The endometrial transcriptome of infertile women with and without implantation failure.

INTRODUCTION: Implantation failure after transferring morphologically "good-quality" embryos in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) may be explained by impaired endometrial receptivity. Analyzing the endometrial transcriptome analysis may reveal the underlying processes and could help in guiding prognosis and using targeted interventions for infertility. This exploratory study investigated whether the endometrial transcriptome profile was associated with short-term or long-term implantation outcomes (ie success or failure). MATERIAL AND METHODS: Mid-luteal phase endometrial biopsies of 107 infertile women with one full failed IVF/ICSI cycle, obtained within an endometrial scratching trial, were subjected to RNA-sequencing and differentially expressed genes analysis with covariate adjustment (age, body mass index, luteinizing hormone [LH]-day). Endometrial transcriptomes were compared between implantation failure and success groups in the short term (after the second fresh IVF/ICSI cycle) and long term (including all fresh and frozen cycles within 12 months). The short-term analysis included 85/107 women (33 ongoing pregnancy vs 52 no pregnancy), excluding 22/107 women. The long-term analysis included 46/107 women (23 'fertile' group, ie infertile women with a live birth after ≤3 embryos transferred vs 23 recurrent implantation failure group, ie no live birth after ≥3 good quality embryos transferred), excluding 61/107 women not fitting these categories. As both analyses drew from the same pool of 107 samples, there was some sample overlap. Additionally, cell type enrichment scores and endometrial receptivity were analyzed, and an endometrial development pseudo-timeline was constructed to estimate transcriptomic deviations from the optimum receptivity day (LH + 7), denoted as ΔWOI (window of implantation). RESULTS: There were no significantly differentially expressed genes between implantation failure and success groups in either the short-term or long-term analyses. Principal component analysis initially showed two clusters in the long-term analysis, unrelated to clinical phenotype and no longer distinct following covariate adjustment. Cell type enrichment scores did not differ significantly between groups in both analyses. However, endometrial receptivity analysis demonstrated a potentially significant displacement of the WOI in the non-pregnant group compared with the ongoing pregnant group in the short-term analysis. CONCLUSIONS: No distinct endometrial transcriptome profile was associated with either implantation failure or success in infertile women. However, there may be differences in the extent to which the WOI is displaced.

An unbiased approach of molecular characterization of the endometrium: toward defining endometrial-based infertility.

Infertility is a complex condition affecting millions of couples worldwide. The current definition of infertility, based on clinical criteria, fails to account for the molecular and cellular changes that may occur during the development of infertility. Recent advancements in sequencing technology and single-cell analysis offer new opportunities to gain a deeper understanding of these changes. The endometrium has a potential role in infertility and has been extensively studied to identify gene expression profiles associated with (impaired) endometrial receptivity. However, limited overlap among studies hampers the identification of relevant downstream pathways that could play a role in the development of endometrial-related infertility. To address these challenges, we propose sequencing the endometrial transcriptome of healthy and infertile women at the single-cell level to consistently identify molecular signatures. Establishing consensus on physiological patterns in endometrial samples can aid in identifying deviations in infertile patients. A similar strategy has been used with great success in cancer research. However, large collaborative initiatives, international uniform protocols of sample collection and processing are crucial to ensure reliability and reproducibility. Overall, the proposed approach holds promise for an objective and accurate classification of endometrial-based infertility and has the potential to improve diagnosis and treatment outcomes.

A systematic review identifying seminal plasma biomarkers and their predictive ability on IVF and ICSI outcomes.

The diverse nature and high molecule concentration of seminal plasma (SP) makes this fluid a good potential source for a potential biomarker that could predict assisted reproductive technology (ART) outcomes. Currently, semen quality parameters cannot accurately predict ART outcomes. A systematic literature search was conducted to identify human SP biomarkers with potential predictive ability for the outcomes of IVF and intracytoplasmic sperm injection. Observational cohort and case-control studies describing the association between biomarkers in human SP and the outcome of infertile men attending for ART were included. Forty-three studies were selected, reporting on 89 potential SP biomarkers (grouped as oxidative stress, proteins glycoproteins, metabolites, immune system components, metals and trace elements and nucleic acids). The present review supports 32 molecules in SP as potentially relevant biomarkers for predicting ART outcomes; 23 molecules were reported once and nine molecules were reported in more than one study; IL-18 and TGF-ß1-IL-18 ratio were confirmed in distinct studies. This review presents the most comprehensive overview of relevant SP biomarkers to predict ART outcomes to date, which is of clinical interest for infertility investigations and assisted reproduction; nevertheless, its potential is under-exploited. This review could serve as starting point for designing an all-encompassing study for biomarkers in SP and their predictive ability for ART outcomes, and for developing a non-invasive diagnostic tool.

The endometrial microbiota of women with or without a live birth within 12 months after a first failed IVF/ICSI cycle.

The endometrial microbiota composition may be associated with implantation success. However, a 'core' composition has not yet been defined. This exploratory study analysed the endometrial microbiota by 16S rRNA sequencing (V1-V2 region) of 141 infertile women whose first IVF/ICSI cycle failed and compared the microbiota profiles of women with and without a live birth within 12 months of follow-up, and by infertility cause and type. Lactobacillus was the most abundant genus in the majority of samples. Women with a live birth compared to those without had significantly higher Lactobacillus crispatus relative abundance (RA) (p = 0.029), and a smaller proportion of them had ≤ 10% L. crispatus RA (42.1% and 70.4%, respectively; p = 0.015). A smaller proportion of women in the male factor infertility group had ≤ 10% L. crispatus RA compared to women in the unexplained and other infertility causes groups combined (p = 0.030). Women with primary infertility compared to secondary infertility had significantly higher L. crispatus RA (p = 0.004); lower proportions of them had ≤ 10% L. crispatus RA (p = 0.009) and > 10% Gardnerella vaginalis RA (p = 0.019). In conclusion, IVF/ICSI success may be associated with L. crispatus RA and secondary infertility with endometrial dysbiosis, more often than primary infertility. These hypotheses should be tested in rigorous well-powered longitudinal studies.

Advanced Technologies for Studying Microbiome-Female Reproductive Tract Interactions: Organoids, Organoids-on-a-Chip, and Beyond.

The female reproductive tract (FRT) is home to diverse microbial communities that play a pivotal role in reproductive health and disorders such as infertility, endometriosis, and cervical cancer. To understand the complex host-microbiota interactions within the FRT, models that authentically replicate the FRT's environment, including the interplay between the microbiota, mucus layer, immune system, and hormonal cycle, are key. Recent strides in organoid and microfluidic technologies are propelling research in this domain, offering insights into FRT-microbiota interactions and potential therapeutic avenues. This review delves into the current state of FRT organoid models and microbe integration techniques, evaluating their merits and challenges for specific research objectives. Emphasis is placed on innovative approaches and applications, including integrating organoids with microfluidics, and using patient-derived biobanks, as this offers potential for deeper mechanistic insights and personalized therapeutic strategies. Modeling various FRT properties in organoids is explored, from encompassing age-related epithelial features, oxygen levels, and hormonal effects to mucus layers, immune responses, and microbial interactions, highlighting their potential to transform reproductive health research and predict possible outcomes.

Organoids can be established reliably from cryopreserved biopsy catheter-derived endometrial tissue of infertile women.

RESEARCH QUESTION: Can organoids be established from endometrial tissue of infertile women and does tissue cryopreservation allow for establishment of organoids comparable to organoids derived from freshly biopsied endometrial tissue? DESIGN: Endometrial tissue was obtained from six infertile women through minimally invasive biopsy using a Pipelle catheter and subjected to organoid development, immediately after biopsy as well as after tissue cryopreservation. Organoid formation efficiency, morphology, expandability potential, endometrial marker expression (immunostaining and reverse transcription quantitative real-time polymerase chain reaction) and hormonal responsiveness (after oestradiol and progesterone treatment) were assessed. RESULTS: Organoids established from both fresh and frozen tissue at comparable efficiency could be passaged long-term and showed similar morphology, i.e. cystic with a central lumen lined by a single epithelial cell layer. They also exhibited comparable expression of endometrial markers and proliferative activity (Ki67 expression). Finally, organoids from freshly biopsied and cryopreserved endometrial tissue showed similar responses to oestradiol and progesterone treatment. CONCLUSIONS: Organoids can be established from cryopreserved endometrial tissue of infertile women and cryopreservation of the biopsy does not affect organoid formation and overall organoid characteristics. Cryopreservation of biopsies for later organoid development facilitates sample collection from any fertility clinic, not just the ones near an organoid laboratory.

Bile-salt stimulated lipase polymorphisms do not associate with HCV susceptibility.

Bile-salt stimulate lipase (BSSL) is a glycoprotein found in human milk and blood that can potently bind DC-SIGN. The BSSL gene is highly polymorphic with a variant number of O-linked glycosylated 11 amino acid repeats at the C-terminus of the protein, encoded in exon 11 of the gene. It has been shown that certain BSSL genotypes associate with decreased HIV-1 transmission in vitro and decreased HIV-1 disease progression. The protein forms dimers and individuals possessing one high (typically 14-21) and one low (typically 7-11) number of repeat domains has been shown to have stronger binding of BSSL to DC-SIGN and HIV-1 inhibitory activity in vitro. Since we previously demonstrated that SNPs within the DC-SIGN gene can associate with risk of HCV sexual transmission and which can be linked to diminished DC-SIGN gene expression we aimed to identify whether BSSL polymorphisms associated similarly through differential binding to DC-SIGN. DNA was isolated from the HIV-1 infected MSM cohort (MOSAIC) composed of HCV multiple exposed uninfected (MEU) (N = 30) and multiple exposed HCV infected (MEI) (N = 32) individuals and from the Amsterdam cohort studies (ACS) intravenous drug using (IDU) cohort (22 MEI and 40 MEU). The numbers of repeats in exon 11 were determined by PCR with repeat distributions compared between MEI and MEU. No statistical significant difference in the copy number of exon 11 repeats, or combinations of, in the BSSL gene was observed when comparing HCV infected MEI with MEU, thus the exon 11 repeat copy number in the BSSL gene does not affect HCV susceptibility.

HIV-1 exposure and immune activation enhance sexual transmission of Hepatitis C virus by primary Langerhans cells.

INTRODUCTION: The significant rise in incidence of Hepatitis C virus (HCV) infection among men-who-have-sex-with-men (MSM) living with HIV-1 suggests that HCV under specific circumstances is transmitted via sexual contact. During sexual transmission HCV has to cross the epithelial barrier to either directly enter the blood stream or indirectly via mucosal immune cells. However, the mechanisms of sexual transmission of HCV remain unclear. We investigated the role of Langerhans cells (LCs) in HCV susceptibility during sexual contact as LCs are among the first cells in mucosal tissues to encounter invading viruses. METHODS: We investigated the phenotype of primary LCs in anal biopsies from MSM living with HIV-1. To investigate the role of primary LCs in HCV infection and transmission, we have used both isolated primary skin LCs and the ex vivo tissue transmission model. RESULTS: Our data identified an important role for mucosal LCs in facilitating HCV transmission after HIV-1 exposure or immune activation. LCs were detected within mucosal anal tissues obtained from HIV-1 positive MSM biopsies. In order to perform functional studies, we used primary LCs from skin, which have a similar phenotype as mucosal LCs. Immature LCs were neither infected nor transmitted HCV to hepatocytes. Notably, exposure to HIV-1 significantly increased HCV transmission by LCs in the ex vivo transmission model. HIV-1 replication was crucial for the increased HCV transmission as HIV-1 inhibitors significantly reduced HIV-1-induced HCV transmission. Moreover, tissue immune activation of LCs also increased HCV transmission to target cells. CONCLUSIONS: Thus, our data strongly indicate that HIV-1 or immune activation in MSM leads to capture of HCV by mucosal LCs, which might facilitate transmission to other cells or allow entry of HCV into the blood. This novel transmission mechanism by LCs also implicates that the activation state of LCs is an important cellular determinant for HCV susceptibility after sexual contact.

SNP rs688 within the low-density lipoprotein receptor (LDL-R) gene associates with HCV susceptibility.

BACKGROUND & AIMS: Despite high-risk behaviour, 10%-20% of HCV multiple exposed individuals remain uninfected (MEU), whilst the remainder become infected (MEI). We hypothesize that host factors play a role in HCV susceptibility. We aimed to identify polymorphisms in host genes that encode for proteins involved in viral entry: CD81, Scavenger receptor 1 (SR-1), Low-density lipoprotein receptor (LDL-R), Claudin-1 (CLDN1), Occludin (OCLN) and Niemann-Pick C1-like 1 (NPC1L1). METHODS: Multiple exposed infected and MEU from two observational cohorts were selected. From the MSM study of acute infection with HCV (MOSAIC), HIV-1 infected MEU cases (n = 30) and HIV-1 infected MEI controls (n = 32) were selected based on reported high-risk behaviour. From the Amsterdam Cohorts Studies (ACS) injecting drug users (IDU) cohort, MEU cases (n = 40) and MEI controls (n = 22) were selected who injected drugs for ≥2 years, in the nineties, when HCV incidence was high. Selected single nucleotide polymorphisms (SNPs) were determined by sequencing or SNP assays. RESULTS: No associations were found for SNPs within genes coding for CD81, SR-1, Claudin-1 or Occludin between the MEU and MEI individuals from either cohort. We did observe a significant association for rs688 within the LDL-R gene with HCV infection (OR: 0.41 P = 0.001), however, LDL cholesterol levels did not vary between individuals carrying the differential SNPs. Additionally, a marginal significant effect was found for rs217434 and rs2072183 (OR: 2.07 P = 0.032 and OR: 1.76 P = 0.039, respectively) within NPC1L1. CONCLUSIONS: Our results demonstrate that the rs688 SNP within the LDL-R gene associates with HCV susceptibility through mucosal as well as intravenous exposure.

DC-SIGN Polymorphisms Associate with Risk of Hepatitis C Virus Infection Among Men who Have Sex with Men but not Among Injecting Drug Users.

We aimed to identify whether genetic polymorphisms within L-SIGN or DC-SIGN correlate with hepatitis C virus (HCV) susceptibility. A men who have sex with men (MSM) and an injecting drug users (IDU) cohort of HCV cases and multiple-exposed uninfected controls were genotyped for numerous L-SIGN and DC-SIGN polymorphisms. DC-SIGN single nucleotide polymorphisms (SNPs) -139, -871, and -939 correlated with HCV acquisition in the MSM cohort only. When the same SNPs were introduced into a transcription activity assay they demonstrated a reduction in expression with predicted alteration in binding of transcription factors. DC-SIGN promoter SNPs correlated with risk of HCV acquisition via sexual but not IDU exposure, likely through modulation of mRNA expression levels.