Biology of Reproduction

Embryo culture, a required step during in vitro fertilization (IVF), exposes developing embryos to altered environmental conditions not normally experienced in vivo, including altered oxygen (O2) tension. Importantly, O2 influences gene expression, metabolism, and the activity of enzymes that sculpt the epigenetic landscape. The lowest O2 tension currently used in clinics during embryo culture is 5%, despite evidence that sections of the mammalian female reproductive tract have O2 levels as low at 2%. Lower O2 may therefore better mimic the in vivo environment and thus lead to improved pre- and postnatal outcomes in IVF-conceived offspring. Using our validated IVF mouse model, we show embryo culture at 2% O2 compared to culture under 5% O2 significantly improves embryo cell number, the chromatin landscape in preimplantation embryos, fetal and placental development during gestation, and metabolic function in adulthood. We further uncover mechanisms by which culture under ultra-low O2 mediates these improvements. Overall, these results suggest embryo culture with 2% O2 ameliorates adverse outcomes after IVF and provide evidence that IVF could be further improved by adjusting culture conditions to model the in vivo environment.

BackgroundPremature ovarian insufficiency (POI) affects approximately 1% of women under 40 and is characterized by elevated levels of gonadotropins, reduced estradiol, impaired folliculogenesis, and infertility. Bone marrow-derived mesenchymal stem cell (BM-MSC)-based therapy has emerged as a promising regenerative strategy in preclinical POI models. This systematic review and meta-analysis evaluated BM-MSC-based interventions, including cell transplantation and secretome/extracellular vesicle administration, in animal models of POI. MethodsA systematic review and meta-analysis was conducted following PRISMA guidelines. PubMed, Web of Science, Scopus, ScienceDirect, and the Cochrane Library were searched from inception to February 19, 2025. Preclinical studies assessing BM-MSC-based interventions in animal models of POI were included. ResultsThirty-four studies comprising 1,357 animals were included. Compared with controls, BM-MSC-based therapy increased serum estradiol (standardized mean difference [SMD] 3.11; 95% confidence interval [CI] 2.38-3.84) and anti-Mullerian hormone (SMD 1.86; 95% CI 1.03-2.69), while reducing follicle-stimulating hormone (SMD -3.54; 95% CI -4.37 to -2.71) and luteinizing hormone (SMD -3.44; 95% CI -5.17 to -1.70). Follicular counts increased across developmental stages, with fewer atretic follicles. Reproductive outcomes improved, including normal estrous cycles (risk ratio [RR] 7.80; 95% CI 3.15-19.34), pregnancy occurrence (RR 3.72; 95% CI 2.14-6.44), and offspring number (SMD 1.57; 95% CI 1.04-2.09). ConclusionBM-MSC-based therapy consistently improved hormonal, follicular, and reproductive outcomes in preclinical POI models. More well-designed, standardized, and adequately controlled studies to confirm these findings are warranted. Systematic review registration: CRD42023449053

Study questionHow does cryptorchidism affect germ cell development and UTF-1-mediated pluripotency potential at the time of orchiopexy? Summary answerCryptorchidism was associated with the following germ cell abnormalities: germ cell clustering with many cords/tubules lacking germ cells and reduced UTF-1 expression, suggesting limited germ cell differentiation into spermatogonia What is known alreadyCryptorchidism, affecting 1.6-9% of male newborns, is known to increase the risk of infertility and testicular cancer due to abnormal germ cell development. Germ cells and pluripotent stem cell gene, UTF-1, play critical roles in spermatogonia differentiation, self-renewal, and spermatogenesis. No prior study has evaluated the testicular development by immunohistochemically mapping of these cell populations, Study design, size, durationA cross-sectional study of 31 postnatal cryptorchid testis and 5 age-matched scrotal testicular biopsies obtained from UCSFs pathology department performed between 1993-2023. Participants/materials, setting, methodsSpecimens were grouped by age at surgery (6-18 months, 19 months-7 years, 8-12 years, and [≥]13 years) and testis location (palpable vs. non-palpable). Scrotal prepubertal testis biopsies were sourced through the Pedi-LIFE program, a fertility preservation research biobank, with at least one control per age group. Immunohistochemistry was performed to stain specimens for germ cell (DDX4, OCT4, TSPY), pluripotent cell marker (UTF-1), as well as other key testis cell markers (A-actin, AR, P450, Sox-9), with staining graded based on control expression levels. The number of germ cells per seminiferous tubule was quantified and compared across anatomical locations using appropriate statistical analyses. Main results and the role of chanceThis study included 36 specimens, comprising 31 cryptorchid testes (86%) and 5 scrotal control testes (16%). The cryptorchid group exhibited testicular dysgenesis and reduced germ cell expression, correlated with increased age and testis location. Qualitative assessment revealed reduced germ cell expression across all ages in cryptorchid testes. The number of germ cells per tubule was markedly reduced in cryptorchid compared with scrotal testes after 19 months of age for DDX4, TSPY, and UTF-1. Germ cell clusters were identified in 15 out of 31 cryptorchid specimens (48%) stained for DDX4 and TSPY. UTF-1 expression was lower in cryptorchid testes across all age groups. No significant differences were noted in other testicular cell markers. Large scale dataNA Limitations, reasons for cautionFirst, the power and generalizability of the study are limited by the availability of specimens within each age group, particularly for scrotal testes, as biopsies of these tissues are not routinely performed. Second, a cross-sectional study design limits a longitudinal comparison to evaluate changes in marker expression, delayed maturation, or irreversible germ cell loss. Third, immunohistochemistry data is semi-quantitative, and protein detection is affected by antibody sensitivity and tissue preservation and influenced by antibody sensitivity. Lastly, scrotal testis used as controls were obtained from cryopreserved tissue from patients with other unrelated pathology, which may influence histological profiles. Wider implications of the findingsCollectively, our findings support a model in which cryptorchidism involves both germ cell depletion and disrupted SSC lineage formation, with UTF-1 downregulation and germ cell clustering as early signatures of testicular dysgenesis. These features may help identify high-risk patients for worsening gonadal dysgenesis and infertility and can provide a rationale for earlier orchiopexy or SSC-preserving strategies. Study funding/competing interest(s)The authors declare no conflicts of interest and received no funding for this study. Data Availability StatementThe data underlying this article cannot be shared publicly due to ethical and legal restrictions related to the use of human tissue specimens, which may compromise donor privacy and confidentiality. Data are available from the corresponding author upon reasonable request and subject to institutional and ethical approvals.

Aberrant changes in circulating cell-free mitochondrial DNA (ccf-mtDNA) across gestation are associated with adverse pregnancy outcomes. Given the inflammatory properties of ccf-mtDNA via pattern recognition receptors such as Toll-like receptor 9 (TLR9), we hypothesized that extracellular mtDNA induces placental inflammation via TLR9 signaling and that this response differs by fetal sex. Pregnant Sprague-Dawley rats were treated intravenously with purified mtDNA (300 g/kg), nuclear DNA (nDNA), saline, and/or the TLR9 antagonist ODN2088 across five studies. Placental responses were evaluated 4 h (Studies 1-3) and 24 h (Study 4) post-treatment; pregnancy and neonatal outcomes were assessed at delivery (Study 5). Exposure to mtDNA, but not nDNA, increased placental il1{beta}, tnf, and il10 mRNA (p < 0.05), establishing response specificity. mtDNA-induced placental inflammation was fetal sex-dependent: mtDNA increased il6 and il1{beta} mRNA in male placentas (p [&le;] 0.0004) but not female placentas, whereas ifn{gamma} was selectively induced in female placentas (p = 0.0004). TLR9 and MyD88 abundance increased in female but not male placentas, and TLR9 antagonism modified selected inflammatory responses with sex-specific patterns. The 4 h inflammatory transcriptional signature resolved by 24 h, whereas mtDNA exposure was associated with a sex-specific shift in antioxidant enzyme expression persisting to 24 h. Despite no effects on gestational length or neonatal biometrics, mtDNA exposure was associated with a higher estimated stillbirth count per litter (IRR = 4.23, 95% CI [0.89, 20.1], p = 0.069). These findings establish extracellular mtDNA as an acute, sex-differentiated placental inflammatory stimulus with partial TLR9 dependence and a potential impact on fetal viability. New & NoteworthyThis study demonstrates that acute exposure to extracellular mtDNA induces placental inflammatory responses in vivo. This response is specific to mtDNA, fetal-sex dependent, and partially mediated by TLR9, with male and female placentas engaging distinct inflammatory signals within hours of exposure. The biological effects extend beyond the initial inflammatory window, with mtDNA exposure producing lasting, sex-specific changes in antioxidant enzyme expression. mtDNA-exposed dams had higher expected stillbirth counts, suggesting extracellular mtDNA may affect fetal viability.

Objective To evaluate adherence to published American Society for Reproductive Medicine (ASRM) infertility evaluation and treatment recommendations among commercially insured infertility patients who subsequently underwent in vitro fertilization (IVF) and to assess whether observed care gaps support the need for a restorative reproductive medical framework. Methods A retrospective claims-based analysis was performed using MarketScan(R) Commercial Claims and Encounter Data between January 1, 2021, and December 31, 2024. Approximately five million commercially insured members were evaluated. Patients with infertility-related diagnoses who subsequently underwent IVF were identified. Claims were analyzed for evidence of diagnostic testing, medical treatment, or surgical intervention recommended by ASRM or AUA/ASRM guidance before IVF initiation. Cumulative adherence rates were assessed over nine months following initial infertility diagnosis. Results IVF initiation rose early and consistently exceeded completion of nearly all guideline-recommended evaluations and treatments. Observed care gaps ranged from approximately 13% to 78% for most recommended evaluations and treatments, with several measures demonstrating gaps exceeding 50 percentage points, suggesting substantial divergence between guideline recommendations and observed clinical practice. By 3 months, IVF initiation ranged from 28% to 39% across cohorts, while adherence to many recommended interventions remained low. Overall, by 9 months, IVF utilization commonly exceeded 70-85%, while many guideline-supported evaluations and treatments remained below 40% adherence, with several interventions remaining below 15%. These findings suggest substantial divergence between published infertility-care recommendations and observed pre-IVF practice patterns. From an RRM perspective, the gaps are clinically important because many recommended steps are directed toward identifying, correcting, restoring, or preserving reproductive function and anatomy before reproductive barriers are bypassed through IVF. Conclusions Many commercially insured infertility patients appeared to progress to IVF without documented evidence of diagnostic evaluation or therapeutic intervention recommended in ASRM and AUA/ASRM guidance. These findings raise important questions regarding the implementation of infertility guidelines before IVF and the extent to which patients receive meaningful opportunities for diagnosis-directed treatment of potentially reversible causes of infertility. The findings further suggest an important role for restorative reproductive medicine as a quality-of-care framework focused on comprehensive evaluation, correction of underlying dysfunction, preservation of reproductive anatomy and physiology, and optimization of patient-centered fertility care prior to attempts with assisted reproduction.

Cryopreservation of ram semen induces structural and functional alterations that compromise sperm fertility. Since seminal plasma contributes to the regulation and preservation of sperm function, increasing attention has been directed toward seminal plasma extracellular vesicles (EVs) that are involved in sperm physiology. EVs act as carriers of proteins that are involved in sperm membrane organization and capacitation, suggesting that they may contribute to the maintenance of sperm stability during cryopreservation.. Thus, the aim of this study was to evaluate the effect of seminal plasma-derived EVs on post-thaw functional parameters of ram sperm. Semen was cryopreserved in the presence or absence of EVs isolated by ultracentrifugation that have been characterized by nanoparticle tracking analysis (NTA) and Western blotting (WB). Post-thaw sperm quality was assessed by evaluating viability, membrane lipid disorder, reactive oxygen species production, protein phosphorylation, acrosome status, intracellular calcium levels, and sperm motility. Sperm cryopreserved with an extender containing EVs showed a significant reduction in membrane lipid disorder and lower intracellular calcium levels compared to control samples (p < 0.05). CASA analysis revealed that EV supplementation did not affect total or progressive motility but modified sperm kinematic patterns, with increased linearity and straightness, indicating improved trajectory efficiency without induction of hyperactivated motility. No differences were detected in viability, ROS content, phosphorylation of proteins in residuous tyrosine (pY) or PKA or acrosome status. These results provide the first evidence that seminal plasma derived extracellular vesicles exert a protective effect during ram semen cryopreservation, preserving membrane organization and calcium homeostasis and improving sperm functional quality after thawing. Highlights- Seminal EVs protect ram sperm during cryopreservation. - EVs reduce membrane lipid disorder and intracellular Ca2+ levels. - EVs modify kinematics, increasing linearity and straightness. - No effects on viability, ROS, phosphorylation or acrosome status. - EVs improve post-thaw sperm functional quality and stability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/732841v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@d1f8a9org.highwire.dtl.DTLVardef@11c3d6aorg.highwire.dtl.DTLVardef@104124forg.highwire.dtl.DTLVardef@4e355f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Oocytes arrested at the dictyate stage of meiosis I must maintain genomic integrity for prolonged periods to preserve female fertility. During this extended arrest, DNA lesions arising from endogenous and exogenous sources threaten oocyte survival, yet the molecular mechanisms coordinating DNA repair in dormant oocytes remain poorly understood. Here, we identify cyclin-dependent kinase 1 (CDK1) as a critical regulator of the oocyte DNA damage response and homologous recombination (HR) repair under genotoxic stress. Using cisplatin-induced DNA damage models in fetal goat ovaries and neonatal mouse ovaries, we investigated repair mechanisms operating within the ovarian reserve. Label-free proteomic profiling revealed significant enrichment of DNA damage response pathways following cisplatin exposure, with CDK1 emerging as one of the most prominently upregulated kinases. Pharmacological inhibition of CDK1 had little effect on follicle survival under physiological conditions but aggravated oocyte and follicle loss following DNA damage, indicating a stress-dependent role for CDK1 in preserving ovarian follicle pool integrity. Mechanistically, DNA damage activated a Chk2-dependent signaling pathway that promoted p63 phosphorylation and altered the WEE1-CDK1 regulatory axis, resulting in reduced inhibitory CDK1 phosphorylation (Thr14/Tyr15) and increased activating phosphorylation (Thr161). Activated CDK1 was associated with enhanced RAD51 phosphorylation and accumulation at DNA damage foci, supporting homologous recombination (HR)-mediated repair in dictyate-arrested oocytes. In contrast, CDK1 inhibition reduced phospho-RAD51 levels, impaired RAD51 localization, increased persistent {gamma}H2AX accumulation, and elevated oocyte apoptosis. Notably, suppression of CDK1 was accompanied by increased expression of the non-homologous end joining (NHEJ) marker Ku80 and the nucleotide excision repair (NER) factor XPA, suggesting increased engagement of alternative DNA repair pathways. Furthermore, inhibition of Chk2 abolished the DNA damage-associated CDK1 activation signature and restored WEE1 expression, supporting a model in which CDK1 functions downstream of Chk2 signaling during the oocyte DNA damage response. Collectively, our findings identify a previously unrecognized Chk2-CDK1-RAD51 signaling axis that coordinates homologous recombination repair in dormant oocytes and safeguards ovarian follicular pool integrity under genotoxic stress. These findings provide new mechanistic insight into how dictyate-arrested oocytes maintain genome stability during prolonged meiotic arrest.

Objective To evaluate the feasibility, safety, patient acceptance, and preliminary clinical relevance of automated self-operated transvaginal ultrasound for ovarian stimulation monitoring. Design Prospective observational pilot study. Subjects Ten women undergoing ovarian stimulation for in vitro fertilization or fertility preservation at a single high-volume private IVF center. Exposure Participants performed investigational self-operated transvaginal ultrasound examinations immediately following standard monitoring visits. Patients inserted and stabilized the ultrasound probe while ovarian and endometrial imaging was acquired through controlled motorized probe rotation without real-time anatomical guidance. Main Outcome Measure(s) The primary outcome was feasibility, defined as the generation of evaluable imaging datasets suitable for ovarian stimulation monitoring. Secondary outcomes included bilateral ovarian visualization, procedural safety, patient-reported outcomes, follicular assessment, and agreement of endometrial thickness measurements with standard transvaginal ultrasound. Result(s) Nineteen investigational scan attempts were performed, yielding 18 evaluable datasets (94.7%). Bilateral ovarian visualization was achieved in 16 of 18 evaluable examinations (88.9%), whereas partial ovarian visualization occurred in 2 examinations (11.1%). No adverse events, adverse device effects, vaginal injury, bleeding, or infection were observed. Patient-reported outcomes demonstrated high procedural acceptability, with all participants expressing willingness to reuse the system. Compared with standard transvaginal ultrasound monitoring, investigational self-operated acquisition significantly improved overall examination experience (Wilcoxon p=0.002). Investigational imaging demonstrated clinically relevant agreement with standard transvaginal ultrasound for follicular categorization and endometrial assessment. Counts of follicles [&ge;]14 mm correlated strongly with mature oocyte recovery for both investigational and standard ultrasound measurements (Spearman {rho}=0.83 and {rho}=0.80, respectively). Endometrial thickness measurements also demonstrated strong correlation between modalities (Spearman {rho}=0.91). Conclusion(s) This prospective pilot study demonstrates the feasibility of automated self-operated transvaginal ultrasound during ovarian stimulation monitoring. Investigational imaging generated clinically relevant monitoring information without observed safety concerns and was associated with high patient acceptance. These findings support further investigation of patient-operated acquisition strategies and standardized imaging workflows in reproductive medicine.

Bovine embryo in vitro production (IVP) is characterised by low efficiency and variable outcomes. Monitoring early embryonic development by videomicroscopy revealed substantial morphokinetic heterogeneity in the first four embryonic cycles (EC, conventionally referred to as the 2-, 4-, 8- and 16-cell stages). Morphokinetic analysis offers a promising approach to characterize divergent developmental trajectories and provide a better understanding of underlying molecular mechanisms. We developed a Random Forest classification system (Bovine Embryo Analyser based on Morphokinetics: BEAM) to predict embryo phenotype. It is based on morphokinetic variables collected from the 1st to the 4th EC and predicts four blastocyst categories (EHB: Early Hatching Blastocyst, HB: Hatching Blastocyst, SSB: Subtle Developmental Shift Blastocyst, ADB: Arrhythmic Development Blastocyst). Classification performance on an independent dataset was good (F1 score = 0.59; Accuracy = 0.78), indicating that the BEAM can be useful for embryo development studies. The BEAM was further applied to embryos submitted to 4.3 days of culture and having completed the 4th EC (16-32 cells). RNA sequencing was performed on sixteen samples (4 x 8 pooled embryos/category). The ADB category was significantly enriched in transcripts involved in the regulation of transcriptional activity compared to the EHB category. In addition, in the ADB category, 22.7% (n = 185/816) of the upregulated genes were of maternal origin while only 6.2% were of embryonic origin (n = 54/816). In conclusion, despite being at a comparable developmental stage and transcriptionally competent, the ADB category showed delayed maternal transcript degradation suggesting delayed transition to embryonic transcriptional autonomy. Illustrated Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=138 SRC="FIGDIR/small/733532v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@13a0958org.highwire.dtl.DTLVardef@13b88a1org.highwire.dtl.DTLVardef@18707c9org.highwire.dtl.DTLVardef@105759_HPS_FORMAT_FIGEXP M_FIG C_FIG Summary SentenceIn vitro produced bovine embryo morphokinetic patterns allow prediction of four blastocyst categories (up to the 4th embryonic cycle) and are associated with distinct transcriptomic profiles at embryonic genome activation in competent embryos.

The adrenal cortex is a critical for life endocrine system. It manages metabolic homeostasis, electrolyte balance, stress response, and sex development. This is accomplished through the release of various steroids from a dynamically changing landscape of concentric cellular zones/layers. Adrenocortical dysfunction is implicated in pathologies ranging from adrenal insufficiency to hypertension. The in-depth investigation of adrenal gland biology, pathology and drug discovery has been hampered by a lack of human, experimentally tractable models. Particularly missing are models that recapitulate the cellular diversity of the adrenal cortex with representation of all fetal and adult cell layers and the capsule. Here, we employ human pluripotent stem cells (hPSCs) to generate cells of all three cortex zones alongside capsular cells in a single 2D platform. This platform mimics the cellular diversity expected from an organoid, yet it provides the simplicity of monolayer cultures, that are better suited for drug discovery and high-throughput settings. These cultures secrete zone-specific steroids (cortisol, aldosterone, and DHEA-S) and exhibit robust, physiologically relevant ACTH stimulation responses. Transcriptomic analysis revealed sequential acquisition of profiles consistent with adrenocortical development, zonation, and signaling programs consistent with zone maintenance. Our platform is validated by a literature meta-analysis that defines transcriptomic signatures for each human cortical cell type, spanning fetal and adult stages. Together, this novel adrenocortical platform enables investigation of adrenal development, disease mechanisms, and therapeutic strategies. Significance StatementWe describe a hPSC-based 2D differentiation strategy with the cell type complexity of an organoid and the technical simplicity necessary for high-throughput assays. The platform contains all cortical subtypes that mediate electrolyte regulation, stress response, sex development and self-maintenance of the tissue. This co-differentiation offers a unique opportunity to study human adrenal development, biology, pathology and enables drug discovery.

Proliferation of cytotrophoblasts (CTBs) and their differentiation into invasive extravillous trophoblasts (EVTs) are critical processes in early placental development. Defects in these processes are associated with adverse pregnancy outcomes, including recurrent pregnancy loss (RPL). There is evidence that reduced expression of the maintenance DNA methyltransferase DNMT1 in the placenta occurs in pregnancy loss and that RPL is associated with aberrant DNA methylation patterns. Therefore, we investigated the role of DNMT1 in human trophoblast growth and differentiation. Using human trophoblast stem cells (hTSCs), an in vitro analog to CTBs, we found that shRNA-mediated knockdown of DNMT1 led to decreased hTSC proliferation, genome-wide reductions in methylation, broad changes in gene expression, and impaired EVT differentiation. Transcriptome profiling of DNMT1-deficient hTSCs and hTSC-derived EVTs highlighted aberrant cytokine expression, drawing a connection to prior reports of immunological dysfunction in RPL. Finally, using a catalytic DNMT1 chemical inhibitor, we demonstrate the canonical methyltransferase activity of DNMT1 is essential for EVT differentiation and invasion. This study identifies new roles for DNMT1 in trophoblasts and addresses the molecular basis of the associations between DNMT1 expression, altered DNA methylation profiles, and RPL.

Leydig cells (LCs) are essential for male reproductive function due to their role in testosterone synthesis, a process critically dependent on mitochondrial cholesterol transport mediated by the Steroidogenic Acute Regulatory protein (StAR). Despite their importance, LCs are highly sensitive to metabolic and exogenous stressors. Ferroptosis, an iron-dependent form of regulated cell death driven by lipid peroxidation, has emerged as a key link between cellular metabolism and cell fate; however, its role in LCs and steroidogenesis remains poorly understood. In this study, we investigated the induction of ferroptosis in LCs and its impact on their steroidogenic capacity. We evaluated cellular responses to canonical ferroptosis inducers (Erastin and RSL3) alongside the transcriptional regulation of key genes. Our results demonstrate that LCs are vulnerable to ferroptotic stress, which significantly downregulates Star expression. Notably, we uncovered a novel endocrine-metabolic crosstalk: hormonal stimulation via hCG effectively rescues LCs from Erastin-induced toxicity and fully sustains maximal steroidogenesis. However, this hormone-driven cytoprotection fails against direct GPX4 inhibition by RSL3, indicating an absolute reliance on functional GPX4. These mechanistic findings highlight the paradoxical dual role of ACSL4 in Leydig cell biology and are further supported by bioinformatic analysis of public transcriptomic profiles from infertile patients, which reveal a detrimental imbalance in the ACSL4/GPX4 axis. Together, our data position ferroptosis as a critical disruptor of male endocrine function and reveal a hormone-mediated metabolic adaptation that could inform novel therapeutic strategies against oxidative stress in the testis. Highlights-Leydig cells exhibit a strong vulnerability to ferroptotic cell death. -Ferroptosis disrupts StAR expression and halts Leydig cell steroidogenesis. -hCG signaling promotes metabolic adaptation against Erastin-induced ferroptosis.

Progestin-IUD use was more frequent among IUA cases (29.9%) than polyp (3.4%) or infertile (11.1%) comparison groups. Compared to infertile comparators, any prior progestin-IUD use was independently associated with IUA (aOR 3.12; 95% CI 2.01, 4.85). There was a duration-response pattern: use of 5 years or less was modestly associated with IUA case status (aOR 1.99; 1.09, 3.64), whereas use >5 years conferred more than a seven-fold increase (aOR 7.26; 3.27, 16.11). The association persisted among surgically naive women (aOR 3.98; 2.44, 6.48) and was concentrated in those who were nulliparous, where use beyond five years conferred an approximately twelve-fold increase in odds (aOR 12.74; 5.25, 30.92). Progestin-IUD use was less frequent in polyp controls relative to IUA and infertile comparators, suggesting a possible role for progestin exposure in preventing endometrial polyp formation. The case control design does not allow for estimation of absolute risk for an individual and cannot inform causation. Further prospective studies are needed to better assess the relationship between progestin-IUD's, particularly when used beyond five years, and adverse fertility outcomes.

CCCTC-binding factor (CTCF) is frequently mutated in endometrial cancer, resulting in genetic haploinsufficiency that contributes to tumour progression. We previously showed that depletion of CTCF disrupted cell polarity in KLE endometrial cancer spheroids; however, the implications for gene dysregulation and endometrial cancer pathophysiology remains poorly understood. ZNF185, an actin-associated and LIM domain-containing protein involved in cytoskeletal remodelling, was identified as a dysregulated target following CTCF haploinsufficiency. In this study, shRNA-mediated knockdown of CTCF was used to model haploinsufficiency in endometrial cancer cells, leading to the identification of a previously unrecognised isoform of ZNF185, named ZNF185B. Unlike the full-length protein, ZNF185B lacked co-localisation with F-actin and exhibited a diffuse cytoplasmic distribution, and ZNF185B was significantly upregulated in CTCF-depleted endometrial cancer cells and in an auxin-inducible degron model in a dose-dependent manner. Functional studies demonstrated that depletion of ZNF185 expression reduced endometrial cancer cell proliferation and clonogenic potential. Together, these findings identify ZNF185B as a novel isoform negatively regulated by CTCF protein dosage and establish ZNF185 as a requirement for endometrial cancer cell proliferation. Our results suggest that dysregulated ZNF185 expression is a crucial downstream consequence of CTCF haploinsufficiency and may contribute to tumour progression in endometrial cancer.

Background Menstrual cycle disturbances have been increasingly reported after COVID-19 vaccination, raising questions about their prevalence and clinical significance among women of reproductive age. Objective This study aimed to investigate the incidence and types of menstrual cycle alterations following different doses of COVID-19 vaccines among Iranian women of reproductive age. Methods A cross-sectional survey was conducted among vaccinated women who reported their menstrual cycle status before and after each vaccine dose. Data on cycle regularity, flow characteristics, and specific menstrual disorders were collected and analyzed. Results Menstrual cycle alterations were reported by 28.8%, 25.4%, 30.3%, and 68.4% of participants after the first, second, third, and fourth vaccine doses, respectively. The most common changes were oligomenorrhea after the first and second doses (8.9% and 5.6%), menorrhagia after the third dose (5.3%), and hypomenorrhea after the fourth dose (8.3%). Comparisons with international studies revealed a wide variation in prevalence (ranging from 25% to 78%), which may be explained by differences in methodology, population characteristics, vaccine types, and pre-vaccination health status. Conclusion A considerable proportion of Iranian women experienced menstrual alterations following COVID-19 vaccination, most commonly oligomenorrhea, menorrhagia, and hypomenorrhea. While generally self-limiting, these findings highlight the need to integrate menstrual health into post-vaccination monitoring and patient counseling. Future research should explore the underlying immune-endocrine mechanisms and long-term clinical implications of these changes.

BACKGROUNDSpontaneous preterm birth (sPTB) remains the foremost cause of neonatal morbidity and mortality worldwide. Although histologic chorioamnionitis (HCA) and placental vascular abnormalities are frequently observed in sPTB, the molecular cascades linking these lesions to labor initiation remain poorly understood. Emerging evidence implicates circadian dysregulation and trophoblast dysfunction as additional drivers of sPTB. OBJECTIVEThis study aims to map placental pathology to distinct transcriptomic functional signatures that may precipitate sPTB, delineate the contribution of circadian regulation - both core-clock genes and circadian transcription-factor target sets (TFTs) - to sPTB, and identify placental cell-type-enriched and developmental pathway signatures that differ between sPTB and term deliveries. STUDY DESIGNWe performed bulk RNA sequencing on 32 formalin fixed, paraffin embedded placental specimens from 12 selected women (9 sPTB and 3 Term) in the POUCH Study cohort. Samples were selected for white ethnicity, maternal age 23-33years, and parity 1-4 to reduce heterogeneity within groups. An extraction-free HTG transcriptome panel assayed 19,398 protein-coding genes. Log2-fold changes of all genes were computed with limma adjusted for maternal age, gestational age, parity, placental region, placental pathology, and POUCHID (a clustering variable) for sPTB vs. Term and HCA/vascular lesion vs. no pathology (no placental pathology adjustment). Gene-set enrichment used 50 Hallmark sets (MSigDB) plus curated placental circadian, circadian TFT, cell-type, and developmental pathways or gene sets. RESULTSsPTB placentas displayed a global suppression of metabolic, secretory, and immune pathways (e.g., protein secretion, oxidative phosphorylation, Interferon responses, Complement, ROS, MYC Targets, TGF {beta}, mTORC1, and Coagulation) while KRAS Signaling Down and EMT were up-regulated. HCA-enriched sets (TNF/NF-{kappa}B, ROS, KRAS Up, IL-2/STAT5, Hypoxia, Interferon-{gamma}) were up-regulated, with EMT and Notch remaining down. Vascular abnormalities alone showed up-regulation of 12 Hallmark sets - including TGF-{beta}, TNF/NF-{kappa}B, ROS, pancreatic {beta}-cell stress, Hypoxia, Oxidative Phosphorylation, EMT, and mTORC1 - while Notch was down-regulated. When HCA co-exists with vascular abnormalities, the Hallmark profile becomes more inflammatory highlighting a synergistic exacerbation of innate immunity, oxidative stress, and programmed cell death with the 12 up-regulated sets (Complement, Interferon /{gamma}, TNF, ROS, Apoptosis, and Heme Metabolism). The exclusive downregulation of DNA Repair suggests compromised genomic integrity. Circadian gene-sets analysis revealed an up-regulated Regulation of Circadian Sleep Wake Cycle in sPTB but down-regulation of core clock pathway and suppressed circadian TF targets. Cell-type enrichment reveals increased trophoblast giant cells and IGFBP1-DKK1 positive fetal cells, with marked suppression of extravillous trophoblasts, syncytiotrophoblasts, villous cytotrophoblasts, and fetal myeloid cells. Placental developmental pathways were downregulated, indicating arrested trophoblast maturation. CONCLUSIONOur pilot analysis demonstrates sPTB placentas exhibit a global suppression of metabolic, secretory, and immune-modulatory programs and maladaptive trophoblast remodeling, whereas HCA and vascular abnormalities drove distinct inflammatory or hypoxic signatures. The shared and opposing Hallmark pathways across phenotypes highlight distinct yet overlapping pathogenic mechanisms. Dysregulated circadian pathways, consistent downregulated transcription factor target gene sets, and trophoblast-specific signatures implicate circadian misalignment and impaired placental maturation as key contributors to preterm parturition. These findings provide a mechanistic atlas linking placental pathology to sPTB and highlight potential targets for chronotherapeutic and cell-type-specific interventions. AJOG at a GlanceO_ST_ABSWhy was this study conducted?C_ST_ABSSpontaneous preterm birth remains a leading cause of neonatal morbidity. Histopathologic lesions of the placenta, particularly chorioamnionitis and vascular abnormalities, are common in preterm deliveries, yet the underlying molecular pathways are poorly understood. We sought to integrate functioning pathway profiles of placental histology, circadian biology, and cell types to identify mechanistic drivers of sPTB. Key findingsO_LIsPTB placentas showed widespread down-regulation of oxidative phosphorylation, mTORC1, hypoxia, interferon, and TNF/NF-{kappa}B pathways. C_LIO_LIHCA placentas up-regulated the same pathways (except androgen response), revealing a reciprocal inflammatory-hypoxic signature. C_LIO_LIVascular abnormalities displayed a distinct mix of up- and down-regulated pathways, suggesting divergent reparative responses. C_LIO_LIPlacentas with co-existing HCA and vascular abnormalities enriched more inflammatory Hallmark pathways: the 12 up-regulated sets (Complement, Interferon /{gamma}, TNF, ROS, Apoptosis, and Heme Metabolism) highlight a synergistic exacerbation of innate immunity, oxidative stress, and programmed cell death and the exclusive down-regulation of DNA Repair suggests compromised genomic integrity, which can contribute to premature placental senescence and preterm labor. C_LIO_LICircadian clock and multiple transcription-factor targets were enriched in sPTB, and trophoblast-specific signatures (giant, extravillous, syncytiotrophoblast) were prominent. C_LI What does this add to what is known?The study demonstrates a clear dichotomy between inflammatory and hypoxic molecular programs in sPTB and HCA, identifies circadian dysregulation as a potential contributor, and highlights trophoblast subpopulations as key players. These insights open avenues for targeted biomarkers and chronotherapy in preterm birth prevention.

Chromosome segregation errors in oocyte meiosis are a leading cause of early miscarriage and congenital disorders in mammals and these errors become more prevalent with advanced maternal age. Although the effects of aging on the functions of critical meiotic proteins and cytoskeletal filaments in oocytes are known, the influence of aging on the force generating capabilities of oocyte spindle components remains largely unexplored. Through the integration of a coarse-grained model and in situ experiments, we compare the long-axis mechanical properties of metaphase I (MI) and II (MII) oocyte spindles from reproductively young and old mice. Increased inter-kinetochore distance in aged MII oocytes agree with a model of age-associated cohesion loss, and kinetochore dynamics in these spindles following laser ablation suggest a similar reduction in inter-kinetochore bridge viscosity. Simultaneously, we find that both cohesive and poleward force generators lose stiffness with advanced age in MI spindles. In total, we quantify the extent to which structural spindle components lose their stiffness and viscosity during maternal aging, highlighting the multifaceted impacts of aging on mouse oocyte spindle mechanics. Significance StatementO_LIMaternal aging influences mammalian oocyte spindles in numerous ways, yet the impacts of aging on the balance of collective spindle forces remain poorly understood. C_LIO_LIIntegrating coarse-grained mechanical modeling with in situ measurements of spindle morphology and kinetochore dynamics, we quantify age-associated changes to the viscosities and elastic stiffnesses of oocyte spindle component parts. C_LIO_LIThis work provides both a characterization of the effects of aging on force production in mammalian oocyte spindles and a blueprint for future studies of spindle force generation in complex biological contexts. C_LI

Background: Prospective pregnancy registries and biobanking infrastructures are essential for future translational studies investigating maternal, placental and offspring health. However, circulating nucleic acid analyses are highly sensitive to preanalytical variability, particularly regarding blood-collection tube type and sample processing conditions. We established a prospective pregnancy registry and biobanking workflow at Cruces University Hospital and evaluated the impact of preanalytical variables on circulating cell-free DNA (cfDNA) and cell-free RNA (cfRNA) preservation in maternal plasma collected at delivery. Methods: The Registry of Pregnant Women at Cruces University Hospital was designed as a prospective infrastructure integrating placental sampling, maternal blood collection and ethically controlled future access to maternal and offspring clinical data. Within this framework, peripheral blood samples from 50 women at delivery were simultaneously collected into EDTA, Norgen and Roche tubes. Plasma samples processed within or after 24 hours following collection underwent cfDNA/cfRNA extraction, electrophoretic profiling, fluorometric quantification and RT-qPCR analyses targeting different stress-related genes. Results: By the end of June 2026, 1,127 women had been prospectively recruited into the registry, with 661 plasma samples, 637 serum samples and 858 sets of four placental biopsies collected, processed and stored in the Basque Biobank. In the preanalytical substudy, EDTA tubes yielded higher cfDNA concentrations, likely reflecting reduced cellular preservation and genomic DNA contamination. In contrast, Roche tubes showed superior cfRNA preservation, with higher cfRNA concentrations and more consistent detection of the characteristic 5S rRNA peak compared with EDTA and Norgen tubes. Processing delays beyond 24 hours reduced cfRNA concentration, while associations between circulating transcripts and gestational age were more consistently detectable in preservative-containing tubes. Conclusions: Prospective infrastructures like ours offer strong foundation for large scale, long-term studies in the framework of the Developmental Origins of Health and Disease hypothesis. Technically, Roche tubes provided superior cfRNA preservation and enhanced sensitivity for detecting subtle biological associations, supporting the importance of standardized preanalytical workflows within prospective pregnancy biobanking resource.

The lactational period requires substantial metabolic and behavioral adaptations, and more than 70% of mothers report weight concerns and attempt weight loss by four months postpartum. Nevertheless, how distinct restrictive paradigms during lactation alter maternal behavior, and the extent to which associated neurochemical changes modulate these behaviors, remains poorly understood. In the current study, we modeled restrictive diets in lactating rats to evaluate caregiving behavior and its relationship to amygdalar redox status. Intermittent fasting (IF) and caloric restriction (CR) administered to lactating Wistar dams from postpartum day 0 to day 28 impaired maternal care, evidenced by delayed pup retrieval, reduced nest building, and decreased nursing frequency relative to ad libitum-fed controls. Both diets reduced body and adipose tissue weight, and energy efficiency. IF and CR increased impulsivity-like phenotype: CR doubled open-arm exploration in the elevated plus maze; IF and CR increased center-zone exploration in the open field by three- and two-fold, respectively; IF doubled time in the light-dark box light compartment. A composite maternal behavioral score showed impairment in dams in both IF and CR groups. At the neurochemical level, both diets reduced amygdalar superoxide dismutase activity, which correlated negatively with the maternal behavioral score. Both restrictive diets produced an underweight phenotype with weakened dam-pup interactions and increased impulsivity. These behavioral changes co-occurred with amygdalar redox imbalance, which correlated with the severity of maternal impairment. Overall, the study refines understanding of the nutritional and behavioral consequences of dietary restriction in lactation and implicates disrupted redox homeostasis as a plausible mechanism.

Identification of signaling networks is an essential goal in systems biology. Here, we use CRISPR/Cas9 knockout screening (employing a whole kinome sgRNA library) to identify functionally critical protein kinases in a well-studied Gs-dependent G-protein coupled receptor (GPCR)-signaling model, namely the vasopressin V2 receptor (V2R) pathway. Screening was done using a specially-designed fluorescence-based reporter cell line with green-fluorescent protein (GFP) co-transcribed with Aqp2, a gene whose transcription is dependent on vasopressin-mediated activation of protein kinase A (PKA). Positive regulators (n=14) included PKA-catalytic subunit (Prkaca) and Dyrk1a (minibrain homolog). Negative regulators (n=12) included PKA-regulatory subunit type I, Stk11 (catalytic subunit of liver kinase B1 [LKB1] complex), and three TGF-{beta} receptor subunits (Tgfbr1, Tgfbr2, Tgfbr3) (see https://esbl.nhlbi.nih.gov/Databases/Kinome-CRISPR-screen/ for full list). Dyrk1a knockout cell lines failed to express AQP2 protein and exhibited a profound decrease in AQP2 mRNA. RNA-sequencing demonstrated widespread increases in cell-cycle transcripts, with a general defect in cell differentiation, accounting for AQP2 loss. TGF-{beta} exposure to un-transformed cells results in a profound decrease in V2R and AQP2 mRNA abundance along with multiple additional transcriptional targets of V2R-PKA signaling, consistent with prior findings in TGF-{beta}-mediated vasopressin escape. Stk11/LKB1 knockout lines displayed marked increases in AQP2 protein and mRNA, even in the absence of vasopressin. RNA-sequencing showed a marked similarity between the responses to Stk11/LKB1 deletion and vasopressin exposure in untransformed cells. Phospho-proteomic data point to opposing roles of Stk11/LKB1 and PKA in the regulation of cAMP-responsive transcriptional coactivator (CRTC) proteins in the transcriptional response to V2R-PKA signaling. Significance StatementCells throughout the body are regulated by extracellular signals like the hormone, vasopressin. Hormonal effects on cellular function are mediated by membrane receptors that trigger biochemical changes, often by inducing chemical modification of the amino acids making up individual proteins, such as addition of function-altering phosphate groups (phosphorylation). Protein phosphorylation is mediated by enzymes known as "protein kinases". Here, we have screened all known protein kinases using modern CRISPR/Cas9 technology to identify those involved in vasopressin action in the kidney. As expected from prior knowledge, the screen identified protein kinase A and one of its regulatory subunits, but also identified several protein kinases not previously implicated in vasopressin action in the kidney.