Causes Of Recurrent Implantation Failure Research Articles

NSUN2-Mediated m5C Methylation Impairs Endometrial Receptivity

Impaired endometrial decidualization is the primary cause of recurrent implantation failure (RIF). RNA methylation modification, especially NSUN family mediated m5C, is crucial for various physiological events, such as maternal-to-zygotic transition, gametogenesis, embryonic development, organismal lifespan, and cell cycle. However, the regulatory mechanisms between NSUN family mediated m5C modification and RIF remain unknown. We acquired NSUN2 expression data of 15 human endometrium samples at proliferative and secretory stages from reproductive cell atlas. The overall pattern of m5C sites and genes was elucidated through m5C-BS-seq, whereas the overall m5C levels in different groups were revealed by dot blot assay. BrdU and western blotting assays were carried out to evaluate the role of NSUN2 in proliferation and autophagy. The effects of NSUN2-mediated m5C modification on embryo attachment were evaluated by an in vitro model of a confluent monolayer of Ishikawa cells cocultured with BeWo spheroids, and its downstream targets were evaluated by real-time reverse-transcription PCR and western blotting in Ishikawa cells. The molecular mechanism for NSUN2 regulating its downstream targets’ expression was determined by Cut&Tag and coimmunoprecipitation assays. NSUN2 was increased in SOX9+ cells and widespread in epithelial cell type at the proliferative stage by previous single-cell RNA sequencing data. NSUN2 overexpression (NSUN2OE) in the Ishikawa cell line elevated m5C levels and promoted cell proliferation and autophagy. NSUN2OE reduced attachment efficiency of BeWo cell spheres. Overexpressed NSUN2 was found to increase STAT1 and MMP14 mRNA expressions by inducing exon skipping. NSUN2 interacted with CLDN4 through m5C modification, and NSUN2OE or NSUN2 knockdown resulted in a similar variation tendency of CLDN4. Overexpression of NSUN2 increased CLDN4 H3K9ac modification by downregulating SIRT4 expression at the protein level, leading to the upregulation of CLDN4 mRNA expression. Our results uncovered a novel intricate regulatory mechanism between NSUN2-mediated m5C and RIF and suggested a potential new therapeutic strategy for RIF.

Debate 6: Microbiome Testing is Indicated for Implantation Failure in 2023

Motion: For Uninvestigated Recurrent Implantation Failure (RIF) has a significant cost, can waste embryos with the potential for life and leads to empirical therapies such as the use of steroids. Chronic Endometritis (CE) is a known cause of RIF is diagnosed by presence of plasma cells and worsens if steroids suppress the protective immune system response. Identifying plasma cells in endometrial tissue samples requires histological expertise, special staining and there is variation in numbers between tissue sections and microscope fields. Furthermore, culturing bacteria from low biomass environments like the endometrium is difficult and will not identify anaerobes and other fastidious organisms. The molecular technique of sequencing the 16S ribosome enables the identification of microbes that can create a hostile environment for embryos. There is clear evidence that some microbes and microbiome signatures are associated with RIF and that changing the microbiome is associated with higher implantation rates. There are established and emerging treatments for an aberrant microbiome that include antibiotic therapy, probiotics, faecal transplantation, and curettage. The more diagnostic information we can obtain to determine the cause of our patients RIF and then design specific, targeted treatments that are effective, the better their pregnancy outcomes will be. It is useful to test the microbiome and will only become more useful as this technology improves. Motion: Against The microbiome is closely related to women’s reproductive health, and therefore microbiome testing is becoming increasingly important in the field of reproductive medicine, especially in guiding assisted reproduction treatments such as infertility. The human microbiome is a collection of all microorganisms that exist on or within human tissues and fluids, as well as the corresponding anatomical sites where they are located, including the female reproductive tract, gastrointestinal tract, skin, semen, saliva, oral mucous membranes, etc., of which the ones that are most relevant to the study of female infertility mainly include the female reproductive tract flora and gastrointestinal tract flora. Several studies have shown that the composition of the microbiota in the female reproductive tract may affect embryo implantation and pregnancy success. An imbalanced or dysbiased microbiome of the female reproductive tract flora may lead to inflammation, altered immune responses, and hinder the optimal conditions needed for successful embryo implantation. Microbiome Testing can be accomplished through a variety of technologies, such as next-generation sequencing, which allows for a comprehensive analysis of microbial composition. Thus, microbiome testing that involves analyzing the microbial community in the female reproductive tract for imbalance or dysbiosis is a breakthrough point with great potential to aid in the treatment of recurrent implantation failure. For recurrent implantation failures, microbiome testing can help fertility specialists tailor a personalized treatment plan. By understanding an individual’s specific microbial profile, physicians can make informed decisions about potential interventions such as antibiotic therapy, probiotics, or other microbial modulation therapies. Of course, the use of microbiome testing should be carefully considered on a case-by-case basis and should be used in conjunction with other diagnostic tools and assessments to provide optimal treatment for patients with implantation failure.

Recurrent Implantation Failure: Reality or a Statistical Mirage?: Consensus Statement From the July 1, 2022 Lugano Workshop on Recurrent Implantation Failure

ABSTRACT Despite advancements in assisted reproductive technologies over the past few decades, a 35% failure rate is still observed for unexplained reasons when transferring euploid embryos to an anatomically normal uterus. Recurrent implantation failure (RIF) is defined based on cumulative ART cycles failing; however, there is an absence in consensus on the diagnostic criteria, which leads to the risk of overdiagnosing and overtreating the condition. This consensus statement by the Lugano RIF Workshop aimed to merge variation in the clinical concept and management of RIF, focusing primarily on euploid blastocyst transfers in hormone replacement-primed cycles. The workshop comprised a panel of 27 international experts selected on the basis of overall research activities and publications on the subject. A literature search as conducted for all relevant titles and abstracts published between January 2015 and May 2022 for review. Consensus conclusions were based on the literature search and expert opinions. Consideration of an RIF diagnosis was determined to focus on failure to achieve sustained implantation (defined as a gestational sac identified on ultrasound). Historically, RIF was defined as the failed transfer of >10 embryos; however, today, a common number used is 3, which is also similar to the rate of failure based on a 95% confidence interval. Based on large clinical data sets, approximately 2%–5% of patients pursuing ART treatment may have RIF, and the decline in future implantation rate with each cycle fits more to an exponential delay curve than a linear decrease. The diagnosis of RIF should not be considered based on a single cohort of embryos, but rather on the number of euploid blastocyst transferred adjusted to patient’s age. In the absence of abnormal uterine bleeding and normal uterine size, further evaluation of asymptomatic adenomyosis is not warranted. Testing for biomarkers of endometrial receptivity and the endometrial microbiome have not been validated using RCTs and should not be used. The role of chronic inflammation in RIF remains unclear, but evidence suggests chronic endometritis does not directly play a role in ART outcome as assessed by sustained implantation rate after euploid embryo transfer. Substantial evidence exists that is low progesterone on the day of embryo transfer results in worse ART outcomes, and that IM progesterone may be superior to vaginal progesterone. There are no effective treatments for RIF currently, and immunomodulatory treatments that may be offered including glucocorticoids, IVIG, and GCSF have a lack of comprehensive data. This consensus document finds that RIF is likely present in fewer than 5% of patients undergoing ART and has been overdiagnosed and overtreated without sufficient critical evaluation of the presumed condition. It also highlights areas where future research is needed, both in the pathophysiologic cause of RIF and hypothetical treatment modalities.

One center experience with a personalized frozen-thawed embryo transfer in patients with recurrent implantation failure

PurposeDisplaced endometrial receptivity has been discussed as a possible cause of recurrent implantation failure in patients undergoing assisted reproductive technology. The aim of this study was to document our experience with the endometrial receptivity analysis in patients with recurrent implantation failure.MethodsThis retrospective cohort study, conducted at the Fertility Centre of the University Hospital, Duesseldorf Germany, presents the results of the endometrial receptivity analysis in 67 patients with recurrent implantation failure and compares the clinical outcome between these 67 patients who underwent a personalized frozen-thawed embryo transfer guided by the results of the endometrial receptivity analysis and 32 patients with recurrent implantation failure who performed a standardized frozen-thawed embryo transfer.ResultsThe data analysis revealed a displaced endometrial receptivity in 73% (49/67) of all tested patients. Out of these patients, 24% (12/49) were early receptive, 74% (36/49) were pre-receptive, and 2% (1/49) were post-receptive. Comparison of pregnancy rate, clinical pregnancy rate, and live-birth rate between personalized (49%, 39%, 27%, respectively) and standardized embryo transfer (44%, 31%, 19%, respectively) reveals no statistically significant difference. In both groups, patients had an average of four unsuccessful embryo transfers.ConclusionIn this cohort of patients with recurrent implantation failure, the endometrial receptivity analysis showed a high incidence of displaced endometrial receptivity. However, a personalized embryo transfer did not increase reproductive outcome. Displaced endometrial receptivity might not be the main cause for recurrent implantation failure in this cohort.

Characterization of epidermal growth factor-like domain 7 (EGFL7) expression in normal endometrium and in the endometrium of women with poor reproductive outcomes.

Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes? EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF). The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL. This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF. Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes. Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not. This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions. Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention. This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose. Not applicable.

Association of Polymorphisms in FSHR, INHA, ESR1, and BMP15 with Recurrent Implantation Failure.

Recurrent implantation failure (RIF) refers to two or more unsuccessful in vitro fertilization embryo transfers in the same individual. Embryonic characteristics, immunological factors, and coagulation factors are known to be the causes of RIF. Genetic factors have also been reported to be involved in the occurrence of RIF, and some single nucleotide polymorphisms (SNPs) may contribute to RIF. We examined SNPs in FSHR, INHA, ESR1, and BMP15, which have been associated with primary ovarian failure. A cohort of 133 RIF patients and 317 healthy controls consisting of all Korean women was included. Genotyping was performed by Taq-Man genotyping assays to determine the frequency of the following polymorphisms: FSHR rs6165, INHA rs11893842 and rs35118453, ESR1 rs9340799 and rs2234693, and BMP15 rs17003221 and rs3810682. The differences in these SNPs were compared between the patient and control groups. Our results demonstrate a decreased prevalence of RIF in subjects with the FSHR rs6165 A>G polymorphism [AA vs. AG adjusted odds ratio (AOR) = 0.432; confidence interval (CI) = 0.206-0.908; p = 0.027, AA+AG vs. GG AOR = 0.434; CI = 0.213-0.885; p = 0.022]. Based on a genotype combination analysis, the GG/AA (FSHR rs6165/ESR1 rs9340799: OR = 0.250; CI = 0.072-0.874; p = 0.030) and GG-CC (FSHR rs6165/BMP15 rs3810682: OR = 0.466; CI = 0.220-0.987; p = 0.046) alleles were also associated with a decreased RIF risk. Additionally, the FSHR rs6165GG and BMP15 rs17003221TT+TC genotype combination was associated with a decreased RIF risk (OR = 0.430; CI = 0.210-0.877; p = 0.020) and increased FSH levels, as assessed by an analysis of variance. The FSHR rs6165 polymorphism and genotype combinations are significantly associated with RIF development in Korean women.

Gene profiling reveals the role of inflammation, abnormal uterine muscle contraction and vascularity in recurrent implantation failure.

Objective: Recurrent implantation failure (RIF) is now disturbing numerous infertile couples accepting assisted reproductive technology (ART). And the endometrial factors are crucial causes of recurrent implantation failure. However, its mechanism is still unclear. Thus, the aim of this study is to identify altered biologic processes in endometrium that may contribute to recurrent implantation failure. Methods: We recruited two microarray datasets (GSE103465, GSE111974) from Gene Expression Omnibus database (GEO), which contain endometrium from RIF and normal women during implantation period. Using the online tools GEO2R and Venny, we identified Differentially Expressed Genes (DEGs) of selected datasets, and obtained common DEGs. Gene Ontology (GO) terms, Kyoto Encyclopedia of Genes and Genomes (KEGG) and BioCatar pathway enrichment were conducted with Enrichr platform, "ssgsea" and "ggplot2" package of RStudio. PPI networks and hub gene related TF-gene interaction and TF-miRNA co-regulation networks were built via online tools STRING and NetworkAnalyst. Immune infiltration analysis was performed by CIBERSORT platform. Recurrent implantation failure subgroup identification was achieved through "ConsensusClusterPlus," "tsne," "ssgsea", and "ggpubr" package in RStudio. Diagnostic characteristic ROC curves were constructed via "pROC" and "ggplot2" package of RStudio. Enrichr platform was utilized to find drugs targeting hub genes. Results: 26 common DEGs were confirmed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes/BioCarta analysis determined common DEGs were mainly enriched in inflammation associated pathways including TNF, NF-κB, IL-4, IL-10, IL-6, and TGF-β signaling pathways. Five hub genes (PTGS2, VCAM1, EDNRB, ACTA2, and LIF) and related TF-gene and TF-miRNA interactions were identified. Immune infiltration analysis indicated the importance of macrophage M2 in recurrent implantation failure patients. Importantly, subgroup identification analysis highlighted that recurrent implantation failure patients can be divided into two subgroups with different phenotypes. Moreover, the ROC curves and drugs may provide new diagnostic and therapeutic thought for recurrent implantation failure.

Role of inflammatory factors in the etiology and treatment of recurrent implantation failure.

Recurrent implantation failure (RIF) is characterized by the absence of implantation after high-grade embryos are transferred to the endometrium by at least three in vitro fertilization cycles. It is one of the most important factors contributing to reproductive failure. After numerous barriers have been overcome to obtain good-quality embryos, RIF causes extreme distress and frustration in women and couples. In recent years, significant progress has been made in understanding how inflammatory factors, which include pro-inflammatory factors, anti-inflammatory factors, chemokines, and other molecules, contribute to RIF. Immunological abnormalities, hypercoagulability, and reproductive diseases are considered potential causes of RIF. In alloimmune disorders, inflammatory factors can affect the success rate of embryo implantation by altering T helper (Th)1/Th2 and Th17/regulatory T cell ratios and causing imbalances of uterine natural killer cells and macrophages. Autoimmune disorders can also lead to RIF. Inflammatory factors also play key roles in RIF-related disorders such as hypercoagulability, chronic endometritis, adenomyosis, hydrosalpinx, and endometriosis. This review focuses on the roles of inflammatory factors in RIF, including immune factors, blood hypercoagulable states, and reproductive diseases such as chronic endometritis, adenomyosis, hydrosalpinx, and endometriosis. It also summarizes the different treatments according to the causes of RIF and discusses the efficacy of sirolimus, peripheral blood mononuclear cells, low-dose aspirin combined with low-molecular-weight heparin, blocking interleukin-22, and gonadotropin-releasing hormone agonists in the treatment of RIF.

A novel lncRNAlncSAMD11-1: 1 interacts with PIP4K2A to promote endometrial decidualization by stabilizing FoxO1 nuclear localization.

Decidualization is essential for a successful pregnancy and determines embryo implantation and pregnancy maintenance. Abnormal decidualization is one of the main causes of recurrent implantation failure (RIF). Studies have shown that large amounts of long noncoding RNAs (lncRNAs) are abnormally expressed in endometrial samples from patients with RIF. However, the functional contributions of lncRNAs to decidualization in RIF have not been explored. In this study, we found that lncSAMD11-1:1 was significantly declined in the endometria of patients with RIF. The knockdown of lncSAMD11-1:1 in human endometrial stromal cells (hESCs) restrained decidualization and embryo implantation in vitro, while the overexpression of lncSAMD11-1:1 facilitated hESC decidualization and embryo implantation in vitro and ameliorated decidualization in RIF patients. Mechanistically, lncSAMD11-1:1 and phosphatidylinositol-5-phosphate 4-kinase type 2 alpha (PIP4K2A) translocated out of nucleus and bound to each other during decidualization, thereby inhibiting the phosphorylation of AKT and promoting FoxO1 nuclear localization. These data suggest that lncSAMD11-1:1 might be a critical novel lncRNA functionally required for human decidualization, and the dysregulation of lncSAMD11-1:1 in the endometrium may be a new predisposing factor of RIF.

P-431 Functionally active microbiota in the receptive-phase endometria in women with recurrent implantation failure

Abstract Study question Does the functionally active microbial composition at the receptive phase endometria differ between women with recurrent implantation failure (RIF) and healthy controls? Summary answer Women with RIF have different functionally active microbiota in the receptive-phase endometria that are involved in distinct metabolical pathways when compared to healthy women. What is known already Recurrent implantation failure (RIF) is a common and burdening diagnosis in infertility treatment. Despite scientific advances in the field, the cause of RIF remains unclear and the treatment options ambiguous. Microorganisms have important role in human physiology and pathophysiology, and their potential involvement in endometrial functions and health are gaining support. Our previous work of microbiota mapping of healthy endometrium identified over 5000 functionally active microorganisms (bacteria, viruses, fungi, archaea) that participate in key metabolic pathways for embryo implantation. In the present work we focus on microbiota mapping in RIF patients, where endometrial dysfunction is suspected. Study design, size, duration A case-control age-match study of 20 RIF and 20 healthy controls was performed. Endometrial samples were obtained at the mid-secretory phase (LH + 7/8) using Pipelle curette. Participants/materials, setting, methods Total RNA sequencing (RNA-seq) was performed. R package MetagenomeSeq was used for metataxonomic microorganismal analyses and HUMAnN2 algorithm was used for functional metagenome assessment. The microbial maps were created using Kraken metagenomic sequence classification. With this approach we were able to identify and map the ‘active’ microorganisms in samples. Main results and the role of chance We identified a total of 4175 microorganisms in the human endometrial samples including different bacteria, fungi, viruses, and archaea. At the taxonomic level, RIF patients demonstrated a total of 180 microorganism with differential abundance when compared to controls (FDR<0.05), where the biggest difference was in Streptomyces, Xanthomonas, Fusarium and Burkholderia that were significantly less detected in RIF. The metabolical functions analysis of the microbes differed significantly between the two groups, with a total of 150 different pathways, where fatty acid biosynthesis, mucin glycosylation, folate pathway and serotonin pathways were influenced in RIF. Limitations, reasons for caution Poly A enrichment could avoid the detection of some microorganism. However, the resolution power in microbial identification was comparable to previous metatranscriptomics published studies. Wider implications of the findings Our study findings are first to demonstrate that alive microbes are involved in the uterine pathology as is RIF, where different metabolic pathways could be dysregulated. This knowledge could help to develop pre-/probiotic strategies to modulate uterine environment for improving endometrial functions in RIF. Trial registration number not applicable

Human Platelet-Rich Plasma Facilitates Angiogenesis to Restore Impaired Uterine Environments with Asherman's Syndrome for Embryo Implantation and Following Pregnancy in Mice.

Asherman’s syndrome (AS) is caused by intrauterine adhesions and inactive endometrium from repeated curettage of the uterine endometrium. AS is a major cause of recurrent implantation failure and miscarriage and is very difficult to treat because of the poor recovery of endometrial basal cells. Platelet-rich plasma (PRP) has abundant growth factors that may induce angiogenesis and cell proliferation. Here, we demonstrate that human PRP (hPRP) significantly enhances angiogenesis to restore embryo implantation, leading to successful pregnancy in mice with AS. In mice with AS, hPRP treatment considerably reduced the expression of fibrosis markers and alleviated oligo/amenorrhea phenotypes. Mice with AS did not produce any pups, but the hPRP therapy restored their infertility. AS-induced abnormalities, such as aberrantly delayed embryo implantation and intrauterine growth retardation, were considerably eliminated by hPRP. Furthermore, hPRP significantly promoted not only the elevation of various angiogenic factors, but also the migration of endometrial stromal cells. It also increased the phosphorylation of STAT3, a critical mediator of wound healing, and the expression of tissue remodeling genes in a fibrotic uterus. PRP could be a promising therapeutic strategy to promote angiogenesis and reduce fibrosis in impaired uterine environments, leading to successful embryo implantation for better clinical outcomes in patients with AS.

Increased expression of HMGB1 in the implantation phase endometrium is related to recurrent implantation failure.

Impaired endometrial receptivity was the main cause of recurrent implantation failure (RIF); however, its underlying mechanisms had not been elucidated. This study aimed to determine the expression level of high-mobility group box protein 1 (HMGB1) in the endometrium with RIF and its effect on endometrial receptivity. Genome-wide expression profiling, real-time reverse transcription PCR, immunohistochemical staining, western blot, and in vitro assays were performed in this study. We found that HMGB1 expression was significantly decreased in the implantation phase endometrium in the control group (patients with tubal infertility and successfully achieve conception after the first embryo transfer) (P = 0.006). However, the expression levels of HMGB1 mRNA and protein were significantly upregulated during the implantation phase in endometrial tissues obtained from patients with RIF compared to that in the control group (P = 0.001), consistent with the results of the genome-wide expression profiling. Moreover, in vitro assays showed that increased expression of HMGB1 in human endometrial epithelial cells dramatically displayed a marked deficiency in supporting blastocysts and human embryonic JAR cells adhesion, which mimic the process of embryo adhesion. These findings strongly indicated that increased HMGB1 levels suppressed the epithelial cell adhesion capability, therefore contributing to impaired endometrial receptivity in patients with recurrent implantation failure, which can be used as a target for the recognition and treatment of recurrent implantation failure in clinical practice.

Recurrent Implantation Failure-Is It the Egg or the Chicken?

Recurrent implantation failure (RIF) is an undefined, quite often, clinical phenomenon that can result from the repeated failure of embryo transfers to obtain a viable pregnancy. Careful clinical evaluation prior to assisted reproduction can uncover various treatable causes, including endocrine dysfunction, fibroid(s), polyp(s), adhesions, uterine malformations. Despite the fact that it is often encountered and has a critical role in Assisted Reproductive Technique (ART) and human reproduction, RIF’s do not yet have an agreed-on definition, and its etiologic factors have not been entirely determined. ART is a complex treatment with a variable percentage of success among patients and care providers. ART depends on several factors that are not always known and probably not always the same. When confronted with repeated ART failure, medical care providers should try to determine whether the cause is an embryo or endometrium related. One of the most common causes of pregnancy failure is aneuploidy. Therefore, it is likely that this represents a common cause of RIF. Other RIF potential causes include immune and endometrial factors; however, with a very poorly defined role. Recent data indicate that the possible endometrial causes of RIF are very rare, thereby throwing into doubt all endometrial receptivity assays. All recent reports indicate that the true origin of RIF is probably due to the “egg”.

CLINICAL UTILITY OF THE NEW ENDOMETRIAL RECEPTIVITY TEST IN ADVANCED MATERNAL AGE PATIENTS WITH RECURRENT IMPLANTATION FAILURE

Although aneuploid embryos account for the major cause of implantation failure, especially in cases of advanced maternal age (AMA), several studies have investigated that even euploid blastocysts fail to implant in about 40% of transfers. Failure of an euploid embryo to implant may suggest the importance of the synchronization between an embryo and the window of implantation as another potential cause of recurrent implantation failure (RIF). In this study, we assessed whether the ERPeakSM endometrial receptivity test is clinically useful even for AMA patients with RIF. A retrospective review was performed for 480 RIF patients who had 3 or more failed embryo transfers and underwent ERPeakSM testing in two private fertility clinics between April 2019 and June 2020. We evaluated clinical outcomes of 306 RIF patients who underwent personalized embryo transfer (pET) in an HRT cycle with morphologically good quality embryos after ERPeakSM testing. A receptive (R) or nonreceptive (NR) endometrial status according to ERPeak SM testing and reproductive outcomes were compared for patients aged 38 years and over (AMA group) versus those under 38 years (non-AMA group). The statistical analysis was performed by using chi-square analysis or Mann-Whitney U test where appropriate with significance determined by a p=0.05. In AMA group (n=326, average age: 41.8±2.6 years, range: 38-51), ERPeakSM testing showed a NR result in 144 patients (44.2%) and a R result in 182 patients (55.8%). In the NRgroup, 66.7% (96/144) indicated a pre-receptive state and 33.3% (48/144) resulted in post-receptive state. In non-AMA group (n=154, average age: 34.0±2.7 years, range: 20-37), ERPeakSM testing showed a NR result in 65 patients (42.2%) and a R result in 89 patients (57.8%). In the NR group, 52.3% (34/65) indicated a pre-receptive state and 47.7% (31/65) resulted in post-receptive state. The rate of pre-receptive state in the AMA group was significantly higher than the non-AMA group (p<0.05). After pET with morphologically good quality embryos for NR patients, we found that the clinical pregnancy rate (CPR) and implantation rate (IR) were similar between NR and R patients (37.4% vs. 37.9% and 30.3% vs. 25.3%, respectively), which is consistent with previous studies of pET based on the ERA test. Among NR patients, pET for AMA group (n=73, average age: 41.0±1.9 years) and non-AMA group (n=50, average age: 34.4±3.2 years) showed similar CPR (34.2% vs. 42.0%, p=0.38), IR (27.4% vs. 31.3%, p=0.58), miscarriage rate (20.0% vs. 19.0%, p=0.94) and live birth rate (75.0% vs. 82.4%, p=0.59), respectively. Although maternal age did not influence the frequency of NR, a pre-receptive status detected by ERPeakSM was more frequently observed in AMA patients. pET after ERPeakSM testing may improve reproductive outcomes of AMA patients with RIF.

Evaluation of Pregnancy Outcomes of Vitrified-Warmed Blastocyst Transfer before and after Endometrial Receptivity Analysis in Identical Patients with Recurrent Implantation Failure

Background: The clinical value of personalized embryo transfer (pET) guided by the endometrial receptivity analysis (ERA) tests for recurrent implantation failure (RIF) cases is still unclear. The aim of this study is to clarify the efficacy of ERA leading to personalization of the day of embryo transfer (ET) in RIF patients. Methods: A retrospective study was performed for 94 patients with RIF who underwent ERA between July 2015 and December 2019. Pregnancy outcomes in a previous vitrified-warmed blastocyst transfer (previous VBT) and a personalized vitrified-warmed blastocyst transfer (pVBT) in identical patients were compared. The details of each pVBT were further analyzed between patients in a non-displaced group, which indicated “receptive” cases in ERA results and those who were in the displaced group, which indicated “non-receptive” cases. Results: When the pregnancy rate, both per patient and per transfer cycle, of previous VBT and pVBT were compared, a significant increase in pVBT was observed between the two methods (5.3% vs. 62.8%, 4.4% vs. 47.9%, respectively). The pregnancy rates, implantation rates, and clinical pregnancy rates of the first pVBT were significantly higher in the displaced group than the non-displaced group. The cumulative ongoing pregnancy rate of the displaced group tended to be higher compared to that of the non-displaced group in the first pVBT, although the difference was not statistically significant (51.0% vs. 31.1%, [Formula: see text]). Conclusions: Our study demonstrates that pVBT guided by ERA tests may improve pregnancy outcomes in RIF patients whose window of implantation (WOI) is displaced, and its effect may be more pronounced at the first pVBT. The displacement of WOI may be considered to be one of the causes of RIF, and its adjustment may contribute to the improvement of pregnancy outcomes in RIF patients.

Endometrial gene expression profiling of recurrent implantation failure after in vitro fertilization.

Recurrent implantation failure (RIF) is diagnosed when good-quality embryos repeatedly fail to implant after transfer in several in vitro fertilization (IVF) treatment cycles. Different expression profiles in maternal mRNAs could be referring to many diseases including RIF. This study aimed to reveal significantly dysregulated selected genes expression between healthy fertile women and RIF patients in the implantation window days of the natural menstrual cycle. MME, WWC1, TNC, and FOXP3 genes were chosen as target genes regarding their possible relations with the implantation process. Pathways with these genes were identified and the relationship between these pathways and RIF was investigated. In this study, the endometrial biopsy samples were collected in the secretory phase (cycle day 20-24) of the menstrual cycle from RIF patients (n = 34) and healthy fertile controls (n = 34). After "Pathway and network-oriented GWAS analysis" (PANOGA) and "Kyoto Encyclopedia of Genes and Genomes" (KEGG) pathway analysis; "Membrane Metalloendopeptidase" (MME), "WW and C2 Domain Containing 1" (WWC1), "Tenascin C" (TNC) and "Forkhead Box P3" (FOXP3) genes were chosen as target genes by regarding their possible relation with implantation process. Detection of differences in mRNA expressions between the control group and RIF patients has been performed with the droplet digital PCR (ddPCR) method. Results of the study showed that MME and WWC1 genes expression levels are significantly (p < 0,05) up-regulated 4.9 and 5.2 times respectively and TNC gene expression level is significantly (p < 0,05) down-regulated 9 times in the RIF samples compared to the control group. However, no statistically significant difference was observed between the patient group and the control group in the expression of the FOXP3 gene (p < 0.05). Changes are observed in the expression of the renin-angiotensin system pathway in which the MME gene is involved in the implantation process. The increase in MME gene expression can be speculated to cause implantation failure by restricting the invasion of trophoblast cells. Increasing WWC1 gene expression in the Hippo signaling pathway inhibits "Yes-associated protein 1" (YAP) expression, which is a transcriptional cofactor. Inhibition of YAP protein expression may impair the implantation process by causing the failure of endometrial decidualization. The TNC gene is located in the focal adhesion pathway and this pathway reduces cell adhesion on the endometrial surface to facilitate the attachment of the embryo to the endometrium. The reason for implantation failure might be that the intercellular connections are not suitable for implantation as a result of decreased expression of the focal adhesion pathway in which the TNC gene is effective. Considering the relations between the pathways of the target genes and the implantation process, changes in the expression of target genes might be a cause of RIF.

Construction of circRNA-miRNA-mRNA network in the pathogenesis of recurrent implantation failure using integrated bioinformatics study.

This research attempted to elucidate the molecular components are involved in the pathogenesis of recurrent implantation failure (RIF). We initially identified that 386 mRNAs, 144 miRNAs and 2548 circRNAs were differentially expressed (DE) in RIF and then investigated the genetic cause of the observed abnormal expression by constructing a circRNA‐miRNA‐mRNA network considering the competing endogenous RNA theory. We further analysed the upstream transcription factors and related kinases of DEmRNAs (DEMs) and demonstrated that SUZ12, AR, TP63, NANOG, and TCF3 were the top five TFs binding to these DEMs. Besides, protein‐protein interaction analysis disclosed that ACTB, CXCL10, PTGS2, CXCL12, GNG4, AGT, CXCL11, SST, PENK, and FOXM1 were the top 10 hub genes in the acquired network. Finally, we performed the functional enrichment analysis and found that arrhythmogenic right ventricular cardiomyopathy (ARVC), hypertrophic cardiomyopathy (HCM), pathways in cancer, TNF signalling pathway and steroid hormone biosynthesis were the potentially disrupted pathways in RIF patients. Optimistically, our findings may deepen our apprehensions about the underlying molecular and biological causes of RIF and provide vital clues for future laboratory and clinical experiments that will ultimately bring a better outcome for patients with RIF.

P73: Macrophage inhibits immune suppression by secreting exosomal PiRNA‐38028: Important cause of recurrent implantation failure

P73: Macrophage inhibits immune suppression by secreting exosomal PiRNA‐38028: Important cause of recurrent implantation failure

ATF3 deficiency impairs the proliferative\u2013secretory phase transition and decidualization in RIF patients

Decidualization is a complex process involving cellular proliferation and differentiation of the endometrial stroma and is required to establish and support pregnancy. Dysregulated decidualization has been reported to be a critical cause of recurrent implantation failure (RIF). In this study, we found that Activating transcription factor 3 (ATF3) expression was significantly downregulated in the endometrium of RIF patients. Knockdown of ATF3 in human endometrium stromal cells (hESCs) hampers decidualization, while overexpression could trigger the expression of decidual marker genes, and ameliorate the decidualization of hESCs from RIF patients. Mechanistically, ATF3 promotes decidualization by upregulating FOXO1 via suppressing miR-135b expression. In addition, the endometrium of RIF patients was hyperproliferative, while overexpression of ATF3 inhibited the proliferation of hESCs through CDKN1A. These data demonstrate the critical roles of endometrial ATF3 in regulating decidualization and proliferation, and dysregulation of ATF3 in the endometrium may be a novel cause of RIF and therefore represent a potential therapeutic target for RIF.

Evaluation of Expression and Phosphorylation of Progesterone Receptor in Endometrial Stromal Cells of Patients with Recurrent Implantation Failure Compared to Healthy Fertile Women

Recurrent implantation failure (RIF) is the repeated failure of good-quality embryos in implantation process following several assisted reproduction cycles. Disruption of the endometrial receptivity is one of the main causes of RIF. Progesterone plays a pivotal role in the endometrial receptivity through the regulation of gene expression pattern by binding to its receptors in the endometrial cells. The aim of this study was to evaluate the expression level of progesterone receptor (PR) and its phosphorylated form in the endometrial stromal cells (eSC) of RIF patients and compare it to the eSC of healthy fertile women as control group. After isolation of the eSC from biopsy samples of RIF patients and healthy fertile women and their characterization, expression levels of PR mRNA, PR protein, and phospho-Ser294 PR protein were evaluated by quantitative real-time PCR and immunofluorescence staining, respectively. The results demonstrated a significant reduction in PR mRNA expression (P < 0.01.) and phospho-Ser294 PR protein (P < 0.05) level in RIF patients compared to the control group. These data for the first time suggest that the expression of PR and its phosphorylated form are impaired in RIF patients. Therefore, designing therapeutic methods for improving PR expression status and its regulation in the endometrium of RIF patients may help in improving the final reproductive outcomes of these cases.