Molecular Human Reproduction

Ovarian disorders, including anovulation, primary ovarian insufficiency (POI), and polyendocrine metabolic ovarian syndrome (PMOS), affect millions of reproductive-age women worldwide; however, mechanistic studies of ovarian biology and pathophysiology remain challenging because current experimental approaches often lack selectivity, tunability, or physiological relevance. Genetically modified animal models are labor-intensive and irreversible; small molecules frequently exhibit off-target effects; and conventional antibodies have limited tissue penetration and restricted temporal control. Designed ankyrin repeat proteins (DARPins) represent a highly modular protein engineering platform with advantages in specificity, size, stability, and extracellular targeting, but their utility in reproductive biology remains largely unexplored. Here, we used epidermal growth factor receptor (EGFR)-targeting DARPins as a proof-of-concept platform to interrogate ovarian signaling during ovulation. Screening of engineered anti-EGFR DARPins identified SX-006, a bispecific tetravalent construct with robust cross-species EGFR binding and potent biological activity. Using an ex vivo murine ovulation system, SX-006 inhibited follicle rupture in a dose-dependent manner with IC50 of 1.21 M without overt cytotoxicity. Lower concentrations of SX-006 preferentially perturbed follicle rupture while largely preserving oocyte meiotic maturation and luteinization, suggesting differential sensitivity of ovulatory processes to extracellular EGFR blockade. Comparative transcriptomic analyses further revealed that extracellular EGFR blockade and small molecule-based intracellular EGFR kinase inhibition produce overlapping but also distinct transcriptional responses, supporting biologically distinct modes of ovulatory signaling pathway perturbation. Together, these findings establish DARPins as a selective, tunable, and physiologically relevant platform for studying ovarian signaling and provide proof-of-concept for extracellular receptor targeting in ovarian biology, infertility research, and non-hormonal contraceptive development. Summary sentenceAn engineered EGFR-targeting DARPin selectively inhibits ovulation through extracellular receptor blockade and establishes a versatile platform for investigating ovarian signaling and reproductive disorders.

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.

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.

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

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.

Capacitation, the process whereby sperm gain the functional competence to fertilize an egg in the absence of de novo transcription and translation, is orchestrated by a hierarchy of kinases driving the phosphorylation of sperm proteins. While increased phosphorylation, in particular tyrosine phosphorylation, is a revered hallmark of fertilization competency in our species, only a limited repository of phosphorylated substrates and kinases have ever been reported from human sperm. To broaden therapeutic targets for sperm targeted contraceptives and infertility therapies, we adapted a contemporary phosphoproteomic technique termed EasyPhos to generate bespoke methodology for the investigation of human sperm signalling. This approach yielded high depth phosphoproteomes of non-capacitated and capacitated human spermatozoa with in silico investigation of the phosphosites revealing 52 kinases with previously uncharacterized roles in sperm capacitation. Investigating the function of the putative sperm capacitation kinases identified yielded several kinases with novel roles in the regulation of sperm function. Of particular interest, polo like kinase 1 (PLK1) inhibition significantly reduced progressive sperm motility, attenuated capacitation-associated tyrosine phosphorylation and reduced the sperm acrosome reaction, an essential step to achieve fertilization. These findings reveal extensive phosphoproteome remodelling during human sperm capacitation, expanding the landscape of molecular targets for fertility control.

Congenital disorders of glycosylation and deglycosylation are rare, serious, and lethal disorders afflicting humans. CDGs and CDDGs result in loss of function enzymes which fail to build or break down oligosaccharides on proteins. This can produce protein aggregates and, in turn, reactive oxygen species that harm the cell eventually leading to autophagy and apoptosis. Because sperm contain high concentrations of polyunsaturated fatty acids, they are especially sensitive to these effects, which is understood as one of the leading factors in human male infertility. Sperm are developed in zebrafish similarly to humans and are useful models to examine human reproductive health, as well as genetic disorders. The combination of these advantages makes the analysis of sperm from zebrafish with heterozygous ALG1 or DPAGT1 CDGs or the NGLY1 CDDG suitable. Analysis of sperm concentration, motility, status, viability, and hypoosmotic swelling demonstrated the effects of these disorders on sperm quality. Results showed a significant decrease in sperm concentration, motility, and hypoosmotic swelling for all mutant zebrafish compared to the wild type. This suggests that CDGs and CDDGs influence the amount of sperm produced, the percentage of sperm cells that are mobile, and the integrity of the plasma membrane.

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.

Background The placenta serves a critical role in nutrient uptake and waste elimination for the developing fetus. The placenta is also responsible for the uptake and/or exchange of xenobiotics, including medications, between the maternal and fetal bloodstreams. An estimated 40-80% of women take medications or drugs during pregnancy for a variety of conditions. Very little is understood about fetal drug and nutrient exposure during pregnancy and how it may change over the course of fetal development. Objective This study aimed to characterize the abundance of transport proteins in placental tissue, which are important in modulating fetal nutrient and drug exposure, over the duration of pregnancy. Mass spectrometry-based global proteomic analysis revealed trends in the expression of thousands of proteins throughout gestation. Focusing on the membrane-associated proteome enabled an increased emphasis on the solute carrier and ATP-binding cassette families of transporter proteins that are critical for nutrient and xenobiotic transport across the maternal-fetal barrier. Study Design Using data-independent acquisition proteomics, relative abundance of proteins in placental tissue samples was profiled across all three trimesters of pregnancy (Trimester 1 = 16, Trimester 2 = 9, and Term = 9). Membrane fractions were generated to enrich membrane-associated proteins for proteomic analysis. Placental samples were grouped into randomized batches for membrane fraction generation and mass spectrometry analysis. Proteomic search results from each batch were imported into the R programming environment from Skyline, concatenated, and normalized as one data set for downstream analysis. Results A total of 6,331 proteins were detected across all samples with 4,210 proteins identified in every sample. Pathway analysis revealed that as gestational age increases, membrane-associated proteins involved in more complex metabolic pathways increase in relative abundance while those involved in extracellular remodeling events and simple organic ion transport tended to decrease. A total of 139 solute carrier and ATP-binding cassette transport proteins were identified in all samples, and 80 were identified in every sample. In general, membrane-associated proteins, including solute carrier and ATP-binding cassette transport proteins, were significantly enriched in placental tissue collected during early gestation compared to term placental tissue. Conclusion This study presents a comprehensive profiling of membrane-associated proteomic changes during gestation and identifies significant gestational age associated abundance changes at the protein level in several transport protein families. The application of data-independent acquisition global proteomic techniques enabled in-depth analysis of thousands of proteomic changes across pregnancy in a single experiment. These data provide critical information to support future studies into the understanding of fetal exposure to xenobiotics and nutrients circulating in the maternal bloodstream.

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.

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

Premature ovarian insufficiency (POI) significantly impacts quality of life, yet the immediate cognitive landscape and lived experience of younger women remain under-researched. In 125 young women (aged 19-48; 66 with idiopathic POI, 59 age-matched controls), we examined self-reported cognitive distress and symptom burden within the POI cohort and compared objective global and domain-specific cognitive performance between groups. Objective accuracy scores were derived from six online tasks (Cognitron) and combined into a robust global measure. Within the POI cohort, there were significant differences in symptom burden domains ({chi}(3) = 61.90, p<0.001), with psychological and sexual symptoms reported at a significantly higher intensity than physical and vasomotor symptoms (all p<0.001). Furthermore, the standardised magnitude of perceived cognitive distress (56.20%) was significantly greater than that of overall symptom burden (42.00%, p<0.001). Case-control comparisons revealed no significant differences in global cognitive performance (p=0.615), yet the POI cohort performed significantly less accurate than controls in verbal analogical reasoning (-0.86 SD, 95% CI: -1.52, -0.20, p = 0.011). The findings highlight an urgent need for comprehensive emotional and psychosexual support in POI care. Additionally, the presence of high cognitive distress alongside localised objective deficits demonstrates that cognitive health monitoring must be proactive in early adulthood, especially given their established long-term risks for later-life cognitive decline and dementia.

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.

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.

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.

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.

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.

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.

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.

Non-obstructive azoospermia (NOA) represents the most severe form of male infertility, severely limiting a patient's prospects for biological fatherhood when surgical retrieval fails. However, the true biological limits of NOA remain obscured by the inherent limitations of conventional gamete recovery protocols: standard centrifugation frequently causes substantial cell loss, masking extremely rare sperm, while surgical interventions are constrained by spatial sampling biases. Here we report SpermSeek, an integrated AI-guided microfluidic platform for real-time, non-destructive isolation of single sperm directly from semen. Operating at scalable throughput (0.36 mL/h), the system achieves 98.3% detection precision and a 95.5% target encapsulation efficiency, suppressing background debris. In a 59-patient NOA cohort, SpermSeek detected morphologically identifiable sperm in 64.4% (38/59) of cases, spanning diverse genetic etiologies, including AZFb/c microdeletions, and severe histopathological phenotypes, such as Sertoli-cell-only syndrome (SCOS). Notably, among a sub-cohort of 41 patients who remained consistently sperm-negative despite prior medical or micro-TESE interventions, our platform identified gametes in 53.7% (22/41) of these cases. Comprehensive safety profiling in healthy human donors and wild-type mice confirmed that processed sperm retain high DNA integrity and epigenomic concordance (r=0.98), supporting transgenerational developmental stability in mice. Furthermore, in a 26-patient validation cohort, SpermSeek recovered rare sperm in 11 cases. Utilizing gametes from a subset (n=5), we demonstrated their capacity to support early human embryogenesis, yielding high-quality cleavage-stage embryos with confirmed genomic euploidy. This work establishes a highly sensitive framework for re-examining the biological limits of human spermatogenesis, laying the foundation to expand autologous reproductive options for patients refractory to conventional retrieval protocols.