The Role of the Physiological Endometrial Microbiota in Assisted Reproduction Outcomes (no exist)

The goal of this observational study is to investigate the influence and contribution of the normal composition and balance of the endometrial microbiota on the achievement of embryo implantation and the maintenance of pregnancy in women undergoing IVF, with comparable clinical profiles, including patients with a history of RIF or RPL, evaluated under identical treatment protocols and hormonal conditions.

The main questions it aims to answer are:

1. Does the normal composition of the endometrial microbiota, particularly the dominance of Lactobacillus species, influence embryo implantation success in women undergoing IVF?
2. Does the endometrial microbiota profile affect pregnancy maintenance, ongoing pregnancies, and live birth rates in women with a history of recurrent implantation failure (RIF) or recurrent pregnancy loss (RPL)

Participants will not undergo any additional interventions beyond standard clinical care.They will provide clinical data already collected as part of routine IVF procedures.

Study Overview

• Status: Enrolling by invitation
• Conditions: Microbiota; Repeated IVF Failure

Detailed Description

Endometrial biopsies were collected from patients using a Pipelle catheter during the designated phase of the menstrual cycle. Each sample was immediately placed in a specialized preservative solution and transported to two external laboratories for analysis.

In the first laboratory, bacterial DNA was extracted using a CE-IVD kit, followed by targeted amplification and barcoded sequencing of seven hypervariable regions (V2, V3, V4, V6, V7, V8, V9) of the 16S rRNA gene using the 16STM metagenomic kit and Ion Torrent sequencing system (Ion GeneStudio S5 with Ion Chef Instrument). Data were analyzed with Ion Reporter™ software and MicroSEC ID, using MicroSEC ID and Greengenes databases for taxonomic classification.

In the second laboratory, total DNA was extracted, and targeted amplification of the 16S rRNA gene was performed using the 16S Barcoding Kit 24 V14 (Nanopore), followed by NGS on the MinION Mk1C analyzer. Bioinformatic analysis was conducted using the EPI2ME Labs 16S workflow. Bacterial composition was classified at the genus level, with results expressed as relative abundance percentages. OTU-level data were not disclosed.

Interpretation of microbiota profiles was performed by the treating physician or clinical geneticist. Patients whose samples showed decreased Lactobacillus abundance or the presence of potentially pathogenic microorganisms received individualized interventions. These interventions included a two-week course of antibiotics tailored to the patient's clinical history, followed by probiotics administered orally or via combined oral and vaginal routes for two weeks to two months. Antifungal agents were administered when clinically indicated.

Embryo transfer schedules were adapted based on microbial profiles and clinical assessment. Patients with normal microbial profiles proceeded with IVF in the next cycle. Patients with abnormal profiles, signs of inflammation, or potential pathogenic colonization had embryo transfer postponed for at least one month following completion of the therapeutic regimen.

The study provides a detailed methodological framework for endometrial microbiota analysis, including sample collection, DNA extraction, sequencing strategies, bioinformatic pipelines, and clinical decision-making based on microbial composition, aiming to support personalized management in assisted reproduction.

• Study Type: Observational
• Enrollment (Estimated): 80

Contacts and Locations

Study Locations

• Greece
  • Thessaloniki, Greece
    • Assisting Nature

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Participants were required to be between 18 and 54 years of age, in accordance with the legally permitted age limits for embryo transfer in Greece

Description

Inclusion Criteria:

1. Individuals who are biologically female.
2. Age between 18 and 54 years, in accordance with the legally permitted age limits for embryo transfer in Greece.
3. Undergoing in vitro fertilization (IVF) treatment.
4. Clinical history consistent with recurrent implantation failure (RIF) or recurrent pregnancy loss (RPL) and comparable IVF treatment profiles.
5. Ability to provide informed consent for the use of clinical and microbiome data for research purposes

Exclusion Criteria:

1. Individuals who are not biologically female.
2. Age below 18 or above 54 years.
3. Absence of endometrial biopsy samples or incomplete clinical data.
4. Presence of significant uterine abnormalities or medical conditions that contraindicate embryo transfer.
5. Participation in another interventional clinical study that may interfere with microbiome assessment or IVF outcomes.

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort
Lactobacillus-dominant microbiota; Participants whose endometrial microbiota is dominated by Lactobacillus species. Data were collected from previously obtained endometrial biopsies, analyzed via 16S rRNA gene sequencing. No interventions were performed beyond standard IVF clinical care.
Non-Lactobacillus-dominant microbiota; Participants whose endometrial microbiota shows depletion of Lactobacillus species or presence of potentially pathogenic microorganisms. Data were collected from previously obtained endometrial biopsies, analyzed via 16S rRNA gene sequencing. In some cases, participants received individualized clinical interventions (e.g., antibiotics or probiotics) based on their microbial profile as part of routine care.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Implantation Rate according to microbiome profile	The study formally assesses differences in embryo implantation rates between women with Lactobacillus-dominant and non-Lactobacillus-dominant endometrial microbiota profiles.	From the day of the embryo transfer to the day of confirmation of implantation (2-4 weeks)
Ongoing Pregnancy/Live Birth Rate according to microbiome profile	Association between endometrial microbiota composition and the likelihood of achieving an ongoing pregnancy or live birth	From the day of embryo transfer up to the end of the first trimester (up to 12 weeks of gestation) and live birth until delivery (up to 40 weeks of gestation)
Biochemical pregnancy rate and microbiome status	Rate of biochemical pregnancies in relation to endometrial microbiota profile	From the day of embryo transfer to the day of confirmation of implantation (2-4 weeks)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Endometrial microbiota composition	Relative abundance of Lactobacillus versus non-Lactobacillus genera in endometrial biopsies analyzed by 16S rRNA sequencing.	At time of biopsy collection (during procedure)

Collaborators and Investigators

• Sponsor: Assisting Nature

Study record dates

Study Major Dates

• Study Start (Actual): January 2, 2025
• Primary Completion (Estimated): May 30, 2026
• Study Completion (Estimated): June 25, 2026

Study Registration Dates

• First Submitted: February 20, 2026
• First Submitted That Met QC Criteria: May 20, 2026
• First Posted (Actual): May 26, 2026

Study Record Updates

• Last Update Posted (Actual): May 26, 2026
• Last Update Submitted That Met QC Criteria: May 20, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: Microbiome; IVF; Microbiota; In vitro fertilization; Embryo; Assisted reproduction; Endometrial Microbiome; Physiological Endometrial Microbiota; Endometrial Microbiota; Implantation success
• Other Study ID Numbers: Microbiome-AN011; Assisting Nature (Other Identifier: Assisting Nature IVF Clinic)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No