Development of a Microbiome Non-invasive Diagnosis Tool (ERA-niMIC)

• ClinicalTrials.gov Identifier: NCT03330444
• Recruitment Status: Completed
• First Posted: November 6, 2017
• Last Update Posted: October 9, 2020
• Sponsor: Igenomix
• Information provided by (Responsible Party): Igenomix

Study Description

Brief 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.

Condition or disease	Intervention/treatment
Infertility of Uterine Origin	Procedure: Endometrial biopsy and endometrial fluid collection

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.

Study Design

• Study Type: Observational
• Actual Enrollment: 452 participants
• Observational Model: Cohort
• Time Perspective: Prospective
• Official Title: Development of a Non-invasive Diagnosis Tool for the Analysis of the Microbiota to Improve Reproductive Outcomes in Infertile Patients.
• Actual Study Start Date: August 4, 2017
• Actual Primary Completion Date: May 17, 2019
• Actual Study Completion Date: June 25, 2020

Groups and Cohorts

Group/Cohort	Intervention/treatment
LD microbiome receptive to the ERA test; Receptive patients by the ERA test with an endometrial microbiota mainly composed of different species of the genus Lactobacillus, determined by NGS sequencing.	Procedure: Endometrial biopsy and endometrial fluid collection; 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.
NLD microbiome receptive to the ERA test; Receptive patients by the ERA test with an endometrial microbiota composed of different pathogenic bacteria such as Streptococcus and Gardnerella, or not dominated by bacteria of the genus Lactobacillus, determined by NGS sequencing.	Procedure: Endometrial biopsy and endometrial fluid collection; 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.

Outcome Measures

Primary Outcome Measures :

1. Development of a new non-invasive diagnosis test of Endometrial Microbiome (EM) in infertile patients [ Time Frame: 24 months ]
   Bacterial DNA analysis by Next Generation Sequencing (NGS) using endometrial fluid for the EM diagnose. This will be measured by the percentage of each bacterial DNA in EF samples

Secondary Outcome Measures :

1. Live birth rate [ Time Frame: 40 weeks ]
   Number of babies born per embryo transfer
2. Implantation rate [ Time Frame: 12 weeks ]
   Number of implanted embryos per total number of embryos transferred
3. Pregnancy rate [ Time Frame: 20 weeks ]
   Number of pregnancies per embryo transfer
4. Biochemical pregnancies [ Time Frame: 20 weeks ]
   Number of biochemical pregnancies per total number of pregnancies
5. Ectopic pregnancies [ Time Frame: 20 weeks ]
   Number of ectopic pregnancies per total number of pregnancies
6. Clinical miscarriages [ Time Frame: 20 weeks ]
   Number of clinical miscarriages per total number of pregnancies
7. Ongoing pregnacy rate [ Time Frame: 40 weeks ]
   Number of ongoing pregnancies per embryo transfer

Biospecimen Retention:   Samples With DNA

DNA from endometrial fluid and biopsy to analyze the endometrial microbiome.

These samples will be completely exhausted during the analysis.

Eligibility Criteria

• Ages Eligible for Study: 18 Years to 50 Years (Adult)
• Sexes Eligible for Study: Female
• Accepts Healthy Volunteers: Yes
• 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).

Criteria

Inclusion Criteria:

• Patients in In Vitro Fertilization (IVF) / IntraCytoplasmic Sperm Injection (ICSI) or OVODON with indication of ERA, with or without Preimplantation genetic screening (PGS) indication, which receive embryo transfer with frozen blastocyst stage embryos (day 5/day 6) on a hormone replacement therapy (HRT) cycle.
• Maternal Age: ≤40 years (IVF/ICSI patients)
• Maternal Age: ≤50 years (OVODONATION patients)
• Body Mass Index (BMI): 18.5 - 30 kg / m2 (both inclusive)
• Negative serological tests for human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), Rapid plasma reagin (RPR) for syphilis
• Women with regular menstrual formula (3-4 / 26- 35 days)
• Sperm concentration: > 2 million spermatozoa/ml

Exclusion Criteria:

• Patients who are Intra-Uterine Device (IUD) carriers in the last 3 months
• Patients who have taken prescribed antibiotics in the last 3 months before the sample collection. (May be accepted those cases where the patient has received previous prophylactic antibiotics to ovarian puncture in the stimulation cycle, according to the standard clinical practice of the centre. The administration of this antibiotic should be at least 1 month before the sample collection).
• Adnexal or uterine pathologies as uterine malformations that may affect the implantation rate (e.g: Polyps, intramural myoma ≥ 4cm or submucosal, septum, hydrosalpinx, etc). Patients affected (before or after of their inclusion) with any of the previously described pathologies, will be allowed to participate in this study as long as they are surgically treated before any study procedure.
• Existence of bacterial, fungal or viral infections severe or uncontrolled that, in the opinion of the principal investigator, could interfere with the patient's participation in the study or the assessments of the study results.
• Any illness or medical condition that is unstable or can put patient safety at risk and compliance in the study.

Contacts and Locations

Locations

United States, Connecticut

RMA Connecticut

Norwalk, Connecticut, United States, 06861

United States, Florida

IVF Florida

Margate, Florida, United States, 33063

United States, Missouri

Missouri Center for Reproductive Medicine (MCRM Fertility)

Chesterfield, Missouri, United States, 63005

United States, Washington

Dominion Fertility

Arlington, Washington, United States, 22203

Argentina

Gestanza Medicina Reproductiva

Rosario, Santa Fe, Argentina

Pregna Medicina Reproductiva

Buenos Aires, Argentina, C1425DGQ

Canada, British Columbia

Pacific Center for Reproductive Medicine

Burnaby, British Columbia, Canada, V5G 4X7

Japan

oak Clinic Sumiyoshi

Osaka, Osaka Prefecture, Japan, 557-0045

Malaysia

Alpha Fertility Center

Petaling Jaya, Selangor, Malaysia, 47810

Mexico

New Hope Fertility Center

Mexico City, Mexico, 11000

Spain

Clinica Fertia

Fuengirola, Malaga, Spain, 29640

ProcreaTec

Madrid, Spain, 28036

Turkey

Bahçeci Fulya IVF Center

Istanbul, Turkey

Sponsors and Collaborators

Igenomix

Investigators

• Study Chair: Carlos Simón, MD PhD; Igenomix

More Information

• Responsible Party: Igenomix
• ClinicalTrials.gov Identifier: NCT03330444
• Other Study ID Numbers: IGX1-MIC-CS-17-05
• First Posted: November 6, 2017
• Last Update Posted: October 9, 2020
• Last Verified: October 2020

Individual Participant Data (IPD) Sharing Statement:

• Plan to Share IPD: No

• Studies a U.S. FDA-regulated Drug Product: No
• Studies a U.S. FDA-regulated Device Product: No

Keywords provided by Igenomix:

Endometrial microbiota; Bacterial pathogens; Assisted reproductive techniques; Dysbiosis; Endometrial receptivity; Endometrial diagnosis

Additional relevant MeSH terms:

Infertility