A functional cellular framework for sex and estrous cycle-dependent gene expression and behavior.

Sex hormones exert a profound influence on gendered behaviors. How individual sex hormone-responsive neuronal populations regulate diverse sex-typical behaviors is unclear. We performed orthogonal, genetically targeted sequencing of four estrogen receptor 1-expressing (Esr1+) populations and identified 1,415 genes expressed differentially between sexes or estrous states. Unique subsets of these genes were distributed across all 137 transcriptomically defined Esr1+ cell types, including estrous stage-specific ones, that comprise the four populations. We used differentially expressed genes labeling single Esr1+ cell types as entry points to functionally characterize two such cell types, BNSTprTac1/Esr1 and VMHvlCckar/Esr1. We observed that these two cell types, but not the other Esr1+ cell types in these populations, are essential for sex recognition in males and mating in females, respectively. Furthermore, VMHvlCckar/Esr1 cell type projections are distinct from those of other VMHvlEsr1 cell types. Together, projection and functional specialization of dimorphic cell types enables sex hormone-responsive populations to regulate diverse social behaviors.

Cellular heterogeneity and dynamics of the human uterus in healthy premenopausal women.

The human uterus is a complex and dynamic organ whose lining grows, remodels, and regenerates every menstrual cycle or upon tissue damage. Here, we applied single-cell RNA sequencing to profile more the 50,000 uterine cells from both the endometrium and myometrium of five healthy premenopausal individuals, and jointly analyzed the data with a previously published dataset from 15 subjects. The resulting normal uterus cell atlas contains more than 167K cells, representing the lymphatic endothelium, blood endothelium, stromal, ciliated epithelium, unciliated epithelium, and immune cell populations. Focused analyses within each major cell type and comparisons with subtype labels from prior studies allowed us to document supporting evidence, resolve naming conflicts, and propose a consensus annotation system of 39 subtypes. We release their gene expression centroids, differentially expressed genes, and messenger Ribonucleic Acid (mRNA) patterns of literature-based markers as a shared community resource. We identify multiple potential progenitor cells: compartment-wide progenitors for each major cell type and potential cross-lineage multipotent stromal progenitors that may replenish the epithelial, stromal, and endothelial compartments. Furthermore, many cell types and subtypes exhibit shifts in cell number and transcriptomes across different phases of the menstrual cycle. Finally, comparisons between premenopausal, postpartum, and postmenopausal samples revealed substantial alterations in tissue composition, particularly in the proportions of stromal, endothelial, and immune cells. The cell taxonomy and molecular markers we report here are expected to inform studies of both basic biology of uterine function and its disorders.

Electroencephalography findings in menstrually-related mood disorders: A critical review.

The female reproductive years are characterized by fluctuations in ovarian hormones across the menstrual cycle, which have the potential to modulate neurophysiological and behavioral dynamics. Menstrually-related mood disorders (MRMDs) comprise cognitive-affective or somatic symptoms that are thought to be triggered by the rapid fluctuations in ovarian hormones in the luteal phase of the menstrual cycle. MRMDs include premenstrual syndrome (PMS), premenstrual dysphoric disorder (PMDD), and premenstrual exacerbation (PME) of other psychiatric disorders. Electroencephalography (EEG) non-invasively records in vivo synchronous activity from populations of neurons with high temporal resolution. The present overview sought to systematically review the current state of task-related and resting-state EEG investigations on MRMDs. Preliminary evidence indicates lower alpha asymmetry at rest being associated with MRMDs, while one study points to the effect being luteal-phase specific. Moreover, higher luteal spontaneous frontal brain activity (slow/fast wave ratio as measured by the delta/beta power ratio) has been observed in persons with MRMDs, while sleep architecture results point to potential circadian rhythm disturbances. In this review, we discuss the quality of study designs as well as future perspectives and challenges of supplementing the diagnostic and scientific toolbox for MRMDs with EEG.

When fertile, women seek status via prestige but not dominance.

Biological predictors of human dominance are hotly contested, with far-reaching implications for psychological sex differences and the placement of men and women in the social hierarchy. Most investigations have focused on dominance in men and testosterone, with diminished attention paid to dominance in women and other biological mechanisms. Investigating biological influences on other routes to status attainment popular among women-such as via prestige in addition to dominance-have also been neglected. Here, I examined whether status seeking via prestige and via dominance covaried with fertility probability in a citizen science project spanning 14 countries and 4 world regions. Across 4,179 observations, participants tracked their menstrual cycle characteristics, motivation for prestige and dominance, dominance contest outcomes, and three domains of self-esteem. Self-esteem is predicted by status within a group and helps individuals navigate social hierarchies. Bayesian mixed models controlling for menstruation indicated that the motivation to obtain status via prestige but not dominance peaked when conception was most likely, as did dominance contest losses and two self-esteem domains. Fertility appears to reorient female psychology toward prestige-based strategies to success, enhancing women's desire for social capital through influence and admiration but not through fear, coercion, or intimidation. These insights fundamentally advance the understanding of the biological correlates of status seeking among women. They further suggest that fertility motivates not only mating competition but gaining rank and positive regard in social hierarchies.

A scoping review of hormonal clinical trials in menstrual cycle-related brain disorders: Studies in premenstrual mood disorder, menstrual migraine, and catamenial epilepsy.

Cyclic variations in hormones during the normal menstrual cycle underlie multiple central nervous system (CNS)-linked disorders, including premenstrual mood disorder (PMD), menstrual migraine (MM), and catamenial epilepsy (CE). Despite this foundational mechanistic link, these three fields operate independently of each other. In this scoping review (N = 85 studies), we survey existing human research studies in PMD, MM, and CE to outline the exogenous experimental hormone manipulation trials conducted in these fields. We examine a broad range of literature across these disorders in order to summarize existing diagnostic practices and research methods, highlight gaps in the experimental human literature, and elucidate future research opportunities within each field. While no individual treatment or study design can fit every disease, there is immense overlap in study design and established neuroendocrine-based hormone sensitivity among the menstrual cycle-related disorders PMD, MM, and CE. SCOPING REVIEW STRUCTURED SUMMARY: Background. The menstrual cycle can be a biological trigger of symptoms in certain brain disorders, leading to specific, menstrual cycle-linked phenomena such as premenstrual mood disorders (PMD), menstrual migraine (MM), and catamenial epilepsy (CE). Despite the overlap in chronicity and hormonal provocation, these fields have historically operated independently, without any systematic communication about methods or mechanisms. OBJECTIVE: Online databases were used to identify articles published between 1950 and 2021 that studied hormonal manipulations in reproductive-aged females with either PMD, MM, or CE. We selected N = 85 studies that met the following criteria: 1) included a study population of females with natural menstrual cycles (e.g., not perimenopausal, pregnant, or using hormonal medications that were not the primary study variable); 2) involved an exogenous hormone manipulation; 3) involved a repeated measurement across at least two cycle phases as the primary outcome variable. CHARTING METHODS: After exporting online database query results, authors extracted sample size, clinical diagnosis of sample population, study design, experimental hormone manipulation, cyclical outcome measure, and results from each trial. Charting was completed manually, with two authors reviewing each trial. RESULTS: Exogenous hormone manipulations have been tested as treatment options for PMD (N = 56 trials) more frequently than MM (N = 21) or CE (N = 8). Combined oral contraceptive (COC) trials, specifically those containing drospirenone as the progestin, are a well-studied area with promising results for treating both PMDD and MM. We found no trials of COCs in CE. Many trials test ovulation suppression using gonadotropin-releasing hormone agonists (GnRHa), and a meta-analysis supports their efficacy in PMD; GnRHa have been tested in two MM-related trials, and one CE open-label case series. Finally, we found that non-contraceptive hormone manipulations, including but not limited to short-term transdermal estradiol, progesterone supplementation, and progesterone antagonism, have been used across all three disorders. CONCLUSIONS: Research in PMD, MM, and CE commonly have overlapping study design and research methods, and similar effects of some interventions suggest the possibility of overlapping mechanisms contributing to their cyclical symptom presentation. Our scoping review is the first to summarize existing clinical trials in these three brain disorders, specifically focusing on hormonal treatment trials. We find that PMD has a stronger body of literature for ovulation-suppressing COC and GnRHa trials; the field of MM consists of extensive estrogen-based studies; and current consensus in CE focuses on progesterone supplementation during the luteal phase, with limited estrogen manipulations due to concerns about seizure provocation. We argue that researchers in any of these respective disciplines would benefit from greater communication regarding methods for assessment, diagnosis, subtyping, and experimental manipulation. With this scoping review, we hope to increase collaboration and communication among researchers to ultimately improve diagnosis and treatment for menstrual-cycle-linked brain disorders.

The importance of translationally evaluating steroid hormone contributions to substance use.

Clinically, women appear to be more susceptible to certain aspects of substance use disorders (SUDs). The steroid hormones 17ß-estradiol (E2) and progesterone (Pg) have been linked to women-specific drug behaviors. Here, we review clinical and preclinical studies investigating how cycling ovarian hormones affect nicotine-, cocaine-, and opioid-related behaviors. We also highlight gaps in the literature regarding how synthetic steroid hormone use may influence drug-related behaviors. In addition, we explore how E2 and Pg are known to interact in brain reward pathways and provide evidence of how these interactions may influence drug-related behaviors. The synthesis of this review demonstrates the critical need to study women-specific factors that may influence aspects of SUDs, which may play important roles in addiction processes in a sex-specific fashion. It is important to understand factors that impact women's health and may be key to moving the field forward toward more efficacious and individualized treatment strategies.

Effect of menstrual cycle and menopause on human gastric electrophysiology.

Chronic gastroduodenal symptoms disproportionately affect females of childbearing age; however, the effect of menstrual cycling on gastric electrophysiology is poorly defined. To establish the effect of the menstrual cycle on gastric electrophysiology, healthy subjects underwent noninvasive Body Surface Gastric Mapping (BSGM; 8x8 array) with the validated symptom logging App (Gastric Alimetry, New Zealand). Participants included were premenopausal females in follicular (n = 26) and luteal phases (n = 18) and postmenopausal females (n = 30) and males (n = 51) were controls. Principal gastric frequency (PGF), body mass index (BMI) adjusted amplitude, Gastric Alimetry Rhythm Index (GA-RI), Fed:Fasted Amplitude Ratio (ff-AR), meal response curves, and symptom burden were analyzed. Menstrual cycle-related electrophysiological changes were then transferred to an established anatomically accurate computational gastric fluid dynamics model (meal viscosity 0.1 Pas) to predict the impact on gastric mixing and emptying. PGF was significantly higher in the luteal versus follicular phase [mean 3.21 cpm, SD (0.17) vs. 2.94 cpm, SD (0.17), P < 0.001] and versus males [3.01 cpm, SD (0.2), P < 0.001]. In the computational model, this translated to 8.1% higher gastric mixing strength and 5.3% faster gastric emptying for luteal versus follicular phases. Postmenopausal females also exhibited higher PGF than females in the follicular phase [3.10 cpm, SD (0.24) vs. 2.94 cpm, SD (0.17), P = 0.01], and higher BMI-adjusted amplitude [40.7 µV (33.02-52.58) vs. 29.6 µV (26.15-39.65), P < 0.001], GA-RI [0.60 (0.48-0.73) vs. 0.43 (0.30-0.60), P = 0.005], and ff-AR [2.51 (1.79-3.47) vs. 1.48 (1.21-2.17), P = 0.001] than males. There were no differences in symptoms. These results define variations in gastric electrophysiology with regard to human menstrual cycling and menopause.NEW & NOTEWORTHY This study evaluates gastric electrophysiology in relation to the menstrual cycle using a novel noninvasive high-resolution methodology, revealing substantial variations in gastric activity with menstrual cycling and menopause. Gastric slow-wave frequency is significantly higher in the luteal versus follicular menstrual phase. Computational modeling predicts that this difference translates to higher rates of gastric mixing and liquid emptying in the luteal phase, which is consistent with previous experimental data evaluating menstrual cycling effects on gastric emptying.

Menstrual cycle-driven hormone concentrations co-fluctuate with white and gray matter architecture changes across the whole brain.

Cyclic fluctuations in hypothalamic-pituitary-gonadal axis (HPG-axis) hormones exert powerful behavioral, structural, and functional effects through actions on the mammalian central nervous system. Yet, very little is known about how these fluctuations alter the structural nodes and information highways of the human brain. In a study of 30 naturally cycling women, we employed multidimensional diffusion and T1-weighted imaging during three estimated menstrual cycle phases (menses, ovulation, and mid-luteal) to investigate whether HPG-axis hormone concentrations co-fluctuate with alterations in white matter (WM) microstructure, cortical thickness (CT), and brain volume. Across the whole brain, 17ß-estradiol and luteinizing hormone (LH) concentrations were directly proportional to diffusion anisotropy (µFA; 17ß-estradiol: ß1 = 0.145, highest density interval (HDI) = [0.211, 0.4]; LH: ß1 = 0.111, HDI = [0.157, 0.364]), while follicle-stimulating hormone (FSH) was directly proportional to CT (ß1 = 0 .162, HDI = [0.115, 0.678]). Within several individual regions, FSH and progesterone demonstrated opposing relationships with mean diffusivity (Diso) and CT. These regions mainly reside within the temporal and occipital lobes, with functional implications for the limbic and visual systems. Finally, progesterone was associated with increased tissue (ß1 = 0.66, HDI = [0.607, 15.845]) and decreased cerebrospinal fluid (CSF; ß1 = -0.749, HDI = [-11.604, -0.903]) volumes, with total brain volume remaining unchanged. These results are the first to report simultaneous brain-wide changes in human WM microstructure and CT coinciding with menstrual cycle-driven hormone rhythms. Effects were observed in both classically known HPG-axis receptor-dense regions (medial temporal lobe, prefrontal cortex) and in other regions located across frontal, occipital, temporal, and parietal lobes. Our results suggest that HPG-axis hormone fluctuations may have significant structural impacts across the entire brain.

Comparison of assay methods for quantifying sex hormone concentrations across the menstrual cycle in rhesus macaques†.

Immunoassays have been the preferred method for steroid hormone analysis for more than 50 years. Automated immunoassays (AIAs) offer high throughput, rapid data turnaround, and low cost for measuring steroid hormone concentrations. The application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for steroid quantification provides greater specificity and selectivity for individual steroids, the ability to simultaneously analyze multiple steroids, and high throughput and automation. We compared AIA and LC-MS/MS for analysis of 17beta-estradiol (E2) and progesterone (P4) over the course of several menstrual cycles in 12 rhesus macaques (Macaca mulatta). Serum samples were collected every 4 days across four menstrual cycles from each monkey. AIAs were performed on a Roche cobas e411 analyzer. LC-MS/MS analysis was performed on a Shimadzu-Nexera-LCMS-8060 instrument. Scatter plots with Passing-Bablok regression showed excellent agreement between AIA and LC-MS/MS for both E2 and P4. Bland-Altman plots revealed no bias for either method; however, AIA overestimated E2 at concentrations >140 pg/ml and underestimated P4 at concentrations >4 ng/ml compared to LC-MS/MS. A comparison of testosterone concentrations measured by AIA and LC-MS/MS in the same samples was also performed. In contrast to E2 and P4, AIA and LC-MS/MS yielded significantly different results for testosterone concentrations, with AIA consistently underestimating concentrations relative to those obtained by LC-MS/MS. Well-characterized automated immunoassays are an excellent tool for daily monitoring of monkey menstrual cycles or providing single data points requiring fast turnaround. In certain situations where AIAs may provide inaccurate estimations of E2 and P4 concentrations, LC-MS/MS assays are preferable.

Early menstrual cycle impacts of oestrogen and progesterone on the timing of the fertile window.

STUDY QUESTION: What is the effect of oestrogen and progesterone at the beginning of the menstrual cycle in delaying entry into the fertile window? SUMMARY ANSWER: Both oestrogen and progesterone contribute to a delay in the onset of the fertile window. WHAT IS KNOWN ALREADY: Oestrogen enhances cervical mucus secretion while progesterone inhibits it. STUDY DESIGN, SIZE, DURATION: Observational study. Daily observation of 220 menstrual cycles contributed by 88 women with no known menstrual cycle disorder. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women recorded cervical mucus daily and collected first-morning urine samples for analysis of oestrone-3-glucuronide, pregnanediol-3-alpha-glucuronide (PDG), FHS, and LH. They underwent serial ovarian ultrasound examinations. The main outcome measure was the timing within the cycle of the onset of the fertile window, as identified by the appearance of mucus felt or seen at the vulva. MAIN RESULTS AND THE ROLE OF CHANCE: Low oestrogen secretion and persistent progesterone secretion during the first week of the menstrual cycle both negatively affect mucus secretion. Doubling oestrogen approximately doubled the odds of entering the fertile window (OR: 1.82 95% CI=1.23; 2.69). Increasing PDG from below 1.5 to 4 µg/mg creatinine was associated with a 2-fold decrease in the odds of entering the fertile window (OR: 0.51 95% CI=0.31; 0.82). Prolonged progesterone secretion during the first week of the menstrual cycle was also statistically significantly associated with higher LH secretion. Finally, the later onset of the fertile window was associated with statistically significant persistently elevated LH secretion during the luteal phase of the previous menstrual cycle. LIMITATIONS, REASONS FOR CAUTION: This post hoc study was conducted to assess the potential impact of residual progesterone secretion at the beginning of the menstrual cycle. It was conducted on an existing data set because of the scarcity of data available to answer the question. Analysis with other datasets with similar hormone results would be useful to confirm these findings. WIDER IMPLICATIONS OF THE FINDINGS: This study provides evidence for residual progesterone secretion in the early latency phase of some menstrual cycles, which may delay the onset of the fertile window. This progesterone secretion may be supported by subtly increased LH secretion during the few days before and after the onset of menses, which may relate to follicular waves in the luteal phase. Persistent progesterone secretion should be considered in predicting the onset of the fertile window and in assessing ovulatory dysfunction. STUDY FUNDING/COMPETING INTEREST(S): The authors declare no conflicts of interest. No funding was provided for this secondary data analysis. TRIAL REGISTRATION NUMBER: N/A.

Impact of a hypocaloric dietary intervention on antral follicle dynamics in eumenorrheic women with obesity.

STUDY QUESTION: Do antral follicle dynamics change in women with obesity and regular ovulatory cycles after a 6-month hypocaloric dietary intervention? SUMMARY ANSWER: After a 6-month hypocaloric dietary intervention, women with obesity and regular ovulatory cycles displayed evidence of improved antral follicle dynamics defined by the emergence of more dominant follicles, larger ovulatory follicle diameter at selection, and increased luteal progesterone concentrations compared to pre-intervention. WHAT IS KNOWN ALREADY: Precise events in antral folliculogenesis must occur in order for natural and regular monthly ovulation. In healthy women of reproductive age, antral follicles are recruited for growth in a wave-like fashion, wherein a subset of follicles are selected for preferential growth, and typically, one dominant follicle culminates in ovulation. Women with obesity and regular ovulatory cycles display evidence of suppressed antral follicle development, as evidenced by fewer recruitment events, fewer selectable and dominant follicles, smaller diameter of the ovulatory follicle at selection, and a higher prevalence of luteal phase defects. While improvements in gonadotropin and ovarian steroid hormone concentrations after weight loss have been documented in eumenorrheic women with obesity, the precise impact of weight loss on antral follicle dynamics has not been evaluated. STUDY DESIGN, SIZE, DURATION: A pre-post pilot study of 12 women who participated in a 6-month hypocaloric dietary intervention. PARTICIPANTS/MATERIALS, SETTING, METHODS: Twelve women with obesity (total body fat ≥35%) underwent transvaginal ultrasonography and venipuncture every-other-day for one inter-ovulatory interval (IOI) both before (baseline) and during the final month (Month 7) of a six-month hypocaloric dietary intervention. Participants were aged 24-34 years and had a self-reported history of regular menstrual cycles (25-35 days). Follicle number and diameter (≥2 mm) were quantified at each study visit, and individual growth profiles for all follicles ≥7 mm were determined. Blood samples were assayed for reproductive hormones. Follicle dynamics and reproductive hormone concentrations were compared pre- and post-intervention. Further, post-intervention follicle and endocrine dynamics (Month 7 IOI) were compared to an age-matched reference cohort of lean women with regular ovulatory cycles (total body fat <35%, N = 21). MAIN RESULTS AND THE ROLE OF CHANCE: Participants lost an average of 11% of their original body weight with the hypocaloric dietary intervention. More dominant follicles were detected (≥10 mm) at Month 7 compared to baseline (0. 3 ± 0.4 versus 0.4 ± 0.5 follicles, P = 0.001), and ovulatory follicles were selected at larger diameters post-intervention (7.3 ± 2.0 versus 10.9 ± 2.6 mm, P = 0.007). Luteal progesterone concentrations were increased at Month 7 compared to baseline (5.3 ± 3.65 versus 6.3 ± 4.74 ng/ml, P < 0.0001). However, risk for luteal phase dysfunction as judged by the prevalence of a luteal phase length <10 days, integrated luteal progesterone levels <80 ng/ml or peak progesterone <10 ng/ml did not differ pre- versus post-intervention (all, P > 0.05). In Month 7, follicle dynamics and endocrine profiles were similar to the reference cohort across all measures. LIMITATIONS, REASONS FOR CAUTION: This study does not inform on the earliest stages of ovarian follicle development and is limited to providing knowledge on the later stages of antral follicle development. This study cannot fully address causation between weight loss and sustained improvements in antral follicle dynamics. The data cannot be extrapolated to comment on potential improvements in fertility and fecundity with weight loss. The small group sizes limit statistical power. WIDER IMPLICATIONS OF THE FINDINGS: The increasing prevalence of obesity necessitates an understanding of the mechanisms that underlie potential improvements in reproductive health outcomes with weight loss. Women with obesity and regular ovulatory cycles who undertook a 6-month hypocaloric dietary intervention demonstrated improvements consistent with benefits of lifestyle intervention on reproductive health even in those without overt signs of reproductive dysfunction. Potential improvements in the cellular makeup of follicles, which may underlie the restoration of normal follicle development and amelioration of subfertility, require further investigation. STUDY FUNDING/COMPETING INTEREST(S): Cornell University, President's Council of Cornell Women, United States Department of Agriculture (Grant No. 8106), and National Institutes of Health (R01-HD0937848). B.Y.J. and H.V.B. were supported by doctoral training awards from the National Institutes of Health (T32-DK007158) and Canadian Institutes of Health Research (Grant No. 146182), respectively. The authors have no competing interests. TRIAL REGISTRATION NUMBER: NCT01927432 and NCT01785719.

Adolescents diagnosed with polycystic ovary syndrome under the Rotterdam criteria but not meeting the diagnosis under the updated guideline.

STUDY QUESTION: What are the characteristics of adolescents diagnosed with polycystic ovary syndrome (PCOS) based on the 2003 Rotterdam criteria, but who do not meet the diagnosis according to the international evidence-based guideline? SUMMARY ANSWER: Adolescents who had features of PCOS but did not meet the evidence-based guideline adolescent criteria exhibited unfavorable metabolic profiles compared to controls and shared considerable metabolic and hormonal features with adolescents who did meet the adolescent criteria. WHAT IS KNOWN ALREADY: The international evidence-based PCOS guideline recommended that ultrasound should not be used for the diagnosis of PCOS in girls with a gynecological age of <8 years. Thus far, few studies have evaluated the clinical characteristics of the girls diagnosed with PCOS based on the Rotterdam criteria but who do not meet the diagnosis according to the updated guideline. STUDY DESIGN, SIZE, DURATION: This is a retrospective study, and subjects attended for care from 2004 to 2022. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adolescent girls with PCOS diagnosed according to the 2003 Rotterdam criteria and healthy controls. All participants were between 2 and 8 years since menarche. MAIN RESULTS AND THE ROLE OF CHANCE: Of the 315 girls diagnosed with PCOS according to the Rotterdam criteria, those with irregular menstruation (IM)/hyperandrogenism (HA)/polycystic ovary (PCO), IM/HA, HA/PCO, and IM/PCO phenotypes accounted for 206 (65.4%), 30 (9.5%), 12 (3.8%), and 67 (21.3%) participants, respectively. According to the evidence-based guideline, 79 girls (25.1%) with the HA/PCO or IM/PCO phenotypes were not diagnosed with PCOS, and aligned to the international guideline; they were designated as the 'at-risk' group. As expected, the girls meeting the evidence-based guideline adolescent criteria showed the worst metabolic profiles (degree of generalized or central obesity, frequency of insulin resistance, prediabetes or diabetes, and metabolic syndrome) and higher hirsutism scores than the at-risk group or controls. Approximately 90% of the at-risk group were not overweight or obese, which was similar to the controls. However, they showed worse metabolic profiles, with higher blood pressure, triglyceride, and insulin resistance parameters than controls; furthermore, these profiles were similar to those of the girls meeting the adolescent criteria. The at-risk group showed similarly elevated serum LH levels and LH/FSH ratio with the girls meeting adolescent criteria. LIMITATIONS, REASONS FOR CAUTION: We could not evaluate hormonal or ultrasound parameters in controls. WIDER IMPLICATIONS OF THE FINDINGS: Compared to the conventional Rotterdam criteria, the recent international evidence-based guideline-avoiding ultrasound in PCOS diagnosis in adolescents-still gives the opportunity to identify young girls at risk, aligned to the findings in this study. A practical approach to this adolescent population would involve establishing IM or HA (with ultrasound not indicated) and designating 'at-risk' PCOS status with regular check-ups for newly developed or worsening PCOS-related symptoms or metabolic abnormalities, with subsequent reassessment including ultrasound or anti-Müllerian hormone, once 8 years post-menarche. STUDY FUNDING/COMPETING INTEREST(S): No funding was received in support of this study. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Deciphering the role of PGRMC2 in the human endometrium during the menstrual cycle and in vitro decidualization using an in vitro approach.

STUDY QUESTION: What is the human endometrial non-classical progesterone receptor (PGR) membrane component 2 (PGRMC2) expression pattern throughout the menstrual cycle and what role does it play during decidualization? SUMMARY ANSWER: Endometrial PGRMC2 expression fluctuates during the human menstrual cycle and is abundantly expressed in human endometrial stromal cells (hEnSCs) during in vitro decidualization, process where PGRMC2 is involved in embryo implantation-related pathways. WHAT IS KNOWN ALREADY: The endometrial response to progesterone is mediated by the classical and non-classical PGRs. We previously demonstrated that PGR membrane component 1 (PGRMC1) is critical for endometrial function, embryo implantation, and future placentation, however, the role(s) of PGRMC2, which is structurally similar to PGRMC1, have not been studied in the human endometrium. STUDY DESIGN, SIZE, DURATION: This prospective study comprehensively evaluated the endometrial expression of PGRMC2 throughout the human menstrual cycle and during in vitro decidualization of hEnSCs (isolated from 77 endometrial biopsies that were collected from 66 oocyte donors), using immunohistochemistry, RT-qPCR, western blot, transcriptomic, and proteomic analyses. In addition, functional analysis was carried out to validate the implication of PGRMC2 in hEnSCs during embryo invasion using an in vitro outgrowth model. PARTICIPANTS/MATERIALS, SETTING, METHODS: In vitro decidualization of hEnSCs was induced using co-treatment with cAMP and medroxyprogesterone 17-acetate progestin, and evaluated by measuring prolactin by ELISA and F-actin immunostaining. RT-qPCR was employed to compare expression with other PGRs. To reveal the function of PGRMC2 during the decidualization process, we specifically knocked down PGRMC2 with siRNAs and performed RNA-seq and quantitative proteomics techniques (SWATH-MS). The common differentially expressed genes (DEGs) and proteins (DEPs) were considered for downstream functional enrichment analysis. Finally, to verify its implication in the trophoblast invasion, an outgrowth model was carried out where hEnSCs with silenced PGRMC2 were co-cultured with human trophoblastic spheroids (JEG-3) following in vitro decidualization. MAIN RESULTS AND THE ROLE OF CHANCE: In contrast to PGRMC1 and classical PGRs, endometrial PGRMC2 gene expression was significantly lower during the late- versus mid-secretory phase (P < 0.05). Accordingly, the elevated PGRMC2 protein abundance observed in the endometrial epithelial glands throughout the menstrual cycle dropped in the late secretory phase, when abundance decreased in all endometrial compartments. Nevertheless, PGRMC2 protein increased during the mid-secretory phase in stromal and glandular cells, and PGRMC2 mRNA (P < 0.0001) and protein (P < 0.001) levels were significantly enhanced in the membranes/organelles of decidualized hEnSCs, compared to non-decidualized hEnSCs. Notably, PGRMC1 and PGRMC2 mRNA were significantly more abundant than classical PGRs throughout menstrual cycle phases and in decidualized and non-decidualized hEnSCs (P < 0.05). RNA-seq and proteomics data revealed 4687 DEGs and 28 DEPs, respectively, in decidualized hEnSCs after PGRMC2 silencing. While functional enrichment analysis showed that the 2420 upregulated genes were mainly associated with endoplasmic reticulum function, vesicular transport, morphogenesis, angiogenesis, cell migration, and cell adhesion, the 2267 downregulated genes were associated with aerobic respiration and protein biosynthesis. The protein enrichment analysis showed that 4 upregulated and 24 downregulated proteins were related to aerobic respiration, cellular response, metabolism, localization of endoplasmic reticulum proteins, and ribonucleoside biosynthesis routes. Finally, PGRMC2 knockdown significantly compromised the ability of the decidualized hEnSCs to support trophoblast expansion in an outgrowth model (P < 0.05). LARGE-SCALE DATA: Transcriptomic data are available via NCBI's Gene Expression Omnibus (GEO) under GEO Series accession number GSE251843 and proteomic data via ProteomeXchange with identifier PXD048494. LIMITATIONS, REASONS FOR CAUTION: The functional analyses were limited by the discrete number of human endometrial biopsies. A larger sample size is required to further investigate the potential role(s) of PGRMC2 during embryo implantation and maintenance of pregnancy. Further, the results obtained in the present work should be taken with caution, as the use of a pure primary endometrial stromal population differentiated in vitro does not fully represent the heterogeneity of the endometrium in vivo, nor the paracrine communications occurring between the distinct endometrial cell types. WIDER IMPLICATIONS OF THE FINDINGS: The repression of endometrial PGRMC2 during the late- versus mid-secretory phase, together with its overexpression during decidualization and multiple implications with embryo implantation not only highlighted the unknown roles of PGRMC2 in female reproduction but also the potential to exploit PGRMC2 signaling pathways to improve assisted reproduction treatments in the future. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by Instituto de Salud Carlos III (ISCIII) granted to F.D. (PI20/00405 and PI23/00860), co-funded by the European Union. Y.M.-L. was supported by a predoctoral research grant from Generalitat Valenciana (ACIF/2019/262). R.G.-M. was supported by Generalitat Valenciana (CIAPOT/2022/15). P.d.C. was supported by a predoctoral grant for training in research into health (PFIS FI20/00086) from the Instituto de Salud Carlos III. I.D.-H. was supported by the Spanish Ministry of Science, Innovation and Universities (FPU18/01550). A.P. was supported by the Instituto de Salud Carlos III (PFIS FI18/00009). This research was also supported by IVI Foundation-RMA Global (1911-FIVI-103-FD). The authors declare no conflict of interest.

Prospective 1-year assessment of within-woman variability of follicular and luteal phase lengths in healthy women prescreened to have normal menstrual cycle and luteal phase lengths.

STUDY QUESTION: What is the relative length variance of the luteal phase compared to the follicular phase within healthy, non-smoking, normal-weight, proven normally ovulatory, premenopausal women with normal-length menstrual cycles? SUMMARY ANSWER: Prospective 1-year data from 53 premenopausal women with two proven normal-length (21-36 days) and normally ovulatory (≥10 days luteal) menstrual cycles upon enrollment showed that, despite 29% of all cycles having incident ovulatory disturbances, within-woman follicular phase length variances were significantly greater than luteal phase length variances. WHAT IS KNOWN ALREADY: Many studies report menstrual cycle variability, yet few describe variability in follicular and luteal phase lengths. Luteal lengths are assumed 'fixed' at 13-14 days. Most studies have described follicular and luteal phase variability between-women. STUDY DESIGN, SIZE, DURATION: This study was a prospective, 1-year, observational cohort study of relative follicular and luteal phase variability both between and within community-dwelling women with two documented normal-length (21-36 days) and normally ovulatory (≥10 days luteal phase) menstrual cycles prior to enrollment. Eighty-one women enrolled in the study and 66 women completed the 1-year study. This study analyzed data from 53 women with complete data for ≥8 cycles (mean 13). PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were healthy, non-smoking, of normal BMI, ages 21-41 with two documented normal-length (21-36 days) and normally ovulatory (≥10 days luteal phase) menstrual cycles prior to enrollment. Participants recorded first morning temperature, exercise durations, and menstrual cycle/life experiences daily in the Menstrual Cycle Diary. We analyzed 694 cycles utilizing a twice-validated least-squares Quantitative Basal Temperature method to determine follicular and luteal phase lengths. Statistical analysis compared relative follicular and luteal phase variance in ovulatory cycles both between-women and within-woman. Normal-length cycles with short luteal phases or anovulation were considered to have subclinical ovulatory disturbances (SOD). MAIN RESULTS AND THE ROLE OF CHANCE: The 1-year overall 53-woman, 676 ovulatory cycle variances for menstrual cycle, follicular, and luteal phase lengths were 10.3, 11.2, and 4.3 days, respectively. Median variances within-woman for cycle, follicular, and luteal lengths were 3.1, 5.2, and 3.0 days, respectively. Menstrual cycles were largely of normal lengths (98%) with an important prevalence of SOD: 55% of women experienced >1 short luteal phase (<10 days) and 17% experienced at least one anovulatory cycle. Within-woman follicular phase length variances were greater than luteal phase length variances (P < 0.001). However, follicular (P = 0.008) and luteal phase length (P = 0.001) variances, without differences in cycle lengths, were greater in women experiencing any anovulatory cycles (n = 8) than in women with entirely normally ovulatory cycles (n = 6). LIMITATIONS, REASONS FOR CAUTION: Limitations of this study include the relatively small cohort, that most women were White, initially had a normal BMI, and the original cohort required two normal-length and normally ovulatory menstrual cycles before enrollment. Thus, this cohort's data underestimated population menstrual cycle phase variances and the prevalence of SOD. WIDER IMPLICATIONS OF THE FINDINGS: Our results reinforce previous findings that the follicular phase is more variable than the luteal phase in premenopausal women with normal-length and ovulatory menstrual cycles. However, our study adds to the growing body of evidence that the luteal phase is not predictably 13-14 days long. STUDY FUNDING/COMPETING INTEREST(S): This medical education project of the University of British Columbia was funded by donations to the Centre for Menstrual Cycle and Ovulation Research. The authors do not have any conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Blunted Cortisol Response to Acute Psychosocial Stress in Women With Premenstrual Dysphoric Disorder.

BACKGROUND: Despite being considered a stress-related condition, it is not known whether the hypothalamic-pituitary-adrenal (HPA) axis is dysfunctional in response to acute psychosocial stress in premenstrual dysphoric disorder (PMDD). This is problematic because many women with PMDD report that they are not able to control their stress levels, and a blunted cortisol output has been identified in women with related psychiatric conditions, such as anxiety and depression. The present study is a part of the Premenstrual Hormonal and Affective State Evaluation (PHASE) project, and it aimed to characterize the cortisol trajectory in response to an acute psychosocial stress challenge. METHODS: Women with PMDD and healthy controls with confirmed ovulatory cycles underwent the Trier Social Stress Test (TSST) procedure in the mid-late luteal phase of the menstrual cycle, throughout which we collected serum samples of cortisol that we analyzed using ultra-performance liquid chromatography tandem mass spectrometry. RESULTS: The linear mixed model analysis indicated a significant time*diagnosis interaction (P = .008) such that women with PMDD displayed significantly lower serum cortisol levels at +40 through +90 minutes from the time of stress induction. CONCLUSION: This is the first study to show that women with PMDD have a blunted cortisol response to psychosocial stress. Combined with our earlier finding showing a greater parasympathetic nervous system withdrawal on heart oscillations in PMDD during acute stress, these and other results show that the dysregulated processing of stress in PMDD may be captured using objective study measures.

Cumulative stressor exposure predicts menstrual cycle affective changes in a transdiagnostic outpatient sample with past-month suicidal ideation.

BACKGROUND: Affective responses to the menstrual cycle vary widely. Some individuals experience severe symptoms like those with premenstrual dysphoric disorder, while others have minimal changes. The reasons for these differences are unclear, but prior studies suggest stressor exposure may play a role. However, research in at-risk psychiatric samples is lacking. METHODS: In a large clinical sample, we conducted a prospective study of how lifetime stressors relate to degree of affective change across the cycle. 114 outpatients with past-month suicidal ideation (SI) provided daily ratings (n = 6187) of negative affect and SI across 1-3 menstrual cycles. Participants completed the Stress and Adversity Inventory (STRAIN), which measures different stressor exposures (i.e. interpersonal loss, physical danger) throughout the life course, including before and after menarche. Multilevel polynomial growth models tested the relationship between menstrual cycle time and symptoms, moderated by stressor exposure. RESULTS: Greater lifetime stressor exposure predicted a more pronounced perimenstrual increase in active SI, along with marginally significant similar patterns for negative affect and passive SI. Additionally, pre-menarche stressors significantly increased the cyclicity of active SI compared to post-menarche stressors. Exposure to more interpersonal loss stressors predicted greater perimenstrual symptom change of negative affect, passive SI and active SI. Exploratory item-level analyses showed that lifetime stressors moderated a more severe perimenstrual symptom trajectory for mood swings, anger/irritability, rejection sensitivity, and interpersonal conflict. CONCLUSION: These findings suggest that greater lifetime stressor exposure may lead to heightened emotional reactivity to ovarian hormone fluctuations, elevating the risk of psychopathology.

Female sex steroids and epilepsy: Part 1. A review of reciprocal changes in reproductive systems, cycles, and seizures.

Seizures, antiseizure medications, and the reproductive systems are reciprocally entwined. In Section 2 of this review, we outline how seizures may affect the hypothalamic-pituitary-gonadal axis, thereby altering sex steroids, and changes in sex steroids across the menstrual cycle and changes in pharmacokinetics during pregnancy may alter seizure susceptibility. The literature indicates that females with epilepsy experience increased rates of menstrual disturbances and reproductive endocrine disorders. The latter include polycystic ovary syndrome, especially for females on valproate. Studies of fertility have yielded mixed results. We aim to summarize and attempt to detangle the existing knowledge on these reciprocal interactions. The menstrual cycle causes changes in seizure intensity and frequency for many females. When this occurs perimenstrually, during ovulation, or in association with an inadequate luteal phase, it is termed catamenial epilepsy. There is a clear biophysiological rationale for how the key female reproductive neurosteroids interact with the brain to alter the seizure threshold, and Section 3 outlines this important relationship. Critically, what remains unknown is the specific pathophysiology of catamenial epilepsy that describes why not all females are affected. There is a need for mechanism-focused investigations in humans to uncover the complexity of the relationship between reproductive hormones, menstrual cycles, and the brain.

Female sex steroids and epilepsy: Part 2. A practical and human focus on catamenial epilepsy.

Catamenial epilepsy is the best described and most researched sex steroid-specific seizure exacerbation. Yet despite this there are no current evidence-based treatments, nor an accepted diagnostic tool. The best tool we currently have is tracking seizures over menstrual cycles; however, the reality of tracking seizures and menstrual cycles is fraught with challenges. In Part 1 of this two-part review, we outlined the often complex and reciprocal relationship between seizures and sex steroids. An adaptable means of tracking is required. In this review, we outline the extent and limitations of current knowledge on catamenial epilepsy. We use sample data to show how seizure exacerbations can be tracked in short/long and even irregular menstrual cycles. We describe how seizure severity, an often overlooked and underresearched form of catamenial seizure exacerbation, can also be tracked. Finally, given the lack of treatment options for females profoundly affected by catamenial epilepsy, Section 3 focuses on current methods and models for researching sex steroids and seizures as well as limitations and future directions. To permit more informative, mechanism-focused research in humans, the need for both a consistent classification of catamenial epilepsy and an objective biomarker is highlighted.

Attention-deficit/hyperactivity disorder and the menstrual cycle: Theory and evidence.

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder that exhibits striking sex differences in symptoms, prevalence, and associated problems across development. Etiological factors and mechanisms underlying these sex differences remain one of the most understudied aspects of this disorder. The current paper seeks to provide a novel theoretical framework for understanding this phenomenon by reviewing evidence that females with ADHD may experience a "double whammy" of organizational and activational pubertal hormonal effects. We propose a novel theory of activational effects of cyclical circulating ovarian hormones on ADHD with increasing risk at times of rapid declines in estrogen. These declines may decrease executive function and trait control at two points of the cycle characterized by biphasic affective risk: (1) increases in approach/risk-taking behaviors at mid-cycle (periovulatory) and (2) increases in avoidance/negative affect perimenstrually. Low estrogen and control may then interact with increases in positive and negative affect, respectively, to increase hyperactivity-impulsivity symptoms post-ovulation and inattention symptoms perimenstrually. These interactions may be exacerbated by organizational pubertal effects on relatively overdeveloped limbic circuitry and adolescent-specific social pressures magnified in females with ADHD.

Mood symptoms and gut function across the menstrual cycle in individuals with premenstrual syndrome.

Gastrointestinal (GI) symptoms such as bloating, constipation, and nausea are common in the days before menstruation, experienced by as many as 73 % of menstruating individuals. Mood may influence the link between menstrual cycle and GI symptoms, with prior studies indicating that even among healthy controls, GI symptoms worsen premenstrually and are associated with worsening mood. Associations between GI symptoms and mood are poorly understood among those with premenstrual syndrome (PMS), a cluster of mood and/or physical symptoms that occur in the week before menses affecting roughly 20 % of menstruators. Our primary aim was to examine associations between GI symptoms and mood symptoms across the menstrual cycle, in those who do and do not report PMS using a menstrual tracking app. We hypothesized that GI symptoms would be reported more frequently in the luteal phase than follicular phase, and that frequency of GI symptoms would be positively associated with mood symptoms in those with PMS. We analyzed data from 33,628 menstrual cycles across 32,241 participants, including n = 27,897 controls (29,137 menstrual cycles) and n = 4344 PMS participants (4491 menstrual cycles). GI symptoms were reported significantly more frequently in the luteal phase than the follicular phase in both control and PMS groups (p < 0.001). Mood symptoms were significantly positively associated with GI symptoms in both groups, in both follicular and luteal phases (p < 0.001). Results suggest that premenstrual GI symptoms are a common issue, and additional work is needed to explore associations between mood and GI symptoms in the context of the menstrual cycle.