Introduction
Endometriosis (EM) is an estrogen-dependent condition in which cells similar to those lining the uterus grow outside of it. It causes painful periods, infertility, and pelvic masses, affecting 10–15% of women in their reproductive years [
1]. However, the prevalence of the disease seems to be significantly higher in infertile women, ranging from 20 to 50% [
2‐
4]. Women under the age of 35 with EM are at twice the risk of infertility [
5]. Although the link between EM and infertility is well established, the exact mechanisms remain incompletely understood. Suspected causes include pelvic adhesions and distortions, which can block the release and transport of eggs or sperm [
6]. EM may also form ovarian cysts, decrease ovarian reserve, and affect egg quality due to harmful substances in cyst fluid that can damage surrounding ovarian tissue, leading to faulty egg development and lower pregnancy chances [
6]. Additional complications may involve ovulation issues and diminished endometrial receptivity [
7].
Recent studies indicate a link between the imbalance of reproductive tract microbiota and EM [
8‐
13], with EM patients showing higher levels of harmful bacteria like Gardnerella in the vagina and cervix and decreased levels of beneficial Lactobacillus [
11,
12]. This dysbiosis extends to the gut and upper reproductive tract, possibly leading to chronic inflammation [
11] and intestinal complications [
13]. Recent research is uncovering a link between infertility and the mix of microbes in the reproductive system [
14‐
18]. Studies have found that infertile women tend to have more asymptomatic vaginal infections [
15] and a different mix of uterine bacteria [
16], including those often linked to conditions like bacterial vaginosis, chronic endometritis, and endometrial polyps [
16]. Fertility dysfunction may be associated with the degradation of immune tolerance due to decreased Treg cells’ function and quantity [
19]. Microbial infections could further weaken the inhibitory ability of maternal Treg cells, causing placental inflammation and leading to abortion [
19]. Also, imbalances in genital tract bacteria and their metabolites can impact plasma metabolite levels, possibly triggering reproductive disorders [
20,
21]. Therefore, it can be seen that female infertility is closely related to the abnormality of the reproductive tract microbiota, but current research mostly focuses on the lower reproductive tract, such as the vagina and cervix.
In brief, the occurrence and development of EM is related to changes in the reproductive tract microbiota, and changes in the reproductive tract microbiota are correlated with infertility. Therefore, we speculate that the infertility caused by EM is related to changes in the reproductive tract microbiota. In the study of endometriosis-related infertility (ERI), selecting an appropriate control group is essential to gain a comprehensive understanding of this condition’s distinct physiological and microbiological features. Tubal obstruction-related infertility (TORI), with its well-defined pathological mechanism of physical blockage impeding ovum transport, serves as an optimal counterpoint in comparative studies. Unlike the pathological underpinnings of ERI, TORI provides a contrasting infertility paradigm that excludes the complication of ectopic endometrial tissues. This juxtaposition allows us to discern not only the innate repercussions of endometriosis on fertility but also the consequential shifts within the reproductive tract’s microbiota attributable to the presence of ectopic endometrial tissue. Currently, there is still no relevant research on whether there is a difference between ERI and TORI. Therefore, we conducted this study to explore the differences in bacterial communities between ERI and TORI patients.
Materials and methods
Patients and sampling
This study was approved by ethics committee of Ningbo Women & Children’s Hospital (EC2023-008). There were a total of 57 cases, and intraoperative samples of uterine cavity fluid and abdominal fluid were collected from the patients. According to the Revised American Society for Reproductive Medicine (rASRM) system, EM is clinically classified to 4 stages. Stage I refers to minimal disease with isolated implants and no significant adhesions. Stage II signifies mild disease with superficial implants and limited adhesions. Stage III constitutes moderate disease, characterized by multiple implants, both superficial and deep, and clear adhesions. Finally, Stage IV, the most severe, includes extensive deep implants, thick adhesions, and notable involvement of the ovaries. Patients with Stage I-II endometriosis are considered to have mild EM, while those in Stages III-IV are categorized as having moderate to severe EM. Consequently, we categorized patients with EM into two groups: the mild group, which includes Stages I-II, and the moderate to severe group, encompassing Stages III-IV. There were 26 ERI patients (8 cases of stage I-II and 18 cases of stage III-IV) and 31 TORI patients. Written informed consent was obtained from the patients to utilize their samples. Inclusion criteria: (a) Meet the diagnostic criteria for infertility; (b) Patients of reproductive age, between 18 and 45 years old; (c) Patients with EM confirmed by intraoperative macroscopic examination or postoperative pathological diagnosis, and patients of TORI group were confirmed to have only tubal obstruction during surgery. If the patients had the following conditions, they needed to be excluded: (a) History of taking antibiotics, probiotics, and hormone medications in the past 8 weeks, (b) Infertility caused by other factors, such as polycystic ovary syndrome, uterine adhesions, endometrial lesions, male factors and so on, (c) Patients with vaginitis, cervical HPV infection, and abnormal cervical TCT screening results, (d) Patients with comorbidities such as hypertension, diabetes, gastrointestinal diseases, and systemic immune system diseases. For the upper reproductive tract samples, uterine fluid (UF) and peritoneal fluid (PF) were taken during the operation. For PF samples, a syringe was used to connect the aspirator, and approximately 5 to 10 milliliters of PF were aspirated from the Douglas pouch. Immediately transfer the liquid to a sterile 15 ml centrifuge tube for subsequent processing. In the case of UF acquisition, a hysteroscope outfitted with a sterile saline infusion system was utilized. Sterile saline was carefully infused into the uterine cavity, and after allowing the solution to interact with the endometrium for a period of one minute, it was then evacuated through the hysteroscope’s outflow channel. A volume of 10 milliliters of this uterine lavage was collected and transferred into a 15 ml centrifuge tube, ensuring minimal contamination and preservation of the sample integrity. All specimens were collected consecutively from July 2022 to June 2023 and stored at -80 ℃ until DNA was extracted.
DNA extraction and PCR amplification
Total DNA of the sample was extracted using MagPure Soil DNA LQ Kit (Magan) and the concentration and purity were determined by NanoDrop 2000 (Thermo Fisher Scientific, USA) and lipopolysaccharide gel electrophoresis. The DNA samples were stored at -20 °C for the further requirements. The V3-V4 region of the 16s rRNA genes was successfully amplified using PCR with the universal primers 343 F and 789R, with a previous study indicating that the reverse read of this amplicon has minimal impact on species classification [
22].
Library construction and sequencing
The Amplicon quality was visualized using agarose gel electrophoresis. The PCR products were purified with AMPure XP beads (Agencourt) and amplified for another round of PCR. After being purified with the AMPure XP beads again, the final amplicon was quantified using Qubit dsDNA Assay Kit (Thermo Fisher Scientific,USA). The concentrations were then adjusted for sequencing. Sequencing was performed on an Illumina NovaSeq 6000 with 250 bp paired-end reads. (Illumina Inc., San Diego, CA; OE Biotech Company; Shanghai, China).
QIIME2 software was used for alpha and beta diversity analysis. The microbial diversity in samples was estimated using the alpha diversity that includes Chao1 index and Shannon index. The unweighted Unifrac distance matrix performed by R package was used for unweighted Unifrac Principal coordinates analysis (PCoA) to estimate the beta diversity. Then the R package was used to analyze the significant differences between different groups using ANOVA statistical test. All data were shown as mean ± SD, and P-values < 0.05 were considered statistically significant.
Discussion
Recent research has linked the progression of EM to changes in the reproductive tract’s microbiota. However, few studies focus on the microbiome in patients with ERI. In this study, we used 16 S rRNA sequencing to profile the microbiome of the PF and UF in patients with ERI and TORI.
Our research found that there was a significant correlation between changes in the microbial communities of the UF and infertility. The results showed that
Lactobacillus,
Pseudomonas, and
Muribaculaceae were key genera in UF. Other research, including Franasiak et al. [
23], also noted
Lactobacillus’s dominance in endometrial samples, and Moreno et al. linked high
Lactobacillus abundance in endometrial fluid of fertile women (over 90%) with better pregnancy outcomes [
24]. Neither patient with EM nor TORI in our study had such high
Lactobacillus levels. According to these findings, we speculate that the decreased abundance of
Lactobacillus in the UF may be associated with adverse reproductive outcomes. Further investigation has revealed a markedly increased level of
Lactobacillus in UF of individuals with primary infertility when compared to those with secondary infertility. Hence, we surmise that a history of parturition may also influence the dynamics of the microbial community within the uterine environment. Therefore, in subsequent studies, further separate analyses should be conducted for patients with and without a history of childbirth.
Considering the link between UF microbiota and pregnancy outcomes, our analysis revealed notable differences in UF microbiota between early-stage EM (I-II) and other groups. We observed an increase in
Bacteroidetes, including
Rikenellaceae,
Blautia, and
Rufibacter. This aligns with previous studies indicating a rise in various bacteria like
Proteobacteria,
Bacteroidetes, and
Actinobacteria in EM patients versus healthy individuals [
25]. Moreover, bacteria like
Enterobacteriaceae,
Streptococcus,
Staphylococcus,
Escherichia coli, and Gram-negative bacteria, commonly found in the cervix and vagina, have been linked to lower implantation success and adverse pregnancy outcomes [
24,
26‐
30].
Phreatobacter, Pontibacter, Aquabacterium, and Rikenellaceae, which were higher in UF of EM patients, were all Gram-negative bacteria. However, we must acknowledge that the smaller sample size and the marked individual variation, especially in the UF of stage I-II EM patients, may have introduced selection bias and reduced the generalizability of our findings.
To delve deeper into the effects of uterine microbiota alterations on pregnancy outcomes, we extended our analysis using the KEGG database. This analysis uncovered significant functional differences in genes within the UF, especially in pathways associated with endometritis and endometrial receptivity. Patients with TORI showed higher levels of the IL-17 signaling pathway, often increased in chronic endometritis [
31]. The Insulin signaling pathway, NOD-like receptor signaling pathway, and PI3K-Akt signaling pathway were all downregulated in patients with stage III-IV EM. NOD-like receptor Pyrins-3 (NLRP3) upregulation can help in endometrial conditioning for embryo implantation [
32,
33], while Insulin signaling is vital for decidualization, a key pregnancy process [
34]. Our data suggest that EM might impact fertility by altering uterine microbiota, potentially affecting receptivity.
In PF,
Pseudomonas and
Lactobacillus were the main dominant genera. Our results align with the study conducted by Chen et al. [
35], where
Pseudomonas was found to be the most abundant microbe in PF, accounting for 13.5%. We found that an increase in the abundance of
Pseudomonas is an important marker in patients with moderate to severe EM. The content of
lactobacilli in PF of TORI patients accounted for 7.6%. In comparison, it accounted for only 5.7% in patients with EM, indicating a significant decrease. Furthermore, the abundance of
lactobacilli in PF was similar between patients with EM in stages I and II (5.9%) and those in stages III and IV (5.4%). These findings were consistent with the results of Wei et al. [
8]. Thus, we speculate that the decreased abundance of
Lactobacillus in the PF may be an important marker for EMs.
In the analysis of microbial abundance differences between patients with stage III-IV and those with TORI. In PF, the abundance of
Pseudomonas,
Enterococcus,
Dubosiella and
Klebsiella were significantly higher in patients with stage III-IV compared to TORI patients.
Enterococcus is commonly found in the intestines of humans. Previous studies have also shown that the altered composition of the intestinal microbiota induced by EM results in the translocation and infiltration of a significant number of Gram-negative bacteria outside the intestinal cavity. This leads to the destruction of intestinal tight junctions and a decrease in the expression of tight junction protein 2 [
36], causing a substantial infiltration of Gram-negative bacteria outside the intestine [
37]. These results suggested that in the early stages of EM, there were only minimal changes observed in the microbiota within the uterine and abdominal cavity. However, as EM progresses, the composition of the microbial community also underwent continued alterations. Our study points to a need for longitudinal studies to verify these implications and investigate causality in microbial shifts and infertility associated with EM.
Conclusion
In summary, the implications of infertility related to EM extend beyond pelvic adhesions, anatomical distortion, ovarian dysfunction, and other direct physiological disruptions. The changes in the microbiota and the subsequent shift in gene functional profiles suggest a significant, yet underexplored, role in reproductive health. We observed dynamic variations in the microbiota associated with the UF and PF as EM progresses, indicating a potential microbial involvement in disease advancement. Recognizing the importance of these findings, it is crucial to discuss their broader implications for future research, clinical practice, and patient management in the field of gynecology and reproductive health. Future studies should aim to elucidate the direct impact of specific microbial alterations on fertility outcomes. Such research endeavors could lead to novel diagnostics and treatments, improving management strategies for patients suffering from infertility associated with EM.
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