Most cleaved anti-müllerian hormone binds its receptor in human follicular fluid but little is competent in serum

Context: Anti-Müllerian hormone (AMH) is an important clinical marker for diagnosing and assessing the reproductive status and/or disorders in men and women. Most studies have not distinguished between levels of inactive AMH precursor and the cleaved noncovalent complex that binds the AMH type II receptor (AMHRII) and initiates signaling. Objective: The objective of the study was to measure the levels of AMH cleavage and bioactivity in human body fluids. Design, Setting, and Patients: AMH cleavage levels and bioactivity were measured in the serum of six boys and in the follicular fluid and serum of nine control women and 13 women with the polycystic ovary syndrome (PCOS). Main Outcome Measures: AMH cleavage levels were measured by capturing AMH with an anti- AMH antibody, followed by Western blotting. The bioactivity of cleaved AMH was assessed with an ELISA that measures the levels of AMH capable of binding AMHRII. Results: PCOS women have an elevated level of AMH cleavage in their follicular fluid (24% vs 8% in controlwomen),andmostofthecleavedAMHcanbindAMHRII.Higherlevels of cleavage areobserved in female (60%) and male (79%) serum, but very little of the cleaved AMH can bind AMHRII. Conclusions: These results support an autocrine role for AMH in the pathophysiology of PCOS in the follicle. In addition,theyindicate thatAMHundergoesinteractions or structuralchangesaftercleavage that prevent receptor binding, meaning, unexpectedly, that the level of cleaved AMH in biological fluids does not always reflect the level of bioactive AMH.Fil: Pierre, Alice. Inserm; Francia. Université Paris Diderot - Paris 7; Francia. Centre National de la Recherche Scientifique; FranciaFil: Racine, Chrystèle. Inserm; Francia. Université Paris Diderot - Paris 7; Francia. Centre National de la Recherche Scientifique; FranciaFil: Rey, Rodolfo Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Gobierno de la Ciudad de Buenos Aires. Centro de Investigaciones Endocrinológicas "Dr. César Bergada". Fundación de Endocrinología Infantil. Centro de Investigaciones Endocrinológicas "Dr. César Bergada"; ArgentinaFil: Fanchin, Renato. Université Paris Diderot - Paris 7; Francia. Centre National de la Recherche Scientifique; FranciaFil: Taieb, Joëlle. Université Paris Diderot - Paris 7; Francia. Centre National de la Recherche Scientifique; FranciaFil: Cohen Tannoudji, Joëlle. Université Paris Diderot - Paris 7; Francia. Centre National de la Recherche Scientifique; FranciaFil: Carmillo, Paul. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Pepinsky, R. Blake. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; ArgentinaFil: Cate, Richard L.. Université Paris Diderot - Paris 7; Francia. Centre National de la Recherche Scientifique; FranciaFil: Di Clemente, Nathalie. Inserm; Francia. Université Paris Diderot - Paris 7; Francia. Centre National de la Recherche Scientifique; Franci

Dysregulation of the anti-mullerian hormone system by steroids in women with polycystic ovary syndrome

International audienceContext: Anti-Mullerian hormone (AMH) and AMH type II receptor (AMHR2) are overexpressed in granulosa cells (GCs) from women with polycystic ovary syndrome (PCOS), the most common cause of female infertility. Objective: The aim of the study was to compare the regulation of the AMH/AMHR2 system by 5adihydrotestosterone (5a-DHT) and estradiol (E2) in GCs from control subjects and women with PCOS. Design, Setting, Patients: Experiments were performed on follicular fluids (FF) and GCs from women undergoing in vitro fertilization. Main Outcome Measures: FF steroid levels were measured by mass spectrometry, and messenger RNA (mRNA) accumulation was quantified by reverse transcription real-time polymerase chain reaction. Results: Total testosterone (T), free T, and 5 alpha-DHT FF levels were significantly higher (P < 0.001) in women with PCOS than in controls. However, E2 and sex hormone-binding globulin concentrations were comparable between the two groups. In GCs from control women, the AMH and AMHR2 expression were not affected by 5a-DHT treatment, whereas AMH mRNA levels were upregulated by 5a-DHT in GCs from patients with PCOS (2.3-fold, P < 0.01) overexpressing the androgen receptor (1.4-fold, P < 0.05). E2 downregulated the AMH and AMHR2 expression in GCs from control women (1.4-fold, P < 0.001 and 1.8-fold, P < 0.01, respectively) but had no effect on these genes in GCs from women with PCOS. This differential effect of E2 was associated with a higher estrogen receptor 1 expression in GCs from women with PCOS (1.9-fold, P < 0.05). Conclusions: In GCs from women with PCOS, the regulation of AMH and AMHR2 expression is altered in a way that promotes the overexpression of the AMH/AMHR2 system, and could contribute to the follicular arrest observed in these patients

The BOC ELISA, a ruminant-specific AMH immunoassay, improves the determination of plasma AMH concentration and its correlation with embryo production in cattle

International audiencePlasma anti-Müllerian hormone (AMH) concentrations have been recently found to be predictive of the number of embryos recovered after FSH superovulatory treatment in the cow. However, the sensitivity of the Active Müllerian-inhibiting substance/AMH ELISA (ref. 10–14400; DSL-Beckman-Coulter) used to make these measurements in bovine plasma samples is low because it was developed to measure human AMH levels. To overcome this limitation, we developed an immunoassay specific for the bovine (B), ovine (O), and caprine (C) species, the bovine-ovine-caprine (BOC) ELISA. For this purpose, we produced recombinant bovine AMH for standardization, and we used monoclonal antibodies raised against bovine AMH, previously prepared by our laboratory. We evaluated the precision, accuracy, specificity, limit of detection, and functional sensitivity of the assay. The intra-assay coefficient of variation ranged between 3.4% and 11.3% for AMH concentrations between 23.68 and 1.74 ng/mL, and the interassay coefficient of variation ranged between 4.8% and 20.5% for concentrations between 25.53 and 1.42 ng/mL, respectively. The assay displayed a good linearity, had a detection limit of 0.4 ng/mL and a functional sensitivity of 1.4 ng/mL. It also cross-reacted with ovine and caprine AMHs. Both the mean and median AMH levels measured in 40 cow plasma samples using the BOC ELISA were approximately 44 fold higher than the mean and median AMH levels measured with the Active Müllerian-inhibiting substance/AMH ELISA. Moreover, a higher correlation was observed between the average number of embryos recovered from each cow after superovulatory treatment and AMH concentrations measured with the BOC ELISA. This BOC ELISA provides a very efficient tool for evaluating the ovarian follicular reserve of cows and predicting their embryo production capacity

Differential regulation of ovarian anti-müllerian hormone (AMH) by estradiol through α- and β-estrogen receptors

Background: Anti-Müllerian hormone (AMH) is a member of the TGF-β family, which limits follicle maturation. Recently serum AMH has been recognized as a useful diagnostic and prognostic tool in human reproductive endocrinology. Objective: The aim of this study was to investigate the regulation of human ovarian AMH by estradiol and FSH. Methods: AMH mRNA were quantified by real time RT-PCR in human granulosa cells (GC). AMH transcription was studied in KK1 GC cotransfected with estrogen receptors (ER)-β or ERα, and normal human AMH promoter-luciferase construct (hAMH-luc) or mutated AMH promoter reporter constructs. Binding sites for estradiol (estrogen response element half-site) and steroidogenic factor 1 were disrupted by targeted mutagenesis. The level of ER in GC was determined by quantitative RT-PCR and Western blotting. Results: In KK1 cells, estradiol up-regulated and inhibited hAMH-luc in the presence of ERα and ERβrespectively. Disruption of estrogen response element half-site and/or steroidogenic factor 1 binding sites did not modify ERβ-mediated effect of estradiol on hAMH-luc, whereas it affected that conveyed by ERα. The FSH enhancement of hAMH-luc was abolished by estradiol in cells overexpressing ERβ. When both ER were transfected, estradiol inhibited hAMH-luc or had no effect. Estradiol repressed AMH mRNAs in human GC, which express a little more ERα than ERβ mRNA. Conclusions: Our results show that AMH expression can be differentially regulated by estradiol depending on the ER and suggest that its decrease in GC of growing follicles, which mainly express ERβ, and during controlled ovarian hyperstimulation is due to the effect of estradiol.Fil: Grynberg, Michaël. Inserm; FranciaFil: Pierre, Alice. Inserm; FranciaFil: Rey, Rodolfo Alberto. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños "Ricardo Gutiérrez"; Argentina. Universidad de Buenos Aires; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Leclerc, Arnaud. Inserm; FranciaFil: Arouche, Nassim. Inserm; FranciaFil: Hesters, Laetitia. Inserm; FranciaFil: Catteau Jonard, Sophie. Universite Lille; FranciaFil: Frydman, René. Inserm; FranciaFil: Picard, Jean Yves. Inserm; FranciaFil: Fanchin, Renato. Inserm; FranciaFil: Veitia, Reiner. Centre National de la Recherche Scientifique; FranciaFil: Di Clemente, Nathalie. Inserm; FranciaFil: Taieb, Joëlle. Inserm; Franci

FSH and Its Second Messenger cAMP Stimulate the Transcription of Human Anti-Müllerian Hormone in Cultured Granulosa Cells

International audienceAnti-Müllerian hormone (AMH), also called Müllerian-inhibiting substance, a member of the TGF-ß family, is responsible for the regression of Müllerian ducts in the male fetus. In females, AMH is synthesized by granulosa cells of preantral and small antral follicles, and production wanes at later stages of follicle maturation. Using RT-PCR in luteal granulosa cells in primary culture and reporter gene techniques in the KK1 granulosa cell line, we show that FSH and cAMP enhance AMH transcription, and LH has an additive effect. Gonadotropins and cAMP act through protein kinase A and p38 MAPK signaling pathways and involve the GATA binding factor-4 and steroidogenic factor-1 transcription factors, among others. The expression profile of AMH and the dynamics of serum AMH after gonadotropin stimulation have been interpreted as a down-regulating effect of FSH upon AMH production by granulosa cells. The specific effect of gonadotropins upon granulosa cells may be obscured in vivo by the effect of FSH upon follicular maturation and by the presence of other hormones and growth factors, acting individually or in concert

Relationship between vitamin D status in pregnancy and the risk for preeclampsia: A nested case-control study.

International audienc

Vitamin D status during pregnancy and in cord blood in a large prospective French cohort.

International audienceDespite the recommended supplementation, vitamin D insufficiency is frequent during pregnancy and in newborns in France

Relationship between vitamin D status in the first trimester of pregnancy and gestational diabetes mellitus - A nested case–control study

International audienceBackground & aims: Gestational diabetes mellitus (GDM) is one of the most frequent medical complications during pregnancy. It has been associated with many adverse pregnancy, fetal and neonatal outcomes, as well as with an increased risk for mothers and children in the long term. There is a growing interest in vitamin D and its potential role in the development of metabolic disorders. However, the medical literature is not consensual. The aim of this study was to assess the risk of GDM according to vitamin D status during the first trimester.Methods: This study is a nested case-control study performed from a multicenter prospective observational cohort of pregnant women assessed for 25-hydroxyvitamin D levels (25OHD). Three hundred ninety-three patients were included in the initial cohort. After applying exclusion criteria, a total of 1191 pregnant women were included. Two hundred fifty women with GDM (cases) were matched to 941 women without GDM (controls) for parity, age, body mass index before pregnancy, the season of conception, and phototype. This study was funded by a grant from the "Programme Hospitalier de Recherche Publique 2010".Results: The GDM risk was significantly greater for patients with 25OHD levels <20 ng/mL (OR = 1∙42, 95% CI 1∙06-1∙91; p = 0∙021). However, there was no significant relationship with other thresholds. The study of 25OHD levels with the more precise cutting of 5 units intervals showed a variable relationship with GDM risk, as the risk was low for very low 25OHD levels, increased for moderated levels, decreased for normal levels, and finally increased for higher levels.Conclusion: According to our study, there seems to be no linear relationship between GDM and 25OHD levels in the first trimester of pregnancy since GDM risk does not continuously decrease as 25OHD concentrations increase. Our results most probably highlight the absence of an association between 25OHD levels and GDM risk.Trial registration: ClinicalTrials.gov NCT01648842

Prognostic Value and Relation with Adjuvant Treatment Duration of ctDNA in Stage III Colon Cancer: a Post Hoc Analysis of the PRODIGE-GERCOR IDEA-France Trial

International audiencePurpose: Circulating tumor DNA (ctDNA) has been suggested as a major prognostic factor in resected stage-III colon cancer. We analyzed ctDNA of patients randomized in the phase III IDEA-France trial.Experimental design: ctDNA was tested for WIF1 and NPY by droplet digital PCR with method developed and validated for colorectal cancer. Disease-free survival (DFS) and overall survival (OS) were analyzed via multivariable analysis in patients with ctDNA samples and in sub-groups according to treatment duration (3/6 months) and disease stage (high/low-risk stage III).Results: Of 2,010 randomized patients, 1,345 had available ctDNA samples (1,017 collected both post-surgery and pre-chemotherapy). More Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 (78% versus 69%) and T4 and/or N2 (40% versus 36%) were observed in patients studied (n = 1017) versus not analyzed (n = 993). There were 877 ctDNA-negative (86.2%) and 140 ctDNA-positive (13.8%) patients; their baseline characteristics were similar. With a median follow-up of 6.6 years, the 3-year DFS rate was 66.39% for ctDNA-positive patients and 76.71% for ctDNA-negative patients (P = 0.015). ctDNA was confirmed as an independent prognostic marker for DFS (adjusted HR = 1.55, 95% CI 1.13-2.12, P = 0.006) and OS (HR = 1.65, 95% CI 1.12-2.43, P = 0.011). ctDNA was prognostic in patients treated for 3 months and with T4 and/or N2 tumors, but not in those treated for 6 months and with T1-3/N1 tumors.Conclusions: In this first ctDNA assessment of a large series of patients with stage III colon cancer enrolled in phase III trial, post-surgery ctDNA was found in 13.8% of them and was confirmed as an independent prognostic marke