BACKGROUND:Polycystic ovary syndrome (PCOS), recently renamed polyendocrine metabolic ovarian syndrome (PMOS), is characterised by neuroendocrine dysfunction with accelerated gonadotrophin-releasing hormone (GnRH)/luteinising hormone (LH) pulsatility driving hyperandrogenism and anovulatory infertility. METHODS:We used the prenatal anti-Müllerian hormone (AMH)-exposed PMOS-like mouse model (PAMH) and a phase I clinical trial in women with PMOS without obesity. PAMH and control mice received acute or intermittent low-dose Ganirelix, and oestrous cyclicity, ovulation, gonadotrophins, and steroids were assessed. In women, two subtherapeutic Ganirelix doses (0.025 mg, n = 8; 0.0625 mg, n = 10) were administered once in early follicular phase, with 10-min blood sampling over 8 h to quantify LH pulsatility and reproductive hormones. FINDINGS:In PMOS-like mice, a single Ganirelix injection normalised exaggerated LH pulsatility, and six-week intermittent treatment restored oestrous cyclicity, ovulation, and testosterone levels without affecting controls. In women with PMOS both Ganirelix doses reduced LH pulse frequency, basal, mean and total LH, and decreased the LH/FSH ratio. D4-androstenedione fell by 25-30% at both doses, AMH declined modestly at 0.0625 mg, while oestradiol remained unchanged. INTERPRETATION:Low-dose GnRH-receptor antagonism with Ganirelix can recalibrate, rather than suppress, GnRH/LH signalling, attenuating hyperandrogenism and, in mice, restoring ovulatory function. These data identify partial GnRHR blockade as a promising neuroendocrine-centred strategy in PMOS and provide a rationale for phase II trials evaluating repeated low-dose regimens, ovulatory restoration, and fertility outcomes. FUNDING:This work was supported by the European Research Council (ERC) Horizon-ERC-POC grant (ERC-2022-POC2, n° 101111874) and the French National Research Agency (ANR-24-CHBS-0002, France 2030).

A growing body of epidemiological evidence links maternal exposure to air pollution with an increased risk of adverse pregnancy outcomes, such as preterm birth and low birth weight. Cerium dioxide nanoparticles (CeO2 NPs or nanoceria) are emerging pollutants, used as additives in diesel fuels and cigarettes for their catalytic properties, and released into the environment. Due to their high surface-to-volume ratio and reactivity, CeO2 NPs develop a surface coating during combustion, which may incorporate other released fuel-borne chemicals, such as benzo[a]pyrene (BaP), a known carcinogen, mutagen and reprotoxicant, raising concerns about their combined impacts on human health. To better reflect environmental reality, we produced BaP-coated CeO2 NPs and exposed primary human trophoblasts and chorionic villi. Our findings show that BaP-coated CeO2 NPs activate the aryl hydrocarbon receptor (AhR) pathway, enhancing trophoblast differentiation and syncytium formation, with effects distinct from those of BaP or CeO₂ NPs alone, or their unbound mixture. Additionally, exposure to CeO2 NPs alone altered homeostasis of mitochondria, affecting their phenotype and function. While individual exposures or BaP-coated CeO2 NPs had no detectable impact, parallel co-exposure resulted in a slight but significant reduction in basal respiration. Finally, uncoated CeO2 NPs altered placental steroidogenesis, increasing estrone level while decreasing dehydroepiandrosterone level, with sex-specific effects. These findings suggest that CeO2 NPs can influence the biological effects of BaP in the human placenta, including modulating trophoblast differentiation, as well as disrupting mitochondria homeostasis and steroid production, with potential implications for pregnancy outcomes in polluted environments.

Introduction While a number of studies have examined the effects of prenatal exposure to per- and polyfluoroalkyl substances (PFAS) on childhood obesity, the results reported have been inconsistent and few studies have integrated biological markers. The aim of this study was to investigate the associations between prenatal exposure to PFAS and cardiometabolic health parameters at age 12, taking pubertal stage into consideration. Method This study included 394 mother-child pairs enrolled in the PELAGIE mother-child cohort (France). Nine PFAS were measured in umbilical cord blood, and the children attended a clinical examination at age 12. Anthropometry, blood metabolic markers, and blood pressure were measured and used to build an internal cardiometabolic score. Linear regression and Quantile G-computation models were used to evaluate individual and mixture PFAS effects, adjusting for confounders and stratifying by sex and pubertal stage. Results No statistically significant association was observed between prenatal exposure to PFAS and cardiometabolic score at age 12. In post-menarche girls, perfluorohexane sulfonate (PFHxS) and perfluorodecanoic acid (PFDA) were statistically significantly associated with a decrease in a number of adiposity parameters (e.g., Body mass index z-score: beta [95%CI] = -0.37 [-0.67; -0.07]), as well as a decrease in low-density lipoproteins (LDL) and leptin levels. Similar results were observed with PFAS mixture, with statistically significantly decreased tricipital skinfolds (beta [95%CI] = -1.30 [(-2.54;-0.06)]). Isolated associations, including higher systolic blood pressure, changes in cholesterol levels, and lower adiponectin levels were observed in specific subgroups. Conclusion There is no clear evidence of an association between prenatal exposure to PFAS and the cardiometabolic health at earlier stage of pubertal development. However, inverse associations between PFAS and anthropometric measures have been observed in post-menarche girls. While the literature on this topic is scarce in pre-adolescents, these results suggest the importance of considering sex and pubertal stage in these associations.

Understanding the tissue-specific distribution and metabolism of sex steroids is critical for elucidating their physiological roles and pathological alterations. However, existing in vivo approaches often require surgical castration to suppress endogenous hormones, disrupting systemic endocrine balance and limiting physiological relevance. Here, we present a novel mass spectrometry method combining systemic administration of deuterium-labeled sex steroids with high-sensitivity gas chromatography-tandem mass spectrometry (GC-MS/MS) to simultaneously quantify exogenous, endogenous, and metabolized steroid species in serum and multiple tissues of non-castrated mice. This approach enables temporally resolved tracking of steroid uptake and biotransformation without perturbing endogenous hormonal status. We demonstrate the method’s high sensitivity, specificity, and capacity to reveal tissue-specific steroid metabolism previously inaccessible in physiological models. Our technique offers a versatile platform for studying steroid dynamics in vivo and has broad applications in endocrinology, neurobiology, and pharmacology. Teaser Deuterium tracing illuminates hidden steroid pathways in living animals. ### Competing Interest Statement The authors have declared no competing interest. Agence Nationale de la Recherche, https://ror.org/00rbzpz17 doctoral school BioSCP (NW, MC).

Polycystic ovary syndrome (PCOS), the most common endocrinopathy in women, causes significant reproductive and metabolic comorbidities, with no current cure. Gestational androgen and anti-Müllerian hormone (AMH) excess are linked to PCOS, and prenatal aberrant exposure to these hormones induces PCOS-like traits in animal models. However, whether the AMH effects on PCOS programming could extend to early postnatal life remains unknown. Clinical observations showed higher AMH levels during minipuberty in infants of mothers with PCOS, but whether this contributes to PCOS development is uncertain. Here, we show that exposure to high AMH levels during minipuberty in mice causes PCOS-like reproductive and metabolic defects in both sexes. A neutralizing antibody targeting AMH receptor 2 (AMHR2) prevented these defects when administered during minipuberty and alleviated symptoms when given in adulthood. These findings highlight the causal role of elevated AMH in PCOS and suggest AMHR2-targeting therapy as a potential preventive or curative approach.

STUDY QUESTION What is the effect of attenuating the physiological hypergonadotropic activity encountered at minipuberty on female reproductive function in a mouse model? SUMMARY ANSWER Decreasing the surge of gonadotropins at minipuberty extended reproductive lifespan, coinciding with alterations in neuroendocrine and ovarian aging. WHAT IS KNOWN ALREADY Minipuberty is characterized by the tremendous activation of the gonadotrope axis, as evidenced by elevated levels of gonadotropins regulating folliculogenesis and the synthesis of ovarian hormones, but its role in fertility remains unclear. STUDY DESIGN, SIZE, DURATION To determine the link between gonadotrope axis activity at minipuberty and reproductive parameters, we used a pharmacological approach to suppress gonadotropin levels in Swiss mice by injecting daily a GnRH receptor antagonist (GnRHR) (Ganirelix, 10 mu g/mouse) or its vehicle between 10 and 16 postnatal days, to cover the entire duration of minipuberty. We analyzed the onset of puberty and estrous cyclicity as well as fertility in young (3-5 months) and middle-aged (11 months) mice from control (CTR) and antagonist-treated groups (n = 17-20 mice/age and treatment group). Ovaries and brains were collected, fixed, and sectioned (for histology, follicle count, and immunohistochemistry) or frozen (for analysis of follicular markers, aging, and inflammation) from adult females, and blood was collected by cardiac puncture for hormonal assays (n = 3-8 mice/age and treatment group). PARTICIPANTS/MATERIALS, SETTING, METHODS To analyze the initiation of puberty, we monitored vaginal opening and performed vaginal smears in CTR and antagonist-treated mice. We studied estrous cyclicity on vaginal smears at the beginning of reproductive life. Mice were mated several times with males to assess fertility rates, delay of conception, and litter size. To evaluate ovarian function, we counted follicles at different stages and corpora lutea, and we determined the relative intra-ovarian abundance of key follicular markers by real-time RT-PCR, as well as the levels of circulating anti-M & uuml;llerian hormone (AMH) and progesterone by ELISA and GC-MS, respectively. We also analyzed features of ovarian aging and inflammation by histology and by measuring the relative intra-ovarian abundance of some markers using real-time RT-PCR. To determine the impact on neuroendocrine determinants related to the CTR of reproduction, we analyzed circulating gonadotropin levels using Luminex assays as well as kisspeptin and GnRH immunoreactivity in the hypothalamus by immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE Our results show that the treatment had no impact on the initiation of puberty, estrous cyclicity, or fertility at the beginning of reproductive life. However, it increased reproductive lifespan, as shown by the higher percentage of antagonist-treated females than CTRs still fertile at 11 months of age (33% versus 6%; P = 0.0471). There were no significant differences in the number of kisspeptin and GnRH neurons, nor in the density of kisspeptin- and GnRH-immunoreactive neurons in the hypothalamic areas involved in reproduction between the two groups of mice studied at either 4 or 11 months. In addition, basal levels of FSH were comparable between the two groups at 4 and 11 months, but not those of LH at 11 months which were much lower in females treated with antagonist than in their age-matched CTRs (237 +/- 59.6 pg/ml in antagonist-treated females versus 1027 +/- 226.3 pg/ml in CTRs, P = 0.0069). Importantly, at this age, antagonist-treated mice had basal LH levels comparable to young mice (e.g. in 4-month-old CTRs: 294 +/- 71.75 pg/ml, P > 0.05). Despite their prolonged reproductive lifespan and delayed neuroendocrine aging, antagonist-treated mice exhibited earlier depletion of their follicles, as shown by lower numbers of primordial, primary, and preantral follicles associated with lower circulating AMH levels and relative intra-ovarian abundance of Amh transcripts than CTR mice. However, they exhibited comparable completion of folliculogenesis, as suggested by the numbers of antral follicles and corpora lutea, relative intra-ovarian abundance of Cyp19a1, Inhba, and Inhbb transcripts, and circulating progesterone levels that all remained similar to those of the CTR group. These observed alterations in ovarian function were not associated with increased ovarian aging or inflammation. LARGE-SCALE DATA None. LIMITATIONS, REASONS FOR CAUTION This study was carried out on mice, which is a validated research model. However, human research is needed for further validation. WIDER IMPLICATIONS OF THE FINDINGS This study, which is the first to investigate the physiological role of minipuberty on reproductive parameters, supports the idea that suppressing the high postnatal levels of gonadotropins may have long-term effects on female fertility by extending the duration of reproductive life. Perturbations in gonadotropin levels during this period of life, such as those observed in infants born prematurely, may thus have profound consequences on late reproductive functions. STUDY FUNDING/COMPETING INTEREST(S) This research was conducted with the financial support of ANR AAPG2020 (ReproFUN), CNRS, Inserm, Universit & eacute; Paris Cit & eacute;, and Sorbonne Universit & eacute;. The authors declare that they have no conflicts of interest.

Although the ovarian reserve is constituted in utero, the literature on the effects of persistent organic pollutants (POPs) during this vulnerable period on the ovarian reserve later in life is limited. We investigated whether cord blood concentrations of POPs were associated with decreased anti-M & uuml;llerian hormone (AMH, a marker of the ovarian reserve) levels in girls at the age of 12. We included 239 girls from the French mother-child PELAGIE cohort. POP concentrations of 14 organochlorine pesticides, 17 polychlorinated biphenyls (PCBs), 5 polybrominated diphenyl ethers, and 9 perpolyfluoroalkyl substances were measured on cord blood sampled at birth. During a follow-up study at 12 years old, blood samples were collected to measure AMH levels. Single-exposure associations were examined with multivariable linear regression models adjusted a priori for potential confounders. Stratification on menarche status was also performed. Mixture effects were investigated using quantile g-computation and Bayesian kernel machine regression. Overall, 16 POPs were measured in at least 30% of samples. No significant associations were found in multivariable linear regressions, except for the third tercile of exposure to PCB 180 which was statistically significantly associated with an increase in AMH levels at 12 years old (Tercile 2 v. Tercile 1: 0.13 ng/mL, 95% CI = -0.29, 0.56; Tercile 3 v. Tercile 1:0.51 ng/mL, 95% CI = 0.02, 0.99). Additionally, in post-menarcheal girls (N = 104) only, the second tercile of p,p'-DDE was statistically significantly associated with decreased AMH levels at 12 years old (Tercile 2 v. Tercile 1: -0.61 ng/mL, 95% CI = -1.16, -0.05, Tercile 3 v. Tercile 1: 0.02 ng/mL, 95% CI = -0.51, 0.54). Both mixture models returned null associations. Despite the limited associations observed in this study, we recommend exploring these associations in larger mother-child cohorts and at older ages.

Background: Advances in chromatography and mass spectrometry have allowed us to develop a novel technique for measuring intraprostatic hormone concentrations directly on prostate needle biopsies, rather than using traditional punch excision. This has significant clinical implications as intraprostatic dihydrotestosterone and testosterone levels could help monitor prostate growth, neoplasia and castration resistance. Methods: Patients undergoing radical cystoprostatectomy for bladder cancer were prospectively included. Each prostate specimen received one 90 mg punch excision and six needle biopsies. Intraprostatic hormones were dosed through gas chromatography-mass spectrometry. Results: We included twenty patients, of which eleven were incidentally diagnosed with prostate cancer; four had ISUP 1 (20%) and seven had ISUP 2 (35%). The prostate biopsy technique was unable to obtain measures for testosterone, Delta-4-androsterone and androstenedione. Tissue concentrations of DHEA, DHT, E1 and E2 can be obtained with no significant difference from the reference established on a punch from a single biopsy core sample. Conclusions: Our study demonstrates that intraprostatic concentrations of DHEA, DHT, E1 and E2 can be measured without significant difference from the reference established on a single punch excision. This finding opens the way to research on the interactions between endocrinology and prostate oncogenesis and particularly on the mechanisms of resistance to hormone therapies in vivo. Level of evidence: 2 (c) 2024 Elsevier Masson SAS. All rights are reserved, including those for text and data mining, AI training, and

Background and objective Failure rates after first-line treatment of localized prostate cancer (PCa) treatment remain high; therefore, it is essential to improve the selection and identification of at-risk patients to reduce mortality. The aim of the ANDROCAN study was to evaluate the biochemical recurrence (BCR) in patients with localized PCa treated by total prostatectomy at 5 yr after surgery, according to their presurgery gonadal status. Methods A prospective cohort study was conducted including 1318 patients undergoing total prostatectomy for localized PCa with a 5-yr postoperative follow-up. Clinical and hormonal data (assays of total testosterone [TT], bioavailable testosterone [BT], dihydrotestosterone, estrone, and estradiol were performed by gas chromatography/mass spectrometry) as well as metabolic syndrome parameters were collected at baseline before surgery. Pathological data (predominant Gleason grade 4 and stage) were collected and cross-referenced centrally. Factors associated with BCR were assessed by a multivariate analysis, and BCR-free survival was assessed by a Kaplan-Meier analysis. Key findings and limitations Among the 1318 patients, 237 had BCR of PCa. Considering demographic characteristics, populations with and without BCR were similar. However, patients with BCR had cancers with a higher Gleason score (p = 0.0001) and higher prostate-specific antigen (PSA) values (p = 0.0005) at baseline. Gleason score, pT >3a, and PSA level at baseline were positively correlated with BCR (p < 0.0001, p < 0.0001, and p = 0.0048, respectively), while BT and TT levels were not associated with BCR. This study includes patients with varying clinical characteristics, such as cancer history and metabolic syndrome, introducing variability that makes it challenging to isolate the specific effects of gonadal status on BCR. Another limitation is the lack of evaluation of long-term BCR beyond 5 yr, potentially overlooking recurrences that occur between 5 and 15 yr after surgery. This could lead to an underestimation of the actual long-term recurrence rates. Conclusions and clinical implications Overall, PSA levels, high Gleason score, and pT >3a are associated with a greater likelihood of disease recurrence following initial treatment and could serve as important prognostic indicators for predicting the risk of BCR. In this prospective study, biochemical hypogonadism was not associated with a higher occurrence of BCR within 5 yr of prostatectomy. The biological gonadal status of preoperative patients could potentially be useful for therapeutic decisions but does not provide an indication for the oncological follow-up. Patient summary Five-year follow up of patients after surgery showed that there is no association between hypogonadism (low levels of total testosterone and bioavailable testosterone) and cancer recurrence. However, cancer recurrence seems to be more associated with aggressiveness of cancer at the time of detection.

BACKGROUND:Industrial progress has led to the omnipresence of chemicals in the environment of the general population, including reproductive-aged and pregnant women. The reproductive function of females is a well-known target of endocrine-disrupting chemicals. This function holds biological processes that are decisive for the fertility of women themselves and for the health of future generations. However, insufficient research has evaluated the risk of combined mixtures on this function. This study aimed to assess the direct impacts of a realistic exposure to eight combined environmental toxicants on the critical process of folliculogenesis. METHODS:Female rabbits were exposed daily and orally to either a mixture of eight environmental toxicants (F group) or the solvent mixture (NE group, control) from 2 to 19 weeks of age. The doses were computed from previous toxicokinetic data to reproduce steady-state serum concentrations in rabbits in the range of those encountered in pregnant women. Ovarian function was evaluated through macroscopic and histological analysis of the ovaries, serum hormonal assays and analysis of the expression of steroidogenic enzymes. Cellular dynamics in the ovary were further investigated with Ki67 staining and TUNEL assays. RESULTS:F rabbits grew similarly as NE rabbits but exhibited higher total and high-density lipoprotein (HDL) cholesterol levels in adulthood. They also presented a significantly elevated serum testosterone concentrations, while estradiol, progesterone, AMH and DHEA levels remained unaffected. The measurement of gonadotropins, androstenedione, pregnenolone and estrone levels yielded values below the limit of quantification. Among the 7 steroidogenic enzymes tested, an isolated higher expression of Cyp19a1 was measured in F rabbits ovaries. Those ovaries presented a significantly greater density/number of antral and atretic follicles and larger antral follicles without any changes in cellular proliferation or DNA fragmentation. No difference was found regarding the count of other follicle stages notably the primordial stage, the corpora lutea or AMH serum levels. CONCLUSION:Folliculogenesis and steroidogenesis seem to be subtly altered by exposure to a human-like mixture of environmental toxicants. The antral follicle growth appears promoted by the mixture of chemicals both in their number and size, potentially explaining the increase in atretic antral follicles. Reassuringly, the ovarian reserve estimated through primordial follicles number/density and AMH is spared from any alteration. The consequences of these changes on fertility and progeny health have yet to be investigated.

Hypercholesterolemia is a major risk factor for coronary artery diseases and cardiac ischemic events. Cholesterol per se could also have negative effects on the myocardium, independently from hypercholesterolemia. Previously, we reported that myocardial ischemia-reperfusion induces a deleterious build-up of mitochondrial cholesterol and oxysterols, which is potentiated by hypercholesterolemia and prevented by translocator protein (TSPO) ligands. Here, we studied the mechanism by which sterols accumulate in cardiac mitochondria and promote mitochondrial dysfunction. We performed myocardial ischemia-reperfusion in rats to evaluate mitochondrial function, TSPO, and steroidogenic acute regulatory protein (STAR) levels and the related mitochondrial concentrations of sterols. Rats were treated with the cholesterol synthesis inhibitor pravastatin or the TSPO ligand 4'-chlorodiazepam. We used Tspo deleted rats, which were phenotypically characterized. Inhibition of cholesterol synthesis reduced mitochondrial sterol accumulation and protected mitochondria during myocardial ischemia-reperfusion. We found that cardiac mitochondrial sterol accumulation is the consequence of enhanced influx of cholesterol and not of the inhibition of its mitochondrial metabolism during ischemia-reperfusion. Mitochondrial cholesterol accumulation at reperfusion was related to an increase in mitochondrial STAR but not to changes in TSPO levels. 4'-Chlorodiazepam inhibited this mechanism and prevented mitochondrial sterol accumulation and mitochondrial ischemia-reperfusion injury, underlying the close cooperation between STAR and TSPO. Conversely, Tspo deletion, which did not alter cardiac phenotype, abolished the effects of 4'-chlorodiazepam. This study reveals a novel mitochondrial interaction between TSPO and STAR to promote cholesterol and deleterious sterol mitochondrial accumulation during myocardial ischemia-reperfusion. This interaction regulates mitochondrial homeostasis and plays a key role during mitochondrial injury.

Study question: What is the role of the physiological hypergonadotropic activity encountered at minipuberty on the implementation of female reproductive function, in a mouse model with manipulated minipubertal gonadotropin levels? Summary answer: Elevated minipubertal levels of gonadotropins may have long-term effects on fertility by mediating neuroendocrine aging and ovarian follicle depletion. What is known already: Minipuberty is characterized by the tremendous activation of the gonadotropin axis, as evidenced by elevated levels of gonadotropins regulating folliculogenesis as well as the synthesis of ovarian hormones including estradiol, testosterone, and AMH. Study design, size, duration: To determine whether hypergonadotropic activity of the gonadotropin axis at mini-puberty could impact reproductive parameters and female fertility, we used a pharmacological approach to suppress gonadotropin levels in Swiss mice by injecting daily a GnRH receptor antagonist (GnRHR) (Ganirelix, 10 ug/mouse) or its vehicle between 10 and 16 postnatal days, to cover the entire duration of minipuberty. We analyzed the onset of puberty and estrous cyclicity as well as fertility in young (3 to 5 months) and middle-aged (11 months) mice from control (CTR) and antagonist-treated groups (n = 17 to 20 mice/age and treatment group). Ovaries and brains were collected, fixed and sectioned (for histology, follicle count and immunohistochemistry) or frozen (for analysis of follicular markers, aging and inflammation) from adult females, and blood was collected by cardiac puncture for hormonal assays (n = 3 to 8 mice/age and treatment group). Participants/materials, setting, methods: To analyze the initiation of puberty, we monitored vaginal opening and performed vaginal smears to detect first estrus and diestrus 2 in control and antagonist-treated mice. We studied estrous cyclicity on vaginal smears to detect the occurrence of the different stages of the cycle at the beginning of reproductive life. Young and middle-aged mice of the two groups were mated several times with males to assess fertility rates, delay of conception and litter size. To evaluate ovarian function, we counted follicles at the primordial, primary, secondary and tertiary stages and corpora lutea by morphometric analyses, and we determined the relative intra-ovarian abundance of follicular markers (Amh, Inhba, Inhbb, Cyp19a1, Lhcgr, Fshr) by real-time RT-PCR, as well as the levels of circulating AMH and progesterone by ELISA and GC/MS, respectively. We also analyzed features of ovarian aging and inflammation (presence of oocyte-depleted follicles and multinucleated giant cells) by histology and by measuring the relative intra-ovarian abundance of Sirt1, Sod2, Tnfa and Il1b using real-time RT-PCR. To determine the impact on neuroendocrine determinants related to the control of reproduction, we analyzed circulating gonadotropin levels using Luminex assays as well as kisspeptin and GnRH immunoreactivity by immunohistochemistry in the hypothalamus, in both young and middle-aged mice. Main results and the role of chance: Our results show that the treatment had no impact on the initiation of puberty, estrous cyclicity, or fertility at the beginning of reproductive life. However, it increased reproductive lifespan, as shown by the higher percentage of antagonist-treated females than controls (33% versus 6%) still fertile at 11 months of age (P=0.0471). There were no significant differences in the number of kisspeptin and GnRH neurons, nor in the density of kisspeptin- and GnRH-immunoreactivity in the hypothalamic areas involved in reproduction between the two groups of mice studied at either 4 or 11 months. In addition, basal levels of LH and FSH were comparable between the two groups at 4 months, but not those of LH at 11 months which were much lower in females treated with antagonist than in their age-matched controls (237 +/- 59.60 pg/mL in antagonist-treated females versus 1027 +/- 226.3 pg/mL in controls, P=0.0069). Importantly, at this age, antagonist-treated mice had basal LH levels comparable to young mice (e.g., in 4-month-old controls: 294 +/- 71.75 pg/mL, P > 0.05), while those of control females were higher (P= 0.0091). Despite their prolonged reproductive lifespan and delayed neuroendocrine aging, antagonist-treated mice exhibited earlier depletion of their follicles, as shown by lower numbers of primordial, primary, and secondary follicles associated with lower circulating AMH levels and relative intra-ovarian abundance of Amh transcripts than control mice. However, they exhibited comparable completion of folliculogenesis, as suggested by the numbers of tertiary follicles and corpora lutea, relative intra-ovarian abundance of Cyp19a1, Inha and Inhb transcripts, and circulating progesterone levels that all remained similar to those of the control group. These observed alterations in ovarian function were not associated with increased ovarian aging or inflammation. Large scale data: none Limitations, reasons for caution: This study was carried out on mice, which is a validated research model. However, human research is needed for further validation. Wider implications of the findings: This study, which is the first to investigate the physiological role of minipuberty on reproductive parameters, supports the idea that high postnatal levels of gonadotropins may have long-term effects on female fertility by regulating the duration of reproductive life. Changes in gonadotropin levels during this period of life, such as those observed in infants born prematurely, may thus have profound consequences on late reproductive functions. ### Competing Interest Statement The authors have declared no competing interest.

Dans le cancer de la prostate localisé, l’impact de l’hypogonadisme biochimique sur l’émergence et la progression du cancer est encore controversée. Notre objectif est de comparer les caractéristiques pathologiques et la récidive biologique (RB) à 5 ans après prostatectomie des patients atteints de cancer de la prostate (CaP) localisé en fonction du statut gonadique évalué par le total (TT) et la biodisponibilité (BT). Une étude de cohorte prospective de 1318 patients (âge 65,0, taille 174 cm, poids 81,5 kg, IMC 26,0 kg/m2, périmètre abdominal 100 cm) atteints de CaP localisé recrutés dans 4 centres urologiques en France, de 6/2013 à 6/2016 ayant tous un suivi de 5 ans postopératoire. Les paramètres du syndrome métabolique (MetS) ont été recueillis. Les dosages de TT, BT, DHT, E1 et E2 ont été réalisés par GC-MS. Un examen croisé centralisé des données pathologiques (grade de Gleason 4 prédominant (PrdGP4), stade) a été effectué. La survie sans RB a été évaluée selon Kaplan-Meier avec comparaisons par test de Log-rank. La cohorte a été divisée en 3 groupes ; le premier (n = 1067 ; 81 %) composé de patients eugonadiques dont la TT et la BT étaient normales (TT ≥ 3 ng/ml et BT ≥ 0,8 ng/ml), le deuxième (n = 251 ; 19 %) de ceux dont la TT et/ou la BT étaient diminuées et le troisième (n = 58 ; 4 %) de ceux dont la TT et la BT étaient diminuées. Les pourcentages de PrdGP4 et de pT ≥ 3 a étaient respectivement de 31 % et 30 % chez les eugonadiques et 41 % et 40 %, et 50 % et 51 % dans les deuxième et troisième groupe (différences significatives). 237 RB ont été observées (fréquence de 17 % chez les eugonadiques contre 21 % chez les hypogonadiques (différence non significative ; Fig. 1) du groupe 2 et 7 % (p = 0,017) chez ceux du groupe 3 (Fig. 1). Cette étude prospective démontre que l’hypogonadisme bioChimique a été associé à des CaP dont les caractéristiques histopathologiques sont plus fréquemment défavorables mais sans survenue significativement supérieure de RB dans les 5 ans après prostatectomie. Par conséquent, le statut gonadique biologique préopératoire des patients est utile pour la décision thérapeutique mais n’indique pas un suivi spécifique sur le plan oncologique.

Estradiol (E2) is a major hormone-controlling folliculogenesis whose dysfunction may participate in polycystic ovary syndrome (PCOS) infertility. To determine whether both the concentration and action of E2 could be impaired in non-hyperandrogenic overweight PCOS women, we isolated granulosa cells (GCs) and follicular fluid (FF) from follicles of women undergoing ovarian stimulation (27 with PCOS, and 54 without PCOS). An analysis of the transcript abundance of 16 genes in GCs showed that androgen and progesterone receptor expressions were significantly increased in GCs of PCOS (by 2.7-fold and 1.5-fold, respectively), while those of the steroidogenic enzymes CYP11A1 and HSD3B2 were down-regulated (by 56% and 38%, respectively). Remarkably, treatment of GC cultures with E2 revealed its ineffectiveness in regulating the expression of several key endocrine genes (e.g., GREB1 or BCL2) in PCOS. Additionally, a comparison of the steroid concentrations (measured by GC/MS) in GCs with those in FF of matched follicles demonstrated that the significant decline in the E2 concentration (by 23%) in PCOS FF was not the result of the E2 biosynthesis reduction. Overall, our study provides novel hallmarks of PCOS by highlighting the ineffective E2 signaling in GCs as well as the dysregulation in the expression of genes involved in follicular growth, which may contribute to aberrant folliculogenesis in non-hyperandrogenic women with PCOS.

In females, androgens contribute to ovarian diseases such as polycystic ovarian syndrome (PCOS), but their action is also crucial for ovarian physiology, i.e., follicular growth and estradiol (E2) synthesis during reproductive life, in interaction with the gonadotropins LH and FSH. However, it is unclear whether androgens already play a role in the ovary at mini-puberty, a phase of postnatal development with active follicular growth and high E2 levels. Therefore, we analyzed the potential actions of androgens on the ovary and their possible interaction with gonadotropins during this period in mice. We used molecular-based studies and pharmacological approaches in vivo and on cultured ovaries. We found that mini-pubertal ovaries produce significant amounts of testosterone and display androgen receptor (AR) expression in growing follicles, both under the control of LH. By blocking AR signaling either in vivo or in ovarian cultures, we found that this pathway may participate in the regulation of prepubertal E2 synthesis and follicular growth, possibly by regulating the expression of a number of key intra-ovarian regulators, including FSH receptor (Fshr), the aromatase enzyme converting androgens into estrogens (Cyp19a1) and the cell cycle inhibitor p27KIP1 (Cdkn1b). We further showed that AR may stimulate FSH-mediated regulation of Cyp19a1 through its action on Fshr mRNA abundance. Overall, this work supports the idea that AR signaling is already activated in mini-pubertal ovaries to regulate E2 synthesis and follicular growth, at the interplay with LH and FSH signaling. Its early action may, thus, contribute to the implementation of early ovarian function with possible impacts on reproductive function.

Abstract Disclosure: M. Devillers: None. C.M. François: None. M. Chester: None. R. Corre: None. V. Cluzet: None. F. Giton: None. J. Cohen-Tannoudji: None. C.J. Guigon: None. Introduction: In females, androgens contribute to ovarian diseases such as polycystic ovary syndrome (PCOS), but their action is also crucial for ovarian physiology, i.e., follicular growth and estradiol (E2) synthesis during reproductive life, in interaction with the gonadotropins LH and FSH. However, it is unclear whether androgens already play a role in the ovary at mini-puberty, a phase of postnatal development with active follicular growth and high E2 levels. Hypothesis/question: Given their role in the adult ovary, we hypothesized that androgens may regulate early ovarian function at mini-puberty. Therefore, we analyzed their potential actions on the ovary and their possible interaction with gonadotropins during this period. Methods: We used the mouse as an experimental model to measure the intra-ovarian content and serum levels of testosterone by gas chromatography-mass spectrometry (GC-MS) at different prepubertal ages, including mini-puberty. We performed molecular-based studies (immunohistochemistry, RT-qPCR), morphometric studies and pharmacological approaches in vivo and in cultured ovaries. Main results: We found that mini-pubertal ovaries produce significant amounts of testosterone and display androgen receptor (AR) expression in growing follicles, both under the control of LH. By blocking AR signaling with flutamide either in vivo or in ovarian cultures, we found that this pathway may participate in the regulation of prepubertal E2 synthesis and follicular growth, possibly by regulating the expression of a number of key intra-ovarian regulators, including FSH receptor (Fshr), the aromatase enzyme converting androgens into estrogens (Cyp19a1) and the cell cycle inhibitor p27KIP1 (Cdkn1b). We further showed that AR may stimulate FSH-mediated regulation of Cyp19a1 through its action on Fshr mRNA abundance. Conclusion: This work supports the idea that AR signaling is already activated in mini-pubertal ovaries to regulate E2 synthesis and follicular growth, in interaction with LH and FSH signaling. This early physiological action of androgens may contribute to the implementation of early ovarian function with possible impacts on reproductive function. Presentation: Friday, June 16, 2023

AROMATASE is encoded by the CYP19A1 gene and is the cytochrome enzyme responsible for estrogen synthesis in vertebrates. In most mammals, a peak of CYP19A1 gene expression occurs in the fetal XX gonad when sexual differentiation is initiated. To elucidate the role of this peak, we produced 3 lines of TALEN genetically edited CYP19A1 knockout (KO) rabbits that were devoid of any estradiol production. All the KO XX rabbits developed as females with aberrantly small ovaries in adulthood, an almost empty reserve of primordial follicles, and very few large antrum follicles. Ovulation never occurred. Our histological, immunohistological, and transcriptomic analyses showed that the estradiol surge in the XX fetal rabbit gonad is not essential to its determination as an ovary, or for meiosis. However, it is mandatory for the high proliferation and differentiation of both somatic and germ cells, and consequently for establishment of the ovarian reserve.

Context Acetaminophen (APAP, paracetamol) is widely used by pregnant women. Although long considered safe, growing evidence indicates that APAP is an endocrine disruptor since in utero exposure may be associated with a higher risk of male genital tract abnormalities. In rodents, fetal exposure has long-term effects on the reproductive function of female offspring. Human studies have also suggested harmful APAP exposure effects. Objective Given that disruption of fetal ovarian development may impact women's reproductive health, we investigated the effects of APAP on fetal human ovaries in culture. Design and Setting Human ovarian fragments from 284 fetuses aged 7 to 12 developmental weeks (DW) were cultivated ex vivo for 7 days in the presence of human-relevant concentrations of APAP (10(-8) to 10(-3) M) or vehicle control. Main Outcome Measures Outcomes included examination of postculture tissue morphology, cell viability, apoptosis, and quantification of hormones, APAP, and APAP metabolites in conditioned culture media. Results APAP reduced the total cell number specifically in 10- to 12-DW ovaries, induced cell death, and decreased KI67-positive cell density independently of fetal age. APAP targeted subpopulations of germ cells and disrupted human fetal ovarian steroidogenesis, without affecting prostaglandin or inhibin B production. Human fetal ovaries were able to metabolize APAP. Conclusions Our data indicate that APAP can impact first trimester human fetal ovarian development, especially during a 10- to 12-DW window of heightened sensitivity. Overall, APAP behaves as an endocrine disruptor in the fetal human ovary.

La folliculogenèse est un processus ovarien complexe qui conduit à la sélection d’un follicule dominant, mature pour l’ovulation. L’œstradiol (E2) secrété par les cellules de la granulosa (CG) des follicules en croissance joue un rôle essentiel dans ce processus. Des troubles de la folliculogenèse sont observés dans le syndrome des ovaires polykystiques (SOPK) qui représente la première cause d’infertilité chez les femmes en âge de procréer (prévalence ∼ 10 %). Il se caractérise par un blocage de la croissance folliculaire associé à un défaut de sélection du follicule dominant. Bien que les critères diagnostiques du SOPK soient clairement établis (dysovulation ± hyperandrogénisme ± ovaires polykystiques), l’étiologie moléculaire du syndrome reste obscure. Étant donné l’importance de l’E2 dans la croissance et la sélection du follicule dominant, nous avons cherché à identifier les possibles perturbations de la signalisation œstrogénique dans le SOPK. Pour cela, nous avons réalisé des cultures primaires de CG provenant de follicules de femmes saines ou atteintes de SOPK qui suivent un protocole de FIV à l’hôpital Antoine-Béclère. Nous avons comparé leur capacité à produire des stéroïdes sexuels par GC-MS et mis en évidence la dérégulation de la chaîne de biosynthèse de la progestérone dans les CG de femmes « SOPK », associée à l’altération de l’expression de gènes codants certaines enzymes de la stéroïdogenèse par RT-qPCR. Le traitement des cultures primaires par l’E2 a également montré la perte de réceptivité œstrogénique dans les CG « SOPK », révélée par l’incapacité de l’E2 à induire l’expression de ses gènes cibles. Nos études complémentaires, utilisant une approche de gènes rapporteurs, sont en faveur d’un défaut d’activation des récepteurs de l’E2. En conclusion, nos résultats apportent des données nouvelles sur les dérégulations hormonales impliquées dans le SOPK et ouvrent des perspectives thérapeutiques prometteuses pour la prise en charge de cette pathologie.

ABSTRACT The binding of 17β-oestradiol to oestrogen receptor alpha (ERα) plays a crucial role in the control of reproduction, acting through both nuclear and membrane-initiated signalling. To study the physiological role of membrane ERα in the reproductive system, we used the C451A-ERα mouse model with selective loss of function of membrane ERα. Despite C451A-ERα mice being described as sterile, daily weighing and ultrasound imaging revealed that homozygous females do become pregnant, allowing the investigation of the role of ERα during pregnancy for the first time. All neonatal deaths of the mutant offspring mice resulted from delayed parturition associated with failure in pre-term progesterone withdrawal. Moreover, pregnant C451A-ERα females exhibited partial intrauterine embryo arrest at about E9.5. The observed embryonic lethality resulted from altered expansion of Tpbpa-positive spiral artery-associated trophoblast giant cells into the utero-placental unit, which is associated with an imbalance in expression of angiogenic factors. Together, these processes control the trophoblast-mediated spiral arterial remodelling. Hence, loss of membrane ERα within maternal tissues clearly alters the activity of invasive trophoblast cells during placentogenesis. This previously unreported function of membrane ERα could open new avenues towards a better understanding of human pregnancy-associated pathologies.