COVID-19 and embryo implantation: EPSTI1 and SUGT1 participate in the dysregulation of SARS-CoV-2 on endometrial receptivity

COVID-19 and embryo implantation: EPSTI1 and SUGT1 participate in the dysregulation of SARS-CoV-2 on endometrial receptivity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article COVID-19 and embryo implantation: EPSTI1 and SUGT1 participate in the dysregulation of SARS-CoV-2 on endometrial receptivity Xi Cheng, Xiting Cai, Hui Wang, Biying Li, Lu Zheng, Jinzhao Ma, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4545088/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To study the potential effect of coronavirus disease 2019 (COVID-19) on the endometrial receptivity. Methods Overlapped differentially expressed genes (DEGs) between the endometrium and the throat swabs of female patients with COVID-19 infection vs. women without COVID-19 were integrated. The expression and distribution of prioritized molecules were detected in mid-secretory phase endometrium from patients infected by COVID-19 within 30 days vs. Individuals without the infection. Serum INF-γ, LPS, TNF-α, IL-1β, IL-1α and IL-6 were detected in female patients infected by COVID-19 within 30 days vs. Individuals without the infection. In vitro decidualization of human endometrial stromal cells (T-hESCs) and human choriocarcinoma (BeWo) spheroids adhesion experiments were employed to explore the functions of the potential molecules EPSTI1 and SUGT1. Cytokines of INF-γ, LPS and IL-1β were administrated in T-hESCs and human endometrial adenocarcinoma (Ishikawa) cells, and the expression of EPSTI1 and SUGT were identified. Results The systemic disease COVID-19 altered endometrial protein expression during peri-implantation window. Epithelial stromal interaction 1 (EPSTI1), S-phase kinase-associated protein 1 (SUGT1) and RCC1-containing protein 5 (HERC5) were up-regulated in mid-secretory phase endometrium of women infected by COVID-19 within 30 days. EPSTI1 and SUGT1 impaired PRL and IGFBP1 expression stimulated by 8-Br-cAMP and MPA in T-hESCs. SUGT1 decreased the adhesion rate of BeWo spheroids to Ishikawa cells induced by E 2 and MPA. Serum INF-γ, LPS, TNF-α, IL-1β and IL-6 were elevated in female patients being infected with Covid-19 with mild or moderate symptoms. IL-1β demonstrated a sustained elevation in women infected with COVID-19 within 8–30 days. EPSTI1 was up-regulated by the administration of IL-1β, INF-γ and LPS in shikawa cells and T-hESCs. SUGT1 was up-regulated by the administration of IL-1β and LPS in shikawa cells and T-hESCs. Conclusions Endometrial decidualization and embryo adhesion function were negatively influenced by EPSTI1 and SUGT1, which were highly expressed in the mid-secretory endometrium of COVID-19-infected women. The elevation of EPSTI1 and SUGT1 in patients infected with Covid-19 maybe related to the increased cytokines of IL-1β, INF-γ and LPS. Investigating the relationship between SARS-CoV-2 and human reproduction may be very helpful in the long run in preventing and controlling COVID-19 and its aftermath. COVID-19 Endometrial receptivity embryo implantation women’s reproductive health EPSTI1 SUGT1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is identified as the causative agent of the COVID-19 pandemic [ 1 ]. Commonly, COVID-19 manifests as pulmonary complications, typically associated with more severe disease and poorer outcomes. However, a range of extrapulmonary symptoms have also been observed, often in cases of mild to moderate severity or even during severe disease stages. Dizziness, skin changes, arrhythmias, liver damage, hematuria, and anosmia or ageusia are some of these symptoms [ 2 ]. As a result, COVID-19 is acknowledged as a multisystemic and heterogeneous illness. Certain COVID-19-related consequences could not be directly caused by the virus itself, but rather are the consequence of a worldwide immunological dysregulation that the virus started [ 3 ]. This raises questions regarding how SARS-CoV-2 could affect organs that have high expression of the COVID-19 gene, as these organs might be more vulnerable to harmful effects after infection. The impact of COVID-19 on pregnancy outcomes has been substantiated by a considerable amount of researches. Notably, SARS-CoV-2 infection during pregnancy is correlated with increased incidences of preeclampsia, preterm birth, and stillbirth [ 4 ]. However, there is uncertainty about how COVID-19 affects endometrial tissue and embryo implantation. Some of the researches have discussed the connection between menstrual cycle and gene expression associated to SARS-CoV-2 infection. One study that looked at scRNA-seq datasets found a low probability of SARS-CoV-2 infection in the endometrium. This conclusion was based on the negligible expression of ACE2 in stromal or unciliated epithelial cells and the absence of concurrent expression of ACE2 and TMPRSS2 during embryo implantation [ 5 ]. Hanerjos-Castillo et al . reported that the endometrium had low ACE2, medium TMPRSS2 expression, and restricted co-expression based on the combination of gene expression data from five investigations, which included 112 individuals [ 6 ]. Around the time of implantation, the research also observed a rise in the expression of MX1, ACE2, TMPRSS4, CTSB, and CTSL [ 6 , 7 ]. Both investigations found significant levels of BSG expression in the endometrium, but they disagreed about how important the BSG apparatus is in the event of a viral infection. According to a meta-analysis, the COVID-19 pandemic undoubtedly had an effect on endometriosis patients, since it intensified symptoms such dysmenorrhea, pelvic discomfort, anxiety, melancholy, and exhaustion [ 8 ]. This suggests that further research on the endometrial tissue of women of reproductive age infected with SARS-CoV-2 will help determine whether the causal implications derived from the molecular characterization of the endometrium translate into actual clinical manifestations. In 2022, Lucía de Miguel-Gomez’s group performed transcriptomic analyses endometrial biopsy samples and identified 235 DEGs from the endometria of COVID-19 patients [ 9 ]. It was the first to look at alterations in the endometrium's global gene expression in women with COVID-19, providing substantial evidence that SARS-CoV-2 infection commonly affects the inflammatory environment, cellular motility, metabolic status, and cytokine production in the endometrium. However, this study had a crucial limitation that the patients’ age, menstrual cycle parameters, fertility status and biopsy collection timing varied greatly, which cannot represent the specific phase of menstruation, not even for the period for window of implantation. In the same year, I. Henarejos-Castillo's group created a model that examined the gene co-expression network of the endometrium in 42 women who had healthy mid-secretory endometrium taken from them without endometrial diseases, together with nasopharyngeal swabs from 231 COVID-19-positive women and 30 negative controls [ 10 ]. 19 important genes that may be affected by COVID-19 were found by topological analysis, and the expression of these genes was confirmed by prospectively collecting an endometrial sample set that included both COVID-19 positive and negative individuals. Nevertheless, we also noted that the basic information and fertility condition of women included in the throat swabs analysis and the endometrial samples study were not comparable. Currently, there are no specific studies to elaborate the mechanisms of COVID-19's effect on endometrial receptivity since getting COVID-19 endometrial samples is a challenging task. In this study, we integrate the co-expression molecules of COVID-19 in endometrium [ 9 ] and female nasopharyngeal swabs [ 10 ] from above two studies, to investigate the comparatively stable genes expressed following SARS-CoV-2 infection in the female population, despite the potential discrepancy in age and menstrual cycle. We experimentally validated the molecules’ expression and distribution in mid-secretory phase endometrial biopsies from women who were diagnosed with COVID-19 within 30 days vs. Individuals without infection, to make predictions of SARS-CoV-2 related genes with endometrial receptivity. Additionally, endometrium-related molecular tests were employed to verify the functions of some of these potential molecules. With these studies, we want to learn more about the molecular processes underlying the changes in uterine endometrial responsiveness to COVID-19. Materials and Methods Specimen collection 8 endometrial samples from patients with COVID-19 infection within 30 days and 9 endometrial samples from healthy women without COVID-19 infection were collected from the Reproductive Center of Nanjing Jinling Hospital during January 2023 to March 2023. These COVID-19 infected patients were symptomatic and had a positive test result for SARS-CoV-2 infection, as indicated by nasopharyngeal swab samples. Patients with mild-to-moderate symptoms were included, whereas asymptomatic or severe cases were not recruited. The healthy women were enrolled from patients who have no COVID-19 infection history. Based on nasopharyngeal swab samples taken within the previous three months, the women in the health group were found to be negative for COVID-19. They also had nasopharyngeal swab tests on a weekly basis. The participants' body mass index (BMI) ranged from 18 to 30 kg/m 2 , and their ages ranged from 27 to 36, and the endometrial thickness ranged from 9 to 16 mm. The patients were infertile with diagnosis of the fallopian tube blockage, male factor or unexplained infertility, excluding endometrial disease such as endometrioma, adenomyosis, endometrial hyperplasia, endometrial polyps and intrauterine adhesion. All the endometiral samples were collected on the 5th day after ovulation.The endometrium thickness was 8–16 mm. Supplemental Table 1 presents the characteristics of the patients with endometrial biopsy. 25 serum samples from female patients with COVID-19 infection within 30 days and 10 serum samples from healthy women without COVID-19 infection were collected from Nanjing Jinling Hospital during January 2023 to December 2023. Among the Covid-19 group, 12 serum samples were drawn from patients 0 to 7 days after diagnosed with COVID-19, 13 serum samples were drawn from patients 8 to 30 days after diagnosed with COVID-19. These COVID-19 infected patients were symptomatic and had a positive test result for SARS-CoV-2 infection, as indicated by nasopharyngeal swab samples. Patients with mild-to-moderate symptoms were included, whereas asymptomatic or severe cases were not recruited. The healthy women were enrolled from patients who have no COVID-19 infection history. Based on nasopharyngeal swab samples taken within the previous three months, the women in the health group were found to be negative for COVID-19. They also had nasopharyngeal swab tests on a weekly basis. All the serum samples were drawn from female patients during nonmenstrual period. The participants' body mass index (BMI) ranged from 18 to 32 kg/m 2 , and their ages ranged from 22 to 40. Supplemental Table 2 presents the characteristics of the patients with serum cytokine examination. The study protocols (2023DZKY-009-01) were authorized by Nanjing Jinling Hospital's Clinical Ethics Review Committee. The Clinical Trials Registry (ID: NCT05685966) has the trial prospectively registered. Every participant endorsed the informed consent form. Immunohistochemistry staining In neutral buffered formalin, the endometrial samples were fixed. The sections were rehydrated and deparaffinized before being exposed to antigen retrieval. Following this, the sections were treated with primary antibodies against either SUGT1 (Proteintech), LRP1 (Abcam), HSPG2 (Abmart), HERC5 (Proteintech), or EPSTI1 (Proteintech). The slides were then exposed to secondary antibody incubation. The sections were counterstained with hematoxylin. The following protocol was used for immunohistochemical scoring, or histoscore: Each patient had five typical areas with stromal cells and endometrial glandular epithelium, at least three of which had glandular epithelium, examined under an Olympus light microscope set to ×200 magnification. With the use of the Fiji (ImageJ) program, the strength of the immunostaining was divided into four categories: 0 = negative; 1 = weak; 2 = moderate; and 3 = strong. Each field's histoscore was computed as follows: histoscore = 0 × percentage of cells that were negatively stained, 1 × proportion of cells with poor staining, 2 × proportion of cells with mild staining, and 3 × proportion of cells with intense staining. The average of the histoscores from five distinct fields was used to get the histoscore of each sample. Cytokine assay Blood samples were collected in 1.5-mL EP tubes, blood clotting was performed for 1 h at 4°C, and the tubes were centrifuged at 500 g at 4°C for 10 min to harvest serum. The levels of Interferon (IFN)-γ (Mlbio), Lipopolysaccharide (LPS) (Mlbio), Tumor Necrosis Factor (TNF)-α (Mlbio), Interleukin (IL)-1β (Mlbio), Interleukin (IL)-1α (Mlbio) and Interleukin (IL)-6 (Mlbio) were determined by enzyme-linked immunosorbent assay kits for human. Cell culture Human endometrial stromal cells (T-hESCs), human choriocarcinoma (BeWo), and human endometrial adenocarcinoma (Ishikawa) cells were cultured in DMEM/F12 media (HyClone) supplemented by 10% (v/v) fetal bovine serum (Gibco) plus 1% (v/v) penicillin/streptomycin. For the purpose of simulating the physiological condition, Ishikawa cells and T-hESCs were cultured in phenol red-free DMEM/F12 medium containing 2.5% (v/v) charcoal/dextranl-treated FBS and 1% (v/v) penicillin/streptomycin, with or without a combination of 10 nM E 2 (Sigma) or 0.5 mM 8Br-cAMP (Sigma), in addition to 1 µM medroxyprogesterone acetate (MPA) (Merck), for the prescribed duration. Western blot The manufacturer's instructions were followed for homogenizing the cells in whole-cell lysis solution with phosphatase inhibitor cocktails 2 and 3 (Sigma). A 6–10% (w/v) SDS–polyacrylamide gel was loaded with equal volumes of total protein (10–30 µg), which were subsequently deposited onto Millipore polyvinylidene fluoride membranes. Following immunoblotting, secondary antibodies conjugated with goat anti-rabbit or rabbit anti-mouse HRP were incubated on the membranes. The primary antibodies used in the immunoblotting process were against EPSTI1, SUGT1, and β-Actin from Proteintech. For detection, the ECL Plus Western Blotting Detection System and Millipore's enhanced chemiluminescence kit were employed. Quantitative real-time PCR Takara's TRIzol reagent was used to extract total RNA in accordance with the manufacturer's instructions. For the cDNA synthesis using the BEI-BEI Biotech Prime Script RT reagent kit, a total of 1 µg of RNA was utilized. Random primers were used for reverse transcription, and a Roche Diagnostics MyiQ Single Color Real-Time PCR Detection System was used for qRT-PCR. Supplementary Table 3 provides an explanation of the primer sequences. Using the 2-∆∆ CT technique, which normalizes gene expression to the level of the endogenous control (18s rRNA), the fold changes in gene expression were determined. In-vitro implantation model In accordance with standard laboratory protocol, BeWo multicellular spheroids were affixed to endometrial Ishikawa cells to serve as an in vitro adhesion model [ 11 ]. Briefly put, a single-cell solution of BeWo cells was placed on a Petri dish coated with the anti-adhesive polymer poly-2-hydroxyethyl methacrylate (Sigma) in order to encourage the growth of 150–200 µm diameter BeWo spheroids. On a 24-well culture plate, confluent monolayers of Ishikawa cells were transfected simultaneously with the chosen vectors. Next, a pre-measured amount of spheroids was added to the confluent monolayers of Ishikawa cells. Following a two-hour incubation period at 37°C, the detached spheroids were extracted using PBS supplemented with 0.1 mg/L of Ca 2+ and 0.1 mg/L of Mg 2+ . The attachment rate to the Ishikawa monolayer was computed by counting the adhering BeWo spheroids under a light microscope. There were three separate, independent attachment tests conducted. Results Overlapped DEGs from endometria and nasopharyngeal swabs between COVID-19 cases and healthy control 235 DEGs between COVID-19 cases (n = 6) and controls (n = 8) of endometrial samples were obtained from RNA-Seq results as Professor Lucía de Miguel-Gomez [ 9 ] described. 2580 DEGs between COVID-19 cases and controls of nasopharyngeal samples were obtained from 231 female transcriptomes (201 COVID-19 cases and 30 controls) as Professor I. Henarejos-Castillo [ 10 ] described. Without distinguishing expression trends, 50 overlapping molecules were identified; however, only 10 showed consistent trends, while 40 showed opposite trends. Eight up-regulated and two down-regulated overlapping molecules were found in both (Supplementary Fig. 1A), and we combined the roles of COVID-19 and fertility-related genes (Table 1 ). The common DEGs with combined scores larger than 0.7 were compiled into a PPI network using GO and KEGG Pathway enrichment analysis (Supplementary Fig. 1B, C). In Fig. 1 , the complete methodological workflow is shown. Table 1 Co-expression genes in the endometria and nasopharyngeal swabs in COVID-19 female patients. Up-regulation in COVID-19 female patients Gene Molecular function Fertility association ATP6V1C1 Regulate V-ATPase activity, energy metabolism and cellar differentiation Association with the expression of androgen receptor CCDC90B Transport calcium ions into the mitochondria The potential common biomarker candidates in the pathospermic conditions of both teratozoospermia and azoospermia EPSTI1 Participate in tumor cell metastasis, epithelial-mesenchymal transition, chronic inflammation, tissue reconstruction, and the regulation of cell apoptosis. Involved in embryonic development and pregnancy establishment FBXO15 Substrate-recognition component of the SCF-type E3 ubiquitin ligase complex. Especially high expressed in fallopian tube HERC5 A critical modulators of the antiviral immune response Association with the abnormal regulation of decidulization IFI44 A crucial regulator of the immune system and an immune evasion biomarker for SARS-CoV-2 Association with pregnancy establishment IFI6 Regulate apoptosis and immune responses Endometrial IFI6 declines during peri-implantation period in pigs SUGT1 Play a role in ubiquitination and subsequent proteasomal degradation of target proteins Endometrial SUGT1 may serve as potential therapeutic targets for patients with endometriosis-induced recurrent pregnancy loss Down-regulation in COVID-19 female patients LRP1 A transmembrane receptor that belongs to the LDLR family, which mediates and regulates the endocytosis Endometrial LRP1 elevates during peri-implantation period in pigs HSPG2 A large multi-domain extracellular matrix proteoglycan, which contributes to the invasion, metastasis and angiogenesis of solid tumor Play a role in the differentiation of granulosa cells. EPSTI1, SUGT1, HERC5, HSPG2 and LRP1 expression in mid-secretory endometrium of women with COVID-19 9 mid-secretory stage endometrial samples from women without COVID-19 infection and a total of 8 mid-secretory stage endometrial samples from patients with COVID-19 infection within 30 days were used to confirm the expression and distribution of potential genes throughout the peri-implantation window. IHC assay was applied to detect EPSTI1, SUGT1, HERC5, HSPG2 and LRP1 expression in mid-secretory endometrium between COVID-19 group and normal control. Separate analyses were conducted for stromal (S) cells, glandular epithelial (GE) cells, and luminal epithelial (LE) cells. As shown in Fig. 2 and Fig. 3 , EPSTI1, SUGT1 and HERC5 were up-regulated in the COVID-19 group, while HSPG2 and LRP1 show no difference in expression between the two groups. EPSTI1 and SUGT1 participate in the regulation of endometrial receptivity. Two prominent indicators of decidualization are PRL and IGFBP-1. When 8Br-cAMP and MPA were stimulated in T-hESCs, plasmid-mediated overexpression of EPSTI1 and SUGT1 led to a prominent reduction in PRL secretion and IGFBP-1 mRNA level for 3 and 6 days (Fig. 4 A, B). HECR5 had no effect on PRL or IGFBP-1 expression during in-vitro decidualization in T-hESCs (Fig. 4 A, B). We also created an in-vitro implantation model that allows us to efficiently investigate the role of embryo adhesion to epithelial cells by co-cultivating Ishikawa cells and human BeWo cells that were incubated in the blastosphere. Overexpressing SUGT1 inhibited the adhesion ability of BeWo spheroids induced by E 2 and MPA, while EPSTI1 and HERC5 had no significant effect on adhesion rate (Fig. 4 C). Based on the aforementioned results, we deduced that SUGT1 dysregulated endometrial stromal cell decidualization as well as endometrial epithelial cell receptivity, and that EPSTI1 hampered endometrial decidualization. Furthermore, we examined the mRNA and protein expression of EPSTI1 and SUGT1 in Ishikawa cells treated with E 2 and MPA, as well as in T-hESCs treated with 8Br-cAMP in conjunction with MPA (Supplementary Fig. 2). We observed an interesting phenomenon that EPSTI1 was up-regulated in Ishikawa cells, yet was down-regulated in T-hESCs in physiological status induced by sexual hormones. The opposite phenotype of EPSTI1 in epithelial cells and stromal cells may be related to the epithelial-to-mesenchymal transition (EMT) characteristics of EPSTI1 itself. As for SUGT1, estrogen and progesterone did not affect its expression significantly in in Ishikawa cells nor T-hESCs. EPSTI1 and SUGT1 expression stimulated by IFN-γ, LPS and IL-1β We continued to investigate the possible mechanism how covid-19 infection lead to the elevated expression of EPSTI1 and SUGT1 in endometrial cells. The cytokine storm is a known factor causing major clinical symptoms in COVID-19 patients [ 12 ]. Consistent with previous studies, we found a significant increasing trend of serum INF-γ, TNF-α, LPS, IL-6 and IL-1β in female patients diagnosed of Covid-19 within 0 to 7 days with mild to moderate symptoms (Fig. 5 A). Previous studies have demonstrated that inflammatory response persist after resolution of COVID-19. This phenomenon has been evaluated in terms of increased levels of several cytokines, including IL-1β [ 13 ]. We also observed that IL-1β demonstrated a sustained elevation in women 8–30 days after COVID-19 infection (Fig. 5 A). As shown in Fig. 5 B, the protein expression of EPSTI1 and SUGT1 were examined in Ishikawa cells treated with recombinant cytokine of IL-1β. Addition of IL-1β significantly upregulated the expression of EPSTI1 and SUGT1. Meanwhile, we detected the EPSTI1 and SUGT1 levels stimulated by INF-γ as well as LPS. EPSTI1 was elevated with the administration of IFN-γ and LPS. SUGT1 was up-regulated with the stimulation of LPS. The above results suggest that the booming cytokines caused by Covid-19 may be responsible for promoting the expression of EPSTI and SUGT1 in the endometrium. Discussion The World Health Organization (WHO) announced on May 5, 2023, that COVID-19 was no more a global public health emergency. This announcement was a major turning point in the global effort to combat the pandemic. In the 'post-epidemic era', we should pay long-term attention to the impact of the new coronavirus on human long-term quality of life. Since 2020, there has been a brief decline in population worldwide, a phenomenon that was alleviated in 2023. In addition to the economic and social factors brought by the epidemic, we also noted the potential impact of the epidemic on reproductive health. ASRM and ESHRE recommended that fertility services be stopped during the early stages of the pandemic, with the exception of urgent cases [ 14 , 15 ]. Further research has revealed a correlation between SARS-CoV-2 infection and some aspects of human fertility. Molecular studies indicated that the ovarian cortex, medulla, oocytes, endometrium, and the membranes of trophectoderm, hypoblast, and epiblast cells in blastocysts have all been demonstrated to co-express ACE2 and TMPRSS2 in females [ 5 , 6 , 16 ]. SARS-CoV-2 infection has been associated with ovarian dysfunction, disruptions in the follicular microenvironment, changes in menstrual volume and cycle length, reproductive outcomes, and potential interference with embryo implantation and pregnancy [ 17 – 19 ]. Clinical assessments of the outcomes of assisted reproductive technology revealed that women who had frozen embryo transfer (ET) within 60 days of infection had reduced pregnancy rates than those who delayed ET [ 20 ]. However, other studies have shown that a history of mild or asymptomatic SARS-CoV-2 infection in females does not appear to have a negative impact on laboratory and clinical outcomes in fresh and frozen ET cycles [ 21 , 22 ]. This suggests that current research on COVID-19's effects on reproduction might only represent the tip of the iceberg, necessitating extensive basic research and clinical observation. To find out if SARS-CoV-2 affects the endometrium during implantation or the embryo in its early stages of development, perhaps influencing implantation or raising the incidence of miscarriages, more research is required. Using total RNA next-generation sequencing, Professor Lucía de Miguel-Gomez explains for the first time how systemic COVID-19 modifies global gene expression in the cyanobacteria of symptomatic women [ 9 ]. The purpose of this endeavor is to look at how COVID-19 affects essential elements of a healthy menstrual cycle and associated reproductive consequences. Professor I. Henarejos-Castillo's MSE gene co-expression network model provides insights into how transcriptome alterations caused by COVID-19, inferred from nasopharyngeal tissue, may impair important endometrial processes [ 10 ]. This model is instrumental in shedding light on the influence of COVID-19 on endometrial processes. These two articles reveal potential mechanisms by which COVID-19 may affect endometrial functions, however the patients’ age, menstrual cycle parameters, fertility status and biopsy collection timing of the selected endometrial specimens and throat swabs in both studies varied greatly. Using the endometrium [ 9 ] and throat swabs [ 10 ] of COVID-19-positive women as our reference, we overlapped the differentially expressed genes to investigate the relatively stable expressed molecule and lessen the discrepancy from the unparallel background of patients. Without distinguishing expression trends, 50 overlapping molecules were identified; however, only 10 showed consistent trends, while 40 showed opposite trends. This strongly indicates the tissue specificity of COVID-19 infection and the impact of potential differences such as age and menstrual timing on the results. It also indirectly highlights the limitations of relying solely on bioinformatics analysis or using throat swab differential gene databases for such analyses. Upon analyzing these 10 molecules, we identified EPSTI1 and SUGT1 as two prioritized molecules. In mixed cultures of human breast cancer cells and fibroblasts, the interferon-responsive gene EPSTI1 was first discovered [ 23 ]. It has been implicated in various biological processes, including tumor cell metastasis, epithelial-mesenchymal transition, chronic inflammation, tissue reconstruction, embryonic development, and the regulation of cell apoptosis [ 24 , 25 ]. Capdevila-Busquets et al . reported that EPSTI1 can modulate the extrinsic apoptotic pathway in both estrogen receptor-positive and triple-negative breast cancer cell lines [ 24 ]. Methylation of EPSTI1 may play a role in SARS-CoV-2 infection processes and influence inflammatory and immune responses by regulating EPSTI1 expression [ 26 ]. Previous studies have reported that the expression of EPSTI1 was significantly increased in BAL cells, PBMCs, leukocytes, and nasopharyngeal tissue of COVID-19 patients [ 27 , 28 ]. In our study, women with COVID-19 infection had considerably higher levels of EPSTI1 in their endometrial cells. The cytokines, such as IFN-γ, IL-1β, IL-6, IL-12, IL-18, IL-33 and TGF-β, are associated with different clinical features of COVID-19. A long-lasting cytokine signature consisting of elevated levels of interleukin IL-1β could potentially underlie many of the clinical symptoms of post Covid-19 infection [ 13 ]. EPSTI1 is an interferon (IFN)-responsive gene and is highly expressed in exposed to virus infection as well as lipopolysaccharide-induced inflammatory [ 29 ]. Here, we further defined that IL-1β, LPS and INF-γ could upregulate EPSTI expression in Ishikawa cells and T-hESCs cells, suggesting that elevated EPSTI1 expression in the endometrium of patients after covid-19 infection may be associated with an inflammatory storm. There have been few studies on the function of EPSTI1 in the endometrium. EPSTI1 levels is lower in endometrial tissues of pregnant mares compared to tissues from non-pregnant mares [ 30 ], suggesting a potential link to pregnancy. In our in vitro decidualization experiment, EPSTI1 inhibited the expression of dPRL, IGFBP-1 in T-hESCs. We also observed that EPSTI1, known as a molecule promotes epithelial-mesenchyma transition (EMT), was down-regulated in T-hESCs during decidualization in vitro . The ability of the mature endometrium to undergo decidualization and cyclic regeneration is essential for successful human reproduction. During the implantation phase, ovarian hormones regulate the mesenchyma-epithelial transition (MET) process, which is consistent with changes in stromal cellular morphology that arise with decidualization [ 31 ]. Therefore, it is reasonable to presume that EPSTI1 restrain decidualization by disturbing the MET process of endometrial stromal cells during the peri-implantation window. By stimulating leucine-rich repeat-containing and nucleotide-binding domain proteins, SUGT1 participates in the innate immune response. When heat shock protein 90 was discovered, SUGT1 was shown to be a co-chaperone. According to reports [ 32 – 34 ], SUGT1 is connected to both HIV-1 infection and colorectal cancer. According to Ata et al ., individuals experiencing recurrent pregnancy loss due to endometriosis may benefit from targeting endometrial SUGT1 as a possible therapeutic target [ 35 ]. Within one month of the COVID-19 infection, we found in this study that there was a elevation of SUGT1 in the endometrial cells of the patients. SUGT was also induced by IL-1β and LPS in Ishikawa cells and T-hESCs. In vitro , SUGT1 suppressed Bewo spheroids adhesion and decidualization while remaining unresponsive to the stimuli of progesterone or estrogen. We speculated that SUGT1 may participate in the dysregulation of endometrial receptivity though inflammatory pathway. More experiments will conducted to explore the concrete mechanism. Although the peak of the COVID-19 epidemic has passed, research on COVID-19 should not be confined to this period. Our research demonstrates that following COVID-19 infection, the endometrium of the peri-implantation window experiences protein-level alterations that impact endometrial receptivity. It is noteworthy that we collected our endometrial samples between 10 and 30 days after the COVID-19 diagnosis, which better captures the relatively long-term consequences of the infection on the endometrium. The immune response is a critical factor in the evolution of COVID-19. There is a growing understanding that the immune system's reaction to COVID-19, rather than the virus itself, is frequently the cause of the morbidity linked to it. With a growing population of recovering patients, it became clear that in 32–87% of patients (including those with mild acute disease), health impairments persist beyond the acute phase of infection [ 12 ]. The start and maintenance in a healthy pregnancy depend on the endometrium's dynamic balance between pro-and anti-inflammatory mediators. SARS-CoV-2 may disrupt the immunological milieu in the uterus, which might impact embryo implantation. The results of this investigation may lead to a reexamination of the connection between COVID-19 and the endometrium and generate theories regarding the emergence of protracted COVID and other post-COVID-19 related adverse effects. The study faces limitations. The clinical sample size is limited. However, we verify potential molecular expression based on analysis from database of high quality and large scale. Our study led to hypotheses concerning the infection of COVID-19 influence the endometrium by regulating key molecules, which could potentially affect endometrial function and implantation. It is now imperative to further investigate this relationship to deepen our understanding. Conclusion Over the past three years, the COVID-19 pandemic has significantly disrupted the provision of assisted reproductive technology treatments. Although we now know more about how SARS-CoV-2 spreads, there are still many unanswered issues about how it sustainably affects reproductive health. In this work, we demonstrate that there were variations in the expression of endometrial proteins during the peri-implantation phase following COVID-19 infection. Notably, in vitro decidualization and embryo adhesion function were restrained by EPSTI1 and SUGT1, which were up-regulated in implantation window endometria of women infected with COVID-19. The long-term prevention and management of COVID-19 and its aftermath may be greatly aided by investigating the relationship between SARS-CoV-2 and human reproduction. Declarations Authors’ roles Xi Cheng designed the study and contributed to writing the article. Xiting Cai, Hui Wang and Biying Li performed the molecular experiments. Lu Zheng and Jinzhao Ma contributed to data analysis. Cheng Zhou, Xuan Huang, Kadiliya Jueraitetibaike, Qin Sun, Meiling Li and Xu Tang contributed to the the collection of clinical samples. Yuming Feng and Hong Zhang contributed to the article revision and editing. Li Chen and Bing Yao contributed to the study design, data interpretation, as well as article revision. All authors have agreed to be listed and have approved the final article. Funding This work is supported by National Natural Science Foundation of China (Grant: 82101757), the Key Research and Development Program of Jiangsu Province (Grant: BE2022712) and Annual Foundation of Nanjing Jinling Hospital (YYQN2021082, 22LCYY-QH5, 22LCYY-QH12). Each of the funding contributed to expenses related to specific procedures carried out in the project. Data Availability All data that support the findings of this study are available from the corresponding author upon reasonable request. Conflicts of interest The authors have declared no conflicts of interest. Ethics approval and consent to participate The study protocols (2023DZKY-009-01) were authorized by Nanjing Jinling Hospital's Clinical Ethics Review Committee. The Clinical Trials Registry (ID: NCT05685966) has the trial prospectively registered. Every participant endorsed the informed consent form. Acknowledgments We thank the other members of Dr. Yao’s laboratory for their discussion and help. References Aliyari R, Mahdavi S, Enayatrad M, Sahab-Negah S, Nili S, Fereidooni M, Mangolian Shahrbabaki P, Ansari-Moghaddam A, Heidarzadeh A, Shahraki-Sanavi F, et al. Study protocol: cohort event monitoring for safety signal detection after vaccination with COVID-19 vaccines in Iran. BMC Public Health. 2022;22:1153. Elrobaa IH, New KJ. COVID-19: Pulmonary and Extra Pulmonary Manifestations. Front Public Health. 2021;9:711616. El Jamal SM, Pujadas E, Ramos I, Bryce C, Grimes ZM, Amanat F, Tsankova NM, Mussa Z, Olson S, Salem F, et al. Tissue-based SARS-CoV-2 detection in fatal COVID-19 infections: Sustained direct viral-induced damage is not necessary to drive disease progression. Hum Pathol. 2021;114:110–19. Jamieson DJ, Rasmussen SA. An update on COVID-19 and pregnancy. Am J Obstet Gynecol. 2022;226:177–86. Vilella F, Wang W, Moreno I, Roson B, Quake SR, Simon C. Single-cell RNA sequencing of SARS-CoV-2 cell entry factors in the preconceptional human endometrium. Hum Reprod. 2021;36:2709–19. Henarejos-Castillo I, Sebastian-Leon P, Devesa-Peiro A, Pellicer A, Diaz-Gimeno P. SARS-CoV-2 infection risk assessment in the endometrium: viral infection-related gene expression across the menstrual cycle. Fertil Steril. 2020;114:223–32. Haouzi D, Entezami F, Tuaillon E, Gala A, Ferrières-Hoa A, Brouillet S, Thierry AR, Hamamah S. SARS-CoV-2 and Implantation Window: Gene Expression Mapping of Human Endometrium and Preimplantation Embryo. Life (Basel) 2021; 11. Kabani Z, Ramos-Nino ME, Ramdass PVAK. Endometriosis and COVID-19: A Systematic Review and Meta-Analysis. Int J Mol Sci 2022; 23. de Miguel-Gómez L, Sebastián-León P, Romeu M, Pellicer N, Faus A, Pellicer A, Díaz-Gimeno P, Cervelló I. Endometrial gene expression differences in women with coronavirus disease 2019. Fertil Steril. 2022;118:1159–69. Henarejos-Castillo I, Devesa-Peiro A, de Miguel-Gomez L, Sebastian-Leon P, Romeu M, Aleman A, Molina-Gil C, Pellicer A, Cervello I, Diaz-Gimeno P. Predicted COVID-19 molecular effects on endometrium reveal key dysregulated genes and functions. Mol Hum Reprod 2022; 28. Cheng X, Zhang Y, Ma J, Wang S, Ma R, Ge X, Zhao W, Xue T, Chen L, Yao B. NLRP3 promotes endometrial receptivity by inducing epithelial-mesenchymal transition of the endometrial epithelium. Mol Hum Reprod 2021; 27. Queiroz MAF, Neves PFMd, Lima SS, Lopes JC, Torres MKS, Vallinoto IMVC, Bichara CDA, Dos Santos EF, de Brito MTFM, da Silva ALS, et al. Cytokine Profiles Associated With Acute COVID-19 and Long COVID-19 Syndrome. Front Cell Infect Microbiol. 2022;12:922422. Schultheiß C, Willscher E, Paschold L, Gottschick C, Klee B, Henkes S-S, Bosurgi L, Dutzmann J, Sedding D, Frese T, et al. The IL-1β, IL-6, and TNF cytokine triad is associated with post-acute sequelae of COVID-19. Cell Rep Med. 2022;3:100663. Gianaroli L, Ata B, Lundin K, Rautakallio-Hokkanen S, Tapanainen JS, Vermeulen N, Veiga A, Mocanu E. The calm after the storm: re-starting ART treatments safely in the wake of the COVID-19 pandemic. Hum Reprod. 2021;36:275–82. Veiga A, Gianaroli L, Ory S, Horton M, Feinberg E, Penzias A. Assisted reproduction and COVID-19: a joint statement of ASRM, ESHRE, and IFFS. Glob Reprod Health 2020; 5. Rajput SK, Logsdon DM, Kile B, Engelhorn HJ, Goheen B, Khan S, Swain J, McCormick S, Schoolcraft WB, Yuan Y, Krisher RL. Human eggs, zygotes, and embryos express the receptor angiotensin 1-converting enzyme 2 and transmembrane serine protease 2 protein necessary for severe acute respiratory syndrome coronavirus 2 infection. F S Sci. 2021;2:33–42. Weinerman R. COVID-19 and the endometrium: inflammation as understanding. Fertil Steril. 2022;118:1170–71. Lebar V, Laganà AS, Chiantera V, Kunič T, Lukanović D. The Effect of COVID-19 on the Menstrual Cycle: A Systematic Review. J Clin Med 2022; 11. Stanley KE, Thomas E, Leaver M, Wells D. Coronavirus disease-19 and fertility: viral host entry protein expression in male and female reproductive tissues. Fertil Steril. 2020;114:33–43. Youngster M, Avraham S, Yaakov O, Landau Rabbi M, Gat I, Yerushalmi G, Baum M, Maman E, Hourvitz A, Kedem A. The impact of past COVID-19 infection on pregnancy rates in frozen embryo transfer cycles. J Assist Reprod Genet. 2022;39:1565–70. Huang J, Liu Y, Xia L, Zhao Y, Tian L, Xu D, Su Q, Hu Y, Xie Q, Chen J, et al. Effect of prior female SARS-CoV-2 infection on IVF outcomes: a prospective cohort study. Front Endocrinol (Lausanne). 2023;14:1239903. Youngster M, Avraham S, Yaakov O, Landau Rabbi M, Gat I, Yerushalmi G, Sverdlove R, Baum M, Maman E, Hourvitz A, Kedem A. IVF under COVID-19: treatment outcomes of fresh ART cycles. Hum Reprod. 2022;37:947–53. Nielsen HL, Rønnov-Jessen L, Villadsen R, Petersen OW. Identification of EPSTI1, a novel gene induced by epithelial-stromal interaction in human breast cancer. Genomics. 2002;79:703–10. Capdevila-Busquets E, Badiola N, Arroyo R, Alcalde V, Soler-López M, Aloy P. Breast cancer genes PSMC3IP and EPSTI1 play a role in apoptosis regulation. PLoS ONE. 2015;10:e0115352. de Neergaard M, Kim J, Villadsen R, Fridriksdottir AJ, Rank F, Timmermans-Wielenga V, Langerød A, Børresen-Dale A-L, Petersen OW, Rønnov-Jessen L. Epithelial-stromal interaction 1 (EPSTI1) substitutes for peritumoral fibroblasts in the tumor microenvironment. Am J Pathol. 2010;176:1229–40. Li Z, Mei Z, Ding S, Chen L, Li H, Feng K, Huang T, Cai Y-D. Identifying Methylation Signatures and Rules for COVID-19 With Machine Learning Methods. Front Mol Biosci. 2022;9:908080. Dong Z, Yan Q, Cao W, Liu Z, Wang X. Identification of key molecules in COVID-19 patients significantly correlated with clinical outcomes by analyzing transcriptomic data. Front Immunol. 2022;13:930866. Shaath H, Vishnubalaji R, Elkord E, Alajez NM. Single-Cell Transcriptome Analysis Highlights a Role for Neutrophils and Inflammatory Macrophages in the Pathogenesis of Severe COVID-19. Cells 2020; 9. Kari A, Aili MZ, Adili Z, Hairula A, Abuduhaer N. A: Knockdown of EPSTI1 alleviates lipopolysaccharide-induced inflammatory injury through regulation of NF-κB signaling in a cellular pneumonia model. Allergol Immunopathol (Madr) 2022; 50:106 – 12. Bauersachs S, Mitko K, Ulbrich SE, Blum H, Wolf E. Transcriptome studies of bovine endometrium reveal molecular profiles characteristic for specific stages of estrous cycle and early pregnancy. Exp Clin Endocrinol Diabetes. 2008;116:371–84. Owusu-Akyaw A, Krishnamoorthy K, Goldsmith LT, Morelli SS. The role of mesenchymal-epithelial transition in endometrial function. Hum Reprod Update. 2019;25:114–33. Allouch A, Di Primio C, Paoletti A, Lê-Bury G, Subra F, Quercioli V, Nardacci R, David A, Saïdi H, Cereseto A, et al. SUGT1 controls susceptibility to HIV-1 infection by stabilizing microtubule plus-ends. Cell Death Differ. 2020;27:3243–57. Iwatsuki M, Mimori K, Sato T, Toh H, Yokobori T, Tanaka F, Ishikawa K, Baba H, Mori M. Overexpression of SUGT1 in human colorectal cancer and its clinicopathological significance. Int J Oncol. 2010;36:569–75. Yan R, Liang X, Hu J. miR-141-3p alleviates ulcerative colitis by targeting SUGT1 to inhibit colonic epithelial cell pyroptosis. Autoimmunity. 2023;56:2220988. Ata B, Vermeulen N, Mocanu E, Gianaroli L, Lundin K, Rautakallio-Hokkanen S, Tapanainen JS, Veiga A. SARS-CoV-2, fertility and assisted reproduction. Hum Reprod Update. 2023;29:177–96. Supplementary Files SFig1.tif Supplemental Figure 1. Analysis of DEGs of endometrium and female nasopharynx with Covid-19. Venn diagram (A) was conducted to obtain the intersection of the DEGs between endometrium and female nasopharynx. PPI network diagram (B), and the enrichment analysis results of GO and KEGG Pathway (C) of the 10 overlapping genes. SFig2.tif Supplemental Figure 2. The expression of EPSTI1 and SUGT1 with the stimulation of estrogen and progesterone. Ishikawa cells were treated with 10 nM E 2 and 1 µM MPA for 24 h or 48 h. T-hESCs were treated with 0.5 mM 8Br-cAMP and 1 µM MPA for 24 h or 72 h. qPCR and western blotting was applied to detect the mRNA (A) and protein (B) expression EPSTI1 and SUGT1. *P <0.05, ***P <0.001. renamed29759.doc STable 1 Table2new.docx STable 2 STable3.doc STable 3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4545088","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":320377250,"identity":"83ebd893-6af2-4285-8dfc-840df77ad9a2","order_by":0,"name":"Xi Cheng","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Cheng","suffix":""},{"id":320377251,"identity":"7003d2de-3f4d-4005-b81e-bee46113fe6a","order_by":1,"name":"Xiting Cai","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Xiting","middleName":"","lastName":"Cai","suffix":""},{"id":320377252,"identity":"66d902ab-d030-439a-b131-8a00112911c5","order_by":2,"name":"Hui Wang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wang","suffix":""},{"id":320377253,"identity":"e5a6bb52-01d8-4cd5-b138-de3e4afa73bb","order_by":3,"name":"Biying Li","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Biying","middleName":"","lastName":"Li","suffix":""},{"id":320377254,"identity":"8cce725a-7e79-47a2-ab24-3747416ba1dc","order_by":4,"name":"Lu Zheng","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Lu","middleName":"","lastName":"Zheng","suffix":""},{"id":320377255,"identity":"d9446234-4875-4470-b07c-3619ca15236f","order_by":5,"name":"Jinzhao Ma","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Jinzhao","middleName":"","lastName":"Ma","suffix":""},{"id":320377256,"identity":"b346abc2-f5c3-452d-b515-b371e1eb5957","order_by":6,"name":"Cheng Zhou","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"","lastName":"Zhou","suffix":""},{"id":320377257,"identity":"3ab745a8-0a0b-4dfb-8726-8069ddd54d63","order_by":7,"name":"Xuan Huang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Huang","suffix":""},{"id":320377258,"identity":"3005f321-5b04-4191-9567-2bab873c2e19","order_by":8,"name":"Kadiliya Jueraitetibaike","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Kadiliya","middleName":"","lastName":"Jueraitetibaike","suffix":""},{"id":320377259,"identity":"7d427592-e7a3-458c-b828-25d7e0ce3d22","order_by":9,"name":"Qin Sun","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Sun","suffix":""},{"id":320377260,"identity":"0131d1fe-f24e-4bf0-bce6-90dab9a76e78","order_by":10,"name":"Meiling Li","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Meiling","middleName":"","lastName":"Li","suffix":""},{"id":320377261,"identity":"06f4c7c0-4ed7-4819-a39e-3ae72353c87c","order_by":11,"name":"Yuming Feng","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Yuming","middleName":"","lastName":"Feng","suffix":""},{"id":320377262,"identity":"9a19f3a3-da61-4abe-8f9a-ee6688aa3efb","order_by":12,"name":"Xu Tang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Xu","middleName":"","lastName":"Tang","suffix":""},{"id":320377263,"identity":"b8c3eb73-8aa1-4056-bfc0-cdda1ea04e8d","order_by":13,"name":"Hong Zhang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zhang","suffix":""},{"id":320377264,"identity":"c96b5794-2651-488e-899e-c888354cb078","order_by":14,"name":"Li Chen","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Chen","suffix":""},{"id":320377265,"identity":"e891f445-55e5-4be4-b72b-fbe82167de1e","order_by":15,"name":"Bing Yao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYHACNoYEBgY5BoYDYDbxWoxJ1AIEiQ0INgFgcPzwsQcPd9Smb2c8Y8DwoewwA//sBgJazqSlGySeOZ67s+GMAeOMc4cZJO4cIKDlQI6ZRGLbsdwNB84YMPO2HWYwkEggoOX8G7CWdAOQlr9EabkBtqUmAayFkRgtkjeeAf3SdsBww4FjBQd7zqXzSNwgoIXvfPKxhz/b6uQNbhze+OBHmbUc/wwCWhQOgKnDDAwSB8CRyYNfPRDIN4CpOgYG/gaCikfBKBgFo2CEAgDhTUyT67RVnAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0002-3420-2220","institution":"Nanjing University","correspondingAuthor":true,"prefix":"","firstName":"Bing","middleName":"","lastName":"Yao","suffix":""}],"badges":[],"createdAt":"2024-06-07 09:21:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4545088/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4545088/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":60854257,"identity":"02183277-07a8-4a27-bf50-84c960980bdc","added_by":"auto","created_at":"2024-07-22 21:48:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2076902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design to explore COVID-19 effects in the endometrium.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA RNA-Seq results of endometrial samples and a transcriptomic dataset of female nasopharynx biopsies from patients with COVID-19 \u003cem\u003evs.\u003c/em\u003e Women without COVID-19 infection were collected. DEGs from endometrial samples overlapping DEGs from female nasopharynx biopsies. Mid-secretory phase endometrial samples from women infected with COVID-19 within 30 days and women without COVID-19 infection were applied to understand the potential endometrial effects of COVID-19 during peri-implantation window. Finally, endometrium-related molecular tests was employed to verify the functions of the potential molecules.\u003c/p\u003e","description":"","filename":"Onlinerenamed576ea.png","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/cd2bae1ba45b2878a1cdc0bf.png"},{"id":60854263,"identity":"fefd9ad8-905b-40de-93cf-d20033479f64","added_by":"auto","created_at":"2024-07-22 21:48:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17064469,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of EPSTI1, SUGT1, HERC5, HSPG2 and LRP1 in mid-secretory phase endometrium of patients infected with COVID-19 and women without COVID-19.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Immunohistochemistry assays were used to detect the expression of EPSTI1, SUGT1, HERC5, HSPG2 and LRP1 in the endometrium of women infected by Covid-19 within 30 days and control groups. Stromal cells are marked as S, glandular epithelium as GE, and luminal epithelium as LE. Scale bar = 100 µm. (B) Histogram of the H-score for EPSTI1, SUGT1, HERC5, HSPG2 and LRP1 in glandular epithelial cells, luminal epithelial cells and stromal cells in the endometrium from Covid-19 group (n=8) control groups (n=9).\u003cem\u003e *P\u003c/em\u003e\u0026lt;0.05, \u003cem\u003e***P\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/d86a8fa64e738736dec55684.png"},{"id":60854264,"identity":"2865a7fe-095a-4a00-9462-21347d3b9680","added_by":"auto","created_at":"2024-07-22 21:48:42","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":38733510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of EPSTI1, SUGT1 and HERC5 in mid-secretory phase endometrium of patients infected with COVID-19 and women without COVID-19.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemistry assays were used to detect the level and distribution of EPSTI (A) and SUGT1 (B) in the endometrium from patients infected by Covid-19 within 30 days (n=2) and women without Covid-19 infection (n=2).\u003c/p\u003e","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/fd1e8ed38c908cc330f2e758.png"},{"id":60854923,"identity":"98e6c18f-ce29-4f9d-8fce-86b1be94745e","added_by":"auto","created_at":"2024-07-22 22:04:42","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":358207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEPSTI1 and SUGT1 participate in the regulation of endometrial receptivity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ehESCs were transfected with plasmids of EPSTI1, SUGT1, HERC5 or vector control. After 24 h, the cells were treated with 0.5 mM 8Br-cAMP and 1µM MPA for an additional 3 or 6 days. Prolactin secretion (A) and IGFBP-1 mRNA level (B) were measured respectively by an enzyme-linked fluorescent assay (ELFA) and qPCR. \u003cem\u003e*P\u003c/em\u003e\u0026lt;0.05, \u003cem\u003e**P\u003c/em\u003e\u0026lt;0.01. (C) Ishikawa cells were transfected with plasmids of EPSTI1, SUGT1, HERC5 or vector control. After 24 h, the cells were treated with 10 nM E\u003csub\u003e2\u003c/sub\u003e and 1 µM MPA for an additional 48 h. \u003cem\u003eIn vitro\u003c/em\u003e experiments of BeWo spheroids attached to Ishikawa cell monolayers. ANOVA test was used to analyse the percentage of the attached spheroids with each treatment. *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/ab1719bcd7eab09de93d3380.png"},{"id":60854261,"identity":"219b4943-3342-43bf-9f38-8c346ffa1e4f","added_by":"auto","created_at":"2024-07-22 21:48:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1438166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEPSTI1 and SUGT1 expression stimulated by IFN-γ, LPS and IL-1β\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Elisa assays were used to detect the expression of serum IL-6, IL-1β, IL-1α, LPS, IFN-γ and TNF-α of healthy women (n=10), female patients diagnosed with Covid-19 within 7 days (n=12) and female patients diagnosed with Covid-19 within 8-30 days (n=13). \u003cem\u003e*P\u003c/em\u003e\u0026lt;0.05, \u003cem\u003e**P\u003c/em\u003e\u0026lt;0.01, \u003cem\u003e***P\u003c/em\u003e\u0026lt;0.001. (B) Ishikawa cells and T-hESCs were treated with recombinant cytokine of IFN-γ, LPS or IL-1β. Western blotting was applied to detect the protein expression EPSTI1 and SUGT1. \u003cem\u003e*P\u003c/em\u003e\u0026lt;0.05, \u003cem\u003e**P\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/27ef1bdd182e8938059c84a2.png"},{"id":65181580,"identity":"841d2a87-eed3-4733-b3b7-d88914299c89","added_by":"auto","created_at":"2024-09-24 13:03:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8455075,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/53485960-acf6-4a32-a27b-dfef12fb8f3f.pdf"},{"id":60854766,"identity":"b2d5aa18-87d4-4ac4-8c65-1fde73577726","added_by":"auto","created_at":"2024-07-22 21:56:43","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":54156800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1. Analysis of DEGs of endometrium and female nasopharynx with Covid-19.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVenn diagram (A) was conducted to obtain the intersection of the DEGs between endometrium and female nasopharynx. PPI network diagram (B), and the enrichment analysis results of GO and KEGG Pathway (C) of the 10 overlapping genes.\u003c/p\u003e","description":"","filename":"SFig1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/f96315fae11bf85d66359287.tif"},{"id":60854265,"identity":"0b7d7965-30bc-4a52-a390-73eb6e20dfec","added_by":"auto","created_at":"2024-07-22 21:48:43","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":20903848,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2. The expression of EPSTI1 and SUGT1 with the stimulation of estrogen and progesterone.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIshikawa cells were treated with 10 nM E\u003csub\u003e2\u003c/sub\u003e and 1 µM MPA for 24 h or 48 h. T-hESCs were treated with 0.5 mM 8Br-cAMP and 1 µM MPA for 24 h or 72 h. qPCR and western blotting was applied to detect the mRNA (A)\u0026nbsp; and protein (B) expression EPSTI1 and SUGT1. \u003cem\u003e*P\u003c/em\u003e\u0026lt;0.05, \u003cem\u003e***P\u003c/em\u003e\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"SFig2.tif","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/865ae06648a3e2ddede34a4d.tif"},{"id":60854764,"identity":"894b2ebf-7989-42d5-a649-06c38e45abff","added_by":"auto","created_at":"2024-07-22 21:56:42","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":43008,"visible":true,"origin":"","legend":"\u003cp\u003eSTable 1\u003c/p\u003e","description":"","filename":"renamed29759.doc","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/f6e76bb4e0fdeeb8e847adb7.doc"},{"id":60854262,"identity":"76f6cfa2-17e8-4e61-8e15-bf036e6fe916","added_by":"auto","created_at":"2024-07-22 21:48:42","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":13818,"visible":true,"origin":"","legend":"\u003cp\u003eSTable 2\u003c/p\u003e","description":"","filename":"Table2new.docx","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/1ca65ed1b0ddc9938e893c73.docx"},{"id":60854260,"identity":"d9c2fe4b-e634-42c5-8cec-c2ca5700657a","added_by":"auto","created_at":"2024-07-22 21:48:42","extension":"doc","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":16896,"visible":true,"origin":"","legend":"\u003cp\u003eSTable 3\u003c/p\u003e","description":"","filename":"STable3.doc","url":"https://assets-eu.researchsquare.com/files/rs-4545088/v1/2ae123eb370d045d13ed7d08.doc"}],"financialInterests":"","formattedTitle":"COVID-19 and embryo implantation: EPSTI1 and SUGT1 participate in the dysregulation of SARS-CoV-2 on endometrial receptivity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is identified as the causative agent of the COVID-19 pandemic [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Commonly, COVID-19 manifests as pulmonary complications, typically associated with more severe disease and poorer outcomes. However, a range of extrapulmonary symptoms have also been observed, often in cases of mild to moderate severity or even during severe disease stages. Dizziness, skin changes, arrhythmias, liver damage, hematuria, and anosmia or ageusia are some of these symptoms [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a result, COVID-19 is acknowledged as a multisystemic and heterogeneous illness. Certain COVID-19-related consequences could not be directly caused by the virus itself, but rather are the consequence of a worldwide immunological dysregulation that the virus started [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This raises questions regarding how SARS-CoV-2 could affect organs that have high expression of the COVID-19 gene, as these organs might be more vulnerable to harmful effects after infection.\u003c/p\u003e \u003cp\u003eThe impact of COVID-19 on pregnancy outcomes has been substantiated by a considerable amount of researches. Notably, SARS-CoV-2 infection during pregnancy is correlated with increased incidences of preeclampsia, preterm birth, and stillbirth [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, there is uncertainty about how COVID-19 affects endometrial tissue and embryo implantation.\u003c/p\u003e \u003cp\u003eSome of the researches have discussed the connection between menstrual cycle and gene expression associated to SARS-CoV-2 infection. One study that looked at scRNA-seq datasets found a low probability of SARS-CoV-2 infection in the endometrium. This conclusion was based on the negligible expression of ACE2 in stromal or unciliated epithelial cells and the absence of concurrent expression of ACE2 and TMPRSS2 during embryo implantation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Hanerjos-Castillo \u003cem\u003eet al\u003c/em\u003e. reported that the endometrium had low ACE2, medium TMPRSS2 expression, and restricted co-expression based on the combination of gene expression data from five investigations, which included 112 individuals [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Around the time of implantation, the research also observed a rise in the expression of MX1, ACE2, TMPRSS4, CTSB, and CTSL [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Both investigations found significant levels of BSG expression in the endometrium, but they disagreed about how important the BSG apparatus is in the event of a viral infection. According to a meta-analysis, the COVID-19 pandemic undoubtedly had an effect on endometriosis patients, since it intensified symptoms such dysmenorrhea, pelvic discomfort, anxiety, melancholy, and exhaustion [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This suggests that further research on the endometrial tissue of women of reproductive age infected with SARS-CoV-2 will help determine whether the causal implications derived from the molecular characterization of the endometrium translate into actual clinical manifestations.\u003c/p\u003e \u003cp\u003eIn 2022, Luc\u0026iacute;a de Miguel-Gomez\u0026rsquo;s group performed transcriptomic analyses endometrial biopsy samples and identified 235 DEGs from the endometria of COVID-19 patients [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. It was the first to look at alterations in the endometrium's global gene expression in women with COVID-19, providing substantial evidence that SARS-CoV-2 infection commonly affects the inflammatory environment, cellular motility, metabolic status, and cytokine production in the endometrium. However, this study had a crucial limitation that the patients\u0026rsquo; age, menstrual cycle parameters, fertility status and biopsy collection timing varied greatly, which cannot represent the specific phase of menstruation, not even for the period for window of implantation. In the same year, I. Henarejos-Castillo's group created a model that examined the gene co-expression network of the endometrium in 42 women who had healthy mid-secretory endometrium taken from them without endometrial diseases, together with nasopharyngeal swabs from 231 COVID-19-positive women and 30 negative controls [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. 19 important genes that may be affected by COVID-19 were found by topological analysis, and the expression of these genes was confirmed by prospectively collecting an endometrial sample set that included both COVID-19 positive and negative individuals. Nevertheless, we also noted that the basic information and fertility condition of women included in the throat swabs analysis and the endometrial samples study were not comparable. Currently, there are no specific studies to elaborate the mechanisms of COVID-19's effect on endometrial receptivity since getting COVID-19 endometrial samples is a challenging task.\u003c/p\u003e \u003cp\u003eIn this study, we integrate the co-expression molecules of COVID-19 in endometrium [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and female nasopharyngeal swabs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] from above two studies, to investigate the comparatively stable genes expressed following SARS-CoV-2 infection in the female population, despite the potential discrepancy in age and menstrual cycle. We experimentally validated the molecules\u0026rsquo; expression and distribution in mid-secretory phase endometrial biopsies from women who were diagnosed with COVID-19 within 30 days \u003cem\u003evs.\u003c/em\u003e Individuals without infection, to make predictions of SARS-CoV-2 related genes with endometrial receptivity. Additionally, endometrium-related molecular tests were employed to verify the functions of some of these potential molecules. With these studies, we want to learn more about the molecular processes underlying the changes in uterine endometrial responsiveness to COVID-19.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSpecimen collection\u003c/h2\u003e \u003cp\u003e8 endometrial samples from patients with COVID-19 infection within 30 days and 9 endometrial samples from healthy women without COVID-19 infection were collected from the Reproductive Center of Nanjing Jinling Hospital during January 2023 to March 2023. These COVID-19 infected patients were symptomatic and had a positive test result for SARS-CoV-2 infection, as indicated by nasopharyngeal swab samples. Patients with mild-to-moderate symptoms were included, whereas asymptomatic or severe cases were not recruited. The healthy women were enrolled from patients who have no COVID-19 infection history. Based on nasopharyngeal swab samples taken within the previous three months, the women in the health group were found to be negative for COVID-19. They also had nasopharyngeal swab tests on a weekly basis. The participants' body mass index (BMI) ranged from 18 to 30 kg/m\u003csup\u003e2\u003c/sup\u003e, and their ages ranged from 27 to 36, and the endometrial thickness ranged from 9 to 16 mm. The patients were infertile with diagnosis of the fallopian tube blockage, male factor or unexplained infertility, excluding endometrial disease such as endometrioma, adenomyosis, endometrial hyperplasia, endometrial polyps and intrauterine adhesion. All the endometiral samples were collected on the 5th day after ovulation.The endometrium thickness was 8\u0026ndash;16 mm. Supplemental Table\u0026nbsp;1 presents the characteristics of the patients with endometrial biopsy.\u003c/p\u003e \u003cp\u003e25 serum samples from female patients with COVID-19 infection within 30 days and 10 serum samples from healthy women without COVID-19 infection were collected from Nanjing Jinling Hospital during January 2023 to December 2023. Among the Covid-19 group, 12 serum samples were drawn from patients 0 to 7 days after diagnosed with COVID-19, 13 serum samples were drawn from patients 8 to 30 days after diagnosed with COVID-19. These COVID-19 infected patients were symptomatic and had a positive test result for SARS-CoV-2 infection, as indicated by nasopharyngeal swab samples. Patients with mild-to-moderate symptoms were included, whereas asymptomatic or severe cases were not recruited. The healthy women were enrolled from patients who have no COVID-19 infection history. Based on nasopharyngeal swab samples taken within the previous three months, the women in the health group were found to be negative for COVID-19. They also had nasopharyngeal swab tests on a weekly basis. All the serum samples were drawn from female patients during nonmenstrual period. The participants' body mass index (BMI) ranged from 18 to 32 kg/m\u003csup\u003e2\u003c/sup\u003e, and their ages ranged from 22 to 40. Supplemental Table\u0026nbsp;2 presents the characteristics of the patients with serum cytokine examination.\u003c/p\u003e \u003cp\u003e The study protocols (2023DZKY-009-01) were authorized by Nanjing Jinling Hospital's Clinical Ethics Review Committee. The Clinical Trials Registry (ID: NCT05685966) has the trial prospectively registered. Every participant endorsed the informed consent form.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry staining\u003c/h2\u003e \u003cp\u003eIn neutral buffered formalin, the endometrial samples were fixed. The sections were rehydrated and deparaffinized before being exposed to antigen retrieval. Following this, the sections were treated with primary antibodies against either SUGT1 (Proteintech), LRP1 (Abcam), HSPG2 (Abmart), HERC5 (Proteintech), or EPSTI1 (Proteintech). The slides were then exposed to secondary antibody incubation. The sections were counterstained with hematoxylin. The following protocol was used for immunohistochemical scoring, or histoscore: Each patient had five typical areas with stromal cells and endometrial glandular epithelium, at least three of which had glandular epithelium, examined under an Olympus light microscope set to \u0026times;200 magnification. With the use of the Fiji (ImageJ) program, the strength of the immunostaining was divided into four categories: 0\u0026thinsp;=\u0026thinsp;negative; 1\u0026thinsp;=\u0026thinsp;weak; 2\u0026thinsp;=\u0026thinsp;moderate; and 3\u0026thinsp;=\u0026thinsp;strong. Each field's histoscore was computed as follows: histoscore\u0026thinsp;=\u0026thinsp;0 \u0026times; percentage of cells that were negatively stained, 1 \u0026times; proportion of cells with poor staining, 2 \u0026times; proportion of cells with mild staining, and 3 \u0026times; proportion of cells with intense staining. The average of the histoscores from five distinct fields was used to get the histoscore of each sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCytokine assay\u003c/h2\u003e \u003cp\u003eBlood samples were collected in 1.5-mL EP tubes, blood clotting was performed for 1 h at 4\u0026deg;C, and the tubes were centrifuged at 500 g at 4\u0026deg;C for 10 min to harvest serum. The levels of Interferon (IFN)-γ (Mlbio), Lipopolysaccharide (LPS) (Mlbio), Tumor Necrosis Factor (TNF)-α (Mlbio), Interleukin (IL)-1β (Mlbio), Interleukin (IL)-1α (Mlbio) and Interleukin (IL)-6 (Mlbio) were determined by enzyme-linked immunosorbent assay kits for human.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman endometrial stromal cells (T-hESCs), human choriocarcinoma (BeWo), and human endometrial adenocarcinoma (Ishikawa) cells were cultured in DMEM/F12 media (HyClone) supplemented by 10% (v/v) fetal bovine serum (Gibco) plus 1% (v/v) penicillin/streptomycin. For the purpose of simulating the physiological condition, Ishikawa cells and T-hESCs were cultured in phenol red-free DMEM/F12 medium containing 2.5% (v/v) charcoal/dextranl-treated FBS and 1% (v/v) penicillin/streptomycin, with or without a combination of 10 nM E\u003csub\u003e2\u003c/sub\u003e (Sigma) or 0.5 mM 8Br-cAMP (Sigma), in addition to 1 \u0026micro;M medroxyprogesterone acetate (MPA) (Merck), for the prescribed duration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eThe manufacturer's instructions were followed for homogenizing the cells in whole-cell lysis solution with phosphatase inhibitor cocktails 2 and 3 (Sigma). A 6\u0026ndash;10% (w/v) SDS\u0026ndash;polyacrylamide gel was loaded with equal volumes of total protein (10\u0026ndash;30 \u0026micro;g), which were subsequently deposited onto Millipore polyvinylidene fluoride membranes. Following immunoblotting, secondary antibodies conjugated with goat anti-rabbit or rabbit anti-mouse HRP were incubated on the membranes. The primary antibodies used in the immunoblotting process were against EPSTI1, SUGT1, and β-Actin from Proteintech. For detection, the ECL Plus Western Blotting Detection System and Millipore's enhanced chemiluminescence kit were employed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTakara's TRIzol reagent was used to extract total RNA in accordance with the manufacturer's instructions. For the cDNA synthesis using the BEI-BEI Biotech Prime Script RT reagent kit, a total of 1 \u0026micro;g of RNA was utilized. Random primers were used for reverse transcription, and a Roche Diagnostics MyiQ Single Color Real-Time PCR Detection System was used for qRT-PCR. Supplementary Table\u0026nbsp;3 provides an explanation of the primer sequences. Using the 2-∆∆ CT technique, which normalizes gene expression to the level of the endogenous control (18s rRNA), the fold changes in gene expression were determined.\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn-vitro\u003c/b\u003e \u003cb\u003eimplantation model\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn accordance with standard laboratory protocol, BeWo multicellular spheroids were affixed to endometrial Ishikawa cells to serve as an \u003cem\u003ein vitro\u003c/em\u003e adhesion model [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly put, a single-cell solution of BeWo cells was placed on a Petri dish coated with the anti-adhesive polymer poly-2-hydroxyethyl methacrylate (Sigma) in order to encourage the growth of 150\u0026ndash;200 \u0026micro;m diameter BeWo spheroids. On a 24-well culture plate, confluent monolayers of Ishikawa cells were transfected simultaneously with the chosen vectors. Next, a pre-measured amount of spheroids was added to the confluent monolayers of Ishikawa cells. Following a two-hour incubation period at 37\u0026deg;C, the detached spheroids were extracted using PBS supplemented with 0.1 mg/L of Ca\u003csup\u003e2+\u003c/sup\u003e and 0.1 mg/L of Mg\u003csup\u003e2+\u003c/sup\u003e. The attachment rate to the Ishikawa monolayer was computed by counting the adhering BeWo spheroids under a light microscope. There were three separate, independent attachment tests conducted.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eOverlapped DEGs from endometria and nasopharyngeal swabs between COVID-19 cases and healthy control\u003c/h2\u003e \u003cp\u003e235 DEGs between COVID-19 cases (n\u0026thinsp;=\u0026thinsp;6) and controls (n\u0026thinsp;=\u0026thinsp;8) of endometrial samples were obtained from RNA-Seq results as Professor Luc\u0026iacute;a de Miguel-Gomez [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] described. 2580 DEGs between COVID-19 cases and controls of nasopharyngeal samples were obtained from 231 female transcriptomes (201 COVID-19 cases and 30 controls) as Professor I. Henarejos-Castillo [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] described. Without distinguishing expression trends, 50 overlapping molecules were identified; however, only 10 showed consistent trends, while 40 showed opposite trends. Eight up-regulated and two down-regulated overlapping molecules were found in both (Supplementary Fig.\u0026nbsp;1A), and we combined the roles of COVID-19 and fertility-related genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The common DEGs with combined scores larger than 0.7 were compiled into a PPI network using GO and KEGG Pathway enrichment analysis (Supplementary Fig.\u0026nbsp;1B, C). In Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the complete methodological workflow is shown.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCo-expression genes in the endometria and nasopharyngeal swabs in COVID-19 female patients.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eUp-regulation in COVID-19 female patients\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMolecular function\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFertility association\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eATP6V1C1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegulate V-ATPase activity, energy metabolism and cellar differentiation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAssociation with the expression of androgen receptor\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCCDC90B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTransport calcium ions into the mitochondria\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThe potential common biomarker candidates in the pathospermic conditions of both teratozoospermia and azoospermia\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEPSTI1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParticipate in tumor cell metastasis, epithelial-mesenchymal transition, chronic inflammation, tissue reconstruction, and the regulation of cell apoptosis.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInvolved in embryonic development and pregnancy establishment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFBXO15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSubstrate-recognition component of the SCF-type E3 ubiquitin ligase complex.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEspecially high expressed in fallopian tube\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHERC5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA critical modulators of the antiviral immune response\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAssociation with the abnormal regulation of decidulization\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIFI44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA crucial regulator of the immune system and an immune evasion biomarker for SARS-CoV-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAssociation with pregnancy establishment\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIFI6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegulate apoptosis and immune responses\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEndometrial IFI6 declines during peri-implantation period in pigs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSUGT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePlay a role in ubiquitination and subsequent proteasomal degradation of target proteins\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEndometrial SUGT1 may serve as potential therapeutic targets for patients with endometriosis-induced recurrent pregnancy loss\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eDown-regulation in COVID-19 female patients\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLRP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA transmembrane receptor that belongs to the LDLR family, which mediates and regulates the endocytosis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEndometrial LRP1 elevates during peri-implantation period in pigs\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHSPG2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eA large multi-domain extracellular matrix proteoglycan, which contributes to the invasion, metastasis and angiogenesis of solid tumor\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePlay a role in the differentiation of granulosa cells.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eEPSTI1, SUGT1, HERC5, HSPG2 and LRP1 expression in mid-secretory endometrium of women with COVID-19\u003c/h2\u003e \u003cp\u003e9 mid-secretory stage endometrial samples from women without COVID-19 infection and a total of 8 mid-secretory stage endometrial samples from patients with COVID-19 infection within 30 days were used to confirm the expression and distribution of potential genes throughout the peri-implantation window. IHC assay was applied to detect EPSTI1, SUGT1, HERC5, HSPG2 and LRP1 expression in mid-secretory endometrium between COVID-19 group and normal control. Separate analyses were conducted for stromal (S) cells, glandular epithelial (GE) cells, and luminal epithelial (LE) cells. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, EPSTI1, SUGT1 and HERC5 were up-regulated in the COVID-19 group, while HSPG2 and LRP1 show no difference in expression between the two groups.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEPSTI1 and SUGT1 participate in the regulation of endometrial receptivity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTwo prominent indicators of decidualization are PRL and IGFBP-1. When 8Br-cAMP and MPA were stimulated in T-hESCs, plasmid-mediated overexpression of EPSTI1 and SUGT1 led to a prominent reduction in PRL secretion and IGFBP-1 mRNA level for 3 and 6 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). HECR5 had no effect on PRL or IGFBP-1 expression during \u003cem\u003ein-vitro\u003c/em\u003e decidualization in T-hESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). We also created an \u003cem\u003ein-vitro\u003c/em\u003e implantation model that allows us to efficiently investigate the role of embryo adhesion to epithelial cells by co-cultivating Ishikawa cells and human BeWo cells that were incubated in the blastosphere. Overexpressing SUGT1 inhibited the adhesion ability of BeWo spheroids induced by E\u003csub\u003e2\u003c/sub\u003e and MPA, while EPSTI1 and HERC5 had no significant effect on adhesion rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Based on the aforementioned results, we deduced that SUGT1 dysregulated endometrial stromal cell decidualization as well as endometrial epithelial cell receptivity, and that EPSTI1 hampered endometrial decidualization.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, we examined the mRNA and protein expression of EPSTI1 and SUGT1 in Ishikawa cells treated with E\u003csub\u003e2\u003c/sub\u003e and MPA, as well as in T-hESCs treated with 8Br-cAMP in conjunction with MPA (Supplementary Fig.\u0026nbsp;2). We observed an interesting phenomenon that EPSTI1 was up-regulated in Ishikawa cells, yet was down-regulated in T-hESCs in physiological status induced by sexual hormones. The opposite phenotype of EPSTI1 in epithelial cells and stromal cells may be related to the epithelial-to-mesenchymal transition (EMT) characteristics of EPSTI1 itself. As for SUGT1, estrogen and progesterone did not affect its expression significantly in in Ishikawa cells nor T-hESCs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eEPSTI1 and SUGT1 expression stimulated by IFN-γ, LPS and IL-1β\u003c/h2\u003e \u003cp\u003eWe continued to investigate the possible mechanism how covid-19 infection lead to the elevated expression of EPSTI1 and SUGT1 in endometrial cells. The cytokine storm is a known factor causing major clinical symptoms in COVID-19 patients [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Consistent with previous studies, we found a significant increasing trend of serum INF-γ, TNF-α, LPS, IL-6 and IL-1β in female patients diagnosed of Covid-19 within 0 to 7 days with mild to moderate symptoms (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Previous studies have demonstrated that inflammatory response persist after resolution of COVID-19. This phenomenon has been evaluated in terms of increased levels of several cytokines, including IL-1β [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. We also observed that IL-1β demonstrated a sustained elevation in women 8\u0026ndash;30 days after COVID-19 infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, the protein expression of EPSTI1 and SUGT1 were examined in Ishikawa cells treated with recombinant cytokine of IL-1β. Addition of IL-1β significantly upregulated the expression of EPSTI1 and SUGT1. Meanwhile, we detected the EPSTI1 and SUGT1 levels stimulated by INF-γ as well as LPS. EPSTI1 was elevated with the administration of IFN-γ and LPS. SUGT1 was up-regulated with the stimulation of LPS. The above results suggest that the booming cytokines caused by Covid-19 may be responsible for promoting the expression of EPSTI and SUGT1 in the endometrium.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe World Health Organization (WHO) announced on May 5, 2023, that COVID-19 was no more a global public health emergency. This announcement was a major turning point in the global effort to combat the pandemic. In the 'post-epidemic era', we should pay long-term attention to the impact of the new coronavirus on human long-term quality of life. Since 2020, there has been a brief decline in population worldwide, a phenomenon that was alleviated in 2023. In addition to the economic and social factors brought by the epidemic, we also noted the potential impact of the epidemic on reproductive health.\u003c/p\u003e \u003cp\u003eASRM and ESHRE recommended that fertility services be stopped during the early stages of the pandemic, with the exception of urgent cases [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Further research has revealed a correlation between SARS-CoV-2 infection and some aspects of human fertility.\u003c/p\u003e \u003cp\u003eMolecular studies indicated that the ovarian cortex, medulla, oocytes, endometrium, and the membranes of trophectoderm, hypoblast, and epiblast cells in blastocysts have all been demonstrated to co-express ACE2 and TMPRSS2 in females [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. SARS-CoV-2 infection has been associated with ovarian dysfunction, disruptions in the follicular microenvironment, changes in menstrual volume and cycle length, reproductive outcomes, and potential interference with embryo implantation and pregnancy [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eClinical assessments of the outcomes of assisted reproductive technology revealed that women who had frozen embryo transfer (ET) within 60 days of infection had reduced pregnancy rates than those who delayed ET [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, other studies have shown that a history of mild or asymptomatic SARS-CoV-2 infection in females does not appear to have a negative impact on laboratory and clinical outcomes in fresh and frozen ET cycles [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. This suggests that current research on COVID-19's effects on reproduction might only represent the tip of the iceberg, necessitating extensive basic research and clinical observation. To find out if SARS-CoV-2 affects the endometrium during implantation or the embryo in its early stages of development, perhaps influencing implantation or raising the incidence of miscarriages, more research is required.\u003c/p\u003e \u003cp\u003eUsing total RNA next-generation sequencing, Professor Luc\u0026iacute;a de Miguel-Gomez explains for the first time how systemic COVID-19 modifies global gene expression in the cyanobacteria of symptomatic women [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The purpose of this endeavor is to look at how COVID-19 affects essential elements of a healthy menstrual cycle and associated reproductive consequences. Professor I. Henarejos-Castillo's MSE gene co-expression network model provides insights into how transcriptome alterations caused by COVID-19, inferred from nasopharyngeal tissue, may impair important endometrial processes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This model is instrumental in shedding light on the influence of COVID-19 on endometrial processes. These two articles reveal potential mechanisms by which COVID-19 may affect endometrial functions, however the patients\u0026rsquo; age, menstrual cycle parameters, fertility status and biopsy collection timing of the selected endometrial specimens and throat swabs in both studies varied greatly. Using the endometrium [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] and throat swabs [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] of COVID-19-positive women as our reference, we overlapped the differentially expressed genes to investigate the relatively stable expressed molecule and lessen the discrepancy from the unparallel background of patients.\u003c/p\u003e \u003cp\u003eWithout distinguishing expression trends, 50 overlapping molecules were identified; however, only 10 showed consistent trends, while 40 showed opposite trends. This strongly indicates the tissue specificity of COVID-19 infection and the impact of potential differences such as age and menstrual timing on the results. It also indirectly highlights the limitations of relying solely on bioinformatics analysis or using throat swab differential gene databases for such analyses. Upon analyzing these 10 molecules, we identified EPSTI1 and SUGT1 as two prioritized molecules.\u003c/p\u003e \u003cp\u003eIn mixed cultures of human breast cancer cells and fibroblasts, the interferon-responsive gene EPSTI1 was first discovered [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. It has been implicated in various biological processes, including tumor cell metastasis, epithelial-mesenchymal transition, chronic inflammation, tissue reconstruction, embryonic development, and the regulation of cell apoptosis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Capdevila-Busquets \u003cem\u003eet al\u003c/em\u003e. reported that EPSTI1 can modulate the extrinsic apoptotic pathway in both estrogen receptor-positive and triple-negative breast cancer cell lines [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Methylation of EPSTI1 may play a role in SARS-CoV-2 infection processes and influence inflammatory and immune responses by regulating EPSTI1 expression [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous studies have reported that the expression of EPSTI1 was significantly increased in BAL cells, PBMCs, leukocytes, and nasopharyngeal tissue of COVID-19 patients [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In our study, women with COVID-19 infection had considerably higher levels of EPSTI1 in their endometrial cells.\u003c/p\u003e \u003cp\u003eThe cytokines, such as IFN-γ, IL-1β, IL-6, IL-12, IL-18, IL-33 and TGF-β, are associated with different clinical features of COVID-19. A long-lasting cytokine signature consisting of elevated levels of interleukin IL-1β could potentially underlie many of the clinical symptoms of post Covid-19 infection [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. EPSTI1 is an interferon (IFN)-responsive gene and is highly expressed in exposed to virus infection as well as lipopolysaccharide-induced inflammatory [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Here, we further defined that IL-1β, LPS and INF-γ could upregulate EPSTI expression in Ishikawa cells and T-hESCs cells, suggesting that elevated EPSTI1 expression in the endometrium of patients after covid-19 infection may be associated with an inflammatory storm.\u003c/p\u003e \u003cp\u003eThere have been few studies on the function of EPSTI1 in the endometrium. EPSTI1 levels is lower in endometrial tissues of pregnant mares compared to tissues from non-pregnant mares [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], suggesting a potential link to pregnancy. In our \u003cem\u003ein vitro\u003c/em\u003e decidualization experiment, EPSTI1 inhibited the expression of dPRL, IGFBP-1 in T-hESCs. We also observed that EPSTI1, known as a molecule promotes epithelial-mesenchyma transition (EMT), was down-regulated in T-hESCs during decidualization \u003cem\u003ein vitro\u003c/em\u003e. The ability of the mature endometrium to undergo decidualization and cyclic regeneration is essential for successful human reproduction. During the implantation phase, ovarian hormones regulate the mesenchyma-epithelial transition (MET) process, which is consistent with changes in stromal cellular morphology that arise with decidualization [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, it is reasonable to presume that EPSTI1 restrain decidualization by disturbing the MET process of endometrial stromal cells during the peri-implantation window.\u003c/p\u003e \u003cp\u003eBy stimulating leucine-rich repeat-containing and nucleotide-binding domain proteins, SUGT1 participates in the innate immune response. When heat shock protein 90 was discovered, SUGT1 was shown to be a co-chaperone. According to reports [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], SUGT1 is connected to both HIV-1 infection and colorectal cancer. According to Ata \u003cem\u003eet al\u003c/em\u003e., individuals experiencing recurrent pregnancy loss due to endometriosis may benefit from targeting endometrial SUGT1 as a possible therapeutic target [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Within one month of the COVID-19 infection, we found in this study that there was a elevation of SUGT1 in the endometrial cells of the patients. SUGT was also induced by IL-1β and LPS in Ishikawa cells and T-hESCs. \u003cem\u003eIn vitro\u003c/em\u003e, SUGT1 suppressed Bewo spheroids adhesion and decidualization while remaining unresponsive to the stimuli of progesterone or estrogen. We speculated that SUGT1 may participate in the dysregulation of endometrial receptivity though inflammatory pathway. More experiments will conducted to explore the concrete mechanism.\u003c/p\u003e \u003cp\u003eAlthough the peak of the COVID-19 epidemic has passed, research on COVID-19 should not be confined to this period. Our research demonstrates that following COVID-19 infection, the endometrium of the peri-implantation window experiences protein-level alterations that impact endometrial receptivity. It is noteworthy that we collected our endometrial samples between 10 and 30 days after the COVID-19 diagnosis, which better captures the relatively long-term consequences of the infection on the endometrium.\u003c/p\u003e \u003cp\u003eThe immune response is a critical factor in the evolution of COVID-19. There is a growing understanding that the immune system's reaction to COVID-19, rather than the virus itself, is frequently the cause of the morbidity linked to it. With a growing population of recovering patients, it became clear that in 32\u0026ndash;87% of patients (including those with mild acute disease), health impairments persist beyond the acute phase of infection [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The start and maintenance in a healthy pregnancy depend on the endometrium's dynamic balance between pro-and anti-inflammatory mediators. SARS-CoV-2 may disrupt the immunological milieu in the uterus, which might impact embryo implantation. The results of this investigation may lead to a reexamination of the connection between COVID-19 and the endometrium and generate theories regarding the emergence of protracted COVID and other post-COVID-19 related adverse effects.\u003c/p\u003e \u003cp\u003eThe study faces limitations. The clinical sample size is limited. However, we verify potential molecular expression based on analysis from database of high quality and large scale. Our study led to hypotheses concerning the infection of COVID-19 influence the endometrium by regulating key molecules, which could potentially affect endometrial function and implantation. It is now imperative to further investigate this relationship to deepen our understanding.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOver the past three years, the COVID-19 pandemic has significantly disrupted the provision of assisted reproductive technology treatments. Although we now know more about how SARS-CoV-2 spreads, there are still many unanswered issues about how it sustainably affects reproductive health. In this work, we demonstrate that there were variations in the expression of endometrial proteins during the peri-implantation phase following COVID-19 infection. Notably, \u003cem\u003ein vitro\u003c/em\u003e decidualization and embryo adhesion function were restrained by EPSTI1 and SUGT1, which were up-regulated in implantation window endometria of women infected with COVID-19. The long-term prevention and management of COVID-19 and its aftermath may be greatly aided by investigating the relationship between SARS-CoV-2 and human reproduction.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; roles\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eXi Cheng designed the study and contributed to writing the article. Xiting Cai, Hui Wang and Biying Li performed the molecular experiments. Lu Zheng and Jinzhao Ma contributed to data analysis. Cheng Zhou, Xuan Huang, Kadiliya Jueraitetibaike, Qin Sun, Meiling Li and Xu Tang contributed to the the collection of clinical samples. Yuming Feng and Hong Zhang contributed to the article revision and editing. Li Chen and Bing Yao contributed to the study design, data interpretation, as well as article revision. All authors have agreed to be listed and have approved the final article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work is supported by National Natural Science Foundation of China (Grant: 82101757), the Key Research and Development Program of Jiangsu Province (Grant: BE2022712) and Annual Foundation of Nanjing Jinling Hospital (YYQN2021082, 22LCYY-QH5, 22LCYY-QH12). Each of the funding contributed to expenses related to specific procedures carried out in the project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared no conflicts of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocols (2023DZKY-009-01) were authorized by Nanjing Jinling Hospital\u0026apos;s Clinical Ethics Review Committee. The Clinical Trials Registry (ID: NCT05685966) has the trial prospectively registered. Every participant endorsed the informed consent form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the other members of Dr. Yao\u0026rsquo;s laboratory for their discussion and help.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAliyari R, Mahdavi S, Enayatrad M, Sahab-Negah S, Nili S, Fereidooni M, Mangolian Shahrbabaki P, Ansari-Moghaddam A, Heidarzadeh A, Shahraki-Sanavi F, et al. Study protocol: cohort event monitoring for safety signal detection after vaccination with COVID-19 vaccines in Iran. BMC Public Health. 2022;22:1153.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElrobaa IH, New KJ. COVID-19: Pulmonary and Extra Pulmonary Manifestations. Front Public Health. 2021;9:711616.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl Jamal SM, Pujadas E, Ramos I, Bryce C, Grimes ZM, Amanat F, Tsankova NM, Mussa Z, Olson S, Salem F, et al. Tissue-based SARS-CoV-2 detection in fatal COVID-19 infections: Sustained direct viral-induced damage is not necessary to drive disease progression. Hum Pathol. 2021;114:110\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJamieson DJ, Rasmussen SA. An update on COVID-19 and pregnancy. Am J Obstet Gynecol. 2022;226:177\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVilella F, Wang W, Moreno I, Roson B, Quake SR, Simon C. Single-cell RNA sequencing of SARS-CoV-2 cell entry factors in the preconceptional human endometrium. Hum Reprod. 2021;36:2709\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenarejos-Castillo I, Sebastian-Leon P, Devesa-Peiro A, Pellicer A, Diaz-Gimeno P. SARS-CoV-2 infection risk assessment in the endometrium: viral infection-related gene expression across the menstrual cycle. Fertil Steril. 2020;114:223\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaouzi D, Entezami F, Tuaillon E, Gala A, Ferri\u0026egrave;res-Hoa A, Brouillet S, Thierry AR, Hamamah S. SARS-CoV-2 and Implantation Window: Gene Expression Mapping of Human Endometrium and Preimplantation Embryo. Life (Basel) 2021; 11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKabani Z, Ramos-Nino ME, Ramdass PVAK. Endometriosis and COVID-19: A Systematic Review and Meta-Analysis. Int J Mol Sci 2022; 23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Miguel-G\u0026oacute;mez L, Sebasti\u0026aacute;n-Le\u0026oacute;n P, Romeu M, Pellicer N, Faus A, Pellicer A, D\u0026iacute;az-Gimeno P, Cervell\u0026oacute; I. Endometrial gene expression differences in women with coronavirus disease 2019. Fertil Steril. 2022;118:1159\u0026ndash;69.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHenarejos-Castillo I, Devesa-Peiro A, de Miguel-Gomez L, Sebastian-Leon P, Romeu M, Aleman A, Molina-Gil C, Pellicer A, Cervello I, Diaz-Gimeno P. Predicted COVID-19 molecular effects on endometrium reveal key dysregulated genes and functions. Mol Hum Reprod 2022; 28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng X, Zhang Y, Ma J, Wang S, Ma R, Ge X, Zhao W, Xue T, Chen L, Yao B. NLRP3 promotes endometrial receptivity by inducing epithelial-mesenchymal transition of the endometrial epithelium. Mol Hum Reprod 2021; 27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQueiroz MAF, Neves PFMd, Lima SS, Lopes JC, Torres MKS, Vallinoto IMVC, Bichara CDA, Dos Santos EF, de Brito MTFM, da Silva ALS, et al. Cytokine Profiles Associated With Acute COVID-19 and Long COVID-19 Syndrome. Front Cell Infect Microbiol. 2022;12:922422.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchulthei\u0026szlig; C, Willscher E, Paschold L, Gottschick C, Klee B, Henkes S-S, Bosurgi L, Dutzmann J, Sedding D, Frese T, et al. The IL-1β, IL-6, and TNF cytokine triad is associated with post-acute sequelae of COVID-19. Cell Rep Med. 2022;3:100663.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGianaroli L, Ata B, Lundin K, Rautakallio-Hokkanen S, Tapanainen JS, Vermeulen N, Veiga A, Mocanu E. The calm after the storm: re-starting ART treatments safely in the wake of the COVID-19 pandemic. Hum Reprod. 2021;36:275\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVeiga A, Gianaroli L, Ory S, Horton M, Feinberg E, Penzias A. Assisted reproduction and COVID-19: a joint statement of ASRM, ESHRE, and IFFS. Glob Reprod Health 2020; 5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajput SK, Logsdon DM, Kile B, Engelhorn HJ, Goheen B, Khan S, Swain J, McCormick S, Schoolcraft WB, Yuan Y, Krisher RL. Human eggs, zygotes, and embryos express the receptor angiotensin 1-converting enzyme 2 and transmembrane serine protease 2 protein necessary for severe acute respiratory syndrome coronavirus 2 infection. F S Sci. 2021;2:33\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeinerman R. COVID-19 and the endometrium: inflammation as understanding. Fertil Steril. 2022;118:1170\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLebar V, Lagan\u0026agrave; AS, Chiantera V, Kunič T, Lukanović D. The Effect of COVID-19 on the Menstrual Cycle: A Systematic Review. J Clin Med 2022; 11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStanley KE, Thomas E, Leaver M, Wells D. Coronavirus disease-19 and fertility: viral host entry protein expression in male and female reproductive tissues. Fertil Steril. 2020;114:33\u0026ndash;43.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoungster M, Avraham S, Yaakov O, Landau Rabbi M, Gat I, Yerushalmi G, Baum M, Maman E, Hourvitz A, Kedem A. The impact of past COVID-19 infection on pregnancy rates in frozen embryo transfer cycles. J Assist Reprod Genet. 2022;39:1565\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang J, Liu Y, Xia L, Zhao Y, Tian L, Xu D, Su Q, Hu Y, Xie Q, Chen J, et al. Effect of prior female SARS-CoV-2 infection on IVF outcomes: a prospective cohort study. Front Endocrinol (Lausanne). 2023;14:1239903.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYoungster M, Avraham S, Yaakov O, Landau Rabbi M, Gat I, Yerushalmi G, Sverdlove R, Baum M, Maman E, Hourvitz A, Kedem A. IVF under COVID-19: treatment outcomes of fresh ART cycles. Hum Reprod. 2022;37:947\u0026ndash;53.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNielsen HL, R\u0026oslash;nnov-Jessen L, Villadsen R, Petersen OW. Identification of EPSTI1, a novel gene induced by epithelial-stromal interaction in human breast cancer. Genomics. 2002;79:703\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCapdevila-Busquets E, Badiola N, Arroyo R, Alcalde V, Soler-L\u0026oacute;pez M, Aloy P. Breast cancer genes PSMC3IP and EPSTI1 play a role in apoptosis regulation. PLoS ONE. 2015;10:e0115352.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ede Neergaard M, Kim J, Villadsen R, Fridriksdottir AJ, Rank F, Timmermans-Wielenga V, Langer\u0026oslash;d A, B\u0026oslash;rresen-Dale A-L, Petersen OW, R\u0026oslash;nnov-Jessen L. Epithelial-stromal interaction 1 (EPSTI1) substitutes for peritumoral fibroblasts in the tumor microenvironment. Am J Pathol. 2010;176:1229\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Z, Mei Z, Ding S, Chen L, Li H, Feng K, Huang T, Cai Y-D. Identifying Methylation Signatures and Rules for COVID-19 With Machine Learning Methods. Front Mol Biosci. 2022;9:908080.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDong Z, Yan Q, Cao W, Liu Z, Wang X. Identification of key molecules in COVID-19 patients significantly correlated with clinical outcomes by analyzing transcriptomic data. Front Immunol. 2022;13:930866.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShaath H, Vishnubalaji R, Elkord E, Alajez NM. Single-Cell Transcriptome Analysis Highlights a Role for Neutrophils and Inflammatory Macrophages in the Pathogenesis of Severe COVID-19. Cells 2020; 9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKari A, Aili MZ, Adili Z, Hairula A, Abuduhaer N. A: Knockdown of EPSTI1 alleviates lipopolysaccharide-induced inflammatory injury through regulation of NF-κB signaling in a cellular pneumonia model. Allergol Immunopathol (Madr) 2022; 50:106\u0026thinsp;\u0026ndash;\u0026thinsp;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBauersachs S, Mitko K, Ulbrich SE, Blum H, Wolf E. Transcriptome studies of bovine endometrium reveal molecular profiles characteristic for specific stages of estrous cycle and early pregnancy. Exp Clin Endocrinol Diabetes. 2008;116:371\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOwusu-Akyaw A, Krishnamoorthy K, Goldsmith LT, Morelli SS. The role of mesenchymal-epithelial transition in endometrial function. Hum Reprod Update. 2019;25:114\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAllouch A, Di Primio C, Paoletti A, L\u0026ecirc;-Bury G, Subra F, Quercioli V, Nardacci R, David A, Sa\u0026iuml;di H, Cereseto A, et al. SUGT1 controls susceptibility to HIV-1 infection by stabilizing microtubule plus-ends. Cell Death Differ. 2020;27:3243\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIwatsuki M, Mimori K, Sato T, Toh H, Yokobori T, Tanaka F, Ishikawa K, Baba H, Mori M. Overexpression of SUGT1 in human colorectal cancer and its clinicopathological significance. Int J Oncol. 2010;36:569\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan R, Liang X, Hu J. miR-141-3p alleviates ulcerative colitis by targeting SUGT1 to inhibit colonic epithelial cell pyroptosis. Autoimmunity. 2023;56:2220988.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAta B, Vermeulen N, Mocanu E, Gianaroli L, Lundin K, Rautakallio-Hokkanen S, Tapanainen JS, Veiga A. SARS-CoV-2, fertility and assisted reproduction. Hum Reprod Update. 2023;29:177\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"COVID-19, Endometrial receptivity, embryo implantation, women’s reproductive health, EPSTI1, SUGT1","lastPublishedDoi":"10.21203/rs.3.rs-4545088/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4545088/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo study the potential effect of coronavirus disease 2019 (COVID-19) on the endometrial receptivity.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eOverlapped differentially expressed genes (DEGs) between the endometrium and the throat swabs of female patients with COVID-19 infection \u003cem\u003evs.\u003c/em\u003e women without COVID-19 were integrated. The expression and distribution of prioritized molecules were detected in mid-secretory phase endometrium from patients infected by COVID-19 within 30 days \u003cem\u003evs.\u003c/em\u003e Individuals without the infection. Serum INF-γ, LPS, TNF-α, IL-1β, IL-1α and IL-6 were detected in female patients infected by COVID-19 within 30 days \u003cem\u003evs.\u003c/em\u003e Individuals without the infection. \u003cem\u003eIn vitro\u003c/em\u003e decidualization of human endometrial stromal cells (T-hESCs) and human choriocarcinoma (BeWo) spheroids adhesion experiments were employed to explore the functions of the potential molecules EPSTI1 and SUGT1. Cytokines of INF-γ, LPS and IL-1β were administrated in T-hESCs and human endometrial adenocarcinoma (Ishikawa) cells, and the expression of EPSTI1 and SUGT were identified.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe systemic disease COVID-19 altered endometrial protein expression during peri-implantation window. Epithelial stromal interaction 1 (EPSTI1), S-phase kinase-associated protein 1 (SUGT1) and RCC1-containing protein 5 (HERC5) were up-regulated in mid-secretory phase endometrium of women infected by COVID-19 within 30 days. EPSTI1 and SUGT1 impaired PRL and IGFBP1 expression stimulated by 8-Br-cAMP and MPA in T-hESCs. SUGT1 decreased the adhesion rate of BeWo spheroids to Ishikawa cells induced by E\u003csub\u003e2\u003c/sub\u003e and MPA. Serum INF-γ, LPS, TNF-α, IL-1β and IL-6 were elevated in female patients being infected with Covid-19 with mild or moderate symptoms. IL-1β demonstrated a sustained elevation in women infected with COVID-19 within 8\u0026ndash;30 days. EPSTI1 was up-regulated by the administration of IL-1β, INF-γ and LPS in shikawa cells and T-hESCs. SUGT1 was up-regulated by the administration of IL-1β and LPS in shikawa cells and T-hESCs.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eEndometrial decidualization and embryo adhesion function were negatively influenced by EPSTI1 and SUGT1, which were highly expressed in the mid-secretory endometrium of COVID-19-infected women. The elevation of EPSTI1 and SUGT1 in patients infected with Covid-19 maybe related to the increased cytokines of IL-1β, INF-γ and LPS. Investigating the relationship between SARS-CoV-2 and human reproduction may be very helpful in the long run in preventing and controlling COVID-19 and its aftermath.\u003c/p\u003e","manuscriptTitle":"COVID-19 and embryo implantation: EPSTI1 and SUGT1 participate in the dysregulation of SARS-CoV-2 on endometrial receptivity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-22 21:48:37","doi":"10.21203/rs.3.rs-4545088/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"dbcd793a-fb16-42a0-b085-517e5f2de238","owner":[],"postedDate":"July 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-09-24T12:55:37+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-22 21:48:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4545088","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4545088","identity":"rs-4545088","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}