-trimester sequential screening using CT together with DIA measurement in week 13 is capable of producing results that are comparable with those of the integrated test, with the additional advantage that the final result is obtained earlier. It is recommended that this strategy be evaluated in large-scale prospective studies.

Suppression of Inhibin A Biological Activity by Alterations in the Binding Site for Betaglycan

Inhibins A and B negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, control ovarian follicle development and steroidogenesis, and act as tumor suppressors in the gonads. Inhibins regulate these reproductive events by forming high affinity complexes with betaglycan and activin or bone morphogenetic protein type II receptors. In this study, the binding site of inhibin A for betaglycan was characterized using inhibin A mutant proteins. An epitope for high affinity betaglycan binding was detected spanning the outer convex surface of the inhibin alpha-subunit. Homology modeling indicates that key alpha-subunit residues (Tyr(50), Val(108), Thr(111), Ser(112), Phe(118), Lys(119), and Tyr(120)) form a contiguous epitope in this region of the molecule. Disruption of betaglycan binding by the simultaneous substitution of Thr(111), Ser(112), and Tyr(120) to alanine yielded an inhibin A variant that was unable to suppress activin-induced follicle-stimulating hormone release by rat pituitary cells in culture. Together these results indicate that a high affinity interaction between betaglycan and residues Val(108)-Tyr(120) of the inhibin alpha-subunit mediate inhibin A biological activity.

High serum concentration of total inhibin in polycystic ovary syndrome

To evaluate serum total inhibin (the sum of precursors, subunits, and mature molecules of inhibin) along with inhibin A and inhibin B in a large sample of women with polycystic ovary syndrome (PCOS) and to investigate whether these parameters differ between lean and overweight PCOS patients. Cross-sectional, controlled study. Academic health centers in Siena and Bologna, Italy. A group of women with PCOS (n = 145) was divided according to body mass index (BMI) into "lean PCOS" (BMI 19-25 kg/m(2), n = 52) and "overweight PCOS" (BMI 26-51 kg/m(2), n = 93). A group of healthy women (n = 90) with BMI 19-24 kg/m(2) and a history of regular menstrual cycles and normal results on physical examination served as controls. Blood samples were drawn between cycle days 3 and 5 in PCOS patients and controls. Serum concentrations of total inhibin, inhibin A, inhibin B, FSH, and LH were measured using commercially available immunoassays. Women with PCOS had serum total inhibin levels twice as high as in the control group (median = 115 vs. 47 pg/mL). Dimeric inhibin A concentration was lower in the PCOS group compared with controls (10 vs. 25 pg/mL), whereas inhibin B concentration did not differ significantly between PCOS and control groups. There was no difference of either total inhibin or inhibin A levels between lean and overweight women with PCOS, whereas the inhibin B level was higher in the lean PCOS subgroup compared with the overweight PCOS subgroup. Total inhibin did not show any linear correlation with inhibin B, inhibin A, or serum gonadotropins. Women with PCOS have high serum concentration of total inhibin but not of inhibin A or inhibin B, thus suggesting that PCOS women have an impaired processing of alpha-inhibin precursor proteins.

439. GDF9 and BMP15 are germ-free regulators of Sertoli cell function

Oocyte-secreted Growth Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are critical regulatory factors in female reproduction. Together they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study, we show that the expression and stage-specific localisation of both factors are limited to the germ cells of the rat seminiferous epithelium; with GDF9 being principally localised in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of the GDF9 and BMP15 signalling receptors (ALK5, ALK6, and BMPRII). Individual receptor types were expressed throughout the seminiferous epithelium, but co-expression of type I and type II receptors was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFβ ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction formation and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localisation of the junctional proteins claudin-11, occludin and ZO-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 upregulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together these results demonstrate that GDF9 and BMP15 are germ cell specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.