Artificial Neural Network Model Using Immune-infiltration Modules for Endometrial Receptivity Assessment of Implantation Failure

Objectives: This study was anchored on the state of local immune-infiltration in the endometrium, which acts as critical factors affecting embryonic implantation, and aimed at establishing novel approaches to assess endometrial receptivity for patients with IVF failure.Methods: Immune-infiltration levels in the GSE58144 dataset (n=115) from GEO were analyzed by digital deconvolution and validated by immunofluorescence (n=30), illustrating that dysregulation of the ratio of Mf1 to Mf2 is an important factor contributing to implantation failure. Then, modules most associated with M1/M2 macrophages (Mfs) and their hub genes were then selected by weighted gene co-expression network and univariate analyses, then validated by GSE5099 macrophage dataset, qPCR analysis (n=16), and western blot. It revealed that closely related gene modules dominated three biological processes in macrophages: antigen presentation, interleukin−1−mediated signalling pathway, and phagosome acidification, respectively. Their hub genes were significantly altered in patients and related with ribosomal, lysosome, and proteasomal pathways. Finally, the artificial neural network (ANN) and nomogram models were established from hub genes, of which efficacy was compared and validated in the GSE165004 dataset (n=72). Models established by the selected hub genes exhibited excellent predictive values in both datasets, and ANN performed best with an accuracy of 98.3% and an AUC of 0.975 (95% CI 0.945-1). Conclusions: Macrophages, proven to be essential for endometrial receptivity, were regulated by gene modules dominating antigen presentation, interleukin−1−mediated signalling pathway, and phagosome acidification. Selected hub genes can effectively assess endometrial dysfunction receptivity for IVF outcomes by the ANN approach.

Transcriptome analysis of eutopic endometrium in adenomyosis after GnRH agonist treatment

Background Adenomyosis is a chronic gynecological disease characterized by invasion of the uterine endometrium into the muscle layer. In assisted reproductive technology (ART), gonadotropin-releasing hormone agonist (GnRHa) is often used to improve pregnancy rates in patients with adenomyosis, but the underlying mechanisms are poorly understood. Methods Eutopic endometrial specimens were collected from patients with adenomyosis before and after GnRHa treatment in the midsecretory phase. RNA sequencing (RNA-Seq) of these specimens was performed for transcriptome analysis. The differentially expressed genes (DEGs) of interest were confirmed by real-time PCR and immunohistochemistry. Results A total of 132 DEGs were identified in the endometrium of patients with adenomyosis after GnRHa treatment compared with the control group. Bioinformatics analysis predicted that immune system-associated signal transduction changed significantly after GnRHa treatment. Chemokine (C-C motif) ligand 21 (CCL21) was found to be highly expressed in the eutopic endometrium after GnRHa treatment, which may be involved in the improvement of endometrial receptivity in adenomyosis. Conclusion This study suggests that molecular regulation related to immune system-associated signal transduction is an important mechanism of GnRHa treatment in adenomyosis. Immunoreactive CCL21 is thought to regulate inflammatory events and participate in endometrial receptivity in adenomyosis.

The Effect of Low Intensity Pulsed Ultrasound on Endometrial Receptivity in Infertile Patients with Adenomyosis

Objective: Adenomyosis (AM) is an important cause of female infertility, and its disease mechanism remains unclear. This study preliminarily investigated the expression of endometrial receptivity markers homeobox A10 (HOXA10) and leukemia inhibitory factor (LIF) in infertile patients with AM and described the effects of low intensity pulsed ultrasound (LIPUS) on it. Methods: In vivo, tissues were obtained from the infertile female AM patient group (AG group, n=10) and healthy control group (CG group, n=11). The expression of HOXA10 and LIF in the two groups was detected by immunohistochemistry (IHC) and western blotting. In vitro, primary cells were extracted and cultured from the two groups, and the expression of HOXA10 and LIF protein was detected by western blotting. Then the AG cells were treated with 15, 30, and 60 mW/cm2 of LIPUS for 7 days (20 min/day), and detected the cell adhesion rate. Finally, treat the AG cells with 30mW/cm2 LIPUS for 7 days (20 min/day), and detect the expression level of ICAM-1 in the cell supernatant by ELISA. The AG cells was treated with 30 mW/cm2 LIPUS for 4 days (20 min/day), and the expression levels of HOXA10 and LIF were detected by western blotting, RT-PCR, and agarose gel electrophoresis. Results: In vivo, IHC staining showed that HOXA10 and LIF proteins were mainly localized in endometrial epithelial cells. Both IHC and western blot showed that the levels of HOXA10 and LIF in the AG group were significantly lower than those in the CG group (P<0.01, P<0.05). In vitro, the expression levels of HOXA10 and LIF protein in the AG cell was significantly lower than those in the CG cell (P<0.001). Then, the cell adhesion ability of the 30 and 60 mW/cm2 groups was higher than that of the 15 mW/cm2 group after LIPUS treatment. Finally, The concentration of ICAM-1 in the supernatant of AG cells treated with LIPUS was significantly higher than that of the control group (P<0.01), and the AG cells were treated with 30 mW/cm2 LIPUS for 4 days (20 min/day), the protein and mRNA expression levels of HOXA10 and LIF were higher than those of the control group (P<0.001). Conclusion: The reduction of HOXA10 and LIF may be one of the reasons for the decreased endometrial receptivity in AM. The LIPUS promoted the adhesion and the expression of HOXA10 and LIF of EEECs from the AM group, thereby increasing endometrial receptivity.

Proteomic analysis of extracellular vesicles secreted by primary human epithelial endometrial cells reveals key proteins related to embryo implantation

Background Successful implantation is dependent on coordination between maternal endometrium and embryo, and the role of EVs in the required cross-talk cell-to-cell has been recently established. In this regard, it has been reported that EVs secreted by the maternal endometrium can be internalized by human trophoblastic cells transferring their contents and enhancing their adhesive and invasive capacity. This is the first study to comprehensively evaluate three EV isolation methods on human endometrial epithelial cells in culture and to describe the proteomic content of EVs secreted by pHEECs from fertile women. Methods Ishikawa cells and pHEECs were in vitro cultured and hormonally treated; subsequently, conditioned medium was collected and EVs isolated. Ishikawa cells were used for the comparison of EVs isolation methods ultracentrifugation, ExoQuick-TC and Norgen Cell Culture Media Exosome Purification Kit (n = 3 replicates/isolation method). pHEECs were isolated from endometrial biopsies (n = 8/replicate; 3 replicates) collected from healthy oocyte donors with confirmed fertility, and protein content of EVs isolated by the most efficient methodology was analysed using liquid chromatography–tandem mass spectrometry. EV concentration and size were analyzed by nanoparticle tracking analysis, EV morphology visualized by transmission electron microscopy and protein marker expression was determined by Western blotting. Results Ultracentrifugation was the most efficient methodology for EV isolation from medium of endometrial epithelial cells. EVs secreted by pHEECs and isolated by ultracentrifugation were heterogeneous in size and expressed EV protein markers HSP70, TSG101, CD9, and CD81. Proteomic analysis identified 218 proteins contained in these EVs enriched in biological processes involved in embryo implantation, including cell adhesion, differentiation, communication, migration, extracellular matrix organization, vasculature development, and reproductive processes. From these proteins, 82 were selected based on their functional relevance in implantation success as possible implantation biomarkers. Conclusions EV protein cargos are implicated in biological processes related to endometrial receptivity, embryo implantation, and early embryo development, supporting the concept of a communication system between the embryo and the maternal endometrium via EVs. Identified proteins may define new biomarkers of endometrial receptivity and implantation success.

Transcriptomics of receptive endometrium in women with sonographic features of adenomyosis

Background Women with uterine adenomyosis seeking assisted reproduction have been associated with compromised endometrial receptivity to embryo implantation. To understand the mechanisms involved in this process, we aimed to compare endometrial transcriptome profiles during the window of implantation (WOI) between women with and without adenomyosis. Methods We obtained endometrial biopsies LH-timed to the WOI from women with sonographic features of adenomyosis (n=10) and controls (n=10). Isolated RNA samples were subjected to RNA sequencing (RNA-seq) by the Illumina NovaSeq 6000 platform and endometrial receptivity classification with a molecular tool for menstrual cycle phase dating (beREADY®, CCHT). The program language R and Bioconductor packages were applied to analyse RNA-seq data in the setting of the result of accurate endometrial dating. To suggest robust candidate pathways, the identified differentially expressed genes (DEGs) associated with the adenomyosis group in the receptive phase were further integrated with 151, 173 and 42 extracted genes from published studies that were related to endometrial receptivity in healthy uterus, endometriosis and adenomyosis, respectively. Enrichment analyses were performed using Cytoscape ClueGO and CluePedia apps. Results Out of 20 endometrial samples, 2 were dated to the early receptive phase, 13 to the receptive phase and 5 to the late receptive phase. Comparison of the transcriptomics data from all 20 samples provided 909 DEGs (p<0.05; nonsignificant after adjusted p value) in the adenomyosis group but only 4 enriched pathways (Bonferroni p value < 0.05). The analysis of 13 samples only dated to the receptive phase provided suggestive 382 DEGs (p<0.05; nonsignificant after adjusted p value) in the adenomyosis group, leading to 33 enriched pathways (Bonferroni p value < 0.05). These included pathways were already associated with endometrial biology, such as “Expression of interferon (IFN)-induced genes” and “Response to IFN-alpha”. Data integration revealed pathways indicating a unique effect of adenomyosis on endometrial molecular organization (e.g., “Expression of IFN-induced genes”) and its interference with endometrial receptivity establishment (e.g., “Extracellular matrix organization” and “Tumour necrosis factor production”). Conclusions Accurate endometrial dating and RNA-seq analysis resulted in the identification of altered response to IFN signalling as the most promising candidate of impaired uterine receptivity in adenomyosis.

Autophagy as a Therapeutic Target of Natural Products Enhancing Embryo Implantation

Infertility is an emerging health issue worldwide, and female infertility is intimately associated with embryo implantation failure. Embryo implantation is an essential process during the initiation of prenatal development. Recent studies have strongly suggested that autophagy in the endometrium is the most important factor for successful embryo implantation. In addition, several studies have reported the effects of various natural products on infertility improvement via the regulation of embryo implantation, embryo quality, and endometrial receptivity. However, it is unclear whether natural products can improve embryo implantation ability by regulating endometrial autophagy. Therefore, we performed a literature review of studies on endometrial autophagy, embryo implantation, natural products, and female infertility. Based on the information from these studies, this review suggests a new treatment strategy for female infertility by proposing natural products that have been proven to be safe and effective as endometrial autophagy regulators; additionally, we provide a comprehensive understanding of the relationship between the regulation of endometrial autophagy by natural products and female infertility, with an emphasis on embryo implantation.

Peritoneal endometriosis and infertility

Peritoneal endometriosis and infertility in most of patients (in 80%) are pathogenetically conjugated. Both peritoneal endometriosis and infertility are based in ovarian failure. These women have a low endometrial receptivity for blastocysts implantation (retardation development of glands, vessels, and stroma; changes in the microrelief of the epithelium). Even at the beginning of the menstruation patients with peritoneal endometriosis and infertility have cells with a great adhesive and proliferative potential in the endometrium. This kind of cells have an ability for long autonomous existence. Ovarian failure in these women is a promotion factor for development of the retrograde menstruation. In these conditions the endometrial cells with adhesive potential are frequently bringing in the abdominal cavity. Active endometrium heterotopias support the ovarian failure and create conditions for uterine infertility (implantation disorders).