Progesterone triggers Rho kinase-cofilin axis during in vitro and in vivo endometrial decidualization.

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 < 0.05) and are further augmented following the 9-day treatment (P < 0.001) with mixtures containing progesterone and 8-Br-cAMP or forskolin. The presence of progesterone is necessary for activation of ROCK, yet it is dispensable in the case of PKA and Akt/PKB; in comparison to controls, PCOS patient-derived ESCs feature dampened response to progesterone. In non-cultured ESCs isolated from secretory vs proliferative phase tissue, only activity of ROCK is increased (P < 0.01). ROCK2 protein levels are slightly elevated in secretory versus proliferative ESCs (relative mean standard deviation < 50%), and ROCK2 mRNA is elevated in mid-secretory versus proliferative full tissue samples (P < 0.05) obtained from controls but not PCOS patients. Activation of ROCK2 downstream signalling results in increase of phospho-S3 CFL1 in secretory endometrium (P < 0.001) as well as in vitro decidualized ESCs (P < 0.01) from controls but not PCOS patients. ROCK2-triggered alterations in the cytoskeleton are reflected by the significantly decreased motility of in vitro decidualized ESCs (P < 0.05). 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.

Chemosensitivity and chemoresistance in endometriosis - differences for ectopic versus eutopic cells.

RESEARCH QUESTION: Endometriosis is a common gynaecological disease defined by the presence of endometrium-like tissue outside the uterus. This complex disease, often accompanied by severe pain and infertility, causes a significant medical and socioeconomic burden; hence, novel strategies are being sought for the treatment of endometriosis. Here, we set out to explore the cytotoxic effects of a panel of compounds to find toxins with different efficiency in eutopic versus ectopic cells, thus highlighting alterations in the corresponding molecular pathways. DESIGN: The effect on cellular viability of 14 compounds was established in a cohort of paired eutopic and ectopic endometrial stromal cell samples from 11 patients. The biological targets covered by the panel included pro-survival enzymes, cytoskeleton proteins, the proteasome and the cell repair machinery. RESULTS: Protein kinase inhibitors GSK690693, ARC-775 and sorafenib, proteasome inhibitor bortezomib, and microtubule-depolymerizing toxin monomethyl auristatin E were more effective in eutopic cells. In contrast, 10 µmol/l of the anthracycline toxin doxorubicin caused cellular death in ectopic cells more effectively than in eutopic cells. The large-scale sequencing of mRNA isolated from doxorubicin-treated and control cells indicated different survival strategies in eutopic versus ectopic endometrium. CONCLUSIONS: Overall, the results confirm evidence of large-scale metabolic reprogramming in endometriotic cells, which underlies the observed differences in sensitivity towards toxins. The enhanced efficiency of doxorubicin interfering with redox equilibria and/or DNA repair mechanisms pinpoints key players that can be potentially used to selectively target ectopic lesions in endometriosis.

Differentially-Expressed miRNAs in Ectopic Stromal Cells Contribute to Endometriosis Development: The Plausible Role of miR-139-5p and miR-375.

microRNA (miRNA) expression level alterations between endometrial tissue and endometriotic lesions indicate their involvement in endometriosis pathogenesis. However, as both endometrium and endometriotic lesions consist of different cell types in various proportions, it is not clear which cells contribute to variability in miRNA levels and the overall knowledge about cell-type specific miRNA expression in ectopic cells is scarce. Therefore, we utilized fluorescence-activated cell sorting to isolate endometrial stromal cells from paired endometrial and endometrioma biopsies and combined it with high-throughput sequencing to determine miRNA alterations in endometriotic stroma. The analysis revealed 149 abnormally expressed miRNAs in endometriotic lesions, including extensive upregulation of miR-139-5p and downregulation of miR-375 compared to eutopic cells. miRNA transfection experiments in the endometrial stromal cell line ST-T1b showed that the overexpression of miR-139-5p resulted in the downregulation of homeobox A9 (HOXA9) and HOXA10 expression, whereas the endothelin 1 (EDN1) gene was regulated by miR-375. The results of this study provide further insights into the complex molecular mechanisms involved in endometriosis pathogenesis and demonstrate the necessity for cell-type-specific analysis of ectopic tissues to understand the interactions between different cell populations in disease onset and progression.

High-throughput mRNA sequencing of stromal cells from endometriomas and endometrium.

The aetiology of endometriosis is still unclear and to find mechanisms behind the disease development, it is important to study each cell type from endometrium and ectopic lesions independently. The objective of this study was to uncover complete mRNA profiles in uncultured stromal cells from paired samples of endometriomas and eutopic endometrium. High-throughput mRNA sequencing revealed over 1300 dysregulated genes in stromal cells from ectopic lesions, including several novel genes in the context of endometriosis. Functional annotation analysis of differentially expressed genes highlighted pathways related to cell adhesion, extracellular matrix-receptor interaction and complement and coagulation cascade. Most importantly, we found a simultaneous upregulation of complement system components and inhibitors, indicating major imbalances in complement regulation in ectopic stromal cells. We also performed in vitro experiments to evaluate the effect of endometriosis patients' peritoneal fluid (PF) on complement system gene expression levels, but no significant impact of PF on C3, CD55 and CFH levels was observed. In conclusion, the use of isolated stromal cells enables to determine gene expression levels without the background interference of other cell types. In the future, a new standard design studying all cell types from endometriotic lesions separately should be applied to reveal novel mechanisms behind endometriosis pathogenesis.

Deep Quantitative Proteomics Reveals Extensive Metabolic Reprogramming and Cancer-Like Changes of Ectopic Endometriotic Stromal Cells.

Endometriosis is a prevalent health condition in women of reproductive age characterized by ectopic growth of endometrial-like tissue in the extrauterine environment. Thorough understanding of the molecular mechanisms underlying the disease is still incomplete. We dissected eutopic and ectopic endometrial primary stromal cell proteomes to a depth of nearly 6900 proteins using quantitative mass spectrometry with a spike-in SILAC standard. Acquired data revealed metabolic reprogramming of ectopic stromal cells with extensive upregulation of glycolysis and downregulation of oxidative respiration, a widespread metabolic phenotype known as the Warburg effect and previously described in many cancers. These changes in metabolism are additionally accompanied by attenuated aerobic respiration of ectopic endometrial stromal cells as measured by live-cell oximetry and by altered mRNA levels of respective enzyme complexes. Our results additionally highlight other molecular changes of ectopic endometriotic stromal cells indicating reduced apoptotic potential, increased cellular invasiveness and adhesiveness, and altered immune function. Altogether, these comprehensive proteomics data refine the current understanding of endometriosis pathogenesis and present new avenues for therapies.

Characterisation of probiotic properties in human vaginal lactobacilli strains.

BACKGROUND: Vaginal lactobacilli offer protection against recurrent urinary infections, bacterial vaginosis, and vaginal candidiasis. OBJECTIVE: To characterise the isolated vaginal lactobacilli strains for their probiotic properties and to compare their probiotic potential. METHODS: The Lactobacillus strains were isolated from vaginal samples by conventional culturing and identified by sequencing of the 16S rDNA fragment. Several functional properties were detected (production of hydrogen peroxide and lactic acid; antagonistic activity against Escherichia coli, Candida albicans, Candida glabrata, and Gardnerella vaginalis; auto-aggregation and adhesiveness) as well as safety (haemolytic activity, antibiotic susceptibility, presence of transferrable resistance genes). RESULTS: A total of 135 vaginal lactobacilli strains of three species, Lactobacillus crispatus (56%), Lactobacillus jensenii (26%), and Lactobacillus gasseri (18%) were characterised using several functional and safety tests. Most of L. crispatus (89%) and L. jensenii (86%) strains produced H2O2. The best lactic acid producers were L. gasseri (18.2±2.2 mg/ml) compared to L. crispatus (15.6±2.8 mg/ml) and L. jensenii (11.6±2.6 mg/ml) (p<0.0001; p<0.0001, respectively). L. crispatus strains showed significantly higher anti-E. coli activity compared to L. jensenii. L. gasseri strains expressed significantly lower anticandidal activity compared to L. crispatus and L. jensenii (p<0.0001). There was no significant difference between the species in antagonistic activity against G. vaginalis. Nearly a third of the strains were able to auto-aggregate while all the tested strains showed a good ability to adhere to HeLa cells. None of the tested lactobacilli caused haemolysis. Although phenotypical resistance was not found to ampicillin, chloramphenicol, erythromycin, gentamycin, tetracycline, and vancomycin, the erm(B), tet(M), and tet(K) were detected in some strains. All strains were resistant to metronidazole, trimethoprim/sulfamethoxazole, and kanamycin. CONCLUSIONS: Our study revealed that the production of different antimicrobial metabolites is highly strain-specific and that the metabolites are not correlated with each other. L. crispatus displays better antagonistic activity against E. coli and Candida spp. than L. gasseri and L. jensenii; therefore; a potential probiotic candidate could be found among L. crispatus strains.

Endocrine disrupting chemicals interfere with decidualization of human primary endometrial stromal cells in vitro.

Multiple studies have shown associations between exposure to endocrine disrupting chemicals (EDCs) and reduced fertility in women. However, little is known about the target organs of chemical disruption of female fertility. Here, we focus on the hormone-sensitive uterine lining, the endometrium, as a potential target. Decidualization is the morphological and functional change that endometrial stromal cells undergo to support endometrial receptivity, which is crucial for successful implantation, placentation, and pregnancy. We investigated the effect of nine selected EDCs on primary human endometrial stromal cell decidualization in vitro. The cells were exposed to a decidualization-inducing mixture in the presence or absence of 1 µM of nine different EDCs for nine days. Extent of decidualization was assessed by measuring the activity of cAMP dependent protein kinase, Rho-associated coiled-coil containing protein kinase, and protein kinase B in lysates using photoluminescent probes, and secretion of prolactin into the media by using ELISA. Decidualization-inducing mixture upregulated activity of protein kinases and prolactin secretion in cells derived from all women. Of the tested chemicals, dichlorodiphenyldichloroethylene (p,p'-DDE), hexachlorobenzene (HCB) and perfluorooctanesulfonic acid (PFOS) significantly reduced decidualization as judged by the kinase markers and prolactin secretion. In addition, bisphenol A (BPA) reduced prolactin secretion but did not significantly affect activity of the kinases. None of the EDCs was cytotoxic, based on the assessment of total protein content or activity of the viability marker casein kinase 2 in lysates. These results indicate that EDCs commonly present in the blood circulation of reproductive-aged women can reduce decidualization of human endometrial stromal cells in vitro. Future studies should focus on detailed hazard assessment to define possible risks of EDC exposure to endometrial dysfunction and implantation failure in women.

Human endometrial cell-type-specific RNA sequencing provides new insights into the embryo-endometrium interplay.

STUDY QUESTION: Which genes regulate receptivity in the epithelial and stromal cellular compartments of the human endometrium, and which molecules are interacting in the implantation process between the blastocyst and the endometrial cells? SUMMARY ANSWER: A set of receptivity-specific genes in the endometrial epithelial and stromal cells was identified, and the role of galectins (LGALS1 and LGALS3), integrin ß1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in embryo-endometrium dialogue among many other protein-protein interactions were highlighted. WHAT IS KNOWN ALREADY: The molecular dialogue taking place between the human embryo and the endometrium is poorly understood due to ethical and technical reasons, leaving human embryo implantation mostly uncharted. STUDY DESIGN SIZE DURATION: Paired pre-receptive and receptive phase endometrial tissue samples from 16 healthy women were used for RNA sequencing. Trophectoderm RNA sequences were from blastocysts. PARTICIPANTS/MATERIALS SETTING METHODS: Cell-type-specific RNA-seq analysis of freshly isolated endometrial epithelial and stromal cells using fluorescence-activated cell sorting (FACS) from 16 paired pre-receptive and receptive tissue samples was performed. Endometrial transcriptome data were further combined in silico with trophectodermal gene expression data from 466 single cells originating from 17 blastocysts to characterize the first steps of embryo implantation. We constructed a protein-protein interaction network between endometrial epithelial and embryonal trophectodermal cells, and between endometrial stromal and trophectodermal cells, thereby focusing on the very first phases of embryo implantation, and highlighting the molecules likely to be involved in the embryo apposition, attachment and invasion. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 499 epithelial and 581 stromal genes were up-regulated in the receptive phase endometria when compared to pre-receptive samples. The constructed protein-protein interactions identified a complex network of 558 prioritized protein-protein interactions between trophectodermal, epithelial and stromal cells, which were grouped into clusters based on the function of the involved molecules. The role of galectins (LGALS1 and LGALS3), integrin ß1 (ITGB1), basigin (BSG) and osteopontin (SPP1) in the embryo implantation process were highlighted. LARGE SCALE DATA: RNA-seq data are available at www.ncbi.nlm.nih.gov/geo under accession number GSE97929. LIMITATIONS REASONS FOR CAUTION: Providing a static snap-shot of a dynamic process and the nature of prediction analysis is limited to the known interactions available in databases. Furthermore, the cell sorting technique used separated enriched epithelial cells and stromal cells but did not separate luminal from glandular epithelium. Also, the use of biopsies taken from non-pregnant women and using spare IVF embryos (due to ethical considerations) might miss some of the critical interactions characteristic of natural conception only. WIDER IMPLICATIONS OF THE FINDINGS: The findings of our study provide new insights into the molecular embryo-endometrium interplay in the first steps of implantation process in humans. Knowledge about the endometrial cell-type-specific molecules that coordinate successful implantation is vital for understanding human reproduction and the underlying causes of implantation failure and infertility. Our study results provide a useful resource for future reproductive research, allowing the exploration of unknown mechanisms of implantation. We envision that those studies will help to improve the understanding of the complex embryo implantation process, and hopefully generate new prognostic and diagnostic biomarkers and therapeutic approaches to target both infertility and fertility, in the form of new contraceptives. STUDY FUNDING/COMPETING INTERESTS: This research was funded by the Estonian Research Council (grant PRG1076); Horizon 2020 innovation grant (ERIN, grant no. EU952516); Enterprise Estonia (grant EU48695); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (IAPP, grant SARM, EU324509); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER) (grants RYC-2016-21199, ENDORE SAF2017-87526-R, and Endo-Map PID2021-127280OB-100); Programa Operativo FEDER Andalucía (B-CTS-500-UGR18; A-CTS-614-UGR20), Junta de Andalucía (PAIDI P20_00158); Margarita Salas program for the Requalification of the Spanish University system (UJAR01MS); the Knut and Alice Wallenberg Foundation (KAW 2015.0096); Swedish Research Council (2012-2844); and Sigrid Jusélius Foundation; Academy of Finland. A.S.-L. is funded by the Spanish Ministry of Science, Innovation and Universities (PRE2018-085440). K.G.-D. has received consulting fees and/or honoraria from RemovAid AS, Norway Bayer, MSD, Gedeon Richter, Mithra, Exeltis, MedinCell, Natural cycles, Exelgyn, Vifor, Organon, Campus Pharma and HRA-Pharma and NIH support to the institution; D.B. is an employee of IGENOMIX. The rest of the authors declare no conflict of interest.

High-throughput sequencing approach uncovers the miRNome of peritoneal endometriotic lesions and adjacent healthy tissues.

Accumulating data have shown the involvement of microRNAs (miRNAs) in endometriosis pathogenesis. In this study, we used a novel approach to determine the endometriotic lesion-specific miRNAs by high-throughput small RNA sequencing of paired samples of peritoneal endometriotic lesions and matched healthy surrounding tissues together with eutopic endometria of the same patients. We found five miRNAs specific to epithelial cells--miR-34c, miR-449a, miR-200a, miR-200b and miR-141 showing significantly higher expression in peritoneal endometriotic lesions compared to healthy peritoneal tissues. We also determined the expression levels of miR-200 family target genes E-cadherin, ZEB1 and ZEB2 and found that the expression level of E-cadherin was significantly higher in endometriotic lesions compared to healthy tissues. Further evaluation verified that studied miRNAs could be used as diagnostic markers for confirming the presence of endometrial cells in endometriotic lesion biopsy samples. Furthermore, we demonstrated that the miRNA profile of peritoneal endometriotic lesion biopsies is largely masked by the surrounding peritoneal tissue, challenging the discovery of an accurate lesion-specific miRNA profile. Taken together, our findings indicate that only particular miRNAs with a significantly higher expression in endometriotic cells can be detected from lesion biopsies, and can serve as diagnostic markers for endometriosis.