Dysregulated miR-124-3p in endometrial epithelial cells reduces endometrial receptivity by altering polarity and adhesion.

The endometrium undergoes substantial remodeling in each menstrual cycle to become receptive to an implanting embryo. Abnormal endometrial receptivity is one of the major causes of embryo implantation failure and infertility. MicroRNA-124-3p is elevated in both the serum and endometrial tissue of women with chronic endometritis, a condition associated with infertility. MicroRNA-124-3p also has a role in cell adhesion, a key function during receptivity to allow blastocysts to adhere and implant. In this study, we aimed to determine the function of microRNA-124-3p on endometrial epithelial adhesive capacity during receptivity and effect on embryo implantation. Using a unique inducible, uterine epithelial-specific microRNA overexpression mouse model, we demonstrated that elevated uterine epithelial microRNA-124-3p impaired endometrial receptivity by altering genes associated with cell adhesion and polarity. This resulted in embryo implantation failure. Similarly in a second mouse model, increasing microRNA-124-3p expression only in mouse uterine surface (luminal) epithelium impaired receptivity and led to implantation failure. In humans, we demonstrated that microRNA-124-3p was abnormally increased in the endometrial epithelium of women with unexplained infertility during the receptive window. MicroRNA-124-3p overexpression in primary human endometrial epithelial cells (HEECs) impaired primary human embryo trophectoderm attachment in a 3-dimensional culture model of endometrium. Reduction of microRNA-124-3p in HEECs from infertile women normalized HEEC adhesive capacity. Overexpression of microRNA-124-3p or knockdown of its direct target IQGAP1 reduced fertile HEEC adhesion and its ability to lose polarity. Collectively, our data highlight that microRNA-124-3p and its protein targets contribute to endometrial receptivity by altering cell polarity and adhesion.

High androgen level during controlled ovarian stimulation cycle impairs endometrial receptivity in PCOS patients.

PCOS is one of the most common endocrine disorders among women of reproductive age. While the mechanism involved is not yet fully characterized. Our study aims to examine the pregnancy outcomes of embryo transfers in women with PCOS after pretreatment, and to explore the possible effect of high androgen levels on endometrial receptivity. Retrospective cohort study was conducted to analyze pregnancy outcomes among 2714 infertile women with tubal factor and 452 PCOS women. Endometrium samples were collected from 6 controls and 6 PCOS patients to detect the expression of endometrial receptivity marks. The implantation rate, clinical and ongoing pregnancy rates and live birth rate in women with PCOS followed fresh embryo transfers were obviously decreased even after the pretreatment. Similar pregnancy outcomes were found in frozen-thawed embryo transfer cycles between women with or without PCOS. Strikingly, serum total testosterone (TT) levels on trigger day were significantly higher in PCOS women. Women with high TT levels presented significantly lower clinical and ongoing pregnancy rates, and the expression of insulin-like growth factor binding protein 1 (IGFBP-1), and leukemia inhibitory factor (LIF) in the endometria decreased significantly as well. High doses of testosterone significantly down-regulated the expression of IGFBP-1 and LIF in Ishikawa cells. Although endocrine abnormalities had been improved before the controlled ovarian stimulation (COS) cycle started, higher serum TT levels were detected on the trigger day of the COS cycle in PCOS patients, which may contribute to the decreased fresh embryo implantation by impairing endometrial receptivity.

Reproductive and transcriptome characteristics of endometrium at the window of im-plantation in patients after fertility-sparing treatment of endometrial intraepithelial neoplasm or cancer.

Background: Progestin therapy is an option for patients with endometrial carcinoma (EC) or endometrial intraepithelial neoplasm (EIN) who fit specific criteria of fertility-sparing treatment. However, the implantation rate remains low among females receiving in vitro fertilization (IVF) even after the complete reversal of endometrial lesions. Methods: Here, ten patients with EC/EIN achieved complete regression (CR) in histology. Their relevant metabolic and IVF parameters were collected. An endometrial sampling at the window of implantation (WOI) and transcriptome analysis were conducted among them, and four healthy controls were analyzed to analyze endometrial receptivity. Results: On average, it took ten patients five months to achieve CR after four curettage procedures. The interquartile range of endometrium thickness on trigger day was between 8.8 and 10.0 mm, while the range was 15.2-18.5 mm for controls. Five patients got pregnant after a frozen-embryo transfer. According to ERA analysis, the endometrial sampling at WOI showed pre-receptive status in four cases. In total, 1458 differential expression genes were identified, and 70 belonged to the ERA genes. ImmuneScore indicated decreased NK cells in the endometrium, affecting endometrial receptivity. Conclusions: Even after EC/EIN reversal in histology, endometrial receptivity has already been compromised regarding altered WOI and immune microenvironment, leading to a low pregnancy rate.

The role of microRNAs in the regulation of critical genes and signalling pathways that determine endometrial receptivity.

Endometrial receptivity is the ability of the endometrium to accept embryos. Thus, endometrial receptivity dysfunction is an important factor leading to embryo implantation failure. A good endometrial receptivity provides a suitable environment for embryo implantation, improving the embryo implantation rate. The "implantation window" stage, or the receptive stage of the endometrium, is regulated by various hormones, genes, proteins and cytokines, among which microRNAs (miRNAs) and their target genes have a regulatory effect on endometrial receptivity. This review outlines the relationship between endometrial receptivity and pregnancy, the mRNAs and related signalling pathways that regulate endometrial receptivity, and the regulatory role of miRNA in endometrial receptivity, providing a deeper understanding of the regulatory mechanisms of miRNA on endometrial receptivity in humans and animals and reference for the endometrial receptivity-related research.

Angiogenic factor-driven improvement of refractory thin endometrium with autologous platelet-rich plasma intrauterine infusion in frozen embryo transfer cycles.

Objective: A beneficial effect on endometrial thickness (EMT) and improvement of pregnancy outcome after intrauterine infusion of platelet-rich plasma (PRP) has been suggested. This study assessed the effect of intrauterine PRP infusion on live birth rate and obstetrical outcomes and analyzed cytokines that can potentially improve pregnancy outcomes through PRP. Method: This study was a prospective cohort study conducted in a university hospital fertility center. The study included ninety-one patients who had a history of two or more failed in vitro fertilization (IVF) attempts and refractory thin endometrium that remained unresponsive after at least two conventional treatments for thin endometrium. Patients were treated with an intrauterine infusion of autologous PRP between days 7 and 14 of their hormone replacement therapy-frozen embryo transfer (HRT-FET) cycle. PRP was administered at 3-day intervals until their EMT reached 7mm. After a maximum of three PRP administrations, embryo transfer (ET) was performed. The primary outcome was the live birth rate. Secondary outcomes included the implantation rate and increase in EMT compared to the previous cycle. We compared the cytokines related to angiogenesis in a patient's whole blood (WB) and PRP by utilizing a commercial screening kit. Results: The live birth rate in the PRP treatment cycle was 20.9% (19 of 91 patients), significantly superior to the previous cycle without PRP infusion (p < 0.001). The implantation rate was also significantly higher during the PRP treatment cycle (16.4%) compared to the previous cycle (3.1%) (p < 0.001). The mean EMT post-PRP treatment was 6.1 mm, showing a significant increase of 0.8 mm (p < 0.001). Nonetheless, an increase in EMT was also observed in the non-pregnancy group. No adverse effects were reported by patients treated with autologous PRP. Cytokine array analysis confirmed marked increases in well-known pro-angiogenic factors such as Ang-1, EGF, LAP (TGF-b1), MMP-8, PDGF-AA, and PDGF-AB/PDGF-BB. Conclusion: Intrauterine PRP infusion offers a safe and effective treatment for patients with refractory thin endometrium and implantation failures. The angiogenic cytokines present in PRP are the primary drivers of this improvement.

Automated endometrial identification and volume calculation in normal uteri using a novel smart ERA technique.

To evaluate the repeatability of a novel automated technique called Smart ERA (Smart Endometrial Receptivity Analysis) for the automated segmentation and volume calculation of the endometrium in patients with normal uteri,, and to compare the agreement of endometrial volume measurements between Smart ERA, the semi-automated Virtual Organ Computer-aided Analysis (VOCAL) technique and manual segmentation. This retrospective study evaluated endometrial volume measurement in infertile patients who underwent frozen-thawed embryo transfer (FET). Transvaginal three-dimensional ultrasound scans were performed using a Resona R9 ultrasound machine. Data was collected from patients between 2021 and 2022. Patients with normal uteri and optimal ultrasound images were included. Endometrial volumes were measured using Smart ERA, VOCAL at 15° rotation, and manual segmentation. Intra-observer repeatability and agreement between techniques were assessed using the intraclass correlation coefficient (ICC) and Bland-Altman analysis. A total of 407 female patients were evaluated (mean age 33.2 ± 4.7 years). The repeatability of Smart ERA showed an ICC of 0.983 (95% CI 0.984-0.991). The agreement between Smart ERA and the manual method, Smart ERA and VOCAL, and VOCAL and the manual method, as assessed by ICC, were 0.986 (95% CI 0.977-0.990), 0.943 (95% CI 0.934-0.963), and 0.951 (95% CI 0.918-0.969), respectively. The Smart ERA technique required approximately 3 s for endometrial volume calculation, while VOCAL took around 5 min and the manual segmentation method took approximately 50 min. The Smart-ERA software, which employs a novel three-dimensional segmentation algorithm, demonstrated excellent intra-observer repeatability and high agreement with both VOCAL and manual segmentation for endometrial volume measurement in women with normal uteri. However, these findings should be interpreted with caution, as the algorithm's performance may not be generalizable to populations with different uterine characteristic. Additionally, Smart ERA required significantly less time compared to VOCAL and manual segmentation.

Optimizing evaluation of endometrial receptivity in recurrent pregnancy loss: a preliminary investigation integrating radiomics from multimodal ultrasound via machine learning.

Background: Recurrent pregnancy loss (RPL) frequently links to a prolonged endometrial receptivity (ER) window, leading to the implantation of non-viable embryos. Existing ER assessment methods face challenges in reliability and invasiveness. Radiomics in medical imaging offers a non-invasive solution for ER analysis, but complex, non-linear radiomic-ER relationships in RPL require advanced analysis. Machine learning (ML) provides precision for interpreting these datasets, although research in integrating radiomics with ML for ER evaluation in RPL is limited. Objective: To develop and validate an ML model that employs radiomic features derived from multimodal transvaginal ultrasound images, focusing on improving ER evaluation in RPL. Methods: This retrospective, controlled study analyzed data from 346 unexplained RPL patients and 369 controls. The participants were divided into training and testing cohorts for model development and accuracy validation, respectively. Radiomic features derived from grayscale (GS) and shear wave elastography (SWE) images, obtained during the window of implantation, underwent a comprehensive five-step selection process. Five ML classifiers, each trained on either radiomic, clinical, or combined datasets, were trained for RPL risk stratification. The model demonstrating the highest performance in identifying RPL patients was selected for further validation using the testing cohort. The interpretability of this optimal model was augmented by applying Shapley additive explanations (SHAP) analysis. Results: Analysis of the training cohort (242 RPL, 258 controls) identified nine key radiomic features associated with RPL risk. The extreme gradient boosting (XGBoost) model, combining radiomic and clinical data, demonstrated superior discriminatory ability. This was evidenced by its area under the curve (AUC) score of 0.871, outperforming other ML classifiers. Validation in the testing cohort of 215 subjects (104 RPL, 111 controls) confirmed its accuracy (AUC: 0.844) and consistency. SHAP analysis identified four endometrial SWE features and two GS features, along with clinical variables like age, SAPI, and VI, as key determinants in RPL risk stratification. Conclusion: Integrating ML with radiomics from multimodal endometrial ultrasound during the WOI effectively identifies RPL patients. The XGBoost model, merging radiomic and clinical data, offers a non-invasive, accurate method for RPL management, significantly enhancing diagnosis and treatment.

Altered Expression of C4BPA and CXCL1 Genes in the Endometrium of Patients with Recurrent Implantation Failure after In Vitro Fertilization and Thin Endometrium.

Currently, recurrent implantation failure (RIF) after in vitro fertilization is a problem that is commonly faced by reproductive specialists. The phenomenon of a thin endometrium in RIF patients is not yet completely understood or sufficiently treated. This study aimed to reveal the dysregulated expression of selected genes between RIF patients with a thin endometrium and fertile women. Endometrial samples were collected in the implantation window (21-24 days of the natural menstrual cycle) from RIF patients (n = 20) and fertile women (n = 14). Ten genes were chosen as target genes regarding their possible relations with the implantation process. The endometrial gene expression levels showed differences in RIF samples compared to fertile samples. Significant downregulation was observed for the CXCL1 (p = 0.005) and C4BPA (p = 0.03) genes. There was no statistically significant difference between the RIF group and the fertile group in the expression of eight genes: CXCL8, HPRT1, MMP10, INFG, VEGFB, HAND2, IL-15, and TNC (p > 0.05). The use of a combination of two markers (C4BPA + CXCL1) allows for the good discrimination of RIF patients from fertile women (AUC 0.806).

ZEB1 Promotes Epithelial-Mesenchymal Transition of Endometrial Epithelial Cells and Plays a Critical Role in Embryo Implantation in Mice.

Zinc finger E-box binding homeobox 1 (ZEB1) promotes epithelial-mesenchymal transition (EMT) in carcinogenesis, but its role in embryo implantation has not yet been well studied. In the present study we evaluated the hypothesis that ZEB1-induced EMT is essential for embryo implantation in vivo. Endometrial epithelium from female Kunming mice (non-pregnant, and pregnant from day 2.5 to 6.5) were collected for assessment of mRNA/protein expression of ZEB1, and EMT markers E-cadherin and vimentin, by employment of real-time quantitative reverse transcription PCR, Western blot, and immunohistochemical staining. To test if knockdown of ZEB1 affects embryo implantation in vivo, mice received intrauterine injection of shZEB1 before the number of embryos implanted was counted. The results showed that, ZEB1 was highly expressed at both mRNA and protein levels in the mouse endometrium on day 4.5 of pregnancy, paralleled with down-regulated E-cadherin and up-regulated vimentin expression (P < 0.05). Intrauterine injection of shZEB1 markedly suppressed embryo implantation in mice (P < 0.01). Conclusively, the present work demonstrated that ZEB1 is essential for embryo implantation under in vivo condition, and is possibly due to its effect on modulation of endometrial receptivity through EMT.

A Comprehensive Review of the Endometrial Receptivity Array in Embryo Transfer: Advancements, Applications, and Clinical Outcomes.

Embryo transfer is a pivotal procedure in assisted reproductive technologies (ART). Yet, the success of this process hinges on multiple factors, with endometrial receptivity playing a critical role in determining the likelihood of successful implantation. The endometrial receptivity array (ERA) is an advanced diagnostic tool designed to personalize embryo transfer timing by assessing the endometrium's receptivity. This review comprehensively examines the ERA, exploring its biological foundation, technological development, and clinical applications. The ERA's ability to analyze the expression of genes associated with endometrial receptivity offers a tailored approach to identifying the optimal window of implantation (WOI), particularly benefiting patients with recurrent implantation failure (RIF) or repeated unsuccessful in vitro fertilization (IVF) cycles. Clinical outcomes from ERA-guided embryo transfers indicate improvements in implantation rates and overall pregnancy success, although challenges such as result variability and cost-effectiveness persist. This review also discusses the latest advancements in ERA technology, including integrating genomic and transcriptomic analyses, non-invasive techniques, and using artificial intelligence (AI). Controversies regarding the widespread application of ERA and its necessity in all IVF cases are critically examined. By summarizing the current state of ERA in embryo transfer, this review aims to inform clinicians, researchers, and patients about its potential to enhance ART outcomes and to highlight areas for future research and innovation.

Glyphosate and a glyphosate-based herbicide dysregulate the epigenetic landscape of Homeobox A10 (Hoxa10) gene during the endometrial receptivity in Wistar rats.

We observed that gestational plus lactational exposure to glyphosate (Gly), as active ingredient, or a glyphosate-based herbicide (GBH) lead to preimplantation losses in F1 female Wistar rats. Here, we investigated whether GBH and/or Gly exposure could impair Hoxa10 gene transcription by inducing epigenetic changes during the receptive stage in rats, as a possible herbicide mechanism implicated in implantation failures. F0 dams were treated with Gly or a GBH through a food dose of 2 mg Gly/kg bw/day from gestational day (GD) 9 up to lactational day 21. F1 female rats were bred, and uterine tissues were analyzed on GD5 (preimplantation period). Transcripts levels of Hoxa10, DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b), histone deacetylases (Hdac-1 and Hdac-3) and histone methyltransferase (EZH2) were assessed by quantitative polymerase chain reaction (qPCR). Four CpG islands containing sites targeted by BstUI methylation-sensitive restriction enzyme and predicted transcription factors (TFs) were identified in Hoxa10 gene. qPCR-based methods were used to evaluate DNA methylation and histone post-translational modifications (hPTMs) in four regulatory regions (RRs) along the gene by performing methylation-sensitive restriction enzymes and chromatin immunoprecipitation assays, respectively. GBH and Gly downregulated Hoxa10 mRNA. GBH and Gly increased DNA methylation levels and Gly also induced higher levels than GBH in all the RRs analyzed. Both GBH and Gly enriched histone H3 and H4 acetylation in most of the RRs. While GBH caused higher H3 acetylation, Gly caused higher H4 acetylation in all RRs. Finally, GBH and Gly enhanced histone H3 lysine 27 trimethylation (H3K27me3) marker at 3 out of 4 RRs studied which was correlated with increased EZH2 levels. In conclusion, exposure to GBH and Gly during both gestational plus lactational phases induces epigenetic modifications in regulatory regions of uterine Hoxa10 gene. We show for the first time that Gly and a GBH cause comparable gene expression and epigenetic changes. Our results might contribute to delineate the mechanisms involved in the implantation failures previously reported. Finally, we propose that epigenetic information might be a valuable tool for risk assessment in the near future, although more research is needed to establish a cause-effect relationship.

Supraphysiological Dose of Testosterone Impairs the Expression and Distribution of Sex Steroid Receptors during Endometrial Receptivity Development in Female Sprague-Dawley Rats.

This study aims to investigate the effect of a supraphysiological dose of testosterone on the levels of sex steroid hormones and the expression and distribution of sex steroid receptors in the uterus during the endometrial receptivity development period. In this study, adult female Sprague-Dawley rats (n = 24) were subcutaneously administered 1 mg/kg/day of testosterone alone or in combination with the inhibitors (finasteride or anastrozole or both) from day 1 to day 3 post-coitus, while a group of six untreated rats served as a control group. The rats were sacrificed on the evening of post-coital day 4 of to measure sex steroid hormone levels by ELISA. Meanwhile, gene expression and protein distribution of sex steroid receptors were analysed by quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC), respectively. In this study, treatment with a supraphysiological dose of testosterone led to a significant reduction in oestrogen and progesterone levels compared to the control. The mRNA expression of the androgen receptor increased significantly in all treatment groups, while the mRNA expression of both the progesterone receptor and the oestrogen receptor-α decreased significantly in all treatment groups. The IHC findings of all sex steroid receptors were coherent with all mRNAs involved. This study shows that a supraphysiological dose of testosterone was able to interrupt the short period of the implantation window. This finding could serve as a basis for understanding the role of testosterone in endometrial receptivity in order to develop further therapeutic approaches targeting androgen-mediated disorders of endometrial receptivity.

Personalized embryo transfer guided by rsERT with hourly precision improves pregnancy outcomes in patients with a receptive window of implantation: a pilot study.

PURPOSE: To investigate whether personalized embryo transfer (pET) predicted by a modified RNA-sequencing-based endometrial receptivity test (rsERT) model can improve intrauterine pregnancy rate (IPR) in patients with a receptive window of implantation (WOI). DESIGN: A retrospective pilot study was conducted in the Center for Reproductive Medicine, Central South University, from January 2018 to December 2023. A total of 524 patients with receptive WOI results from rsERT were assigned into two groups based on whether they underwent conventional embryo transfer (conventional ET) or pET. Patients in the conventional ET were matched with those in the pET group at a 1:1 ratio using propensity score matching (PSM). RESULTS: Before PSM, the IPR (55.73% vs. 46.19%, P = 0.032) and implantation rate (IR) (47.51% vs. 34.03%, P = 0.000) in the pET group were significantly higher than that in the conventional ET group. However, the number and types of transferred embryos differed significantly between the two groups. After adjusting for confounding factors, IPR (57.38% vs. 44.81, P = 0.016) and IR (46.81% vs. 33.10%, P = 0.001) remained significantly higher in the pET group compared to the conventional ET group. The implantation failure rate was significantly lower in the pET group compared to controls (42.62% vs. 55.19%, P = 0.016). Additionally, the multiple-pregnancy rate was significantly higher in the pET group compared to the conventional ET group (10.29% vs. 1.68%, P = 0.001). CONCLUSIONS: Women with receptive WOI results could benefit from the receptivity-timed pET predicted by the newly refined rsERT. These findings provide a basis for future research in precision medicine for embryo transfer.

Improvement of endometrial thickness and in vitro fertilization outcomes in patients with Asherman's refractory endometrium using autologous mesenchymal stem cells from the stromal vascular fraction.

OBJECTIVE: Patients with Asherman's Syndrome (AS) and an endometrial thickness (EMT) less than 7 mm are infertile women with suboptimal endometrium due to uterine scarring or endometrial atrophy. This study aimed to examine the effect of intrauterine injections of adipose-derived mesenchymal stem cells (ADMSC) from the Stromal Vascular Fraction (SVF) of adipose tissue on EMT and in vitro fertilization (IVF) outcomes: which are improvements in EMT and pregnancy rates. METHODS: This double-arm retrospective study included 41 AS patients with hysteroscopic adhesiolysis. Twenty-one patients with AS refractory endometrium (Group 2) were given ADMSC to improve EMT, and 20 non-treated, age-matched patients served as controls (Group 1). For Group 2, SVF was isolated from 15 ml of adipose tissue and transmyometrial injected into the patient's uterine cavity. For all patients, EMT was examined using ultrasound before embryo transfer. RESULTS: In Group 2, after ADMSC treatment, EMT significantly improved (3.2 ± 1.8 mm, P<0.001). Afterward, three patients spontaneously became pregnant, and eighteen underwent frozen embryo transfer. A significant increase in implantation (66.7% vs. 4.8%, P = 0.002) and live birth rates (0.0% vs. 47.6%, P = 0.001) were recorded. No significant difference was observed in EMT, cycle implantation, or clinical pregnancy between the two groups, but the live birth rate in Group 2 after ADMSC treatment was higher than in Group 1. CONCLUSION: The results demonstrate that autologous intrauterine ADMSC injection can improve EMT, implantation, and pregnancy rates in AS patients with refractory endometrium. This research underscores the life-changing potential of autologous ADMSC treatment for patients with refractory endometrium, providing a promising avenue for future treatments.

Expression of Endometrial Receptivity Markers throughout the Menstrual Cycle in Women with and without Uterine Adenomyosis.

Background/Objectives: While it is known that adenomyosis is associated with poor reproductive outcomes, the underlying mechanisms are unclear, and to date, there is no standard treatment protocol for these patients. Endometrium from adenomyosis patients is characterized by several abnormalities, potentially resulting in impaired receptivity and subsequent implantation failure. Methods: Endometrial biopsies were collected from 26 women with adenomyosis and 26 control subjects. Immunohistochemistry was performed to evaluate the expression of markers of endometrial receptivity, namely the progesterone receptor (PR), glycodelin, leukemia inhibitory factor (LIF), homeobox A10 (HOXA10), integrin beta chain beta 3 (integrin ß3) and osteopontin. Scanning electron microscopy was used to observe pinopodes on the surface of mid-secretory endometrial epithelium. Results: PR, LIF and osteopontin expression were all found to be weaker in secretory-phase stroma from adenomyosis patients than in healthy controls. HOXA10 expression was decreased in adenomyosis during the secretory phase, and also the proliferative phase, where it reached statistical significance in both epithelial and stromal compartments. Glycodelin and integrin ß3 levels did not differ between diseased and healthy tissues in any of the cycle phases. Pinopodes were fewer and at later developmental stages in adenomyosis compared to those on the surface of healthy endometrium from the same time period of the menstrual cycle. Conclusions: Endometrium from adenomyosis patients is characterized by abnormal expression of various receptivity markers. The stromal compartment appears to be affected most, showing reduced expression of PR, LIF and osteopontin in the secretory phase and lower levels of HOXA10 during both proliferative and secretory phases. Decreased receptivity due to impaired stromal decidualization may contribute to poor reproductive outcomes in adenomyosis patients.

Endometrial E-cadherin and N-cadherin Expression during the Mid-Secretory Phase of Women with Ovarian Endometrioma or Uterine Fibroids.

BACKGROUND: Endometriosis and uterine fibroids are benign conditions frequently linked to subfertility/infertility. Recent research has highlighted the importance of epithelial-mesenchymal transition between embryonic and endometrial cells in the context of embryo implantation. Additionally, the adverse endometrial environment during implantation has been proposed as a mechanism contributing to infertility in endometriosis. Nevertheless, the role of cadherin molecule alterations in relation to endometrial receptivity and embryo invasion remains a subject of controversy. METHODS: We investigated the expression patterns of E-cadherin and N-cadherin in the endometria of women with ovarian endometrioma or uterine fibroids and assessed whether they differed from those of healthy women. We enrolled 17 women with ovarian endometrioma, 16 with uterine fibroids, and 6 healthy women. Endometrial tissues were obtained at the mid-secretory phase on days 19-24 of the menstrual cycle. The E-cadherin and N-cadherin mRNA and protein expression levels were measured using quantitative reverse transcriptase polymerase chain reaction and Western blot analysis, respectively. RESULTS: The E-cadherin and N-cadherin mRNA expression levels were higher and lower, respectively, in the endometrium of women with ovarian endometrioma than in those of the controls. In the endometrium of women with uterine fibroids, similar patterns with higher E-cadherin and lower N-cadherin levels were observed compared with that of the controls. Protein expression showed similar patterns. CONCLUSIONS: Our findings revealed higher E-cadherin expression and lower N-cadherin expression in the endometria of women with infertility-related diseases than in those of healthy women in the mid-secretory phase. This suggests a resistance to endometrial receptivity, potentially reflecting mesenchymal-epithelial transition properties.

Generation of epithelial-stromal assembloids as an advanced in vitro model of impaired adenomyosis-related endometrial receptivity.

OBJECTIVE: To create a novel, more advanced in vitro model of human endometrium, using so-called assembloids, looking to explore endometrial receptivity in adenomyosis. DESIGN: Evaluation of assembloid responsiveness to hormonal stimulation by immunohistochemistry, enzyme-linked immunosorbent assay, and scanning electron microscopy. SETTING: University-based research unit in gynecology. PATIENT(S): Twelve women, six of whom were affected by adenomyosis. INTERVENTION(S): Organoids (in the form of glandular fragments) and stromal fibroblasts were collected from endometrial biopsies. The two populations were combined inside an extracellular matrix to create 3D assembloids, which were then exposed to hormonal stimulation (ß-estradiol for 48 hours, followed by ß-estradiol/progesterone/cyclic adenosine monophosphate for 72 hours) to mimic the window of implantation. MAIN OUTCOME MEASURE(S): Glycodelin, leukemia inhibitory factor (LIF), and homeobox A10 (HOXA10) expression, prolactin secretion, and pinopode development. RESULT(S): Endometrial organoids and stromal cells were successfully isolated from women with and without adenomyosis and combined to generate the assembloid model. On stimulation, assembloids from both groups acquired a more secretory phase-like phenotype, as demonstrated by histology, and were shown to be positive for glycodelin, LIF, and HOXA10 by immunohistochemistry. Adenomyotic assembloids expressed significantly lower levels of LIF and HOXA10 within the stromal compartment after stimulation than did healthy assembloids in the same condition. Enzyme-linked immunosorbent assay revealed prolactin secretion in vitro, showing an upward trend in hormonally treated assembloids from both healthy and affected women. By scanning electron microscopy, fully formed pinopodes were discerned on the epithelial surface of healthy assembloids after stimulation, but they were absent in case of adenomyosis. CONCLUSION(S): Primary assembloids can be generated from endometrial biopsies from both healthy subjects and women affected by adenomyosis. These assembloids are amenable to hormonal stimulation and mimic secretory phase-specific characteristics of endometrial tissue in vivo, including glycodelin, LIF, and HOXA10 expression, and pinopode formation. Assembloids from adenomyosis appear to be less sensitive to hormonal treatment, showing reduced expression of LIF and HOXA10 in the stromal compartment and failing to form pinopodes. All in all, endometrial assembloids may serve as an advanced preclinical model of adenomyosis-related impaired endometrial receptivity, opening up new horizons in understanding and treating the condition.

Adiponectin receptor 1 regulates endometrial receptivity via the adenosine monophosphate­activated protein kinase/E­cadherin pathway.

Endometrial receptivity is essential for successful embryo implantation and pregnancy initiation and is regulated via various signaling pathways. Adiponectin, an important adipokine, may be a potential regulator of reproductive system functions. The aim of the present study was to elucidate the regulatory role of adiponectin receptor 1 (ADIPOR1) in endometrial receptivity. The endometrial receptivity between RL95­2 and AN3CA cell lines was confirmed using an in vitro JAr spheroid attachment model. 293T cells were transfected with control or short hairpin (sh)ADIPOR1 vectors and RL95­2 cells were transduced with lentiviral particles targeting ADIPOR1. Reverse transcription­quantitative PCR and immunoblot assays were also performed. ADIPOR1 was consistently upregulated in the endometrium during the mid­secretory phase compared with that in the proliferative phase and in receptive RL95­2 cells compared with that in non­receptive AN3CA cells. Stable cell lines with diminished ADIPOR1 expression caused by shRNA showed reduced E­cadherin expression and attenuated in vitro endometrial receptivity. ADIPOR1 regulated AMP­activated protein kinase (AMPK) activity in endometrial epithelial cells. Regulation of AMPK activity via dorsomorphin and 5­aminoimidazole­4­carboxamide ribonucleotide affected E­cadherin expression and in vitro endometrial receptivity. The ADIPOR1/AMPK/E­cadherin axis is vital to endometrial receptivity. These findings can help improve fertility treatments and outcomes.

The Current Understanding of Molecular Mechanisms in Adenomyosis-Associated Infertility and the Treatment Strategy for Assisted Reproductive Technology.

Adenomyosis, endometriosis of the uterus, is associated with an increased likelihood of abnormal endometrial molecular expressions thought to impair implantation and early embryo development, resulting in disrupted fertility, including the local effects of sex steroid and pituitary hormones, immune responses, inflammatory factors, and neuroangiogenic mediators. In the recent literature, all of the proposed pathogenetic mechanisms of adenomyosis reduce endometrial receptivity and alter the adhesion molecule expression necessary for embryo implantation. The evidence so far has shown that adenomyosis causes lower pregnancy and live birth rates, higher miscarriage rates, as well as adverse obstetric and neonatal outcomes. Both pharmaceutical and surgical treatments for adenomyosis seem to have a positive impact on reproductive outcomes, leading to improved pregnancy and live birth rates. In addition, adenomyosis has negative impacts on reproductive outcomes in patients undergoing assisted reproductive technology. This association appears less significant after patients follow a long gonadotropin-releasing hormone agonist (GnRHa) protocol, which improves implantation rates. The pre-treatment of GnRHa can also be beneficial before engaging in natural conception attempts. This review aims to discover adenomyosis-associated infertility and to provide patient-specific treatment options.

Perinatal Exposure to Glyphosate or a Commercial Formulation Alters Uterine Mechanistic Pathways Associated with Implantation Failure in Rats.

Perinatal exposure to a glyphosate-based herbicide (GBH) or its active ingredient, glyphosate (Gly), has been demonstrated to increase implantation failure in rats. This study investigates potential mechanisms of action, analyzing uterine preparation towards the receptive state. Pregnant Wistar rats (F0) were treated orally with GBH or Gly (3.8 and 3.9 mg Gly/kg/day, respectively) from gestational day (GD) 9 until weaning. Adult F1 females became pregnant and uterine samples were collected on GD5 (preimplantation period). Histomorphological uterine parameters were assessed. Immunohistochemistry was applied to evaluate cell proliferation and protein expression of estrogen receptors (ERα and ERß), cell cycle regulators (PTEN, cyclin G1, p27, and IGF1R-α), and the Wnt5a/ß-catenin/FOXA2/Lif pathway. Both GBH and Gly females showed increased stromal proliferation, associated with a high expression of ERs. Dysregulation of PTEN and cyclin G1 was also observed in the Gly group. Reduced gland number was observed in both groups, along with decreased expression of Wnt5a/ß-catenin/FOXA2/Lif pathway in the glandular epithelium. Overall, GBH and Gly perinatal exposure disrupted intrinsic uterine pathways involved in endometrial proliferation and glandular function, providing a plausible mechanism for glyphosate-induced implantation failure by compromising uterine receptivity. Similar effects between GBH and Gly suggest the active principle mainly drives the adverse outcomes.