Our study investigated the presence of bacteria in endometrial samples from patients with recurrent pregnancy loss (RPL) and explored potential correlations between bacterial presence and immunological response of the endometrium. Using RT-PCR identification techniques, we detected at least one of 10 selected bacterial species in 25.6% of endometrial samples. Notably, our results revealed a significant correlation between the presence of PCR-identified bacteria and a higher number of pregnancy losses, adding to the growing body of evidence that these bacteria may play a role in influencing reproductive outcomes.

In a subgroup of patients, we performed, in addition to PCR, two other types of endometrial biopsy examination: microbial culture and immunohistochemistry (IHC) for plasma cell identification. We observed no correlation between bacterial presence in PCR examination and the number of plasma cells in endometrial sections. However, a significant correlation was found between bacterial detection by PCR and microbial culture.

The role of endometrial factors in infertility and RPL has been the subject of extensive research, focusing on the immunological changes associated with chronic endometritis (Takimoto et al. 2023; Cicinelli et al. 2021; McQueen et al. 2015; Espinós et al. 2021). Studies have demonstrated associations between chronic endometritis and adverse pregnancy outcomes; however, the absence of standardised diagnostic criteria remains a contentious issue, potentially compromising the robustness of accrued evidence (Margulies et al. 2021; Klimaszyk et al. 2023; Huang et al. 2020).

Among several researchers addressing this diagnostic challenge, McQueen et al. conducted a study comparing CE prevalence between women with RPL and controls using expanded histopathological criteria, which notably included endometrial stromal changes alongside plasma cell assessment (McQueen et al. 2021). Their approach yielded optimal diagnostic accuracy when CE was defined by the presence of at least one plasma cell per 10 HPFs combined with stromal changes. A recent survey of pathologists’ clinical practices further highlights the global inconsistency in CE diagnostic criteria, underscoring the urgent need for standardisation, particularly as histopathological examination serves as a verification method for emerging diagnostic techniques (Margulies et al. 2021).

The aetiology of CE and its underlying immunological mechanisms remain incompletely understood. The initial concept of pathogenic bacteria as causative agents in CE development emerged from two key lines of evidence. First, the pioneering work of Cicinelli’s group identified bacteria in endometrial samples from CE patients using traditional microbial culture techniques (Cicinelli et al. 2009). Subsequently, Song et al. provided additional support for the infectious aetiology through a prospective randomised control trial demonstrating that antibiotic treatment effectively resolves histopathologically confirmed CE (Song et al. 2021).

Early microbial culture studies by Cicinelli and Kitaya’s research groups identified several common pathogens in CE patients, including Streptococcus species, Escherichia coli, Enterococcus faecalis, Staphylococcus species, Mycoplasma/Ureaplasma species, Proteus species, Pseudomonas aeruginosa, Klebsiella pneumoniae, Gardnerella vaginalis, Corynebacterium species, and yeast (Cicinelli et al. 2009; Kitaya et al. 2017; Cicinelli et al. 2008).

However, our understanding of endometrial microbiology has evolved significantly. When these initial studies were conducted, the uterine cavity was widely considered a sterile environment, leading researchers to conclude that any bacterial presence must be pathogenic and responsible for the inflammatory response seen in CE.

Our study design was inspired by the work of Moreno et al., who compared various diagnostic methods for chronic endometritis and introduced RT-PCR as a molecular detection technique in the CE diagnostic process (Moreno et al. 2018). Interestingly, Moreno et al. reported a higher rate of bacterial detection (55.79%) compared to our study (24.4%). This discrepancy in bacterial detection rates could be attributed to significant differences in the study populations. Our study focused exclusively on RPL patients. In contrast, Moreno et al. investigated infertile patients undergoing in vitro fertilisation (IVF) procedures. The bacterial profiles identified in our study and Moreno’s research reveal similarities and differences. Streptococci emerged as a common finding in both investigations. Moreno’s study identified Streptococci as the predominant pathogens, while our research found Streptococcus species to be the second most prevalent, present in 5 out of 22 positive samples. Notably, our study identified Staphylococcus species as the most frequently detected bacteria, found in 11 samples. Our investigation found isolated instances of Enterococcus species, Escherichia coli, and Mycoplasma hominis. Moreno’s study, however, did not provide a detailed breakdown of less prevalent pathogens, making a direct comparison challenging. Interestingly, neither study detected Chlamydia trachomatis or Neisseria gonorrhoeae.

Interestingly, we did not observe a correlation between the presence of bacteria detected by RT-PCR and chronic endometritis (CE) as defined by at least one plasma cell per 10 high-power fields (HPF) of the endometrial sample. This finding contrasts with a previous study by Liu et al. that compared the microbiota of CE and non-CE patients (Liu et al. 2019). However, it is essential to note that those studies employed different microbial analysis techniques, specifically culture-independent massively parallel sequencing of the 16S ribosomal RNA gene. Additionally, they used a different cut-off point for CE definition, which was > 5.15 cells per 10 mm2. Our study investigates the role of pathogenic bacteria and cannot be extrapolated to information on the uterus microbiome, that is most likely why we did not obtain similar results as Liu’s group.

Results from classical microbial culture techniques demonstrated a significant correlation with PCR findings. However, culture methods detected bacterial presence in fewer cases than RT-PCR. This discrepancy likely stems from the low abundance of bacteria in endometrial samples, which often falls below the threshold required for successful culture-based detection. This makes microbial culture a possible but unreliable method for detecting bacteria in endometrial samples.

It is essential to acknowledge that while PCR identification of specific bacteria provides valuable insights, it may not fully capture the complex relationship between the endometrial microbiome, chronic endometritis, and RPL. Although sensitive for detecting specific bacterial species, PCR-based methods have limitations in representing the full diversity and interactions within the microbiome.

Recent research has emphasised the importance of considering the entire microbial community rather than focusing on individual pathogens. For instance, Moreno et al. demonstrated that the overall composition of the endometrial microbiota, particularly the dominance of Lactobacillus species, could influence implantation success in IVF patients (Moreno et al. 2016). Chen et al. similarly found associations between altered endometrial microbiota and adverse reproductive outcomes (Chen et al. 2022).

These findings underscore the need for more comprehensive microbiome studies to elucidate the relationship between endometrial bacteria and RPL fully. Next-generation sequencing techniques, such as 16S rRNA gene sequencing, offer a more holistic approach to characterising the endometrial microbiome (Peuranpää et al. 2022). These methods can provide a more complete picture of microbial diversity and abundance, potentially revealing essential patterns or imbalances that may be missed by targeted PCR or culture-based techniques.

A compelling example of the importance of overall microbial community structure comes from a study by Tine Wronding et al., which demonstrated that vaginal microbiota transplantation, shifting from a Gardnerella-dominated to a Lactobacillus-dominant microbiome, resulted in successful pregnancies in RPL patient (Wrønding et al. 2023). This highlights the potential significance of the overall microbial community structure rather than the presence or absence of specific bacterial species. The case report presented by Moreno gives us the first glimpse into the microbiome of early pregnancy (Moreno et al. 2020). It shows that microbial profiles of endometrium differ in cases before miscarriage and before a successful pregnancy. Lactobacillus iners was the most prevalent microbe found in the endometrium during the fourth week of a successful pregnancy.

When studying endometrial microbiota using PCR, contamination poses significant challenges. This includes environmental contamination from high-biomass sites like the cervix and vagina, reagent contamination from extraction kits, and cross-contamination during sample handling. Given the low-biomass nature of endometrial samples, even minor contamination can significantly alter results (Reschini et al. 2022; Winters et al. 2019).

In our study, we conducted diagnostic hysteroscopy prior to endometrial sampling, following a similar approach used by several other research groups (Cicinelli et al. 2008; Moreno et al. 2018). Although a sterile medium was used during the hysteroscopy procedures, bacterial transfer through the cervical canal could still occur. However, since our microbial analysis was performed on endometrial tissue samples rather than uterine fluid, and we adhered to strict sterile protocols, we believe that any potential impact on our results was minimal.

While our study’s finding of a correlation between PCR-identified bacteria and increased frequency of pregnancy losses provides a valuable point, the future of research is more comprehensive microbiome analysis techniques. This approach could lead to a deeper understanding of the role of the endometrial microbiome in recurrent pregnancy loss and potentially inform new diagnostic and therapeutic strategies for women experiencing RPL.