Does the uterine microbiota affect the reproductive outcomes in women with recurrent implantation failures?

BACKGROUND: Inefficiency of in vitro fertilization (IVF) programs can be caused by implantation failures. The uterine microbiota can influence the implantation process. However, it still remains unclear whether opportunistic microorganisms detected in the endometrium have a negative impact on the implantation success. The aim of our study was to evaluate the influence of the uterine microbiota on the embryo implantation success in patients undergoing assisted reproductive technologies. METHODS: The study included 130 women diagnosed with infertility. The patients were divided into three groups: group I included women with the first IVF attempt (n = 39); group II included patients with recurrent implantation failure following embryo transfer with ovarian stimulation (n = 27); group III consisted of women with recurrent implantation failure following frozen-thawed embryo transfer (n = 64). We performed microbiological examination of the embryo transfer catheter which was removed from the uterine cavity after embryo transfer; cervical discharge of all the patients was studied as well. Thirty patients were selected for metagenomic sequencing. RESULTS: The study showed that the uterine cavity is not free of microorganisms. A total of 44 species of microorganisms were detected: 26 species of opportunistic organisms and 18 species of commensals (14 species of lactobacilli and 4 species of bifidobacteria). Obligate anaerobic microorganisms and Gardnerella vaginalis were detected more frequently in group I compared to group III (strict anaerobes-15.4 and 1.6%; G. vaginalis-12.8 and 1.6%, respectively) (p < 0.05). However, this fact did not have a negative influence on the pregnancy rate: it was 51.3% in group I, it was 29.6% and 35.9% in women with recurrent implantation failures, respectively. CONCLUSION: Opportunistic microorganisms which were revealed in low or moderate titers (103-105 CFU/ml) in the uterine cavity and cervical canal did not affect the pregnancy rate in the women in the study groups. The microflora of the uterine cavity and cervical canal differed in qualitative composition in 87.9% of patients, therefore, we can suggest that the uterine cavity may form its own microbiota. The microbiota of the uterine cavity is characterized by fewer species diversity compared to the microbiota of the cervical canal.

LC-MS Analysis Revealed the Significantly Different Metabolic Profiles in Spent Culture Media of Human Embryos with Distinct Morphology, Karyotype and Implantation Outcomes.

In this study we evaluated possible differences in metabolomic profiles of spent embryo culture media (SECM) of human embryos with distinct morphology, karyotype, and implantation outcomes. A total of 153 samples from embryos of patients undergoing in vitro fertilization (IVF) programs were collected and analyzed by HPLC-MS. Metabolomic profiling and statistical analysis revealed clear clustering of day five SECM from embryos with different morphological classes and karyotype. Profiling of day five SECM from embryos with different implantation outcomes showed 241 significantly changed molecular ions in SECM of successfully implanted embryos. Separate analysis of paired SECM samples on days three and five revealed 46 and 29 molecular signatures respectively, significantly differing in culture media of embryos with a successful outcome. Pathway enrichment analysis suggests certain amino acids, vitamins, and lipid metabolic pathways to be crucial for embryo implantation. Differences between embryos with distinct implantation potential are detectable on the third and fifth day of cultivation that may allow the application of culture medium analysis in different transfer protocols for both fresh and cryopreserved embryos. A combination of traditional morphological criteria with metabolic profiling of SECM may increase implantation rates in assisted reproductive technology programs as well as improve our knowledge of the human embryo metabolism in the early stages of development.

The role of abnormal hypermethylation of the HOXA10 and HOXA11 promoters in implantation failures in IVF programs.

Objective: The objective of this study is to investigate the association between methylation levels of HOXA10 and HOXA11 promoters, clinical parameters, and implantation outcomes after in vitro fertilization-embryo transfer (IVT-ET) cycles in women with repeated implantation failures and tubal infertility. Methods: Endometrium samples were collected from 34 women during implantation window before IVF-ET cycle to assess methylation status of HOXA10 and HOXA11 promoters using bisulfite sequencing. All participants had a tubal factor of infertility and at least two implantation failures in the anamnesis. A logistic regression model was used to predict the implantation outcome depending on methylation status and clinical parameters. Results: The methylation of CpG-islands of HOXA10 and HOXA11 promoters was identified in 76.5 and 100% of participants, respectively. The median methylation levels did not differ significantly between the groups with different implantation outcomes, but a logistic regression model based on HOXA10 and HOXA11 methylation and clinical parameters allowed to classify the implantation outcomes with the total percentage of correct predictions of 85.19%. Conclusions: Abnormal methylation levels of the HOXA10 and HOXA11 promoters were found in the endometrium of women with tubal infertility and repeated implantation failures. The findings suggest that methylation status could be an important factor of implantation failure during IVF-ET cycles.

The Link Between Seminal Cytokine Interleukin 18, Female Circulating Regulatory T Cells, and IVF/ICSI Success.

Seminal plasma (SP) is thought to be a crucial factor which affects the expansion of regulatory T cells (Tregs) in female reproductive tract during embryo implantation. We propose that seminal transforming growth factor (TGF) ß1 is responsible for local accumulation of circulating Tregs, which manifests as changes in Treg frequency in peripheral blood, whereas seminal interleukin (IL) 18 interferes with TGF-ß1-dependent cellular reactions. The purpose of the present study is to determine whether the frequency of circulating Tregs is associated with the levels of seminal cytokines and pregnancy establishment in women exposed to partner's SP during in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) cycle. Twenty-nine women were exposed to SP via timed intercourse before the day of ovum pickup (day-OPU) and also subjected to intravaginal SP application just after OPU. Measurements of seminal TGF-ß1 and IL-18 were made by FlowCytomix technology. The percentage of CD4+CD25+CD127low+/- Tregs among total circulating CD4+ T cells was determined by flow cytometry and the difference between Treg values on the day of embryo transfer and day-OPU was calculated. The percentage of Tregs on the day-OPU, identified as a predictive factor of clinical pregnancy after IVF/ICSI, showed a positive correlation with IL-18 concentration and content of this cytokine per ejaculate (P < .001 and P < .004, respectively) and negative correlation with the TGF-ß1/IL-18 ratio (P < .014).These findings indicate that the adverse effect of seminal IL-18 excess on implantation may be realized by the prevention of postcoital TGF-ß1-related migration of circulating Tregs, which clearly manifests as elevated level of Treg frequency in peripheral blood.

Cumulus cell mitochondrial activity in relation to body mass index in women undergoing assisted reproductive therapy.

Most studies have considered the negative influence of obesity on fertility in both genders. In the present study, we assessed mitochondrial activity expressed as the mitochondrial potential index (MPI) in cumulus cells from obese women and women with a normal body mass index (BMI) during assisted reproductive therapy. The results revealed a significant reduction of MPI with increased body mass. The lower MPI levels in cumulus cells from obese women may reflect mitochondrial dysfunction caused by oxidative stress, which can affect the cumulus-oocyte complex and have an impact on oocyte development.

The relationship of seminal transforming growth factor-ß1 and interleukin-18 with reproductive success in women exposed to seminal plasma during IVF/ICSI treatment.

It has been proposed that the transforming growth factor (TGF)-ß1 present in seminal plasma (SP) triggers a female immune response favorable for implantation. We hypothesize that seminal interleukin (IL)-18, a cytokine that can potentially cause implantation failure, interferes with the beneficial effect of TGF-ß1. This study aims to determine whether the levels of seminal TGF-ß1 and IL-18 are associated with reproductive outcomes in patients exposed to SP during in vitro fertilization (IVF) or IVF with intracytoplasmic sperm injection (ICSI). A prospective study, which included 71 couples undergoing IVF/ICSI was carried out. Female patients were exposed to their partners' SP via timed intercourse before the day of ovum pick-up (OPU) and also subjected to intravaginal SP application just after OPU. Quantitative measurements of total TGF-ß1 (active plus latent) as well as IL-18 were determined by FlowCytomix™ technology in the SP to be used for intravaginal applications. Comparison of SP cytokine profiles between pregnant and non-pregnant groups revealed that pregnancy was correlated with a lower concentration of IL-18 (P=0.018) and lower content per ejaculate for both of IL-18 (P=0.0003) and TGF-ß1 (P=0.047). The ratio of TGF-ß1-to-IL-18 concentration was significantly higher in the pregnant than in the non-pregnant group (P=0.026). This study supports the notion that two key cytokines TGF-ß1 and IL-18, both present in SP are associated with reproductive outcomes in female patients exposed to SP during IVF/ICSI treatment.