Development of a Non-invasive Diagnosis Tool for the Analysis of the Microbiota to Improve Reproductive Outcomes in Infertile Patients.

Embryonic implantation is a critical process to the survival of the species that is relatively inefficient, especially in humans. Key elements are the embryo, the maternal endometrium, and the cross-communication between them using different scientific perspectives. Endometrial receptivity describes the phenotype that allows embryo adhesion and placentation to occur. Due to the need of an accurate and objective molecular diagnostic test for the receptivity status of endometrium, the Endometrial Receptivity Analysis (ERA) was developed. ERA test is a customized expression microarray that identifies the transcriptomic expression profile signature of the personalized window of implantation (WOI). Its clinical usefulness has been demonstrated in patients with implantation failure in whom 25% of them have a displaced WOI becoming pregnant once the embryo transfer is performed at the proper WOI predicted by ERA. However, this procedure requires an endometrial biopsy (EB), which is the major drawback of this diagnosis test, because embryo transfer has to be delayed to the next cycle, and also the possible discomforts caused to the patients. For this reason, a previous pilot study was conducted in order to confirm whether endometrial receptivity can be predicted through non-invasive methods in endometrial fluid (EF) samples. The gene panel of ERA test was interrogated on epithelial and stromal cells from endometrial biopsies and cells isolated from the EF at the single cell level, demonstrating that the major cells on the EF classifies together with the epithelial population.

On the other hand, to try to better understand the mechanisms that connect endometrial receptivity and/or implantation of the embryo with an altered endometrial microbiome, another pilot study was conducted to determine the composition of the endometrial microbiome after the analysis of the bacterial 16S ribosomal RNA by NGS. Interestingly, in patients with receptive endometrium, diagnosed by Endometrial Receptivity Array (ERA) who had endometrial microbiome with pathogens, or not dominated by bacteria of the genus Lactobacillus (NLD) showed significantly lower implantation (23.1% vs 60.7% p = 0.02), pregnancy (33.3% vs 70.6%, p = 0.03), ongoing pregnancy per embryo transfer (13.3% vs 58.8%, p = 0.03), and live birth (6.7% vs 58.8%, p = 0.002) rates than those with a healthy endometrial microbiome dominated by Lactobacillus (LD). Moreover, this relationship was much more significant when pathogenic species found belonged to the genus Gardnerella and Streptococcus. This is in line with other published studies that analyze the impact of endometrial pathogens in IVF treatments, highlighting the importance of the study of bacterial communities for reproductive health.

Given these preliminary results, the present study aims to validate in a larger sample set, the relationship between the imbalance of endometrial microbiome and the decline in reproductive success in patients undergoing ART. To do this, the experimental design will be improved in the following areas: (i) a higher sample size will be analysed to validate the previous results obtained in the previous pilot study on endometrial microbiome, (ii) paired samples from endometrial fluid and endometrial biopsy will be analysed for the microbial profile to study whether there is bacterial variability associated to the sample type, (iii) the endometrial microbiota will be simultaneously assessed with ERA as a supplementary diagnosis for this tool, and (iv) the analysis of the endometrial microbiota will be performed using the most advanced technology on bacterial metagenome sequencing to widen the information of the microorganisms identified in each sample.