Developmental programming of production and reproduction in dairy cows: IV. Association of maternal milk fat and protein percentage and milk fat to protein ratio with offspring's birth weight, survival, productive and reproductive performance and AMH concentration from birth to the first lactation period.

Although the association of maternal milk production with developmental programming of offspring has been investigated, there is limited information available on the relationship of maternal milk components with productive and reproductive performance of the offspring. Therefore, the present study was conducted to analyze the association of maternal milk fat and protein percentage and milk fat to protein ratio with birth weight, survival, productive and reproductive performance and AMH concentration in the offspring. In study I, data of birth weight, milk yield and reproductive variables of offspring born to lactating dams (n = 14,582) and data associated with average maternal milk fat percentage (MFP), protein percentage (MPP) and fat to protein ratio (MFPR) during 305-day lactation were retrieved. Afterwards, offspring were classified in various categories of MFP, MPP and MFPR. In study II, blood samples (n = 339) were collected from offspring in various categories of MFP, MPP and MFPR for measurement of serum AMH. Maternal milk fat percentage was positively associated with birth weight and average percentage of milk fat (APMF) and protein (APMP) and milk fat to protein ratio (FPR) during the first lactation, but negatively associated with culling rate during nulliparity in the offspring (P < 0.05). Maternal milk protein percentage was positively associated with birth weight, APMF, APMP, FPR and culling rate, but negatively associated with milk yield and fertility in the offspring (P < 0.05). Maternal FPR was positively associated with APMF and FPR, but negatively associated with culling rate, APMP and fertility in the offspring (P < 0.05). However, concentration of AMH in the offspring was not associated with MFP, MPP and MFPR (P > 0.05). In conclusion, the present study revealed that maternal milk fat and protein percentage and their ratio were associated with birth weight, survival, production and reproduction of the offspring. Yet it was a preliminary research and further studies are required to elucidate the mechanisms underlying these associations.

O hormônio antimulleriano e a reserva ovariana: uma revisão integrativa da literatura / The anti-mullerian hormone and the ovarian reserve: an integrative literature review

O hormônio antimulleriano é secretado pelas células da granulosa dos folículos que estão em desenvolvimento no ovário. Por meio da sua dosagem, é possível avaliar a reserva ovariana. A mulher tem seu número máximo de oócitos no perío- do fetal, mas, conforme o tempo passa, existe uma queda do número de células germinativas. Desse modo, para mulheres que têm o desejo de engravidar, a dosa- gem de hormônios e a avaliação da reserva ovariana podem ajudar no processo. O objetivo do estudo foi encontrar evidências na literatura que comprovem que o hormônio antimulleriano é o melhor marcador da reserva ovariana. Para isso, foi realizada uma revisão integrativa, classificada como qualitativa; a busca de da- dos foi realizada no PubMed, utilizando a seguinte palavra-chave: "hormônio anti- mulleriano (HAM)". Foram encontrados oito artigos que abordavam diretamente o tema, e há evidências que corroboram a hipótese de que o hormônio antimulleria- no é um bom marcador da reserva ovariana, sendo necessários mais estudos para determinar a sua superioridade.

The anti-mullerian hormone is secreted by the granulosa cells of follicles that are developing in the ovary. Though its dosage is possible to evaluate the ovarian re- serve. Women have their maximum number of oocytes in the fetal period, but there is a decrease in the number of germinative cells as time goes by. Thus, women that desire to get pregnant can have hormones dosed and the ovarian reserve evalua- ted to help them with this process. The objective of this study was to find evidence in the literature that proves that the anti-mullerian hormone is the best marker of ovarian reserve. For this purpose, an integrative review was conducted, using the key word: "anti-mullerian hormone (AMH)". Eight articles were found on the subject and there is evidence that proves the hypothesis of the anti-mullerian hormone as a good marker, however more studies are needed to determine its superiority.

A novel method for purification of biologically active C-terminal fragment of human recombinant anti-mullerian hormone.

Anti-mullerian hormone (AMH) is one of the least studied members of transforming growth factor beta superfamily showing pro-apoptotic activity against cells positive for hormone type II receptor overexpressed by malignant cells in many cancer cases. Here, we propose an improved method for isolation of recombinant C-terminal AMH fragment (C-rAMH) to obtain homogeneous preparations of this protein with high biological activity. In contrast to our previously developed C-rAMH purification technology based on reversed-phase HPLC, the key stage of the new approach is hydrophobic interaction chromatography using Toyopearl Butyl-650S resin performed under more benign conditions. This modification of the previously developed method allowed highly purified C-rAMH to be obtained that is characterized by twice the specificity estimated as the ability to bind to the recombinant analog of AMH type II receptor and by significantly higher biological activity, that is, the ability to induce the death of target cells. Thus, we made the purification technology even more cost-effective and suitable for the production of drug forms based on C-rAMH.

Functional Activity of Recombinant Forms of Amh and Synergistic Action with Fsh in European Sea Bass Ovary.

Although anti-Müllerian hormone (AMH) has classically been correlated with the regression of Müllerian ducts in male mammals, involvement of this growth factor in other reproductive processes only recently come to light. Teleost is the only gnathostomes that lack Müllerian ducts despite having amh orthologous genes. In adult teleost gonads, Amh exerts a role in the early stages of germ cell development in both males and females. Mechanisms involving the interaction of Amh with gonadotropin- and growth factor-induced functions have been proposed, but our overall knowledge regarding Amh function in fish gonads remains modest. In this study, we report on Amh actions in the European sea bass ovary. Amh and type 2 Amh receptor (Amhr2) are present in granulosa and theca cells of both early and late-vitellogenic follicles and cannot be detected in previtellogenic ovaries. Using the Pichia pastoris system a recombinant sea bass Amh has been produced that is endogenously processed to generate a 12-15 kDa bioactive mature protein. Contrary to previous evidence in lower vertebrates, in explants of previtellogenic sea bass ovaries, mature Amh has a synergistic effect on steroidogenesis induced by the follicle-stimulating hormone (Fsh), increasing E2 and cyp19a1a levels.

Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-ß family.

Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-ß) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-ß family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.

Challenges in Measuring AMH in the Clinical Setting.

Serum anti-Mullerian hormone (AMH) is a widely used marker of functional ovarian reserve in the assessment and treatment of infertility. It is used to determine dosing of gonadotropins used for superovulation prior to in vitro fertilization, as well as to determine the degree of damage to ovarian reserve by cytotoxic treatments such as chemotherapy. AMH is also now used to predict proximity to menopause and potentially provides a sensitive and specific test for polycystic ovarian syndrome. Twenty one different AMH immunoassay platforms/methods are now commercially available. Of those compared, the random-access platforms are the most reliable. However, to date there has not been an agreed common international AMH reference preparation to standardize calibration between the various immunoassays. Recently, a purified human AMH preparation (code 16/190) has been investigated by the World Health Organization as a potential international reference preparation. However, this was only partially successful as commutability between it and serum samples was observed only in some but not all immunoassay methods. Development of a second generation reference preparation with wider commutability is proposed.

Molecular characterization and expression analysis of anti-Müllerian hormone in common carp (Cyprinus carpio) adult testes.

Anti-Müllerian hormone (Amh) is a member of the transforming growth factor-ß (Tgf-ß) superfamily required in the regression of Müllerian ducts during gonadal sex differentiation of higher vertebrates. Teleost fish lack Müllerian ducts, but identified Amh orthologs have been shown to exert crucial functions during sex determination and differentiation of several species of teleosts. However, the function of Amh during gametogenesis in adult fish remains poorly investigated. Therefore, to expand present knowledge on the role of Amh in teleosts, the present study aimed to isolate and clone full-length amh cDNA in the common carp, Cyprinus carpio, and examine its expression levels throughout the male reproductive cycle and in response to different hormone treatments of testicular explants. Molecular cloning and characterization showed that the common carp Amh precursor amino acid sequence shared common features to other fish Amh precursors, including a conserved C-terminus (Tgf-ß domain) and a double proteolytic cleavage site (R-X-X-R-X-X-R) upstream to the Tgf-ß domain. Expression analysis showed amh dimorphic expression in the adult gonads with higher expression in the testes than ovaries. In testes, amh mRNA was detected in Sertoli cells contacting different types of germ cells, although the expression was greatest in Sertoli cells associated with type A undifferentiated spermatogonia. Expression analysis during the reproductive cycle showed that amh transcripts were down-regulated during the developing phase, which is characterized by an increased proliferation of type A undifferentiated spermatogonia and Sertoli cells and appearance of spermatocytes (meiosis) in the testes. Furthermore, ex vivo experiments showed that a 7 day exposure to Fsh or estrogens was required to decrease amh mRNA levels in common carp testicular explants. In summary, this study provided information on the molecular characterization and transcript abundance of amh in common carp adult testes. Altogether, these data will be useful for further investigations on sex determination and differentiation in this species, and also to improved strategies for improved carp aquaculture, such as inhibiting precocious maturation of males.

The proportion of cleaved anti-Müllerian hormone is higher in serum but not follicular fluid of obese women independently of polycystic ovary syndrome.

RESEARCH QUESTION: Does the relative distribution of anti-Müllerian hormone (AMH) isoforms differ between patients depending on their body mass index (BMI) and polycystic ovary syndrome (PCOS) status in serum and follicular fluid? DESIGN: Obese and normal weight patients (PCOS [n = 70]; non-PCOS [n = 37]) were selected for this case-control study in the serum. Between 2018 and 2019, obese (n = 19) and normal weight (n = 20) women with or without PCOS who were receiving IVF treatment were included in the follicular fluid study. The bio-banked serums and follicular fluid were tested for total AMH (proAMH and AMHN,C combined) and proAMH using an automatic analyzer. The AMH prohormone index (API = [proAMH]/[total AMH]x 100) was calculated as an inverse marker of conversion of proAMH to AMHN,C, with only the latter isoform that could bind to the AMH receptor complex. RESULTS: The API was not significantly different between controls and women with PCOS, whereas obese women had a lower API compared with their normal weight counterparts. Grouping PCOS and controls, a lower API was found in obese versus normal weight women, suggesting a greater conversion of proAMH to AMHN,C. The API in the serum was significantly correlated with metabolic parameters. In the follicular fluid, API is not different between obese and normal weight women independently of PCOS and is higher than in the concomitant serum. CONCLUSIONS: The proportion of inactive form of AMH in the serum is higher in normal weight versus obese women but not in the follicular fluid, independently of PCOS. The conversion of proAMH into the cleaved isoform is likely to occur in extra-ovarian tissues and to exacerbate in obese individuals.

A mutation inactivating the distal SF1 binding site on the human anti-Müllerian hormone promoter causes persistent Müllerian duct syndrome.

The persistent Müllerian duct syndrome (PMDS) is a 46,XY disorder of sexual development characterized by the persistence of Müllerian duct derivatives, uterus and tubes, in otherwise normally masculinized males. The condition, transmitted as a recessive autosomal trait, is usually due to mutations in either the anti-Müllerian hormone (AMH) gene or its main receptor. Many variants of these genes have been described, all targeting the coding sequences. We report the first case of PMDS due to a regulatory mutation. The AMH promoter contains two binding sites for steroidogenic factor 1 (SF1), one at -102 and the other at -228. Our patient carries a single base deletion at -225, significantly decreasing its capacity for binding SF1, as measured by the electrophoresis mobility shift assay. Furthermore, by linking the AMH promoter to the luciferase gene, we show that the transactivation capacity of the promoter is significantly decreased by the mutation, in contrast to the disruption of the -102 binding site. To explain the difference in impact we hypothesize that SF1 could partially overcome the lack of binding to the -102 binding site by interacting with a GATA4 molecule linked to a nearby response element. We show that disruption of both the -102 SF1 and the -84 GATA response elements significantly decreases the transactivation capacity of the promoter. In conclusion, we suggest that the distance between mutated SF1 sites and potentially rescuing GATA binding motifs might play a role in the development of PMDS.

Vitamin D and Anti-Müllerian Hormone Levels in Infertility Treatment: The Change-Point Problem.

BACKGROUND: Anti-Müllerian hormone (AMH) is considered to be one of the most significant indicators of women's fertility. Many studies have shown that vitamin D may modify human reproductive functions; however, the results are conflicting. The composition of follicular fluid (FF) creates the biochemical environment of the oocyte and affects its quality, which later determines the embryo quality. In this study, we aimed to revise with advanced statistical techniques the relationship between AMH and vitamin D in FF. METHODS: The study was designed as a prospective single-center study in infertile patients with AMH ≥ 0.7 ng/mL who underwent controlled ovarian hyperstimulation for in vitro fertilization. AMH and vitamin D levels in FF were measured. Next, the standard and advanced statistical (including segmented regression) techniques were applied. RESULTS: We observed a negative linear correlation between levels of AMH in serum and FF and total vitamin D concentrations up to approximately 30 ng/ml; with a statistically significant relationship in FF. Beyond that concentration, the trend was positive but statistically insignificant. CONCLUSIONS: As an existing "change-point problem" was noticed, we suggest segmentation in the relationship between vitamin D and AMH during infertility treatment.

Oligopeptides for Immunotherapy Approaches in Ovarian Cancer Treatment.

BACKGROUND: Anti-ovarian cancer vaccines based on minimal immune determinants uniquely expressed in ovarian cancer biomarkers appear to promise a high level of sensitivity and specificity for ovarian cancer immunodiagnostics, immunoprevention, and immunotherapy. METHODS: Using the Pir Peptide Match program, three ovarian cancer biomarkers - namely, sperm surface protein Sp17, WAP four-disulfide core domain protein 2, and müllerian-inhibiting substance - were searched for unique peptide segments not shared with other human proteins. Then, the unique peptide segments were assembled to define oligopeptides potentially usable as synthetic ovarian cancer antigens. RESULTS AND CONCLUSION: This study describes a methodology for constructing ovarian cancer biomarkerderived oligopeptide constructs that might induce powerful, specific, and non-crossreactive immune responses against ovarian cancer.

Quantification of Müllerian Inhibiting Substance/Anti-Müllerian Hormone polypeptide by isotope dilution mass spectrometry.

Measurement of serum concentrations of Müllerian inhibiting substance (MIS), also known as anti-Müllerian Hormone (AMH) by immunoassay is gaining clinical acceptance and widespread use for the diagnosis of ovarian conditions and for prediction of the response to ovarian stimulation protocols as part of assisted reproductive therapies. Provision of an International Standard to harmonize immunoassay methods is required. It is desirable for the content of a future International Standard to be assigned in mass units for consistency with the units reported by current methods. Isotope dilution mass spectrometry (IDMS), a physicochemical method with traceability to the SI (Système International d'Unités) unit of mass, is a candidate approach to provide orthogonal data to support this mass assignment. Here, we report on the development of an IDMS method for quantitation of AMH using three peptides from different regions of the AMH monomer as surrogates for the measurement of AMH. We show the sensitivity and linearity of the standard peptides and demonstrate the reproducibility and consistency of the measurement amongst the three peptides for determining the AMH content in buffered preparations and in trial preparations of recombinant AMH, lyophilised in the presence of an excess of bovine casein.

Potential therapeutic applications of human anti-Müllerian hormone (AMH) analogues in reproductive medicine.

Members of the transforming growth factor-beta (TGF-beta) superfamily are key regulators of various physiological processes. Anti-Müllerian hormone (AMH) which is also commonly known as Müllerian-inhibiting substance (MIS) is a member of the TGF-beta superfamily and an important regulator of reproductive organ differentiation and ovarian follicular development. While AMH has been used for diagnostic purposes as a biomarker for over 15 years, new potential therapeutic applications of recombinant human AMH analogues are now emerging as pharmacologic agents in reproductive medicine. Therapeutic uses of AMH in gonadal tissue may provide a unique opportunity to address a broad range of reproductive themes, like contraception, ovulation induction, onset of menopause, and fertility preservation, as well as specific disease conditions, such as polycystic ovarian syndrome (PCOS) and cancers of the reproductive tract. This review explores the most promising therapeutic applications for a novel class of drugs known as AMH analogues with agonist and antagonist functions.

The Persistent Müllerian Duct Syndrome: An Update Based Upon a Personal Experience of 157 Cases.

Male sex differentiation is driven by 2 hormones, testosterone and anti-müllerian hormone (AMH), responsible for the regression of müllerian ducts in male fetuses. Mutations inactivating AMH or its receptor AMHRII lead to the persistent müllerian duct syndrome (PMDS) in otherwise normally virilized 46,XY males. Our objective was to review the clinical, anatomical, and molecular features of PMDS based upon a review of the literature and upon 157 personal cases. Three clinical presentations exist: bilateral cryptorchidism, unilateral cryptorchidism with contralateral hernia, and transverse testicular ectopia. Abnormalities of male excretory ducts are frequent. Testicular malignant degeneration occurs in 33% of adults with the disorder, while cancer of müllerian derivatives is less frequent. Fertility is rare but possible if at least one testis is scrotal and its excretory ducts are intact. Eighty families with 64 different mutations of the AMH gene have been identified, mostly in exons 1, 2, and 5. AMHRII gene mutations representing 58 different alleles have been discovered in 75 families. The most common mutation, a 27-bp deletion in the kinase domain, was found in 30 patients of mostly Northern European origin. In 12% of cases, no mutation of AMH or AMHRII has been detected, suggesting a disruption of other pathways involved in müllerian regression.

Anti-Müllerian hormone signaling is influenced by Follistatin 288, but not 14 other transforming growth factor beta superfamily regulators.

The hypothesis that, in contrast to other transforming growth factor-beta (TGFß) superfamily ligands, the dose-response curve of Anti-Müllerian hormone (AMH) is unmodulated was tested by examining whether known TGFB superfamily modulators affect AMH signaling, using a P19/BRE luciferase reporter assay. AMHC and AMHN,C activated the reporter with an EC50 of approximately 0.5 nM. Follistatins (FS) produced concentration-dependent increases in AMHC - and AMHN,C -initiated reporter activity, with FS288 being more potent than FS315; however, the maximum bioactivity of AMH was not altered by either follistatin. Thirteen other TGFß regulators (Chordin, Chordin-like 1, Chordin-like 2, Differential screening-selected gene aberrative in neuroblastoma [DAN], Decorin, Endoglin, Follistatin-like 1, Follistatin-like 3, Follistatin-like 4, Noggin, α2 macroglobulin, TGFß receptor 3, Von Willebrand factor C domain-containing 2) had little or no effect. Surface plasmon resonance analysis showed no significant association between FS288 and AMHC , suggesting that FS288 indirectly regulates AMH signaling. Activin A, a direct target of FS288, did not itself induce reporter activity in P19 cells, but did prevent the FS288-induced increase in AMH signaling. Hence, local concentrations of FS288 and Activin A may influence the response of some cell types to AMH.

Developmental effects of imatinib mesylate on follicle assembly and early activation of primordial follicle pool in postnatal rat ovary.

Imatinib mesylate is an anti-cancer agent that competitively inhibits several receptor tyrosine kinases (RTKs). RTKs play important roles in the regulation of primordial follicle formation, the recruitment of primordial follicles into the pool of growing follicles and maturation of the follicles. In the present study, we investigated the effects of the tyrosine kinase inhibitor imatinib on primordial follicle assembly and early folliculogenesis in postnatal rats. Female Sprague-Dawley rats were treated with either imatinib (150mg/kg) or placebo (water) on postnatal days 2-4. Bilateral ovariectomy was performed on postnatal day 2 and 5. Histology, immunohistochemistry, and mRNA analysis were performed. Imatinib treatment was associated with increased density of the multi-oocyte follicles (P<0.01), oogonia (p<0.01) and germline clusters (P<0.05), decreased activation of primordial follicles, increased expression of c-Kit and AMH, and decreased protein expression of Kit-ligand and GDF9 when compared to age-matched controls. In conclusion, imatinib affects folliculogenesis in postnatal rat ovaries by delaying the cluster breakdown, follicular assembly and early activation of the primordial follicle pool.

Molecular changes in the equine follicle in relation to variations in antral follicle count and anti-Müllerian hormone concentrations.

REASONS FOR PERFORMING STUDY: The wide variation in circulating anti-Müllerian hormone (AMH) concentrations between mares is attributed to differences in antral follicle count (AFC) which may reflect follicular function. There are few data regarding variations in AFC and associated regulatory factors for AMH in the equine follicle during follicular development. OBJECTIVES: To examine molecular and hormonal differences in the equine follicle in relation to variations in AFC and circulating AMH concentrations during follicular development and to identify genes co-expressed with AMH in the equine follicle. STUDY DESIGN: Observational study. METHODS: Plasma AMH concentrations and AFC were determined in 30 cyclic mares. Granulosa cells, theca cells and follicular fluid were recovered from growing (n = 17) or dominant follicles (n = 13). The expression of several genes, known to be involved in folliculogenesis and steroidogenesis, was examined using real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. Intrafollicular oestradiol and AMH concentrations were determined by immunoassay. RESULTS: Within growing follicles, the expression of AMH, AMHR2, ESR2 and INHA in granulosa cells was positively correlated with AFC and plasma AMH concentrations. In addition, the expression of ESR1 and FSHR was positively associated with plasma AMH concentrations. No significant associations were detected in dominant follicles. Furthermore, there was no association between AMH or oestradiol concentrations in follicular fluid and variations in AFC. Finally, the expression of AMH and genes co-expressed with AMH (AMHR2, ESR2 and FSHR) in granulosa cells as well as intrafollicular AMH concentrations decreased during follicular development while intrafollicular oestradiol concentrations increased and were inversely related to intrafollicular AMH concentrations. CONCLUSIONS: This study indicates that variations in AFC and circulating AMH concentrations are associated with molecular changes in the growing equine follicle.

Anti-mullerian hormone is expressed by endometriosis tissues and induces cell cycle arrest and apoptosis in endometriosis cells.

BACKGROUND: The anti-mullerian hormone (AMH) is a member of the transforming growth factor ß (TGF-ß) superfamily, which is responsible of the regression of the mullerian duct. AMH is expressed in the normal endometrium, where, acting in a paracrine fashion, negatively regulates cellular viability. Our objective was to evaluate the in vitro effects of the treatment with AMH of endometriosic cells. METHODS: AMH expression in human endometriosis glands was evaluated by immunohistochemistry. RT-PCR has been used to quantify the expression levels of AMH and AMH RII isoforms, as well as of cytochrome P450 in both endometriosis epithelial and stromal cells Effects of AMH and AMH-cleaved treatment in endometriosis cells were evaluated by flow-cytometry analysis. Finally, it has been evaluated the effect of plasmin-digested AMH on cytochrome P450 activity. RESULTS: AMH and AMH RII isoforms, as well as cytochrome P450, were expressed in both endometriosis epithelial and stromal cells. Treatment of endometriosis stromal and epithelial cell growth with AMH was able to induce a decrease in the percentage of cells in S phase and increase percentage of cells in G1 and G2 phase; coherently, decreased cell viability and increased percentage of cells death fraction was observed. The plasmin-digested AMH was able to suppress most of the cytochrome P450 activity, causing an increase of pre-G1 phase and of apoptosis induction treating with plasmin-digested AMH in both cell lines, most marked in the epithelial cells. CONCLUSIONS: The data produced suggest a possible use of AMH as therapeutic agents in endometriosis.

Decreasing quality of the new generations of anti-Müllerian hormone assays.

Anti-Müllerian hormone (AMH) measurements are widely used to optimize the stimulation protocols. First generation AMH kits correlated well with ovarian reserve and response to stimulation. In the present study we aimed to asses if the new generation kits share the same accurate correlations. Retrospective data were collected from 8323 blood samples. For comparison we used Immunotech I generation kit (ImI 4035 samples), Beckman Coulter II generation kit RUO (BCII RUO 3449, samples) and Beckman Coulter II generation kit with IVD certificate (BCII IVD 839 samples). We compared average AMH concentrations measured with different kits, as well as correlation between kits. We also compared average AMH concentrations in sera collected on different cycle days and samples of different quality of preservation. AMH serum concentrations differed for each kit, ranging 4.4 ± 4.12 (mean ± SD) for the ImI, 2.68 ± 3.15 for the BCII RUO, and 1.64 ± 2.85 for BCII IVD. The mean differences from an adjusted regression model were -48.7%, -40%, and -69.2%, respectively. In conclusion, the changes of the BC AMH kits are unpredictable; however, the improvement of them is still possible. It would be very dangerous to use elaborated stimulation protocol (based on the Ist generation AMH results) with the results from the IInd generation assays.

Müllerian inhibiting substance in the caudate amphibian Pleurodeles waltl.

Müllerian inhibiting substance (MIS, also known as anti-Müllerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Müllerian duct regression in male embryos. In fish, despite the absence of Müllerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Müllerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF-ß family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Müllerian ducts occurred secondarily during the course of evolution.