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Activin A, a dimer of the βA-subunit of inhibin, has been shown to have multiple biological activities and sites of production. Follistatin is a high-affinity binding protein for activin, which neutralizes its activity. This study provides the first data, using a cross-sectional design, on the measurement of both these proteins in the maternal circulation of a large cohort of women (6-39 weeks of gestation, n = 2-20 women/time point) during normal pregnancies, and confirms that similar patterns are seen in nine women studied longitudinally during pregnancy. The concentrations of total activin A were measured using a specific two-site enzyme-linked immunosorbent assay (ELISA), and a new radioimmunoassay for measuring total follistatin in serum utilizing dissociating reagents to eliminate the interference of activin is described. At 38-39 weeks gestation, both activin A and follistatin concentrations rose to a peak (4.59 ± 0.54 ng/ml and 72.7 ± 3.31 ng/ml, respectively). The activin A and follistatin concentrations were highly correlated both in the cross-sectional study (P < 0.0001) and in individual women in the longitudinal study (P < 0.05-0.0001). Concentrations of follistatin showed a greater increase in the second trimester of pregnancy relative to activin A concentrations. The parallel increase in the secretion of these two proteins throughout pregnancy probably reflects feto-placental secretion. [ABSTRACT FROM PUBLISHER]

Maternal serum concentrations of inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC-related inhibin forms, total follistatin, steroids and gonadotrophins were measured longitudinally in six normal singleton pregnancies. Maternal venous blood was collected randomly during a spontaneous follicular phase prior to donor insemination, at 5, 7, 9, 11, 16, 20, 24, 28, 32 and 36 weeks after the first missed menses and in the early puerperium. Steroid and gonadotrophin profiles conformed to previous reports. While at week 5 of gestation inhibin-A, activin-A and follistatin concentrations were similar to those at the follicular phase, all three increased progressively (P < 0.001) to maximal concentrations in week 36: approximately 48-fold (3740 +/- 1349 ng inhibin-A/ml), approximately 22-fold (6109 +/- 1443 ng activin-A/ml) and approximately 10-fold (3563 +/- 418 ng follistatin/ml) higher. Pro-alphaC concentrations reached a maximum in weeks 5 (approximately 5-fold, P < 0.001) and 36 (1027 +/- 174 pg/ml, P < 0.01). Inhibin-B (71 +/- 23 pg/ml prior to pregnancy) was undetectable (<12 pg/ml) between week 5-16 of gestation but increased slightly in the third trimester (26 +/- 7 pg/ml in week 36). Activin-AB was undetectable throughout pregnancy. Post-partum concentrations of inhibin-A (41 +/- 12 ng/ml), inhibin-B (<12 pg/ml), activin-A (950 +/- 149 pg/ml), pro-alphaC (128 +/- 22 pg/ml) and follistatin (990 +/- 79 ng/ml) were substantially lower than at week 36 of gestation. The activin-A:follistatin ratio increased from 0.5 in week 5 to 1.8 in week 36, suggesting that more free activin-A is available in the maternal circulation during late pregnancy. [ABSTRACT FROM AUTHOR]

Follistatin is a binding protein for the activin and inhibin family of hormones, regulating their biological activity. In the male reproductive tract, the interaction of these factors is likely to be involved in the regulation of the proliferation of several cell types. We have investigated the presence of follistatin and activin A in seminal plasma using specific immunoassays and have localized follistatin and activin/inhibin subunits in the adult human testis, prostate and seminal vesicle to establish their likely sources. High concentrations of immunoreactive follistatin were present in seminal plasma in normal men (mean 97.9 ng/ml; 1.43 ng/ml in peripheral plasma) and were similar in men with oligo/azoospermia and following vasectomy. Follistatin immunoreactivity was localized to both Leydig and Sertoli cells of the testis, and to epithelial cells of the prostate gland and seminal vesicle, which are likely to be the predominant sources of the hormone in seminal plasma. Activin A was also present in seminal plasma in normal men but was undetectable following vasectomy, thus deriving from the testis. Consistent with this finding, the βA-subunit was immunolocalized in Sertoli and Leydig cells but was not present in seminal vesicle or prostate gland. The functional significance of the high concentrations of follistatin secreted into seminal plasma by the prostate gland and/or seminal vesicle is uncertain, but they may regulate the biological activity of testis-derived activin A and inhibin B. [ABSTRACT FROM PUBLISHER]

Follistatin is a specific binding protein which controls bioavailability of activins and inhibins which have an important role in fetal development. In the first trimester of pregnancy bioactive dimeric inhibins are found at high concentrations in the extra-embryonic coelomic fluid, but the distribution of follistatin and activins is not known. We have used recently developed immunoassays for follistatin, activin A and activin AB to determine their presence in the intrauterine compartments during early pregnancy. Follistatin was present in highest concentrations in the extra-embryonic coelomic fluid (11.72 ± 1.70 ng/ml; median ± SEM), with less in maternal serum (6.35 ± 4.58) and lowest amounts in amniotic fluid (0.97 ± 0.52). Follistatin concentrations in extra- embryonic coelomic fluid were highly correlated with both dimeric inhibin isoforms. Activin A was present in only barely detectable amounts in some samples of extra-embryonic coelomic fluid (41% of samples) and maternal serum (26%) and was undetectable in all amniotic fluid samples. Activin AB was undetectable in all compartments. The presence of follistatin in the amniotic and extra-embryonic coelomic fluids may regulate the availability of bioactive activins and inhibins which are released into the intrauterine compartments during the development of the fetus and placenta in early pregnancy. [ABSTRACT FROM PUBLISHER]

In men, inhibin B is the circulating isoform involved in the regulation of follicle stimulating hormone (FSH) secretion. Within the testis, inhibin B may have a role in Sertoli and germ cell interactions, thus secretion into seminal plasma may reflect seminiferous tubule function. Using specific immunoassays, inhibin B was present in seminal plasma in fertile men (n = 105) and in unselected men attending an infertility clinic (n = 174) with a wide range in concentration from undetectable (<15 pg/ml) up to 54,100 pg/ml (geometric mean 280 pg/ml). There was a highly significant correlation between seminal plasma inhibin B concentration and sperm concentration (r = 0.46, P < 0.001), but no correlation with percentages of spermatozoa with progressive motility or normal morphology. Inhibin A and isoforms containing pro and alphaC immunoreactivity were not detectable. In post-vasectomy seminal plasma samples (18 of 20) inhibin B was undetectable, indicating that the testis is the predominant source. In unselected men attending an infertility clinic, inhibin B was undetectable in 17% (present in remainder; maximum concentration 26,200 pg/ml; mean 263 pg/ml), with a highly significant correlation between seminal plasma inhibin B and sperm concentration (r = 0.55, P < 0.0001). In men with oligo/ azoospermia (sperm concentration <20 x 10(6)/ml), seminal plasma inhibin B concentrations were lower in those with elevated plasma FSH concentrations (mean values 42 and 205 pg/ml, P < 0.05). Inhibin alpha and betaB subunits were localized predominantly in Sertoli and Leydig cells, using immunohistochemistry. We conclude that inhibin B of testicular origin is present in normal human seminal plasma, but with a very wide range in concentration, and may reflect the functional state of the seminiferous epithelium. [ABSTRACT FROM AUTHOR]

Tubal pregnancy is now commonly managed by laparoscopic salpingostomy or systemic methotrexate. A disadvantage of such conservative management is the need for appropriate follow-up, with serial measurement of serum concentrations of human chorionic gonadotrophin (HCG), to exclude persistent ectopic pregnancy (PEP). Concentrations of inhibin A, also a placental product, are significantly increased during pregnancy and the half-life of inhibin A is significantly shorter than that of HCG. To assess the suitability of inhibin A as a marker of PEP, we studied 16 women who had undergone surgery for a tubal pregnancy, measuring HCG and inhibin during follow-up. The mean ± SEM time taken to achieve non-pregnant concentrations of inhibin A was significantly shorter than for HCG (4.2 ± 0.8 days versus 21.6 ± 4.4 days respectively; P < 0.001 Wilcoxon signed rank test). However, in all women the inhibin A concentration increased rapidly after reaching a nadir, reflecting the return of ovarian function, complicating the interpretation of results. In four women inhibin A was almost undetectable preoperatively, while the corresponding HCG concentration was high. These data suggest that inhibin A will not be a useful marker for PEP but that it may provide a more accurate preoperative assessment of trophoblast viability than HCG, thereby improving management. [ABSTRACT FROM PUBLISHER]

Background: Immunoregulatory genes encoding activin A (Inhba) and B (Inhbb), and indolamine 2,3-dioxygenase-1 (Ido1) are highly expressed in the murine caput epididymidis, which also has a network of intraepithelial mononuclear phagocytes. This environment is postulated to promote immunological tolerance to epididymal sperm. The factors regulating the immunoregulatory agents in the epididymal caput are poorly understood., Objectives: This study aimed to investigate the potential role of testicular lumicrine factors in regulating activin and other immune-related genes in the caput epididymidis., Materials and Methods: The efferent ducts in adult C57/Bl6 mice were exposed and ligated bilaterally. Serum and tissues were collected seven days later. Animals with bilateral sham ligation and animals with no ligations (collectively referred to as the "intact" group) were used as controls., Results: Pressure-induced seminiferous epithelial damage due to intratubular fluid accumulation was observed in all ligated testes. Testicular inhibin was significantly increased and testosterone was elevated in some animals following bilateral ligation, but serum testosterone, serum LH, and serum inhibin were normal. Ligation caused epithelial regression in the initial segment, with similar but less severe effects in other caput segments. Activin A staining by immunohistochemistry in the epithelium was reduced in bilateral ligation, particularly in the initial segment, with moderately reduced staining intensity in the rest of the caput. Inhba expression within the caput was not significantly affected by bilateral ligation, but Inhbb was reduced by more than 60%. Transcripts encoding the macrophage-specific receptor Cx3cr1 were significantly reduced following bilateral ligation, but other immune cell markers, Ido1, and inflammatory genes were unaffected., Conclusion: These data indicate that testicular lumicrine secretion regulates several genes that are preferentially expressed in the initial segment, but has marginal effects on genes such as those encoding activin A and IDO1, which are expressed more widely in the caput., (© 2021 American Society of Andrology and European Academy of Andrology.)

Noncovalent complexes of transforming growth factor-β family growth/differentiation factors with their prodomains are classified as latent or active, depending on whether the complexes can bind their respective receptors. For the anti-Müllerian hormone (AMH), the hormone-prodomain complex is active, and the prodomain is displaced upon binding to its type II receptor, AMH receptor type-2 (AMHR2), on the cell surface. However, the mechanism by which this displacement occurs is unclear. Here, we used ELISA assays to measure the dependence of prodomain displacement on AMH concentration and analyzed results with respect to the behavior expected for reversible binding in combination with ligand-induced receptor dimerization. We found that, in solution, the prodomain has a high affinity for the growth factor (GF) (K d  = 0.4 pM). Binding of the AMH complex to a single AMHR2 molecule does not affect this K d and does not induce prodomain displacement, indicating that the receptor binding site in the AMH complex is fully accessible to AMHR2. However, recruitment of a second AMHR2 molecule to bind the ligand bivalently leads to a 1000-fold increase in the K d for the AMH complex, resulting in rapid release of the prodomain. Displacement occurs only if the AMHR2 is presented on a surface, indicating that prodomain displacement is caused by a conformational change in the GF induced by bivalent binding to AMHR2. In addition, we demonstrate that the bone morphogenetic protein 7 prodomain is displaced from the complex with its GF by a similar process, suggesting that this may represent a general mechanism for receptor-mediated prodomain displacement in this ligand family., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Study Question: Does activin A contribute to testicular fibrosis under inflammatory conditions?, Summary Answer: Our results show that activin A and key fibrotic proteins are increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis and in murine experimental autoimmune orchitis (EAO) and that activin A stimulates fibrotic responses in peritubular cells (PTCs) and NIH 3T3 fibroblasts., What Is Known Already: Fibrosis is a feature of EAO. Activin A, a regulator of fibrosis, was increased in testes of mice with EAO and its expression correlated with severity of the disease., Study Design, Size, Duration: This is a cross-sectional and longitudinal study of adult mice immunized with testicular homogenate (TH) in adjuvant to induce EAO, collected at 30 (n = 6), 50 (n = 6) and 80 (n = 5) days after first immunization. Age-matched mice injected with adjuvant alone (n = 14) and untreated mice (n = 15) were included as controls. TH-immunized mice with elevated endogenous follistatin, injected with a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of follistatin (rAAV-FST315; n = 3) or vector with an empty cassette (empty vector controls; n = 2) 30 days prior to the first immunization, as well as appropriate adjuvant (n = 2) and untreated (n = 2) controls, were also examined.Human testicular biopsies showing focal inflammatory lesions associated with impaired spermatogenesis (n = 7) were included. Biopsies showing intact spermatogenesis without inflammation, from obstructive azoospermia patients, served as controls (n = 7).Mouse primary PTC and NIH 3T3 fibroblasts were stimulated with activin A and follistatin 288 (FST288) to investigate the effect of activin A on the expression of fibrotic markers. Production of activin A by mouse primary Sertoli cells (SCs) was also investigated., Participants/materials, Setting, Methods: Testicular RNA and protein extracts collected from mice at days 30, 50 and 80 after first immunization were used for analysis of fibrotic marker genes and proteins, respectively. Total collagen was assessed by hydroxyproline assay and fibronectin; collagen I, III and IV, α-smooth muscle actin (α-SMA) expression and phosphorylation of suppressor of mothers against decapentaplegic (SMAD) family member 2 were measured by western blot. Immunofluorescence was used to detect fibronectin. Fibronectin (Fn), αSMA (Acta2), collagen I (Col1a2), III (Col3a1) and IV (Col4a1) mRNA in PTC and NIH 3T3 cells treated with activin A and/or FST288 were measured by quantitative RT-PCR (qRT-PCR). Activin A in SC following tumour necrosis factor (TNF) or FST288 stimulation was measured by ELISA. Human testicular biopsies were analysed by qRT-PCR for PTPRC (CD45) and activin A (INHBA), hydroxyproline assay and immunofluorescence., Main Results and the Role of Chance: Production of activin A by SC was stimulated by 25 and 50 ng/ml TNF (P < 0.01, P < 0.001, respectively) as compared to untreated cells. INHBA mRNA was increased in human testicular biopsies with leukocytic infiltrates and impaired spermatogenesis, compared with control biopsies (P < 0.05), accompanied by increased total collagen (P < 0.01) and fibronectin deposition. Total testicular collagen (P < 0.0001) and fibronectin protein expression (P < 0.05) were also increased in EAO, and fibronectin expression was correlated with the severity of the disease (r = 0.9028). In animals pre-treated with rAAV-FST315 prior to immunization with TH, protein expression of fibronectin was comparable to control. Stimulation of PTC and NIH 3T3 cells with activin A increased fibronectin mRNA (P < 0.05) and the production of collagen I (P < 0.001; P < 0.01) and fibronectin (P < 0.05). Moreover, activin A also increased collagen IV mRNA (P < 0.05) in PTC, while αSMA mRNA (P < 0.01) and protein (P < 0.0001) were significantly increased by activin A in NIH 3T3 cells., Large Scale Data: N/A., Limitations, Reasons for Caution: A limited number of human testicular specimens was available for the study. Part of the study was performed in vitro, including NIH 3T3 cells as a surrogate for testicular fibroblasts., Wider Implications of the Findings: Resident fibroblasts and PTC may contribute to the progression of testicular fibrosis following inflammation, and activin A is implicated as a key mediator of this process., Study Funding/competing Interest(s): This work was supported by the National Health and Medical Research Council of Australia, the Victorian Government's Operational Infrastructure Support Program and the International Research Training Group between Justus Liebig University (Giessen) and Monash University (Melbourne) (GRK 1871/1-2) on `Molecular pathogenesis on male reproductive disorders' funded by the Deutsche Forschungsgemeinschaft and Monash University. The authors declare no competing financial interests., (© The Author(s) 2019. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Regionalised interaction of the activins, follistatin and inhibin was investigated in the male reproductive tract of mice lacking the inhibin α-subunit (Inha -/- ). Serum and intratesticular activin B, although not activin A and follistatin, were increased in Inha -/- mice at 25 days of age, but all three proteins were elevated at 56 days. None of these proteins were altered within the epididymis and vas deferens at either age. At 25 days, histology of the epididymis and vas deferens was similar to wild-type. At 56 days, the testis contained extensive somatic cell tumours, leading to Leydig cell regression and testosterone deficiency. The epididymis and vas deferens showed epithelial regression and increased prominence of the interstitial stroma. Immunoregulatory and fibrotic gene expression in the epididymis and vas deferens were unchanged. Thus, absence of the inhibin α-subunit has marginal effects on activins in the epididymis and vas deferens, and regression of these tissues is associated with androgen deficiency., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is clinically defined and characterised by persistent disabling tiredness and exertional malaise, leading to functional impairment., Methods: This study introduces the weighted standing time (WST) as a proxy for ME/CFS severity, and investigates its behaviour in an Australian cohort. WST was calculated from standing time and subjective standing difficulty data, collected via orthostatic intolerance assessments. The distribution of WST for healthy controls and ME/CFS patients was correlated with the clinical criteria, as well as pathology and cytokine markers. Included in the WST cytokine analyses were activins A and B, cytokines causally linked to inflammation, and previously demonstrated to separate ME/CFS from healthy controls. Forty-five ME/CFS patients were recruited from the CFS Discovery Clinic (Victoria) between 2011 and 2013. Seventeen healthy controls were recruited concurrently and identically assessed., Results: WST distribution was significantly different between ME/CFS participants and controls, with six diagnostic criteria, five analytes and one cytokine also significantly different when comparing severity via WST. On direct comparison of ME/CFS to study controls, only serum activin B was significantly elevated, with no significant variation observed for a broad range of serum and urine markers, or other serum cytokines., Conclusions: The enhanced understanding of standing test behaviour to reflect orthostatic intolerance as a ME/CFS symptom, and the subsequent calculation of WST, will encourage the greater implementation of this simple test as a measure of ME/CFS diagnosis, and symptom severity, to the benefit of improved diagnosis and guidance for potential treatments.

Background and Objective: There are currently no sensitive and specific assays for activin B that could be utilized to study human biological fluids. The aim of this project was to develop and validate a 'total' activin B ELISA for use with human biological fluids and establish concentrations of activin B in the circulation and fluids from the reproductive organs., Design: The new ELISA was validated and then used to measure activin B levels in the circulation of healthy participants, IVF patients, pregnant women and in ovarian follicular fluid and seminal plasma., Patients and Measurements: Healthy adult subjects (n = 143), subjects from an IVF clinic (n = 27) and pregnancy groups (n = 29) were sampled., Results: The sensitivity of the assay was 0.019 ng/ml. Validation of the activin B ELISA showed good recovery (90.7 +/- 9.8%) and linearity in biological fluid and cell culture media and low cross-reactivity with related analytes (inhibin B = 0.077% and activin A = 0.0034%). There was a negative correlation between activin B concentration (r = -0.281, P < 0.011) and females with increasing age. Patients attending IVF clinics had significantly lower levels of activin B compared with gender-matched control subjects. Ovarian follicular fluid and seminal plasma had 50-80 fold higher levels of activin B (mean = 5.35 and 3.66 ng/ml respectively) than sera (mean = 0.071 ng/ml)., Conclusions: This fully validated ELISA for activin B offers a tremendous utility for measuring this protein in a variety of normal physiological processes and in various clinical pathologies.

Purpose: Previous conflicting results about the prognostic significance of estrogen receptor (ER)-beta in breast cancer may be explained by contribution of isoforms, of which five exist. Our aim was to elucidate the prognostic significance of ERbeta1, ERbeta2, and ERbeta5 by immunohistochemistry in a large cohort of breast carcinomas with long-term follow-up., Experimental Design: Tissue microarrays were stained with ERbeta1, ERbeta2, and ERbeta5 antibodies and scored as percentage of positive tumor cells and using the Allred system. Nuclear and cytoplasmic staining was evaluated and correlated with histopathologic characteristics, overall survival (OS), and disease-free survival (DFS)., Results: Nuclear ERbeta2 and ERbeta5, but not ERbeta1, significantly correlated with OS (P = 0.006, P = 0.039, and P = 0.099, respectively), and ERbeta2 additionally with DFS (P = 0.013). ERbeta2 also predicted response to endocrine therapy (P = 0.036); correlated positively with ERalpha, progesterone receptor, androgen receptor, and BRCA1; and correlated inversely with metastasis and vascular invasion. Tumors coexpressing ERbeta2 and ERalpha had better OS and DFS. Cytoplasmic ERbeta2 expression, alone or combined with nuclear staining, predicted significantly worse OS. Notably, patients with only cytoplasmic ERbeta2 expression had significantly worse outcome (P = 0.0014)., Conclusions: This is the first study elucidating the prognostic role of ERbeta1, ERbeta2, and ERbeta5 in a large breast cancer series. ERbeta2 is a powerful prognostic indicator in breast cancer, but nuclear and cytoplasmic expression differentially affect outcome. Measuring these in clinical breast cancer could provide a more comprehensive picture of patient outcome, complementing ERalpha.

Context: Insulin resistance, which associates with levels of retinol-binding protein 4 (RBP4) and adiponectin, is implicated in the development of polycystic ovary syndrome (PCOS)., Objective: The objective of the study was to explore the potential contribution of RBP4 and adiponectin in the etiology of PCOS and their relationships with specific fat depot measurements., Design: This was a cross-sectional study., Setting and Participants: Serum RBP4 and adiponectin levels were compared between 50 PCOS cases and 28 female controls (including 22 body mass index/fat mass-matched pairs) and correlated with specific fat depot (including visceral) axial magnetic resonance imaging cross-sectional area measurements. All subjects were of U.K. British/Irish origin., Main Outcome Measure(s): Serum levels of RBP4 (automated immunonephelometric assay) and adiponectin [immunoassay: total and high molecular weight (HMW)]. Data are reported as geometric mean (sd, range) and optionally adjusted for fat mass and age., Results: Between the 50 PCOS cases and 28 controls, serum RBP4 levels were indistinguishable [39.0 microg/ml (31.0, 49.0) vs. 41.6 microg/ml (32.7, 52.9), respectively, unadjusted P = 0.24; adjusted P = 0.55]. Total (and HMW) adiponectin levels were lower in PCOS cases [total adiponectin 19.9 microg/ml (14.2, 27.8) vs. 25.8 microg/ml (17.7, 37.7), respectively, unadjusted P = 2.4 x 10(-3); adjusted P = 0.10]. For the paired-sample analyzes, there were no differences in RBP4 (P = 0.09), total adiponectin (P = 0.06), HMW adiponectin (P =0.19), or HMW to total adiponectin ratio (P = 0.98). In PCOS cases, L4-visceral fat area was associated positively with RBP4 (r(2) = 0.34, P = 0.01) and negatively with HMW to total adiponectin ratio (r(2) = -0.44, P = 1.3 x 10(-3)). Controls showed similar relationships., Conclusions: Although associated with visceral fat, serum RBP4 and adiponectin levels do not play important, fat-mass-independent primary roles in the development of PCOS.

Objective: To investigate the relationship between oxidative stress and the underlying causes of infertility, preovulatory ovarian hormones, and ovarian response to gonadotropin stimulation in patients undergoing assisted reproductive techniques., Design: Prospective, cross-sectional study., Setting: Assisted conception unit, university hospital., Patient(s): One hundred thirty women presenting with infertility, of the following types: male factor (n = 56), unexplained (n = 36), tubal factor (n = 16), polycystic ovary syndrome (n = 15), and endometriosis (n = 7)., Intervention(s): Follicular fluid (FF) and peripheral blood samples were collected at oocyte retrieval., Main Outcome Measure(s): Blood and FF samples were analyzed for inhibin A, inhibin B, activin A, anti-Müllerian hormone, and E(2) by using ELISA. Total antioxidant capacity (TAC) was measured in plasma and FF by using a calorimetric microplate assay., Result(s): There was no significant relationship between plasma or FF TAC and the underlying etiology of infertility. There was a statistically significant positive association between FF E(2) levels and TAC (r = 0.26). Higher antral follicle count, delta E(2) (day 3 E(2) minus day 2 E(2)), preovulatory serum anti-Müllerian hormone, inhibin B, and E(2) were associated with good ovarian response, whereas higher FF E(2) was associated with a statistically significant poor response. No significant direct relationship was observed between TAC and ovarian response as well as between TAC or any of the parameters measured and pregnancy outcome., Conclusion(s): Oxidative stress has an impact on the production of granulosa cell steroid hormones, in particular E(2), which is an important predictor of ovarian response. The positive association between FF E(2) and total antioxidant capacity suggests that E(2) may play a role in the ovarian antioxidant-oxidant balance.

Inhibin B has emerged as a clinically useful analyte for studies of reproductive function in both men and women. The antibody to the betaB subunit (C5) used in current commercially available assays (DSL and OBI) was raised in this laboratory to a synthetic peptide from the betaB subunit. These assays require pre-treatment of samples with hydrogen peroxide to oxidise two methionines in the epitope to the sulfoxide for full immunoreactivity. It was also claimed that this antibody cross-reacted significantly with inhibin A leading to a 0.5% cross-reaction of inhibin A in the current generation of immunoassays. Both of the above immunoassays required overnight incubation with sample. The development of improved antibodies to the betaB subunit has proved difficult due to the conservation of the betaB subunit between species. We describe the development of new monoclonal antibodies to the betaB subunit, by immunisation of mice with recombinant X. laevis activin B using the RIMMS method of immunisation. The result has been the development of highly specific antibodies in a short time period. One of these antibodies 46A/F is shown to be a highly effective capture antibody in a human inhibin B ELISA, without any sample pre-treatment. The results of the validation of an improved inhibin B assay using 46A/F as the capture antibody are shown, with comparison to one of the commercially available inhibin B assays. Overall, the inhibin B assay is simplified and the performance improved by using this new antibody 46A/F. It was further shown that the cross-reaction of inhibin A in both the OBI and DSL inhibin B ELISAs is ten fold less than previously reported. This can be attributed to the poor quality of recombinant inhibin B available for use as standard in 1996. Although the present generation of inhibin B assays has proved adequate to enable the physiological function of inhibin to be determined and novel clinical applications found, the simplification of the assay made possible by the improved antibody should make possible a new generation of more rapid, sensitive, convenient and robust tools for routine use.

Background: Declining human reproductive health over the last 60 years has been proposed to be due to effects of environmental chemicals, especially endocrine disrupting compounds, on fetal development. We investigated whether a model pesticide, dieldrin, at concentrations within both maternal circulation and environmental ranges (1 pmol/l = 0.0004 p.p.b. = 380.9 pg/l), could disrupt the human fetal testis., Methods: Human fetal testes were collected during the second trimester, a critical period of male sexual differentiation (development and masculinization). Testis explants were cultured for 24 h in the presence and absence of LH (10-1000 IU LH/l) and dieldrin (1 pmol and 1 nmol/l). Endocrine, immunohistological and proteome characteristics of the tissues were investigated., Results: Exposure to dieldrin reduced LH-induced testosterone secretion (P < 0.05) and tissue protein concentrations of LH receptor and steroid acute regulatory protein (P < 0.05). Dieldrin altered proteins associated with cancer, apoptosis, transcription and development. Wnt-2b was reduced 3-fold and immunolocalized to Leydig and Sertoli cells. Dieldrin also reversed some LH-induced changes in protein expression, supporting the conclusion that Leydig cell function is at risk from environmental chemicals., Conclusions: Our findings indicate that exposure to very low, biologically relevant, concentrations of environmental chemicals could affect the fetal human Leydig cell, reducing testosterone secretion and potentially leading to subtle dysregulation of reproductive development and adult fecundity.

Rising serum FSH levels is one of the earliest signs of human female reproductive aging. Whether or not elevated FSH remains a passive reflection of a diminishing ovarian follicle pool or actively contributes to declining female fertility with age has not been established. We therefore investigated female reproduction in mice expressing progressively rising serum levels of transgenic human FSH (Tg-FSH, 2.5-10 IU/liter) independently of follicle depletion. We show that serum LH and estradiol levels and uterine size remained normal in Tg-FSH females, whereas ovarian weight and corpora lutea number were significantly increased up to 1.3- and 5-fold, respectively. Furthermore, the monotrophic FSH rise produced a striking biphasic effect on female fertility. Tg-FSH females less than 22 wk old delivered increased litter sizes, then beyond 23 wk, litter sizes decreased rapidly culminating in premature infertility despite continued ovary follicle development, and increased ovulation and uterine embryo implantation sites as well as normal serum levels of anti-Mullerian hormone, a marker of ovarian follicle reserve. We found that rising circulating Tg-FSH produced premature infertility by increasing embryo-fetal resorption and parturition failure with age. Thus, our Tg-FSH mice present a novel paradigm to investigate selective contributions of elevated FSH to age-related female infertility, which revealed that rising FSH levels, despite no exhaustion of ovarian reserve, actively accelerates female reproductive aging primarily by postimplantation reduction of embryo-fetal survival.

Ovarian reserve can be diminished following treatment for breast cancer. This study evaluated biochemical and biophysical parameters of ovarian reserve in these patients. Biochemical and biophysical tests of ovarian reserve were performed simultaneously in young (age 22-42 years), regularly menstruating women with breast cancer (n=22) and age-matched controls (n=24). All tests were performed before (baseline) and after transient ovarian stimulation in the early follicular phase. Patients were recruited both before and after completion of chemotherapy, with some patients being followed up prospectively. Serum samples were analysed for follicle-stimulating hormone (FSH), luteinising hormone (LH), oestradiol (E(2)), inhibins A and B, and antimullerian hormone (AMH). Biophysical (ultrasound) tests included ovarian volume, antral follicle count (AFC), ovarian stromal blood flow and uterine dimensions. Significant differences were revealed (when compared with the controls) for basal FSH (11.32+/-1.48 vs 6.62+/-0.42 mIU ml(-1), P<0.001), basal AMH (0.95+/-0.34 vs 7.89+/-1.62 ng ml(-1), P<0.001) and basal inhibin B (19.24+/-4.56 vs 83.61+/-13.45 pg ml(-1), P<0.001). Following transient ovarian stimulation, there were significant differences in the increment change (Delta) for inhibin B (3.02+/-2.3 vs 96.82+/-16.38 pg ml(-1), P<0.001) and E(2) (107.8+/-23.95 vs 283.2+/-40.34 pg ml(-1), P<0.01). AFC was the only biophysical parameter that was significantly different between patients and the controls (7.80+/-0.85 vs 16.77+/-1.11, P<0.001). Basal and stimulated biochemical (serum AMH, FSH, inhibin B and E(2)) and biophysical (AFC) tests may be potential markers of ovarian reserve in young women with breast cancer.

Background/aims: Activins A and E negatively regulate hepatic cell number by inhibiting cell replication and inducing apoptosis. Follistatin and follistatin-like 3 bind activins and antagonise their biological activities. Aim of our study was to investigate, whether activins and follistatins may play a role in hepatocarcinogenesis., Methods: Expression levels of follistatin, follistatin-like 3, and activin subunits beta(A) as well as beta(E) were investigated in chemically induced rat and human liver tumours by real-time PCR and immunohistochemistry. In addition, the effects of follistatin and activin A on DNA synthesis of normal as well as preneoplastic hepatocytes and hepatoma cells were analysed., Results: Follistatin was overexpressed while both activin subunits were downregulated in the majority of rat and human liver tumours. Follistatin-like 3 expression was low in normal but enhanced in malignant rat liver. In human normal liver, in contrast, it was abundantly expressed but downregulated in liver cancer. Administration of follistatin to normal and preneoplastic hepatocytes stimulated DNA synthesis preferentially in preneoplastic rat hepatocytes, whereas activin A repressed it., Conclusions: The balanced expression of follistatins and activins becomes deregulated during hepatocarcinogenesis. The sensitivity of preneoplastic hepatocytes to activin signals suggests the activin/follistatin system as promising target for therapeutic intervention.

Background: Reproductive function following cancer treatment is of increasing importance with improving survival rates. We therefore assessed the markers of the ovarian reserve in premenopausal women, to investigate and compare the effects of chemotherapy and long-term gonadotrophin withdrawal on ovarian function., Methods: Fifty premenopausal (age range 28-52 years) women with early breast cancer were recruited. Serum hormone and ovarian ultrasound measurements were taken before treatment and at intervals up to 1 year during chemotherapy or gonadotrophin suppressive therapy., Results: Pretreatment samples indicated a fall in anti-Müllerian hormone (AMH) concentration with age before changes in other hormone concentrations. AMH concentration showed a rapid and marked fall during chemotherapy, with undetectable concentrations in many women (P<0.0001). Inhibin B concentration showed a lesser fall (P<0.0001), whereas estradiol (E2) concentrations were maintained. Both antral follicle count (AFC) and ovarian volume fell (P<0.0001 and P<0.05 respectively). Regimens containing taxanes in addition to cyclophosphamide showed increased gonadotoxicity. Gonadotrophin suppression resulted in expected falls in E2 (P<0.05) and inhibin B (P<0.001) levels, but also resulted in a delayed fall in AMH level after 6 months (P<0.0001)., Conclusions: These data confirm the value of AMH concentration as an early indicator of ovarian ageing including assessment of chemotherapy-induced ovarian follicle loss. FSH and AMH concentration measurements may be useful for the comparison of ovarian toxicity of different chemotherapy regimens.