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.

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.

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.

Participation is limited to individuals with female biological sex, due to the requirement for endometrial tissue sampling and pregnancy-related outcomes.

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