Does ulipristal acetate (UPA) used for emergency contraception (EC) interfere with the human embryo implantation process? UPA, at the dosage used for EC, does not affect human embryo implantation process, in vitro. A single pre-ovulatory dose of UPA (30 mg) acts by delaying or inhibiting ovulation and is recommended as first choice among emergency contraceptive pills due to its efficacy. The compound has also been demonstrated to have a dose-dependent effect on the endometrium, which theoretically could impair endometrial receptivity but its direct action on human embryo implantation has not yet been studied. Effect of UPA on embryo implantation process was studied in an in vitro endometrial construct. Human embryos were randomly added to the cultures and cultured for 5 more days with UPA (n = 10) or with vehicle alone (n = 10) to record the attachment of embryos. Endometrial biopsies were obtained from healthy, fertile women on cycle day LH+4 and stromal and epithelial cells were isolated. A three-dimensional in vitro endometrial co-culture system was constructed by mixing stromal cells with collagen covered with a layer of epithelial cells and cultured in progesterone containing medium until confluence. The treatment group received 200 ng/ml of UPA. Healthy, viable human embryos were placed on both control and treatment cultures. Five days later the cultures were tested for the attachment of embryos and the 3D endometrial constructs were analysed for endometrial receptivity markers by real-time PCR. There was no significant difference in the embryo attachment rate between the UPA treated group and the control group as 5 out of 10 human embryos exposed to UPA and 7 out of 10 embryos in the control group attached to the endometrial cell surface (P = 0.650). Out of 17 known receptivity genes studied here, only 2 genes, HBEGF (P = 0.009) and IL6 (P = 0.025) had a significant up-regulation and 4 genes, namely HAND2 (P = 0.003), OPN (P = 0.003), CALCR (P = 0.016) and FGF2 (P = 0.023) were down-regulated with the exposure of UPA, compared with control group. This proof of concept study was conducted with a few human embryos, as their availability was limited. Although the 3D model used for this study is well established and the artificial endometrial luminal epithelium shown to express progesterone regulated markers of endometrial receptivity it is still an in vitro model, lacking all cell types that constitute the receptive endometrium in vivo. This study provides new insights on the mechanism of action of UPA on human embryo implantation, demonstrating that UPA in a dosage used for EC does not affect embryo viability and the implantation process of embryo. Progesterone receptor modulators (PRMs) hold the potential to be attractive estrogen- and gestagen-free contraceptives and thus may be made available to a larger proportion of women globally due to these findings. Swedish Research Council (K2010-54X-14212-09-3) and support provided through the regional agreement on medical training and clinical research (ALF) between Stockholm County Council and Karolinska University Hospital.

Is the composition of microRNAs (miRNAs) in uterine fluid (UF) of women with recurrent implantation failure (RIF) different from that of healthy fertile women? 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. 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. 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. 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). 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 <0.05 and a fold change (FC) of $≤$−2 or $≥$2. When analyzed with published literature, we found that several of the differentially abundant miRNAs are expressed in endometrial epithelial cells and have been reported in endometrial extracellular vesicles and in association with endometrial receptivity and RIF. Their predicted target genes were further expressed both in the trophectodermal cells of blastocyst-stage embryos and endometrial mid-secretory epithelial cells, as assessed by publicly available single-cell transcriptome-sequencing studies. Pathway analysis further revealed that 25 pathways, having key roles in endometrial receptivity and implantation, were significantly enriched. Hsa-miR-486-5p (FC −20.32; P-value = 0.004) and hsa-miR-92b-3p (FC −9.72; P-value = 0.004) were successfully technically validated with RT-PCR. The data are available in Gene Expression Omnibus (GEO) at https://www.ncbi.nlm.nih.gov/geo/ with GEO accession number: GSE173289. 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. 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. 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.

Cell properties, such as attachment, adhesion and invasion, are important for the normal function of the endometrium. However, it is believed that the same properties may also be involved in the development of gynaecological diseases, such as endometriosis. Endometrial cells, shed by retrograde menstruation, may have an aberrant expression of molecules involved in these functions, leading to endometriosis. Therefore, the aim of this study was to investigate the expression of proteins involved in adhesion, attachment and invasion in eutopic and ectopic endometrium. Endometrial biopsy specimens were collected from healthy volunteers (controls: proliferative phase, n = 10; secretory phase, n = 15) and from endometriosis patients (proliferative phase: n = 9, secretory phase: n = 10). Biopsy specimens from endometriomas were also collected (proliferative phase: n = 9, secretory phase: n = 10). Expression of apolipoprotein E (ApoE), integrin β-2 (ITGB2), integrin β-7 (ITGB7), Laminin γ-1 (LAMC1), CD24 molecule (CD24) and junctional adhesion molecule-1 (JAM-1) was evaluated with real-time reverse transcriptase polymerase chain reaction and immunohistochemistry. The endometrium from controls and women with endometriosis expressed ApoE, ITGB2, ITGB7, LAMC1, CD24 and JAM-1. Gene expression of ApoE and JAM-1 was decreased in both proliferative and secretory phase in the endometrium from women with endometriosis compared with control endometrium. Also, mRNA expression of LAMC1 was reduced in the endometrium from endometriosis patients compared with controls in the proliferative phase. An altered gene expression of CD24 was seen between the endometrium from endometriosis patients and endometriomas in the secretory phase. The ITGB2 protein expression was altered in epithelia cells between the endometrium from healthy volunteers and endometriosis patients in the secretory phase. We have shown differential expression of adhesion, attachment and invasion proteins in proliferative and secretory endometrium from controls and endometriosis patients and in endometriomas. This study suggests that molecules with these properties may have a role in the anchoring of endometrial cells at ectopic sites, thus initiating the development of endometriosis.

The complexity of endometrial receptivity at the molecular level needs to be explored in detail to improve the management of infertility. Here, differential expression of transcriptomes in receptive endometrial glands and stroma revealed Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a progesterone regulated factor and confirmed by various methods, both at mRNA and protein level. The involvement of ENPP3 in embryo attachment was tested in an in vitro model for human embryo implantation. Interestingly, there was high expression of ENPP3 mRNA in stroma but not protein. Presence of N-glycosylated ENPP3 in receptive phase uterine fluid in women confirms its regulation by progesterone and makes it possible to use in a non-invasive test of endometrial receptivity.