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BACKGROUND The beneficial effects of hormonal therapy in stimulating spermatogenesis in patients with non-obstructive azoospermia (NOA) and either normal gonadotrophins or hypergonadotropic hypogonadism prior to surgical sperm retrieval (SSR) is controversial. Although the European Association of Urology guidelines state that hormone stimulation is not recommended in routine clinical practice, a significant number of patients undergo empiric therapy prior to SSR. The success rate for SSR from microdissection testicular sperm extraction is only 40–60%, thus hormonal therapy could prove to be an effective adjunctive therapy to increase SSR rates. OBJECTIVE AND RATIONALE The primary aim of this systematic review and meta-analysis was to compare the SSR rates in men with NOA (excluding those with hypogonadotropic hypogonadism) receiving hormone therapy compared to placebo or no treatment. The secondary objective was to compare the effects of hormonal therapy in normogonadotropic and hypergonadotropic NOA men. SEARCH METHODS A literature search was performed using the Medline, Embase, Web of Science and Clinicaltrials.gov databases from 01 January 1946 to 17 September 2020. We included all studies where hormone status was confirmed. We excluded non-English language and animal studies. Heterogeneity was calculated using I2 statistics and risk of bias was assessed using Cochrane tools. We performed a meta-analysis on all the eligible controlled trials to determine whether hormone stimulation (irrespective of class) improved SSR rates and also whether this was affected by baseline hormone status (hypergonadotropic versus normogonadotropic NOA men). Sensitivity analyses were performed when indicated. OUTCOMES A total of 3846 studies were screened and 22 studies were included with 1706 participants. A higher SSR rate in subjects pre-treated with hormonal therapy was observed (odds ratio (OR) 1.96, 95% CI: 1.08–3.56, P = 0.03) and this trend persisted when excluding a study containing only men with Klinefelter syndrome (OR 1.90, 95% CI: 1.03–3.51, P = 0.04). However, the subgroup analysis of baseline hormone status demonstrated a significant improvement only in normogonadotropic men (OR 2.13, 95% CI: 1.10–4.14, P = 0.02) and not in hypergonadotropic patients (OR 1.73, 95% CI: 0.44–6.77, P = 0.43). The literature was at moderate or severe risk of bias. WIDER IMPLICATIONS This meta-analysis demonstrates that hormone therapy is not associated with improved SSR rates in hypergonadotropic hypogonadism. While hormone therapy improved SSR rates in eugonadal men with NOA, the quality of evidence was low with a moderate to high risk of bias. Therefore, hormone therapy should not be routinely used in men with NOA prior to SSR and large scale, prospective randomized controlled trials are needed to validate the meta-analysis findings.

Hypogonadism can be present in up to 40% in men who present with couple infertility. Testosterone is the major androgen regulating-spermatogenesis in men; as a result, men with either primary or secondary hypogonadism may be subfertile because of impaired spermatogenesis. The clinical impact of hypogonadism on fertility potential depends on the timing of its onset (fetal, prepubertal, or postpubertal) and effect on semen parameters. Treatment pathways and success rates differ according to the cause of hypogonadism and the time of its onset. When medical therapy fails to induce sufficient sperm, assisted reproductive technologies are considered.

This study compared the cost per live birth and cost-effectiveness of the originator recombinant human follicle-stimulating hormone follitropin alfa (r-hFSH-alfa) and r-hFSH-alfa biosimilars for ovarian stimulation prior to assisted reproductive technology treatment in Spain. A decision tree model was developed, comprising pregnancy and live birth for one treatment cycle with fresh embryo transfer. Clinical inputs were based on a recent meta-analysis by Chua et al. [4]. Cost inputs were extracted from publicly available Spanish sources. The costs per live birth were lower with originator r-hFSH-alfa (€18,138) versus r-hFSH-alfa biosimilars (€20,377). The incremental cost-effectiveness ratio was €7208 for originator r-hFSH-alfa versus biosimilars. Drug acquisition costs for originator r-hFSH-alfa represented 10.5% of total costs in the base case analysis, and 6.2% in a treatment cycle resulting in live birth with one fresh embryo transfer. Results from the sensitivity analyses confirmed the robustness of the findings. ispartof: BEST PRACTICE & RESEARCH CLINICAL OBSTETRICS & GYNAECOLOGY vol:85 issue:Pt B pages:203-216 ispartof: location:Netherlands status: published

ObjectivesMost of the currently available ovulation prediction kits provide a relatively rough estimation of ovulation time with a short fertility window. This is due to their focus on the maximum probability of conception occurring one day before ovulation, with no follow-up after LH surge until ovulation nor during the subsequent days thereafter. Earlier studies have shown that urine of reproductive age women contains at least 3 different molecular forms of luteinizing hormone (LH); 1) intact LH, 2) LH beta-subunit (LHβ) and a 3) small molecular weight fragment of LHβ, LHβ core fragment (LHβcf). The proportion of these LH forms in urine varies remarkably during the menstrual cycle, particularly in relation to the mid-cycle LH surge. In this exploratory study, we studied the potential implications of determining the periovulatory course of total LH immunoreactivity in urine (U-LH-ir) and intact LH immunoreactivity in serum (S-LH-ir) in the evaluation of the fertility window from a broader aspect with emphasis on the post-surge segment.MethodsWe determined total U-LH-ir in addition to intact S-LH-ir, follicle-stimulating hormone (FSH), progesterone, and estradiol in 32 consecutive samples collected daily from 10 women at reproductive age. Inference to the non-intact U-LH-ir levels was made by calculating the proportion of total U-LH-ir to intact S-LH-ir.ResultsTotal U-LH-ir increased along with LH surge and remained at statistically significantly higher levels than those in serum for 5 consecutive days after the surge in S-LH-ir. S-LH-ir returned to follicular phase levels immediately on the following day after the LH surge, whereas the same took 7 days for total U-LH-ir.ConclusionsThe current exploratory study provides preliminary evidence of the fact that U-LH-ir derived from degradation products of LH remains detectable at peak levels from the LH surge until ovulation and further during the early postovulatory period of fecundability. Thus, non-intact (or total) U-LH-ir appears to be a promising marker in the evaluation of the post-surge segment of the fertility window. Future studies are needed to unravel if this method can improve the prediction of ovulation time and higher rates of fecundability in both natural and assisted conception.

Objective: Klinefelter syndrome (KS) (47,XXY and variants, KS) is the most common sex chromosome disorder in humans. However, little is known about the onset and progression of puberty in patients with KS. In this study, we describe the onset and progression of puberty in a large series of boys with KS in a single tertiary centre. Design and Patients: Retrospective data (Tanner stages, testicular length, testosterone supplementation, levels of luteinizing hormone [LH] and testosterone) before possible testosterone treatment on 72 KS patients with 47,XXY karyotype were reviewed, and G (n = 59 patients) and P (n = 56 patients) stages were plotted on puberty nomograms. Measurements and Results: One boy had a delayed onset of puberty, as he was at the G1 stage at the age of 13.8 years (-2.2 SDs). No observations of delay were made of boys at Stage G2. The progression of G stages was within normal limits in the majority of patients; only few boys were late at G3 (4.1%; 1 out of 24) and G4 (7.4%; 2 out of 27). Testosterone supplementation was started at the average age of 15.5 years to 35 boys (47%), 2 of whom were over 18 years old. LH level was on average 18.2 IU/L (SD: 6.3 IU/L) and testosterone 9.1 nmol/L (SD: 3.1 nmol/L) when testosterone supplementation was started. Conclusions: Our results suggest that puberty starts within the normal age limits in boys with KS, and testosterone supplementation is not needed for the initial pubertal progression in the majority of patients.

Background and Aims Patients suffering from polycystic liver disease (PLD) can develop large liver volumes, leading to physical and psychological complaints, reducing quality of life. There is an unmet need for new therapies in these patients. Estrogen seems to be a promising target for new therapies. In this review, we summarize the available experimental and epidemiological evidence to unravel the role of estrogens and other female hormones in PLD, to answer clinical questions and identify new targets for therapy. Methods We identified all experimental and epidemiologial studies concerning estrogens or other female hormones and PLD, to answer pre-defined clinial questions. Results Female sex is the most important risk factor for the presence and severity of disease; estrogen supplementation enhances liver growth and after menopause, liver growth decreases. Experimental studies show the presence of the estrogen receptors alfa and beta on cystic cholangiocytes, and increased in vitro growth after administration of estrogen. Conclusions Based on the available evidence, female PLD patients should be discouraged from taking estrogen-containing contraceptives or hormone replacement therapy. Since liver growth rates decline after menopause, treatment decisions should be based on measured liver growth in postmenopausal women. Finally, blockage of estrogen receptors or estrogen production is a promising target for new therapies.

Although the full primary structures of the alfa and beta subunits of reference r-hFSH-alfa and its biosimilars are identical, cell context-dependent differences in the expressing cell lines and manufacturing process can lead to variations in glycosylation profiles. In the present study, we compared the structural features of reference r-hFSH-alfa with those of five biosimilar preparations approved in different global regions outside Europe (Primapur®, Jin Sai Heng®, Follitrope®, Folisurge®, and Corneumon®) with respect to glycosylation, macro- and microheterogeneity, and other post-translational modifications and higher order structure. The mean proportion of N-glycosylation-site occupancy was highest in reference r-hFSH-alfa, decreasing sequentially in Primapur, Jin Sai Heng, Corneumon, Follisurge and Follitrope, respectively. The level of antennarity showed slightly higher complexity in Corneumon, Primapur and Follitrope versus reference r-hFSH-alfa, whereas Jin Sai Heng and Folisurge were aligned with reference r-hFSH-alfa across all N-glycosylation sites. Sialylation level was higher in Corneumon and Follitrope, but small differences were detected in other biosimilar preparations compared with reference r-hFSH-alfa. Jin Sai Heng showed higher levels of N-glyconeuramic acid than the other preparations. Minor differences in oxidation levels were seen among the different products. Therefore, in summary, we identified var ious differences in N-glycosylation occupancy, antennarity, sialylation and oxidation between reference r-hFSH-alfa and the biosimilar preparations analyzed. ispartof: INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES vol:23 issue:12 ispartof: location:Switzerland status: published

Background Recombinant human follitropin alfa (r-hFSH) is used for ovarian stimulation as part of medically assisted reproduction. There is a risk for ovarian hyperstimulation syndrome (OHSS) with r-hFSH treatment, and an increased risk for thromboembolic events in the presence of pregnancy with OHSS. Objectives To report the frequency of OHSS and thromboembolism with originator follitropin alfa (GONAL-f) based on the Global Safety Database of Merck KGaA, Darmstadt, Germany and a systematic review of published data. Data Sources Reports of OHSS and thromboembolism were obtained from the Global Safety Database of Merck KGaA, Darmstadt, Germany from 20 October 1995 to 19 October 2018. The systematic review was based on MEDLINE and Embase searches from inception to 19 October 2018. Study Eligibility Criteria Patients receiving GONAL-f for ovulation induction or ART, with a starting dose within the range included in the prescribing information and providing information on the occurrence of OHSS and/or thromboembolism. Study Appraisal and Synthesis Matches In the Global Safety Database of Merck KGaA, Darmstadt, Germany there were an estimated 16,525,975 treatment cycles since 20 October 1995; 1110 reported cases of OHSS and 80 reported cases of thromboembolic events (reporting rates 6.7 and 0.48 per 100,000 treatment cycles, respectively). The systematic review identified 45 studies (5186 patients exposed to GONAL-f; 5240 treatment cycles). There were 272 reports of OHSS (5190 [5.19%] per 100,000 treatment cycles), including 10 cases of severe OHSS (191 [0.19%] per 100,000 treatment cycles). Limitations There may be the potential for under-reporting of safety outcomes in the literature, and under-reporting is a well-known phenomenon in spontaneous reporting databases. Conclusion and Implications of Key Findings Our analyses demonstrate low rates of OHSS and thromboembolism with GONAL-f. Electronic Supplementary Material The online version of this article (10.1007/s12325-020-01512-w) contains supplementary material, which is available to authorized users.

To evaluate the expression of AMH and its receptor AMHRII, ovaries of 33 p cats were investigated by western blot and immunohistochemistry. After ovariohysterectomy, the cats were grouped according to pregnancy stages and ovarian/placental endocrine activity: group I (n = 3, 24–29 days), II (n = 8, 32–40 days), III (n = 4, 41–46 days), IV (n = 6, 53–61 days) and according to cycle stages: V (n = 6, interestrus) and VI (n = 6, estrus). Serum progesterone- and AMH-concentration was measured. Follicle numbers did not differ between groups. The number of corpora lutea was higher in pregnant cats than in the non-pregnant cats. Serum AMH concentration was at maximum between day 30 and 50 of gestation, and was higher than in non-pregnant cats, then decreased towards term (p < 0.05). In the ovaries, AMH immunopositivity was observed in granulosa cells of secondary and antral follicles, and in interstitial cells of corpora lutea; highest percentage of immunopositive areas was detected in group III (p < 0.05). A positive correlation between the number of corpora lutea and the positive AMH signals in ovarian tissue was determined (r2 = 0.832, p < 0.05); however, only during mid-gestation (group II). Expression of AMHRII was in close co-localization with AMH and strong in the interstitial cells surrounding follicles undergoing atresia. AMHRII expression did not differ between pregnant groups but was higher compared to estrus cats (p ˂ 0.05). We conclude that AMH and AMHRII expression in the feline ovary is comparable to other species. The high serum AMH concentration and ovarian AMHRII expression between day 30 and 50 of gestation are probably related to ovarian activity and follicular atresia.

International audience; Estradiol (E2) is a major hormone controlling women fertility, in particular folliculogenesis. This steroid, which is locally produced by granulosa cells (GC) within ovarian follicles, controls the development and selection of dominant preovulatory follicles. E2 effects rely on a complex set of nuclear and extra-nuclear signal transduction pathways principally triggered by its nuclear receptors, ER alpha and ER beta. These transcription factors are differentially expressed within follicles, with ER beta being the predominant ER in GC. Several ER beta splice isoforms have been identified and display specific structural features, which greatly complicates the nature of ER beta-mediated E2 signaling. This review aims at providing a concise overview of the main actions of E2 during follicular growth, maturation, and selection in human. It also describes the current understanding of the various roles of ER beta splice isoforms, especially their influence on cell fate. We finally discuss how E2 signaling deregulation could participate in two ovarian pathogeneses characterized by either a follicular arrest, as in polycystic ovary syndrome, or an excess of GC survival and proliferation, leading to granulosa cell tumors. This review emphasizes the need for further research to better understand the molecular basis of E2 signaling throughout folliculogenesis and to improve the efficiency of ovarian-related disease therapies.

Reproduction is an energetically costly event across vertebrates and tightly linked to nutritional status and en-ergy reserves. In mammals, the hormone leptin is considered as a link between energy homeostasis and repro-duction. However, its role in fish reproduction is still unclear. In this study, we investigated the possible role of leptin in the regulation of reproduction in zebrafish, using a loss of function leptin receptor (lepr) strain. Impaired leptin signaling resulted in severe reproductive deficiencies in female zebrafish. lepr mutant females laid significantly fewer eggs, with low fertilization rates compared to wild-type females. Folliculogenesis was not affected, but oocyte maturation and ovulation were disrupted in lepr mutants. Interestingly, the expression of luteinizing hormone beta (lhb) in the pituitary was significantly lower in mutant females. Analysis of candidate genes in the ovaries and isolated fully grown follicles revealed differential expression of genes involved in ste-roidogenesis, oocyte maturation and ovulation in the mutants, which are known to be regulated by LH signaling. Moreover, subfertility in lepr mutants could be partially restored by administration of human chorionic gonad-otropin. In conclusion, our results show that leptin deficiency does not affect early stages of follicular devel-opment, but leptin might be essential in later steps, such as in oocyte maturation and ovulation. To our knowledge, this is the first time that leptin is associated to reproductive deficiencies in zebrafish.

Introduction Aberrant function of granulosa cells has been implicated in the pathophysiology of PCOS. Materials & methods Granulosa lutein (GL) cells were collected during oocyte retrieval for IVF/ICSI. RT-qPCR was used to compare gene expression between 12 control women, 12 with ovulatory PCO and 12 with anovulatory PCOS. To examine which genes are directly regulated by androgens, GL cells from an additional 12 control women were treated in-vitro with 10 nM dihydrotestosterone (DHT). Results GL cells from women with PCOS showed reduced expression of CYP11A1 3-fold (p = 0.005), HSD17B1 1.8-fold (p = 0.02) and increased expression of SULT1E1 7-fold (p = 0.0003). Similar results were seen in ovulatory women with PCO. GL cells treated with 10 nM DHT showed a 4-fold (p = 0.03) increase in expression of SULT1E1 and a 5-fold reduction in SRD5A1 (p = 0.03). Conclusions These findings support the notion that aberrant regulation of steroid metabolism or action play a part in ovarian dysfunction in PCOS.

The increased global prevalence of obesity over the last 40-years has driven a rise in prevalence of obesity-related co-morbidities, including polycystic ovary syndrome (PCOS). On a background of genetic susceptibility, PCOS often becomes clinically manifest following weight gain, commonly during adolescence. A common endocrinopathy affecting between 6%-10% of reproductive-age women, PCOS presents with the cardinal features of hyperandrogenism, reproductive and metabolic dysfunction. PCOS associates with insulin resistance, independently of (but amplified by) obesity. Insulin resistance in PCOS is characterized by abnormal post-receptor signalling within the phosphatidylinositol-kinase (PI3-K) pathway. Multiple factors (including most notably, weight gain) contribute towards the severity of insulin resistance in PCOS. Compensatory hyperinsulinaemia ensues, resulting in over-stimulation of the (intact) post-receptor mitogen-activated protein kinase (MAP-K) insulin pathway, with consequent implications for steroidogenesis and ovarian function. In this concise review, we explore the effects of weight gain and obesity on the pathogenesis of PCOS from the perspective of its three cardinal features of hyperandrogenism, reproductive and metabolic dysfunction, with a focus on the central mediating role of the insulin pathway. We also consider key lifestyle strategies for the effective management of obese and overweight women with PCOS.

Contains fulltext : 238413.pdf (Publisher’s version ) (Open Access) BACKGROUND AND AIMS: Patients suffering from polycystic liver disease (PLD) can develop large liver volumes, leading to physical and psychological complaints, reducing quality of life. There is an unmet need for new therapies in these patients. Estrogen seems to be a promising target for new therapies. In this review, we summarize the available experimental and epidemiological evidence to unravel the role of estrogens and other female hormones in PLD, to answer clinical questions and identify new targets for therapy. METHODS: We identified all experimental and epidemiologial studies concerning estrogens or other female hormones and PLD, to answer pre-defined clinial questions. RESULTS: Female sex is the most important risk factor for the presence and severity of disease; estrogen supplementation enhances liver growth and after menopause, liver growth decreases. Experimental studies show the presence of the estrogen receptors alfa and beta on cystic cholangiocytes, and increased in vitro growth after administration of estrogen. CONCLUSIONS: Based on the available evidence, female PLD patients should be discouraged from taking estrogen-containing contraceptives or hormone replacement therapy. Since liver growth rates decline after menopause, treatment decisions should be based on measured liver growth in postmenopausal women. Finally, blockage of estrogen receptors or estrogen production is a promising target for new therapies.

Anti-Müllerian hormone (AMH), also called Müllerian inhibiting substance, was shown to be synthesized by the ovary in the 1980s. This article reviews the main findings of the past 20 years on the regulation of the expression of AMH and its specific receptor AMHR2 by granulosa cells, the mechanism of action of AMH, the different roles it plays in the reproductive organs, its clinical utility, and its involvement in the principal pathological conditions affecting women. The findings in respect of regulation tell us that AMH and AMHR2 expression is mainly regulated by bone morphogenetic proteins, gonadotropins, and estrogens. It has now been established that AMH regulates the different steps of folliculogenesis and that it has neuroendocrine effects. On the other hand, the importance of serum AMH as a reliable marker of ovarian reserve and as a useful tool in the prediction of the polycystic ovary syndrome (PCOS) and primary ovarian failure has also been acknowledged. Last but not least, a large body of evidence points to the involvement of AMH in the pathogenesis of PCOS.

International audience; The glycoprotein hormones (GPH) are heterodimers composed of a common alpha subunit and a specific beta subunit. They act by activating specific leucine-rich repeat G protein-coupled receptors. However, individual subunits have been shown to elicit responses in cells devoid of the receptor for the dimeric hormones. The alpha subunit is involved in prolactin production from different tissues. The human chorionic gonadotropin beta subunit (beta hCG) plays determinant roles in placentation and in cancer development and metastasis. A truncated form of the thyrotropin (TSH) beta subunit is also reported to have biological effects. The GPH alpha- and beta subunits are derived from precursor genes (gpa and gpb, respectively), which are expressed in most invertebrate species and are still represented in vertebrates as GPH subunit paralogs (gpa2 and gpb5, respectively). No specific receptor has been found for the vertebrate GPA2 and GPB5 even if their heterodimeric form is able to activate the TSH receptor in mammals. Interestingly, GPA and GPB are phylogenetically and structurally related to cysteine-knot growth factors (CKGF) and particularly to a group of antagonists that act independently on any receptor. This review article summarizes the observed actions of individual GPH subunits and presents the current hypotheses of how these actions might be induced. New approaches are also proposed in light of the evolutionary relatedness with antagonists of the CKGF family of proteins.

Menstrual irregularities account for up to 30% of outpatient visits to gynecologists. We present a case of a 29-year-old nulliparous woman with abnormal vaginal bleeding for the last five years. A transvaginal ultrasound revealed both ovaries enlarged with multiple cysts. Blood tests showed mildly elevated levels of estradiol and prolactin, and a suppressed LH. Cerebral MRI showed a pituitary macroadenoma. The patient was submitted to transsphenoidal surgery. The final diagnosis was pituitary adenoma with FSH and LH expression. Cases of pituitary adenomas causing clinical symptoms are unusual This diagnosis should be considered in women with menstrual irregularities and hyperstimulated ovaries.

Intracytoplasmic sperm injection (ICSI), combined with surgical sperm retrieval (SR) techniques, is the sole option for patients with non-obstructive azoospermia to achieve fertility; however, with suboptimal results. Given the variability in clinical presentation, the potential role of factors that can predict the likelihood of successful testicular SR needs to be clarified. This article summarizes the current evidence concerning the variables predicting SR success in non-obstructive azoospermic patients with spermatogenic failure. For this, we used 60 articles, including 46 original papers and six meta-analyses. Clinical and laboratory factors, as well as adjuvant therapies and surgical retrieval methods, were the factors most commonly investigated. We found that Klinefelter syndrome, Y chromosome microdeletions in regions AZFa/b, and Sertoli cell-only histopathology were associated with reduced SR success. By contrast, testis volume > 12.5 ml, history of cryptorchidism, use of micro-TESE as the sperm retrieval method, and adjuvant therapy were associated with improved SR success. None of the predictors, alone or combined, provide definitive information about the chances of harvesting sperm in men with non-obstructive azoospermia, except for Y chromosome microdeletions in regions AZFa/b. In the latter, SR success is virtually nil. We conclude that SR outcomes in men with non-obstructive azoospermia are difficult to predict based on the existing variables. Although several predictors can be used for patient counseling, their clinical value is limited to either ensure SR success or discourage reproductive urologists from recommending SR to men with non-obstructive azoospermia seeking fertility. A notable exception includes the deletions involving the regions AZFa and/or AZFb of the Y chromosome; the affected patients should be counseled against undergoing SR.

Adult-type granulosa cell tumors (AGCTs) are sex-cord derived neoplasms with a propensity for late relapse. Hormonal modulators have been used empirically in the treatment of recurrent AGCT, albeit with limited success. To provide a more rigorous foundation for hormonal therapy in AGCT, we used a multimodal approach to characterize the expressions of key hormone biomarkers in 175 tumor specimens and 51 serum samples using RNA sequencing, immunohistochemistry, RNA in situ hybridization, quantitative PCR, and circulating biomarker analysis, and correlated these results with clinical data. We show that FSH receptor and estrogen receptor beta (ERβ) are highly expressed in the majority of AGCTs, whereas the expressions of estrogen receptor alpha (ERα) and G-protein coupled estrogen receptor 1 are less prominent. ERβ protein expression is further increased in recurrent tumors. Aromatase expression levels show high variability between tumors. None of the markers examined served as prognostic biomarkers for progression-free or overall survival. In functional experiments, we assessed the effects of FSH, estradiol (E2), and the aromatase inhibitor letrozole on AGCT cell viability using 2 in vitro models: KGN cells and primary cultures of AGCT cells. FSH increased cell viability in a subset of primary AGCT cells, whereas E2 had no effect on cell viability at physiological concentrations. Letrozole suppressed E2 production in AGCTs; however, it did not impact cell viability. We did not find preclinical evidence to support the clinical use of aromatase inhibitors in AGCT treatment, and thus randomized, prospective clinical studies are needed to clarify the role of hormonal treatments in AGCTs.

STUDY QUESTION What is the early luteal phase hormonal profile in patients undergoing ovarian stimulation for IVF/ICSI followed by hCG trigger and a freeze-all strategy without luteal phase support? SUMMARY ANSWER The peak concentration of progesterone occurred 4 days after oocyte pick-up (OPU + 4), with an average 35% fall from OPU + 4 to OPU + 6, and progesterone levels before and 12 h after hCG administration predicted levels during the early luteal phase. WHAT IS KNOWN ALREADY The luteal phase during IVF differs from that during normal cycles, particularly with respect to the serum progesterone level profile. This can cause asynchrony between the embryo and the endometrium, potentially resulting in implantation failure and poor reproductive outcomes. STUDY DESIGN, SIZE, DURATION This prospective study included 161 women with normal ovarian reserve receiving GnRH antagonist co-treatment during ovarian stimulation with FSH who were followed up to 6 days after OPU in a single IVF cycle. PARTICIPANTS/MATERIALS, SETTING, METHODS Women aged 18–42 years undergoing IVF with ovarian stimulation using FSH were included. Ovulation was triggered with recombinant hCG 250 μg. Hormone levels were determined from blood samples taken on the day of trigger, before hCG, at 12, 24 and 36 h after hCG and at 1, 2, 3, 4, 5 and 6 days after OPU. The primary endpoint was early luteal phase serum concentrations of progesterone, LH, estradiol and hCG. MAIN RESULTS AND THE ROLE OF CHANCE One outlier with a pre-hCG serum progesterone level of 11.42 ng/mL was excluded, so all analyses included 160 subjects. Progesterone levels began to increase 1 day after OPU, peaked 4 days after OPU (114 ng/mL), then declined from OPU + 5 onwards. Peak progesterone levels were at OPU + 4, OPU + 5 or OPU + 6 in 38.8, 29.4 and 13.8% of patients, respectively. Approximately two-thirds of patients had a fall in serum progesterone from OPU + 4 to OPU + 6. Pre-hCG progesterone levels correlated significantly with those at 24 h after hCG (r2 = 0.28; P LIMITATIONS, REASONS FOR CAUTION The influence of different profiles of serum progesterone on reproductive outcomes could not be determined because a freeze-all strategy was used in all patients. In addition, data were not available to relate serum hormone level findings with endometrial histology or endometrial receptivity analysis to clearly identify the relationship between serum hormones and the window of implantation. WIDER IMPLICATIONS OF THE FINDINGS Detailed information about early luteal phase hormone levels could be used to optimize and individualize luteal phase support to improve reproductive outcomes. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by My Duc Hospital, Ho Chi Minh City, Vietnam. All authors state that they have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER NCT02798146; NCT03174691.

Background Sperm chromatin integrity is essential for normal embryo development and pregnancy outcome. Sperm DNA fragmentation (SDF) testing constitutes a diagnostic tool to measure the proportion of spermatozoa with damaged chromatin in the ejaculate. SDF is associated with potentially treatable conditions, including varicocele, male accessory gland infections, inadequate lifestyle, and gonadotoxin exposure, thus prompting their treatment as a means of improving sperm DNA quality and the reproductive outcomes. Objective To provide an up-to-date review of the role of clinical and surgical interventions on SDF values in subfertile men. Materials and methods An extensive search of studies examining the relationship between male infertility conditions associated with SDF was performed using PubMed and MEDLINE, with a focus on interventional therapy. The start date for the search was not defined, whereas the end date was March 2019. Randomized and non-randomized controlled trials, observational studies, systematic and narrative reviews, and case series were evaluated. Results Treating the underlying male infertility factor seems a promising way to alleviate SDF and to increase the likelihood of achieving natural and assisted conception, but data remain limited. The best evidence relates to varicocele repair and hormonal therapy with the follicle-stimulating hormone. Antioxidant therapy and lifestyle changes might alleviate oxidative sperm markers and decrease SDF but their effects on pregnancy outcomes are still unclear. Among men with high SDF undergoing assisted reproductive technology, the use of testicular spermatozoa in preference over ejaculated spermatozoa for intracytoplasmic sperm injection (ICSI) has been shown to improve pregnancy rates possibly owing to the better sperm chromatin quality in testicular spermatozoa than in ejaculated spermatozoa. Conclusion Current evidence supports interventional therapy as a means to alleviate sperm DNA damage. Identification of the conditions associated with SDF remains important to enable treatment to potentially improve pregnancy outcomes but given the limited data further research is needed to determine the exact role of specific interventional therapy for subfertile men with impaired sperm chromatin.

Context Application of ultrasound (US) to evaluate attainment and morphology of glandular tissue provides a new rationale for evaluating onset and progression of female puberty, but currently no hormone references complement this method. Furthermore, previous studies have not explored the predictive value of endocrine profiling to determine female puberty onset. Objective To integrate US breast staging with hypothalamic-pituitary-gonadal hormone references and test the predictive value of an endocrine profile to determine thelarche. Design Setting and Participants Cross-sectional sample of 601 healthy Norwegian girls, ages 6 to 16 years. Main Outcome Measures Clinical and ultrasound breast evaluations were performed for all included girls. Blood samples were analyzed by immunoassay and ultrasensitive liquid chromatography–tandem mass spectrometry (LC-MS/MS) to quantify estradiol (E2) and estrone (E1) from the subpicomolar range. Results References for E2, E1, luteinizing hormone, follicle-stimulating hormone, and sex hormone–binding globulin were constructed in relation to chronological age, Tanner stages, and US breast stages. An endocrine profile index score derived from principal component analysis of these analytes was a better marker of puberty onset than age or any individual hormone, with receiver-operating characteristic area under the curve 0.91 (P Conclusions We provide the first hormone references for use with US breast staging and demonstrate the application of endocrine profiling to improve detection of female puberty onset.

STUDY QUESTION: Does a reduced FSH dose in women with a predicted hyper response, apparent from a high antral follicle count (AFC), who are scheduled for IVF/ICSI lead to a different outcome with respect to cumulative live birth rate and safety?SUMMARY ANSWER: Although in women with a predicted hyper response (AFC > 15) undergoing IVF/ICSI a reduced FSH dose (100 IU per day) results in similar cumulative live birth rates and a lower occurrence of any grade of ovarian hyperstimulation syndrome (OHSS) as compared to a standard dose (150 IU/day), a higher first cycle cancellation rate and similar severe OHSS rate were observed.WHAT IS KNOWN ALREADY: Excessive ovarian response to controlled ovarian stimulation (COS) for IVF/ICSI may result in increased rates of cycle cancellation, the occurrence of OHSS and suboptimal live birth rates. In women scheduled for IVF/ICSI, an ovarian reserve test (ORT) can be used to predict response to COS. No consensus has been reached on whether ORT-based FSH dosing improves effectiveness and safety in women with a predicted hyper response.STUDY DESIGN SIZE, DURATION: Between May 2011 and May 2014, we performed an open-label, multicentre RCT in women with regular menstrual cycles and an AFC > 15. Women with polycystic ovary syndrome (Rotterdam criteria) were excluded. The primary outcome was ongoing pregnancy achieved within 18 months after randomization and resulting in a live birth. Secondary outcomes included the occurrence of OHSS and cost-effectiveness. Since this RCT was embedded in a cohort study assessing over 1500 women, we expected to randomize 300 predicted hyper responders.PARTICIPANTS/MATERIALS, SETTING, METHODS: Women with an AFC > 15 were randomized to an FSH dose of 100 IU or 150 IU/day. In both groups, dose adjustment was allowed in subsequent cycles (maximum 25 IU in the reduced and 50 IU in the standard group) based on pre-specified criteria. Both effectiveness and cost-effectiveness were evaluated from an intention-to-treat perspective.MAIN RESULTS AND THE ROLE OF CHANCE: We randomized 255 women to a daily FSH dose of 100 IU and 266 women to a daily FSH dose of 150 IU. The cumulative live birth rate was 66.3% (169/255) in the reduced versus 69.5% (185/266) in the standard group (relative risk (RR) 0.95 [95%CI, 0.85-1.07], P = 0.423). The occurrence of any grade of OHSS was lower after a lower FSH dose (5.2% versus 11.8%, RR 0.44 [95%CI, 0.28-0.71], P = 0.001), but the occurrence of severe OHSS did not differ (1.3% versus 1.1%, RR 1.25 [95%CI, 0.38-4.07], P = 0.728). As dose reduction was not less expensive (€4.622 versus €4.714, delta costs/woman €92 [95%CI, -479-325]), there was no dominant strategy in the economic analysis.LIMITATIONS, REASONS FOR CAUTION: Despite our training programme, the AFC might have suffered from inter-observer variation. Although strict cancellation criteria were provided, selective cancelling in the reduced dose group (for poor response in particular) cannot be excluded as observers were not blinded for the FSH dose and small dose adjustments were allowed in subsequent cycles. However, as first cycle live birth rates did not differ from the cumulative results, the open design probably did not mask a potential benefit for the reduced dosing group. As this RCT was embedded in a larger cohort study, the power in this study was unavoidably lower than it should be. Participants had a relatively low BMI from an international perspective, which may limit generalization of the findings.WIDER IMPLICATIONS OF THE FINDINGS: In women with a predicted hyper response scheduled for IVF/ICSI, a reduced FSH dose does not affect live birth rates. A lower FSH dose did reduce the incidence of mild and moderate OHSS, but had no impact on severe OHSS. Future research into ORT-based dosing in women with a predicted hyper response should compare various safety management strategies and should be powered on a clinically relevant safety outcome while assessing non-inferiority towards live birth rates.STUDY FUNDING/COMPETING INTEREST(S): This trial was funded by The Netherlands Organization for Health Research and Development (ZonMW, Project Number 171102020). SCO, TCvT and HLT received an unrestricted research grant from Merck Serono (the Netherlands). CBL receives grants from Merck, Ferring and Guerbet. BWJM is supported by a NHMRC Practitioner Fellowship (GNT1082548) and reports consultancy for OvsEva, Merck and Guerbet. FJMB receives monetary compensation as a member of the external advisory board for Ferring pharmaceutics BV and Merck Serono for consultancy work for Gedeon Richter (Belgium) and Roche Diagnostics (Switzerland) and for a research cooperation with Ansh Labs (USA). All other authors have nothing to declare.TRIAL REGISTRATION NUMBER: Registered at the ICMJE-recognized Dutch Trial Registry (www.trialregister.nl). Registration number: NTR2657.TRIAL REGISTRATION DATE: 20 December 2010.DATE OF FIRST PATIENT’S ENROLMENT: 12 May 2011.

The first commercially available gonadotropin product was a human chorionic gonadotropin (hCG) extract, followed by animal pituitary gonadotropin extracts. These extracts were effective, leading to the introduction of the two-step protocol, which involved ovarian stimulation using animal gonadotropins followed by ovulation triggering using hCG. However, ovarian response to animal gonadotropins was maintained for only a short period of time due to immune recognition. This prompted the development of human pituitary gonadotropins; however, supply problems, the risk for Creutzfeld-Jakob disease, and the advent of recombinant technology eventually led to the withdrawal of human pituitary gonadotropin from the market. Urinary human menopausal gonadotropin (hMG) preparations were also produced, with subsequent improvements in purification techniques enabling development of products with standardized proportions of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) activity. In 1962 the first reported pregnancy following ovulation stimulation with hMG and ovulation induction with hCG was described, and this product was later established as part of the standard protocol for ART. Improvements in immunopurification techniques enabled the removal of LH from hMG preparations; however, unidentified urinary protein contaminants remained a problem. Subsequently, monoclonal FSH antibodies were used to produce a highly purified FSH preparation containing

Study question What is the peripubertal outcome of recombinant human FSH (r-hFSH) treatment during minipuberty in boys with congenital hypogonadotropic hypogonadism (CHH)? Summary answer Sertoli-cell response to r-hFSH, given during the minipuberty of infancy, appears insufficient to maintain Sertoli cell function throughout childhood, as evaluated by inhibin B measurements. What is known already Severe CHH in boys can be diagnosed during the minipuberty of infancy. Combined gonadotropin treatment at that age is suggested to improve testicular endocrine function and future fertility, yet long-term evidence is lacking. Study design, size, duration In this retrospective cohort study, we describe five CHH boys treated with r-hFSH in Helsinki University Hospital or Kuopio University Hospital between 2004 and 2018. Immediate follow-up data (0.1-1.4 months after cessation of the gonadotropin therapy) was available for four boys and long-term observations (at the age of 10.0-12.8 years) was available for three boys. As a retrospective control cohort, we provide inhibin B values of eight untreated CHH boys at the age of 12.7-17.8 years. Participants/materials, setting, methods Four patients had combined pituitary hormone deficiency, and one had CHARGE syndrome due to a CHD7 mutation. The patients were treated at the age of 0.7-4.2 months with r-hFSH (3.4 IU/kg-7.5 IU/kg per week in 2 or 3 s.c. doses for 3-4.5 months) combined with T (25 mg i.m. monthly for three months for the treatment of micropenis). Inhibin B was chosen as the primary outcome measure. Main results and the role of chance During the r-hFSH + T treatment, inhibin B increased from 76 ± 18 ng/l to 176 ± 80 ng/l (P = 0.04) and penile length increased by 81 ± 50% (P = 0.04). Unexpectedly, two boys with robust inhibin B responses in infancy demonstrated low inhibin B values in peripuberty: declining from 290 ng/l (4 months) to 16 ng/l (12.4 years), and from 207 ng/l (6 months) to 21 ng/l (12.8 years). All boys underwent orchiopexy at 2.0 ± 0.7 years of age. Inhibin B values in long-term follow-up, available for the three boys, did not significantly differ from the untreated CHH controls. Limitations, reasons for caution Limitations of this retrospective study are the small number and heterogeneity of the patients and their treatment schemes. Wider implications of the findings We describe the first long-term follow-up data on CHH boys treated with r-hFSH and T as infants. The results from this small patient series suggest that the effects of infant r-hFSH treatment may be transient, and further longitudinal studies are required to determine the efficacy of this treatment approach to optimise the fertility potential in this patient population. Study funding/competing interest(s) This work was supported by the Finnish foundation for Pediatric Research, the Academy of Finland and the Emil Aaltonen Foundation. The authors have no competing interests. Trial registration number Non-applicable.

The initiation and maintenance of reproductive capacity in humans is dependent on pulsatile secretion of the hypothalamic hormone GnRH. Congenital hypogonadotropic hypogonadism (CHH) is a rare disorder that results from the failure of the normal episodic GnRH secretion, leading to delayed puberty and infertility. CHH can be associated with an absent sense of smell, also termed Kallmann syndrome, or with other anomalies. CHH is characterized by rich genetic heterogeneity, with mutations in >30 genes identified to date acting either alone or in combination. CHH can be challenging to diagnose, particularly in early adolescence where the clinical picture mirrors that of constitutional delay of growth and puberty. Timely diagnosis and treatment will induce puberty, leading to improved sexual, bone, metabolic, and psychological health. In most cases, patients require lifelong treatment, yet a notable portion of male patients (approximate to 10% to 20%) exhibit a spontaneous recovery of their reproductive function. Finally, fertility can be induced with pulsatile GnRH treatment or gonadotropin regimens in most patients. In summary, this review is a comprehensive synthesis of the current literature available regarding the diagnosis, patient management, and genetic foundations of CHH relative to normal reproductive development.

Anti-Müllerian hormone (AMH) regulates ovarian function in cyclic females, notably by preventing premature follicle-stimulating hormone (FSH)-mediated follicular growth and steroidogenesis. Its expression in growing follicles is controlled by FSH and by estradiol (E2). In infantile females, there is a transient increase in the activity of the gonadotrope axis, as reflected by elevated levels of both gonadotropins and E2. We previously demonstrated in mice that elevated FSH concentrations are necessary to induce E2 production by preantral/early antral follicles through the stimulation of aromatase expression without supporting their growth. However, whether this action of FSH could involve AMH is unknown. Here, we show that Amh mRNA and protein abundance and serum AMH levels are elevated in infantile mouse females, compared with those in adults. By experimentally manipulating FSH and E2 levels in infantile mice, we demonstrate that high FSH concentrations lower Amh expression specifically in preantral/early antral follicles, whereas E2 has no effect. Importantly, treatment of infantile ovaries in organotypic cultures with AMH decreases FSH-mediated expression of Cyp19a1 aromatase, but it does not alter the expression of cyclin D2-mediating granulosa cell proliferation. Overall, our data indicate that the infantile elevation in FSH levels suppresses Amh expression in preantral/early antral follicles, thereby favoring Cyp19a1 aromatase expression and E2 production. Together with recent discoveries that AMH can act on both the hypothalamus and the pituitary to increase gonadotropin levels, this work suggests that AMH is a critical regulator of the gonadotrope axis during the infantile period, thereby contributing to adult reproductive function programming.

Androgen production by the ovary is an essential requirement for normal cyclical secretion of oestradiol but its physiological role extends to important actions on both preantral and antral follicle development, including promotion of granulosa cell proliferation. It is likely only in mature antral follicles that androgens encourage apoptosis and consequent follicle atresia, and this may be an important mechanism to ensure mono-follicular ovulation in primates, including humans. Recent studies have provided new insight into the mechanism of androgen signalling in the ovary which involves both genomic and non-genomic effects that are complementary in effecting a cellular response. In polycystic ovary syndrome, a condition characterised by intra-ovarian androgen excess, aberrant development of both preantral and antral follicles is a salient feature. We present evidence that local action of androgens plays a part in such abnormalities. Finally, we review the role of androgens in follicle atresia and conclude that the effects are part of the normal physiology of follicle maturation.

Primary ovarian insufficiency (POI) affects similar to 1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of > 60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful.

Background: Male testosterone levels decline by 1% per year from the age of 40yrs. Whilst a primary testicular deficit occurs, hypothalamic or pituitary dysregulation may also coexist. This study aimed to compare the hypothalamic response to kisspeptin and the pituitary response to GnRH of older men with those of young men. Methods: Following 1 hour of baseline sampling, healthy older men (n=5, mean age 59.3±2.9yrs) received a 3 hour intravenous infusion (IVI) of either: vehicle, kisspeptin-54 0.1, 0.3, 1.0nmol/kg/h, or GnRH 0.1nmol/kg/h on 5 different study days. Serum gonadotropins and total testosterone were measured every 10 minutes and compared to young men (n=5/group) (mean age 28.9±2.0yrs) with a similar BMI (24 kg/m2) who underwent the same protocol. Results: Kisspeptin and GnRH significantly stimulated serum gonadotropin release in older men compared to vehicle (P

Testicular function and future fertility may be affected by cancer treatment during childhood. Whilst survival of the germ (stem) cells is critical for ensuring the potential for fertility in these patients, the somatic cell populations also play a crucial role in providing a suitable environment to support germ cell maintenance and subsequent development. Regulation of the spermatogonial germ-stem cell niche involves many signalling pathways with hormonal influence from the hypothalamo-pituitary-gonadal axis. In this review, we describe the somatic cell populations that comprise the testicular germ-stem cell niche in humans and how they may be affected by cancer treatment during childhood. We also discuss the experimental models that may be utilized to manipulate the somatic environment and report the results of studies that investigate the potential role of somatic cells in the protection of the germ cells in the testis from cancer treatment.

International audience; Primary ovarian insufficiency (POI) affects similar to 1% of women before 40 years of age. The recent leap in genetic knowledge obtained by next generation sequencing (NGS) together with animal models has further elucidated its molecular pathogenesis, identifying novel genes/pathways. Mutations of > 60 genes emphasize high genetic heterogeneity. Genome-wide association studies have revealed a shared genetic background between POI and reproductive aging. NGS will provide a genetic diagnosis leading to genetic/therapeutic counseling: first, defects in meiosis or DNA repair genes may predispose to tumors; and second, specific gene defects may predict the risk of rapid loss of a persistent ovarian reserve, an important determinant in fertility preservation. Indeed, a recent innovative treatment of POI by in vitro activation of dormant follicles proved to be successful.

International audience; The objective of this study was to examine the effect of nutrition during the first 18 weeks of life on the physiological and transcriptional functionality of the hypothalamic (arcuate nucleus region), anterior pituitary and testes in Holstein-Friesian bull calves. Holstein-Friesian bull calves with a mean (+/- S.D.) age and bodyweight of 19 (+/- 8.2) days and 47.5 (+/- 5.3) kg, respectively, were assigned to either a HIGH (n=10) or LOW (n=10) plane of nutrition, to achieve an overall target growth rate of 1.2 or 0.5 kg/day, respectively. At 126 +/- 1.1 days of age, all calves were euthanised. Animal performance (weekly) and systemic concentrations of metabolic (monthly) and reproductive hormones (fortnightly) were assessed. Testicular histology, targeted gene and protein expression of the arcuate nucleus region, anterior pituitary and testes were also assessed using qPCR and immunohistochemistry, respectively. The expression of candidate genes in testicular tissue from post pubertal 19-month-old Holstein-Friesian bulls (n=10) was compared to that of the 18-week-old calves. Metabolite and metabolic hormone profiles generally reflected the improved metabolic status of the calves on the HIGH (P < 0.001). Calves offered a HIGH plane of nutrition were heavier at slaughter (P

International audience; Objective: Ovarian reserve evaluation has been the focus of substantial clinical research for several years. This study aimed to examine the associations between markers of ovarian reserve and ovarian response. Methods: This prospective study included 132 infertile women aged 24-48 years undergoing routine exploration during unstimulated cycles prior to the start of assisted reproductive technology (ART) treatments at our center from July 2015 to January 2017. Descriptive parameters and patient characteristics were reported as mean (SD) or median (range) values depending on the data distribution pattern. Student's t-test was performed for continuous variables; the Wilcoxon and Pearson's test were used for data not following a normal distribution; and Fisher's test was used for categorical variables. p

Item does not contain fulltext CONTEXT: Previously we showed that pubertal children born small for gestational age (SGA) with a poor adult height (AH) expectation can benefit from treatment with GH 1 mg/m(2) per day ( approximately 0.033 mg/kg/d) in combination with 2 years of GnRH analog (GnRHa) and even more so with a double GH dose. GnRHa treatment is thought to have negative effects on body composition and blood pressure. Long-term effects and GH-dose effects on metabolic health in children treated with combined GH/GnRHa are unknown. OBJECTIVE: This study aimed to investigate body composition, blood pressure, and lipid profile during GH treatment, either with or without 2 years of additional GnRHa. To assess whether GH 2 mg/m(2) per day ( approximately 0.067 mg/kg/d) results in a similar or even more favorable metabolic health at AH than GH 1 mg/m(2) per day. METHODS: This was a longitudinal, randomized, dose-response GH trial involving 107 short SGA children (58 girls) treated with GH until AH (GH randomized 1 or 2 mg/m(2)/d during puberty). Sixty-four children received additional GnRHa. At AH, metabolic parameters were compared between children treated with combined GH/GnRHa and those with only GH. The GH dose effect on metabolic health was evaluated in a subgroup of 47 children who started GH treatment in early puberty (randomized 1 or 2 mg/m(2)/d) with 2 years of GnRHa. RESULTS: At AH, fat mass percentage (FM%) SD score (SDS), lean body mass (LBM) SDS, blood pressure SDS, and lipid profile were similar between children treated with combined GH/GnRHa and those with only GH. In the pubertal subgroup, FM% SDS was lower during treatment with GH 2 mg/m(2) per day. There was no GH dose-dependent effect on LBM SDS, blood pressure, and lipid profile. CONCLUSIONS: Combined GH/GnRHa treatment has no long-term negative effects on metabolic health compared with only GH. Started in early puberty, a GH dose of 2 mg/m(2) per day results in a similar metabolic health at AH and a more favorable FM% than GH 1 mg/m(2) per day.

Background: GPCRs form complex oligomers whose role in signaling is poorly understood. Results: Super-resolution imaging of functionally asymmetric oligomers reveals diverse functional and structural organizations and the ability to alter signal responses. Conclusion: GPCR oligomers may fine-tune receptor signaling by altering the functional role of individual protomers. Significance: Distinct oligomers could be exploited pharmacologically to improve efficacy, selectivity, and/or specificity., Formation of G protein-coupled receptors (GPCRs) into dimers and higher order oligomers represents a key mechanism in pleiotropic signaling, yet how individual protomers function within oligomers remains poorly understood. We present a super-resolution imaging approach, resolving single GPCR molecules to ∼8 nm resolution in functional asymmetric dimers and oligomers using dual-color photoactivatable dyes and localization microscopy (PD-PALM). PD-PALM of two functionally defined mutant luteinizing hormone receptors (LHRs), a ligand-binding deficient receptor (LHRB−) and a signaling-deficient (LHRS−) receptor, which only function via intermolecular cooperation, favored oligomeric over dimeric formation. PD-PALM imaging of trimers and tetramers revealed specific spatial organizations of individual protomers in complexes where the ratiometric composition of LHRB− to LHRS− modulated ligand-induced signal sensitivity. Structural modeling of asymmetric LHR oligomers strongly aligned with PD-PALM-imaged spatial arrangements, identifying multiple possible helix interfaces mediating inter-protomer associations. Our findings reveal that diverse spatial and structural assemblies mediating GPCR oligomerization may acutely fine-tune the cellular signaling profile.

Secretion of 17-β-estradiol (E2) by human granulosa cells can be disrupted by various environmental toxicants. In the current study, we investigated whether carbon black nanoparticles (CB NPs) affect the steroidogenic activity of cultured human granulosa cells. The human granulosa cell line KGN and granulosa cells from patients undergoing in vitro fertilization were treated with increasing concentrations of CB NPs (1 to 100 µg/mL) together or not with follicle-stimulating hormone (FSH). We observed that CB NPs are internalized in KGN cells without affecting cell viability. CB NPs could be localized in the cytoplasm, within mitochondria and in association with the outer face of the endoplasmic reticulum membrane. In both cell types, CB NPs reduced in a dose-dependent manner the activity of aromatase enzyme, as reflected by a decrease in E2 secretion. A significant decrease was observed in response to CB NPs concentrations from 25 and 50 µg/mL in KGN cell line and primary cultures, respectively. Furthermore, CB NPs decreased aromatase protein levels in both cells and reduced aromatase transcript levels in KGN cells. CB NPs rapidly activated extracellular signal-regulated kinase 1 and 2 in KGN cells and pharmacological inhibition of this signaling pathway using PD 98059 significantly attenuated the inhibitory effects of CB NPs on CYP19A1 gene expression and aromatase activity. CB NPs also inhibited the stimulatory effect of FSH on aromatase expression and activity. Altogether, our study on cultured ovarian granulosa cells reveals that CB NPs decrease estrogens production and highlights possible detrimental effect of these common NPs on female reproductive health.

Background: Maintaining optimal reproductive and litter performance is essential for meeting economic targets in commercial pig production. Treatment with exogenous gonadotropins in sows after weaning or in gilts after altrenogest treatment has been used to stimulate follicular development leading to more piglets born and eventually higher birth weights. The effect of peforelin on litter performance was investigated in 212 gilts, primi- and pluriparous sows in three herds. Animals were randomly allocated to three treatments 24 h after weaning: peforelin (P group), eCG (E group), and physiological saline solution (C group). Numbers of total, liveborn and stillborn piglets and mortality rate during lactation were recorded. Birth weights and coefficient of variation in weights within litter were assessed. All parameters were compared among treatments. Results: Over all parities, no difference was found among treatments in litter size nor mortality rate, but birth weights were significantly lower in the E group. Stillbirth numbers in pluriparous sows were 2.2, 0.9 and 1.4 for P, E and C groups, respectively (p = 0.04). Piglets in the P group had significantly higher live born birth weights in gilts, compared to the E group (1.36, 1.26, 1.32 kg (p < 0.02) for P, E and C group, respectively). No significant differences were found for the other investigated parameters. Conclusions: Peforelin treatment showed no improvement of litter performance compared to no treatment.

Determination of biological activity of gonadotropin hormones is essential in reproductive medicine and pharmaceutical manufacturing of the hormonal preparations. The aim of the study was to adopt a G-protein coupled receptor (GPCR)-mediated signal transduction pathway based assay for quantification of biological activity of gonadotropins. We focussed on studying human chorionic gonadotropin (hCG) and follicle-stimulating hormone (FSH), as these hormones are widely used in clinical practice. Receptor-specific changes in cellular cyclic adenosine monophosphate (cAMP, second messenger in GPCR signalling) were monitored by a Förster resonance energy transfer (FRET) biosensor protein TEpacVV in living cells upon activation of the relevant gonadotropin receptor. The BacMam gene delivery system was used for biosensor protein expression in target cells. In the developed assay only biologically active hormones initiated GPCR-mediated cellular signalling. High assay sensitivities were achieved for detection of hCG (limit of detection, LOD: 5 pM) and FSH (LOD: 100 pM). Even the small-scale conformational changes caused by thermal inactivation and reducing the biological activity of the hormones were registered. In conclusion, the proposed assay is suitable for quantification of biological activity of gonadotropins and is a good alternative to antibody- and animal-testing-based assays used in pharmaceutical industry and clinical research.

OBJECTIVE Melatonin synthesis and secretion are partly modulated by estrogen and progesterone. Changes in melatonin concentrations, possibly related to the menopausal transition, may be associated with climacteric mood, sleep, and vasomotor symptoms. The aims of this study were to compare the serum concentrations of melatonin in perimenopausal and postmenopausal women and to evaluate melatonin's influence on mood, sleep, vasomotor symptoms, and quality of life. METHODS We analyzed the data of 17 healthy perimenopausal women (aged 43-51 y) and 18 healthy postmenopausal women (aged 58-71 y) who participated in a prospective study. On study night (9:00 pm-9:00 am), serum melatonin was sampled at 20-minute (9:00 pm-12:00 midnight; 6:00-9:00 am) and 1-hour (12:00 midnight-6:00 am) intervals. Questionnaires were used to assess depression (Beck Depression Inventory), anxiety (State-Trait Anxiety Inventory), insomnia and sleepiness (Basic Nordic Sleep Questionnaire [BNSQ]), subjective sleep quality, vasomotor symptoms, and quality of life (EuroQoL). RESULTS Postmenopausal women had lower nighttime serum melatonin concentrations than perimenopausal women. The duration of melatonin secretion tended to be shorter in postmenopause, whereas melatonin peak time did not differ. Mean melatonin concentrations and exposure levels did not correlate with follicle-stimulating hormone level, estradiol level, body mass index, Beck Depression Inventory score, State-Trait Anxiety Inventory score, BNSQ insomnia score, BNSQ sleepiness score, subjective sleep score, climacteric vasomotor score, or quality of life. In perimenopause, the later is the melatonin peak, the higher is the level of anxiety (P = 0.022), and the longer is the melatonin secretion, the better is the quality of life (P < 0.001). CONCLUSIONS Longitudinal research is needed to better understand the possible contributory role of menopause in lower melatonin levels.

The ability of peforelin (l-GnRH-III) to stimulate follicular growth, FSH release, and estrus in gilts after altrenogest treatment and in sows after weaning was investigated. In three farrow-to-wean herds, with at least 600 sows and average production performance, 216 gilts, 335 primiparous, and 1299 pluriparous sows were randomly allocated to three treatments: peforelin (M group: Maprelin), eCG (F group: Folligon), and physiological saline solution (C group). Animals were treated 48 hours after their last altrenogest treatment (gilts) or 24 hours after weaning (sows). The weaning-to-estrus interval, estrus duration, estrus rate (ER), pregnancy rate, and total born (TB), live born, and stillborn (SB) numbers were recorded and compared between treatments for the different parity groups (gilts and primiparous and pluriparous sows). Follicle sizes were measured in representative animals from each group on the occasion of their last altrenogest treatment or at weaning, and also on the occasions of their first (FS1) and second (FS2) attempted inseminations. Blood samples were taken to determine FSH concentrations at weaning and 2 hours after injection, and progesterone concentrations 10 days after the first insemination attempt. The relative change in FSH concentrations was calculated. Significant differences were found for ER within 7 days of weaning in pluriparous sows (95%, 91%, and 90% for the M, F, and C groups, respectively, P = 0.005). Gilts in the F-group had high TB numbers, and pluriparous sows in the M group had high SB numbers (TB gilts = 13.6, 15.4, and 14.9 [P = 0.02] and SB pluriparous sows = 1.8, 1.4, and 1.7 [P = 0.05] for the M, F, and C groups, respectively). The M group had the highest FS1 (for gilts) and FS2 (for pluriparous sows) values: FS1 = 5.4, 4.9, and 4.9 mm [P = 0.02] and FS2 = 6.8, 5.3, and 6.3 mm [P = 0.03] for the M, F, and C groups, respectively. There were no significant differences between the different treatments within each parity group with respect to any of the other variables. Overall, peforelin treatment had small but positive effects on the ER and follicle growth in certain parity groups but did not seem to affect litter sizes or FSH and progesterone levels in sows on the occasions of the corresponding examinations.

FSH inactivity due to secondary hypoglycosylation has been suggested as a potential mechanism for primary ovarian insufficiency in classic galactosemia. To investigate the role of FSH and to gain insight in the timing of the damage, ovarian stimulation tests were performed and data on ovarian imaging collected. Fifteen patients with primary ovarian insufficiency underwent ovarian stimulation with gonadotropins. Only one patient showed a normal increase in estradiol level, all the others had a low or no estradiol response. Anti-Mullerian hormone measurement in all girls and women showed levels below the detection limit of 0.10 mu g/l. Ovarian volumes were evaluated by MRI in 14 patients and compared to age matched controls, prepubertal controls and postmenopausal controls. The ovarian volumes of the galactosemic girls were smaller than those of the age matched controls (p = 0.001) and the prepubertal ovaries (p = 0.008), and did not differ significantly from postmenopausal ovarian volumes (p = 0.161). In conclusion we found no evidence that FSH inactivity plays a role in primary ovarian insufficiency in classic galactosemia. Moreover, ovarian imaging results point to an early onset of ovarian failure in this disease.

International audience; The ovine cervix relaxes at estrus allowing easier entry of spermatozoa into the uterus. The mechanism responsible for this relaxation is not fully elucidated and we hypothesized that cervical relaxation at estrus is induced by ovarian and pituitary hormones stimulating the local production of prostaglandin E-2 Via a biosynthetic pathway involving a number Of mediators including oxytocin, phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor gamma (PPAR gamma). The aim of this study was to investigate the cervical expression of estradiol receptor alpha (ER alpha), oxytocin receptor (OTR), cPLA(2), COX-2, and PPAR gamma at three stages of the estrous cycle (the luteal phase and two times during the follicular phase, just before and just after the LH surge). An experiment was conducted during the breeding season, in 25 ewes to test this hypothesis. Samples of cervical tissue were collected from groups of ewes at three stages of the estrous cycle: the luteal (N = 8), "pre-LH surge" (N = 8), and "post-LH surge" (N = 9) stages. Cervical tissue from uterine, mid, and vaginal regions of the cervix were analyzed by Western immunoblot analysis for ER alpha, OTR, cPLA(2), COX-2, and PPAR gamma. The results showed that the levels of all five proteins were lowest during the luteal phase of the estrous cycle in all regions of the cervix. The levels of all except cPLA(2), increased significantly during the "pre-LH surge" stage. The levels of cPLA(2) and ER alpha increased in the "post-LH surge" stage and those for OTR and PPARy were unchanged and those for COX-2 were lower. These data show that the cervical levels of all five of the intermediates in the synthesis of prostaglandin E-2 that were examined in this study were higher in the "pre-" and "post-LH surge" stages compared with the luteal phase of the estrous cycle and these findings are consistent with our hypothesis. (C) 2013 Elsevier Inc. All rights reserved.

This study aimed to explore if natural dizygotic (DZ) twinning is associated with earlier menopause and lower anti-Mullerian hormone (AMH) values. We investigated if advanced biological reproductive aging, which can be responsible for the multiple follicle growth in familial twinning, is similar to mechanisms that occur in normal ovarian aging, reflected by earlier menopause in mothers of DZ twins and lower levels of AMH. A total of 16 mothers of DZ twins enrolled with the Netherlands Twin Register (average age at first assessment: 35.9 ± 3.0 years) and 14 control mothers (35.1 ± 3 years) took part in a prospective study. Fifteen years after entry into the study, which included follicle-stimulating hormone (FSH) assessment, AMH was measured in stored serum samples and menopause status was evaluated. Average AMH levels were not significantly different between DZ twin mothers and controls (2.1 ± 2.4 μg/L vs. 1.9 ± 1.9 μg/L). Among the 16 mothers of twins, 7 had an elevated (FSH) value over 10 U/L at first assessment. Their AMH levels were lower than the nine twin mothers with normal FSH values: 0.6 ± 0.4 versus 3.4 ± 2.6 μg/L (p = .01). Of the mothers of twins, eight mothers had entered menopause at the second assessment compared with only one control mother (p = .07). Thus, slightly more DZ mothers were in menopause than the control mothers, although this difference was not significant. The subgroup of DZ twin mothers who had an increased FSH concentration 15 years ago had a limited ovarian reserve as reflected by lower AMH levels. These data indicate that advanced ovarian aging can be a feature in familial DZ twinning, particularly with elevated early follicular phase FSH.

Background: 1-chloro-4-[2,2,2-trichloro-1-(4-chlorophenyl)ethyl]benzene (p,p′-DDT) is a persistent environmental endocrine disruptor (ED). Several studies have shown an association between p,p′-DDT exposure and reproductive abnormalities. Objectives: To investigate the putative effects of p,p′-DDT on the human follitropin receptor (FSHR) function. Methods and Results: We used Chinese hamster ovary (CHO) cells stably expressing human FSHR to investigate the impact of p,p′-DDT on FSHR activity and its interaction with the receptor. At a concentration of 5 μM p,p′-DDT increased the maximum response of the FSHR to follitropin by 32 ± 7.45%. However, 5 μM p,p′-DDT decreased the basal activity and did not influence the maximal response of the closely related LH/hCG receptor to human chorionic gonadotropin (hCG). The potentiating effect of p,p′-DDT was specific for the FSHR. Moreover, in cells that did not express FSHR, p,p′-DDT had no effect on cAMP response. Thus, the potentiating effect of p,p′-DDT was dependent on the FSHR. In addition, p,p′-DDT increased the sensitivity of FSHR to hCG and to a low molecular weight agonist of the FSHR, 3-((5methyl)-2-(4-benzyloxy-phenyl)-5-{[2-[3-ethoxy-4-methoxy-phenyl)-ethylcarbamoyl]-methyl}-4-oxo-thiazolidin-3-yl)-benzamide (16a). Basal activity in response to p,p′-DDT and potentiation of the FSHR response to FSH by p,p′-DDT varied among FSHR mutants with altered transmembrane domains (TMDs), consistent with an effect of p,p′-DDT via TMD binding. This finding was corroborated by the results of simultaneously docking p,p′-DDT and 16a into the FSHR transmembrane bundle. Conclusion: p,p′-DDT acted as a positive allosteric modulator of the FSHR in our experimental model. These findings suggest that G protein–coupled receptors are additional targets of endocrine disruptors. Citation: Munier M, Grouleff J, Gourdin L, Fauchard M, Chantreau V, Henrion D, Coutant R, Schiøtt B, Chabbert M, Rodien P. 2016. In vitro effects of the endocrine disruptor p,p′-DDT on human follitropin receptor. Environ Health Perspect 124:991–999; http://dx.doi.org/10.1289/ehp.1510006

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy affecting women, and a leading cause of female infertility worldwide. Defined clinically by the presence of hyperandrogenemia and oligomenorrhoea, PCOS represents a state of hormonal dysregulation, disrupted ovarian follicle dynamics, and subsequent oligo- or anovulation. The syndrome’s prevalence is attributed at least partly to a well-established association with obesity and insulin resistance (IR). Indeed, the presence of severe PCOS in human genetic obesity and IR syndromes supports a causal role for IR in the pathogenesis of PCOS. The molecular mechanisms underlying this causality, however, as well as the important role of hyperandrogenemia, remain poorly elucidated. As such, treatment of PCOS is necessarily empirical, focusing on symptom alleviation. The generation of knockout and transgenic rodent models of obesity and IR offer a promising platform in which to address mechanistic questions about reproductive dysfunction in the context of metabolic disease. The impact of primary perturbations in rodent gonadotrophin or androgen signaling has been similarly interrogated. The insights gained from such models, however, have been limited by the relatively poor fidelity of rodent models to human PCOS. In this minireview we evaluate the ovarian phenotypes associated with rodent models of obesity and IR, including the extent of endocrine disturbance, ovarian dysmorphology and subfertility. We compare them to both human PCOS and other animal models of the syndrome (genetic and hormonal), explore reasons for their discordance and consider the new opportunities that are emerging to better understand and treat this important condition.

Polyspermy or the penetration of more than one sperm cell remains a problem during porcine in vitro fertilization (IVF). After in vitro culture of porcine zygotes, only a low percentage of blastocysts develop and their quality is inferior to that of in vivo derived blastocysts. It is unknown whether the cytoplasmic maturation of the oocyte is sufficiently sustained in current in vitro maturation (IVM) procedures. The complex interplay between oocyte and cumulus cells during IVM is a key factor in this process. By focusing on this bidirectional communication, it is possible to control the coordination of cumulus expansion, and nuclear and cytoplasmic maturation during IVM to some extent. Therefore, this review focuses on the regulatory mechanisms between oocytes and cumulus cells to further the development of new in vitro embryo production (IVP) procedures, resulting in less polyspermy and improved oocyte developmental potential. Specifically, we focused on the involvement of cAMP in maturation regulation and function of oocyte-secreted factors (OSFs) in the bidirectional regulatory loop between oocyte and cumulus cells. Our studies suggest that maintaining high cAMP levels in the oocyte during the first half of IVM sustained improved oocyte maturation, resulting in an enhanced response after IVF and cumulus matrix disassembly. Recent research indicated that the addition of OSFs during IVM enhanced the developmental competence of small follicle-derived oocytes, which was stimulated by epidermal growth factor (EGF) via developing EGF-receptor signaling.

Context: GH treatment is effective in improving height in short children born small for gestational age (SGA). GH is thought to have limited effect when started during adolescence.Objective: The aim of this study was to investigate GH treatment efficacy in short SGA children when treatment was started during adolescence; to assess whether GH 2 mg/m(2).d during puberty improves adult height (AH) compared with 1 mg/m(2).d; and to assess whether an additional 2-yr postponement of puberty by GnRH analog (GnRHa) improves AH in children who are short at the start of puberty (Patients and Design: In this longitudinal, randomized, dose-response GH trial, we included 121 short SGA children (60 boys) at least 8 yr of age. We performed intention-to-treat analyses on all children and uncensored case analyses on 84 children who reached AH. Besides, we evaluated growth during 2 yr of combined GH/GnRHa and subsequent GH treatment until AH in a subgroup of 40 pubertal children with a height of less than 140 cm at the start.Results: Short SGA children started treatment at a median age of 11.2 yr, when 46% had already started puberty. Median height increased from -2.9 at start to -1.7 SD score (SDS) at AH (P Conclusion: When started in adolescence, GH treatment significantly improves AH in short SGA children, particularly with GH 2 mg/m(2).d during puberty. When SGA children are short at the start of puberty, they can benefit from combined GH/GnRHa treatment. (J Clin Endocrinol Metab 97: 4096-4105, 2012)