Polycystic ovary syndrome (PCOS) and its underlying features remain poorly understood. In this genetic study (n = 544,513), we expand the number of genetic loci from 16 to 29, and additionally identify 31 associated plasma proteins. Many risk-increasing loci were associated with later age at menopause, underscoring the reproductive longevity related to an increased oocyte number and/or availability across the lifespan. Hormonal regulation in the etiology of this condition, through metabolic and reproductive features, was emphasized. The proteomic analysis highlighted metabolic biology known to be related to PCOS. A polygenic risk score (PRS) was associated with adverse cardiometabolic outcomes, with differing relevance of testosterone and body mass index in women and men. Finally, while oligo-anovulation and anovulatory infertility are features of PCOS, we observed no impact of PCOS susceptibility on childlessness. We suggest that PCOS susceptibility confers balanced pleiotropic influences on fertility in women, and life-long adverse metabolic consequences in both sexes.

Current risk prediction models for ischemic heart disease in clinical use are relatively simple and use a limited collection of well-known risk factors. Using machine learning to integrate a broader panel of features from electronic health records (EHRs) may improve post-angiography prognostication. This retrospective model development and validation study was based on Danish EHR data. Icelandic EHR data were used for external test. Patients with a coronary angiography-confirmed diagnosis of coronary atherosclerosis between 2006 and 2016 were included for model development (n = 39,746). Time to all-cause mortality, the prediction target, was tracked until 2019, or up to 5 years, whichever came first. To model time-to-event data and deal with censoring, neural network-based discrete-time survival models were used. The model, PMHnet, uses 584 different features including clinical characteristics, laboratory tests, and diagnosis and procedure codes. Model performance was evaluated using time-dependent AUC (tdAUC) and the Brier score. PMHnet was benchmarked against the updated GRACE2.0 risk score and less feature-rich neural network models. Models were evaluated using hold-out data (n = 5000) and external validation data from Iceland. Feature importance and model explainability were assessed using SHAP analysis. On the test set (n = 5000), the tdAUC of PMHnet was 0.88 [ 0.86–0.90] (case count = 196) at six months, 0.88 [0.86–0.90] (cc = 261) at one year, 0.84 [0.82–0.86] (cc = 395) at three years, and 0.82 [0.80–0.84] (cc = 763) at five years. PMHnet showed similar performance in the Icelandic data. Compared to the GRACE2.0 score and intermediate models limited to GRACE2.0 features or single data modalities, PMHnet had significantly better model discrimination across all evaluated prediction timepoints. More complex and feature-rich machine learning models can better predict all-cause mortality in ischemic heart disease and may be used by clinicians and patients to inform and guide treatment and management.

BACKGROUND:A vaginal microbiota dominated by Lactobacillus species is associated with reduced risk of infection and adverse reproductive outcomes. Effective interventions to restore healthy microbiota remain scarce. In this study, we aimed to assess the efficacy of vaginal microbiota transplants (VMTs) without antibiotic pretreatment in achieving conversion to a Lactobacillus-dominated vaginal microbiome. METHODS:This single-centre, double-blind, randomised controlled trial was done at Copenhagen University Hospital (Hvidovre, Denmark) between June 1, 2021, and March 1, 2023. We enrolled women aged 18-40 years with asymptomatic or symptomatic molecular vaginal dysbiosis (<10% total relative abundance of Lactobacillus spp and >20% relative abundance of Gardnerella spp, Fannyhessea vaginae, and Prevotella spp) who were otherwise healthy premenopausal women and not pregnant as recipients; donors were healthy women aged 18-40 years with a Lactobacillus-dominated vaginal microbiota (>80%) and a low (<5%) abundance of Gardnerella spp, F vaginae, and Prevotella spp, and negative screening for sexually transmitted infections. Participants were randomly assigned (3:1) to the intervention or placebo through a computer-generated schedule with block randomisation and stratification by hormonal contraception. Participants and investigators were masked to the group. Up to three administrations of VMT or placebo were given across three menstrual cycles, with follow-up for six cycles. The primary endpoint was resolution of dysbiosis at any timepoint during follow-up, defined as at least 70% relative abundance of Lactobacillus spp and less than 10% combined abundance of Gardnerella spp, F vaginae, and Prevotella spp, as assessed by shotgun metagenomic sequencing of vaginal samples. This analysis was done in the intention-to-treat population, excluding any participants who withdrew consent. An extension study assessed the effect of antiseptic pretreatment before additional VMT in refractory participants. This study was registered with ClinicalTrials.gov (NCT04855006) and is completed. FINDINGS:A total of 302 women were screened, of whom 49 were enrolled. 37 women were randomly assigned to the VMT group (mean age 26·1 years [SD 3·8]) and 12 to the placebo group (27·3 years [4·8]). The primary outcome showed no significant difference in dysbiosis resolution between active and placebo groups (HR 0·65; 95% CI 0·20-2·16, p=0·49). In an extension study of refractory participants, five (50%) of the ten women who received antiseptic pretreatment followed by VMT had a microbiome conversion. Adverse events occurred in 15 (42%) VMT participants and five (42%) placebo participants; none were serious or led to withdrawal. A single pregnancy and one new human papillomavirus infection occurred, both unrelated to treatment. INTERPRETATION:VMT without antibiotics did not significantly improve microbiome conversion in this trial. However, findings from the extension study suggest that antiseptic pretreatment might enhance efficacy. Future trials should explore optimised dosing and use donor engraftment as a primary outcome. FUNDING:Freya Biosciences.

Intrahepatic cholestasis of pregnancy, which affects 0.2-2% of pregnancies, is characterized by pruritus, increased aminotransferase activity and elevated serum bile acids. Previous studies have implicated liver-enriched genes in intrahepatic cholestasis of pregnancy. We conducted a meta-analysis of intrahepatic cholestasis of pregnancy genome-wide association studies in the FinnGen study, deCODE, Estonian Biobank, the Danish Blood Donor Study and Copenhagen Hospital Biobank with 4,738 women with prior ICP and 436,834 female controls. The analysis found 26 genome-wide significant associations of which 10 were novel. Genes in the associated loci were prioritized using lead SNP expression quantitative trait loci associations and colocalization analysis to assess potential causality. The associated loci implicate bile acid synthesis, LDL cholesterol, and lipid metabolism. Additionally, comorbidity, genetic correlation and polygenic risk score analyses further indicated a link between intrahepatic cholestasis of pregnancy and pancreatitis, suggesting shared genetic underpinnings.

Abstract Purpose Endometriosis is associated with pain, cardiometabolic, psychiatric, and immune comorbidities. We tested whether the genetic liability captured by an endometriosis polygenic risk score (PRS) extends to these comorbidities through shared pathways or operates independently of the disease. Methods In 168,238 women and 155,304 men of European ancestry from two Danish genetic studies linked to national health registers, we constructed an endometriosis PRS using LDpred2 from an independent GWAS meta-analysis (23,112 cases, 429,677 controls). Entropy balancing with doubly robust adjustment addressed selection bias. Comorbidity analyses were performed in both sexes to distinguish shared from endometriosis-specific pathways. Results The PRS was associated with endometriosis (OR 1.55 per SD, 95% CI 1.50–1.61; AUC 0.73) and with 20 of 29 comorbidities in women. Pain conditions (fibromyalgia, migraine, chronic back pain) and cardiometabolic conditions replicated in men, indicating shared pathways, whereas immune-mediated conditions associated in women only. Seventeen associations persisted in women without diagnosed endometriosis. High genetic risk was associated with increased healthcare utilisation and all-cause mortality (HR 1.05, 95% CI 1.01–1.10). Conclusion The endometriosis PRS captures a pain-inflammatory-metabolic genetic axis operating in both sexes, indicating that the genetic liability extends beyond the uterine disease and providing a rationale for targeted investigation and risk stratification.

Germline mutations are heritable; they occur before the formation of a fertilized egg and are found in all cells. They can be detected through somatic tissue sampling, and de novo mutations (DNMs) are well-studied. The majority of known DNMs originate in paternal cells, but some include maternal contributions as well. Certain kinds of DNMs prevent a fertilized egg from developing to term, and these are much less well-characterized. Some also lack sequence variants in certain genes and some are never observed in a homozygous form; these are also not well studied. Recombination failure can cause aneuploidies (trisomies or monosomies), and an estimated half of pregnancy losses are explained by this phenomenon. Early pregnancy loss is understudied, and there are few therapeutic interventions. This study, the Copenhagen Pregnancy Loss (COPL) study, was designed to contribute to the understanding of pregnancy loss through trios of patients (mother, father, and fetus) with clinically diagnosed pregnancy loss, attempting to document sequence diversity and interplay between meiotic recombination and point mutations. This study included 664 cases of early pregnancy loss with 1439 fetal samples (multiple were collected from each loss, where possible). In 467 of the 664 cases, there was at least 1 fetal sample and 1 sample from both parents. A total of 59 losses indicated a higher-than-expected kinship with the mother, and 11 indicated a higher kinship with the father. Whole-genome sequencing (WGS) was used to assess aneuploidies, and detected them in 206 cases. Of these, monosomy X and trisomy 16 were the most common. In addition, 19 large de novo copy number variants (CNVs) were detected in 14 loss cases, none of which were near a common fragile site. Of these 14 cases, 11 were euploid losses and 6 contained aneuploidies. Failure at meiosis I results in the presence of both homologous chromosomes from the same parent. An estimated 27.2% of paternal and 32.3% of maternal triploidies occur at recombination hotspots, supporting the idea of meiosis failure. A total of 15,086 DNMs were pinpointed as paternal and 5967 as maternal, consistent with previous literature supporting a high paternal contribution to DNMs. Consistent with this, paternal triploidies showed a proportionally higher paternal fraction of phased mutations. DNMs shown in maternal triploidies indicated a lower paternal fraction than euploid fetuses. In addition, there was no correlation between sister/homologous state differences for high-AB DNMs in paternal triploidies. When searching for pathogenic single-site variants (SSVs) in the DNMs, 26 genotypes were found that were pathogenic or likely pathogenic; a total of 23 were DNMs and 3 were biallelic predicted loss-of-function variants (pLoF). The frequency of pathogenic SSVs in early pregnancy loss was higher compared with controls [odds ratio (OR) 2.98, P=5.7×10-6), and this effect remained after correction for parental age. These results showed probable genetic causes for pregnancy loss in 254 of 467 cases, including aneuploidies, triploidies, pathogenic SSVs, and de novo CNVs. Most of the genetic causes of loss originated on maternal chromosomes, and fetuses with triploidies had significantly more DNMs than fetuses that were euploid. These results indicate significant sequence diversity in early pregnancy loss, with additional diversity likely present but unidentified in the stages between implantation and clinically recognized pregnancy. Future research should focus on potential explanations for early pregnancy loss that cannot be explained by genetic causes, as well as on potential interventions for these cases.

OBJECTIVE:To investigate associations between endocrine diseases and pregnancy loss, including associations according to the number of pregnancy losses, temporal patterns, and familial clustering independent of the woman's own endocrine diagnosis. DESIGN:Nationwide population-based cohort study. SUBJECTS:366,539 women born in Denmark between 1977 and 1993 with documented pregnancies in the Danish national registers. EXPOSURE:Presence of endocrine disease diagnosed before or after pregnancy. Subgroup analyses of major endocrine diseases, including hypothyroidism, hyperthyroidism, polycystic ovary syndrome (PCOS), type 1 and 2 diabetes, and gestational diabetes. MAIN OUTCOME MEASURES:Pregnancy loss, defined as the number of pregnancy losses before 22 weeks of gestation (categorized as 0, 1, 2, or ≥3 losses) in association with endocrine disease. Multivariable multinomial logistic regression models calculated odds ratios (ORs) with 95% confidence interval adjusted for maternal age and year of pregnancy. Additional analyses examined associations according to timing of endocrine disease diagnosis relative to the first pregnancy and evaluated familial clustering. RESULTS:Endocrine disease was diagnosed in 56,618 women (15%). In analyses that did not account for temporal order, endocrine disease showed progressively stronger associations with increasing number of pregnancy losses: OR 1.15 (95% CI: 1.12-1.17) for one, OR 1.30 (95% CI: 1.24-1.37) for two, and OR 1.81 (95% CI: 1.70-1.93) for three or more (p<0.001). All major endocrine diseases were significantly associated with pregnancy loss. Women diagnosed with an endocrine disease after their first pregnancy showed a stronger association with pregnancy loss in the first pregnancy (OR 1.24 [95% CI: 1.20-1.29]) than those diagnosed before the first pregnancy (OR 1.11 [95% CI: 1.07-1.15]). Familial endocrine disease in a parent or sister was also positively associated with pregnancy loss (OR 1.07 [95% CI: 1.05-1.09] and OR 1.08 [95% CI: 1.03-1.13], respectively), independent of the woman's own endocrine diagnosis. CONCLUSION:Endocrine diseases and pregnancy loss were strongly associated regardless of temporal order, with progressively stronger associations across increasing numbers of pregnancy losses, with significant familial clustering independent of personal diagnosis, suggesting shared genetic or environmental factors. Our findings support consideration of targeted endocrine evaluation in women with pregnancy loss. Further research should determine optimal screening strategies and timing.

STUDY QUESTION:What is the association between periconceptional GLP-1 receptor agonist exposure and risk of obstetric complications? SUMMARY ANSWER:Periconceptional GLP-1 receptor agonist exposure was associated with increased preterm birth risk when used for diabetes treatment (liraglutide aOR 1.70, 95% CI 1.17-2.48; semaglutide aOR 1.84, 95% CI 1.24-2.7) but not for weight management, suggesting the underlying diabetes rather than the medication may be the causal factor. WHAT IS KNOWN ALREADY:GLP-1 receptor agonists are rapidly expanding in use among reproductive-age women for diabetes and obesity treatment. While not approved for use in pregnancy, inadvertent periconceptional exposure occurs frequently. Limited safety data exist, with recent small studies suggesting no increased risk of major congenital malformations, but comprehensive obstetric outcome data remain lacking. STUDY DESIGN SIZE DURATION:This nationwide observational cohort study used data from Danish health registries from October 2009 through December 2023. We analyzed 756 636 singleton pregnancies among 480 231 women, with 529 pregnancies having periconceptional GLP-1 receptor agonist exposure. PARTICIPANTS/MATERIALS SETTING METHODS:We identified periconceptional liraglutide or semaglutide exposure (prescription redemption within 8 weeks before/after last menstrual period) using the National Prescription Register. Exposed pregnancies were stratified by maternal pre-existing diabetes status and compared with propensity score-matched unexposed controls. Propensity score matching incorporated maternal age, BMI, smoking, geographic region, education, pre-existing diabetes, parity, and temporal factors. MAIN RESULTS AND THE ROLE OF CHANCE:Before adjustment, exposed women had higher rates of multiple obstetric complications. After propensity score matching, only the risk of preterm birth remained elevated for exposed women. This increased risk was confined to women using GLP-1 receptor agonists for diabetes treatment (liraglutide aOR 1.70, 95% CI 1.17-2.48; semaglutide aOR 1.84, 95% CI 1.24-2.71). Among women without pre-existing diabetes using these medications for weight management, no association with preterm birth was observed (liraglutide aOR 1.01, 95% CI 0.58-1.76; semaglutide aOR 0.71, 95% CI 0.30-1.70). LIMITATIONS REASONS FOR CAUTION:Study limitations include the absence of data regarding medication compliance post prescription redemption, potential misclassification due to parallel importation, and the inability to control for unmeasured confounding factors. The observational design cannot establish causality. Most semaglutide weight-loss prescriptions occurred late in the study period, limiting long-term follow-up data. WIDER IMPLICATIONS OF THE FINDINGS:The results are compatible with the hypothesis that diabetes-related factors, rather than GLP-1 receptor agonist exposure itself, may contribute to the increased preterm birth risk. If so, there are important implications for preconception counselling and may inform future guidelines for GLP-1 receptor agonist use in reproductive-age women. STUDY FUNDING/COMPETING INTERESTS:KVRH, KB, DW, and HSN acknowledge funding from the Novo Nordisk Foundation (NNF21OC0069257 and NNF220C0077221) and the AP Moller Foundation. LMH was supported in part by grants from NordForsk (id: 105545), the Novo Nordisk Foundation (NNF17OC0027594 and NNF17OC0027812), and the Villum Foundation ('Nation-Scale Social Networks'). KSL was supported by the Independent Research Fund Denmark (8045-00047B), NordForsk (id: 156298), and Centre for Childhood Health (id: 72 2024_F_008 and 2024_I_001). SM: Advisory boards: AstraZeneca, Boehringer Ingelheim, Intarcia Therapeutics, Novo Nordisk, Sanofi, Abbott Lab, Bayer, Amgen; Lecture fees: AstraZeneca, Novo Nordisk, MSD; Research grant recipient: Novo Nordisk; Novo Nordisk foundation, Boehringer Ingelheim; Support for attending meetings and/or travel: Novo Nordisk, Boehringer-Ingelheim, Bayer. Grants were paid to the institution, Hvidovre Hospital, University of Copenhagen, with no personal fee. None of the grants has any relation to the work presented in the paper. SM is also a consultant for Netdoktor and has served as principal investigator in relation to the development of drugs for the treatment of type 2 diabetes and obesity in collaboration with Novo Nordisk and Bayer, with funds paid to the institution where he is employed, with no personal fee and with no relation to the work reported in this article. HSN: lecture fees on own research: Novo Nordisk A/S, Ferring Pharmaceuticals, Merck A/S, Astra Zeneca, Cook Medical, Gedeon Richter and Ibsa Nordic. K.B holds stock/share or stock/share options with Novonesis, Genmab, and Novo Nordisk (held in personal investment portfolio). The remaining authors have no other disclosures. TRIAL REGISTRATION NUMBER:N/A.

STUDY QUESTION:How fast does cell-free fetal DNA (cffDNA) decline after early pregnancy loss and which factors affect the decline? SUMMARY ANSWER:After pregnancy loss cffDNA declines gradually with detectable levels persisting up to 3 days post-tissue passage correlating with β-hCG decline. WHAT IS KNOWN ALREADY:Postpartum clearance of cffDNA occurs within hours, but little is known about its decline following early pregnancy loss. Initial results from the Copenhagen Pregnancy Loss (COPL) study showed slower clearance in relation to pregnancy loss with detectable levels found up to 24 h after tissue passage. STUDY DESIGN, SIZE, DURATION:This prospective cohort study included 1463 women from the COPL cohort, enrolled between 12 November 2020 and 19 December 2022. Participants were divided into three groups based on sampling time: the standard group (samples collected before tissue passage), the delayed sample group (samples collected after tissue passage at maximum 24 h), and the repeated sample group (samples collected at multiple time points after medical and surgical treatment). PARTICIPANTS/MATERIALS, SETTING, METHODS:Eligible women were 18 years old with a confirmed intrauterine pregnancy loss before 22 gestational weeks. Exclusion criteria included ectopic, molar, or unknown-location pregnancies and inability to consent. For the repeated sample group, additional exclusions were vaginal bleeding at diagnosis, anembryonic pregnancies, and opting for expectant management. Blood samples were analyzed for β-hCG and cffDNA, with fetal fraction measured using the sequencing-based fetal fraction (SeqFF) method. In the repeated sampling group, analyses were performed on Days 2 and 3 for surgically treated and Days 7 and 14 for medically treated. MAIN RESULTS AND THE ROLE OF CHANCE:After pregnancy tissue passage, both cffDNA and β-hCG levels declined consistently over time with a corresponding increase in no-call rates. The decline in SeqFF following pregnancy loss occurred more gradually than the immediate clearance previously reported after delivery, remaining measurable for up to 3 days after pregnancy loss, though 30% of samples became inconclusive at this timepoint. The fetal fraction of cffDNA was highest when tissue remained in utero and declined significantly beyond 12-h post-passage (median 4.7% <6 h vs 2.8% >12 h). The no call rate was 9.1% when the tissue was still in situ but increased to 27.1% in the 12- to 24-h post-passage group. Higher β-hCG levels correlated with increased odds of a conclusive cffDNA test (OR 1.30, 95% CI: 1.20-1.43, P < 0.001). β-hCG is accounting for 20% of the variability in fetal fraction measurements. Variability in the decline of cffDNA between groups reflects the differences in the timing of sampling and treatment approaches. LIMITATIONS, REASONS FOR CAUTION:The small size of the repeated sample group may limit the generalizability of the findings. Reliance on the SeqFF method introduces variability, as it may not perform consistently. Adjustment for key confounders including BMI, gestational age, type of pregnancy loss, and timing of sampling was performed, but residual confounding cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS:As a non-invasive method, cffDNA testing offers critical genetic insights for couples facing time-sensitive reproductive decisions or those with recurrent pregnancy loss. The correlation between β-hCG and fetal fraction suggests β-hCG screening could optimize sequencing decisions. These findings support refining cffDNA diagnostics to enhance pregnancy loss evaluation and reproductive care. STUDY FUNDING/COMPETING INTEREST(S):The study was funded by the Ole Kirks Foundation, the BioInnovation Institute Foundation (BII21SG1020554, NNF20SA0066125, and NNF15OC0016662), the Novo Nordisk Foundation (NNF22OC0077221 and NNF23OC0087269), and the A.P. Møller Foundation. The authors have declared their COI. TRIAL REGISTRATION NUMBER:N/A.

Systemic inflammation underlies many chronic diseases, yet its sex-specific genetic architecture remains under-explored. We developed sex-specific polygenic scores (PGSs) for 47 inflammation and vascular stress biomarkers using 10,000 healthy individuals from the Danish Blood Donor Study and evaluated their associations across 12 chronic diseases in the Copenhagen Hospital Biobank (N ≈ 300,000). Genome-wide association studies identified significant associations for 83% of biomarkers, yielding 112 independent loci and 30 significant sex-by-genotype interactions. Individual sex-specific PGSs constructed from these discovery analyses explained an average of 2.6% of phenotypic variance upon validation. We then partitioned genetic risk into four functional domains (innate proinflammation, growth factors/vascular stress, chemokines, and T-cell-associated inflammation). Association mapping across the 12 diseases revealed distinct shared and sex-specific patterns, with genome-wide PGSs capturing broad systemic risk profiles whilst local, cis-restricted PGSs isolated unconfounded aetiological mechanisms. Subsequent clinical classification using these PGSs yielded modest absolute incremental gains in the area under the curve (ΔAUC). However, non-linear machine learning (XGBoost) optimised the added predictive value in over half of the disease-sex groups. These PGSs establish a validated, open-access resource to map baseline inflammation-related genetic liabilities and clarify sex-divergent aetiological mechanisms at a biobank scale.

OBJECTIVE:To study whether a machine learning algorithm can effectively predict fetal genetic status, aneuploid or euploid, in cases of pregnancy loss, based solely on readily available clinical data. Accurate early prediction may enable improved clinical decision-making and personalized patient management. DESIGN:Prospective multicenter cohort study within the Copenhagen Pregnancy Loss study, with a development cohort (n = 788) from Copenhagen University Hospital Hvidovre and external validation cohorts from Copenhagen University Hospitals Herlev (n = 229) and North Zealand (n = 199). Enrollment was from November 2020 to May 2022. SUBJECTS:Women ≥18 years with confirmed intrauterine pregnancy loss before 22 weeks at three Danish Copenhagen University Hospitals. EXPOSURE:Ploidy status was determined using cell-free fetal deoxyribonucleic acid analysis from maternal blood. Forty-five clinical predictors, including anthropometric data, medical history, lifestyle factors, and partner information, were analyzed using a gradient boosted model. Feature importance was interpreted through SHapley Additive exPlanations analysis to elucidate key prognostic indicators. MAIN OUTCOME MEASURES:Model discrimination capability assessed by area under the receiver operating characteristic curve and area under the precision-recall curve, sensitivity, specificity, and calibration across cohorts. RESULTS:The machine learning model demonstrated moderate discriminative ability with a cross-validated area under the receiver operating characteristic curve of 0.69 (95% confidence interval 0.65-0.73) in the development cohort, maintaining equivalent performance in external validations. At a 90% specificity threshold, sensitivity was 54.4% in the development cohort (Hvidovre), 47% at Herlev, and 48% at North Zealand. Key predictors included maternal age, gestational age, paternal age, body mass index, vitamin D supplementation, and vitamin E supplementation. CONCLUSION:This study represents the first machine learning approach to distinguish euploid from aneuploid pregnancy losses using clinical data alone, providing a framework for earlier recognition of women with treatable conditions before multiple losses occur. Novel associations between vitamin supplementation and ploidy status invite further mechanistic investigation. However, moderate discriminative performance and population-specific factors highlight the need for additional validation and exploration before clinical integration.

Abstract Menstrual symptoms vary across the cycle, yet most research assumes a normative 28-day cycle with fixed phase durations, obscuring the physiological relevance of natural cycle variation. Using the mcPHASES dataset, we characterised cycle and phase length variation across 96 menstrual cycles from 37 participants, with ovulation timing estimated from daily urinary luteinizing hormone measurements using a Bayesian hierarchical model, and examined associations with daily symptoms in a subset of 64 cycles from 35 participants with complete symptom data. Twelve physical, mental, and behavioural symptom domains were modelled using Bayesian ordinal regression, with posterior uncertainty in phase-length predictors propagated via a measurement error framework. Total cycle length was not associated with daily symptom burden, except sleep disturbances. By contrast, phase length decomposition revealed systematic associations across multiple domains: longer menstrual phase length was broadly associated with greater symptom intensity spanning physical, gastrointestinal, affective, and sleep domains; longer luteal phase duration was associated with greater fatigue and more frequent headaches, but lower sore breast intensity and lower stress; and longer follicular phase duration and later ovulation were each associated with greater sore breast intensity and more frequent mood swings. These associations require knowledge of actual ovulation timing and cannot be recovered from cycle length alone, indicating that the common assumption of a fixed 14-day luteal phase introduces systematic misclassification of hormonal exposure. Daily symptom intensity was also predominantly person-specific, with cycle phase explaining little of the between-person variance across most symptoms. These findings indicate that calendar-based phase assignment is insufficient for research and clinical assessment of hormone-sensitive conditions, and that person-specific baselines, rather than population-level phase averages, are needed for clinically meaningful symptom monitoring.

Research question Can microchimerism be detected in menstrual blood and is it associated with recurrent pregnancy loss (RPL) or infertility? Design Exploratory cross-sectional study including 48 healthy women (controls), 30 women with RPL and eight women with infertility. All participants collected menstrual blood during the first 48 hours of one menstrual cycle. A peripheral blood sample was collected on cycle day three. Peripheral blood mononuclear cells and menstrual blood mononuclear cells were isolated by density gradient centrifugation. 287 samples from 86 women were examined for microchimerism with a Y-chromosome marker (DYS14) and with the indel method. Additionally, 100 buccal swabs from male partners (n=66) and live children (n=34) were analyzed for microchimerism. Results Microchimerism was detected in menstrual blood in a proportion of women in each study group,and additionally, in peripheral blood with the exception of nulliparous homosexual women. The prevalence of microchimerism detected with DYS14 was higher in menstrual blood compared to peripheral blood for nulliparous heterosexual controls (75% (15/20) vs. 4.8% (1/21)), women with primary RPL (53.3% (8/15) vs. 6.3% (1/16)), and women with infertility (62.5% (5/8) vs. 0% (0/7)). These comparisons were not formally tested.The prevalence of microchimerism in menstrual blood detected with both DYS14 and indel was similar in both condom users and non-users in nulliparous heterosexual controls. Conclusion Microchimerism can be detected in menstrual blood across all groups of investigated women, irrespective of parity. Additionally, we found that microchimerism detected in menstrual blood cannot exclusively be explained by sperm remains. The higher prevalence of DYS14 microchimerism in menstrual blood compared to peripheral blood for some study groups could reflect different accumulation of microchimerism.

• Does adenomyosis increase risk of euploid pregnancy loss (PL)? • Is there a correlation of euploid PL and direct and/or indirect features of adenomyosis? No increased risk of euploid PL was observed in women with ultrasonographic signs of adenomyosis according to the revised MUSA (Morphological Uterus Sonographic Assessment) criteria. Adenomyosis is defined by benign invasion of the endometrium into the myometrium and disruption of the uterine anatomy. Diagnosis by histology is the gold standard, usually after hysterectomy. Recently, focus on non-invasive diagnostics has increased with the revised MUSA criteria for using transvaginal ultrasound for the diagnosis of adenomyosis. The impact of adenomyosis on fertility is conflicting as current literature range from showing no effect to lower live birth rates after in vitro fertilization and increased risk of PL in women with adenomyosis. The included patients are from an ongoing prospective cohort study in Denmark focused on PL from year 2020-2025, n = 2800. In a random sample of 375 women, two trained sonographers conducted a retrospective analysis of 3D ultrasound uterine volumes without Doppler. A questionnaire on physical health symptoms was answered 14 days after the pregnancy loss including questions on menstrual pain Women were included at the time of PL diagnosis. The ploidy status of the fetus, aneuploid or euploid, was determined using cell free fetal DNA analysis or direct sequencing of fetal tissue. 3D ultrasound uterine volumes without Doppler were performed four to eight weeks post-pregnancy loss. The revised MUSA-criteria were applied to describe adenomyosis by 3D ultrasonography by two sonographers. Women in the cohort were aged 20-45 years, with a mean BMI of 23.7 and with a mean gestational age of 9.3 weeks at time of the PL. In the evaluated 375 3D ultrasonographic scans, we did not observe any significant difference in the amount of direct and indirect MUSA features between women with euploid and aneuploid PL. Women with two or more PLs had a higher prevalence of irregular (OR = 1.57, p = 0.04) and disrupted junction zones (OR = 1.63, p = 0.03), compared to women with only one PL. Globular uterus tended to be more frequent in women with more than one PL (OR 1.7, p = 0.09), however insignificantly. Higher cumulative number of MUSA features was associated with higher age of the women, r-value 0.16 (p = 0.001). Women with severe menstrual pain more often experienced euploid PL, OR 1.44 (p = 0.03). The 3D-volumes were without Doppler, resulting in unassessed translesional vascularity, risking underestimating the effect of adenomyosis. Junctional-zone changes are more frequent in women with more than two PLs, indicating that changes in the junctional zone could be involved in the pathogenesis of some PL increasing the recurrence risk. The amount of direct and indirect MUSA features were comparable in women with euploid vs. aneuploid PL. No

STUDY QUESTION:Are there any differences in embryo development and morphokinetics between ICSI and conventional IVF (c-IVF) in first cycle patients without severe male factor infertility? SUMMARY ANSWER:ICSI resulted in fewer usable and high-quality blastocysts on Day 5 compared to c-IVF, with no observed differences in morphokinetics or cleavage patterns among patients without severe male factor infertility. WHAT IS KNOWN ALREADY:Recent randomized controlled trials (RCTs) comparing ICSI and c-IVF have found that ICSI does not improve live birth rates compared to c-IVF when there is no severe male factor. Data on embryo development and morphokinetics between ICSI and c-IVF are lacking. STUDY DESIGN, SIZE, DURATION:This was a secondary analysis of an open-label, multicentre, RCT comparing ICSI and c-IVF in 824 patients without severe male factor infertility, with participants recruited between 29 November 2019 and 14 December 2022. In this secondary study, we aimed to explore potential differences in embryo development between ICSI and c-IVF by using registered data on embryo quality and destiny, along with time-lapse data on embryo morphokinetics and cleavage patterns. All analyses were carried out comparing per protocol results between the ICSI group and the c-IVF group. PARTICIPANTS/MATERIALS, SETTING, METHODS:Eligible participants were women with a male partner who did not have severe male factor infertility, defined as a minimum of 2 million progressively motile spermatozoa, and women/couples using donor sperm. Data were collected on all retrieved oocytes from the study cycles, along with information on embryo development and utilization. Additionally, time-lapse data were extracted and analysed from three trial sites, allowing for the evaluation of morphokinetic parameters and cleavage patterns. MAIN RESULTS AND THE ROLE OF CHANCE:The analysis included 388 participants in the ICSI group and 378 in the c-IVF group. There was no significant difference in the number of retrieved oocytes between the two groups. Compared to c-IVF, ICSI resulted in fewer 1PN and >2PN embryos. However, ICSI was also associated with fewer cleaved embryos on Day 2, lower total number of blastocysts, fewer blastocysts utilized for transfer or vitrification, and fewer high-quality blastocysts on Day 5 (based on Gardner's criteria) (P < 0.05 for all). No significant differences were observed in the number of high-quality embryos on Day 2, high-quality blastocysts on Day 6 or vitrified Day 6 blastocysts. Blastocyst quality in the first embryo transfer did not differ between groups. The time-lapse imaging subgroup analysis included 482 participants (247 in the ICSI group and 235 in the c-IVF group), and assessment of 1846 ICSI-treated and 1900 c-IVF-treated oocytes. When accounting for an expected c-IVF delay, there were no differences in timing of embryo development between ICSI and c-IVF. No significant differences were found in the incidence of multinucleation at the two-cell or four-cell stage, direct cleavage, rolling or reverse cleavage. LIMITATIONS, REASONS FOR CAUTION:Traditional sperm parameters, including sperm count and motility, were used in this study. We did not assess sperm DNA fragmentation or other advanced sperm characteristics, which have been suggested to influence early embryonic development. WIDER IMPLICATIONS OF THE FINDINGS:This is the first study based on data from a randomized trial in which patients were assigned to either ICSI or c-IVF to examine the effect of fertilization method on embryo morphokinetics, cleavage patterns and blastocyst formation. ICSI resulted in fewer available blastocysts and high-quality blastocysts on Day 5 compared to c-IVF, while most other outcomes were comparable between groups. These findings are clinically relevant and add to the growing body of evidence that ICSI should not be preferred over c-IVF in the absence of severe male factor infertility. STUDY FUNDING/COMPETING INTEREST(S):The INVICSI study was supported by an unrestricted grant from Gedeon Richter (to S.B.) and funded by the Capital Region of Denmark (A6606), Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis' Legat (4101466; to S.B.) and Amager/Hvidovre Hospital. The funders had no involvement in the study design, data collection, analysis, interpretation of results, manuscript preparation or the decision to submit the manuscript for publication. S.B. has received a grant from Læge Sofus Carl Emil Friis og Hustru Olga Doris Friis' Fond outside the current work. A.P. has received grants from Gedeon Richter, Ferring Pharmaceuticals, Merck A/S and Cryos, all paid to her institution; consulting fees from IBSA, Ferring Pharmaceuticals, Gedeon Richter, Merck A/S and Cryos; honoraria from Gedeon Richter, Ferring Pharmaceuticals, Merck A/S and Organon; and travel support from Gedeon Richter (paid directly to institution). M.L.G. has received grants from Gedeon Richter (via her institution) and Merck, and consulting fees from Cooper Surgical. B.N. has received grants from Gedeon Richter, Merck, and Ferring, all paid to his institution, and has received support for attending meetings and/or travel from Gedeon Richter and Merck. N.L.C.F. has received grants from Gedeon Richter, Merck and Cryos (all paid to her institution); consulting fees from Merck; and support for attending meetings from Gedeon Richter, Merck, Ferring Pharmaceuticals and IBSA. E.L. has received honoraria from Pfizer (lecture), hospital compensation from Radiometer (equipment validation) and support for attending meetings and/or travel from Gedeon Richter and Merck. She also serves on the advisory board for Astellas Pharma Nordic. H.S.N. has received grants from Freya Biosciences, Ferring Pharmaceuticals, BioInnovation Institute, Ministry of Education, Novo Nordisk Foundation, Augustinus Fonden, Oda og Hans Svenningsens Fond, Demant Fonden, Independent Research Fund Denmark and Ole Kirks Fond; and honoraria from Ferring Pharmaceuticals, Merck, AstraZeneca, Cook Medical, Gedeon Richter, Novo Nordisk and IBSA. A.L.E., L.P., A.Z., M.R.P., A.V.G., D.F.S. and D.W. declare no competing interests. TRIAL REGISTRATION NUMBER:NCT04128904.

The Copenhagen Hospital Biobank-chronic inflammatory disease—inflammatory bowel disease (CHB-CID: IBD) cohort contributes to genetic research in inflammatory bowel disease, including Crohn’s disease and ulcerative colitis. Of the 327,084 enrolled and genotyped individuals in the cohort, 10,626 have been diagnosed with IBD as of May 2023. The CHB-CID: IBD cohort includes both patients without IBD and healthy blood donors as control groups. Clinical data is collected from Danish registries and patient records, including details on hospital contacts, co-morbidities, medication, surgical procedures, and laboratory investigations. The cohort features a wide age range (> 18 years), extensive population coverage representative of Danish adults, and validated IBD diagnoses. Finally, the cohort benefits from continuous recruitment and regular updates of clinical information. The aim is to enhance IBD management and ultimately improve patients’ quality of life.

Every generation, the human genome is shuffled during meiosis and a single fertilized egg gives rise to all of the cells of the body1. Meiotic errors leading to chromosomal abnormalities are known causes of pregnancy loss2,3, but genetic aetiologies of euploid pregnancy loss remain largely unexplained4. Here we characterize sequence diversity in early pregnancy loss through whole-genome sequencing of 1,007 fetal samples and 934 parental samples from 467 trios affected by pregnancy loss (fetus, mother and father). Sequenced parental genomes enabled us to determine both the parental and meiotic origins of chromosomal abnormalities, detected in half of our set. It further enabled us to assess de novo mutations on both homologous chromosomes from parents transmitting extra chromosomes, and date them, revealing that 6.6% of maternal mutations occurred before sister chromatid formation in fetal oocytes. We find a similar number of de novo mutations in the trios affected by pregnancy loss as in 9,651 adult trios, but three times the number of pathogenic small (<50 bp) sequence variant genotypes in the loss cases compared with adults. Overall, our findings indicate that around 1 in 136 pregnancies is lost due to a pathogenic small sequence variant genotype in the fetus. Our results highlight the vast sequence diversity that is lost in early pregnancy.

Ischemic heart disease (IHD) is heterogeneous with respect to onset, burden of symptoms, and disease progression. We hypothesized that unsupervised clustering analysis could facilitate identification of distinct and clinically relevant multimorbidity clusters. We included IHD patients who underwent coronary angiography (CAG) or coronary computed tomography angiography (CCTA) between 2004 and 2016 and used the earliest procedure as the index date. Patient health records were obtained from the Danish National Patient Registry, the Danish National Prescription Registry, and two in-hospital laboratory database systems. Genetic data were obtained from the Copenhagen Hospital Biobank. Using registered pre-index diagnosis codes (n = 3046), patients were clustered by application of the Markov Cluster algorithm. Multimorbidity clusters were then characterized using Cox regressions (new ischemic events, non-IHD mortality, and all-cause mortality) and enrichment analysis to explore both risks and phenotypical characteristics. In a cohort of 72,249 patients with IHD (mean age 63.9 years, 63.1

What is the prevalence of stress, depression and anxiety among couples experiencing pregnancy loss and is this affected by reproductive history? Depression, stress, and anxiety are common after pregnancy loss, especially among women. Couples with prior losses and failed primary treatment are especially vulnerable. Studies of mental health consequences of pregnancy loss present a complex and inconsistent picture and reported rates of depression, stress, and anxiety varies significantly in prior research. Several factors likely contribute to this variability: many studies are relatively old and may not reflect current experiences and cultural norms and samples sizes are generally small which may impact reliability and generalizability of the results. Furthermore, research often focuses solely on the mother, overlooking potential mental health impacts on partners. Prospective cohort study with 2,085 women and 1,212 partners included between November 2020 and December 2024. Women with miscarriage before 22 weeks’ gestation and their partners. Two to eight weeks after the loss, participants completed psychometric scales including Major Depression Index (MDI), Perceived Stress Scale (PSS) and Generalized Anxiety Disorder-7 (GAD-7). Univariate comparison of the psychometric scales to eight covariates were performed on women and partners separately and adjusted for age where relevant. Odds-ratios were found using a proportional-odds model, and confidence intervals and p-values were estimated using a Wald’s test. For women, median (IQR) on the MDI was 15 (9; 25), 253 (12.2%) had moderate/severe depression. Median on the PSS was 18 (13; 24), 1030 women (49.4%) were stressed (PSS>18). Median on the GAD-7 was 6 (2; 9), 499 women (24.1%) had moderate/severe anxiety. Having children was correlated with lower scores, OR (95% CI): MDI: 0.67 (0.57; 0.78), p < 0.001; PSS: 0.75 (0.64; 0.87), p < 0.001; GAD-7: 0.78 (0.67; 0.91), p = 0.004. Prior losses correlated with higher scores: MDI 1.17 (1.10;1.23), p < 0.001; PSS 1.15 (1.09; 1.22), p < 0.001; GAD-7: 1.15 (1.09; 1.22), p < 0.001, as did repeated treatment due to retained tissue: MDI: 1.41 (1.13; 1.76), p = 0.005 and PSS: 1.31 (1.05; 1.63), p = 0.03. Among partners, median score (IQR) on the MDI was 8 (4, 15), 46 (3.8%) had moderate/severe depression. PSS median: 14 (9, 18), 286 (23.6%) were stressed; GAD-7: 3 (0, 6) and 99 (8.3%) had moderate/severe anxiety. Prior losses correlated with higher scores on the MDI: 1.14 (1.06; 1.23), p = 0.007, PSS: 1.12 (1.05; 1.20), P = 0.01, and GAD7: 1.10 (1.03; 1.19), p = 0.03. Studies based on self-reported symptoms have inherent limitations in that participants may over- or under-report symptoms due to recall bias and differences in interpretations of symptom severity. However, the rating scales used are validated and widely used internationally. The results of this study imply that pregnancy loss care could be improved by including mental health assessments for both women and their partners. Couples with prior losses and women where primary treatment is insufficient are especially at risk and tailored care should be provided. No