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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 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.

BackgroundThe period of time when the embryo and the endometrium undergo significant morphological alterations to facilitate a successful implantation—known as “window of implantation”—is a critical moment in human reproduction. Embryo and the endometrium communicate extensively during this period, and lipid bilayer bound nanoscale extracellular vesicles (EVs) are purported to be integral to this communication.MethodsTo investigate the nature of the EV-mediated embryo-maternal communication, we have supplemented trophoblast analogue spheroid (JAr) derived EVs to an endometrial analogue (RL 95–2) cell layer and characterized the transcriptomic alterations using RNA sequencing. EVs derived from non-trophoblast cells (HEK293) were used as a negative control. The cargo of the EVs were also investigated through mRNA and miRNA sequencing.ResultsTrophoblast spheroid derived EVs induced drastic transcriptomic alterations in the endometrial cells while the non-trophoblast cell derived EVs failed to induce such changes demonstrating functional specificity in terms of EV origin. Through gene set enrichment analysis (GSEA), we found that the response in endometrial cells was focused on extracellular matrix remodelling and G protein-coupled receptors’ signalling, both of which are of known functional relevance to endometrial receptivity. Approximately 9% of genes downregulated in endometrial cells were high-confidence predicted targets of miRNAs detected exclusively in trophoblast analogue-derived EVs, suggesting that only a small proportion of reduced expression in endometrial cells can be attributed directly to gene silencing by miRNAs carried as cargo in the EVs.ConclusionOur study reveals that trophoblast derived EVs have the ability to modify the endometrial gene expression, potentially with functional importance for embryo-maternal communication during implantation, although the exact underlying signalling mechanisms remain to be elucidated.

OBJECTIVE The study aimed to validate a whole-genome sequencing-based NIPT laboratory method and our recently developed NIPTmer aneuploidy detection software with the potential to integrate the pipeline into prenatal clinical care in Estonia. METHOD In total, 424 maternal blood samples were included. Analysis pipeline involved cell-free DNA extraction, library preparation and massively parallel sequencing on Illumina platform. Aneuploidies were determined with NIPTmer software, which is based on counting pre-defined per-chromosome sets of unique k-mers from sequencing raw data. SeqFF was implemented to estimate cell-free fetal DNA (cffDNA) fraction. RESULTS NIPTmer identified correctly all samples of non-mosaic trisomy 21 (T21, 15/15), T18 (9/9), T13 (4/4) and monosomy X (4/4) cases, with the 100% sensitivity. However, one mosaic T18 remained undetected. Six false-positive (FP) results were observed (FP rate of 1.5%, 6/398), including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). The level of cffDNA of

Although chromosomal instability (CIN) is a common phenomenon in cleavage-stage embryogenesis following in vitro fertilization (IVF)(1-3), its rate in naturally conceived human embryos is unknown. CIN leads to mosaic embryos that contain a combination of genetically normal and abnormal cells, and is significantly higher in in vitro-produced preimplantation embryos as compared to in vivo-conceived preimplantation embryos(4). Even though embryos with CIN-derived complex aneuploidies may arrest between the cleavage and blastocyst stages of embryogenesis(5,6), a high number of embryos containing abnormal cells can pass this strong selection barrier(7,8). However, neither the prevalence nor extent of CIN during prenatal development and at birth, following IVF treatment, is well understood. Here we profiled the genomic landscape of fetal and placental tissues postpartum from both IVF and naturally conceived children, to investigate the prevalence and persistence of large genetic aberrations that probably arose from IVF-related CIN. We demonstrate that CIN is not preserved at later stages of prenatal development, and that de novo numerical aberrations or large structural DNA imbalances occur at similar rates in IVF and naturally conceived live-born neonates. Our findings affirm that human IVF treatment has no detrimental effect on the chromosomal constitution of fetal and placental lineages.

Background Balanced translocation carriers are burdened with fertility issues due to improper chromosome segregation in gametes, resulting in either implantation failure, miscarriage or birth of a child with chromosomal disorders. At the same time, these individuals are typically healthy with no signs of developmental problems, hence they often are unaware of their condition. Yet, because of difficulties in conceiving, balanced translocation carriers often turn to assisted reproduction, some of whom may also undergo preimplantation genetic testing for aneuploidy (PGT-A) to improve the likelihood of achieving a successful pregnancy. Case report We describe a female patient, who pursued in vitro fertilization (IVF) treatment coupled with PGT-A following two consecutive miscarriages, unaware of her genetic condition. PGT-A was performed on blastocyst-stage embryos and the results of comprehensive chromosome screening from a first IVF cycle demonstrated reciprocal segmental aberrations on chromosome 7 and chromosome 10 in two out of four embryos. Due to distinct embryo profiles, the couple was then referred for genetic counselling and subsequent parental karyotyping revealed the presence of a previously undetected balanced translocation in the mother. Conclusions These results confirm previous reports that genome-wide PGT-A can facilitate the identification of balanced translocation carriers in IVF patients, providing explanation for poor reproductive outcome and allowing adjustments in treatment strategies.

Vaginal discharge is one of the common reasons for gynaecologist consultation, as bacterial vaginosis and candidiasis are the main causes of discharge. These patients frequently experience numerous problems due to recurrent infections, side effects and drug resistance therefore alternative drugs are needed. Our primary aim was to evaluate safety and tolerability of the potentially probiotic Lactobacillus crispatus strains in volunteer women considering themselves healthy. We also monitored the effects of these strains on vaginal health parameters and lactobacilli counts in vagina and intestine. Forty women were recruited into trial. Absence of chronic diseases was confirmed by questionnaire and blood analysis at screening visit. In randomised double-blind placebo-controlled crossover study the eligible participants were randomly allocated to one of four groups and had to consume one of the two study products (Pro I or Pro II) – a capsule containing 3 strains, 109 cfu per strain, or placebo for 1 week. Treatment period was followed by 2-week washout period and continued with second treatment and washout period. Individuals receiving firstly probiotic, received later placebo and vice versa. Blood, vaginal and faecal samples were collected, and self-reported questionnaires were applied. Thirty subjects completed the trial. The probiotic capsules were well-tolerated. The Pro II intake resulted in a significant decrease in Nugent score (from median 3.0 to 2.0, mean 3.9 to 2.6, P=0.002) and reduction in Gardnerella vaginalis counts (log10 3.57 to 2.38; P=0.027). Reduction of total vaginal bacterial counts was revealed in Pro I group (log10 7.99 to 7.72; P=0.048). In conclusion, the selected vaginal L. crispatus strains are well tolerable and Pro II mixture is prospectively effective in reducing Nugent score and vaginal counts of G. vaginalis. Therefore, these strains seem to be promising candidates for development of novel evidence-based well-focused probiotics to target female urogenital tract disorders.

Background: Usage of sexed semen that allows to choose the gender of the calves, is commonly practiced in livestock industry as a profitable breeding alternative, especially in dairy farming. The flow cytometric cell sorting is the only commercially available method for bovine sperm sexing. For validation of the sexing procedure several methods have been developed including sperm fluorescence in situ hybridisation techniques. Latter usually include the use of pre-labelled nucleotides for probe synthesis which is relatively expensive approach compared to combined application of aminoallyl-dUTP and chemical binding of fluorescent dyes. Here a sex determining dual colour bovine sperm fluorescence in situ hybridisation method is presented which is considered more cost-effective technique than the previously reported approaches. Results: The reliability of sex chromosome identifying probes, designed in silico, was proven on bovine metaphase plate chromosomes and through comparison with a commercially available standard method. In the dual colour FISH experiments of unsexed and sexed bovine sperm samples the hybridisation efficiency was at least 98%, whereas the determined sex ratios were not statistically different from the expected. Very few cells carried both of the sex chromosome-specific signals (less than 0.2%). Conclusions: A protocol for a dual colour bovine sperm FISH method is provided which is cost-effective, simple and fast for sex determination of spermatozoa in bull semen samples.

Non-invasive prenatal testing (NIPT) is a powerful screening method for fetal aneuploidy detection, relying on laboratory and computational analysis of cell-free DNA. Although several published computational NIPT analysis tools are available, no prior comprehensive, head-to-head accuracy comparison of the various tools has been published. Here, we compared the outcome accuracies obtained for clinically validated samples with five commonly used computational NIPT aneuploidy analysis tools (WisecondorX, NIPTeR, NIPTmer, RAPIDR, and GIPseq) across various sequencing depths (coverage) and fetal DNA fractions. The sample set included cases of fetal trisomy 21 (Down syndrome), trisomy 18 (Edwards syndrome), and trisomy 13 (Patau syndrome). We determined that all of the compared tools were considerably affected by lower sequencing depths, such that increasing proportions of undetected trisomy cases (false negatives) were observed as the sequencing depth decreased. We summarised our benchmarking results and highlighted the advantages and disadvantages of each computational NIPT software. To conclude, trisomy detection for lower coverage NIPT samples (e.g. 2.5M reads per sample) is technically possible but can, with some NIPT tools, produce troubling rates of inaccurate trisomy detection, especially in low-FF samples., Author summary Non-invasive prenatal testing analysis relies on computational algorithms that are used for inferring chromosomal aneuploidies, such as chromosome 21 triploidy in the case of Down syndrome. However, the performance of these algorithms has not been compared on the same clinically validated data. Here we conducted a head-to-head comparison of WGS-based NIPT aneuploidy detection tools. Our findings indicate that at and below 2.5M reads per sample, the least accurate algorithm would miss detection of almost a third of trisomy cases. Furthermore, we describe and quantify a previously undocumented aneuploidy risk uncertainty that is mainly relevant in cases of very low sequencing coverage (at and below 1.25M reads per sample) and could, in the worst-case scenario, lead to a false negative rate of 245 undetected trisomies per 1,000 trisomy cases. Our findings underscore the importance of the informed selection of NIPT software tools in combination with sequencing coverage, which directly impacts NIPT sequencing cost and accuracy.

Context Despite the gut microbiome being widely studied in metabolic diseases, its role in polycystic ovary syndrome (PCOS) has been scarcely investigated. Objective Compare the gut microbiome in late fertile age women with and without PCOS and investigate whether changes in the gut microbiome correlate with PCOS-related metabolic parameters. Design Prospective, case–control study using the Northern Finland Birth Cohort 1966. Setting General community. Participants A total of 102 PCOS women and 201 age- and body mass index (BMI)-matched non-PCOS control women. Clinical and biochemical characteristics of the participants were assessed at ages 31 and 46 and analyzed in the context of gut microbiome data at the age of 46. Intervention (s): None Main outcome measure(s) Bacterial diversity, relative abundance, and correlations with PCOS-related metabolic measures. Results Bacterial diversity indices did not differ significantly between PCOS and controls (Shannon diversity P = .979, unweighted UniFrac P = .175). Four genera whose balance helps to differentiate between PCOS and non-PCOS were identified. In the whole cohort, the abundance of 2 genera from Clostridiales, Ruminococcaceae UCG-002, and Clostridiales Family XIII AD3011 group, were correlated with several PCOS-related markers. Prediabetic PCOS women had significantly lower alpha diversity (Shannon diversity P = .018) and markedly increased abundance of genus Dorea (false discovery rate = 0.03) compared with women with normal glucose tolerance. Conclusion PCOS and non-PCOS women at late fertile age with similar BMI do not significantly differ in their gut microbial profiles. However, there are significant microbial changes in PCOS individuals depending on their metabolic health.

Hyperandrogenic women with PCOS show disrupted decidualization (DE) and placentation. Dihydrotestosterone (DHT) is reported to enhance DE in non-PCOS endometrial stromal cells (eSCCtrl); however, this has not been assessed in PCOS cells (eSCPCOS). Therefore, we studied the transcriptome profile of non-decidualized (non-DE) and DE eSCs from women with PCOS and Ctrl in response to short-term estradiol (E2) and/or progesterone (P4) exposure with/without (±) DHT. The non-DE eSCs were subjected to E2 ± DHT treatment, whereas the DE (0.5 mM 8-Br-cAMP, 96 h) eSCs were post-treated with E2 and P4 ± DHT, and RNA-sequenced. Validation was performed by immunofluorescence and immunohistochemistry. The results showed that, regardless of treatment, the PCOS and Ctrl samples clustered separately. The comparison of DE vs. non-DE eSCPCOS without DHT revealed PCOS-specific differentially expressed genes (DEGs) involved in mitochondrial function and progesterone signaling. When further adding DHT, we detected altered responses for lysophosphatidic acid (LPA), inflammation, and androgen signaling. Overall, the results highlight an underlying defect in decidualized eSCPCOS, present with or without DHT exposure, and possibly linked to the altered pregnancy outcomes. We also report novel factors which elucidate the mechanisms of endometrial dysfunction in PCOS.

The diagnosis of vulvo-vaginal complaints has always been enigmatic in obstetrics and gynecology. Patients with clear, pathognomonic symptoms end up with a proper diagnosis and treatment most of the time, but unfortunately we are now living in a world where women reach out to the Internet and readily get all information as to which disease their symptoms correspond to and also find the appropriate treatment "over-the-counter." Because of this trend, we as specialists are increasingly confronted with patients with complex and combined conditions. At the same time, extremely sensitive and accurate diagnostic tools are now being developed at a rapid pace, allowing the physicians to diagnose vulvo-vaginal disease with a substantially increased precision. Moreover, many of these molecular biology (MB)-based tests have become so common and affordable that self-sampling and self-testing are no longer utopia. On the other hand, too much information that is too readily available and that is too affordable also encompasses pitfalls, leading to gross overtreatment and psychological burden. As experienced caregivers, we should supervise these evolutions, define their place and proper use as diagnostic tools, utilize their potential as ad hoc tools to follow-up treatment efficacy and guide how such tools can be used for responsible self-testing. In the present paper, responding to the need for appropriate, quality assured and accessible tests for vulvovaginitis and the huge potential delivered by the rapidly developing MB methods, we recommend the need for a broad and regular discussion forum, composed of both clinical and technical experts and opinion makers, in order to match the needs with the huge opportunities and ideally combine the initiatives and forces into the same direction. This forum should then translate conceived strategies into regularly updated, evidence-based national and international guidelines. (C) 2017 S. Karger AG, Basel

Cell-free RNAs have the potential to act as a means of gene expression regulation between cells and are therefore used as diagnostic markers describing the state of tissue environment. The origin and functions of such RNAs in human ovarian follicle, the environment of oocyte maturation, are unclear. The current study investigates the difference in the microRNA profiles of fertile women and polycystic ovary syndrome (PCOS) patients in three compartments from the same preovulatory follicle: mural granulosa cells (MGC), cell-free follicular fluid (FF), and extracellular vesicles (EV) of the FF by small RNA sequencing. In silico analysis was used for the prediction and over-representation of targeted pathways for the detected microRNAs. PCOS follicles were distinguished from normal tissue by the differential expression of 30 microRNAs in MGC and 10 microRNAs in FF (FDR &lt, 0.1) that commonly regulate cytokine signaling pathways. The concentration of EV-s was higher in the FF of PCOS patients (p = 0.04) containing eight differentially expressed microRNAs (p &lt, 0.05). In addition, we present the microRNA profiles of MGC, FF, and EV in the fertile follicle and demonstrate that microRNAs loaded into EVs target mRNAs of distinct signaling pathways in comparison to microRNAs in FF. To conclude, the three follicular compartments play distinct roles in the signaling disturbances associated with PCOS.

The capability of spermatozoa to directly influence maternal gene expression is already established. Indeed, some of the changes induced by spermatozoa may have a direct functional importance in the pre-conceptional period. Although the mechanisms underlying these sperm-maternal interactions are not well characterized, it is possible that they could involve ligands that are released from the spermatozoa. This study therefore aimed to test whether physical contact between bovine spermatozoa and bovine oviductal epithelial cells (BOECs) is a prerequisite for spermatozoa-induced gene expression changes. We used two co-culture models: a contact co-culture model in which spermatozoa interact directly with BOECs, and a non-contact co-culture model in which an insert with the pore size of 0.4 μm was placed between spermatozoa and BOECs. Messenger RNA sequencing analysis of BOECs by RNA-seq revealed ten differentially expressed genes in contact system and 108 differentially expressed genes in the non-contact system after 10 h of co-culture. Retinol metabolism pathway and ovarian steroidogenesis pathway were significantly enriched in the non-contact co-culture system. Q-PCR analysis revealed that transcriptional responses can be rapid, with increased expression of four genes (DHRS3, CYP1B1, PTGS2, and ATF3) detectable within just 90 min of co-incubation, but with expression levels highly dependent on the type of co-culture system. The findings from our study demonstrate that direct contact with spermatozoa is not necessary to induce changes in gene expression of oviductal epithelial cells, suggesting that spermatozoa may be able to signal to maternal tissues in advance of their arrival. Electronic supplementary material The online version of this article (10.1007/s12079-020-00575-2) contains supplementary material, which is available to authorized users.

SARS-CoV-2 infection risks developing into life-threatening COVID-19 disease. Whereas age, hypertension, and chronic inflammatory conditions are risk factors, underlying host factors and markers for disease severity, e.g. requiring intensive care unit (ICU) treatment, remain poorly defined. To this end, we longitudinally profiled blood inflammation markers, antibodies, and 101 plasma proteins of hospitalized COVID-19 patients who did or did not require (ICU admission. Whereas essentially all patients displayed SARS-CoV-2-specific antibodies and virus-neutralization capacity within 12-15 days, a rapid, mostly transient upregulation of selective inflammatory markers including IL-6, CXCL10, CXCL11, IFNg, IL-10, and monocyte-attracting CCL2, CCL7 and CCL8, was particularly evident in ICU patients. In addition, there was consistent and sustained upregulation of apoptosis-associated proteins CASP8, TNFSF14, HGF, and TGFB1, with HGF discriminating between ICU and non-ICU cohorts. Thus, COVID-19 is associated with a selective inflammatory milieu within which the apoptotic pathway is a cardinal feature with potential to aid risk-based patient stratification.

Miscarriage is a common, complex trait affecting ~15% of clinically confirmed pregnancies. Here we present the results of large-scale genetic association analyses with 69,054 cases from five different ancestries for sporadic miscarriage, 750 cases of European ancestry for multiple (≥3) consecutive miscarriage, and up to 359,469 female controls. We identify one genome-wide significant association (rs146350366, minor allele frequency (MAF) 1.2%, P = 3.2 × 10−8, odds ratio (OR) = 1.4) for sporadic miscarriage in our European ancestry meta-analysis and three genome-wide significant associations for multiple consecutive miscarriage (rs7859844, MAF = 6.4%, P = 1.3 × 10−8, OR = 1.7; rs143445068, MAF = 0.8%, P = 5.2 × 10−9, OR = 3.4; rs183453668, MAF = 0.5%, P = 2.8 × 10−8, OR = 3.8). We further investigate the genetic architecture of miscarriage with biobank-scale Mendelian randomization, heritability, and genetic correlation analyses. Our results show that miscarriage etiopathogenesis is partly driven by genetic variation potentially related to placental biology, and illustrate the utility of large-scale biobank data for understanding this pregnancy complication., Miscarriage affects around 15% of clinically confirmed pregnancies. Here the authors carry out a large genome-wide association study for sporadic and multiple consecutive miscarriage and suggest links with placental biology.

Embryo-derived extracellular vesicles (EVs) may play a role in mediating the embryo-maternal dialogue at the oviduct, potentially carrying signals reflecting embryo quality. We investigated the effects of bovine embryo-derived EVs on the gene expression of bovine oviductal epithelial cells (BOECs), and whether these effects are dependent on embryo quality. Presumptive zygotes were cultured individually in vitro in culture medium droplets until day 8 while their development was assessed at day 2, 5 and 8. Conditioned medium samples were collected at day 5 and pooled based on embryo development (good quality embryo media and degenerating embryo media). EVs were isolated from conditioned media by size exclusion chromatography and supplemented to primary BOEC monolayer cultures to evaluate the effects of embryo-derived EVs on gene expression profile of BOEC. Gene expression was quantified by RNA-seq and RT-qPCR. A total of 7 upregulated and 18 downregulated genes were detected in the BOECs supplemented with good quality embryo-derived EV compared to the control. The upregulated genes included interferon-τ-induced genes, such as OAS1Y, MX1 and ISG15, which have previously been reported as upregulated in the oviductal epithelial cells in the presence of embryos. Of the upregulated genes, OAS1Y and MX1 were validated with RT-qPCR. In contrast, only one differentially expressed gene was detected in BOECs in response to degenerating embryo-derived EVs, suggesting that oviductal responses are dependent on embryo quality. Our results support the hypothesis that embryo-derived EVs are involved in embryo-maternal communication at the oviduct and the oviductal response is dependant on the embryo quality. KEY MESSAGES: • Extracellular vesicles (EVs) released by individually cultured pre-implantation bovine embryos can alter the gene expression of primary oviductal epithelial cells. • The oviductal response, in terms of gene expression, to the bovine embryo-derived EVs varied depending on the embryo quality. • In vivo, the oviduct may have the ability to sense the quality of the pre-implantation embryos. • The observed effect of embryo-derived EVs on oviductal epithelial cells could serve as a non-invasive method of evaluating the embryo quality.

Modern living challenges female reproductive health. We are witnessing a rise in reproductive disorders and drop in birth rates across the world. The reasons for these manifestations are multifaceted and most likely include continuous exposure to an ever-increasing number of chemicals. The cause–effect relationships between chemical exposure and female reproductive disorders, however, have proven problematic to determine. This has made it difficult to assess the risks chemical exposures pose to a woman’s reproductive development and function. To address this challenge, this review uses the adverse outcome pathway (AOP) concept to summarize current knowledge about how chemical exposure can affect female reproductive health. We have a special focus on effects on the ovaries, since they are essential for lifelong reproductive health in women, being the source of both oocytes and several reproductive hormones, including sex steroids. The AOP framework is widely accepted as a new tool for toxicological safety assessment that enables better use of mechanistic knowledge for regulatory purposes. AOPs equip assessors and regulators with a pragmatic network of linear cause–effect relationships, enabling the use of a wider range of test method data in chemical risk assessment and regulation. Based on current knowledge, we propose ten putative AOPs relevant for female reproductive disorders that can be further elaborated and potentially be included in the AOPwiki. This effort is an important step towards better safeguarding the reproductive health of all girls and women.

Introduction The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has exposed vulnerable populations to an unprecedented global health crisis. The knowledge gained from previous human coronavirus outbreaks suggests that pregnant women and their fetuses are particularly susceptible to poor outcomes. The objective of this study was to summarize the clinical manifestations and maternal and perinatal outcomes of COVID‐19 during pregnancy. Material and methods We searched databases for all case reports and series from 12 February to 4 April 2020. Multiple terms and combinations were used including COVID‐19, pregnancy, maternal mortality, maternal morbidity, complications, clinical manifestations, neonatal morbidity, intrauterine fetal death, neonatal mortality and SARS‐CoV‐2. Eligibility criteria included peer‐reviewed publications written in English or Chinese and quantitative real‐time polymerase chain reaction (PCR) or dual fluorescence PCR‐confirmed SARS‐CoV‐2 infection. Unpublished reports, unspecified date and location of the study or suspicion of duplicate reporting, cases with suspected COVID‐19 that were not confirmed by a laboratory test, and unreported maternal or perinatal outcomes were excluded. Data on clinical manifestations, maternal and perinatal outcomes including vertical transmission were extracted and analyzed. Results Eighteen articles reporting data from 108 pregnancies between 8 December 2019 and 1 April 2020 were included in the current study. Most reports described women presenting in the third trimester with fever (68%) and coughing (34%). Lymphocytopenia (59%) with elevated C‐reactive protein (70%) was observed and 91% of the women were delivered by cesarean section. Three maternal intensive care unit admissions were noted but no maternal deaths. One neonatal death and one intrauterine death were also reported. Conclusions Although the majority of mothers were discharged without any major complications, severe maternal morbidity as a result of COVID‐19 and perinatal deaths were reported. Vertical transmission of the COVID‐19 could not be ruled out. Careful monitoring of pregnancies with COVID‐19 and measures to prevent neonatal infection are warranted.

PURPOSE: To evaluate the association of objectively measured physical activity (PA) and sedentary behaviour before and during in vitro fertilization (IVF) with controlled ovarian stimulation (COS) and pregnancy outcomes. METHODS: This longitudinal study involved 107 infertile women undergoing IVF treatment. PA and sedentary behaviour were measured for 14 consecutive days using accelerometry as follows: (1) before IVF treatment, (2) during IVF at the implantation time, immediately after embryo transfer, and (3) after positive pregnancy test. Total screen time was assessed by questionnaires. COS results were measured as the number of oocytes and embryos obtained, and the study outcomes included positive hCG, clinical pregnancy, and live birth. RESULTS: Compared with baseline activity levels, women significantly reduced their PA and increased sedentary behaviour during IVF (p ≤ 0.001). Higher average PA, light PA, and ratio between breaks in every ≥ 30-min blocks of sedentary time showed positive associations, while sedentary time, number, and time accumulated in blocks of ≥ 30 min of sedentary time associated negatively with oocyte and embryo counts (all p

Extracellular vesicles (EVs) are membrane-bound biological nanoparticles (NPs) and have gained wide attention as potential biomarkers. We aimed to isolate and characterize EVs from media conditioned by individually cultured preimplantation bovine embryos and to assess their relationship with embryo quality. Presumptive zygotes were cultured individually in 60 μl droplets of culture media, and 50 μl of media were collected from the droplets either on day 2, 5 or 8 post-fertilization. After sampling, the embryo cultures were continued in the remaining media until day 8, and the embryo development was evaluated at day 2 (cleavage), day 5 (morula stage) and day 8 (blastocyst stage). EVs were isolated using qEVsingle® columns and characterized. Based on EV Array, EVs isolated from embryo conditioned media were strongly positive for EV-markers CD9 and CD81 and weakly positive for CD63 and Alix among others. They had a cup-like shape typical to EVs as analyzed by transmission electron microscopy and spherical shape in scanning electron microscopy, and hence regarded as EVs. However, the NPs isolated from control media were negative for EV markers. Based on nanoparticle tracking analysis, at day 2, the mean concentration of EVs isolated from media conditioned by embryos that degenerated after cleaving (8.25 × 108/ml) was higher compared to that of embryos that prospectively developed to blastocysts (5.86 × 108/ml, p < 0.05). Moreover, at day 8, the concentration of EVs isolated from media conditioned by degenerating embryos (7.17 × 108/ml) was higher compared to that of blastocysts (5.68 × 108/ml, p < 0.05). Furthermore, at day 8, the mean diameter of EVs isolated from media conditioned by degenerating embryos (153.7 nm) was smaller than EVs from media conditioned by blastocysts (163.5 nm, p < 0.05). In conclusion, individually cultured preimplantation bovine embryos secrete EVs in the culture media and their concentration and size are influenced by embryo quality and may indicate their prospective development potential.

While follicular fluid (FF) is well known to provide an optimal environment for oogenesis, its functional roles following its release into the oviduct during ovulation are currently elusive. We hypothesized that FF and FF-derived extracellular vesicles (EVs) may be conveyors of signals capable of inducing functionally-relevant transcriptional responses in oviductal cells. The aim of this study was, therefore, to evaluate the effect of FF and FF-derived EVs on the transcriptome of primary bovine oviductal epithelial cells (BOECs). We examined the gene expression of BOECs in three conditions: BOECs cultured with FF, FF-derived EVs, and without supplementations. For each condition, cells were cultured for 6 and 24 h. RNA sequencing results revealed that FF had a stronger effect on BOECs gene expression compared to EVs. We detected 488 and 1998 differentially expressed genes (DEGs) with FF treatment in 6 and 24 h, respectively, whereas only 41 DEGs were detected at 6 h following EV treatment. Pathway analysis of the FF-induced DEGs showed that several pathways were highly enriched, notably oxidative phosphorylation, thermogenesis, arachidonic acid metabolism, and steroid hormone biosynthesis. Some of these pathways have a role in sperm survival, fertilization, and early embryo development. In conclusion, the findings of our study demonstrate for the first time that bovine FF and FF-derived EVs can induce changes in the gene expression of the bovine oviductal cells which, although observed in vitro, may be reflective of in vivo responses which may contribute to a favorable periconceptional microenvironment for sperm survival, fertilization, and early embryo development.

International audience; Currently available test methods are not well-suited for the identification of chemicals that disturb hormonal processes involved in female reproductive development and function. This renders women's reproductive health at increasing risk globally, which, coupled with increasing incidence rates of reproductive disorders, is of great concern. A woman's reproductive health is largely established during embryonic and fetal development and subsequently matures during puberty. The endocrine system influences development, maturation, and function of the female reproductive system, thereby making appropriate hormone levels imperative for correct functioning of reproductive processes. It is concerning that the effects of human-made chemicals on the endocrine system and female reproductive health are poorly addressed in regulatory chemical safety assessment, partly because adequate test methods are lacking. Our EU-funded project FREIA aims to address this need by increasing understanding of how endocrine disrupting chemicals (EDCs) can impact female reproductive health. We will use this information to provide better test methods that enable fit-for-purpose chemical regulation and then share our knowledge, promote a sustainable society, and improve the reproductive health of women globally.

MicroRNAs (miRNAs) are known post-transcriptional regulators of various biological processes including ovarian follicle development. We have previously identified miRNAs from human pre-ovulatory ovarian granulosa cells that are expressed from the intronic regions of two key genes in normal follicular development: FSH receptor (FSHR) and CYP19A1, the latter encoding the aromatase enzyme. The present study aims to identify the target genes regulated by these miRNAs: hsa-miR-548ba and hsa-miR-7973, respectively. The miRNAs of interest were transfected into KGN cell line and the gene expression changes were analyzed by Affymetrix microarray. Potential miRNA-regulated genes were further filtered by bioinformatic target prediction algorithms and validated for direct miRNA:mRNA binding by luciferase reporter assay. LIFR, PTEN, NEO1 and SP110 were confirmed as targets for hsa-miR-548ba. Hsa-miR-7973 target genes ADAM19, PXDN and FMNL3 also passed all verification steps. Additionally, the expression pattern of the miRNAs was studied in human primary cumulus granulosa cell culture in relation to the expression of their host genes and FSH stimulation. Based on our findings we propose the involvement of hsa-miR-548ba in the regulation of follicle growth and activation via LIFR and PTEN. Hsa-miR-7973 may be implicated in the modulation of extracellular matrix and cell-cell interactions by regulating the expression of its identified targets.

Stanniocalcin-1 (STC-1) is a pro-survival factor that protects tissues against stressors, such as hypoxia and inflammation. STC-1 is co-expressed with the endometrial receptivity markers, and recently endometrial STC-1 was reported to be dysregulated in endometriosis, a condition linked with endometrial progesterone resistance and inflammation. These features are also common in the endometrium in women with polycystic ovary syndrome (PCOS), the most common endocrine disorder in women. Given that women with PCOS present with subfertility, pregnancy complications, and increased risk for endometrial cancer, we investigated endometrial STC-1 expression in affected women. Endometrial biopsy samples were obtained from women with PCOS and controls, including samples from overweight/obese women with PCOS before and after a 3-month lifestyle intervention. A total of 98 PCOS and 85 control samples were used in immunohistochemistry, reverse-transcription polymerase chain reaction, or in vitro cell culture. STC-1 expression was analyzed at different cycle phases and in endometrial stromal cells (eSCs) after steroid hormone exposure. The eSCs were also challenged with 8-bromo-cAMP and hypoxia for STC-1 expression. The findings indicate that STC-1 expression is not steroid hormone mediated although secretory-phase STC-1 expression was blunted in PCOS. Lower expression seems to be related to attenuated STC-1 response to stressors in PCOS eSCs, shown as downregulation of protein kinase A activity. The 3-month lifestyle intervention did not restore STC-1 expression in PCOS endometrium. More studies are warranted to further elucidate the mechanisms behind the altered endometrial STC-1 expression and rescue mechanism in the PCOS endometrium., Summary sentence Endometrial expression of STC-1 in the secretory phase is blunted in women with PCOS, suggesting impaired protection against stress.

Background Only a few microbial studies have conducted in IVF (in vitro fertilization), showing the high-variety bacterial contamination of IVF culture media to cause damage to or even loss of cultured oocytes and embryos. We aimed to determine the prevalence and counts of bacteria in IVF samples, and to associate them with clinical outcome. Methods The studied samples from 50 infertile couples included: raw (n = 48), processed (n = 49) and incubated (n = 50) sperm samples, and IVF culture media (n = 50). The full microbiome was analyzed by 454 pyrosequencing and quantitative analysis by real-time quantitative PCR. Descriptive statistics, t-, Mann-Whitney tests and Spearman’s correlation were used for comparison of studied groups. Results The study involved normozoospermic men. Normal vaginal microbiota was present in 72.0% of female partners, while intermediate microbiota and bacterial vaginosis were diagnosed in 12.0 and 16.0%, respectively. The decreasing bacterial loads were found in raw (35.5%), processed (12.0%) and sperm samples used for oocyte insemination (4.0%), and in 8.0% of IVF culture media. The most abundant genera of bacteria in native semen and IVF culture media were Lactobacillus, while in other samples Alphaproteobacteria prevailed. Staphylococcus sp. was found only in semen from patients with inflammation. Phylum Bacteroidetes was in negative correlation with sperm motility and Alphaproteobacteria with high-quality IVF embryos. Conclusion Our study demonstrates that IVF does not occur in a sterile environment. The prevalent bacteria include classes Bacilli in raw semen and IVF culture media, Clostridia in processed and Bacteroidia in sperm samples used for insemination. The presence of Staphylococcus sp. and Alphaproteobacteria associated with clinical outcomes, like sperm and embryo quality.

PURPOSE: The purpose of this study was to develop a feasible approach for single sperm isolation and chromosome analysis by next-generation sequencing (NGS). METHODS: Single sperm cells were isolated from semen samples of normozoospermic male and an infertile reciprocal translocation (RcT) carrier with the 46,XY,t(7;13)(p12;q12.1) karyotype using the optimized fluorescence-activated cell sorting (FACS) technique. Genome profiling was performed using NGS. RESULTS: Following whole-genome amplification, NGS, and quality control, the final chromosome analysis was performed on 31 and 6 single cell samples derived from the RcT carrier and normozoospermic male, respectively. All sperm cells from normozoospermic male showed a normal haploid 23-chromosome profile. For the RcT carrier, the sequencing data revealed that 64.5% of sperm cells harbored different variants of chromosome aberrations, involving deletion of 7p or 7q, duplication of 7p, and duplication of 13q, which is concordant with the expected chromosome segregation patterns observed in balanced translocation carriers. In one sample, a duplication of 9q was also detected. CONCLUSIONS: We optimized FACS protocol for simple and efficient isolation of single human sperm cells that subsequently enabled a successful genome-wide chromosome profiling and identification of segmental aneuploidies from these individual cells, following NGS analysis. This approach may be useful for analyzing semen samples of infertile men or chromosomal aberration carriers to facilitate the reproductive risk assessment. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-018-1340-0) contains supplementary material, which is available to authorized users.

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.

Background: Assisted reproductive technology (ART) has been associated with low birth weight of fresh embryo transfer (FRESH) derived and increased birth weight of frozen embryo transfer (FET)-derived newborns. Owing to that, we focused on imprinted insulin-like growth factor 2 (IGF2)/H19 locus known to be important for normal growth. This locus is regulated by H19 imprinting control region (ICR) with seven binding sites for the methylation-sensitive zinc finger regulatory protein (CTCF). A polymorphism rs10732516 G/A in the sixth binding site for CTCF, associates with a genotype-specific trend to the DNA methylation. Due to this association, 62 couples with singleton pregnancies derived from FRESH (44 IVF/18 ICSI), 24 couples from FET (15 IVF/9 ICSI), and 157 couples with spontaneously conceived pregnancies as controls were recruited in Finland and Estonia for genotype-specific examination. DNA methylation levels at the H19 ICR, H19 DMR, and long interspersed nuclear elements in placental tissue were explored by MassARRAY EpiTYPER (n = 122). Allele-specific changes in the methylation level of H19 ICR in placental tissue (n = 26) and white blood cells (WBC, n = 8) were examined by bisulfite sequencing. Newborns' (n = 243) anthropometrics was analyzed by using international growth standards.Results: A consistent trend of genotype-specific decreased methylation level was observed in paternal allele of rs10732516 paternal A/maternal G genotype, but not in paternal G/maternal A genotype, at H19 ICR in ART placentas. This hypomethylation was not detected in WBCs. Also genotype-specific differences in FRESH-derived newborns' birth weight and head circumference were observed (P = 0.04, P = 0.004, respectively): FRESH-derived newborns with G/G genotype were heavier (P = 0.04) and had larger head circumference (P = 0.002) compared to newborns with A/A genotype. Also, the placental weight and birth weight of controls, FRESH- and FET-derived newborns differed significantly in rs10732516 A/A genotype (P = 0.024, P = 0.006, respectively): the placentas and newborns of FET-derived pregnancies were heavier compared to FRESH-derived pregnancies (P = 0.02, P = 0.004, respectively).Conclusions: The observed DNA methylation changes together with the phenotypic findings suggest that rs10732516 polymorphism associates with the effects of ART in a parent-of-origin manner. Therefore, this polymorphism should be considered when the effects of environmental factors on embryonic development are studied.

Non-invasive prenatal testing (NIPT) is a recent and rapidly evolving method for detecting genetic lesions, such as aneuploidies, of a fetus. However, there is a need for faster and cheaper laboratory and analysis methods to make NIPT more widely accessible. We have developed a novel software package for detection of fetal aneuploidies from next-generation low-coverage whole genome sequencing data. Our tool – NIPTmer – is based on counting pre-defined per-chromosome sets of unique k-mers from raw sequencing data, and applying linear regression model on the counts. Additionally, the filtering process used for k-mer list creation allows one to take into account the genetic variance in a specific sample, thus reducing the source of uncertainty. The processing time of one sample is less than 10 CPU-minutes on a high-end workstation. NIPTmer was validated on a cohort of 583 NIPT samples and it correctly predicted 37 non-mosaic fetal aneuploidies. NIPTmer has the potential to reduce significantly the time and complexity of NIPT post-sequencing analysis compared to mapping-based methods. For non-commercial users the software package is freely available at http://bioinfo.ut.ee/NIPTMer/.

Several studies have demonstrated that human embryonic stem cells [hESC] can be differentiated into trophoblast-like cells if exposed to bone morphogenic protein 4 [BMP4] and/or inhibitors of fibroblast growth factor 2 [FGF2] and the transforming growth factor beta [TGF-beta]/activin/nodal signalling pathways. The goal of this study was to investigate how the inhibitors of these pathways improve the efficiency of hESC differentiation when compared with basic BMP4 treatment. RNA sequencing was used to analyse the effects of all possible inhibitor combinations on the differentiation of hESC into trophoblast-like cells over 12 days. Genes differentially expressed compared with untreated cells were identified at seven time points. Additionally, expression of total human chorionic gonadotrophin [HCG] and its hyperglycosylated form [HCG-H] were determined by immunoassay from cell culture media. We showed that FGF2 inhibition with BMP4 activation up-regulates syncytiotrophoblast-specific genes [CGA, CGB and LGALS16], induces several molecular pathways involved in embryo implantation and triggers HCG-H production. In contrast, inhibition of the TGF-beta/activin/nodal pathway decreases the ability of hESC to form trophoblast-like cells. Information about the conditions needed for hESC differentiation toward trophoblast-like cells helps us to find an optimal model for studying the early development of human trophoblasts in normal and in complicated pregnancy. (C) 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

Publisher's version (útgefin grein), Polycystic ovary syndrome (PCOS) is a disorder characterized by hyperandrogenism, ovulatory dysfunction and polycystic ovarian morphology. Affected women frequently have metabolic disturbances including insulin resistance and dysregulation of glucose homeostasis. PCOS is diagnosed with two different sets of diagnostic criteria, resulting in a phenotypic spectrum of PCOS cases. The genetic similarities between cases diagnosed based on the two criteria have been largely unknown. Previous studies in Chinese and European subjects have identified 16 loci associated with risk of PCOS. We report a fixed-effect, inverse-weighted-variance meta-analysis from 10,074 PCOS cases and 103,164 controls of European ancestry and characterisation of PCOS related traits. We identified 3 novel loci (near PLGRKT, ZBTB16 and MAPRE1), and provide replication of 11 previously reported loci. Only one locus differed significantly in its association by diagnostic criteria; otherwise the genetic architecture was similar between PCOS diagnosed by self-report and PCOS diagnosed by NIH or non-NIH Rotterdam criteria across common variants at 13 loci. Identified variants were associated with hyperandrogenism, gonadotropin regulation and testosterone levels in affected women. Linkage disequilibrium score regression analysis revealed genetic correlations with obesity, fasting insulin, type 2 diabetes, lipid levels and coronary artery disease, indicating shared genetic architecture between metabolic traits and PCOS. Mendelian randomization analyses suggested variants associated with body mass index, fasting insulin, menopause timing, depression and male-pattern balding play a causal role in PCOS. The data thus demonstrate 3 novel loci associated with PCOS and similar genetic architecture for all diagnostic criteria. The data also provide the first genetic evidence for a male phenotype for PCOS and a causal link to depression, a previously hypothesized comorbid disease. Thus, the genetics provide a comprehensive view of PCOS that encompasses multiple diagnostic criteria, gender, reproductive potential and mental health., This work has been supported by MRC grant MC_U106179472 (FD, KO, JRBP), Samuel Oschin Comprehensive Cancer Institute Developmental Funds, Center for Bioinformatics and Functional Genomics and Department of Biomedical Sciences Developmental Funds (MRJ), NCI P30CA177558 (CH), NCI UM1CA186107 (PK), European Regional Development Fund (Project No. 2014-2020.4.01.15-0012) and the European Union’s Horizon 2020 research and innovation program under grant agreements No 692065 (TL, RM, AS) and 692145 (RM), NICHD R01HD065029 (RS), Estonian Ministry of Education and Research (grant IUT34-16 to TL), NICHD R01HD057450 (MU), NICHD P50HD044405 (AD), NICHD R01HD057223 (AD), R01HD085227 (MGH, AD), deCode Genetics (GT, UT, KS, US), Raine Medical Research Foundation Priming Grant (BHM), SCGOPHCG RAC 2015-16/034 (SGW, BGAS), 2016-17/018 (BGAS), NIHR BRC, Wellcome Trust, MRC (TDS), Eris M. Field Chair in Diabetes Research (MOG), NIDDK P30 DK063491 (MOG), NIDDK U01DK094431, U01DK048381 (DE), NICHD U10HD38992 (RL), Estonian Ministry of Education and Research (grant IUT34-16), Enterprise Estonia (grant EU48695); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, grant SARM, EU324509 to AS), Wellcome (090532, 098381, 203141); European Commission (ENGAGE: HEALTH-F4-2007-201413 to MIM), MRC G0802782, MR/M012638/1 (SF), Li Ka Shing Foundation, WT-SSI/John Fell Funds, NIHR Biomedical Research Centre, Oxford, Widenlife and NICHD 5P50HD028138-27 (CML), NICHD R01HD065029, ADA 1-10-CT-57, Harvard Clinical and Translational Science Center, from the National Center for Research Resources 1UL1 RR025758 (CKW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Background Successful establishment of pregnancy hinges on appropriate communication between the embryo and the uterus prior to implantation, but the nature of this communication remains poorly understood. Here, we tested the hypothesis that the endometrium is receptive to embryo-derived signals in the form of RNA. Methods We have utilized a non-contact co culture system to simulate the conditions of pre implantation environment of the uterus. We bioorthogonally tagged embryonic RNA and tracked the transferred transcripts to endometrium. Transferred transcripts were separated from endometrial transcripts and sequenced. Changes in endometrial transcripts were quantified using quantitative PCR. Results We show that three specific transcripts are transferred to endometrial cells. We subsequently demonstrate a role of extracellular vesicles (EVs) in this process, as EVs obtained from cultured trophoblast spheroids incubated with endometrial cells induced down-regulation of all the three identified transcripts in endometrial cells. Finally, we show that EVs/nanoparticles captured from conditioned culture media of viable embryos as opposed to degenerating embryos induce ZNF81 down-regulation in endometrial cells, hinting at the functional importance of this intercellular communication. Conclusion Ultimately, our findings demonstrate the existence of an RNA-based communication which may be of critical importance for the establishment of pregnancy.

Chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) has revolutionized our understanding of chromatin-related biological processes. The method, however, requires thousands of cells and has therefore limited applications in situations where cell numbers are limited. Here we describe a novel method called Restriction Assisted Tagmentation Chromatin Immunoprecipitation (RAT-ChIP) that enables global histone modification profiling from as few as 100 cells. The method is simple, cost-effective and takes a single day to complete. We demonstrate the sensitivity of the method by deriving the first genome-wide maps of histone H3K4me3 and H3K27me3 modifications of inner cell mass and trophectoderm of bovine blastocyst stage embryos.

Introduction: The goal of this study was to identify the profile of genital tract infections and their relationship with clinical and demographic parameters as well as tubal diseases among infertile women in Vietnam. Methodology: In this cross-sectional descriptive study, we enrolled 597 women undergoing infertility treatment at the Center for Reproductive Endocrinology & Infertility, Hue University Hospital, Vietnam. All of the study participants were interviewed and examined by a gynecologist. Consecutive tests were then conducted including direct microscopy examination (wet mount and Gram stain), vaginal culture, polymerase chain reaction (PCR) for chlamydia diagnosis from a cervical canal swab, and a blood test for syphilis detection. A hysterosalpingogram (HSG) was carried out to examine the uterine cavity and Fallopian tubes. Results: A gynecologic infection was diagnosed in 43.4% (259/597) of the infertile women. Bacterial vaginosis was the most common condition at 19.6%of the cases. Candida spp., Chlamydia trachomatis, and Trichomonas vaginalis infections accounted for 17.4%, 3.7%, and 0.3%, respectively. Normal HSG results accounted for 87.4% of the women while 5.5% had 2-sided tubal occlusions, 5.4% had 1-sided tubal occlusions, 1.0% had 1-sided hydrosalpinx, and 0.7% had 2-sided hydrosalpinx. There was no significant association between tubal diseases and current infections; however, aerobic vaginitis increased the risk of tubal diseases by 2.4 times. Conclusions: A marked proportion of infertile Vietnamese women have genital tract infections that can significantly influence their reproductive function and performance. These infections should be routinely screened and treated properly to prevent their consequences, such as infertility, which is especially important in developing countries.

The corpus of bioinformatics resources is huge and expanding rapidly, presenting life scientists with a growing challenge in selecting tools that fit the desired purpose. To address this, the European Infrastructure for Biological Information is supporting a systematic approach towards a comprehensive registry of tools and databases for all domains of bioinformatics, provided under a single portal (https://bio.tools). We describe here the practical means by which scientific communities, including individual developers and projects, through major service providers and research infrastructures, can describe their own bioinformatics resources and share these via bio.tools.

Endometriosis is a benign chronic condition characterized by the existence of endometrial-like stroma and glandular tissue in extrauterine locations. The molecular mechanisms of its pathogenesis have not been elucidated. We have studied the role of EXTL3 (exostosin-like 3) in endometriosis and found that it is expressed in endometrial tissue as well as endometriosis lesions. We have found that serum from endometriosis patients contains a factor or factors, which interact with EXTL3 resulting in strongly increased colony formation in regenerating cell culture. We also found increased anti-EXTL3 antibodies in endometriosis patients’ sera. EXTL3 is an N-acetyl glucosamine (GlcNAc) transferase, performing a key step in heparan sulfate (HS) glucosaminoglycan synthesis. Many viruses replicate in regenerating epithelial cells and use HS as a receptor for cell entry. We measured antibody titres to viruses, which use HS as a receptor for cell entry, and found rarely increased titres for these viruses in endometriosis sera, whereas titres to viruses using other receptors were equally distributed in study groups. The data indicate that perturbation of HS metabolism is associated with endometriosis.

Background: Ovarian insufficiency is a major concern for long-term cancer survivors. Although semen freezing is well established to preserve male fertility, the possibilities to secure post-cancer female fertility are mostly limited to oocyte or embryo freezing. These methods require time-consuming ovarian stimulation with or without in vitro fertilization (IVF) that evidently delays cancer therapy. Ovarian tissue cryopreservation and subsequent thawed tissue autotransplantation are considered the most promising alternative strategy for restoring the fertility of oncology patients, which has not yet received the full clinical acceptance. Therefore, all successful cases are needed to prove its reliability and safety.Case presentation: Here we report a single case in Estonia, where a 28-year-old woman with malignant breast neoplasm had ovarian cortex cryopreserved before commencing gonadotoxic chemo- and radiotherapy. Two years after cancer therapy, the patient underwent heterotopic ovarian tissue transplantation into the lateral pelvic wall. The folliculogenesis was stimulated in the transplanted tissue by exogenous follicle-stimulating hormone and oocytes were collected under ultrasound guidance for IVF and embryo transfer. The healthy boy was born after full-term gestation in 2014, first in Eastern Europe.Conclusion: Despite many countries have reported the first implementation of the ovarian tissue freezing and transplantation protocols, the data is still limited on the effectiveness of heterotopic ovarian transplant techniques. Thus, all case reports of heterotopic ovarian tissue transplantation and long-term follow-ups to describe the children's health are valuable source of clinical experience.

MicroRNAs (miRNAs) have known roles in the post-transcriptional regulation of various biological processes including ovarian follicle development. We have previously identified miRNAs from human pre-ovulatory granulosa cells that are expressed from the intronic regions of two key genes in normal follicular development: FSH receptor (FSHR) and CYP19A1, the latter encoding the aromatase enzyme. In the present study, we aim to identify the targets regulated by those two miRNAs: hsa-miR-548ba and hsa-miR-7973, respectively. The miRNAs of interest were endogenously expressed in KGN cell-line, gene expression changes were analyzed by Affymetrix microarray and confirmed by RT-qPCR. Potential miRNA-regulated sequences were further filtered from the obtained results by bioinformatic target prediction algorithms and validated for direct miRNA:mRNA binding by luciferase reporter assay. Our results verified Leukemia inhibitory factor receptor (LIFR), Phosphatase and tensin homolog (PTEN), Neogenin 1 (NEO1) and SP110 nuclear body protein (SP110) as target genes for hsa-miR-548ba. Hsa-miR-7973 target genes ADAM metallopeptidase domain 19 (ADAM19), Peroxidasin (PXDN) and Formin like 3 (FMNL3) also passed all verification steps. In conclusion we propose that hsa-miR-548ba may be involved in the regulation of follicle growth and activation via LIFR and PTEN. Hsa-miR-7973 may be implicated in the modulation of extracellular matrix and cell-cell interactions. Taken together, our results suggest that those two miRNAs of interest have important regulatory roles in granulosa cells and in follicle development in general.Summary sentenceConfirmed targets of miRNAs hsa-miR-548ba and hsa-miR-7973 are involved in follicle recruitment, apoptosis, intercellular interactions and extracellular matrix remodeling pathways in KGN cells.

Miscarriage is a common complex trait that affects 10-25% of clinically confirmed pregnancies1,2. Here we present the first large-scale genetic association analyses with 69,118 cases from five different ancestries for sporadic miscarriage and 750 cases of European ancestry for recurrent miscarriage, and up to 359,469 female controls. We identify one genome-wide significant association on chromosome 13 (rs146350366, minor allele frequency (MAF) 1.2%,Pmeta=3.2×-8(CI) 1.2-1.6) for sporadic miscarriage in our European ancestry meta-analysis (50,060 cases and 174,109 controls), located nearFGF9involved in pregnancy maintenance3and progesterone production4. Additionally, we identified three genome-wide significant associations for recurrent miscarriage, including a signal on chromosome 9 (rs7859844, MAF=6.4%,Pmeta=1.3×-8in controlling extravillous trophoblast motility5. We further investigate the genetic architecture of miscarriage with biobank-scale Mendelian randomization, heritability and, genetic correlation analyses. Our results implicate that miscarriage etiopathogenesis is partly driven by genetic variation related to gonadotropin regulation, placental biology and progesterone production.

RESEARCH QUESTION: How does mucin MUC20 expression change during the menstrual cycle in different cell types of human endometrium? DESIGN: Study involved examination of MUC20 expression in two previously published RNA-seq datasets in whole endometrial tissue (n = 10), sorted endometrial epithelial (n = 44) or stromal (n = 42) cell samples. RNA-Seq results were validated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) in whole tissue (n = 10), sorted epithelial (n = 17) and stromal (n = 17) cell samples. MUC20 protein localization and expression were analysed in human endometrium by immunohistochemical analysis of intact endometrial tissue (n = 6) and also Western blot of cultured stromal and epithelial cells (n = 2). RESULTS: MUC20 is differentially expressed in the endometrium between the pre-receptive and receptive phases. We show that MUC20 is predominantly expressed by epithelial cells of the receptive endometrium, both at the mRNA (RNA-Seq, P = 0.005; qRT-PCR, P = 0.039) and protein levels (Western blot; immunohistochemistry, P = 0.029). CONCLUSION: Our results indicate MUC20 as a novel marker of mid-secretory endometrial biology. We propose a model of MUC20 function in the hepatocyte growth factor (HGF)-activated mesenchymal-epithelial transition (MET) receptor signalling specifically in the receptive phase. Further investigations should reveal the precise function of MUC20 in human endometrium and the possible connection between MUC20 and HGF-activated MET receptor signalling. MUC20 could potentially be included in the list of endometrial receptivity markers after further clinical validation.

Non-invasive prenatal testing (NIPT) enables accurate detection of fetal chromosomal trisomies. The majority of publicly available computational methods for sequencing-based NIPT analyses rely on low-coverage whole-genome sequencing (WGS) data and are not applicable for targeted high-coverage sequencing data from cell-free DNA samples. Here, we present a novel computational framework for a targeted high-coverage sequencing-based NIPT analysis. The developed framework uses a hidden Markov model (HMM) in conjunction with a supplemental machine learning model, such as decision tree (DT) or support vector machine (SVM), to detect fetal trisomy and parental origin of additional fetal chromosomes. These models were developed using simulated datasets covering a wide range of biologically relevant scenarios with various chromosomal quantities, parental origins of extra chromosomes, fetal DNA fractions, and sequencing read depths. Developed models were tested on simulated and experimental targeted sequencing datasets. Consequently, we determined the functional feasibility and limitations of each proposed approach and demonstrated that read count-based HMM achieved the best overall classification accuracy of 0.89 for detecting fetal euploidies and trisomies on simulated dataset. Furthermore, we show that by using the DT and SVM on the HMM classification results, it was possible to increase the final trisomy classification accuracy to 0.98 and 0.99, respectively. We demonstrate that read count and allelic ratio-based models can achieve a high accuracy (up to 0.98) for detecting fetal trisomy even if the fetal fraction is as low as 2%. Currently, existing commercial NIPT analysis requires at least 4% of fetal fraction, which can be possibly a challenge in case of early gestational age (35 kg/m2). More accurate detection can be achieved at higher sequencing depth using HMM in conjunction with supplemental models, which significantly improve the trisomy detection especially in borderline scenarios (e.g., very low fetal fraction) and enables to perform NIPT even earlier than 10 weeks of pregnancy.

Bioinformaticians and biologists rely increasingly upon workflows for the flexible utilization of the many life science tools that are needed to optimally convert data into knowledge. We outline a pan-European enterprise to provide a catalogue (https://bio.tools) of tools and databases that can be used in these workflows. bio.tools not only lists where to find resources, but also provides a wide variety of practical information. Electronic supplementary material The online version of this article (10.1186/s13059-019-1772-6) contains supplementary material, which is available to authorized users.

Ovarian function is central to female fertility, and several genome-wide association studies (GWAS) have been carried out to elucidate the genetic background of traits and disorders that reflect and affect ovarian physiology. While GWAS have been successful in reporting numerous genetic associations and highlighting involved pathways relevant to reproductive aging, for ovarian disorders, such as premature ovarian insufficiency and polycystic ovary syndrome, research has lagged behind due to insufficient study sample size. Novel approaches to study design and analysis methods that help to fit GWAS findings into biological context will improve our knowledge about genetics governing ovarian function in fertility and disease, and provide input for clinical tools and better patient management.

Little consensus has been reached on the best protocol for endometrial preparation for frozen embryo transfer (FET). It is not known how, and to what extent, hormone supplementation in artificial cycles influences endometrial preparation for embryo implantation at a molecular level, especially in patients who have experienced recurrent implantation failure. Transcriptome analysis of 15 endometrial biopsy samples at the time of embryo implantation was used to compare two different endometrial preparation protocols, natural versus artificial cycles, for FET in women who have experienced recurrent implantation failure compared with fertile women. IPA and DAVID were used for functional analyses of differentially expressed genes. The TRANSFAC database was used to identify oestrogen and progesterone response elements upstream of differentially expressed genes. Cluster analysis demonstrated that natural cycles are associated with a better endometrial receptivity transcriptome than artificial cycles. Artificial cycles seemed to have a stronger negative effect on expression of genes and pathways crucial for endometrial receptivity, including ESR2, FSHR, LEP, and several interleukins and matrix metalloproteinases. Significant overrepresentation of oestrogen response elements among the genes with deteriorated expression in artificial cycles (P < 0.001) was found; progesterone response elements predominated in genes with amended expression with artificial cycles (P = 0.0052).

Genomic imprinting is an epigenetic feature characterized by parent-specific monoallelic gene expression. The aim of this study was to compare the DNA methylation status of imprinted genes and imprinting control regions (ICRs), harboring differentially methylated regions (DMRs) in a comprehensive panel of 18 somatic tissues. The germline DMRs analyzed were divided into ubiquitously imprinted and placenta-specific DMRs, which show identical and different methylation imprints in adult somatic and placental tissues, respectively. We showed that imprinted genes and ICR DMRs maintain methylation patterns characterized by intermediate methylation levels in somatic tissues, which are pronounced in a specific region of the promoter area, located 200–1500 bp from the transcription start site. This intermediate methylation is concordant with gene expression from a single unmethylated allele and silencing of a reciprocal parental allele through DNA methylation. The only exceptions were seen for ICR DMRs of placenta-specific imprinted genes, which showed low levels of methylation, suggesting that these genes escape parent-specific epigenetic regulation in somatic tissues.

Biological fluid sample collection often includes the risk of blood contamination that may alter the proteomic profile of biological fluid. In proteomics studies, exclusion of contaminated samples is usually based on visual inspection and counting of red blood cells in the sample; analysis of specific blood derived proteins is less used. To fill the gap, we developed a fast and sensitive method for ascertainment of blood contamination in crude biological fluids, based on specific blood-derived protein, hemoglobin detection by MALDI-TOF MS. The MALDI-TOF MS based method allows detection of trace hemoglobin with the detection limit of 0.12 nM. UV-spectrometry, which was used as reference method, was found to be less sensitive. The main advantages of the presented method are that it is fast, effective, sensitive, requires very small sample amount and can be applied for detection of blood contamination in various biological fluids collected for proteomics studies. Method applicability was tested on human cerebrospinal and follicular fluid, which proteomes generally do not contain hemoglobin, however, which possess high risk for blood contamination. Present method successfully detected the blood contamination in 12 % of cerebrospinal fluid and 24 % of follicular fluid samples. High percentage of contaminated samples accentuates the need for initial inspection of proteomic samples to avoid incorrect results from blood proteome overlap.

Human granulosa cells acquired as leftover from IVF treatment can be used to study infertility problems and are a valuable tool in the research of follicle maturation and ovulation. There is a need for more defined and long-term culture protocols for studying the response of granulosa cells upon treatment with selected hormones/chemicals. In the current study, we tested the effect of adding FGF2, IGF2 and FSH into defined basal medium in order to find culture conditions that would support proliferation of cumulus and mural granulosa cells along with the expression of common granulosa cell type markers such as FSHR, AMHR2, LHR and CYP19A1. We found that FGF2, IGF2 together with FSH helped to retain granulosa cell marker expression while supporting cell survival at least for two weeks of culture. The defined serum-free culture conditions for long-term culturing will be valuable in providing new standards in the research of human granulosa cells.

The endometrium undergoes extensive changes to prepare for embryo implantation and microRNAs (miRNAs) have been described as playing a significant role in the regulation of endometrial receptivity. However, there is no consensus about the miRNAs involved in mid-secretory endometrial functions. We analysed the complete endometrial miRNome from early secretory (pre-receptive) and mid-secretory (receptive) phases from fertile women and from patients with recurrent implantation failure (RIF) to reveal differentially expressed (DE) miRNAs in the mid-secretory endometrium. Furthermore, we investigated whether the overall changes during early to mid-secretory phase transition and with RIF condition could be reflected in blood miRNA profiles. In total, 116 endometrial and 114 matched blood samples collected from two different population cohorts were subjected to small RNA sequencing. Among fertile women, 91 DE miRNAs were identified in the mid-secretory vs. early secretory endometrium, while no differences were found in the corresponding blood samples. The comparison of mid-secretory phase samples between fertile and infertile women revealed 21 DE miRNAs from the endometrium and one from blood samples. Among discovered novel miRNAs, chr2_4401 was validated and showed up-regulation in the mid-secretory endometrium. Besides novel findings, we confirmed the involvement of miR-30 and miR-200 family members in mid-secretory endometrial functions., This research was funded by Estonian Ministry of Education and Research (grant IUT34-16); Enterprise Estonia (grant EU48695); the EU-FP7 Eurostars program (grant NOTED, EU41564); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, grant SARM, EU324509); Horizon 2020 innovation programme (WIDENLIFE, 692065); grant from the University of Granada (Incorporación de jóvenes doctores) and grant from the Spanish Ministry of Economy, Industry and Competitiveness–MINECO–(RYC-2016-21199 and grant ENDORE SAF2017-87526).

ObjectiveThe study aimed to validate a whole-genome sequencing-based NIPT method and our newly developed NIPTmer analysis software with the potential to integrate the pipeline into prenatal clinical care in Estonia.MethodIn total, 447 maternal blood samples were included to the study. Analysis pipeline involved whole-genome library preparation and massively parallel sequencing on Illumina NextSeq 500. Aneuploidy status was determined with NIPTmer software, which is based on counting pre-defined per-chromosome sets of unique k-mers from raw sequencing data. To estimate fetal fraction (FF) from total cell-free DNA SeqFF was implemented.ResultsNIPTmer software allowed to identify correctly all samples of non-mosaic T21 (15/15), T18 (9/9) and T13 (4/4) cases. However, one mosaic T18 remained undetected. Six false positive results were observed, including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). FF < 4% (2.8-3.99%) was estimated in eight samples, including two samples with T13 and T18. Despite low FF, these two samples were determined as aneuploid with NIPTmer software.ConclusionOur NIPT analysis pipeline proved to perform efficiently in detecting common fetal aneuploidies T21, T18 and T13 and is feasible for implementation into clinical service in Estonia.