Uncovering hidden connections: the role of the male reproductive system microbiome and gut microorganisms in implantation: a critical review

Despite more than a century of intensive study, the mechanisms of successful pregnancy remain unclear. Recent research suggests that NF-κB (nuclear factor kappa B) plays an important role in embryo implantation. In the current study, we aimed to identify SNPs that contribute to genetic susceptibility for recurrent implantation failure (RIF). Thus, we examined the potential associations between RIF and ten SNPs (rs28362491, rs3774932, rs1598856, rs230528, rs230521, rs3774956, rs4648055, rs3774964, rs4648068, and rs3774968) of the NF-κB gene. Participants included 209 patients with RIF and 395 controls. Our results revealed that there were statistically significant differences observed in the allelic and genotypic frequencies of the rs28362491 promoter in the NF-κB gene. The frequency of the del/ del genotype was significantly higher in RIF patients than in healthy controls (P = 0.004). Compared with healthy controls, the RIF patients carried a higher frequency of the rs28362491 del allele (P = 0.010). Furthermore, strong linkage disequilibrium was observed in the three identified haplotype blocks (D' > 0.9). Particularly, in block 1 (rs230528-rs230521), the A-C haplotype occurred significantly more frequently (P = 0.029) in subjects with RIF (P = 0.0003). In contrast, the A-G haplotype occurred significantly less frequently (P = 0.008) in RIF subjects. These findings support an important role for G-712A polymorphisms of NF-κB in RIF, and may guide future studies that aim to characterize genetic risk factors for RIF.

CircSTK40 contributes to recurrent implantation failure via modulating the HSP90/AKT/FOXO1 axis

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.

The endometrium becomes receptive to the embryo only in the mid-luteal phase, but not in the other stages of the menstrual cycle. Endometrial factors play an important role in implantation. Women with recurrent miscarriage and recurrent implantation failure have both been reported to have altered expression of receptivity markers during the window of implantation. We aimed to compare the gene expression profiles of the endometrium in the window of implantation among women with unexplained recurrent implantation failures (RIF) and unexplained recurrent miscarriages (RM) by RNA sequencing (RNA-Seq). In total 20 patients (9 RIF and 11 RM) were recruited. In addition 4 fertile subjects were included as reference. Endometrium samples were precisely timed on the 7th day after luteal hormone surge (LH + 7). All the 24 endometrium samples were extracted for total RNA. The transcriptome was determined by RNA-Seq in the first 14 RNA samples (5 RIF, 6 RM and 3 fertile). Differentially expressed genes between RM and RIF were validated by quantitative real-time PCR (qPCR) in all 24 RNA samples (9 RIF, 11 RM and 4 fertile). Transcriptomic profiles of RM and RIF, but not control samples, were separated from each other by principle component analysis (PCA) and support vector machine (SVM). Complementary and coagulation cascades pathway was significantly up-regulated in RIF while down-regulated in RM. Differentially expressed genes C3, C4, C4BP, DAF, DF and SERPING1 in complement and coagulation cascade pathway between RM and RIF were further validated by qPCR. This study compared endometrial transcriptome among patients with RIF and RM in the window of implantation; it identified differential molecular pathways in endometrium between RIF and RM, which potentially affect the implantation process.

Editor: In June 2010, a workshop entitled “Mucosal Immunity in the Male and Female Reproductive Tract and Prevention of HIV Transmission” was organized by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) in collaboration with the Dartmouth Medical School at the Dartmouth Hitchcock Medical Center. This meeting was a first of its kind since it brought together leading scientists from around the world who are actively working in the area of heterosexual transmission of HIV, HIV mucosal immunity, and reproductive immunology. In a workshop/presentation format, researchers discussed HIV transmission and immune responses in the male and female reproductive tracts (RTs) and explored ways to harness this knowledge for the development of novel and more comprehensive approaches to prevention. Despite the fact that HIV is mainly transmitted sexually through the genital tract, the immune systems of the male and female genital mucosa and the ways in which they are influenced by hormonal balance have received very little attention. Compared with other mucosae, the male and female RTs are covered by distinct epithelial cell layers and types of mucus, have different commensal flora, and have unique innate and adaptive immune mechanisms. The meeting at Dartmouth aimed to fill this gap by discussing the most recent developments in basic, clinical, and translational studies in the field of reproductive immunology as they relate to the transmission of HIV, highlighting the potential of immunity in the male and female RTs in preventing HIV infection, and facilitating cross-fertilization between HIV researchers and those in the field of reproductive immunology. The organizing committee composed of Drs. Charles R. Wira, Dartmouth Medical School; Fulvia Veronese, NIAID; Geetha Bansal, NIAID; Susan Cu-Uvin, the Warren Alpert Medical School of Brown University; Charu Kaushic, McMaster University; Jiri Mestecky, University of Alabama at Birmingham; and Robin Shattock, St. Georges, University of London, developed a scientific agenda that focused on a broad range of cross-cutting topics to be addressed by leaders in the field. Topics included the unique characteristics of the immune system in the female and male RTs, which have relevance to HIV transmission, and early virological and immunological events in male-to-female and female-to-male transmission of HIV. Functional and immunological distinctions between the gastrointestinal and RTs were highlighted and contrasted to male and female RT biology and HIV transmission. Lessons learned from reproductive immunology were discussed, and the unique relationship between factors that influence sexual HIV transmission and human reproductive biology were brought together to identify novel approaches to prevent HIV transmission and to measure genital immune responses to HIV. Lively discussions resulted in new understandings of the complexities of the male and female RTs as they pertain to susceptibility to infection and protection against HIV, and highlighted the gaps that remain to be answered. The scientific meeting was followed by a public session entitled “The Forgotten Epidemic: AIDS in the 21st Century” for the at-large scientific faculty at Dartmouth and the general public, which focused on the impact HIV/AIDS is still having and the challenges we are confronting for its prevention and treatment. The public session was well-attended and highlighted the importance of the prevention and treatment of AIDS to the general public in juxtaposition to our scientific understanding of the processes involved in HIV transmission, treatment, and prevention. Given the groundbreaking nature of this meeting, the American Journal of Reproductive Immunology is publishing on March 1, 2011, a special issue dedicated exclusively to the immunology of the female and male RTs and HIV sexual transmission and its prevention. This special issue includes review articles written by invited speakers and two letters to the editors by Dr. C. Everett Koop, former Surgeon General of the United States, who delivered the keynote address; and Ms. Dawn Averitt Bridge, founder of The Well Project; who spoke about the impact that HIV/AIDS is still having on different populations of women in the United States.

BACKGROUNDUterine natural killer cells (uNK) are the most abundant lymphocytes found in the decidua during implantation and in first trimester pregnancy. They are important for early placental development, especially trophoblast invasion and transformation of the spiral arteries. However, inappropriate uNK function has been implicated in reproductive failure, such as recurrent miscarriage (RM) or recurrent implantation failure (RIF). Previous studies have mainly focussed on peripheral NK cells (pNK), despite the well-documented differences in pNK and uNK phenotype and function. In recent years, there has been an explosion of studies conducted on uNK, providing a more suitable representation of the immune environment at the maternal–foetal interface. Here, we summarize the evidence from studies published on uNK in women with RM/RIF compared with controls.OBJECTIVE AND RATIONALEThe objectives of this systematic review and meta-analysis are to evaluate: differences in uNK level in women with RM/RIF compared with controls; pregnancy outcome in women with RM/RIF stratified by high and normal uNK levels; correlation between uNK and pNK in women with RM/RIF; and differences in uNK activity in women with RM/RIF compared with controls.SEARCH METHODSMEDLINE, EMBASE, Web of Science and Cochrane Trials Registry were searched from inception up to December 2020 and studies were selected in accordance with PRISMA guidelines. Meta-analyses were performed for uNK level, pregnancy outcome and uNK/pNK correlation. Narrative synthesis was conducted for uNK activity. Risk of bias was assessed by ROBINS-I and publication bias by Egger’s test.OUTCOMESOur initial search yielded 4636 articles, of which 60 articles were included in our systematic review. Meta-analysis of CD56+ uNK level in women with RM compared with controls showed significantly higher levels in women with RM in subgroup analysis of endometrial samples (standardized mean difference (SMD) 0.49, CI 0.08, 0.90; P = 0.02; I2 88%; 1100 women). Meta-analysis of CD56+ uNK level in endometrium of women with RIF compared with controls showed significantly higher levels in women with RIF (SMD 0.49, CI 0.01, 0.98; P = 0.046; I2 84%; 604 women). There was no difference in pregnancy outcome in women with RM/RIF stratified by uNK level, and no significant correlation between pNK and uNK levels in women with RM/RIF. There was wide variation in studies conducted on uNK activity, which can be broadly divided into regulation and receptors, uNK cytotoxicity, cytokine secretion and effect of uNK on angiogenesis. These studies were largely equivocal in their results on cytokine secretion, but most studies found lower expression of inhibitory receptors and increased expression of angiogenic factors in women with RM.WIDER IMPLICATIONSThe observation of significantly increased uNK level in endometrium of women with RM and RIF may point to an underlying disturbance of the immune milieu culminating in implantation and/or placentation failure. Further research is warranted to elucidate the underlying pathophysiology. The evidence for measuring pNK as an indicator of uNK behaviour is sparse, and of limited clinical use. Measurement of uNK level/activity may be more useful as a diagnostic tool, however, a standardized reference range must be established before this can be of clinical use.

Despite a thorough work-up, the underlying cause remains unknown in a large proportion of cases with recurrent implantation failure. Sperm plays an incontrovertible role in initiating and maintaining a successful pregnancy. Thus, male factor evaluation could provide insight into the etiology of unexplained implantation failure. Sperm genetics and epigenetics are burgeoning areas in infertility research, providing new insights into the diagnosis and management of recurrent implantation failure. This chapter reviews the current evidence on sperm chromosomal aneuploidy and sperm DNA damage, with a focus on recurrent pregnancy loss and implantation failure. Potential causes of sperm DNA damage, including oxidative stress, and different diagnostic methods for detection of sperm DNA fragmentation are summarized. Roles of modifiable risk factors such as alcohol, smoking, varicocele, testicular heat stress, medications, and environmental toxins are discussed, and recommendations are made regarding potential therapeutic options and interventions. Finally, alternative and newer methods for sperm retrieval and selection for assisted reproductive technology are discussed, and opportunities for future assessment and treatment of male factors in recurrent implantation failure are covered.

Antiphospholipid antibodies in women with recurrent embryo implantation failure: A systematic review and meta-analysis

BackgroundGlobally, there is no conclusive data on the pregnancy outcomes of patients with recurrent implantation failure (RIF) who received the next embryo transfer. The purpose of this study is to summarize the pregnancy outcomes of patients with RIF after embryo transfer and understand the disease burden of patients with RIF.MethodsWe searched for literature from databases such as PubMed, Web of Science, Embase, and Cochrane Central Register of Controlled Trials from inception to May 19, 2024, and extracted the pregnancy outcomes of patients with RIF in the blank control group, including implantation rate (IR), clinical pregnancy rate (CPR), ongoing pregnancy rate (OPR), miscarriage rate (MR), live birth rate (LBR), and ectopic pregnancy rate (EPR). Subsequently, meta-analyses of the rates were summarized and subgroup analyses were performed based on implantation failures, embryo type, fresh/frozen embryo transfer and regions.ResultsA total of 110 studies (14,159 patients) were included in the meta-analysis. Globally, the overall IR, CPR, OPR, MR, LBR, and EPR of patients with RIF were 19.3%, 29.4%, 24.6%, 19.9%, 23.0%, and 0.9%, respectively. No differences in pregnancy outcomes were found between RIF patients with three or more implantation failures and those with two or more implantation failures. RIF patients who transferred blastocyst achieved significantly higher IR, higher CPR, higher OPR, higher LBR and lower EPR, but no lower MR. There are differences in IR and CPR among patients with RIF in different regions, and no significant differences in other pregnancy outcomes.ConclusionThis study summarizes the global pregnancy outcomes of patients with RIF who undergo subsequent embryo transfer. Pregnancy outcomes in patients with RIF may not be related to the number of implantation failures. Frozen blastocyst transfer is recommended for patients with RIF. Pregnancy outcomes in patients with RIF vary across regions.Protocol registrationThis study has been registered on PROSPERO (CRD 42024539968).Supplementary InformationThe online version contains supplementary material available at 10.1186/s12884-025-08372-8.

Study question What is the cumulative ongoing pregnancy rate within one year after visiting the RIF outpatient clinic in patients with RIF after IVF/ICSI treatment? Summary answer The cumulative ongoing pregnancy rate was 42.9% with a mean time to pregnancy of 8.8 months (95% CI 7.5-10.1 months). What is known already An estimated portion of 10-15% of IVF patients experience RIF. Multiple add-on treatments have been proposed, however, evidence for effective clinical therapeutic options still remain scarce. Although it is described that RIF patients might eventually benefit most from a ‘keep calm and carry on’ approach, in practice patients often seek clearer answers on their pregnancy chance and request further investigations/interventions. It would be helpful to give patients insight in their chances of achieving pregnancy after RIF, but at this moment little is known about their prognosis. This is further complicated by the lack of consistency in the definition of RIF. Study design, size, duration A prospective cohort study on 42 RIF patients after one-year follow-up was performed, as part of the MURIM (Multidisciplinary Research on Repeated Implantation Failure and Recurrent Miscarriages) study. Baseline characteristics including an endometrial assessment and ReceptIVFity (vaginal microbiome) test were collected at the RIF outpatient clinic. After a follow-up duration of minimal one year participants were asked to complete a questionnaire regarding fertility treatment and pregnancy outcome. Missing information was received via medical files. Participants/materials, setting, methods RIF patients aged 18 to 38 years old visiting the RIF outpatient clinic at Maastricht University Medical Centre+ between April 2019 until September 2021 were included. RIF was defined as consecutive implantation failure of three high quality embryos or ten embryos without a quality criterion. Clinical characteristics, pregnancy outcome and time to pregnancy was analyzed by survival analysis and cox hazard regression analysis via SPSS version 20.0. A p-value of < 0.05 was considered statistically significant. Main results and the role of chance Forty-two out of 44 contacted patients responded to the questionnaire (response rate = 95.5%). The ongoing pregnancy rate was 42.9% (Standard Deviation (SD): 0.50%) during the first year after visiting the RIF outpatient clinic. Mean time to ongoing pregnancy was 8.8 months (95%-CI: 7.5-10.1 months). The mean amount of embryo transfers (ETs) during this year was 2 (SD: 1.78). The ongoing pregnancy rate per ET (by survival analysis) for the first, third and sixth ET was 33.1%, 49.3% and 63.8%, respectively. To correct for a possible overestimation of the cumulative ongoing pregnancy rate per ET by normal survival analysis, pregnancy rates were calculated again using the number at risk of the total group during the first ET (pessimistic cumulative pregnancy rate). The pessimistic cumulative ongoing pregnancy rates were 33.1%, 42.9% and 48.6%, respectively. When comparing baseline characteristics by univariable cox hazard regression between women with and without ongoing pregnancy, no significant differences were found. Nineteen pregnancies were reported during the one-year follow-up, one conceived spontaneously (5.3%); four via fresh ET (IVF=15.8% and ICSI=5.3%); and 14 by frozen ET (IVF=26.4% and ICSI=47.4%). The pregnancies resulted in 14 livebirths (73.7%); one miscarriage (5.3%); one stillbirth (5.3%); and three ongoing pregnancies (15.8%). Limitations, reasons for caution Although this is the first study that prospectively describes pregnancy prognosis after RIF, it is limited by the short follow-up period and relatively small sample size. The RIF outpatient clinic consultation might have contributed to the obtained pregnancy results but the proportionality is unclear as there is no control group. Wider implications of the findings The obtained ongoing pregnancy prognosis after RIF is encouraging and justifies a conservative approach after three failed ETs. Furthermore, the high pregnancy rate indicates the need of well-defined, individualized diagnostic criteria to define RIF and to be able to deviate between couples with 'bad luck' and an underlying (treatable) cause. Trial registration number NL66835.068.18/METC18-040

Should patients be screened for chronic endometritis before assisted reproductive technology?

Endometrial injury to overcome recurrent embryo implantation failure: a systematic review and meta-analysis

Endometrial integrin expression in women with recurrent implantation failure after in vitro fertilization and its relationship to pregnancy outcome

Recurrent implantation failure (RIF) poses a significant challenge in assisted reproductive technology (ART) outcomes. The endometrium plays a crucial role in embryo implantation, and its protein expression profile is integral in determining receptivity. Proteomics has emerged as a valuable tool in unraveling the molecular intricacies underlying endometrial receptivity and RIF. The aim of the present review is to analyze the contribution of proteomics to the understanding of endometrial protein expression in women with RIF, based on the results of significant proteomic studies. Medline/Pubmed databases were searched using keywords pertaining to proteomics combined with terms related to RIF. 15 studies were included in the present review. Several proteins have been found to exbibit differential expression in endometrial biopsies and fluid samples between fertile women and women with RIF during the receptive endometrial phase. The profile of endometrial proteins varied significantly among the studies. Nevertheless, similar changes in the expression levels of annexin-6, progesterone receptor, MMP-2, and MMP-9 in the endometrium of women with RIF, were found in more than one study indicating that certain proteins could potentially be effective biomarkers of endometrial receptivity. Proteomics contributes significantly to the understanding of protein expression in the endometrium of women with RIF and the analysis of proteins in endometrial fluid are promising for improving the clinical management of RIF.

Despite numerous advances in fertility techniques, some individuals experience implantation failure. One of the therapeutic approaches is the study of immunological aspects of the implantation process in recurrent implantation failure (RIF) patients. Peripheral blood mononuclear cell (PBMC) therapy and platelet-rich plasma are currently available cell therapies. The aim of this study was to determine the expressions of the FGFR-2 and LIF genes that are regulated by miR-199a-5p and miR-125b-5p. These genes play a fundamental role in implantation in RIF patients treated with PBMCs. 20 patients clinically diagnosed with RIF were randomly assigned to a RIF patient with PBMCs intrauterine infusion group (n = 10) and RIF group (n = 10). Normal, healthy females (n = 10) comprised the control group. In order to examine the efficacy of the PBMCs injection in the treatment group, expressions of miR-199a-5p and miR-125-5p and FGFR-2 and LIF as their target genes, were evaluated in all three groups and were compared the results. We discovered that the RIF group had higher expressions of miR-199a-5p and miR-125-5p along with decreased expressions of their target genes. However, both FGFR-2 and LIF gene had elevated expressions in the RIF patients with PBMCs intrauterine infusion group compared to the RIF group, with significant decrease in miR-199a-5p and miR-125b-5p reciprocally. The treatment with PBMCs can be effective in changing the expression of microRNAs and genes associated with endometrial receptivity and by changes in the expression of them and their role during embryo development improve this process.