Viral infection of the ovaries compromises pregnancy and reveals innate immune mechanisms protecting fertility.

Viral infections during pregnancy are a considerable cause of adverse outcomes and birth defects, and the underlying mechanisms are poorly understood. Among those, cytomegalovirus (CMV) infection stands out as the most common intrauterine infection in humans, putatively causing early pregnancy loss. We employed murine CMV as a model to study the consequences of viral infection on pregnancy outcome and fertility maintenance. Even though pregnant mice successfully controlled CMV infection, we observed highly selective, strong infection of corpus luteum (CL) cells in their ovaries. High infection densities indicated complete failure of immune control in CL cells, resulting in progesterone insufficiency and pregnancy loss. An abundance of gap junctions, absence of vasculature, strong type I interferon (IFN) responses, and interaction of innate immune cells fully protected the ovarian follicles from viral infection. Our work provides fundamental insights into the effect of CMV infection on pregnancy loss and mechanisms protecting fertility.

Meiotic Chromosome Structure, the Synaptonemal Complex, and Infertility.

In meiosis, homologous chromosome synapsis is mediated by a supramolecular protein structure, the synaptonemal complex (SC), that assembles between homologous chromosome axes. The mammalian SC comprises at least eight largely coiled-coil proteins that interact and self-assemble to generate a long, zipper-like structure that holds homologous chromosomes in close proximity and promotes the formation of genetic crossovers and accurate meiotic chromosome segregation. In recent years, numerous mutations in human SC genes have been associated with different types of male and female infertility. Here, we integrate structural information on the human SC with mouse and human genetics to describe the molecular mechanisms by which SC mutations can result in human infertility. We outline certain themes in which different SC proteins are susceptible to different types of disease mutation and how genetic variants with seemingly minor effects on SC proteins may act as dominant-negative mutations in which the heterozygous state is pathogenic.

Bi-allelic variants in the ESAM tight-junction gene cause a neurodevelopmental disorder associated with fetal intracranial hemorrhage.

The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies."

Comprehensive Proteomic Analysis Reveals Distinct Features and a Diagnostic Biomarker Panel for Early Pregnancy Loss in Histological Subtypes.

Early pregnancy loss (EPL) is a common event in human reproduction and is classified into histological subtypes such as hydropic abortion (HA) and hydatidiform moles, including complete hydatidiform moles (CHMs) and partial hydatidiform moles (PHMs). However, accurate diagnosis and improved patient management remain challenging due to high rates of misdiagnosis and diverse prognostic risks. Therefore, diagnostic biomarkers for EPL are urgently needed. Our study aimed to identify biomarkers for EPL through comprehensive proteomic analysis. Ten CHMs, six PHMs, ten HAs, and 10 normal control products of conception were used to obtain a proteomic portrait. Parallel reaction monitoring-targeted proteomic and regression analyses were used to verify and select the diagnostic signatures. Finally, 14 proteins were selected and a panel of diagnostic classifiers (DLK1, SPTB/COL21A1, and SAR1A) was built to represent the CHM, PHM, and normal control groups (area under the receiver operating characteristic curve = 0.900, 0.804/0.885, and 0.991, respectively). This high diagnostic power was further validated in another independent cohort (n = 148) by immunohistochemistry (n = 120) and Western blot analyses (n = 28). The protein SPTB was selected for further biological behavior experiments in vitro. Our data suggest that SPTB maintains trophoblast cell proliferation, angiogenesis, cell motility, and the cytoskeleton network. This study provides a comprehensive proteomic portrait and identifies potential diagnostic biomarkers. These findings enhance our understanding of EPL pathogenesis and offer novel targets for diagnosis and therapeutic interventions.

The metabolic characteristic of decidual immune cells and their unique properties in pregnancy loss.

Maternal tolerance to semi- or fully allograft conceptus is a prerequisite for the maintenance of pregnancy. Once this homeostasis is disrupted, it may result in pregnancy loss. As a potential approach to prevent pregnancy loss, targeting decidual immune cells (DICs) at the maternal-fetal interface has been suggested. Although the phenotypic features and functions of DIC have been extensively profiled, the regulatory pathways for this unique immunological adaption have yet to be elucidated. In recent years, a pivotal mechanism has been highlighted in the area of immunometabolism, by which the changes in intracellular metabolic pathways in DIC and interaction with the adjacent metabolites in the microenvironment can alter their phenotypes and function. More inspiringly, the manipulation of metabolic profiling in DIC provides a novel avenue for the prevention and treatment of pregnancy loss. Herein, this review highlights the major metabolic programs (specifically, glycolysis, ATP-adenosine metabolism, lysophosphatidic acid metabolism, and amino acid metabolism) in multiple immune cells (including decidual NK cells, macrophages, and T cells) and their integrations with the metabolic microenvironment in normal pregnancy. Importantly, this perspective may help to provide a potential therapeutic strategy for reducing pregnancy loss via targeting this interplay.

Frequency of Gene Polymorphisms in Admixed Venezuelan Women with Recurrent Pregnancy Loss: Microsomal Epoxy Hydroxylase (rs1051740) and Enos (rs1799983).

Recurrent pregnancy loss (RPL) affects around 2% of women of reproductive age. Primary RPL is defined by ≥2 pregnancy losses and no normal birth delivery. In secondary RPL, the losses are after a normal pregnancy and delivery. Most cases have no clear aetiology, although primary cases are the most complex. Several gene single nucleotide polymorphisms (SNPs) have been associated with RPL. The frequency of some SNPs is increased in women suffering from RLP from Asian or Caucasian races; however, in admixed populations, the information on possible genetic links is scarce and contradictory. This study aimed to assess the frequency of two SNPs present in two different enzymes involved in medical conditions observed during pregnancy. It is a case-control study. Microsomal epoxy hydrolase (mEPH) is involved in detoxifying xenobiotics, is present in the ovaries, and is hormonally regulated. The endothelial nitric oxide synthase (NOS3) that forms nitric is involved in vascular tone. Two SNPs, rs1051740 (mEPH) and rs1799983 (NOS3), were assessed. The study included 50 controls and 63 primary RPL patients. The frequency of mutated alleles in both SNPs was significantly higher in patients (p < 0.05). Double-mutated homozygotes were encountered only in RPL patients (p < 0.05). Genetic polymorphisms rs1051740 and rs1799983 may be involved in primary RPL in the Venezuelan admix population. Genetic studies could provide crucial information on the aetiology of primary RPL.

Decoding recurrent pregnancy loss: insights from comparative proteomics studies.

Recurrent pregnancy loss (RPL) represents a common disorder that affects up to 2% of the women aiming at childbirth with long-term consequences on family and society. Factors contributing to it in more than half of the cases are still unknown. Comparative proteomic analysis can provide new insights into the biological pathways underlining the pathogenesis of RPL. Until now, chorionic villi, decidua, placenta, endometrium, and maternal blood from women with RPL have been analyzed by comparative proteomics studies. In this review, we aimed to provide a critical evaluation of the published comparative studies of RPL on human samples, gathered by systematic literature search using PubMed and Google Scholar databases. We provide a detailed overview of the analyzed materials, proteomics platforms, proposed candidate biomarkers and altered pathways and processes linked with RPL. The top, most identified and validated biomarker candidates from all studies are discussed, followed by bioinformatics analysis of the available high-throughput data and presentation of common altered processes and pathways in RPL. Finally, future directions aimed at developing new and efficient therapeutic strategies are discussed as well.

Estrogen to progesterone ratio is associated with conceptus attachment in dairy cows receiving artificial insemination after Double-Ovsynch but not estrus†.

Prediction of pregnancy survival in lactating dairy cows can be determined by the conceptus attachment timeframe via daily pregnancy-specific protein B (PSPB) monitoring. All factors contributing to reduced fertility in dairy cows receiving AI following estrus detection remain unclear. This study aimed to determine differences in time to conceptus attachment in lactating cows treated with the fertility program Double-Ovsynch compared to cows that were detected in estrus. Additionally, we investigated various pre- and post-conception factors potentially influencing fertility outcomes. We hypothesized that AI following a natural estrus detected with automated activity monitors would lead to an extended time to conceptus attachment and lower PSPB concentrations post-attachment compared to Double-Ovsynch. There were no differences in the average time to conceptus attachments between treatments. However, cows inseminated post-estrus that experienced pregnancy loss between conceptus attachment and 60-66 days post-AI exhibited diminished PSPB concentrations on Days 2 and 3 following conceptus attachment. Steroid hormone interactions were assessed with radioimmunoassay to determine the ratios of estrogen to progesterone concentrations on the day of the luteinizing hormone (LH) surge. Notably, estrogen to progesterone ratio proved to predict conceptus attachment in cows subjected to Double-Ovsynch but not in those inseminated post-estrus detection surge. In conclusion, the estrogen to progesterone ratio measured around the time of the pre-ovulatory LH surge emerges as a potentially effective tool for estimating the fertility potential of lactating dairy cows undergoing timed AI, particularly in the context of the Double-Ovsynch program.

Interleukin-10: a novel metabolic inducer of macrophage differentiation and subsequently contributing to improved pregnancy outcomes of mice by orchestrating oxidative phosphorylation metabolism†.

Metabolism regulates the phenotype and function of macrophages. After recruitment to local tissues, monocytes are influenced by the local microenvironment and differentiate into various macrophages depending on different metabolic pathways. However, the metabolic mechanisms underlying decidual macrophage differentiation remain unknown. Interleukin-10 (IL-10) is an important decidual macrophage inducer and promotes oxidative phosphorylation (OXPHOS) of bone marrow-derived macrophages. In this study, we mainly investigate the metabolic changes involved in IL-10-generated macrophages from monocytes using in vitro models. We demonstrate that exposure of monocytes (either peripheral or THP-1) to IL-10 altered the phenotype and function of resultant macrophages that are linked with OXPHOS changes. Interleukin-10 enhanced the mitochondrial complex I and III activity of THP-1 cell-differentiated macrophages and increased the mitochondrial membrane potential, intracellular adenosine triphosphate, and reactive oxygen species levels. Oxidative phosphorylation blockage with oligomycin changed the cell morphology of IL-10-generated macrophages and the expression levels of cytokines, such as transforming growth factor beta, tumor necrosis factor-alpha, interferon gamma, and IL-10, apart from changes in the expression level of the surface markers CD206, CD209, and CD163. Moreover, in vivo IL-10 administration reduced the lipopolysaccharide (LPS)-induced embryo resorption rate, and this effect was diminished when OXPHOS was inhibited, demonstrating that OXPHOS is important for the improved pregnancy outcomes of IL-10 in LPS-induced abortion-prone mice. Our findings provide deep insights into the roles of IL-10 in macrophage biology and pregnancy maintenance. Nevertheless, the direct evidence that OXPHOS is involved in decidual macrophage differentiation needs further investigations.

Updates to Male Infertility: AUA/ASRM Guideline (2024).

PURPOSE: In 2023 the American Urological Association (AUA) requested an Update Literature Review (ULR) to incorporate new evidence generated since the 2020 publication of this Guideline. The resulting 2024 Guideline Amendment addresses updated recommendations to provide guidance on the appropriate evaluation and management of the male partner in an infertile couple. MATERIALS AND METHODS: In 2023, the Male Infertility Guideline was updated through the AUA amendment process in which newly published literature is reviewed and integrated into previously published guidelines. An updated literature search identified 4093 new abstracts. Following initial abstract screening, 125 eligible study abstracts met inclusion criteria. On data extraction, 22 studies of interest were included in the final evidence base to inform the Guideline amendment. RESULTS: The Panel developed evidence- and consensus-based statements based on an updated review to provide guidance on evaluation and management of male infertility. These updates are detailed herein. CONCLUSIONS: This update provides several new insights, including revised thresholds for Y-chromosome microdeletion testing, indications for pelvic MRI imaging in infertile males, and guidance regarding the use of testicular sperm in nonazoospermic males. This Guideline will require further review as the diagnostic and treatment options in this space continue to evolve.

The epidemiology of pregnancy loss: global burden, variable risk factors, and predictions.

STUDY QUESTION: Is the incidence of pregnancy loss correlated with various geographic, socio-demographic, and age stratifications at the societal and national levels, and what are the risk factors associated with pregnancy loss at the individual level? SUMMARY ANSWER: The epidemiological trends and disease burden of pregnancy loss were correlated with various geographic, socio-demographic, and age stratifications, and we identified that poor health condition, smoking, sedentary behaviour, lower educational level, and lower maternal birth weight may significantly increase the risk of pregnancy loss. WHAT IS KNOWN ALREADY: Several studies have used national, regional, or single-centre data to describe trends in the burden of pregnancy loss, and previous observational studies have identified some variable factors possibly associated with pregnancy loss. However, a comprehensive analysis of global trends and predictions of pregnancy loss are lacking, and the conclusions have been inconsistent. STUDY DESIGN, SIZE, DURATION: We have utilized the data from Global Burden of Disease (GBD) 2019 to provide an overview of the trends in pregnancy loss in 204 countries and regions worldwide from 1990 to 2019, and have made a forecast for the next 10 years. Moreover, we applied a variety of statistical genetics methods to analyse 34 239 pregnancy loss and 89 340 non-pregnancy loss cases from the FinnGen consortium to comprehensively assess the bidirectional causality of variable factors with pregnancy loss from an individual perspective. PARTICIPANTS/MATERIALS, SETTING, METHODS: We analysed trends in the incidence, disability-adjusted life years (DALYs), and maternal mortality of pregnancy loss at global, regional, national, socio-demographic index (SDI), and age levels. The autoregressive integrated moving average (ARIMA) model was used to predict trends by 2030. Finally, we used two-sample Mendelian randomization (MR) and multivariate MR (MVMR) analyses to explore the relationship between the pregnancy loss and variables closely related to physical condition, physical activity, lifestyle, sleep conditions, basic conditions. MAIN RESULTS AND THE ROLE OF CHANCE: In 2019, there were approximately 42.39 million cases of pregnancy loss worldwide. Globally, the incidence, DALYs, and mortality of pregnancy loss showed a decreasing trend between 1990 and 2019, although the number was increasing in some countries. The age-standardized incidence, DALYs, and mortality rate were negatively correlated with SDI level and show a further decline by 2030. Based on MR analyses, we confirmed that genetically predicted overall health rating (inverse-variance weighted (IVW) odds ratio (OR), 1.68; 95% CI, 1.34-2.13; P = 5.10 × 10-6), smoking initiation (IVW OR, 1.26; 95% CI, 1.16-1.38; P = 1.90 × 10-9), sedentary behaviour (IVW OR, 1.56; 95% CI, 1.20-2.01; P = 2.76 × 10-5), educational level (IVW OR, 0.64; 95% CI, 0.55-0.73; P = 6.56 × 10-10), and maternal birth weight (IVW OR, 0.70; 95% CI, 0.58-0.85; P = 2.98 × 10-4) were significantly related to the risk of pregnancy loss, whereas body mass index (IVW OR, 1.10; 95% CI, 1.03-1.17; P = 5.31 × 10-3), alcohol consumption (IVW OR, 1.74; 95% CI, 1.03-2.95; P = 0.04), insomnia (IVW OR, 1.66; 95% CI, 1.14-2.42; P = 7.00 × 10-3), and moderate-to-vigorous physical activity (IVW OR, 0.59; 95% CI, 0.37-0.95; P = 2.85 × 10-2) were suggestively associated with the risk of pregnancy loss. These results were supported by sensitivity and directional analyses. LIMITATIONS, REASONS FOR CAUTION: Despite efforts to standardize GBD data from all over the world, uncertainties in data quality control regarding ascertainment of pregnancy loss, medical care accessibility, cultural differences, and socioeconomic status still exist. Furthermore, the population in the MRstudy was limited to Europeans, which means that the results may not be extrapolated to people of other origins. WIDER IMPLICATIONS OF THE FINDINGS: Our study provides for the first time an overview of the epidemiological trends and disease burden of pregnancy loss related with SDI, region, country, and age, and predicts changes in future trends up to 2030. In addition, findings support that genetic susceptibility, smoking, health condition, and sedentary behaviour may be powerful indicators of an increased risk of pregnancy loss. These results would be beneficial for policy makers of different countries and regions to improve prevention implementation. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants 2021JH2/10300093, from the Science and Technology Projects of Liaoning Province, China. All authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Hydroxychloroquine in recurrent pregnancy loss: data from a French prospective multicenter registry.

STUDY QUESTION: What are the outcomes of pregnancies exposed to hydroxychloroquine (HCQ) in women with a history of recurrent pregnancy loss (RPL), and what factors predict the course of these pregnancies beyond the first trimester? SUMMARY ANSWER: In our cohort of pregnancies in women with a history of RPL exposed to HCQ early in pregnancy, we found that the only factor determining the success of these pregnancies was the number of previous miscarriages. WHAT IS KNOWN ALREADY: Dysregulation of the maternal immune system plays a role in RPL. HCQ, with its dual immunomodulating and vascular protective effects, is a potential treatment for unexplained RPL. STUDY DESIGN, SIZE, DURATION: The FALCO (Facteurs de récidive précoce des fausses couches) registry is an ongoing French multicenter infertility registry established in 2017 that includes women (aged from 18 to 49 years) with a history of spontaneous RPL (at least three early miscarriages (≤12 weeks of gestation (WG)) recruited from several university hospitals. PARTICIPANTS/MATERIALS, SETTING, METHODS: Spontaneous pregnancies enrolled in the FALCO registry with an exposure to HCQ (before conception or at the start of pregnancy) were included. Pregnancies concomitantly exposed to tumor necrosis factor inhibitors, interleukin-1 and -2 inhibitors, intravenous immunoglobulin, and/or intravenous intralipid infusion, were excluded. Concomitant treatment with low-dose aspirin (LDA), low-molecular weight heparin (LMWH), progesterone, and/or prednisone was allowed. All patients underwent the recommended evaluations for investigating RPL. Those who became pregnant received obstetric care in accordance with French recommendations and were followed prospectively. The main endpoint was the occurrence of a pregnancy continuing beyond 12 WG, and the secondary endpoint was the occurrence of a live birth. MAIN RESULTS AND THE ROLE OF CHANCE: One hundred pregnancies with HCQ exposure in 74 women were assessed. The mean age of the women was 34.2 years, and the median number of previous miscarriages was 5. Concomitant exposure was reported in 78 (78%) pregnancies for prednisone, 56 (56%) pregnancies for LDA, and 41 (41%) pregnancies for LMWH. Sixty-two (62%) pregnancies ended within 12 WG, the other 38 (38%) continuing beyond 12 WG. The risk of experiencing an additional early spontaneous miscarriage increased with the number of previous miscarriages, but not with age. The distributions of anomalies identified in RPL investigations and of exposure to other drugs were similar between pregnancies lasting ≤12 WG and those continuing beyond 12WG. The incidence of pregnancies progressing beyond 12 WG was not higher among pregnancies with at least one positive autoantibody (Ab) (i.e. antinuclear Ab titer ≥1:160, ≥1 positive conventional and/or non-conventional antiphospholipid Ab, and/or positive results for ≥1 antithyroid Ab) without diminished ovarian reserve (18/51, 35.3%) than among those without such autoantibody (18/45, 40.0%) (P = 0.63). Multivariate analysis showed that having ≤4 prior miscarriages was the only factor significantly predictive for achieving a pregnancy > 12 WG, after adjustment for age and duration of HCQ use prior to conception (adjusted odds ratio (OR) = 3.13 [1.31-7.83], P = 0.01). LIMITATIONS, REASONS FOR CAUTION: Our study has limitations, including the absence of a control group, incomplete data for the diagnostic procedure for RPL in some patients, and the unavailability of results from endometrial biopsies, as well as information about paternal age and behavioral factors. Consequently, not all potential confounding factors could be considered. WIDER IMPLICATIONS OF THE FINDINGS: Exposure to HCQ in early pregnancy for women with a history of RPL does not seem to prevent further miscarriages, suggesting limited impact on mechanisms related to the maternal immune system. STUDY FUNDING/COMPETING INTEREST(S): The research received no specific funding, and the authors declare no competing interests. TRIAL REGISTRATION NUMBER: clinicaltrial.gov NCT05557201.

A nomogram and risk stratification to predict subsequent pregnancy loss in patients with recurrent pregnancy loss.

STUDY QUESTION: Could the risk of subsequent pregnancy loss be predicted based on the risk factors of recurrent pregnancy loss (RPL) patients? SUMMARY ANSWER: A nomogram, constructed from independent risk factors identified through multivariate logistic regression, serves as a reliable tool for predicting the likelihood of subsequent pregnancy loss in RPL patients. WHAT IS KNOWN ALREADY: Approximately 1-3% of fertile couples experience RPL, with over half lacking a clear etiological factor. Assessing the subsequent pregnancy loss rate in RPL patients and identifying high-risk groups for early intervention is essential for pregnancy counseling. Previous prediction models have mainly focused on unexplained RPL, incorporating baseline characteristics such as age and the number of previous pregnancy losses, with limited inclusion of laboratory and ultrasound indicators. STUDY DESIGN, SIZE, DURATION: The retrospective study involved 3387 RPL patients who initially sought treatment at the Reproductive Immunology Clinic of Renji Hospital, Shanghai Jiao Tong University School of Medicine, between 1 January 2020 and 31 December 2022. Of these, 1153 RPL patients met the inclusion criteria and were included in the analysis. PARTICIPANTS/MATERIALS, SETTING, METHODS: RPL was defined as two or more pregnancy losses (including biochemical pregnancy loss) with the same partner before 28 weeks of gestation. Data encompassing basic demographics, laboratory indicators (autoantibodies, peripheral immunity coagulation, and endocrine factors), uterine and endometrial ultrasound results, and subsequent pregnancy outcomes were collected from enrolled patients through initial questionnaires, post-pregnancy visits fortnightly, medical data retrieval, and telephone follow-up for lost patients. R software was utilized for data cleaning, dividing the data into a training cohort (n = 808) and a validation cohort (n = 345) in a 7:3 ratio according to pregnancy success and pregnancy loss. Independent predictors were identified through multivariate logistic regression. A nomogram was developed, evaluated by 10-fold cross-validation, and compared with the model incorporating solely age and the number of previous pregnancy losses. The constructed nomogram was evaluated using the AUC, calibration curve, decision curve analysis (DCA), and clinical impact curve analysis (CICA). Patients were then categorized into low- and high-risk subgroups. MAIN RESULTS AND THE ROLE OF CHANCE: We included age, number of previous pregnancy losses, lupus anticoagulant, anticardiolipin IgM, anti-phosphatidylserine/prothrombin complex IgM, anti-double-stranded DNA antibody, arachidonic acid-induced platelet aggregation, thrombin time and the sum of bilateral uterine artery systolic/diastolic ratios in the nomogram. The AUCs of the nomogram were 0.808 (95% CI: 0.770-0.846) in the training cohort and 0.731 (95% CI: 0.660-0.802) in the validation cohort, respectively. The 10-fold cross-validated AUC ranged from 0.714 to 0.925, with a mean AUC of 0.795 (95% CI: 0.750-0.839). The AUC of the nomogram was superior compared to the model incorporating solely age and the number of previous pregnancy losses. Calibration curves, DCAs, and CICAs showed good concordance and clinical applicability. Significant differences in pregnancy loss rates were observed between the low- and high-risk groups (P < 0.001). LIMITATIONS, REASONS FOR CAUTION: This study was retrospective and focused on patients from a single reproductive immunology clinic, lacking external validation data. The potential impact of embryonic chromosomal abnormalities on pregnancy loss could not be excluded, and the administration of medication to all cases impacted the investigation of risk factors for pregnancy loss and the model's predictive efficacy. WIDER IMPLICATIONS OF THE FINDINGS: This study signifies a pioneering effort in developing and validating a risk prediction nomogram for subsequent pregnancy loss in RPL patients to effectively stratify their risk. We have integrated the nomogram into an online web tool for clinical applications. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Natural Science Foundation of China (82071725). All authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

The endometrial microbiota and early pregnancy loss.

The human endometrium is a dynamic entity that plays a pivotal role in mediating the complex interplay between the mother and developing embryo. Endometrial disruption can lead to pregnancy loss, impacting both maternal physical and psychological health. Recent research suggests that the endometrial microbiota may play a role in this, although the exact mechanisms are still being explored, aided by recent technological advancements and our growing understanding of host immune responses. Suboptimal or dysbiotic vaginal microbiota, characterized by increased microbial diversity and reduced Lactobacillus dominance, has been associated with various adverse reproductive events, including miscarriage. However, the mechanisms linking the lower reproductive tract microbiota with pregnancy loss remain unclear. Recent observational studies implicate a potential microbial continuum between the vaginal and endometrial niche in patients with pregnancy loss; however, transcervical sampling of the low biomass endometrium is highly prone to cross-contamination, which is often not controlled for. In this review, we explore emerging evidence supporting the theory that a dysbiotic endometrial microbiota may modulate key inflammatory pathways required for successful embryo implantation and pregnancy development. We also highlight that a greater understanding of the endometrial microbiota, its relationship with the local endometrial microenvironment, and potential interventions remain a focus for future research.

A history of recurrent pregnancy loss is associated with increased perinatal complications, but not necessarily a longer birth interval: a population study spanning 18 years.

STUDY QUESTION: Is there a difference in the time interval between the first and second live births among individuals with and without recurrent pregnancy loss (RPL)? SUMMARY ANSWER: Primary RPL (two or more pregnancy losses before the first live birth) is associated with a shorter time interval between the first and second live births compared with individuals without RPL, but this association is reversed in patients with secondary RPL (RPL patients with no or one pregnancy loss before the first live birth). WHAT IS KNOWN ALREADY: There is limited information regarding the ability to have more than one child for patients with RPL. Previous studies have investigated the time to live birth and the live birth rate from the initial presentation to clinical providers. Most of the previous studies have included only patients treated at specialized RPL clinics and thus may be limited by selection bias, including patients with a more severe condition. STUDY DESIGN, SIZE, DURATION: We conducted a population-based retrospective cohort study of 184 241 participants who delivered in British Columbia, Canada, and had at least two recorded live births between 2000 and 2018. The aim was to study the differences in the time interval between the first and second live births and the prevalence of pregnancy complications in patients with and without RPL. Additionally, 198 319 individuals with their first live birth between 2000 and 2010 were studied to evaluate cumulative second live birth rates. PARTICIPANTS/MATERIALS, SETTING, METHODS: Among individuals with at least two recorded live births between 2000 and 2018, 12 321 patients with RPL and 171 920 participants without RPL were included. RPL was defined as at least two pregnancy losses before 20 weeks gestation. Patients with primary RPL had at least two pregnancy losses occurring before the first live birth, while patients with secondary RPL had no or one pregnancy loss before the first live birth. We compared the time interval from the first to second live birth in patients with primary RPL, those with secondary RPL, and participants without RPL using generalized additive models to allow for a non-linear relationship between maternal age and time interval between first and second live births. We also compared prevalence of pregnancy complications at the first and second live births between the groups using non-parametric Kruskal-Wallis H test and Fisher's exact test for continuous and categorical variables, respectively. We assessed the cumulative second live birth rates in patients with primary RPL and those without RPL, among participants who had their first live birth between 2000 and 2010. Cox proportional hazards model was used to estimate and compare hazard ratios between the two groups using a stratified modelling approach. MAIN RESULTS AND THE ROLE OF CHANCE: The adjusted time interval between the first and second live births was the longest in patients with secondary RPL, followed by individuals without RPL, and the shortest time interval was observed in patients with primary RPL: 4.34 years (95% CI: 4.09-4.58), 3.20 years (95% CI: 3.00-3.40), and 3.05 years (95% CI: 2.79-3.32). A higher frequency of pregnancy losses was associated with an increased time interval between the first and second live births. The prevalence of pregnancy complications at the first and second live births, including gestational diabetes, hypertensive disorder of pregnancy, preterm birth, and multiple gestations was significantly higher in patients with primary RPL compared with those without RPL. The cumulative second live birth rate was significantly lower in patients with primary RPL compared with individuals without RPL. LIMITATIONS, REASONS FOR CAUTION: This study may be limited by its retrospective nature. Although we adjusted for multiple potential confounders, there may be residual confounding due to a lack of information about pregnancy intentions and other factors, including unreported pregnancy losses. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study provide information that will help clinicians in the counselling of RPL patients who desire a second child. STUDY FUNDING/COMPETING INTEREST(S): This study was supported in part by a grant from the Canadian Institutes of Health Research (CIHR): Reference Number W11-179912. M.A.B. reports research grants from CIHR and Ferring Pharmaceutical. He is also on the advisory board for AbbVie, Pfizer, and Baxter. The other authors report no conflict of interest. TRIAL REGISTRATION NUMBER: NCT04360564.

Is biochemical pregnancy loss associated with embryo or endometrium? A retrospective cohort study in frozen single embryo transfer of own and donated oocytes.

STUDY QUESTION: Does the use of preimplantation genetic testing for aneuploidies (PGT-A), personalized embryo transfer with endometrial receptivity assay (pET-ERA), or the use of donated oocytes modify the incidence of biochemical pregnancy loss (BPL) in frozen single embryo transfer (FSET)? SUMMARY ANSWER: Following FSET, BPL incidence does not differ between own and donated oocytes, and the use of PGT-A with euploid embryo transfer or pET-ERA results in a similar incidence of BPL compared to cycles without embryo or endometrial analysis. WHAT IS KNOWN ALREADY: BPL occurs frequently after IVF, and many factors have been associated with its incidence. The etiology of BPL is not well known, but the most probable cause seems to be either a low-quality embryo or impaired endometrial maintenance. The impact of techniques diagnosing embryonic ploidy or endometrial receptivity on BPL incidence and the BPL incidence between own and donated oocytes have not been analyzed. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort study analyzing the incidence of BPL over 3741 cycles of FSET derived from own (2399 cycles) and donated (1342 cycles) oocytes between January 2013 and January 2022 in 1736 of which PGT-A, pET-ERA, or both were applied. PARTICIPANTS/MATERIALS, SETTING, METHODS: We defined BPL as a pregnancy diagnosed only by serum ß-hCG > 10 UI/l followed by a decrease that does not result in a clinical pregnancy. Clinical pregnancy was defined as the presence of gestational sac on transvaginal ultrasound. We compared BPL rates among patients undergoing 2399 FSETs from own oocytes, which comprised 1310 cycles of embryos analyzed by PGT-A, 950 cycles of untested embryos, 30 cycles of untested embryos with pET-ERA, and a subgroup of 109 cycles analyzed by both PGT-A and pET-ERA. We also included a total of 1342 FSET cycles from donated oocytes comprising 132, 1055, 140, and 15 cycles in the same groups, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: In FSET from own oocytes, the overall BPL rate per embryo transfer was 8.2% (95% CI [7.09-9.33]). In untested embryo transfers, the BPL rate was 7.5% [5.91-9.37]. In the PGT-A group, the BPL rate was 8.8% [7.32-10.47]. In the pET-ERA group, the rate was 6.7% [0.82-22.07]. In the PGT-A+ERA group, the rate was 6.5% [2.65-12.90]. No significant differences were found (P = 0.626). A multivariate analysis considering clinically meaningful variables that were significantly different among groups, taking the untested embryos/endometrium group as a reference, showed comparable incidences among groups. For PGT-A, the adjusted odds ratio (AdjOR) was 1.154 [0.768-1.735] (P = 0.49) and for PGT-A+ERA 0.885 [0.330-2.375] (P = 0.808). Because of a low number of registered cases in the pET-ERA group, and to prevent statistical errors and convergence issues, this group was excluded from further analysis. In FSET of donated oocytes, the overall BPL rate per embryo transfer was 4.9% [3.76-6.14]. In the PGT-A group, the BPL rate was 6.8% [3.16-12.55]. In the pET-ERA group, the rate was 5.0% [2.03-10.03]. In untested embryo transfers, the rate was 4.7% [3.46-6.10]. No cases occurred in the PGT-A+ERA group, and no significant differences were found (P = 0.578). The multivariate analysis showed comparable incidences among groups. For PGT-A the AdjOR was 1.669 [0.702-3.972] (P = 0.247) and for pET-ERA 1.189 [0.433-3.265] (P = 0.737). The PGT-A+ERA group was eliminated from the model to prevent statistical errors and convergence issues because no BPL cases were registered in this group. In the multivariate analysis, when the sources of oocytes were compared, own versus donated, no significant differences were found in the incidence of BPL. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective cohort study with potential biases. In addition, we were unable to control differences among groups due to modifications in medical or laboratory protocols during this long time period, which may modify the relationships being addressed. Factors previously associated with BPL, such as immunological conditions other than thyroid autoimmunity, were not considered in this study. Limited sample sizes of some groups may limit the statistical power for finding differences that can be present in the general population. WIDER IMPLICATIONS OF THE FINDINGS: BPL may be related to a mechanism not associated with the chromosomal constitution of the embryo or the transcriptome of the endometrium. More studies are needed to explore the factors associated with this reproductive issue. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was available for this study. None of the authors have a conflict of interest to declare with regard to this study. TRIAL REGISTRATION NUMBER: This trial was registered at clinicaltrials.gov (NCT04549909).

The importance of the 'uterine factor' in recurrent pregnancy loss: a retrospective cohort study on women screened through 3D transvaginal ultrasound.

STUDY QUESTION: What is the prevalence of congenital and acquired anomalies of the uterus in women with recurrent pregnancy loss (RPL) of unknown etiology examined using 3D transvaginal ultrasound (US)? SUMMARY ANSWER: Depending on the adopted diagnostic criteria, the prevalence of partial septate uterus varies between 7% and 14% and a T-shaped uterus is 3% or 4%, while adenomyosis is 23%, at least one of type 0, type 1 or type 2 myoma is 4%, and at least one endometrial polyp is 4%. WHAT IS KNOWN ALREADY: ESHRE and the Royal College of Obstetricians and Gynaecologists guidelines on RPL recommend the adoption of the 3D transvaginal US to evaluate the 'uterine factor'. Nevertheless, there are no published studies reporting the prevalence of both congenital and acquired uterine anomalies as assessed by 3D transvaginal US and diagnosed according to the criteria proposed by the most authoritative panels of experts in a cohort of women with RPL. STUDY DESIGN, SIZE, DURATION: This was a retrospective cohort study including 442 women with at least two previous first-trimester spontaneous pregnancy losses (i.e. non-viable intrauterine pregnancies), who referred to the obstetrics and gynecology unit of two university hospitals between July 2020 and July 2023. PARTICIPANTS/MATERIALS, SETTING, METHODS: Records of eligible women were reviewed. Women could be included in the study if: they were between 25 and 42 years old; they had no relevant comorbidities; they were not affected by infertility, and they had never undergone ART; they and their partner tested negative to a comprehensive RPL diagnostic work-up; and they had never undergone metroplasty, myomectomy, minimally invasive treatments for uterine fibroids or adenomyomectomy. Expert sonographers independently re-analyzed the stored 2- and 3D transvaginal US images of all included patients. Congenital uterine anomalies (CUAs) were reported according to the American Society for Reproductive Medicine (ASRM) 2021, the ESHRE/European Society for Gynaecological Endoscopy (ESGE) and the Congenital Uterine Malformation by Experts (CUME) criteria. Acquired uterine anomalies were reported according to the International Federation of Gynecology and Obstetrics (FIGO) and the Morphological Uterus Sonographic Assessment (MUSA) criteria. MAIN RESULTS AND THE ROLE OF CHANCE: The partial septate uterus was diagnosed in 60 (14%; 95% CI: 11-17%), 29 (7%; 95% CI: 5-9%), and 47 (11%; 95% CI: 8-14%) subjects, according to the ESHRE/ESGE, the ASRM 2021, and the CUME criteria, respectively. The T-shaped uterus was diagnosed in 19 women (4%; 95% CI: 3-7%) according to the ESHRE/ESGE criteria and in 13 women (3%; 95% CI: 2-5%) according to the CUME criteria. The borderline T-shaped uterus (diagnosed when two out of three CUME criteria for T-shaped uterus were met) was observed in 16 women (4%; 95% CI: 2-6%). At least one of FIGO type 0, type 1, or type 2 myoma was detected in 4% of included subjects (95% CI: 3-6%). Adenomyosis was detected in 100 women (23%; 95% CI: 19-27%) and was significantly more prevalent in women with primary RPL and in those with three or more pregnancy losses. At least one endometrial polyp was detected in 4% of enrolled women (95% CI: 3-7%). LIMITATIONS, REASONS FOR CAUTION: The absence of a control group prevented us from investigating the presence of an association between both congenital and acquired uterine anomalies and RPL. Second, the presence as well as the absence of both congenital and acquired uterine anomalies detected by 3D US was not confirmed by hysteroscopy. Finally, the results of the present study inevitably suffer from the intrinsic limitations of the adopted classification systems. WIDER IMPLICATIONS OF THE FINDINGS: The prevalence of CUAs in women with RPL varies depending on the classification system used. For reasons of clarity, the US reports should always state the name of the uterine anomaly as well as the adopted classification and diagnostic criteria. Adenomyosis seems to be associated with more severe forms of RPL. The prevalence rates estimated by our study as well as the replicability of the adopted diagnostic criteria provide a basis for the design and sample size calculation of prospective studies. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was used. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Factors affecting biochemical pregnancy loss (BPL) in preimplantation genetic testing for aneuploidy (PGT-A) cycles: machine learning-assisted identification.

PURPOSE: To determine the factors influencing the likelihood of biochemical pregnancy loss (BPL) after transfer of a euploid embryo from preimplantation genetic testing for aneuploidy (PGT-A) cycles. METHODS: The study employed an observational, retrospective cohort design, encompassing 6020 embryos from 2879 PGT-A cycles conducted between February 2013 and September 2021. Trophectoderm biopsies in day 5 (D5) or day 6 (D6) blastocysts were analyzed by next generation sequencing (NGS). Only single embryo transfers (SET) were considered, totaling 1161 transfers. Of these, 49.9% resulted in positive pregnancy tests, with 18.3% experiencing BPL. To establish a predictive model for BPL, both classical statistical methods and five different supervised classification machine learning algorithms were used. A total of forty-seven factors were incorporated as predictor variables in the machine learning models. RESULTS: Throughout the optimization process for each model, various performance metrics were computed. Random Forest model emerged as the best model, boasting the highest area under the ROC curve (AUC) value of 0.913, alongside an accuracy of 0.830, positive predictive value of 0.857, and negative predictive value of 0.807. For the selected model, SHAP (SHapley Additive exPlanations) values were determined for each of the variables to establish which had the best predictive ability. Notably, variables pertaining to embryo biopsy demonstrated the greatest predictive capacity, followed by factors associated with ovarian stimulation (COS), maternal age, and paternal age. CONCLUSIONS: The Random Forest model had a higher predictive power for identifying BPL occurrences in PGT-A cycles. Specifically, variables associated with the embryo biopsy procedure (biopsy day, number of biopsied embryos, and number of biopsied cells) and ovarian stimulation (number of oocytes retrieved and duration of stimulation), exhibited the strongest predictive power.

Transcriptome analysis of adenomyosis eutopic endometrium reveals molecular mechanisms involved in adenomyosis-related implantation failure and pregnancy disorders.

BACKGROUND: Women with adenomyosis are characterized by having defective decidualization, impaired endometrial receptivity and/or embryo-maternal communication, and implantation failure. However, the molecular mechanisms underlying adenomyosis-related infertility remain unknown, mainly because of the restricted accessibility and the difficult preservation of endometrial tissue in vitro. We have recently shown that adenomyosis patient-derived endometrial organoids, maintain disease-specific features while differentiated into mid-secretory and gestational endometrial phase, overcoming these research barriers and providing a robust platform to study adenomyosis pathogenesis and the associated molecular dysregulation related to implantation and pregnancy disorders. For this reason, we aim to characterize the dysregulated mechanisms in the mid-secretory and gestational endometrium of patients with adenomyosis by RNA-sequencing. METHODS: Endometrial organoids were derived from endometrial biopsies collected in the proliferative phase of women with adenomyosis (ADENO) or healthy oocyte donors (CONTROL) (n = 15/group) and differentiated into mid-secretory (-SECorg) and gestational (-GESTorg) phases in vitro. Following RNA-sequencing, the significantly differentially expressed genes (DEGs) (FDR < 0.05) were identified and selected for subsequent functional enrichment analysis and QIAGEN Ingenuity Pathway Analysis (IPA). Statistical differences in gene expression were evaluated with the Student's t-test or Wilcoxon test. RESULTS: We identified 1,430 DEGs in ADENO-SECorg and 1,999 DEGs in ADENO-GESTorg. In ADENO-SECorg, upregulated genes included OLFM1, FXYD5, and RUNX2, which are involved in impaired endometrial receptivity and implantation failure, while downregulated genes included RRM2, SOSTDC1, and CHAC2 implicated in recurrent implantation failure. In ADENO-GESTorg, upregulated CXCL14 and CYP24A1 and downregulated PGR were related to pregnancy loss. IPA predicted a significant inhibition of ID1 signaling, histamine degradation, and activation of HMGB1 and Senescence pathways, which are related to implantation failure. Alternatively, IPA predicted an inhibition of D-myo-inositol biosynthesis and VEGF signaling, and upregulation of Rho pathway, which are related to pregnancy loss and preeclampsia. CONCLUSIONS: Identifying dysregulated molecular mechanisms in mid-secretory and gestational endometrium of adenomyosis women contributes to the understanding of adenomyosis-related implantation failure and/or pregnancy disorders revealing potential therapeutic targets. Following experimental validation of our transcriptomic and in silico findings, our differentiated adenomyosis patient-derived organoids have the potential to provide a reliable platform for drug discovery, development, and personalized drug screening for affected patients.

Increased levels of villus-derived exosomal miR-29a-3p in normal pregnancy than uRPL patients suppresses decidual NK cell production of interferon-γ and exerts a therapeutic effect in abortion-prone mice.

OBJECTIVE: Recurrent pregnancy loss (RPL) patients have higher absolute numbers of decidual natural killer (dNK) cells with elevated intracellular IFN-γ levels leading to a pro-inflammatory cytokine milieu, which contributes to RPL pathogenesis. The main objective of this study was twofold: first to explore the regulatory effects and mechanisms of villus-derived exosomes (vEXOs) from induced abortion patients or RPL patients at the level of intracellular IFN-γ in dNK cells; second to determine the validity of application of vEXOs in the treatment of unexplained RPL (uRPL) through in vitro experiments and mouse models. METHODS: Exosomes were isolated from villus explants by ultracentrifugation, co-cultured with dNK cells, and purified by enzymatic digestion and magnetically activated cell sorting. Flow cytometry, enzyme-linked immunosorbent assays, and RT-qPCR were used to determine IFN-γ levels. Comparative miRNA analysis of vEXOs from induced abortion (IA) and uRPL patients was used to screen potential candidates involved in dNK regulation, which was further confirmed by luciferase reporter assays. IA-vEXOs were electroporated with therapeutic miRNAs and encapsulated in a China Food and Drug Administration (CFDA)-approved hyaluronate gel (HA-Gel), which has been used as a clinical biomaterial in cell therapy for > 30 years. In vivo tracking was performed using 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyaine iodide (DiR) labelling. Tail-vein and uterine horn injections were used to evaluate therapeutic effects of the engineered exosomes in an abortion-prone mouse model (CBA/J × DBA/2 J). Placental growth was evaluated based on placental weight. IFN-γ mRNA levels in mouse placentas were measured by RT-qPCR. RESULTS: IFN-γ levels were significantly higher in dNK cells of uRPL patients than in IA patients. Both uRPL-vEXOs and IA-vEXOs could be efficiently internalized by dNK cells, whereas uRPL-vEXOs could not reduce the expression of IFN-γ by dNK cells as much as IA-vEXOs. Mechanistically, miR-29a-3p was delivered by vEXOs to inhibit IFN-γ production by binding to the 3' UTR of IFN-γ mRNA in dNK cells. For in vivo treatment, application of the HA-Gel effectively prolonged the residence time of vEXOs in the uterine cavity via sustained release. Engineered vEXOs loaded with miR-29a-3p reduced the embryo resorption rate in RPL mice with no signs of systemic toxicity. CONCLUSION: Our study provides the first evidence that villi can regulate dNK cell production of IFN-γ via exosome-mediated transfer of miR-29a-3p, which deepens our understanding of maternal-fetal immune tolerance for pregnancy maintenance. Based on this, we developed a new strategy to mix engineered vEXOs with HA-Gel, which exhibited good therapeutic effects in mice with uRPL and could be used for potential clinical applications in uRPL treatment.