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.

Proteomic Profiling Identifies Candidate Diagnostic Biomarkers of Hydrosalpinx in Endometrial Fluid: A Pilot Study.

Hydrosalpinx is a fluid occlusion and distension of the fallopian tubes, often resulting from pelvic inflammatory disease, which reduces the success of artificial reproductive technologies (ARTs) by 50%. Tubal factors account for approximately 25% of infertility cases, but their underlying molecular mechanisms and functional impact on other reproductive tissues remain poorly understood. This proteomic profiling study applied sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) to study hydrosalpinx cyst fluid and pre- and post-salpingectomy endometrial fluid. Among the 967 proteins identified, we found 19 and 17 candidate biomarkers for hydrosalpinx in pre- and post-salpingectomy endometrial fluid, respectively. Salpingectomy significantly affected 76 endometrial proteins, providing insights into the enhanced immune response and inflammation present prior to intervention, and enhanced coagulation cascades and wound healing processes occurring one month after intervention. These findings confirmed that salpingectomy reverses the hydrosalpinx-related functional impairments in the endometrium and set a foundation for further biomarker validation and the development of less-invasive diagnostic strategies for hydrosalpinx.

The effect of vitrification on blastocyst mitochondrial DNA dynamics and gene expression profiles.

PURPOSE: Does vitrification/warming affect the mitochondrial DNA (mtDNA) content and the gene expression profile of blastocysts? METHODS: Prospective cohort study in which 89 blastocysts were obtained from 50 patients between July 2017 and August 2018. mtDNA was measured in a total of 71 aneuploid blastocysts by means of real-time polymerase chain reaction (RT-PCR). Transcriptomic analysis was performed by RNA sequencing (RNA-seq) in an additional 8 aneuploid blastocysts cultured for 0 h after warming, and 10 aneuploid blastocysts cultured for 4-5 h after warming. RESULTS: A significant decrease in mtDNA content just during the first hour after the warming process in blastocysts was found (P < 0.05). However, mtDNA content experimented a significantly increased along the later culture hours achieving the original mtDNA levels before vitrification after 4-5 h of culture (P < 0.05). Gene expression analysis and functional enrichment analysis revealed that such recovery was accompanied by upregulation of pathways associated with embryo developmental capacity and uterine embryo development. Interestingly, the significant increase in mtDNA content observed in blastocysts just after warming also coincided with the differential expression of several cellular stress response-related pathways, such as apoptosis, DNA damage, humoral immune responses, and cancer. CONCLUSION: To our knowledge, this is the first study demonstrating in humans, a modulation in blastocysts mtDNA content in response to vitrification and warming. These results will be useful in understanding which pathways and mechanisms may be activated in human blastocysts following vitrification and warming before a transfer.

Bioaccumulation of Non-Essential Trace Elements Detected in Women's Follicular Fluid, Urine, and Plasma Is Associated with Poor Reproductive Outcomes following Single Euploid Embryo Transfer: A Pilot Study.

This study aims to determine the association of non-essential trace elements present in follicular fluid, plasma, and urine with reproductive outcomes of women undergoing intracytoplasmic sperm injection (ICSI), preimplantation genetic testing for aneuploidies (PGT-A) and single frozen euploid embryo transfer (SET/FET). This single-center, prospective cohort study included sixty women undergoing ICSI with PGT-A and SET/FET between 2018 and 2019. Urine, plasma and follicular fluid samples were collected on the vaginal oocyte retrieval day to simultaneously quantify ten non-essential trace elements (i.e., Ba, Sr, Rb, Sn, Ti, Pb, Cd, Hg, Sb, and As). We found several associations between the levels of these non-essential trace elements and clinical IVF parameters. Specifically, the increased levels of barium in follicular fluid were negatively associated with ovarian function, pre-implantation development and embryo euploidy, while elevated strontium concentrations in this biofluid were negatively associated with impaired blastulation and embryo euploidy. Elevated plasma strontium levels were negatively associated with ovarian function, fertilization and blastulation. Enhanced presence of other trace elements in plasma (i.e., rubidium and arsenic) were associated with a diminished ovarian function and limited the number of recovered oocytes, mature oocytes and zygotes, respectively. Fully adjusted models suggested significantly lower odds of achieving a live birth when increased concentrations of barium and tin were found in urine.

The Impact of Essential Trace Elements on Ovarian Response and Reproductive Outcomes following Single Euploid Embryo Transfer.

Essential trace elements are required in extremely small amounts and obtained through diet. This research focuses on detecting major trace elements in different biofluids of sixty women undergoing ICSI with PGT-A and SET/FET at IVI-RMA, New Jersey, and assessing their impact on their IVF outcomes. Urine, plasma, and follicular fluid samples were collected on the vaginal oocyte retrieval day to measure the concentrations of eight essential trace elements (copper, zinc, molybdenum, lithium, selenium, manganese, chromium, and iron) using ICP-MS. After analysis, ovarian response and preimplantation outcomes had significant positive associations with both copper alone and the copper/zinc ratio in the follicular fluid and plasma, in addition to plasma manganese. Alternatively, elevated follicular fluid lithium concentrations were significantly associated with poor preimplantation outcomes while the urinary molybdenum concentration was significantly associated with a lower probability of implantation, clinical pregnancy, and live birth. Urinary lithium and chromium concentrations were significantly associated with a lower probability of achieving a live birth. Our results suggest that the essential trace elements present in follicular fluid, plasma, and urine of women are directly associated with their reproductive outcomes, with copper and manganese exerting positive effects and lithium and molybdenum exerting negative effects.

Phytoestrogens Present in Follicular Fluid and Urine Are Positively Associated with IVF Outcomes following Single Euploid Embryo Transfer.

The impact and safety of phytoestrogens, plant-derived isoflavones with estrogenic activity predominantly present in soy, on female reproductive health and IVF outcomes continues to be hotly debated. In this prospective cohort study, 60 women attending IVI-RMA New Jersey undergoing IVF with single frozen embryo transfer (SET/FET) of good-quality euploid blastocyst after PGT-A analysis were recruited. Concentrations of two phytoestrogens (daidzein and genistein) in follicular fluid (FF) and urine (U) were measured by UPLC-MSMS, both collected on vaginal oocyte retrieval day. These measurements correlated with IVF clinical outcomes. In models adjusted for age, BMI, race/ethnicity, and smoking status, higher FF phytoestrogen concentrations were significantly associated with higher serum estradiol, enhanced probability of implantation, clinical pregnancy, and live birth. Moreover, higher urine phytoestrogen concentrations were significantly associated with improved oocyte maturation and fertilization potential and increased probability of clinical pregnancy and live birth. Finally, higher FF and urine phytoestrogen concentrations were associated with a higher probability of live birth from a given IVF cycle. Our results suggest that dietary phytoestrogens improved reproductive outcomes of women undergoing IVF treatment. However, additional prospective studies are needed to optimize the use of phytoestrogens to further enhance reproductive outcomes and/or protect against reproductive insults.

Antioxidant Supplementation Alleviates Mercury-Induced Cytotoxicity and Restores the Implantation-Related Functions of Primary Human Endometrial Cells.

Mercury (Hg) cytotoxicity, which is largely mediated through oxidative stress (OS), can be relieved with antioxidants. Thus, we aimed to study the effects of Hg alone or in combination with 5 nM N-Acetyl-L-cysteine (NAC) on the primary endometrial cells' viability and function. Primary human endometrial epithelial cells (hEnEC) and stromal cells (hEnSC) were isolated from 44 endometrial biopsies obtained from healthy donors. The viability of treated endometrial and JEG-3 trophoblast cells was evaluated via tetrazolium salt metabolism. Cell death and DNA integrity were quantified following annexin V and TUNEL staining, while the reactive oxygen species (ROS) levels were quantified following DCFDA staining. Decidualization was assessed through secreted prolactin and the insulin-like growth factor-binding protein 1 (IGFBP1) in cultured media. JEG-3 spheroids were co-cultured with the hEnEC and decidual hEnSC to assess trophoblast adhesion and outgrowth on the decidual stroma, respectively. Hg compromised cell viability and amplified ROS production in trophoblast and endometrial cells and exacerbated cell death and DNA damage in trophoblast cells, impairing trophoblast adhesion and outgrowth. NAC supplementation significantly restored cell viability, trophoblast adhesion, and outgrowth. As these effects were accompanied by the significant decline in ROS production, our findings originally describe how implantation-related endometrial cell functions are restored in Hg-treated primary human endometrial co-cultures by antioxidant supplementation.

Comparative single-cell transcriptomic profiles of human androgenotes and parthenogenotes during early development.

OBJECTIVE: To unravel the differential transcriptomic behavior of human androgenotes (AGs) and parthenogenotes (PGs) throughout the first cell cycles, analyze the differential expression of genes related to key biologic processes, and determine the time frame for embryonic genome activation (EGA) in AGs and PGs. DESIGN: Laboratory study. SETTING: Private fertility clinic. PATIENT(S): Mature oocytes were retrieved from healthy donors and subjected to artificial oocyte activation using calcium ionophore and puromycin to generate PGs (n = 6) or enucleated and subjected to intracytoplasmic sperm injection to generate AGs (n = 10). INTERVENTION(S): Uniparental constructs at different early stages of development were disaggregated into constituent single cells (we suggest the terms parthenocytes and androcytes) to characterize the single-cell transcriptional landscape using next-generation sequencing. MAIN OUTCOMES MEASURE(S): Transcriptomic profiles comparison between different stages of early development in AGs and PGs. RESULT(S): The uniparental transcriptomic profiles at the first cell cycle showed 68 down-regulated and 26 up-regulated differentially expressed genes (DEGs) in PGs compared with AGs. During the third cell cycle, we found 60 up-regulated and 504 down-regulated DEGs in PGs compared with AGs. In the fourth cell cycle, 1,771 up-regulated and 1,171 down-regulated DEGs were found in PGs compared with AGs. The AGs and PGs had reduced EGA profiles during the first 3 cell cycles, and a spike of EGA at the fourth cell cycle was observed in PGs. CONCLUSION(S): Transcriptomic analysis of AGs and PGs revealed their complementary behavior until the fourth cell cycle. Androgenotes undergo a low wave of transcription during the first cell cycle, which reflects the paternal contribution to cell cycle coordination, mechanics of cell division, and novel transcription regulation. Maternal transcripts are most prominent in the third and fourth cell cycles, with amplification of transcription related to morphogenic progression and embryonic developmental competence acquisition. Regarding EGA, in PGs, a primitive EGA begins at the 1-cell stage and gradually progresses until the 4-cell stage, when crucial epigenetic reprogramming (through methylation) is up-regulated. In addition, our longitudinal single-cell transcriptomic analysis challenges that the zygote and early cleavage stages are the only totipotent entities, by revealing potential totipotency in cleavage-stage AGs and implications of paternal transcripts.

Endometrial Cells Acutely Exposed to Phthalates In Vitro Do Not Phenocopy Endometriosis.

Environmental factors that have been linked to an increased endometriosis risk include exposure to di-(2-ethylhexyl)-phthalate (DEHP), an endocrine disruptor. This study aims to investigate whether DEHP in vitro exposure in primary endometrial stromal cells (EnSC), primary endometrial epithelial cells (EnEC), and the human endometrial adenocarcinoma cell line Ishikawa properly mimics alterations described in the eutopic endometrium of women with endometriosis. Primary EnSC and EnEC, isolated from six fertile egg donors, and Ishikawa cells were exposed to DEHP (0.1, 1, and 10 µM) and were assessed for viability, endometriosis markers (IL-6, VEGF-A, HOXA10, EZH2, and LSD1), steroid receptor gene expressions (ER-1, ER-2, PR-T, PR-B, and PGRMC1), and invasive capacity. Viability after 72 h of DEHP exposure was not significantly affected. None of the endometriosis markers studied were altered after acute DEHP exposure, nor was the expression of steroid receptors. The invasive capacity of EnSC was significantly increased after 10 µM of DEHP exposure. In conclusion, acute DEHP exposure in primary endometrial cells does not fully phenocopy the changes in the viability, expression of markers, or steroidal receptors described in endometriosis. However, the significant increase in EnSC invasiveness observed after DEHP exposure could be a link between DEHP exposure and increased endometriosis likelihood.

Integrative analysis of the DNA methylome and transcriptome in uterine leiomyoma shows altered regulation of genes involved in metabolism, proliferation, extracellular matrix, and vesicles.

Uterine leiomyomas (ULs) are the most common benign tumors in women of reproductive age. Despite the high prevalence, tumor pathology remains unclear, which hampers the development of safe and effective treatments. Epigenetic mechanisms appear to be involved in UL development, particularly via DNA methylation that regulates gene expression. We aimed to determine the relationship between DNA methylation and gene expression in UL compared with adjacent myometrium (MM) to identify molecular mechanisms involved in UL formation that are under epigenetic control. Our results showed a different DNA methylation profile between UL and MM, leading to hypermethylation of UL, and a different global transcriptome profile. Integration of DNA methylation and whole-transcriptome RNA-sequencing data identified 93 genes regulated by methylation, with 22 hypomethylated/upregulated and 71 hypermethylated/downregulated. Functional enrichment analysis showed dysregulated biological processes and molecular functions involved in metabolism and cell physiology, response to extracellular signals, invasion, and proliferation, as well as pathways related to uterine biology and cancer. Cellular components such as cell membranes, vesicles, extracellular matrix, and cell junctions were dysregulated in UL. In addition, we found hypomethylation/upregulation of oncogenes (PRL, ATP8B4, CEMIP, ZPMS2-AS1, RIMS2, TFAP2C) and hypermethylation/downregulation of tumor suppressor genes (EFEMP1, FBLN2, ARHGAP10, HTATIP2), which are related to proliferation, invasion, altered metabolism, deposition of extracellular matrix, and Wnt/ß-catenin pathway dysregulation. This confirms that key processes of UL development are under DNA methylation control. Finally, inhibition of DNA methyltransferases by 5-aza-2'-deoxycitidine increased the expression of hypermethylated/downregulated genes in UL cells in vitro. In conclusion, gene regulation by DNA methylation is implicated in UL pathogenesis, and reversion of this methylation could offer a therapeutic option for UL. © 2022 The Pathological Society of Great Britain and Ireland.

Mercury impairs human primary endometrial stromal cell function†.

Heavy metal exposures could compromise endometrial cells. Although studies assessed mercury toxicity in cell lines, limited data are available on the concentration of mercury that damage human endometrial stromal cells (hEnSCs) and alter endometrial function. This research aims to study the effects of mercury exposure on cell viability and functional features of hEnSCs. Primary hEnSCs were isolated from 23 endometrial biopsies obtained from healthy donors. After in vitro mercury exposure cell viability of hEnSCs was evaluated via tetrazolium salt metabolism and oxidative stress was assessed by 2', 7'-dichlorofluorescin diacetate assay. hEnSCs were decidualized in vitro in the presence of mercury (0, 25, 50, 75, 250, and 350 nM). Decidualization was evaluated based on prolactin and insulin-like growth factor-binding protein (IGFBP1) secretion and cytoskeletal rearrangement (F-actin staining). Cell proliferation and apoptosis were evaluated by Ki67 immunostaining and TUNEL assay. Mercury doses of 250 nM (P = 0.028) and 500 nM (P = 0.026) increased reactive oxygen species production in hEnSCs after 24 h. Cell viability significantly decreased after 48 h and 72 h (P < 0.05) of mercury exposure at 500 nM. After in vitro decidualization and mercury treatment, decidual hEnSCs showed a dose-dependent decrease in prolactin and IGFBP1 secretion, particularly at 350 nM (P = 0.016). Cell proliferation was decreased in hEnSCs treated with 350 nM mercury (P < 0.001); an increase in apoptosis followed a dose-dependent trend in non-decidual and decidual hEnSCs. These findings support that mercury-induced damage could be due to an increase in ROS production.

Deciphering the Role of PGRMC1 During Human Decidualization Using an In Vitro Approach.

CONTEXT: Non-classical membrane progesterone receptor (mPRs) and progesterone receptor membrane component 1 (PGRMC1) expression have been detected in endometrium, but their role in decidualization had not yet been investigated. We previously demonstrated PGRMC1 downregulation in receptive endometrium and that its overexpression inhibits decidualization. Furthermore, during decidualization, PGRMC1 mainly interacts with proteins involved in biosynthesis, intracellular transport, and mitochondrial activity. OBJECTIVE: To determine PGRMC1 and mPRs signaling role during decidualization. METHODS: Isolated primary endometrial stromal cells (EnSC) were decidualized in vitro in the presence of classic stimuli (E2 + P4), PGRMC1 inhibitor (AG205), or membrane-impermeable P4 (P4-BSA). Endometrial biopsies were obtained from 19 fertile oocyte donors attending the IVI-Valencia in vitro fertilization (IVF) clinic. EnSC decidualization was evaluated by prolactin ELISA and F-actin immunostaining. Progesterone receptor localization was evaluated by immunofluorescence. EnSC transcriptomic profiles were analyzed by microarray technology. RESULTS: PGRMC1 inhibition during EnSC decidualization (AG205dEnSC) does not interfere with EnSC cytoskeletal rearrangements and prolactin secretion. However, global transcriptional profiling revealed more differentially expressed genes in AG205dEnSC than in dEnSC, compared with nondecidualized EnSC (ndEnSC). In silico analysis showed that PGRMC1 inhibition upregulated more genes related to metabolism, molecular transport, and hormonal biosynthesis compared with control dEnSC. EnSC decidualized in the presence of P4-BSA showed a similar behavior as ndEnSC in terms of morphological features, absence of prolactin secretion, and transcriptomic pattern. CONCLUSION: Our findings associate PGRMC1 to hormonal biosynthesis, metabolism, and vesicular transport-important cellular functions for dEnSC supporting pregnancy. Activation of membrane P4 receptor signaling alone was unable to induce downstream effects needed for proper decidualization.

An artificial intelligence model based on the proteomic profile of euploid embryos and blastocyst morphology: a preliminary study.

RESEARCH QUESTION: The study aimed to develop an artificial intelligence model based on artificial neural networks (ANNs) to predict the likelihood of achieving a live birth using the proteomic profile of spent culture media and blastocyst morphology. DESIGN: This retrospective cohort study included 212 patients who underwent single blastocyst transfer at IVI Valencia. A single image of each of 186 embryos was studied, and the protein profile was analysed in 81 samples of spent embryo culture medium from patients included in the preimplantation genetic testing programme. The information extracted from the analyses was used as input data for the ANN. The multilayer perceptron and the back-propagation learning method were used to train the ANN. Finally, predictive power was measured using the area under the curve (AUC) of the receiver operating characteristic curve. RESULTS: Three ANN architectures classified most of the embryos correctly as leading (LB+) or not leading (LB-) to a live birth: 100.0% for ANN1 (morphological variables and two proteins), 85.7% for ANN2 (morphological variables and seven proteins), and 83.3% for ANN3 (morphological variables and 25 proteins). The artificial intelligence model using information extracted from blastocyst image analysis and concentrations of interleukin-6 and matrix metalloproteinase-1 was able to predict live birth with an AUC of 1.0. CONCLUSIONS: The model proposed in this preliminary report may provide a promising tool to select the embryo most likely to lead to a live birth in a euploid cohort. The accuracy of prediction demonstrated by this software may improve the efficacy of an assisted reproduction treatment by reducing the number of transfers per patient. Prospective studies are, however, needed.

Tacrolimus Improves the Implantation Rate in Patients with Elevated Th1/2 Helper Cell Ratio and Repeated Implantation Failure (RIF).

Introduction An abnormal endometrial immune response is involved in the pathogenesis of repeated implantation failure (RIF), so we investigated the effectiveness of tacrolimus treatment on the endometrium of RIF patients. Materials and Methods Ten RIF patients with elevated T-helper 1/T-helper 2 (Th1/Th2) cell ratios were recruited into a clinical study. The expression of p53, leukemia inhibitory factor (LIF), interleukin (IL)-4, IL-10, IL-17, and interferon gamma (IFN-γ) in the endometrium of patients with and without tacrolimus treatment and the association of these factors with assisted reproductive technology (ART) outcomes were investigated. Results Tacrolimus significantly increased the expression of LIF, IL-10, and IL-17 and decreased the expression of IL-4, IFN-γ, and the IFN-γ/IL-10 ratio in RIF patients. Tacrolimus treatment resulted in an implantation rate of 40%, a clinical pregnancy rate of 50%, and a live birth rate of 35% in RIF patients with elevated Th1/Th2 ratios who had previously failed to become pregnant despite at least three transfers of embryos. We also found a significant positive correlation between IL-10 levels and the implantation rate. Conclusions Our findings suggest that RIF patients with a higher Th1/Th2 ratio could be candidates for tacrolimus therapy and that this immunosuppressive drug could be acting through upregulation of LIF, IL-10, and IL-17.

Novel nonclassic progesterone receptor PGRMC1 pulldown-precipitated proteins reveal a key role during human decidualization.

OBJECTIVE: To investigate PGRMC1-precipitating proteins in human endometrial stromal cells (ESC) to understand its role during in vitro decidualization. DESIGN: Prospective observational study. SETTING: Academic fertility center. PATIENT(S): Fifteen fertile oocyte donors. INTERVENTION(S): Isolated ESCs decidualized in vitro and used in pulldown assays. MAIN OUTCOME MEASURE(S): GST-PGRMC1-precipitated proteins identified in nondecidualized ESC (ndESC) and ESC decidualized via a long (8 days) or short (4 days) decidualization protocol (dESC). RESULT(S): Using pulldown assays and mass spectrometry, decidualization was evaluated by prolactin secretion (ELISA) and cytoskeleton morphology (F-actin staining). The protein interactions were validated by colocalization and coimmunoprecipitation. The pulldown and mass spectrometry analysis identified 21, 24, and 24 new significant GST-PGRMC1-precipitated proteins in ndESC, long dESC, and short dESC, respectively, compared with controls. The functional annotation analysis categorized these proteins mainly into endomembrane system and mitochondria cellular components, both related to adenosine triphosphate (ATP) generation and transport activity, protein biosynthesis and posttranslational processing, vesicle trafficking, and protection against oxidative stress activities. Monoamine oxidase B (MAOB) and B-cell receptor-associated protein 31 (BAP31) were identified in dESC from both decidualization protocols. PGRMC1-MAOB/BAP31 interactions were confirmed by immunofluorescence and coimmunoprecipitation in dESC. CONCLUSION(S): Novel GST-PGRMC1-precipitated proteins discovered in ESC suggest that this protein is implicated in deep remodeling of ESC during decidualization and aggregates mainly with proteins involved in biosynthesis, intracellular transport, and mitochondrial activity.

Preeclampsia: a defect in decidualization is associated with deficiency of Annexin A2.

BACKGROUND: Decidualization defects in the endometrium have been demonstrated at the time of delivery in women with severe preeclampsia and to linger for years, which suggests a maternal contribution to the pathogenesis of this condition. Global transcriptional profiling reveals alterations in gene expression, which includes down-regulation of Annexin A2 in severe preeclampsia patients with decidualization resistance. OBJECTIVE: We investigated the functional role of Annexin A2 deficiency during endometrial decidualization and its potential contribution to shallow trophoblast invasion during implantation and subsequent placentation using in vitro and in vivo modeling. STUDY DESIGN: Annexin A2 gene and protein levels were assessed during in vitro decidualization of human endometrial stromal cells isolated from biopsy specimens that were collected from women with previous severe preeclampsia (n=5) or normal obstetric outcomes (n=5). Next, Annexin A2 was inhibited with small interference RNA in control human endometrial stromal cells that were isolated from endometrial biopsy specimens (n=15) as an in vitro model to analyze decidualization defects at the morphologic level and the secretion of prolactin and insulin-like growth binding protein-1. Annexin A2-inhibited cells were used to evaluate motility and promotion of embryo invasion. Decidualization and placentation defects of Annexin A2 deficiency were confirmed with the use of an Annexin A2-null mouse model. RESULTS: Annexin A2 gene and protein levels were down-regulated during in vitro decidualization of human endometrial stromal cells from women with previous severe preeclampsia compared with control individuals. To assess its role in the endometrial stroma, we inhibited Annexin A2 expression and detected decidualization failure as evidenced by impaired morphologic transformation, which was associated with altered actin polymerization and low prolactin and insulin-like growth binding protein-1 secretions. Functionally, in vitro models demonstrated that Annexin A2 inhibition failed to support embryo invasion. This finding was corroborated by reduced trophoblast spreading through human endometrial stromal cells, lack of motility of these cells, and reduced trophoblast invasion in the presence of conditioned media from Annexin A2-inhibited cells. Extending our discovery to an animal model, we detected that Annexin A2-null mice have a functional deficiency in decidualization and placentation that impairs fetal growth as a feature that is associated with severe preeclampsia. CONCLUSION: Together, in vitro and in vivo results suggest that endometrial defects in Annexin A2 expression impair decidualization of endometrial stromal cells as well as the uterine microenvironment that promotes embryo implantation and placentation. Our findings highlight the maternal contribution to the pathogenesis of severe preeclampsia and suggest that evaluation of Annexin A2 may provide a novel strategy to assess a woman's risk of experiencing this disease and perhaps discover therapeutic interventions to improve decidualization.

Phytoestrogen exposure alters endometrial stromal cells and interferes with decidualization signaling.

OBJECTIVE: To investigate whether phytoestrogens (genistein and daidzein) alter in vitro decidualization of human endometrial stromal cells (ESCs). DESIGN: Isolated primary ESCs were exposed to phytoestrogens and decidualized in vitro. SETTING: Academic fertility center. PATIENT(S): Twenty fertile oocyte donors attending the IVI Valencia clinic. INTERVENTION(S): Treatment of ESC with phytoestrogens at 0, 10, 20, 50, and 100 µM. MAIN OUTCOME MEASURE(S): The ESC proliferation was analyzed by MTS assay. In vitro decidualization was induced in the presence of phytoestrogens by medroxyprogesterone acetate/cyclic adenosine 3':5' monophosphate and evaluated by prolactin (PRL) ELISA and F-actin immunostaining. The Ki67 proliferative marker was analyzed by immunofluorescence. The ESC apoptosis was assessed by annexin V/propidium iodide detection using flow cytometry. Estrogen (ERß) and P receptor (PR) localization were evaluated by immunofluorescence. RESULT(S): The ESC exposed to 0, 19, 20, 50, and 100 µM of genistein, daidzein, and genistein + daidzein showed a dose-dependent proliferation decrease. After 48-96 hours of culture, this reduction was significant in the presence of 50 µM of phytoestrogens versus 10 µM untreated ESC. The ESC decidualized in the presence of phytoestrogens did not rearrange their cytoskeletons and showed a significant decrease in PRL secretion compared with untreated decidualized ESCs (dESCs). However, phytoestrogens did not alter proliferative status or the percentage of viable/apoptotic cells in dESC compared with untreated dESC. During decidualization, phytoestrogens induced the same nuclear translocation of ERß and PR as the control dESC. CONCLUSION(S): This study reveals that high doses of phytoestrogens could affect the in vitro decidualization process.

Single-cell RNA sequencing of oocytes from ovarian endometriosis patients reveals a differential transcriptomic profile associated with lower quality.

STUDY QUESTION: Do oocytes from women with ovarian endometriosis (OE) have a different transcriptomic profile than those from healthy women? SUMMARY ANSWER: Oocytes from endometriosis patients, independently of whether they came from the affected ovary, exhibited a differential transcriptomic profile compared to oocytes from healthy egg donors. WHAT IS KNOWN ALREADY: Studies of endometriosis have sought to determine whether OE affects oocyte quality. While many reports indicate that oocytes recovered from endometriotic ovaries may be affected by the disease, other studies have found no significant differences among oocyte/embryo quality and fertilization, implantation and pregnancy rates in women with endometriosis. STUDY DESIGN, SIZE, DURATION: This prospective study compared metaphase II (MII) oocytes (n = 16) from endometriosis patients (n = 7) to oocytes (n = 16) from healthy egg donors (n = 5) by single-cell RNA sequencing (scRNA-seq). Participants were recruited between December 2016 and February 2018 at IVI-RMA Valencia and Vigo clinics. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human MII oocytes were collected from healthy egg donors and OE patients aged 18-34 years, with a body mass index of <30 and >6 pre-antral follicles. RNA was extracted, cDNA was generated and libraries were constructed and sequenced. scRNA-seq data libraries were processed and statistically analysed. Selected genes were validated by quantitative real-time PCR. MAIN RESULTS AND THE ROLE OF CHANCE: Our scRNA-seq results revealed an effect of endometriosis on global transcriptome behaviour in oocytes from endometriotic ovaries. The highest number of differentially expressed genes (DEGs) was found when oocytes from women with OE were compared to oocytes from healthy donors [520 DEGs (394 upregulated and 126 downregulated)], independently of whether oocytes came from an affected or unaffected ovary. Among the top 20 significant DEGs in this comparison, most were upregulated, including APOE, DUSP1, G0S2, H2AFZ, ID4, MGST1 and WEE1. PXK was the only downregulated gene. Subsequently, functional analysis showed 31 enriched functions deregulated in endometriosis patients (Benjamini P < 0.1), being 16 significant enriched functions considering Benjamini P < 0.05, which involved in biological processes and molecular functions, such as steroid metabolism, response to oxidative stress and cell growth regulation. In addition, our functional analysis showed enrichment for mitochondria, which are an important cellular component in oocyte development. Other functions important in embryo development, such as angiogenesis and methylation, were also significantly enriched. LARGE SCALE DATA: All raw sequencing data are submitted in Gene Expression Omnibus (GEO) under accession number (PRJNA514416). LIMITATIONS, REASONS FOR CAUTION: This study was restricted only to OE and thereby other anatomical entities, such as peritoneal and deep infiltrating endometriosis, were not considered. This is a descriptive study with a limited number of samples reflecting the difficulty to recruit human oocytes, especially from women with endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that OE exhibits a global transcriptomic effect on oocytes of patients in OE, independently if they come from an affected or unaffected ovary and alters key biological processes and molecular functions related to steroid metabolism, response to oxidative stress and cell growth regulation, which reduce oocyte quality. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by IVI Foundation, the Spanish Ministry of Economy and Competitiveness through the Miguel Servet programme (CPII018/00002 to F.D.), the Sara Borrell Program (CD15/00057 to H.F.) and the VALi+d Programe (Generalitat Valenciana); ACIF/2016/444 to A.C.). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: None.

iTRAQ comparison of proteomic profiles of endometrial receptivity.

Endometrial receptivity is a limiting step in human reproduction. A disruption in the development of endometrial receptivity is responsible for recurrent implantation failures (RIF) of endometrial origin. To understand the molecular mechanisms behind the endometrial receptivity process, we used the isobaric tag for relative and absolute quantitation (iTRAQ) method to compare three different endometrial statuses: fertile women, intrauterine device (IUD) carriers, and RIF patients. Overall, iTRAQ allowed identified 1889 non-redundant proteins. Of these, 188 were differentially expressed proteins (DEP) (p-value < .05). Pairwise comparisons revealed 133 significant DEP in fertile vs. IUD carriers and 158 DEP in RIF vs. IUD carriers. However, no DEP were identified between fertile and RIF patients. Western blot validation of three DEP involved in endometrial receptivity (plastin 2, lactotransferrin, and lysozyme) confirmed our iTRAQ results. Moreover, functional KEGG enrichment revealed that complement and coagulation cascades and peroxisome were the two most significant pathways for the RIF vs. IUD comparison and ribosome and spliceosome for the fertile vs. IUD comparison, as possible important pathways involved in the endometrial receptivity acquisition. The lack of DEP between fertile and RIF patient endometria suggest that idiopathic RIF may not have an endometrial origin, with other as-yet-unknown factors involved. SIGNIFICANCE: A pilot study where a comparison of the endometrial protein profile from women with different endometrial receptive grade (fertile women, IUD carriers and RIF patients) during the same period of time (overlapping with the window of implantation) of a hormone replacement therapy was performed using a high-throughput proteomic technique. This approach lead us to better understand the molecular mechanisms undergoing endometrial receptivity, a time-limiting step to achieve pregnancy in humans. Moreover, the number of samples per group (10 Fertile women, 10 IUD carriers and 8 RIF patients) according to the methodology here employed (8plex iTRAQ), give more robustness to our results. Our findings confirm that an IUD introduces numerous changes in the endometrial protein profile when compared to fertile and RIF endometria, revealing some key proteins involved in endometrial receptivity. Finding no significant differences between Fertile and RIF patient endometria could suggest that other as-yet-unknown factors could be involved in the etiology of idiopathic RIF.

Transcriptomic behavior of genes associated with chromosome 21 aneuploidies in early embryo development.

OBJECTIVE: To analyze how chromosome 21 (HSA21) ploidy affects global gene expression of early human blastocysts. DESIGN: Prospective study. SETTING: University-affiliated in vitro fertilization clinic. PATIENT(S): A total of 26 high-quality donated embryos from in vitro fertilization (IVF) patients: trisomy 21 (n = 8), monosomy 21 (n = 10), and euploid (n = 8) blastocysts. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Blastocyst transcriptome changes and its associated functions. RESULT(S): Trisomy 21, monosomy 21, and euploid blastocysts were classified by comparative genomic hybridization. The global transcriptome of whole blastocysts was analyzed with small cell number RNA sequencing, and they were compared to understand the gene expression behavior at early development and its implications for embryo implantation. We identified 1,232 differentially expressed genes (false discovery rate <0.05) in monosomy 21 compared with euploid blastocysts associated with dysregulated functions in embryo development as the Rap1 signaling pathway. Curiously, Down syndrome in early development revealed fewer transcriptomic changes than expected. In addition, Down syndrome gene expression in neonates, children, and adults revealed that the number of deregulated genes increases across life stages from blastocysts to adults, suggesting a potential dosage-compensation mechanism for human chromosome 21. CONCLUSION(S): At the transcriptomic level, early development in Down syndrome is mainly dosage compensated. However, monosomy 21 is strongly transcriptionally affected because early development involving main functions is associated with embryo implantation.