Polycystic Ovary Syndrome (PCOS), the leading cause of infertility worldwide, is characterised by oligo-anovulation, hyperandrogenism, polycystic ovarian morphology and high Anti-Müllerian hormone (AMH) levels, associated with severe metabolic disturbances. However, the role of AMH in the physiopathology of this syndrome remains poorly understood and strategies to block its effects have never been investigated in animal models of PCOS. We used Western-blotting, ELISA and gene reporter approaches to evaluate the blocking efficacy, interspecificity and mechanism of action of an antibody against human AMH, Mab22A2. Then, we investigated the ability of a rat version of Mab22A2, rMab22A2, to alleviate reproductive dysfunction in Goto-Kakizaki (GK) rats, which spontaneously exhibit all the features of women with PCOS. We showed that Mab22A2 was interspecific, did not prevent AMH from binding to its receptor and was able to block the effects of AMH in gonadal cell lines. In addition, treatment of anovulatory GK rats with rMab22A2 reduced their bioavailable serum AMH levels and normalised their androgen concentrations. Finally, this treatment also induced ovulation in 84% of the rats and resulted in 66% of pregnancies. Our results show that AMH is a major driver of reproductive and hormonal dysfunction in PCOS and provide proof of concept that a blocking antibody against AMH can reverse the major reproductive dysfunction observed in PCOS, opening up promising avenues for the treatment of patients with PCOS. Inserm, Sorbonne University, Inserm Transfert, the French Endocrine Society and the Medical Research Foundation (grant agreement n°EQU201903007868).

Children undergoing cancer treatments are at risk for impaired fertility. Cryopreserved prepubertal testicular biopsies could theoretically be later matured in vitro to produce spermatozoa for assisted reproductive technology. A complete in vitro spermatogenesis has been obtained from mouse prepubertal testicular tissue, although with low efficiency. Steroid hormones are essential for the progression of spermatogenesis, the aim of this study was to investigate steroidogenesis and steroid signaling in organotypic cultures. Histological, RT-qPCR, western blot analyses, and steroid hormone measurements were performed on in vitro cultured mouse prepubertal testicular tissues and age-matched in vivo controls. Despite a conserved density of Leydig cells after 30 days of culture (D30), transcript levels of adult Leydig cells and steroidogenic markers were decreased. Increased amounts of progesterone and estradiol and reduced androstenedione levels were observed at D30, together with decreased transcript levels of steroid metabolizing genes and steroid target genes. hCG was insufficient to facilitate Leydig cell differentiation, restore steroidogenesis, and improve sperm yield. In conclusion, this study reports the failure of adult Leydig cell development and altered steroid production and signaling in tissue cultures. The organotypic culture system will need to be further improved before it can be translated into clinics for childhood cancer survivors.

Stability of adrenocorticotropic hormone in whole blood samples: effects of storage conditions

In vitro spermatogenesis appears to be a promising approach to restore the fertility of childhood cancer survivors. The rat model has proven to be challenging, since germ cell maturation is arrested in organotypic cultures. Here, we report that, despite a meiotic entry, abnormal synaptonemal complexes were found in spermatocytes, and in vitro matured rat prepubertal testicular tissues displayed an immature phenotype. RNA-sequencing analyses highlighted up to 600 differentially expressed genes between in vitro and in vivo conditions, including genes involved in blood-testis barrier (BTB) formation and steroidogenesis. BTB integrity, the expression of two steroidogenic enzymes, and androgen receptors were indeed altered in vitro. Moreover, most of the top 10 predicted upstream regulators of deregulated genes were involved in inflammatory processes or immune cell recruitment. However, none of the three anti-inflammatory molecules tested in this study promoted meiotic progression. By analysing for the first time in vitro matured rat prepubertal testicular tissues at the molecular level, we uncovered the deregulation of several genes and revealed that defective BTB function, altered steroidogenic pathway, and probably inflammation, could be at the origin of meiotic arrest.

Background: The measurement of serum Anti-Mullerian hormone (AMH) is used in daily practice to estimate the ovarian reserve in women. Methods: The aim of this study was to evaluate the new Lumipulse AMH (R) immunoassay (Fujirebio) with regard to the reliability with two preexisting assays: Elecsys AMH Plus (R) (cobas (R), Roche) and Vidas AMH (R) (bioMerieux). Precision of Lumipulse AMH was evaluated on the Lumipulse G600 II using the manufacturer's quality controls. Thirty-three samples were used for method comparison. Results: Lumipulse AMH repeatability and intermediate precision did not exceed 4.1 %. There was a proportional bias between Lumipulse and Cobas method and between Lumipulse and Vidas whereas a good agreement was found between Cobas and Vidas. Conclusions: Lumipulse AMH assay demonstrated good precision and good agreement with the two other methods up to 6 ng/mL. Nevertheless, this study shows the need to standardize AMH assays to improve the comparability of the methods.