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STUDY QUESTION Are there specific autoantibody profiles in patients with endometriosis that are different from those in controls? SUMMARY ANSWER This study did not reveal a significantly higher prevalence of autoantibodies in the studied groups of patients. WHAT IS KNOWN ALREADY Various inflammatory factors are postulated to be involved in the pathomechanisms of endometriosis, and a potential link exists with autoimmune diseases, which may also play an important role. As the diagnosis of endometriosis remains invasive, it can only be confirmed using laparoscopy with histopathological examination of tissues. Numerous studies have focused on identifying useful biomarkers to confirm the disease, but without unequivocal effects. Autoantibodies are promising molecules that serve as potential prognostic factors. STUDY DESIGN, SIZE, DURATION A multicentre, cross-sectional study was conducted over 18 months (between 2018 and 2019), at eight Departments of Obstetrics and Gynaecology in several cities across Poland on 137 patients undergoing laparoscopic examination for the diagnosis of endometriosis. PARTICIPANTS/MATERIALS, SETTINGS, METHODS During laparoscopy, we obtained plasma samples from 137 patients and peritoneal fluid (PF) samples from 98 patients. Patients with autoimmune diseases were excluded from the study. Autoantibody profiling was performed using HuProt v3.1 human proteome microarrays. MAIN RESULTS AND THE ROLE OF CHANCE We observed no significant differences in the expression of autoantibodies in the plasma or PF between the endometriosis and control groups. The study revealed that in the PF of women with Stage II endometriosis, compared with other stages, there were significantly higher reactivity signals for ANAPC15 and GABPB1 (adj. P 1 in both cases). Comparison of the luteal and follicular phases in endometriosis patients revealed that levels of NEIL1 (adj. P LIMITATIONS, REASONS FOR CAUTION Although this study was performed using the highest-throughput protein array available, it does not cover the entire human proteome and cannot be used to study potentially promising post-translational modifications. Autoantibody levels depend on numerous factors, such as infections; therefore the autoantibody tests should be repeated for more objective results. WIDER IMPLICATIONS OF THE FINDINGS Although endometriosis has been linked to different autoimmune diseases, it is unlikely that autoimmune responses mediated by specific autoantibodies play a pivotal role in the pathogenesis of this inflammatory disease. Our study shows that in searching for biomarkers of endometriosis, it may be more efficient to use higher-throughput proteomic microarrays, which may allow the detection of potentially new biomarkers. Only research on such a scale, and possibly with different technologies, can help discover biomarkers that will change the method of endometriosis diagnosis. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by a grant from the Polish Ministry of Health (grant no. 6/6/4/1/NPZ/2017/1210/1352). It was also funded by the Estonian Research Council (grant PRG1076) and the Horizon 2020 Innovation Grant (ERIN; grant no. EU952516), Enterprise Estonia (grant no. EU48695), and MSCA-RISE-2020 project TRENDO (grant no. 101008193). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER N/A.

STUDY QUESTION Can a combination of the focussed protein kinase assays and a wide-scale proteomic screen pinpoint novel, clinically relevant players in decidualization in vitro and in vivo? SUMMARY ANSWER Rho-dependent protein kinase (ROCK) activity is elevated in response to the combined treatment with progesterone and 8-Br-cAMP during in vitro decidualization, mirrored by increase of ROCK2 mRNA and protein levels and the phosphorylation levels of its downstream target Cofilin-1 (CFL1) in secretory versus proliferative endometrium. WHAT IS KNOWN ALREADY Decidualization is associated with extensive changes in gene expression profile, proliferation, metabolism and morphology of endometrium, yet only a few underlying molecular pathways have been systematically explored. In vitro decidualization of endometrial stromal cells (ESCs) can be reportedly induced using multiple protocols with variable physiological relevance. In our previous studies, cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA)/prolactin axis that is classically upregulated during decidualization showed dampened activation in ESCs isolated from polycystic ovary syndrome (PCOS) patients as compared to controls. STUDY DESIGN, SIZE, DURATION In vitro decidualization studies were carried out in passage 2 ESCs isolated from controls (N = 15) and PCOS patients (N = 9). In parallel, lysates of non-cultured ESCs isolated from proliferative (N = 4) or secretory (N = 4) endometrial tissue were explored. The observed trends were confirmed using cryo-cut samples of proliferative (N = 3) or secretory endometrium (N = 3), and in proliferative or secretory full tissue samples from controls (N = 8 and N = 9, respectively) or PCOS patients (N = 10 for both phases). PARTICIPANTS/MATERIALS, SETTING, METHODS The activities of four target kinases were explored using kinase-responsive probes and selective inhibitors in lysates of in vitro decidualized ESCs and non-cultured ESCs isolated from tissue at different phases of the menstrual cycle. In the latter lysates, wide-scale proteomic and phosphoproteomic studies were further carried out. ROCK2 mRNA expression was explored in full tissue samples from controls or PCOS patients. The immunofluorescent staining of phosphorylated CFL1 was performed in full endometrial tissue samples, and in the in vitro decidualized fixed ESCs from controls or PCOS patients. Finally, the cellular migration properties were explored in live in vitro decidualized ESCs. MAIN RESULTS AND THE ROLE OF CHANCE During in vitro decidualization, the activities of PKA, protein kinase B (Akt/PKB), and ROCK are increased while the activity of casein kinase 2 (CK2) is decreased; these initial trends are observable after 4-day treatment (P LARGE SCALE DATA Proteomic and phosphoproteomic data are available via ProteomeXchange with identifier PXD026243. LIMITATIONS, REASONS FOR CAUTION The number of biological samples was limited. The duration of protocol for isolation of non-cultured ESCs from tissue can potentially affect phosphorylation pathways in cells, yet the possible artefacts were minimized by the identical treatment of proliferative and secretory samples. WIDER IMPLICATIONS OF THE FINDINGS The study demonstrated the benefits of combining the focussed kinase activity assay with wide-scale phosphoproteomics and showed the need for detailed elaboration of the in vitro decidualization protocols. ROCK was identified as the novel target of interest in decidualization, which requires closer attention in further studies—including the context of decidualization-related subfertility and infertility. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the Estonian Ministry of Education and Research, and the Estonian Research Council (PRG1076, PRG454, PSG230 and PSG608), Enterprise Estonia (EU48695), Horizon 2020 innovation grant (ERIN, Grant no. EU952516) of the European Commission, the COMBIVET ERA Chair, H2020-WIDESPREAD-2018-04 (Grant agreement no. 857418), the Academy of Finland (Project grants 315921 and 321763), the Finnish Medical Foundation and The Sigrid Juselius Foundation. The authors confirm that they have no conflict of interest with respect to the content of this article.

Study question Is there a difference in the proportions of preovulatory follicle cell types between normo- and hyporesponders? Summary answer Human preovulatory follicles consist of 14 distinct cell types, 3 of them are significantly different between normo- and hyporesponder patients. What is known already Human ovarian follicles are a diverse dynamic environment for oocytes to mature and ovulate. Follicles consist of multiple cell types in layers and altogether they are responsible for a repertoire of biological processes. Dysregulations in follicular cells could result in hyporesponsiveness to ovarian stimulation. Hyporesponse might lead to the failure of IVF, due to a low number of retrieved oocytes. Its pathophysiological mechanisms have not been fully elucidated so far. This condition affects up to 25% of patients and investigating their follicular cell populations in detail is highly informative for increasing their prognosis for the live birth outcome. Study design, size, duration Study groups consisted of IVF patients with normoresponse (n = 10) and hyporesponse (n = 9) to rFSH stimulation according to antagonist protocol undergoing in vitro fertilization treatment at Nova Vita Clinic, Estonia. Hyporesponse to treatment was diagnosed if ≥ 200 IU of rFSH was administered to retrieve an oocyte. All study participants were ≤40 years of age with matched BMI and normal ovarian reserve. Women with polycystic ovary morphology and ovarian abnormalities observed by ultrasound examination were excluded. Participants/materials, setting, methods Oocyte pick-up was performed for patients after 36 hours of hCG trigger. Cumulus-oocyte complex was removed from the follicular fluid for IVF and the remaining cells were collected by centrifugation and treated with hyaluronidase and DNase. Genome-wide single-cell RNA-seq was performed for >6000 individual cells of 3 normoresponder patients each. RNA-seq was performed for pooled follicular cells of each participant. CIBERTSORTx software was used to deconvolute the proportion of cell types from bulk RNA-seq data. Main results and the role of chance By analyzing a total number of 24 213 single cells from preovulatory follicles, we identified 4 immune cell lineages (CD45+), including neutrophils, T cells, M1 and M2 macrophages along with 10 non-immune cell lineages (CD45-): epithelial cells and theca cells, cumulus cells and 7 subtypes of granulosa cells with different functional transcriptomic profiles (p Limitations, reasons for caution The small sample size limits the study. Wider implications of the findings Our work advances understanding the heterogeneity of cell types in human preovulatory follicles. The differences in the proportions of cell types of preovulatory follicles of hyporesponders could provide valuable insight for assisting IVF treatment by introducing potential therapeutic targets to improve their live birth outcome. Trial registration number not applicable

Study question Does the functionally active microbial composition at the receptive phase endometria differ between women with recurrent implantation failure (RIF) and healthy controls? Summary answer Women with RIF have different functionally active microbiota in the receptive-phase endometria that are involved in distinct metabolical pathways when compared to healthy women. What is known already Recurrent implantation failure (RIF) is a common and burdening diagnosis in infertility treatment. Despite scientific advances in the field, the cause of RIF remains unclear and the treatment options ambiguous. Microorganisms have important role in human physiology and pathophysiology, and their potential involvement in endometrial functions and health are gaining support. Our previous work of microbiota mapping of healthy endometrium identified over 5000 functionally active microorganisms (bacteria, viruses, fungi, archaea) that participate in key metabolic pathways for embryo implantation. In the present work we focus on microbiota mapping in RIF patients, where endometrial dysfunction is suspected. Study design, size, duration A case-control age-match study of 20 RIF and 20 healthy controls was performed. Endometrial samples were obtained at the mid-secretory phase (LH + 7/8) using Pipelle curette. Participants/materials, setting, methods Total RNA sequencing (RNA-seq) was performed. R package MetagenomeSeq was used for metataxonomic microorganismal analyses and HUMAnN2 algorithm was used for functional metagenome assessment. The microbial maps were created using Kraken metagenomic sequence classification. With this approach we were able to identify and map the ‘active’ microorganisms in samples. Main results and the role of chance We identified a total of 4175 microorganisms in the human endometrial samples including different bacteria, fungi, viruses, and archaea. At the taxonomic level, RIF patients demonstrated a total of 180 microorganism with differential abundance when compared to controls (FDR Limitations, reasons for caution Poly A enrichment could avoid the detection of some microorganism. However, the resolution power in microbial identification was comparable to previous metatranscriptomics published studies. Wider implications of the findings Our study findings are first to demonstrate that alive microbes are involved in the uterine pathology as is RIF, where different metabolic pathways could be dysregulated. This knowledge could help to develop pre-/probiotic strategies to modulate uterine environment for improving endometrial functions in RIF. Trial registration number not applicable

Study question Does the endometrial transcriptome profile differ between infertile women with or without a recurrent implantation failure (RIF)? Summary answer Although two different clusters emerged from the endometrial transcriptome data, these were not associated with clinical phenotype (RIF vs non-RIF). What is known already Despite the transfer of morphologically ‘good-quality’ embryos in IVF/ICSI, implantation failure often occurs, which may be explained by impaired endometrial receptivity. In order to guide prognosis and use effective therapeutic interventions, identifying a gene expression profile predictive of endometrial receptivity as well as implantation failure, would be of great value. Additionally, transcriptome analysis may also shed light on alterations in biological processes responsible for the implantation failure. Thousands of potential biomarkers for endometrial receptivity have already been identified by transcriptomic approach, however due to differences in study methodology, there is little overlap of markers between studies. Study design, size, duration Endometrial tissue was obtained from a cohort of 141 infertile women undergoing endometrial scratching within a randomised controlled trial (RCT) (SCRaTCH trial, NL5193/NTR5342). Briefly, women aged 18-44 years with failed implantation after one full IVF/ICSI cycle and planning a subsequent IVF/ICSI cycle, were eligible. Participants were followed-up until 12 months after randomisation, with the primary outcome being live birth, defined as the delivery of at least one live foetus after 24 weeks of gestation. Participants/materials, setting, methods Endometrial tissue was obtained with an endometrial biopsy catheter in the midluteal phase of a natural cycle preceding subsequent IVF/ICSI. Biopsies were snap-frozen and stored at -80 °C until use. After thawing, total RNA isolation, library preparation and paired-end RNA-sequencing were performed. Raw data was preprocessed and mapped to GRCh38. Reads (counts per million) were normalised using library size. Differential gene expression (DGE) analysis was conducted using the EdgeR package with significance threshold FDR Main results and the role of chance Out of 141 endometrium samples, 107 were included in the RNA-sequencing based on RNA quality. For DGE analysis, data of two groups were compared: the ‘fertile’ group, women with a live birth after ≤3 good quality embryo(s) transfers (n = 23), and the RIF group, women with no live birth after ≥3 good quality embryo(s) transfers (n = 23). Two clusters were visible in the principle component analysis (PCA) plot showing transcriptome data of the fertile and RIF samples (cluster 1, n = 29; cluster 2, n = 10), which was not explained by clinical phenotype, as both clusters contained samples of both the fertile and RIF group. DGE analysis between the fertile and RIF group resulted in respectively 3 significantly upregulated and 0 significantly downregulated genes, whereas DGE analysis between the two clusters resulted in 2,235 significantly upregulated and 2,162 significantly downregulated genes. Enrichment analysis of differentially expressed genes between both clusters demonstrated upregulation of enriched terms mainly annotated to cell migration and downregulation of enriched terms mainly annotated to lipid and mitochondrial metabolism. Limitations, reasons for caution A strength of the study is the large number of samples included. Bulk RNA-sequencing was conducted and there was a variation in LH-based timing of the biopsies (5-8 days after LH surge) for which adjustments of the transcriptome data for tissue cellular composition and menstrual cycle were performed. Wider implications of the findings Future studies investigating underlying biological mechanisms in the endometrium in (in)fertility by a (multi-)omics analysis approach with standardised methodology are required to obtain consistencies in relevant biomarkers/pathways, and in due course create possibilities to improve and personalise care for infertile couples. Trial registration number NL5193/NTR5342

Study question What changes occur in the endometrium during ageing and how they may affect fertility? Summary answer The endometrial transcriptome of women of advanced maternal age is significantly different from the young women, indicating specific pathways involved in endometrial ageing. What is known already A woman’s peak reproductive years are considered in her twenties. Trending postponed family planning, unfortunately, brings more women in their late forties to fertility specialists to seek for assisted conception. In vitro fertilization (IVF) using donated oocytes is a common approach to overcome the impact of maternal age on ovarian reserve. However, even with the implementation of embryo that underwent pre-implantation genetic testing, the IVF success rate drops significantly in the late forties. It still remains unclear which age-related molecular processes take place in the endometrium and whether it may impact the ability to support embryo implantation. Study design, size, duration Endometrial transcriptome profiling was done in 44 women undergoing endometrial receptivity evaluation at hormonal replacement therapy before IVF. Patients younger than 29 were considered as young maternal age group (YMA, age 23-27) and women older than 45 were considered as advanced maternal age group (AMA, age 47-50). Participants/materials, setting, methods Endometrial biopsies were obtained on day 5 of progesterone treatment and RNA was extracted. All endometrial samples were evaluated as receptive based on the expression of 57 common endometrial receptivity markers. Study group samples (12 YMA + 12 AMA) were subject to Illumina RNA sequencing. The sequences were annotated using the RefSeq database and differential expression analysis was performed using DeSeq2.We validated our results (10 YMA + 10 AMA) usingquantitative-PCR and histological validation. Main results and the role of chance A total of 37228 mRNA transcripts were expressed in the analyzed endometrial samples. After multiple testing corrections, 144 significantly differentially expressed(DE) transcripts (92 up-regulated, 52 down-regulated) were identified in the endometrium of the AMA versus YMAgroup. Overexpressed genes were associated with decidualization (ALDH3A1), endometrial receptivity (EML5, GALNT12), cell cycle (CDKN2A) and signal transduction, while down-regulated genes included sugar metabolism and inflammation (C2CD4B, NFKB), cellular motility (SPAG6)and progesterone signaling (RPL9). The pathways most affected by age were cellular remodeling, cell motility and migration, and immune response. Interestingly, some of the identified DE genes have been previously associated with ageing. Our results suggest the involvement of p16-associated cellular senescence and the suppression of metabolic and inflammatory processes essential for endometrial preparation for embryo transfer. Limitations, reasons for caution The study includes only patients undergoing hormonal replacement therapy and it is unclear whether the same processes are affected by age in the natural cycles. Wider implications of the findings These findings allow us to explain the age-related molecular changes that take place in the endometrial tissue. Understanding these alterations and using them in assisted reproductive technology may help to improve infertility management in women with advanced reproductive age. Trial registration number None

STUDY QUESTION What is the physiological role of transforming growth factor-beta (TGF-β1) and syndecans (SDC1, SDC4) in endometriotic cells in women with endometriosis? SUMMARY ANSWER We observed an abnormal, pro-invasive phenotype in a subgroup of samples with ovarian endometriosis, which was reversed by combining gene silencing of SDC1 with the TGF-β1 treatment. WHAT IS KNOWN ALREADY Women with endometriosis express high levels of TGF-β1 and the proteoglycan co-receptors SDC1 and SDC4 within endometriotic cysts. However, how SDC1 and SDC4 expression is regulated by TGF-β1 and the physiological significance of the high expression in endometriotic cysts remains unknown as does the potential role in disease severity. STUDY DESIGN, SIZE, DURATION We utilized a pre-validated panel of stem- and cancer cell-associated markers on endometriotic tissue (n = 15) to stratify subgroups of women with endometriosis. Furthermore, CD90+CD73+CD105+ (SC+) endometriotic stromal cells from these patient subgroups were explored for their invasive behaviour in vitro by transient gene inhibition of SDC1 or SDC4, both in the presence or absence of TGF-β1 treatment. PARTICIPANTS/MATERIALS, SETTING, METHODS Endometriotic cyst biopsies (n = 15) were obtained from women diagnosed with ovarian endometriosis (ASRM Stage III–IV). Gene expression variability was assessed on tissue samples by applying gene clustering tools for the dataset generated from the pre-validated panel of markers. Three-dimensional (3D) spheroids from endometriotic SC+ were treated in vitro with increasing doses of TGF-β1 or the TGFBRI/II inhibitor Ly2109761 and assessed for SDC1, SDC4 expression and in vitro 3D-spheroid invasion. Transcriptomic signatures from the invaded 3D spheroids were evaluated upon combining transient gene silencing of SDC1 or SDC4, both in presence or absence of TGF-β1 treatment. Furthermore, nanoscale changes on the surface of endometriotic cells were analysed after treatment with TGF-β1 or TGFBRI/II inhibitor using atomic force microscopy. MAIN RESULTS AND THE ROLE OF CHANCE Gene clustering analysis revealed that endometriotic tissues displayed variability in their gene expression patterns; a small subgroup of samples (2/15, Endo-hi) exhibited high levels of SDC1, SDC4 and molecules involved in TGF-β signalling (TGF-β1, ESR1, CTNNB1, SNAI1, BMI1). The remaining endometriotic samples (Endo-lo) showed a uniform, low gene expression profile. Three-dimensional spheroids derived from Endo-hi SC+ but not Endo-lo SC+ samples showed an aberrant expression of SDC1 and exhibited enhanced 3D-spheroid invasion in vitro, upon rhTGF-β1 treatment. However, this abnormal, pro-invasive response of Endo-hi SC+ was reversed upon gene silencing of SDC1 with the TGF-β1 treatment. Interestingly, transcriptomic signatures of 3D spheroids silenced for SDC1 and consecutively treated with TGF-β1, showed a down-regulation of cancer-associated pathways such as WNT and GPCR signalling. LARGE SCALE DATA Transcriptomic data were deposited in NCBI’s Gene Expression Omnibus (GEO) and could be retrieved using GEO series accession number: GSE135122. LIMITATIONS, REASONS FOR CAUTION It is estimated that about 2.5% of endometriosis patients have a potential risk for developing ovarian cancer later in life. It is possible that the pro-oncogenic molecular changes observed in this cohort of endometriotic samples may not correlate with clinical occurrence of ovarian cancer later in life, thus a validation will be required. WIDER IMPLICATIONS OF THE FINDINGS This study emphasizes the importance of interactions between syndecans and TGF-β1 in the pathophysiology of endometriosis. We believe that this knowledge could be important in order to better understand endometriosis-associated complications such as ovarian cancer or infertility. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by Cancerfonden (CAN 2016/696), Radiumhemmets Forskningsfonder (Project no. 154143 and 184033), EU MSCA-RISE-2015 project MOMENDO (691058), Estonian Ministry of Education and Research (IUT34-16), Enterprise Estonia (EU48695) and Karolinska Institute. Authors do not have any conflict of interest.

STUDY QUESTION Is the composition of microRNAs (miRNAs) in uterine fluid (UF) of women with recurrent implantation failure (RIF) different from that of healthy fertile women? SUMMARY ANSWER The composition of miRNAs in UF of women with RIF is different from that of healthy fertile women and the dysregulated miRNAs are associated with impaired endometrial receptivity and embryo implantation. WHAT IS KNOWN ALREADY It has previously been demonstrated that the miRNAs secreted from endometrial cells into the UF contribute to the achievement of endometrial receptivity. Endometrial miRNAs are dysregulated in women with RIF. STUDY DESIGN, SIZE, DURATION In this descriptive laboratory case–control study, miRNA abundancy was compared between UF collected during implantation phase from healthy fertile women (n = 17) and women with RIF (n = 34), which was defined as three failed IVF cycles with high-quality embryos. PARTICIPANTS/MATERIALS, SETTING, METHODS Recruitment of study subjects and sampling of UF were performed at two university clinics in Stockholm, Sweden and Tartu, Estonia. The study participants monitored their menstrual cycles using an LH test kit. The UF samples were collected on Day LH + 7–9 by flushing with saline. Samples were processed for small RNA sequencing and mapped for miRNAs. The differential abundance of miRNAs in UF was compared between the two groups using differential expression analysis (DESeq2). Further downstream analyses, including miRNA target gene prediction (miRTarBase), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (g:Profiler) and external validation using relevant published data, were performed on the dysregulated miRNAs. Two miRNAs were technically validated with quantitative real-time PCR (RT-PCR). MAIN RESULTS AND THE ROLE OF CHANCE After processing of the sequencing data, there were 15 samples in the healthy fertile group and 33 samples in the RIF group. We found 61 differentially abundant UF miRNAs (34 upregulated and 27 downregulated) in RIF compared to healthy women with a false discovery rate of LARGE SCALE DATA The data are available in Gene Expression Omnibus (GEO) at https://www.ncbi.nlm.nih.gov/geo/ with GEO accession number: GSE173289. LIMITATIONS, REASONS FOR CAUTION This is a descriptive study with a limited number of study participants. Moreover, the identified differentially abundant miRNAs should be validated in a larger study cohort, and the predicted miRNA target genes and enriched pathways in RIF need to be confirmed and further explored in vitro. WIDER IMPLICATIONS OF THE FINDINGS RIF is a major challenge in the current IVF setting with no diagnostic markers nor effective treatment options at hand. For the first time, total miRNAs have been extensively mapped in receptive phase UF of both healthy women with proven fertility and women diagnosed with RIF. Our observations shed further light on the molecular mechanisms behind RIF, with possible implications in future biomarker and clinical treatment studies. STUDY FUNDING/COMPETING INTEREST(S) This work was financially supported by the Swedish Research Council (2017-00932), a joint grant from Region Stockholm and Karolinska Institutet (ALF Medicine 2020, FoUI-954072), Estonian Research Council (PRG1076), Horizon 2020 innovation (ERIN, EU952516) and European Commission and Enterprise Estonia (EU48695). The authors have no competing interests to declare for the current study.

Study question To investigate the prevalence and effect of (mosaic) de novo genomic aberrations in recurrent pregnancy loss (RPL) and sporadic abortion (SA). Summary answer Prevalence of maternal uniparental disomies (UPDs) was high in both cohorts. While chromosomal UPDs were found in both cohorts, genome wide UPDs were RPL specific. What is known already Spontaneous abortion occurs in 10–15% of clinically recognized pregnancies and recurrent pregnancy loss in 1–3%. SA and RPL are associated with reduced quality of life. Multiple factors contribute to SA and RPL, such as uterine malformations and parental/fetal chromosomal abnormalities. However, in ∼60% of SA and RPL the cause remains unknown. UPD is defined as the presence of two homologues chromosomes originating from a single parent. This phenomenon can lead to imprinting disorders that are characterised by clinical features affecting growth, development and metabolism in liveborn offspring. However, it could also be responsible for pregnancy loss. Study design, size, duration We recruited 32 families with pregnancy loss (n = 16 RPL cohort, n = 16 SA cohort) with no known genetic predispositions and normal karyotyping results in both parents and the fetus. Average maternal age was 28.68 years (SD = 5.43), paternal age 30.3 years (SD = 5.53), and the gestational age at pregnancy loss was 8.65 weeks (SD = 2.47). The average number of miscarriages in the RPL group was 3.57 (SD = 0.84). We profiled the genomic landscape of both cohorts using SNP typing. Participants/materials, setting, methods We isolated DNA from blood of both parents and the placental tissues from the miscarried products of conception. The placenta tissues were sampled from two distinct extraembryonic and embryonic germ layers, the extraembryonic mesoderm and the chorionic villi cytotrophoblast. Subsequently, we performed SNP-genotyping using Illumina’s Global-Screening Array–24 v2.0 BeadChips and applied haplarithmisis to delineate allelic architecture of fetal tissues of both cohorts. This allowed us to detect large de novo copy-number and -neutral (>10kb) changes. Main results and the role of chance In this pilot study, we have analyzed 132 DNA samples (n = 32 families), of which 16 families were in the RPL cohort and 16 in the SA cohort. Within the RPL cohort, we found: one family with mosaic genome wide hexaploidy both in the extraembryonic mesoderm and chorionic villi, one family with a non-mosaic genome wide hetero UPD of the chorionic villi tissue, one family with a mosaic UPD of chromosome 14 in both tissues and tetraploidy exclusively in the chorionic villi, one family with a mosaic UPD of chromosome 16 in both tissues, one family with a mosaic UPD of chromosome 6 in both tissues, and another family with a mosaic UPD of chromosome 5 in the extraembryonic mesoderm. Within the SA group, one family showed a UPD of chromosome 7 and another family showed a segmental UPD of chromosome 5 in both tissues. Strikingly, all the UPDs found in this study were maternal in origin. Limitations, reasons for caution The main limitation of this study is the resolution of detecting copy-neutral and copy-number variations, which is an inherent limiting factor of SNP-array technology. In addition, in the sample in which we observed non-mosaic genome wide UPD, maternal contamination is likely that can be investigated by other technologies. Wider implications of the findings: Multiple genome wide UPDs are found in the RPL group but none in the SA group, indicating an association between genome wide mosaic UPD and RPL. These findings could lead to a better understanding of causative factors for SA and RPL and the need for a SNP-based non-invasive prenatal testing. Trial registration number Not applicable

Study question What is the qualitative and quantitative profile of microRNAs (miR) and their sequence variants - isomiRs, and how it changes during the menstrual-cycle phase transitions? Summary answer Time-course analysis of endometrial miR/isomiR profiles has shown that menstrual-phase transitions cause widespread and complex changes in miR gene expression and processing. What is known already Embryo implantation depends on the receptivity of the endometrium during the window of implantation, when ovarian hormones and genetic factors coordinate the development of the uterine lining and prepare it for embryo implantation. The most important factors for successful implantation studied so far are the embryo itself, the histological dating of the endometrium and its molecular genetic characteristics, including miRs. With the rapid development of next-generation sequencing technologies, it has become clear that miR genes have the potential to produce not only miR but also variants (isomiRs) thereof, which can differ in sequence and length and can be functionally significant. Study design, size, duration miR/isomiR landscape was assessed by small RNA sequencing of endometrial biopsy samples at 4 time points of endometrial cycle covering the proliferative and secretory phases. Healthy, fertile, female volunteers took part in the study lasting one and a half years. For accurate phase dating, human chorionic gonadotropin (hCG) was administrated, and ultrasonic, histological and hormonal assessments were done at each time point. Statistically significant data of miR/isomiR identification and expression dynamics was considered for analysis. Participants/materials, setting, methods Participant choice criteria - at least one child born, problem-free pregnancies, no diseases or allergies; hCG application time determined according follicle and endometrium ultrasound scanning, and ovarian hormone levels; endometrial biopsies taken at hGC (before hormone application), hGC+2, hGC+7, hGC+9 time points; small RNAseq completed by Karolinska Institute, Sweden; miR/isomiR identified using local Galaxy instance with an in-built workflow and tools developed by our laboratory; differential expression and target prediction evaluated with DESeq2 and miRDB,resp. Main results and the role of chance Within the cohort of patients, across the four study time points, the small RNAseq data revealed numbers of miRs and isomiRs to be changed. The largest statistically significant changes in their expression were found at LH + 9. The miR families that showed the largest number of members with altered expression were miR125a, miR30d, miR449c, miR92a/b and miR99a. The expression levels tended to decrease in the miR125a and miR92a families and to increase in the miR10a and miR449c families during the three studied time points of the cycle compared to the proliferative phase. Among those affected, the number of isomiRs, including templated and non-templated isomiRs, was much higher than that of miRs. For example, the ratio of the significantly altered miRs/templated isomiRs/non-templated isomiRs was 6/16/11 at LH + 9. Templated isomiRs of hsa-miR–148a–3p, hsa-miR–30d–5p and hsa-miR–449c–5p were among the most upregulated, while several templated and non-templated isomiRs of hsa-miR–125–5p were the most downregulated at LH + 9. Of particular interest are those isomiRs, in which the seed site is shifted compared to the reference miRs and results in altered target transcripts. Target prediction of the most affected isomiR of hsa-miR–449c–5p identified new targets of target scores much higher than of the reference miR. Limitations, reasons for caution IsomiRs are a source of novel biomarkers for clinical diagnosis. An important next step is the validation of the in-silico predicted miRs/isomiRs and their target transcripts by RT-qPCR in larger number of individuals. Expression profiles should be associated with the dominant cell type in the endometrial biopsy preparation. Wider implications of the findings: MiR/isomiR signatures, together with those of their target mRNAs, can be applied to distinguish the endometrial phases, especially the implantation window, as well as for diagnosing endometrial dysfunction. It is worth investigating the possibility of miRs/isomiRs being used as biomarkers not only in endometrial biopsy but also in liquid biopsy. Trial registration number The Bulgarian National Science Fund КП–06 Н31/2

Study question Does cellular composition of the endometrial biopsy affect the gene expression profile of endometrial whole-tissue samples? Summary answer The differences in epithelial and stromal cell proportions in endometrial biopsies modify the whole-tissue gene expression profiles and affect the results of differential expression analyses. What is already known Each cell type has its unique gene expression profile. The proportions of epithelial and stromal cells vary in endometrial tissue during the menstrual cycle, along with individual and technical variation due to the method and tools used to obtain the tissue biopsy. Study design, size, duration Using cell-population specific transcriptome data and computational deconvolution approach, we estimated the epithelial and stromal cell proportions in whole-tissue biopsies taken during early secretory and mid-secretory phases. The estimated cellular proportions were used as covariates in whole-tissue differential gene expression analysis. Endometrial transcriptomes before and after deconvolution were compared and analysed in biological context. Participants/material, setting, methods Paired early- and mid-secretory endometrial biopsies were obtained from 35 healthy, regularly cycling, fertile volunteers, aged 23-36 years, and analysed by RNA sequencing. Differential gene expression analysis was performed using two approaches. In one of them, computational deconvolution was applied as an intermediate step to adjust for the proportions of epithelial and stromal cells in the endometrial biopsy. The results were then compared to conventional differential expression analysis. Ten paired endometrial samples were analysed with qPCR to validate the results. Main results and the role of chance The estimated average proportions of stromal and epithelial cells in early secretory phase were 65% and 35%, and during mid-secretory phase, 46% and 54%, respectively, correlating well with the results of histological evaluation (r = 0.88, P = 1.1 × 10-6). Endometrial tissue transcriptomic analysis showed that approximately 26% of transcripts (n = 946) differentially expressed in receptive endometrium in cell-type unadjusted analysis also remain differentially expressed after adjustment for biopsy cellular composition. However, the other 74% (n = 2645) become statistically non-significant after adjustment for biopsy cellular composition, underlining the impact of tissue heterogeneity on differential expression analysis. The results suggest new mechanisms involved in endometrial maturation, involving genes like LINC01320, SLC8A1 and GGTA1P, described for the first time in context of endometrial receptivity. Large-scale data The RNA-seq data presented in this study is deposited in the Gene Expression Omnibus database with accession number GSE98386. Limitations reasons for caution Only dominant endometrial cell types were considered in gene expression profile deconvolution; however, other less frequent endometrial cell types also contribute to the whole-tissue gene expression profile. Wider implications of the findings The better understanding of molecular processes during transition from pre-receptive to receptive endometrium serves to improve the effectiveness and personalization of assisted reproduction protocols. Biopsy cellular composition should be taken into account in future endometrial 'omics' studies, where tissue heterogeneity could potentially influence the results. Study funding/competing interest(s) This study was funded by: Estonian Ministry of Education and Research (grant IUT34-16); Enterprise Estonia (EU48695); the EU-FP7 Eurostars program (NOTED, EU41564); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (SARM, EU324509); Horizon 2020 innovation program (WIDENLIFE, EU692065); MSCA-RISE-2015 project MOMENDO (No 691058) and the Miguel Servet Program Type I of Instituto de Salud Carlos III (CP13/00038); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526. Authors confirm no competing interests.

What is the measured prevalence and phenotype of spontaneous premature ovarian failure (POF) in the general population?Spontaneous POF occurs in ∼1% of the general population with unique phenotype of post-menopausal ageing distinct from surgically induced premature menopause.POF is multifactorial ovarian quiescence before the age of 40. The clinical features of POF are diverse and the population prevalence of POF is still not known.This population-depictive registry-based case-cohort study included 34 041 women from the Estonian Genome Center registered between 2003 and 2013.Spontaneous POF was selected retrospectively by excluding other causes for premature menopause under the age of 40 (N = 310) and women with surgically induced premature menopause participated as a reference group (N = 242).The prevalence of spontaneous POF was 0.91% (0.81-1.02%) among women of the general population in Estonia. In women with POF, menarche occurred a few months later than in the reference group and a significantly higher number of live births during their reproductive life was recorded. Women with POF also consumed less alcohol and had smaller waist-to-hip ratios than those in the reference group, although both groups of women were similar in body mass index a decade after menopause. The prevalence of concomitant diseases was similar between two groups of women by their fifties, but the pattern of onset of these diseases was different. Surgically induced premature menopause associated with faster development of osteoporosis, hypertension, and connective tissue diseases, but slower development of allergies, compared with spontaneous POF. The age of menopause was determined by irregular menstrual cycles, but not by the length of regular menstrual cycles, the age of menarche, the number of pregnancies or live births, smoking or alcohol consumption, or the use of oral contraceptives for some time during the reproductive period.POF is rarely stated in medical records and cannot be diagnosed retrospectively by standard procedures. Therefore the data on all cases of women with primary amenorrhea or premature menopause before the age of 40 were requested from the registry and spontaneous POF was predicted retrospectively by excluding other extraovarian causes for premature menopause. Since the current study is retrospective registry-based data analysis, no genetic evaluation concerning possible candidate genes and no blood analysis concerning immunologic disorders could be performed to describe etiopathogenesis of POF.Spontaneous POF most likely comprises several diseases with different etiopathologies and there may be a unique phenotype of post-menopausal ageing distinct from that in surgically induced premature menopause. Irregular menstrual cycles may be a prospective risk for developing spontaneous POF. Compared with spontaneous POF, surgically induced premature menopause associates with faster development of age-related diseases. The data point to new ideas and hypotheses for further studies on etiopathologies and treatment options for spontaneous POF.The study was funded by grant SF0180044s09, SF0180027s10 and IUT20-43 from the Estonian Ministry of Education and Research, Enterprise Estonia, grant no EU30020, Eureka's EUROSTARS programme grant (NOTED, EU41564). No competing interests are declared.

BACKGROUND: The reduction of multiple pregnancies by using elective single embryo transfers (eSET) requires critical and careful selection of the embryo for transfer. The current study was undertaken to assess whether early cleavage could be used as a marker of embryo competence in eSET procedures. METHODS: The study included analysis of 178 eSET procedures. All embryos were checked for early cleavage at 25‐27 h post insemination or ICSI. The embryos that possessed two cells at 25‐27 h post insemination or ICSI were designated as ‘early cleavage’ (EC) embryos and those that had not yet cleaved were classified as ‘no early cleavage’ (NEC) embryos. Selection of the embryo for transfer was based on embryo morphology and growth rate on day 2 and not early cleavage. Clinical parameters were compared between 72 EC and 106 NEC single embryo transfers. RESULTS: A significantly higher clinical pregnancy rate was observed after transfer of EC (50%) than NEC (26.4%) embryos. CONCLUSIONS: The current study provides compelling evidence that EC embryos possess significantly higher developmental competence than NEC embryos.