Development of a Microbiome Non-invasive Diagnosis Tool
Condition: Infertility of Uterine Origin
MeSH terms: Infertility
Study type: Observational

Summary: 
      Although the endometrium tissue has been traditionally considered free of bacteria, recent
      studies have documented the presence of an endometrial microbiome. In a pilot study conducted
      by our research team, the microorganisms present in the endometrium were analysed in samples
      of endometrial fluid (EF) using next generation sequencing (NGS).Consistent with previously
      published studies, in normal conditions the endometrium is mainly composed of different
      species of the genus Lactobacillus. It was further noted that the presence of other
      pathogenic bacteria such as Streptococcus, and/or Gardnerella may alter the endometrial
      microbiome and can disrupt the uterine environment, affecting implantation rates and
      pregnancy success.

      This project aims to validate the microbiome found in the endometrium of women of
      reproductive age and try to corroborate the relationship between the endometrial microbiome
      and the reproductive outcomes in patients undergoing assisted reproduction treatment (ART).
      The use of Endometrial Receptivity Analysis (ERA) tool, together with the analysis of the
      endometrial flora before the embryo transfer will allow to evaluate the impact of the
      presence of endometrial pathogens on implantation in receptive patients.

      Therefore, the focus of this project is the development and clinical validation of a
      non-invasive diagnosis tool to analyse the microbiota, adding the microbiome study to the ERA
      analysis.
    

Detailed description: 
      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.
    

Intervention name(s): Endometrial biopsy and endometrial fluid collection
Intervention type(s): Procedure
Intervention description(s): On the same day that the EB for ERA is indicated, in day 5 of a HRT cycle with 5 days of progesterone, according to the common clinical practice, a sample of EF will be aspirated immediately prior to EB for ERA. This EF sample will be used for the non-invasive diagnosis of the microbiome, also a small portion of the endometrial tissue obtained for the endometrial receptivity diagnosis by ERA will be used as a control for the study of the endometrial microbiome. Those patients with a receptive endometrium will continue their ART according to the standard clinical protocol. Those patients with non-receptive endometrium will follow the recommendation derived of the ERA test for the election of the day for a second EB sample, in which the collection of EF sample will be repeated. Finally, the receptivity and microbiome results in EF will be correlated with that on the endometrial tissue, and with the reproductive outcomes of these patients after the embryo transfer.
Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        ART patients, with indication of diagnosis of endometrial receptivity by ERA that will
        receive embryo transfer with frozen blastocyst stage embryos (day 5/day 6).
      
Biospecimen description: 
      DNA from endometrial fluid and biopsy to analyze the endometrial microbiome.

      These samples will be completely exhausted during the analysis.
    
Keywords: Endometrial microbiota; Bacterial pathogens; Assisted reproductive techniques; Dysbiosis; Endometrial receptivity; Endometrial diagnosis