Deciphering the role of PGRMC2 in the human endometrium during the menstrual cycle and in vitro decidualization using an in vitro approach.

STUDY QUESTION: What is the human endometrial non-classical progesterone receptor (PGR) membrane component 2 (PGRMC2) expression pattern throughout the menstrual cycle and what role does it play during decidualization? SUMMARY ANSWER: Endometrial PGRMC2 expression fluctuates during the human menstrual cycle and is abundantly expressed in human endometrial stromal cells (hEnSCs) during in vitro decidualization, process where PGRMC2 is involved in embryo implantation-related pathways. WHAT IS KNOWN ALREADY: The endometrial response to progesterone is mediated by the classical and non-classical PGRs. We previously demonstrated that PGR membrane component 1 (PGRMC1) is critical for endometrial function, embryo implantation, and future placentation, however, the role(s) of PGRMC2, which is structurally similar to PGRMC1, have not been studied in the human endometrium. STUDY DESIGN, SIZE, DURATION: This prospective study comprehensively evaluated the endometrial expression of PGRMC2 throughout the human menstrual cycle and during in vitro decidualization of hEnSCs (isolated from 77 endometrial biopsies that were collected from 66 oocyte donors), using immunohistochemistry, RT-qPCR, western blot, transcriptomic, and proteomic analyses. In addition, functional analysis was carried out to validate the implication of PGRMC2 in hEnSCs during embryo invasion using an in vitro outgrowth model. PARTICIPANTS/MATERIALS, SETTING, METHODS: In vitro decidualization of hEnSCs was induced using co-treatment with cAMP and medroxyprogesterone 17-acetate progestin, and evaluated by measuring prolactin by ELISA and F-actin immunostaining. RT-qPCR was employed to compare expression with other PGRs. To reveal the function of PGRMC2 during the decidualization process, we specifically knocked down PGRMC2 with siRNAs and performed RNA-seq and quantitative proteomics techniques (SWATH-MS). The common differentially expressed genes (DEGs) and proteins (DEPs) were considered for downstream functional enrichment analysis. Finally, to verify its implication in the trophoblast invasion, an outgrowth model was carried out where hEnSCs with silenced PGRMC2 were co-cultured with human trophoblastic spheroids (JEG-3) following in vitro decidualization. MAIN RESULTS AND THE ROLE OF CHANCE: In contrast to PGRMC1 and classical PGRs, endometrial PGRMC2 gene expression was significantly lower during the late- versus mid-secretory phase (P < 0.05). Accordingly, the elevated PGRMC2 protein abundance observed in the endometrial epithelial glands throughout the menstrual cycle dropped in the late secretory phase, when abundance decreased in all endometrial compartments. Nevertheless, PGRMC2 protein increased during the mid-secretory phase in stromal and glandular cells, and PGRMC2 mRNA (P < 0.0001) and protein (P < 0.001) levels were significantly enhanced in the membranes/organelles of decidualized hEnSCs, compared to non-decidualized hEnSCs. Notably, PGRMC1 and PGRMC2 mRNA were significantly more abundant than classical PGRs throughout menstrual cycle phases and in decidualized and non-decidualized hEnSCs (P < 0.05). RNA-seq and proteomics data revealed 4687 DEGs and 28 DEPs, respectively, in decidualized hEnSCs after PGRMC2 silencing. While functional enrichment analysis showed that the 2420 upregulated genes were mainly associated with endoplasmic reticulum function, vesicular transport, morphogenesis, angiogenesis, cell migration, and cell adhesion, the 2267 downregulated genes were associated with aerobic respiration and protein biosynthesis. The protein enrichment analysis showed that 4 upregulated and 24 downregulated proteins were related to aerobic respiration, cellular response, metabolism, localization of endoplasmic reticulum proteins, and ribonucleoside biosynthesis routes. Finally, PGRMC2 knockdown significantly compromised the ability of the decidualized hEnSCs to support trophoblast expansion in an outgrowth model (P < 0.05). LARGE-SCALE DATA: Transcriptomic data are available via NCBI's Gene Expression Omnibus (GEO) under GEO Series accession number GSE251843 and proteomic data via ProteomeXchange with identifier PXD048494. LIMITATIONS, REASONS FOR CAUTION: The functional analyses were limited by the discrete number of human endometrial biopsies. A larger sample size is required to further investigate the potential role(s) of PGRMC2 during embryo implantation and maintenance of pregnancy. Further, the results obtained in the present work should be taken with caution, as the use of a pure primary endometrial stromal population differentiated in vitro does not fully represent the heterogeneity of the endometrium in vivo, nor the paracrine communications occurring between the distinct endometrial cell types. WIDER IMPLICATIONS OF THE FINDINGS: The repression of endometrial PGRMC2 during the late- versus mid-secretory phase, together with its overexpression during decidualization and multiple implications with embryo implantation not only highlighted the unknown roles of PGRMC2 in female reproduction but also the potential to exploit PGRMC2 signaling pathways to improve assisted reproduction treatments in the future. STUDY FUNDING/COMPETING INTEREST(S): This research was funded by Instituto de Salud Carlos III (ISCIII) granted to F.D. (PI20/00405 and PI23/00860), co-funded by the European Union. Y.M.-L. was supported by a predoctoral research grant from Generalitat Valenciana (ACIF/2019/262). R.G.-M. was supported by Generalitat Valenciana (CIAPOT/2022/15). P.d.C. was supported by a predoctoral grant for training in research into health (PFIS FI20/00086) from the Instituto de Salud Carlos III. I.D.-H. was supported by the Spanish Ministry of Science, Innovation and Universities (FPU18/01550). A.P. was supported by the Instituto de Salud Carlos III (PFIS FI18/00009). This research was also supported by IVI Foundation-RMA Global (1911-FIVI-103-FD). The authors declare no conflict of interest.

Uterine natural killer cells: from foe to friend in reproduction.

BACKGROUND: Recurrent miscarriage and pre-eclampsia are common reproductive disorders, but their causes are often unknown. Recent evidence has provided new insight into immune system influences in reproductive disorders. A subset of lymphocytes of the innate immune system known as uterine natural killer (uNK) cells are now recognized as fundamental to achieving embryo implantation and successful pregnancy, but were initially attributed a bad reputation. Indeed, immune therapies have been developed to treat the 'exaggerated' immune response from uNK cells. These treatments have been based on studies of peripheral blood natural killer (pbNK) cells. However, uNK cells and pbNK cells have different phenotypic and functional characteristics. The functions of uNK cells are closely related to their interactions with the extravillous trophoblast cells (EVTs) and spiral arteries, which underlie an essential role in regulating vascular function, controlling trophoblast invasion and promoting placental development. EVTs express MHC molecules of class I HLA-C/E/G/F, while uNK cells express, among other receptors, killer cell immunoglobulin-like receptors (KIRs) that bind to HLA-C or CD94/NKG2A inhibitory receptors, and then bind HLA-E. Associations of certain KIR/HLA-C combinations with recurrent miscarriage, pre-eclampsia, and foetal growth restriction and the interactions between uNK cells, trophoblasts and vascular cells have led to the hypothesis that uNK cells may play a role in embryo implantation. OBJECTIVE AND RATIONALE: Our objective was to review the evolution of our understanding of uNK cells, their functions, and their increasingly relevant role in reproduction. SEARCH METHODS: Relevant literature through June 2020 was retrieved using Google Scholar and PubMed. Search terms comprised uNK cells, human pregnancy, reproductive failure, maternal KIR and HLA-C, HLA-E/G/F in EVT cells, angiogenic cytokines, CD56+ NK cells, spiral artery, oestrogen and progesterone receptors, KIR haplotype and paternal HLA-C2. OUTCOMES: This review provides key insights into the evolving conceptualization of uNK cells, from their not-so-promising beginnings to now, when they are considered allies in reproduction. We synthesized current knowledge about uNK cells, their involvement in reproduction and their main functions in placental vascular remodeling and trophoblast invasion. One of the issues that this review presents is the enormous complexity involved in studying the immune system in reproduction. The complexity in the immunology of the maternal-foetal interface lies in the great variety of participating molecules, the processes and interactions that occur at different levels (molecular, cellular, tissue, etc.) and the great diversity of genetic combinations that are translated into different types of responses. WIDER IMPLICATIONS: Insights into uNK cells could offer an important breakthrough for ART outcomes, since each patient could be assessed based on the combination of HLA and its receptors in their uNK cells, evaluating the critical interactions at the materno-foetal interface. However, owing to the technical challenges in studying uNK cells in vivo, there is still much knowledge to gain, particularly regarding their exact origin and functions. New studies using novel molecular and genetic approaches can facilitate the identification of mechanisms by which uNK cells interact with other cells at the materno-foetal interface, perhaps translating this knowledge into clinical applicability.