ETS transcription factors regulate precise matrix metalloproteinase expression and follicle rupture in Drosophila.

Drosophila matrix metalloproteinase 2 (MMP2) is specifically expressed in posterior follicle cells of stage-14 egg chambers (mature follicles) and is crucial for the breakdown of the follicular wall during ovulation, a process that is highly conserved from flies to mammals. The factors that regulate spatiotemporal expression of MMP2 in follicle cells remain unknown. Here, we demonstrate crucial roles for the ETS-family transcriptional activator Pointed (Pnt) and its endogenous repressor Yan in the regulation of MMP2 expression. We found that Pnt is expressed in posterior follicle cells and overlaps with MMP2 expression in mature follicles. Genetic analysis demonstrated that pnt is both required and sufficient for MMP2 expression in follicle cells. In addition, Yan was temporally upregulated in stage-13 follicle cells to fine-tune Pnt activity and MMP2 expression. Furthermore, we identified a 1.1 kb core enhancer that is responsible for the spatiotemporal expression of MMP2 and contains multiple pnt/yan binding motifs. Mutation of pnt/yan binding sites significantly impaired the Mmp2 enhancer activity. Our data reveal a mechanism of transcriptional regulation of Mmp2 expression in Drosophila ovulation, which could be conserved in other biological systems.

Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.

Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.

A spatiotemporal molecular atlas of the ovulating mouse ovary.

Ovulation is essential for reproductive success, yet the underlying cellular and molecular mechanisms are far from clear. Here, we applied high-resolution spatiotemporal transcriptomics to map out cell type- and ovulation stage-specific molecular programs as function of time during follicle maturation and ovulation in mice. Our analysis revealed dynamic molecular transitions within granulosa cell types that occur in tight coordination with mesenchymal cell proliferation. We identified molecular markers for the emerging cumulus cell fate during the preantral-to-antral transition. We describe transcriptional programs that respond rapidly to ovulation stimulation and those associated with follicle rupture, highlighting the prominent roles of apoptotic and metabolic pathways during the final stages of follicle maturation. We further report stage-specific oocyte-cumulus cell interactions and diverging molecular differentiation in follicles approaching ovulation. Collectively, this study provides insights into the cellular and molecular processes that regulate mouse ovarian follicle maturation and ovulation with important implications for advancing therapeutic strategies in reproductive medicine.

The bHLH-PAS transcriptional complex Sim:Tgo plays active roles in late oogenesis to promote follicle maturation and ovulation.

Across species, ovulation is a process induced by a myriad of signaling cascades that ultimately leads to the release of encapsulated oocytes from follicles. Follicles first need to mature and gain ovulatory competency before ovulation; however, the signaling pathways regulating follicle maturation are incompletely understood in Drosophila and other species. Our previous work has shown that the bHLH-PAS transcription factor Single-minded (Sim) plays important roles in follicle maturation downstream of the nuclear receptor Ftz-f1 in Drosophila. Here, we demonstrate that Tango (Tgo), another bHLH-PAS protein, acts as a co-factor of Sim to promote follicle cell differentiation from stages 10 to 12. In addition, we discover that re-upregulation of Sim in stage-14 follicle cells is also essential to promote ovulatory competency by upregulating octopamine receptor in mushroom body (OAMB), matrix metalloproteinase 2 (Mmp2) and NADPH oxidase (NOX), either independently of or in conjunction with the zinc-finger protein Hindsight (Hnt). All these factors are crucial for successful ovulation. Together, our work indicates that the transcriptional complex Sim:Tgo plays multiple roles in late-stage follicle cells to promote follicle maturation and ovulation.

Loss of bmp15 function in the seasonal spawner Atlantic salmon results in ovulatory failure.

Bone morphogenetic protein 15 (BMP15) is an oocyte-specific growth factor important for successful female reproduction in mammals. While mutations in BMP15/Bmp15 cause ovulatory deficiency and/or infertility in certain mammalian species, loss of bmp15 in zebrafish, a continuous spawner and the only bmp15 knockout model in fish to date, results in complete arrest of follicle development and later female-to-male sex reversal, preventing to examine effects on ovulation/fertilization. Here, we used Atlantic salmon, a seasonal spawner, and generated bmp15 mutants to investigate ovarian development and fertility. Histological and morphometric analyses revealed that in biallelic frameshift (bmp15 fs/fs) mutant ovaries, folliculogenesis started earlier, resulting in an advanced development compared to wild-type (WT) controls, accompanied by a weaker expression of the (early) oocyte-specific factor figla. This precocious ovarian development was followed in bmp15 fs/fs females by enhanced follicle atresia during vitellogenic stages. Although genes involved in steroid synthesis and signaling (star, cyp11b, cyp17a1 and esr1) were dramatically higher in late vitellogenic bmp15 fs/fs mutant ovaries, estradiol-17ß plasma levels were lower than in WT counterparts, potentially reflecting compensatory changes at the level of ovarian gene expression. At spawning, bmp15 fs/fs females displayed lower gonado-somatic index values and reduced oocyte diameter, and the majority (71.4%), showed mature non-ovulating ovaries with a high degree of atresia. The remaining (28.6%) females spawned eggs but they either could not be fertilized or, upon fertilization, showed severe malformations and embryonic mortality. Our results show that Bmp15 is required for proper follicle recruitment and growth and later ovulatory success in Atlantic salmon, providing an alternative candidate target to induce sterility in farmed salmon. Moreover, since loss of bmp15 in salmon, in contrast to zebrafish, does not result in female-to-male sex change, this is the first mutant model in fish allowing further investigations on Bmp15-mediated functions in the ovulatory period.

Guideline for the management of conception and pregnancy in thalassaemia syndromes: A British Society for Haematology Guideline.

This comprehensive guideline, developed by a representative group of UK-based medical experts specialising in haemoglobinopathies, addresses the management of conception and pregnancy in patients with thalassaemia. A systematic search of PubMed and EMBASE using specific keywords, formed the basis of the literature review. Key terms included "thalassaemia," "pregnancy," "Cooley's anaemia," "Mediterranean anaemia," and others, covering aspects such as fertility, iron burden and ultrasonography. The guideline underwent rigorous review by prominent organisations, including the Endocrine Society, the Royal College of Obstetricians and Gynaecologists (RCOG), the United Kingdom Thalassaemia Society and the British Society of Haematology (BSH) guideline writing group. Additional feedback was solicited from a sounding board of UK haematologists, ensuring a thorough and collaborative approach. The objective of the guideline is to equip healthcare professionals with precise recommendations for managing conception and pregnancy in patients with thalassaemia.

Slit1 inhibits ovarian follicle development and female fertility in mice.

Previous in vitro studies have suggested that SLIT ligands could play roles in regulating ovarian granulosa cell proliferation and gene expression, as well as luteolysis. However, no in vivo study of Slit gene function has been conducted to date. Here we investigated the potential role of Slit1 in ovarian biology using a Slit1-null mouse model. Female Slit1-null mice were found to produce larger litters than their wild-type counterparts due to increased ovulation rates. Increased ovarian weights in Slit1-null animals were found to be due to the presence of greater numbers of healthy antral follicles with similar numbers of atretic ones, suggesting both an increased rate of follicle recruitment and a decreased rate of atresia. Consistent with this, treatment of cultured granulosa cells with exogenous SLIT1 induced apoptosis in presence or absence of FSH, but had no effect on cell proliferation. Although few alterations in the mRNA levels of FSH-responsive genes were noted in granulosa cells of Slit1-null mice, LH target gene mRNA levels were greatly increased. Finally, increased phospho-AKT levels were found in granulosa cells isolated from Slit1-null mice, and SLIT1 pretreatment of cultured granulosa cells inhibited the ability of both FSH and LH to increase AKT phosphorylation, suggesting a mechanism whereby SLIT1 could antagonize gonadotropin signaling. These findings therefore represent the first evidence for a physiological role of a SLIT ligand in the ovary, and define Slit1 as a novel autocrine/paracrine regulator of follicle development.

Luteinizing hormone stimulates ingression of mural granulosa cells within the mouse preovulatory follicle†.

Luteinizing hormone (LH) induces ovulation by acting on its receptors in the mural granulosa cells that surround a mammalian oocyte in an ovarian follicle. However, much remains unknown about how activation of the LH receptor modifies the structure of the follicle such that the oocyte is released and the follicle remnants are transformed into the corpus luteum. The present study shows that the preovulatory surge of LH stimulates LH receptor-expressing granulosa cells, initially located almost entirely in the outer layers of the mural granulosa, to rapidly extend inwards, intercalating between other cells. The cellular ingression begins within 30 min of the peak of the LH surge, and the proportion of LH receptor-expressing cell bodies in the inner half of the mural granulosa layer increases until the time of ovulation, which occurs at about 10 h after the LH peak. During this time, many of the initially flask-shaped cells appear to detach from the basal lamina, acquiring a rounder shape with multiple filipodia. Starting at about 4 h after the LH peak, the mural granulosa layer at the apical surface of the follicle where ovulation will occur begins to thin, and the basolateral surface develops invaginations and constrictions. Our findings raise the question of whether LH stimulation of granulosa cell ingression may contribute to these changes in the follicular structure that enable ovulation.

Proposal for targeted, neo-evolutionary-oriented, secondary prevention of early-onset endometriosis and adenomyosis. Part I: pathogenic aspects.

The potential for repeated ovulation and menstruation is thought to have provided a Darwinian advantage during the Palaeolithic. Reproductive conditions remained relatively stable until the pre-industrial era, characterized by late menarche, very young age at first birth, multiple pregnancies, and prolonged periods of lactational amenorrhoea. For hundreds of thousands of years, menstruators experienced few ovulatory cycles, even though they were genetically adapted to ovulate and menstruate every month. In the post-industrial era, the age at menarche gradually declined, the age at first birth progressively increased, and breastfeeding became optional and often of short duration. This created a mismatch between genetic adaptation and socio-environmental evolution, so that what was initially a probable reproductive advantage subsequently contributed to increased susceptibility to diseases associated with lifetime oestrogen exposure, such as ovarian, endometrial and breast cancer and, hypothetically, also those associated with the number of ovulatory menstruations, such as endometriosis and adenomyosis. The incidence of endometriosis shows a steep and progressive increase around the age of 25 years, but given the consistently reported delay in diagnosis, the actual incidence curve should be shifted to the left, supporting the possibility that the disease has its roots in adolescence. This raises the question of whether, from an evolutionary point of view, anovulation and amenorrhoea should not still be considered the physiological state, especially in the postmenarchal period. However, an increase in the frequency of endometriosis in recent decades has not been demonstrated, although this deserves further epidemiological investigation. In addition, as endometriosis occurs in a minority of individuals exposed to retrograde menstruation, other important pathogenic factors should be scrutinised. Research should be resumed to explore in more detail the transtubal reflux of not only blood, but also endometrial cells, and whether they are systematically present in the peritoneal fluid after menstruation. If repetitive ovulatory menstruation during the early reproductive years is shown to increase the risk of endometriosis and adenomyosis development and progression in susceptible individuals, hormonal interventions could be used as secondary prevention in symptomatic adolescents.

Proposal for targeted, neo-evolutionary-oriented secondary prevention of early-onset endometriosis and adenomyosis. Part II: medical interventions.

According to consistent epidemiological data, the slope of the incidence curve of endometriosis rises rapidly and sharply around the age of 25 years. The delay in diagnosis is generally reported to be between 5 and 8 years in adult women, but it appears to be over 10 years in adolescents. If this is true, the actual onset of endometriosis in many young women would be chronologically placed in the early postmenarchal years. Ovulation and menstruation are inflammatory events that, when occurring repeatedly for years, may theoretically favour the early development of endometriosis and adenomyosis. Moreover, repeated acute dysmenorrhoea episodes after menarche may not only be an indicator of ensuing endometriosis or adenomyosis, but may also promote the transition from acute to chronic pelvic pain through central sensitization mechanisms, as well as the onset of chronic overlapping pain conditions. Therefore, secondary prevention aimed at reducing suffering, limiting lesion progression, and preserving future reproductive potential should be focused on the age group that could benefit most from the intervention, i.e. severely symptomatic adolescents. Early-onset endometriosis and adenomyosis should be promptly suspected even when physical and ultrasound findings are negative, and long-term ovulatory suppression may be established until conception seeking. As nowadays this could mean using hormonal therapies for several years, drug safety evaluation is crucial. In adolescents without recognized major contraindications to oestrogens, the use of very low-dose combined oral contraceptives is associated with a marginal increase in the individual absolute risk of thromboembolic events. Oral contraceptives containing oestradiol instead of ethinyl oestradiol may further limit such risk. Oral, subcutaneous, and intramuscular progestogens do not increase the thromboembolic risk, but may interfere with attainment of peak bone mass in young women. Levonorgestrel-releasing intra-uterine devices may be a safe alternative for adolescents, as amenorrhoea is frequently induced without suppression of the ovarian activity. With regard to oncological risk, the net effect of long-term oestrogen-progestogen combinations use is a small reduction in overall cancer risk. Whether surgery should be considered the first-line approach in young women with chronic pelvic pain symptoms seems questionable. Especially when large endometriomas or infiltrating lesions are not detected at pelvic imaging, laparoscopy should be reserved to adolescents who refuse hormonal treatments or in whom first-line medications are not effective, not tolerated, or contraindicated. Diagnostic and therapeutic algorithms, including self-reported outcome measures, for young individuals with a clinical suspicion of early-onset endometriosis or adenomyosis are proposed.

Resumption of ovulation in anovulatory women with PCOS and obesity is associated with reduction of 11ß-hydroxyandrostenedione concentrations.

STUDY QUESTION: Is resumption of ovulation after a 6-month lifestyle intervention in women with PCOS and obesity associated with differential changes in endocrine and metabolic parameters (weight, insulin resistance, anti-Müllerian hormone (AMH), and androgens) compared to women with PCOS who remained anovulatory? SUMMARY ANSWER: Resumption of ovulation after a 6-month lifestyle intervention in women with PCOS and obesity is associated with changes in serum 11ß-hydroxyandrostenedione (11OHA4) concentrations. WHAT IS KNOWN ALREADY: Lifestyle interventions have been shown to reduce clinical and biochemical hyperandrogenism in women with PCOS. Weight loss of 5-10% may reverse anovulatory status, thereby increasing natural conception rates. However, the mechanisms underlying why some women with PCOS remain anovulatory and others resume ovulation after weight loss are unclear. Reproductive characteristics at baseline and a greater degree of change in endocrine and metabolic features with lifestyle intervention may be crucial for ovulatory response. STUDY DESIGN, SIZE, DURATION: We used data and samples originating from an earlier randomized controlled trial (RCT), which examined the efficacy of a 6-month lifestyle intervention prior to infertility treatment compared to prompt infertility treatment on live birth rate in women with obesity. A total of 577 women with obesity (BMI > 29 kg/m2) were randomized between 2009 and 2012. Anovulatory women with PCOS who were allocated to the intervention arm of the original RCT (n = 95) were included in the current analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: We defined women as having resumed ovulation (RO+) based on the following criteria: spontaneous pregnancy; or assignment to expectant management; or IUI in natural cycles as the treatment strategy after lifestyle intervention. Steroid hormones were measured using liquid chromatography tandem mass spectrometry. Generalized estimating equations with adjustment for baseline measures and interaction between group and time was used to examine differences in changes of endocrine and metabolic parameters between RO+ (n = 34) and persistently anovulatory women (RO-, n = 61) at 3 and 6 months after intervention. MAIN RESULTS AND THE ROLE OF CHANCE: At baseline, the mean ± SD age was 27.5 ± 3.6 years in the RO+ group and 27.9 ± 4.1 years in the RO- group (P = 0.65), and the mean ± SD weights were 101.2 ± 9.5 kg and 105.0 ± 14.6 kg, respectively (P = 0.13). Baseline AMH concentrations showed significant differences between RO+ and RO- women (median and interquartile range [IQR] 4.7 [3.2; 8.3] versus 7.2 [5.3; 10.8] ng/ml, respectively). Baseline androgen concentrations did not differ between the two groups. During and after lifestyle intervention, both groups showed weight loss; changes in 11OHA4 were significantly different between the RO+ and RO groups (P-value for interaction = 0.03). There was a similar trend for SHBG (interaction P-value = 0.07), and DHEA-S (interaction P-value = 0.06), with the most pronounced differences observed in the first 3 months. Other parameters, such as AMH and FAI, decreased over time but with no difference between the groups. LIMITATIONS, REASONS FOR CAUTION: No high-resolution transvaginal ultrasonography was used to confirm ovulatory status at the end of the lifestyle program. The small sample size may limit the robustness of the results. WIDER IMPLICATIONS OF THE FINDINGS: Reduction of androgen concentrations during and after lifestyle intervention is associated with recovery of ovulatory cycles. If our results are confirmed in other studies, androgen concentrations could be monitored during lifestyle intervention to provide individualized recommendations on the timing of resumption of ovulation in anovulatory women with PCOS and obesity. STUDY FUNDING/COMPETING INTEREST(S): The study was supported by a grant from ZonMw, the Dutch Organization for Health Research and Development (50-50110-96-518). The Department of Obstetrics and Gynecology of the UMCG received an unrestricted educational grant from Ferring Pharmaceuticals BV, The Netherlands. A.H. reports consultancy for the development and implementation of a lifestyle App MyFertiCoach developed by Ferring Pharmaceutical Company. All other authors have no conflicts to declare. TRIAL REGISTRATION NUMBER: The LIFEstyle RCT was registered at the Dutch trial registry (NTR 1530).

The intrafollicular concentrations of biologically active cortisol in women rise abruptly shortly before ovulation and follicular rupture.

STUDY QUESTION: What is the temporal activity and the concentration in follicular fluid (FF) of the anti-inflammatory steroid cortisol during the ovulatory process in humans? SUMMARY ANSWER: Intrafollicular concentrations of cortisol become massively upregulated close to ovulation concomitant with an exceptionally high biological activity securing a timely and efficient termination of inflammatory processes. WHAT IS KNOWN ALREADY: Ovulation has been described as a local, controlled inflammatory process resulting in the degeneration of the follicle wall which facilitate oocyte extrusion. Ovulation also affects the glucocorticoid metabolism of granulosa cells (GCs) and although de novo synthesis of cortisol only occurs in the adrenal cortex, the mid-cycle surge has been shown to induce a change from high expression of HSD11B2, inactivating cortisol to cortisone, to high expression of HSD11B1 which reversibly catalyses cortisol production from cortisone. Furthermore, high concentrations of progesterone and 17OH-progesterone within follicles may cause dislodging of cortisol from cortisol binding protein (CBP) thereby activating the biological activity of cortisol. STUDY DESIGN, SIZE, DURATION: This prospective cohort study included 50 women undergoing fertility treatment according to a standard antagonist protocol at a university hospital-affiliated fertility clinic in Denmark. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women donated FF and GCs from one follicle for research purpose aspirated at one of four time points during the process of final maturation of follicles: T = 0 h, T = 12 h, T = 17 h, T = 32 h. A second sample was collected at oocyte pick up at T = 36 h. The concentration of cortisol and cortisone together with a range of sex steroids was measured by LC-MS/MS in FF collected at the five time points mentioned above. Whole genome microarray data, validated by q-PCR analysis, was used to evaluate gene expression of CYP11B1, CYP21A2, HSD11B1, HSD11B2, and NR3C1 in GCs at the same time points. MAIN RESULTS AND THE ROLE OF CHANCE: The concentration of cortisol was significantly increased from a few nM at 0 h to around 100-140 nM (P ≤ 0.0001) at 32-36 h, whilst cortisone was almost constant from 0 to 17 h at a concentration of between 90 and 100 nM being significantly reduced to 25-40 nM (P ≤ 0.0001) at 32-36 h. This was paralleled by a 690-fold upregulation of HSD11B1 from 0 to 12 h increasing to a more than 20.000-fold change at 36 h. HSD11B2 was quickly downregulated 15- to 20-fold after ovulation induction. Concentrations of progesterone and 17OH-progesterone increased during the ovulatory process to high levels which in essence displaces cortisol from its binding protein CBP due to similar binding affinities. Furthermore, a significant decrease in 11-deoxycortisol expression was seen, but CYP11B1 expression was below detection limit in GCs. LIMITATIONS, REASONS FOR CAUTION: The study included women undergoing ovarian stimulation and results may differ from the natural cycle. More observations at each specific time point may have strengthened the conclusions. Furthermore, we have not been able to measure the actual active biological concentration of cortisol. WIDER IMPLICATIONS OF THE FINDINGS: For the first time, this study collectively evaluated the temporal pattern of cortisol and cortisone concentrations during human ovulation, rendering a physiological framework for understanding potential dysregulations in the inflammatory reaction of ovulation. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the University Hospital of Copenhagen, Rigshospitalet, and Novo Nordisk Foundation grant number NNF21OC00700556. Interreg V ÔKS through ReproUnion (www.reprounion.eu); Region Zealand Research Foundation. The funders had no role in study design, collection of data, analyses, writing of the article, or the decision to submit it for publication. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Granulosa cell metabolism at ovulation correlates with oocyte competence and is disrupted by obesity and aging.

STUDY QUESTION: Is oocyte developmental competence associated with changes in granulosa cell (GC) metabolism? SUMMARY ANSWER: GC metabolism is regulated by the LH surge, altered by obesity and reproductive aging, and, in women, specific metabolic profiles are associated with failed fertilization versus increased blastocyst development. WHAT IS KNOWN ALREADY: The cellular environment in which an oocyte matures is critical to its future developmental competence. Metabolism is emerging as a potentially important factor; however, relative energy production profiles between GCs and cumulus cells and their use of differential substrates under normal in vivo ovulatory conditions are not well understood. STUDY DESIGN, SIZE, DURATION: This study identified metabolic and substrate utilization profiles within ovarian cells in response to the LH surge, using mouse models and GCs of women undergoing gonadotropin-induced oocyte aspiration followed by IVF/ICSI. PARTICIPANTS/MATERIALS, SETTING, METHODS: To comprehensively assess follicular energy metabolism, we used real-time metabolic analysis (Seahorse XFe96) to map energy metabolism dynamics (mitochondrial respiration, glycolysis, and fatty acid oxidation) in mouse GCs and cumulus-oocyte complexes (COCs) across a detailed time course in the lead up to ovulation. In parallel, the metabolic profile of GCs was measured in a cohort of 85 women undergoing IVF/ICSI (n = 21 with normal ovarian function; n = 64 with ovarian infertility) and correlated with clinical parameters and cycle outcomes. MAIN RESULTS AND THE ROLE OF CHANCE: Our study reveals dynamic changes in GC energy metabolism in response to ovulatory LH, with mitochondrial respiration and glycolysis differentially affected by obesity versus aging, in both mice and women. High respiration in GCs is associated with failed fertilization (P < 0.05) in a subset of women, while glycolytic reserve and mitochondrial ATP production are correlated with on-time development at Day 3 (P < 0.05) and blastocyst formation (P < 0.01) respectively. These data provide new insights into the cellular mechanisms of infertility, by uncovering significant associations between metabolism within the ovarian follicle and oocyte developmental competence. LIMITATIONS, REASONS FOR CAUTION: A larger prospective study is needed before the metabolic markers that were positively and negatively associated with oocyte quality can be used clinically to predict embryo outcomes. WIDER IMPLICATIONS OF THE FINDINGS: This study offers new insights into the importance of GC metabolism for subsequent embryonic development and highlights the potential for therapeutic strategies focused on optimizing mitochondrial metabolism to support embryonic development. STUDY FUNDING/COMPETING INTEREST(S): National Health and Medical Research Council (Australia). The authors have no competing interests. TRIAL REGISTRATION NUMBER: N/A.

A single-cell gene expression atlas of human follicular aspirates: Identification of leukocyte subpopulations and their paracrine factors.

Leukocytes are in situ regulators critical for ovarian function. However, little is known about leukocyte subpopulations and their interaction with follicular cells in ovulatory follicles, especially in humans. Single-cell RNA sequencing (scRNA-seq) was performed using follicular aspirates obtained from four IVF patients and identified 13 cell groups: one granulosa cell group, one thecal cell group, 10 subsets of leukocytes, and one group of RBC/platelet. RNA velocity analyses on five granulosa cell populations predicted developmental dynamics denoting two projections of differentiation states. The cell type-specific transcriptomic profiling analyses revealed the presence of a diverse array of leukocyte-derived factors that can directly impact granulosa cell function by activating their receptors (e.g., cytokines and secretory ligands) and are involved in tissue remodeling (e.g., MMPs, ADAMs, ADAMTSs, and TIMPs) and angiogenesis (e.g., VEGFs, PGF, FGF, IGF, and THBS1) in ovulatory follicles. Consistent with the findings from the scRNA-seq data, the leukocyte-specific expression of CD68, IL1B, and MMP9 was verified in follicle tissues collected before and at defined hours after hCG administration from regularly cycling women. Collectively, this study demonstrates that this data can be used as an invaluable resource for identifying important leukocyte-derived factors that promote follicular cell function, thereby facilitating ovulation and luteinization in women.

Salivary testosterone across the menstrual cycle.

Testosterone production in women is thought to systematically shift across the menstrual cycle, peaking during the mid-cycle ovulatory window, and potentially influencing women's behavior. Testosterone is a molecular intermediary to the production of estradiol, which is necessary for ovulation to occur, but the amount of testosterone escape and exposure to the peripheral tissues is not fully understood. Salivary testosterone is a common biomarker in behavioral neuroendocrinological studies and is thought to reflect the bioactive portions in serum. In N = 339 women with confirmed ovulation via luteinizing hormone tests, salivary testosterone, assayed with LC-MS/MS, was sampled four times across the mid-cycle ovulatory window the luteal phase. Within-subject analysis revealed a significant but small pattern of a mid-cycle peak and a luteal decrease at the aggregate level. However, at the individual level, there was substantial variability in the direction and magnitude of the testosterone-cycle pattern. We discuss the relevant underlying physiology, background research, issues with assay methodolody, and considerations for researchers studying testosterone levels in women.

Longitudinal changes in sexual desire and attraction among women who started using the Natural Cycles app.

Many women experience sexual side effects, such as decreased libido, when taking hormonal contraceptives (HCs). However, little is known about the extent to which libido recovers after discontinuing HCs, nor about the timeframe in which recovery is expected to occur. Given that HCs suppress the activities of multiple endogenous hormones that regulate both the ovulatory cycle and women's sexual function, resumption of cycles should predict libido recovery. Here, using a combination of repeated and retrospective measures, we examined changes in sexual desire and partner attraction (among partnered women) across a three-month period in a sample of Natural Cycles users (Survey 1: n = 1596; Survey 2: n = 550) who recently discontinued HCs. We also tested whether changes in these outcomes coincided with resumption of the ovulatory cycle and whether they were associated with additional factors related to HC use (e.g., duration of HC use) or relationship characteristics (e.g., relationship length). Results revealed that both sexual desire and partner attraction, on average, increased across three months after beginning to use Natural Cycles. While the prediction that changes in sexual desire would co-occur with cycle resumption was supported, there was also evidence that libido continued to increase even after cycles resumed. Together, these results offer new insights into relationships between HC discontinuation and women's sexual psychology and lay the groundwork for future research exploring the mechanisms underlying these effects.

Effects of SARS-CoV-2 infection during ovarian stimulation on ART outcomes.

RESEARCH QUESTION: Does severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection during ovarian stimulation affect assisted reproductive technology outcomes? DESIGN: This retrospective cohort study conducted at the Reproductive Medicine Centre of The First Affiliated Hospital of Anhui Medical University aimed to assess the effects of acute SARS-CoV-2 infection during IVF on treatment outcomes and the reproductive system. The study included 151 treatment cycles involving couples with coronavirus disease 2019 (COVID-19) during ovarian stimulation, along with 224 cycles of non-infected couples as a control group. Clinical characteristics and laboratory parameters were analysed, including total gonadotrophin dosage, duration of ovarian stimulation, number of oocytes retrieved, fertilization method, fertilization rate, and number of blastocyst embryos available. Forty-six follicular fluid samples, 38 semen samples and 78 embryo culture medium samples from patients with COVID-19 were tested for SARS-CoV-2 RNA using reverse transcription polymerase chain reaction assay. RESULTS: The treatment and control groups showed similar cycle characteristics, including fertilization method, total gonadotrophin dosage and duration of ovarian stimulation. The mean number of oocytes retrieved per cycle and rate of mature oocytes in intracytoplasmic sperm injection cycles were comparable. No significant difference was observed in the total number of blastocyst embryos available between the groups. Furthermore, no SARS-CoV-2 RNA was detected in any of the samples of patients with COVID-19. CONCLUSIONS: In conclusion, acute SARS-CoV-2 infection during ovarian stimulation does not have a significant impact on IVF treatment outcomes. Additionally, no risk to the reproductive system was observed in patients infected with SARS-CoV-2. Therefore, individuals with asymptomatic or mild COVID-19 can safely continue IVF treatment. Future research is needed to investigate the long-term effects of COVID-19 on fertility and reproductive outcomes.

Effect of vitamin D on pregnancy in women with polycystic ovary syndrome: retrospective and prospective studies.

RESEARCH QUESTION: Does vitamin D affect the pregnancy rate of patients with polycystic ovary syndrome (PCOS) receiving ovulation-induction therapy? DESIGN: The retrospective study included 200 patients with PCOS and 200 healthy women. The prospective study included 160 patients with PCOS receiving vitamin D or placebo supplementation. Pregnancy rates were assessed after a maximum of three cycles of ovulation induction. Serum concentrations of 25-hydroxycalciferol [25-(OH)D3], LH, FSH, progesterone, oestradiol, testosterone and fasting insulin; LH/FSH ratio; and body mass index were evaluated. RESULTS: In the retrospective study, patients with PCOS had lower 25-(OH)D3 concentrations than healthy women, pregnant patients with PCOS had higher 25-(OH)D3 concentrations than non-pregnant patients with PCOS (both P = 0.000), and the pregnancy rate was lower in the vitamin-D-deficient group compared with the non-vitamin-D-deficient group (P = 0.022). In the prospective study, compared with placebo supplementation, vitamin D supplementation increased the serum concentration of 25-(OH)D3 (P = 0.000), and reduced the LH/FSH ratio, and concentrations of LH and testosterone significantly (all P ≤ 0.049). After the intervention, it was found that the LH/FSH ratio, and concentrations of LH and testosterone were significantly lower in both groups compared with pre-intervention (P = 0.000). After ovulation induction, the pregnancy rate was higher in patients in the vitamin D supplementation group compared with the placebo supplementation group (P = 0.049). CONCLUSIONS: Vitamin D deficiency is common in patients with PCOS, and vitamin-D-deficient patients with PCOS have lower pregnancy rates after ovulation induction compared with non-vitamin-D-deficient patients with PCOS. Vitamin D supplementation can improve the pregnancy rate and mitigate basic hormone disorders. Therefore, monitoring vitamin D supplementation and checking vitamin D concentrations before and during interventions are essential for patients with PCOS.

Dapagliflozin Ameliorates Ovulation Disorders via Attenuating Activated Microglia-Mediated Hypothalamic Inflammation in HFD-Fed Mice.

INTRODUCTION: Sodium-glucose cotransporter 2 inhibitors (SGLT2is) have shown neuroprotective effects in obese mice. However, whether SGLT2i can ameliorate high-fat diet (HFD)-related ovulation disorders remains unknown. The aim of this research was to investigate whether dapagliflozin improves HFD-induced ovulatory dysfunction by attenuating microglia-mediated hypothalamic inflammation. METHODS: C57BL/6J female mice fed HFD were treated with dapagliflozin (1 mg/kg) for 22 weeks. Plasma insulin, leptin, luteinizing hormone (LH), estradiol (E2), and IL-1ß levels were also tested. Microglial morphology, cell numbers, and SGLT2 expression were evaluated using immunofluorescence. The expression of IL-1ß, NLRP3, kisspeptin, gonadotropin-releasing hormone (GnRH), SGLT2, insulin, and leptin receptors in the hypothalamus was determined using immunohistochemical staining. We also examined the effects of dapagliflozin on glucose metabolism and the release of inflammatory factor in palmitic acid (PA)-treated HMC3 cells. RESULTS: As expected, dapagliflozin improved HFD-induced metabolic disturbances, peripheral versus central insulin and leptin resistance and also restored the regular estrous cycle. Furthermore, dapagliflozin blunted microglia activation, NLRP3 inflammasome priming, hypothalamic inflammation, and increased the expression of GnRH and kisspeptin at proestrus in the hypothalamus. Additionally, dapagliflozin markedly reduced IL-6 and NO release and fat accumulation, decreased lactic acid production and glucose consumption, and inhibited mammalian target of rapamycin (mTOR) and hexokinase 2 (HK2) expression in PA-treated HMC3 cells. These effects suggest that dapagliflozin reduced the mTOR/HK2-mediated aerobic glycolysis. CONCLUSIONS: Dapagliflozin improved HFD-related ovulation disorders by regulating glucose metabolism through mTOR/HK2 signaling and attenuating microglia-mediated hypothalamic inflammation. These results validate the novel role for the neuroprotection of SGLT2i in HFD-induced obesity and ovulation disorders.

Ibuprofen delays ovulation by several hours: prospective controlled study in natural cycles with HCG-triggered ovulation.

RESEARCH QUESTION: Does ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), delay ovulation? DESIGN: Two-stage, proof-of-concept, controlled study, assessing the percentage of non-ovulated follicles 42 h after HCG injection in patients taking ibuprofen. The intervention group consisted of women undergoing natural cycle IVF treatment taking ibuprofen 3 × 400 mg per day. The control group consisted of women undergoing timed sexual intercourse or intrauterine insemination. The proportion of patients with non-ovulated follicles in the ibuprofen group was first compared against a reference of 50% using a one-sample binomial test, and second against the proportion observed in the control group using an adjusted logistic regression. RESULTS: A total of 26 women were recruited in the ibuprofen intervention group. Twenty-five patients were recruited in the control group. The proportion of patients with delayed ovulation observed (22/26 [84.6%]; 95% CI 65.1% to 95.6%) was significantly higher than the reference of 50% (P < 0.001). In the control group, the proportion of patients with delayed ovulation was 20.0% ([5/25], 95% CI 6.8% to 40.7%). Compared with the ibuprofen group, a significantly increased probability of a delayed ovulation was found in the ibuprofen intervention group (adjusted OR 22.72, 95% CI 5.77 to 115; P < 0.001). Of the 22 women with delayed ovulation, oocytes were retrieved in 20 women (90.9%) and all oocytes were mature (metaphase II). CONCLUSIONS: Women trying to conceive should avoid non-selective NSAIDs around the time of ovulation. Ibuprofen or other NSAID can be used to delay ovulation for several hours in assisted reproductive technology and other infertility treatments if required.