STUDY QUESTION:How do unoperated ovarian endometriomas affect ovarian response during ovarian stimulation cycles? SUMMARY ANSWER:Ovaries affected by unoperated ovarian endometriomas show similar outcomes to unaffected ovaries following controlled ovarian stimulation. WHAT IS KNOWN ALREADY:Ovarian endometriomas are estimated to affect between 17% and 44% of women with endometriosis. The heterogeneity of the published literature from IVF/ICSI cycles aggregated in meta-analyses has shown both lower and similar reproductive outcomes in women with ovarian endometriomas compared with control women. To overcome these problems in studying reproductive outcomes in presence of ovarian endometriomas, some observational studies have been performed, comparing the affected unoperated ovary with the unaffected contralateral ovary in women with unilateral disease. This approach allows to investigate both the quantitative and qualitative aspects of the ovarian response in presence of a unilateral ovarian cyst. STUDY DESIGN, SIZE, DURATION:A systematic search for all eligible studies in the PubMed and Cochrane databases until 1 May 2025 was conducted. Eligible studies met the following criteria: (i) original studies comparing unoperated unilateral endometriomas with contralateral healthy ovaries in the same patients, (ii) clear description of diagnostic methods for ovarian endometriosis, and (iii) available data on ovarian response, oocyte collection, and reproductive outcomes following ART from each ovary. PARTICIPANTS/MATERIALS, SETTING, METHODS:Studies were screened, data were extracted, and the risk of bias was assessed independently by two investigators. The main outcome was the number of oocytes retrieved per ovary. Secondary outcomes included the number of MII oocytes, fertilization rate, the number of embryos formed, the number of good-quality embryos, and implantation rates. MAIN RESULTS AND THE ROLE OF CHANCE:Two thousand nine hundred and three studies were identified and 13 observational studies met the inclusion criteria and were included in this systematic review and meta-analysis. The meta-analysis of the number of oocytes retrieved showed comparable results between affected and unaffected ovaries in women with unilateral ovarian endometrioma (MD -0.72, 95% CI -1.45 to 0.02; I² = 97%; 11 studies, 560 patients). Similarly, the number of MII oocytes and good-quality embryos were comparable between affected and unaffected ovaries (MD -0.60, 95% CI -1.86 to 0.66; I² = 81%; 6 studies, 149 patients and MD -0.18, 95% CI -0.53 to 0.18; I² = 0%; 3 studies, 74 affected vs 74 unaffected ovaries, respectively). Conversely, the number of embryos formed was significantly lower in affected ovaries (MD -0.72, 95% CI -1.39 to -0.05; I² = 68%; 4 studies, 192 affected vs 193 unaffected ovaries, respectively). LIMITATIONS, REASONS FOR CAUTION:Most of the included studies were observational, with eight being retrospective, which increases susceptibility to bias. In addition, the outcome reporting was inconsistent, and none of the studies was preregistered, raising the risk of selective reporting. WIDER IMPLICATIONS OF THE FINDINGS:Unoperated ovarian endometriomas appear to have little or no impact on ovarian stimulation outcomes, however, the impact of larger endometriomas remains uncertain. These findings suggest that gynecologists should carefully consider whether to recommend surgery prior to IVF/ICSI, particularly for small endometriomas, which may not adversely impact ovarian stimulation outcomes. FUNDING:No funding was used to conduct this study. DISCLOSURES:D.R.K. received research grants from Gottfried und Julia Bangerter-Rhyner and Bouriez foundations. E.S. declares speakers' fees from IPSEN and Gedeon Richter, fees from consultations from Ferring and grants for research from IBSA and Ferring. P.V. has received honoraria as Co-Editor-in-Chief of the Journal of Endometriosis and Uterine Disorders. M.C. has nothing to disclose. C.B. reports grants and personal fees from MSD, Ferring, Abbott, and Gedeon Richter. The remaining authors have nothing to disclose. REGISTRATION NUMBER:CRD42024507807 (https://www.crd.york.ac.uk/PROSPERO/view/CRD42024507807).
STUDY QUESTION:What are the individual and combined effects of a common genetic cause of human male infertility (loss of M1AP function) and a negative lifestyle factor (a Western diet) on male fertility, spermatogenesis, and sperm quality in mice? SUMMARY ANSWER:The loss of M1AP and a Western diet separately led to subfertility (a form infertility) in male mice and in combination subfertility was worsened. WHAT IS KNOWN ALREADY:Genetics and lifestyle factors individually play important roles in male infertility aetiology. How lifestyle factors influence common genetic causes of infertility are unknown. STUDY DESIGN, SIZE, DURATION:We generated knockout M1ap mice (M1ap-/--) and investigated the effects of genotype (wild type or knockout) and diet (control or Western diet) or genotype and age (young or aged) on male fertility and sperm parameters. PARTICIPANTS/MATERIALS, SETTINGS, METHODS:C57BL6/J mice: wild type (M1ap+/+) and M1ap knockout (M1ap-/-) at 10-12 (young) weeks or 26-28 weeks old (aged), on a control of Western (high-fat and high-sugar) diet and age. We evaluated male fertility, testis and epididymis histology, and sperm quality (motility, morphology, and count). MAIN RESULTS AND THE ROLE OF CHANCE:Compared to the individual gene deletion or diet treatments, the combined M1ap-/- and Western diet group had more abnormal germ cells, sloughed germ cells and resulted in fewer pups per litter. In accordance, sperm motility and morphology in the combination group were more significantly compromised compared to individual experimental groups. LARGE SCALE DATA:N/A. LIMITATIONS, REASONS FOR CAUTION:While the mutant mouse mimics the genetic variants seen in many infertile men, this is a mouse model study, meaning findings should be validated in a human cohort where possible. WIDER IMPLICATIONS OF THE FINDINGS:Taken together, our data highlight that genetic and lifestyle factors can interact to exacerbate male infertility. This study sets a precedent for an improved consideration of how genetic, lifestyle and environmental factors contribute to infertility aetiology. These data inform a clinical foundation for lifestyle interventions for men presenting at infertility clinics prior to medically assisted reproduction. FUNDING:This project was supported by a Society for Reproductive Biology Career Development Award to B.J.H., National Health and Medical Research Council project grant (APP1120356) awarded to M.K.O.B., an Australia-Germany Joint Research Co-operation Scheme (UA-DAAD) grant (57511796) awarded to M.K.O.B., J.E.M.D., and F.T., and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the Clinical Research Unit 'Male Germ Cells' (CRU326, project number 329621271) awarded to F.T. and C.F. DISCLOSURES:N/A.
STUDY QUESTION:Does an ultra-sensitive anti-Müllerian hormone (UD-AMH) assay enhance the accuracy of ovarian dysfunction assessment in premenopausal women with breast cancer (PBC) during and after chemotherapy? SUMMARY ANSWER:Low AMH levels at pre-treatment and end-of-chemotherapy, measured by the UD-AMH assay, are strong predictors for the development of premature ovarian insufficiency (POI) at 12 months post-chemotherapy. WHAT IS KNOWN ALREADY:Anti-Müllerian hormone (AMH) is a recognized biomarker for assessing ovarian reserve and serves as an early indicator of ovarian dysfunction in female cancer patients. However, post-chemotherapy AMH levels frequently fall below detectable limits of conventional assays, hindering rapid and accurate evaluation of residual ovarian reserve. STUDY DESIGN, SIZE, DURATION:Prospective cohort study; 145 total subjects enrolled; follow-up conducted from pre-treatment through 12 months post-chemotherapy. PARTICIPANTS/MATERIALS, SETTING, METHODS:Premenopausal women (aged 18-46 years) with breast cancer recruited at diagnosis; 83 scheduled to receive chemotherapy and 62 who did not. Serum samples were collected pre-treatment (T0), after each chemotherapy cycle, at end-of-chemotherapy (T1), 3 (T2), 6 (T3), and 12 (T4) months post-chemotherapy. AMH levels were quantified using the UD-AMH assay (detection limit 0.13 pg/ml, offering 100-fold higher sensitivity than conventional methods). POI was defined as oligo/amenorrhea with follicle-stimulating hormone (FSH) levels >25 mIU/ml. MAIN RESULTS AND THE ROLE OF CHANCE:AMH was quantifiable in all samples by using the UD-AMH assay. At T4, 30% of PBC exhibited POI. AMH levels at T0 were significantly higher in the non-POI group compared to the POI group (median 5425 pg/ml vs 1103 pg/ml, P < 0.001), as were levels at T1 (median 127 pg/ml vs 10 pg/ml, P < 0.001). T0 AMH predicted POI with an AUC of 0.86 (sensitivity 67%, specificity 95%), while T1 AMH showed superior predictive value with an AUC of 0.89 (sensitivity 81%, specificity 93%). LIMITATIONS, REASONS FOR CAUTION:This trial was a single-center study with sample size attrition due to the COVID-19 pandemic. Additionally, the study focused on hormonal dynamics up to 1 months post-chemotherapy, lacking long-term data on clinical fertility outcomes in the identified risk groups. WIDER IMPLICATIONS OF THE FINDINGS:The application of the UD-AMH assay enables early prediction of POI and offers a valuable tool to guide personalized treatment decisions and fertility preservation strategies in young female cancer patients. FUNDING:This work was supported by the National Key Research and Development Program of China (2023YFB3210301), National Natural Science Foundation of China (82271664), Shanghai Municipal Council for Science and Technology (20JC1412100), the Shanghai Municipal Health Commission (202240345), the interdisciplinary program of Shanghai Jiao Tong University (YG2022ZD028), and the Shanghai Key Laboratory of Embryo Original Diseases (Shelab2022ZD01). DISCLOSURES:R.A.A. has received research support from Roche Diagnostics; all other authors declare no competing interests. TRIAL REGISTRATION NUMBER:ClinicalTrials.gov Identifier: NCT04767607.
STUDY QUESTION:Are young women with anti-Müllerian hormone (AMH) levels suggesting polycystic ovarian morphology (PCOM) at baseline better protected against chemo-induced ovarian damages? SUMMARY ANSWER:Compared with women with lower baseline AMH levels, women with pre-treatment AMH levels suggesting PCOM experienced a similar degree of follicular loss and only partial recovery of baseline values after treatment but maintained significantly higher AMH levels at 12 and 24 months of follow-up and had a faster menstrual function resumption. WHAT IS KNOWN ALREADY?:Longitudinal studies on AMH variations in women treated by chemotherapy have shown that pre-treatment AMH levels is one of the factors influencing the degree of the ovarian recovery. Recombinant AMH administration recently demonstrated a protective effect on ovarian follicles exposed to alkylating agents in mouse models. High AMH levels in women with polycystic ovaries have been associated with decreasing apoptosis of granulosa cells in small follicles and altered initial follicular growth. STUDY DESIGN, SIZE, DURATION:This retrospective analysis of prospective collected data was carried out at the OncoFertility Observatory of the Lille University Hospital from January 2012 to February 2022. All women eligible for chemotherapy are prospectively recruited for ovarian function indicators follow-up until 24 months after the end of treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS:467 women treated by alkylating agents for breast cancer (BC) or hematological malignancies (HM) were included in our Onco-Fertility Observatory and benefited from an onco-fertility consultation with discussion on fertility preservation options. The study population was divided into two groups according to basal AMH level at time of diagnosis: PCOM-like AMH levels ≥35 pmol/l before 2016 and ≥ 30 pmol/l after 2026 (PCOM group), and 8 ≥ AMH <35 pmol/l (non-PCOM group). Serial AMH measurements were performed at baseline before chemotherapy initiation (AMH0), 15 days after the start of chemotherapy (AMH1), 15 days before the last chemotherapy cycle (AMH2), and at time 3, 6, 12, 18, and 24 months from the end of chemotherapy. Menstrual function was assessed in parallel and antral follicle count (AFC) was performed at baseline, 12- and 24-month post-treatment. Serum AMH levels were measured using the second-generation enzyme immunoassay EIA AMH/MIS kit from 2012 to 2016, and by an automated AMH immunoassay on an Access Dxi analyzer thereafter. Results were secondarily homogenized using a validated conversion formula. MAIN RESULTS AND THE ROLE OF CHANCE:261 BC women aged 31.4 years, and 206 HM patients aged 24.4 years eligible for alkylating regimen were included before commencing treatment. Sixty-six women in the BC population were excluded from this study (death n = 4, recurrence n = 7, lost to follow-up n = 55) and 81 in the HM population (death n = 6, lost of follow-up n = 46, pregnancies n = 2, oophorectomy for cryopreservation n = 27). A total of 195 BC and 125 HM patients completed the whole follow-up and were finally analyzed. In BC group, women with PCOM were significantly younger (P = 0.002), had a higher incidence of menstrual disturbances at baseline (P = 0.003) and had more frequently a hormone receptor-positive (HR+) tumor (P = 0.04). In HM group, PCOM women were not significantly different from non-PCOM women concerning general characteristics. In BC and HM groups, pre-treatment AFC was significantly higher in PCOM group versus non-PCOM (37.8 ± 14.7 vs 17.3 ± 9.4; P ≤ 0.001 and 33.6 ± 12.4 vs 19.3 ± 8.4; P ≤ 0.001, respectively). In BC, as in HM population, a depletion phase can be distinguished from the beginning to the end of chemotherapy (AMH0-AMH2) and a recovery phase from the end of chemotherapy (AMH2) to the time +12 months followed by a sustaining phase between time +12 and 24 months. In BC as in HM population, before and after adjustment for the confounding variables (age, BMI, smoking and oral contraceptive (OC) use prior to chemotherapy), AMH values in the PCOM group remained significantly higher at time +12 and +24 months of follow-up. In BC PCOM subgroup, the AMH depletion slope was steeper and followed by a faster and higher AMH increase after the end of treatment, while no difference was observed between PCOM and non-PCOM HM women. In both cancer groups and in both subgroups of AMH levels, AMH values plateaued between Time +12 and Time +24 months (adjusted mean difference in log AMH (95% CI) 0.2 (-0.2 to 0.5) in BC population; 0.5 (-0.1 to 1.1) in HM population). At the end of follow-up, the frequency of undetectable AMH values in BC was 8.3% in PCOM versus 30.4% in non-PCOM (P = 0.0072) whereas, in HM group, there was no significant difference (18.7% vs 29.9%; P = 0.55). In HM, only three women, all from the PCOM group, recovered their pre-treatment values at the end of follow-up. In BC, none of the women recovered their initial AMH values. Clinically, the incidence of persistent amenorrhea at time +12months in HM subgroup tended to be lower in PCOM than in non-PCOM women but did not reach significance (13.3% vs 30.4%; P = 0.22). In BC subgroup, the incidence of persistent amenorrhea was significantly higher in non-PCOM women at time +12 months (2% vs 12%; P = 0.043) but no longer at time + 24 months. LIMITATIONS, REASONS FOR CAUTION:the retrospective design of this study did not allow us to differentiate women with PCOS from women with PCOM only, as androgen levels were not routinely measured at baseline in this oncologic setting. The high incidence of women under OC at diagnosis (50%) may have underestimated the number of patients with AMH levels higher than 35 pmol/l. Two methods of AMH assays were used due to the long duration of the inclusion period but with validated and adapted cut-offs levels for the diagnosis of PCOM. WIDER IMPLICATIONS OF THE FINDINGS:Fertility preservation and biological/clinical follow-up should be systematically offered to all women undergoing alkylating regimen, irrespective of their pre-treatment AMH level as very high ovarian follicular content does not preclude to significant follicle loss. A better understanding of the role of AMH in ovaries facing chemotherapy may ultimately help in developing ovarioprotective drugs. FUNDING:This work was supported by Agence Régionale de Santé Hauts de France and Agence Onco Hauts-de-France who provided finances for AMH dosages (no. DOS/SDES/AR/FIR/2019/282). DISCLOSURES:No competing interests. TRIAL REGISTRATION NUMBER:DC-2008-642 and CNIL DEC2015-112.
STUDY QUESTION:Are periconception maternal modifiable lifestyle factors (i.e. smoking, alcohol consumption, caffeine intake, folic acid supplement use, and physical activity) associated with embryonic morphologic development and growth in a large population-based cohort study? SUMMARY ANSWER:Inadequate folic acid supplement use and insufficient serum folate levels were associated with delayed embryonic morphologic development and reduced growth, while no associations were observed for smoking, alcohol, caffeine intake, or physical activity. WHAT IS KNOWN ALREADY:Modifiable maternal lifestyle factors during the periconception period may influence embryonic growth and development. Prior studies suggest associations between smoking, folic acid use, and first trimester growth, but evidence on morphologic development remains limited. STUDY DESIGN, SIZE, DURATION:This study was embedded in the Generation R Next Study, a population-based periconception cohort in Rotterdam, the Netherlands, conducted between 2017 and 2021. Of the 2173 included spontaneous singleton pregnancies, 993 had reliable pregnancy dating, 3D ultrasound data up to 10 + 2 weeks of gestation, and available information on periconception lifestyle risk factors. PARTICIPANTS/MATERIALS, SETTING, METHODS:Embryonic development and growth were assessed in vivo at 7 and 9 weeks of gestation using 3D transvaginal ultrasound and virtual reality techniques. Morphologic development was determined using the Carnegie staging system based on limb position, embryonic curvature, and brain morphology; crown-rump length (CRL) was used to assess growth. Periconception lifestyle factors, including smoking, alcohol and caffeine intake, physical activity, and folic acid supplement use, were assessed using early pregnancy questionnaires. Linear mixed models were used to examine the association between lifestyle factors and embryonic Carnegie stages and CRL. Analyses were adjusted for gestational age at ultrasound, maternal age, ethnicity, education level, parity, fetal sex, and the other lifestyle factors. MAIN RESULTS AND THE ROLE OF CHANCE:Inadequate folic acid supplement use was associated with a 0.51-day delay in embryonic development and a 0.72-mm reduction in CRL (-1.6%) at 10 + 2 weeks of gestation. Insufficient serum folate levels (<25.5 nmol/l) were associated with a 1.01-day developmental delay and a 1.24-mm reduction in CRL (-3.7%). No significant associations were observed for smoking, alcohol or caffeine intake, or physical activity. Results remained robust after adjustment for confounders. LIMITATIONS, REASONS FOR CAUTION:Lifestyle exposures were self-reported, which may have introduced recall or social desirability bias. Gestational age determination was based on the last menstrual period, which can result in dating discrepancies due to variability in cycle length or inaccurate recall, but strict inclusion criteria were applied to minimize misclassification. The relatively healthy study population could limit the generalizability of our results to higher-risk groups. WIDER IMPLICATIONS OF THE FINDINGS:Our findings emphasize the importance of initiating folic acid supplementation in the preconception period to ensure sufficient maternal folate levels, which may support optimal embryonic morphologic development and growth during the first trimester. These results reinforce public health recommendations advocating timely folic acid supplementation to promote healthy early embryonic development. STUDY FUNDING/COMPETING INTEREST(S):The Generation R Next Study is financially supported by the Erasmus University Medical Centre Rotterdam, the Erasmus University Rotterdam, and the Netherlands Organization for Health Research and Development. R.G. received funding from the Netherlands Organization for Health Research and Development (NWO, ZonMw VIDI 09150172110034, and NWO, ZonMW, grant number 05430052110007) and an European Research Council Starting Grant (ERC-2024-STG 101161004). V.W.V.J. received a grant from the Netherlands Organization for Health Research and Development (NWO, ZonMw 05430052110007) and a European Research Council Consolidator Grant (ERC-2014-CoG-648916). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER:N/A.
STUDY QUESTION:Does vanillin, at concentrations relevant to maternal exposures during vaping, disrupt gastrulation-related processes in human embryonic stem cells (hESCs) by activating TRPV4 channels? SUMMARY ANSWER:Activation of TRPV4 channels by nanomolar concentrations of vanillin promoted an exit from pluripotency and a shift toward endodermal gene expression, while micromolar concentrations induced developmental toxicity, identifying a concentration-dependent mechanism by which vanillin, a flavoring agent in electronic cigarette (EC) aerosols, may pose risks to embryonic development during pregnancy. WHAT IS KNOWN ALREADY:The use of ECs during pregnancy is increasing, potentially driven by perceptions of reduced harm and the appeal of flavoring agents, such as vanillin. However, vanillin activates transient receptor potential (TRP) channels, raising concerns about its impact on early embryonic development. STUDY DESIGN, SIZE, DURATION:hESCs, which model the epiblast stage of development, were treated in vitro for various times with vanillin, a TRPV4 antagonist, a TRPV4 function-blocking antibody, or vanillin combined with either the antagonist or antibody. All experiments were done three times with different passages of stem cells. PARTICIPANTS/MATERIALS, SETTING, METHODS:After variable periods of exposure, the hESC colonies were evaluated for activation of TRPV4 channels and calcium influx, colony growth, colony detachment, increased cell death, mitochondrial dysfunction, colony morphology, gap formation, downregulation of EpCAM, loss of pluripotency, and initiation of differentiation. MAIN RESULTS AND THE ROLE OF CHANCE:We demonstrate that vanillin, at concentrations relevant to maternal EC exposure, significantly (P < 0.05) disrupted gastrulation-related processes in hESCs. hESCs, which model the epiblast during Weeks 2-3 of human development, exhibited TRPV4 channel expression and showed a significant increase in intracellular calcium upon nanomolar-to-micromolar vanillin exposure, as detected by the Fluo-8 calcium dye. Time-lapse imaging and quantitative analysis indicated that micromolar vanillin impaired colony expansion, induced colony detachment, and increased cell death over 72 h, while concurrently reducing mitochondrial reductase activity in the MTT assay. At nanomolar concentrations, vanillin promoted TRPV4 activation, an exit from pluripotency, downregulation of EpCAM cell adhesion protein, intercellular gap formation, and a shift toward endodermal gene expression. All effects were blocked by a TRPV4 antagonist (HC067047), identifying activation of this channel as a critical mediator of vanillin-induced toxicity. LARGE SCALE DATA:N/A. LIMITATIONS, REASONS FOR CAUTION:Our data are derived from in vitro experiments, as direct investigation of early human development is ethically and practically limited. Although our findings do not establish embryo lethality or malformation in vivo, the observed loss of pluripotency and SOX17 upregulation, consistent with endodermal induction, at nanomolar concentrations of vanillin are signs of early cellular perturbations that could compromise human development. WIDER IMPLICATIONS OF THE FINDINGS:Nanomolar vanillin concentrations, which our exposure model estimates reach the embryo during early pregnancy in EC users, were sufficient to activate TRPV4 channels in hESCs and alter germ layer specification. Micromolar concentrations produced cytotoxic effects and increased cell death. These findings indicate that vanillin exposure at levels attainable during EC use disrupts intracellular Ca2+ homeostasis and compromises core processes of early human development. Maternal EC use during pregnancy therefore represents a significant and previously under-appreciated risk to early embryogenesis. Clinicians should counsel patients who are pregnant or planning pregnancy about this potential harm, and regulatory agencies should consider mandatory ingredient disclosure on EC packaging. FUNDING:This research was supported by grant number T32IR4848 from the Tobacco-Related Disease Research Program (TRDRP), by grant number EDUC4-12752 from the California Institute of Regenerative Medicine (CIRM), and by UCR Yvonne Danielson Endowed Graduate and Dissertation Completion Fellowship Awards. The content is solely the responsibility of the authors and does not necessarily represent the official view of the TRDRP, CIRM, or UCR. DISCLOSURES:The authors have no conflicts of interest to declare.
STUDY QUESTION:Does the endometrium of oligo-ovulatory women with polyendocrine metabolic ovarian syndrome (PMOS) exhibit menstrual cycle phase-specific transcriptomic alterations compared to women without PMOS? SUMMARY ANSWER:Compared to healthy controls, women with PMOS exhibit endometrial transcriptomic dysregulations which are menstrual cycle phase-specific and, most notably, related to mitochondrial function. WHAT IS KNOWN ALREADY:Infertility and gestational dysfunction in women with PMOS are increasingly attributed to endometrial abnormalities, yet the underlying mechanisms behind PMOS-related endometrial dysfunction remain unclear. Previous studies have focused on anovulatory phenotypes, resulting in a poor understanding of cycle phase-specific defects in the endometria of women with PMOS. STUDY DESIGN, SIZE, DURATION:Endometrial biopsies were obtained from oligo-ovulatory women with PMOS and healthy controls at four distinct menstrual cycle phases (proliferative, early-, mid-, and late-secretory phases) and subjected to RNA sequencing. Parallel in vitro experiments were conducted using cultured endometrial stromal cells (ESCs) and epithelial organoids (EEOs) derived from an additional endometrial sample cohort. PARTICIPANTS/MATERIALS, SETTING, METHODS:RNA sequencing was performed on biopsies from 78 (oligo-)ovulatory women, age- and BMI-matched between PMOS (n = 39) and control (n = 39) groups. Phase-specific transcriptomic profiles and pathway enrichment analyses were performed. For functional assays, endometrial cells were isolated from four PMOS and four control donors per BMI group (lean: < 25 kg/m2; obese: ≥30 kg/m2). Mitochondrial function, including respiration, ATP and reactive oxygen species production, and membrane potential, was assessed in both cell types before and after hormonal exposure. MAIN RESULTS AND THE ROLE OF CHANCE:Endometrial transcriptomes clustered primarily by cycle phase; however, PMOS-specific dysregulation was observed related to extracellular matrix organization, estrogen receptor (ESR) signaling, ion transport, and mitochondrial respiratory function. Gene trajectory analyses revealed phase-specific alterations in progesterone-, insulin signaling-, and mitochondria-related gene expression during the early-secretory phase, and epithelial function during the mid-secretory phase. Functionally, decidualized ESCs from obese PMOS women displayed reduced mitochondrial respiration, whereas EEOs from lean PMOS patients exhibited increased mitochondrial activity. LARGE SCALE DATA:Sequencing data are available from the corresponding author upon request. LIMITATIONS, REASONS FOR CAUTION:Women with PMOS were oligo-ovulatory while controls exhibited regular cycles. The sample size for in vitro studies was modest, warranting validation in larger cohorts. Additionally, the use of bulk RNA sequencing may obscure cell-type-specific transcriptomic signatures, emphasizing the need for future single-cell approaches to dissect cellular heterogeneity in PMOS endometrium. WIDER IMPLICATIONS OF THE FINDINGS:This study uncovers dynamic, cycle phase-dependent molecular and bioenergetic alterations in the endometrium of oligo-ovulatory women with PMOS. Mitochondrial dysfunction, particularly during the implantation window, emerges as a key pathological feature. The observed BMI-dependent divergence in mitochondrial function suggests that personalized therapeutic strategies may be necessary to improve reproductive outcomes in women with PMOS. FUNDING:Funded by the Jusélius Foundation, Novo Nordisk Foundation, Research Council of Finland, and the Horizon 2020 Marie-Curie MATER Innovative Training Network (all to T.T.P.), with additional support from the University of Oulu Scholarship Foundation (L.L.), the Estonian Research Council (E.V.M. and I.R.), and the Estonian Research Council grant no. PRG1076, Swedish Research Council grant no. 2024-02530, Novo Nordisk Foundation grant no. NNF24OC0092384 and Horizon Europe grant NESTOR, grant no. 101120075 (A.S.). DISCLOSURES:The authors declare no competing interests.
STUDY QUESTION:Is the preconception 1-h plasma glucose level during the oral glucose tolerance test (OGTT) more strongly associated with subsequent gestational diabetes mellitus than the fasting or 2-h glucose levels in women with PMOS/PCOS undergoing IVF treatment? SUMMARY ANSWER:Preconception 1-h plasma glucose showed a stronger and more consistent association with gestational diabetes mellitus than the fasting or 2-h glucose levels in women with PMOS/PCOS undergoing IVF treatment. WHAT IS KNOWN ALREADY:Women with PMOS/PCOS have an increased risk of gestational diabetes mellitus. International guidelines recommend a preconception OGTT in women with PMOS/PCOS without pre-existing diabetes because of the risk of hyperglycaemia and pregnancy complications. However, the comparative predictive value of different preconception OGTT time points, particularly 1-h glucose, remains unclear. STUDY DESIGN, SIZE, DURATION:This was a multi-centre retrospective cohort study including 2571 women with PMOS/PCOS who underwent their first IVF treatment at three reproductive medicine centres in China and had a preconception 75-g OGTT between March 2018 and December 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS:Women with PMOS/PCOS who completed a preconception 75-g OGTT before initiation of the IVF treatment were included. The primary outcome was GDM diagnosed at 24-28 weeks of gestation. Generalized linear mixed-effects models were used to assess associations between OGTT glucose levels and GDM, adjusting for age, BMI, infertility duration, hormonal profiles, and treatment protocols. Stratified analyses by BMI and PMOS/PCOS phenotype were conducted, and multiple imputation was applied for missing data. MAIN RESULTS AND THE ROLE OF CHANCE:Preconception fasting (aOR 1.97 [1.49-2.61]), 1-h (aOR 1.23 [1.13-1.34]) and 2-h glucose (aOR 1.30 [1.17-1.45]) were all significantly associated with GDM (all P < 0.001). Within the diabetic range, only the 1-h glucose measurement of ≥11.6 mmol/l remained a significant indicator. The association of 1-h glucose with GDM was significant in normal-weight and obese women but not in overweight women. The associations were consistent across PMOS/PCOS phenotypes. LIMITATIONS, REASONS FOR CAUTION:The retrospective design may have introduced residual confounding and limits causal inference. Missing data required imputation, and subgroup analyses may have limited statistical power. WIDER IMPLICATIONS OF THE FINDINGS:These findings suggest that preconception 1-h glucose may provide additional value for identifying women who are a higher risk of GDM among normal-weight and obese women with PMOS/PCOS undergoing IVF treatment, and support its consideration in preconception metabolic assessments. FUNDING:This work was supported by the National Natural Science Foundation of China (82522038, 82471670 and 82401908), the Natural Science Foundation of Fujian Province (2025J011428), and the "Yongjiang Science and Technology Innovation 2035" Key Research and Development Program (2025Z151). DISCLOSURES:The authors declared no competing interests. TRIAL REGISTRATION NUMBER:N/A.
STUDY QUESTION:What is the sustained impact of master's programs in reproductive science and medicine in meeting the growing global demand for expertise in this field? SUMMARY ANSWER:This study provides quantitative data supporting the value of master's degree programs in providing students with educational experiences to meet the increasing demands for a well-trained workforce of reproductive scientists. WHAT IS KNOWN ALREADY:Master's programs focused on reproduction-ranging from animal science, clinical embryology, and reproductive health and biology-are expanding around the globe, providing students with a degree beyond the bachelor's or its equivalent and are an alternative or bridge to other advanced degrees. However, the impact of these programs in creating a well-trained next generation of reproductive scientists to support workforce needs and advance the field has not been systematically investigated. STUDY DESIGN, SIZE, DURATION:We performed a web search to define the landscape of master's programs offered across the globe. We also convened a meeting (Global Perspectives: A Think Tank for MS Programs in Reproductive Science and Medicine at Northwestern University on 24-25 June 2024) to establish a network between four master's programs (two in the United States and two in Europe), including the Master at Northwestern University (USA), at University of Dundee (United Kingdom), at Colorado State University (USA), and at the University of Murcia (Spain). These programs are established at public and private institutions, are varied with respect to class size and duration, and span focus areas ranging from veterinary to human reproductive health to Assisted Reproductive Technologies (ART). We obtained and analyzed enrollment and outcome data from the four featured master's programs to determine the profile of students who pursue these master's degrees in reproductive science and determine how they contribute to the field post-degree. PARTICIPANTS/MATERIALS, SETTING, METHODS:The study relied on self-reported data from each participating program. The programs were selected because they represent a cross-section of established master's programs in reproduction that have collectively trained almost 1000 students since 2004. We analyzed enrollment and outcome data from these four established master's programs. Programs reported on student outcomes post-degree, including job categories, job titles, employers, and other impact (e.g. publications, awards and honors, and grants and independent funding). MAIN RESULTS AND THE ROLE OF CHANCE:Master's programs in reproduction and related fields are on the rise, with the number of programs having almost doubled from 73 in 2022 to 145 in 2025. The four featured programs reported on at least 9 years of retrospective data. The University of Murcia program started in 2004, Colorado State University in 2013, University of Dundee in 2014, and Northwestern in 2016. To date, these programs have trained 366, 210, 244, and 108 individuals, respectively, with average class sizes for each program ranging between 12 and 22 students. Most students across programs (81% cumulatively) are female. The programs based in the USA primarily attract US students, whereas the programs in the United Kingdom and Spain have a more global footprint. Students matriculate into programs with academic backgrounds in life sciences and related fields and have obtained prior degrees in broadly defined fields including biology, chemistry, animal sciences, and medicine. After students complete their master's degrees, they are generally continuing within the reproductive science and medicine pipeline with careers spanning: advanced training (e.g. PhD, MD, and DVM programs), clinical careers (e.g. embryology and andrology), research careers (e.g. scientist or faculty), and careers in industry and education. LIMITATIONS, REASONS FOR CAUTION:Our study is limited to four master's programs that were invited to the Global Perspectives: A Think Tank for MS Programs in Reproductive Science and Medicine meeting. The analysis performed here can serve as a basic template for tracking student profiles and outcomes across all existing programs, so that we can glean a more wholistic understanding of the impact of master's degrees programs in reproductive science and medicine. WIDER IMPLICATIONS OF THE FINDINGS:This study provides quantitative data supporting the value of master's degree programs in providing students with educational experiences to meet the increasing demands for a well-trained workforce of reproductive scientists. This work lays the foundation for establishing a formal consortium of master's programs in reproductive science whereby prospective data can be collected in a standardized way across programs, alumni outcomes tracked, and an educational network created. This could be of value in disciplines where there is an increasing trend toward accreditation and adherence to legal frameworks, such as reproductive medicine and embryology. FUNDING:This work was supported by institutional funds from the Center for Reproductive Science, the Thomas J. Watkins Endowment, and the Department of Obstetrics and Gynecology at Northwestern University (F.E.D.) and the Makowski Family Endowment, Department of Obstetrics and Gynecology, CU Anschutz School of Medicine (T.R.K.). DISCLOSURES:C.L.R.B. is Co-Editor-in-Chief of Human Reproduction; he is a faculty member and deputy program lead for one of the courses discussed in this article (Dundee) for which salary is paid by the University of Dundee. All other authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER:Not applicable.
Mitochondria are central to oocyte competence and early embryonic development, with roles that extend beyond energy production to include regulation of redox homeostasis, apoptosis and cellular aging. Mitochondrial dysfunction is increasingly recognized as a key contributor to diminished ovarian reserve, impaired embryo development, and accelerated reproductive aging. Mitochondria-targeted therapeutic strategies, including pharmacological approaches such as Coenzyme Q10, mitoquinone, resveratrol, rapamycin, and NAD+ precursors, as well as mitochondrial replacement techniques such as maternal spindle and pronuclear transfer, have shown promise in preclinical models; however, clinical outcomes remain heterogeneous and often inconclusive. This translational gap likely reflects critical limitations, including variability in therapeutic targets, suboptimal timing of intervention relative to oocyte development, and insufficiently powered or standardized clinical studies. Greater emphasis on well-defined, physiologically justified therapeutic targets, along with the use of physiologically relevant experimental systems, may improve therapeutic precision and efficacy. Rigorous evaluation of safety, particularly for interventions with pleiotropic effects or heritable consequences, remains essential. A more targeted, developmentally informed and systematically validated approach is needed to advance mitochondria-based therapies toward meaningful improvements in reproductive outcomes.
STUDY QUESTION:Are higher ambient temperatures before oocyte retrieval associated with adverse oocyte-related outcomes in women undergoing their first IVF or ICSI cycle? SUMMARY ANSWER:Higher pre-retrieval temperatures are associated with reduced oocyte yield, an effect partially mediated through LH and partially counteracted by compensatory increases in FSH and estradiol (E2). WHAT IS KNOWN ALREADY:Evidence on the impact of ambient temperature on IVF/ICSI outcomes is emerging but limited. A recent study reported weak negative associations between higher ambient temperatures during folliculogenesis and oocyte yield, though the effect was modest and findings across populations remain inconsistent. However, these findings are further limited by small sample sizes, specific clinical subpopulations or geographic regions, which prevents the establishment of a generalizable dose-response relationship and leaves the underlying biological mechanisms largely unexplored. STUDY DESIGN, SIZE, DURATION:A retrospective cohort study of 58 468 women undergoing their first IVF/ICSI cycle with oocyte retrieval between 1 January 2018 and 31 May 2024. PARTICIPANTS/MATERIALS, SETTING, METHODS:Women undergoing their first IVF/ICSI cycle were included. The primary outcomes were total oocytes retrieved, mature oocytes retrieved, and 2PN (two pronuclei) fertilized oocytes. Secondary outcomes included rates of oocyte maturation, normal fertilization, and blastocyst formation. Basal sex hormone levels were measured during the early follicular phase (cycle days 2-4). Environmental exposure data, including both ambient temperature and apparent temperature (integrating air temperature and humidity), were assessed over biologically relevant exposure windows. Generalized linear mixed-effects models were used to estimate associations between temperature exposures and oocyte-related outcomes. Restricted cubic spline models assessed nonlinearity. A counterfactual causal mediation analysis examined the roles of basal sex hormones. Stratified analyses by age, BMI, antral follicle count, anti-Müllerian hormone, season, COVID-19 period, and the distance to ART center, along with sensitivity analyses, were performed to evaluate robustness. MAIN RESULTS AND THE ROLE OF CHANCE:Each 1°C increase in apparent temperature was associated with significant decreases in total oocytes retrieved (-0.20%; 95% CI: -0.28%, -0.12%), mature oocytes retrieved (-0.17%; 95% CI: -0.26%, -0.09%), and 2PN fertilized oocytes (-0.19%; 95% CI: -0.28%, -0.09%); consistent associations were observed for ambient temperature (-0.25%, -0.22%, and -0.22% per 1°C, respectively). For rate-based outcomes, no significant associations were observed for the normal fertilization rate or blastocyst formation rate; the mature oocyte rate showed small positive associations in longer-term exposure windows (3-week and 90-day). Mediation analyses indicated that LH partially mediated these associations, accounting for up to 5.23% of the total effect, while both FSH and E2 exhibited suppression effects. LIMITATIONS, REASONS FOR CAUTION:The observational design cannot definitively establish causality. Unmeasured confounding (e.g. individual indoor climate control, occupational heat exposure) and residential address-based exposure assessment are potential sources of bias. Restricting the primary analysis to completed cycles may introduce selection bias, although sensitivity analyses incorporating canceled cycles yielded consistent estimates. WIDER IMPLICATIONS OF THE FINDINGS:Higher ambient temperatures represent a modifiable environmental risk factor for oocyte yield in ART treatment, with basal sex hormone pathways, including LH as a partial mediator and compensatory shifts in FSH and E2, providing a plausible biological basis for this effect. In the context of rising global temperatures, these findings suggest that ambient thermal exposure during the peri-folliculogenesis period warrants clinical attention, and that monitoring basal sex hormone levels may help identify patients at risk of a suboptimal ovarian response in high-temperature settings. FUNDING:This study was supported in part by the Technology Innovation and Research & Development Project of the Chengdu Science and Technology Bureau (No. 2024-YF05-02159-SN), the Open Fund of Chongqing Maternal and Child Disease Control and Public Health Research Center (No. CQFYSJ01001), the Program of Inheritance and Innovation of Traditional Chinese Medicine in Chongqing (Chongqing Traditional Chinese Medicine [2022] No. 33), and Chongqing Yuzhong District Natural Science Foundation Project (No. 20240118). DISCLOSURES:All authors declare that they have no conflicts of interest related to this study. TRIAL REGISTRATION NUMBER:N/A.
STUDY QUESTION:How do the three most widely accessible large language models perform in terms of accuracy, consistency, and reliability when answering clinically relevant questions derived from the 2022 ESHRE guideline on endometriosis? SUMMARY ANSWER:Model A achieved the highest accuracy scores, model C demonstrated significantly superior consistency across repeated queries, and all three models showed comparable but suboptimal reliability under free-tier access, while under official API access median accuracy converged across providers and reliability increased. WHAT IS KNOWN ALREADY:Large language models are increasingly consulted by both clinicians and patients as readily accessible sources of medical information. In reproductive medicine, preliminary evidence suggests that individual platforms may retrieve endometriosis-related content with mixed fidelity. However, no study has simultaneously benchmarked multiple models against a single, internationally recognized endometriosis guideline. The extent to which these tools can be trusted to faithfully reproduce evidence-based recommendations on endometriosis diagnosis and management remains largely unexplored. STUDY DESIGN, SIZE, DURATION:Cross-sectional, multi-platform comparative study. Fifty clinically relevant questions covering the diagnostic and therapeutic domains of the 2022 ESHRE endometriosis guideline were simultaneously submitted to all three models during December 2025. Each question was entered in duplicate using independent sessions to assess response consistency and reliability. Subgroup analyses were carried out by submitting the questions to the API version and to the free-tier version of the platforms available in May 2026. PARTICIPANTS/MATERIALS, SETTING, METHODS:The three models were accessed through their respective free web interfaces using new accounts, without prompt engineering, prior training, or retrieval-augmented generation. A zero-shot prompting approach was adopted. Accuracy was evaluated by two independent experts using the Global Quality Score (GQS); consistency was defined as identical responses across the three iterations; reliability was defined as the alignment of each response with the ESHRE guideline. Discrepancies were settled by a third reviewer. MAIN RESULTS AND THE ROLE OF CHANCE:Significant differences in accuracy were observed across models (Kruskal-Wallis H = 37.10, P < 0.001). Model A achieved the highest median GQS (5, interquartile range [IQR] 4-5), followed by model C (4, IQR 3-5) and model B (3, IQR 3-4). Post-hoc analysis confirmed that model A significantly outperformed model B (P < 0.001) but not model C (P = 0.123), while model C also scored significantly higher than model B (P < 0.001). For consistency, model C demonstrated a significantly higher rate of reproducible responses (92.0%) compared with model A (72.0%, P = 0.028) and model B (68.0%, P = 0.008). No significant between-model differences were found for reliability (χ2 = 1.029, P = 0.598), with rates of 76.0% for model C, 68.0% for model A, and 68.0% for model B. In API and free-tier May 2026 subgroup analyses, median GQS converged to 4 across all three providers, and reliability rose to at least 76% for each provider. LIMITATIONS, REASONS FOR CAUTION:Model outputs may change with subsequent updates. GQS retains a degree of subjectivity despite expert adjudication. The study tested factual recall rather than complex clinical reasoning, limiting generalizability to real-world decision-making scenarios. WIDER IMPLICATIONS OF THE FINDINGS:These findings provide the first multi-platform benchmark of large language models against the latest endometriosis guideline. While models A and C retrieved guideline-concordant information with acceptable fidelity, none of the models achieved a level of reliability required for unsupervised clinical use. The dissociation between accuracy and consistency at the free tier, and its attenuation under API and more recent accesses, indicates that both model capability and commercial tier shape user-facing outputs. The dissociation between accuracy and consistency underscores that a model producing high-quality answers does not necessarily do so in a reproducible manner. Expert oversight remains crucial when interpreting their output regarding endometriosis care. Future research should extend this framework to additional endometriosis guidelines and to longitudinal monitoring of models' performance with their upgrades. FUNDING:No external funding was received for this study. DISCLOSURES:The authors declare no competing interests. TRIAL REGISTRATION NUMBER:N/A.
STUDY QUESTION:What is the incidence of premature ovarian insufficiency (POI) and its associated factors in Ontario, Canada, between 1995 and 2019? SUMMARY ANSWER:The incidence of POI increased after 2011, driven primarily by non-iatrogenic POI, and was highest among women with a history of endometriosis. WHAT IS KNOWN ALREADY:Recent estimates suggest a global POI prevalence of ∼3.7%, with increasing rates over time. POI has heterogeneous etiologies, including genetic, autoimmune, infectious, and iatrogenic causes; however, the cause remains unknown in ∼70% of non-iatrogenic cases. Existing incidence estimates are limited by recall bias, cross-sectional designs, small sample sizes, and limited assessment of associated factors. STUDY DESIGN, SIZE, DURATION:This population-based cohort study included all women aged ≤ 39 years residing in Ontario between 1995 and 2019. Follow-up continued until POI diagnosis, age 40 years, death, loss of provincial health insurance, or end of the study period. PARTICIPANTS/MATERIALS, SETTING, METHODS:POI was defined using administrative health data as a diagnosis occurring before age 40 years identified by either (i) surgical menopause or (ii) ≥1 physician consultation coded as menopause (ICD-9 627). Annual incidence rates (IRs) were calculated using census data, with the Ontario female population aged ≤ 39 years as the denominator. POI was classified as non-iatrogenic, surgical, post-chemotherapy and/or radiotherapy, or combined surgical with chemotherapy and/or radiotherapy. Associations between clinical factors and POI were estimated using age-adjusted modified Poisson regression to derive incidence rate ratios (IRRs). MAIN RESULTS AND THE ROLE OF CHANCE:Among 3 635 702 eligible women, 168 173 developed POI from any etiology (IR 389.4 per 100 000 person-years). Median follow-up was 11 years (IQR 6-18), and median age at POI diagnosis was 31 years (IQR 25-36). IRs were 351.4 per 100 000 person-years for non-iatrogenic POI, 30.2 for surgical POI, 5.5 for post-chemotherapy and/or radiotherapy POI, and 2.3 for combined surgical with chemotherapy and/or radiotherapy POI. Overall POI incidence declined between 2002 and 2008, followed by a sustained increase through 2019, largely attributable to non-iatrogenic POI. The incidence of surgically induced POI decreased from 60 per 100 000 person-years in 1995 to 20 in 2019, while combined surgical with chemotherapy and/or radiotherapy POI increased steadily over the study period. Factors associated with higher POI incidence included endometriosis (IRR 3.45, 95% CI 3.39, 3.52), cancer treated with chemotherapy or radiotherapy (IRR 2.01, 95% CI 1.89, 2.14), drug or tobacco use (IRR 1.59, 95% CI 1.51, 1.67), chromosomal disorders (IRR 1.50, 95% CI 1.42, 1.59), and autoimmune diseases (IRR 1.19, 95% CI 1.17, 1.22). LIMITATIONS, REASONS FOR CAUTION:Use of administrative health data may result in outcome misclassification and limited availability of detailed clinical and demographic information. WIDER IMPLICATIONS OF THE FINDINGS:These findings provide contemporary population-based estimates of POI incidence and identify endometriosis as a major associated factor. The marked decline in surgically induced POI over time suggests increased adoption of ovarian-preserving surgical approaches, while rising non-iatrogenic POI incidence underscores the need for improved risk stratification, counselling, and further studies to elucidate underlying mechanisms and modifiable risk factors. FUNDING:Canadian Menopause Society. DISCLOSURES:Nothing to disclose. TRIAL REGISTRATION NUMBER:N/A.
Infertility affects approximately one in six people globally and demand for ARTs, is expected to rise as parenthood is increasingly delayed. However, ART success remains constrained by gamete and embryo quality, female age, and biological factors beyond chromosomal status alone, highlighting the need for non-invasive methods that can complement current morphology-based and genetic approaches. Because gamete and embryo developmental competence is tightly coupled to cellular metabolism, label-free metabolic imaging has emerged as a promising strategy to assess developmental potential through endogenous autofluorescence of reduced nicotinamide adenine dinucleotide/phosphate [NAD(P)H] and oxidized flavins (FAD), which provide optical proxies of redox balance, mitochondrial activity, and oxidative metabolism. This invited mini-review synthesizes the biochemical basis of NAD(P)H/FAD autofluorescence signals, relates these readouts to the unique metabolic programs of oocytes, embryos across preimplantation development, and sperm, and reviews reproductive studies using fluorescence lifetime imaging microscopy, hyperspectral microscopy, and emerging light-sheet fluorescence microscopy. We discuss practical and interpretive challenges, including modality-dependent signal biases and recent consensus efforts towards standardization. Evidence linking metabolic signatures to reproductive ageing, developmental potential, and embryo ploidy status suggests that metabolic imaging, particularly when paired with artificial intelligence (AI), could enable automated, objective decision support for gamete and embryo selection. Finally, we briefly outline how AI could convert complex metabolic imaging data into clinically interpretable decision-support outputs for embryo ranking and risk stratification. Advances in rapid volumetric imaging, microsystems, and AI may support future ART workflows aimed at improving efficiency, accessibility, and cost-effectiveness.
Stress granules (SGs) are stress-induced ribonucleoprotein condensates assembled around untranslated mRNAs and RNA-binding proteins. G3BP1 is a central regulator of SG formation, yet the molecular events that initiate G3BP1-mediated condensation remain poorly understood. Current models propose that condensation is initiated by RNA-RNA interactions, G3BP1 self-association, or RNA-dependent assembly of G3BP1 into higher-order networks. To define the earliest steps of condensate formation, we employed high-speed atomic force microscopy (HS-AFM) to monitor G3BP1-RNA assembly at the nanometer scale. HS-AFM revealed that G3BP1 first associates with RNA to form discrete nascent assemblies that progressively recruit additional RNA and G3BP1 molecules. These assemblies subsequently grow into higher-order RNA-protein condensates through stepwise assembly. Together, these observations identify RNA-bound G3BP1 assemblies as the initiating structures of condensate formation and provide a framework for understanding the early stages of stress granule assembly.
