Prevalence of Pathogenic Microbes within the Endometrium in Normal Weight vs. Obese Women with Infertility

: This study investigates the association between body mass index (BMI) and the composition of the endometrial microbiota in infertile women of childbearing age. This is a retrospective clinical study comparing the endometrial microbiota across body weight in 132 patients presenting for care at an infertility clinic. The reason for infertility was recurrent pregnancy loss (RPL) or implantation failure with a prior IVF cycle. Microbe analysis was completed by Igenomix Laboratory (Valencia, Spain) using two comprehensive panels. Patients were separated into three groups based on their results: normal, dysbiotic, and pathogenic. Prevalence of these groups was compared across BMI categories and statistical analysis was used to determine significance. Of the 132 endometrial samples collected, 80 (60.6%) were normal, 16 (12.1%) were dysbiotic, and 36 (27.3%) were pathogenic. Patients with a BMI ≥ 30 showed a statistically significant increase in pathogenic endometrium compared to normal weight controls ( p = 0.029). Our conclusion is that the prevalence of pathogenic endometrium was significantly higher in the obese group compared with normal weight controls. There is a possible association between obesity and the endometrial microbiome.


Introduction
The prevalence of obesity (body mass index [BMI] of 30.0 and over) in the United States has risen in recent decades among women of reproductive age [1].Nearly 25% of women in the US who become pregnant have obesity [1].It is recognized as a multifactorial condition influenced by genetic, environmental, and behavioral factors.In addition to its established associations with cardiovascular disease, diabetes, and certain cancers, there is evidence that obesity also impacts reproductive function [2].
The primary effect of a BMI > 30.0 in women with obesity is increased adiposity, which leads to inflammation, oxidative stress, and the development of metabolic dysfunction, which has a negative impact on overall health [3].Additionally, obesity causes hormonal imbalance through the disruption of the hypothalamic-pituitary-gonadal axis [4].Due to these factors, obesity increases the risks for infertility, impaired implantation, and recurrent pregnancy loss [5][6][7][8][9].The inflammatory response caused by obesity, as well as this hormonal disruption, also puts affected women at risk for other pathologies including endometrial cancer and endometritis [10,11].
The Human Microbiome project has revealed that approximately 9% of the total human microbiome is found in the female reproductive tract, and recent studies have proved the existence of the endometrial microbiome [12,13].These bacterial communities play a crucial role in pregnancy maintenance, with disruptions potentially affecting in vitro fertilization (IVF) success through immune responses and metabolic fluctuations [14].For instance, shifts in endometrial microbiota have been associated with recurrent implantation failure (RIF) [15][16][17], with RIF patients showing significant enrichment of specific genera including Atopobium, Bifidobacterium, Delftia, Gardnerella, and Prevotella, alongside different bacterial compositions compared to controls [15,16].Additionally, positive cultures of H 2 O 2 -producing Lactobacillus have been associated with increased live birth rates (LBRs) in IVF treatments, whereas recovery of Streptococcus viridans, Gardnerella, and Staphylococcus have been linked to lower LBRs [18,19].
Also related to the endometrial microbiome, chronic endometritis is a disorder of prolonged, continuous, mild endometrial inflammation associated with infertility conditions such as RIF and recurrent pregnancy loss (RPL) [20].Microorganisms that are thought to cause chronic endometritis include Chlamydia trachomatis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Mycoplasma, Neisseria gonorrhoeae, Staphylococcus aureus, Streptococcus viridans, and Ureaplasma urealyticum [21].
The relationship between obesity and the microbiome of the female reproductive system is still being explored.Recent studies have shown that the vaginal microbiome differs in reproductive-aged women with obesity compared with normal-weight women [22], but a similar relationship involving the endometrial microbiome remains largely unexplored.The studies that have looked for an association between the uterine microbiome and BMI have focused on postmenopausal women with endometrial cancer.One such study reported that microbial diversity was significantly increased in obese vs. non-obese white women with endometrial cancer [23].Another study found that obesity may influence microbiota types in the endometrium [24].
Given the established influence of BMI on the gut microbiome [25], coupled with evidence suggesting BMI's role in endometrial microbiome composition from the aforementioned cancer studies [23,24], there is compelling rationale to investigate the correlation between obesity and the endometrial microbiome in reproductive-aged women.Therefore, the goal of this study was to evaluate the prevalence of pathogenic endometrium across body weight in women of reproductive age presenting for care at a fertility clinic.

Materials and Methods
This is a retrospective study carried out at our fertility center (Reproductive Endocrinology and Infertility, University of Kansas Medical Center).Female patients undergoing in vitro fertilization (IVF) treatment with scheduled frozen embryo transfers at our facility were included.These patients were recruited for the study because they had recurrent pregnancy loss (RPL), defined as the occurrence of 3 or more consecutive pregnancy losses, or ≥1 implantation failure with a prior IVF cycle.Exclusion criteria included (1) a known cause for infertility, including intrauterine lesions, endocrine diseases, chromosomal abnormalities, and thrombophilia; (2) antibiotic use within 1 month prior to biopsy; and (3) ongoing antibiotic treatment.A total of 132 patients were included in the study.Of these women, 73 of these women had ≥1 IVF cycle resulting in implantation failure and 59 had RPL.The average age of the participants was 36 years with a range of 25-46 years.The data for this project was previously collected as part of a prospective cohort study which was approved by The School of Medicine Institutional Review Board at the University of Kansas Medical Center (IRB #STUDY00148252).
Endometrial biopsies (EBs) were taken between days 15 and 25 of the menstrual cycle for patients with regular menstrual cycles and between days 5 and 7 in the case of medicated cycles.EBs were collected using a Pipelle endometrial biopsy cannula with transabdominal ultrasound guidance.EB samples were decanted into sterile tubes containing RNAlater solution, and each tube was vigorously shaken for approximately 4 s.After the tubes were stored at 4 • C for 4-72 h, they were shipped to Igenomix headquarters (Valencia, Spain) at room temperature.
This study was conducted using endometrial microbiome metagenomic analysis (EMMA) (accessed on 20 June 2021: https://www.igenomix.com/genetic-solutions/emmaclinics/)as a tool for assessing the uterine microbiome, using next-generation sequencing (NGS sequencing) of the bacterial 16S rRNA genes.Analysis of infectious chronic endometritis (ALICE) (accessed on 20 June 2021: https://www.igenomix.net/geneticsolutions/alice-clinics/)was also used to analyze the endometrium for chronic endometritis-causing pathogens.Detailed EMMA and ALICE protocols have been previously described [19,26].EMMA comprehensively detects a variety of bacterial genera and ALICE detects endometritis-causing pathogens present in the uterus and together they determine whether the uterine microbial environment is optimal for pregnancy.This molecular method is based on detecting and quantifying the amount of bacterial DNA present in EB samples.
Reports were received that outlined the results of the EMMA and ALICE panels.Patients were separated into 3 groups based on the results of these panels.Group (1) Normal: presence of ≥1 Lactobacillus (LB) spp.within normal range and no pathogens detected in EMMA or ALICE; Group (2) Dysbiotic: presence of 0 LB spp.within normal range and no pathogens detected in EMMA or ALICE; Group (3) Pathogenic: presence of ≥1 pathogen out of normal range in either EMMA or ALICE.
Statistical analysis was performed comparing the endometrial microbiota between the BMI groups (normal < 25, overweight 25-29, and obese ≥ 30).Demographic information was compared between the microbial groups.The Chi-square test and Fisher's exact test were used for qualitative variables and the non-parametric Mann-Whitney U test for quantitative variables, considering a p < 0.05 as statistically significant.

Results
This retrospective study evaluates the prevalence of pathogenic endometrium in overweight and obese individuals compared to normal weight controls.There were 132 total patients included in this study.A total of 53 patients had a normal BMI, 32 had an overweight BMI, and 47 had an obese BMI.Out of the 132 endometrial samples, 80 (60.6%) had normal results, 16 (12.1%)were dysbiotic, and 36 (27.3%) were pathogenic (Table 1).The average age of the patients with a normal BMI was similar to the patients with an overweight or obese BMI (35.35 years, 36.53 years, and 36.46 years, respectively).Over half (n = 73; 55.3%) of the women included in this study had a history of failed implantation with IVF.There was no significant difference in the etiology of infertility across the three BMI groups (p = 0.28).The average number of previously failed IVF cycles was similar across all groups (p = 0.46) (Table 1).There was a significant difference between the prevalence of pathogenic endometrium in obese patients compared with normal weight controls.A total of 17 patients (36.2%) with an obese BMI (≥30) and 9 patients (17%) with a normal BMI (<25) had a pathogenic endometrium (p = 0.029) (Figure 1).Out of all patients who had a pathogenic endometrium, 74.3% of these patients had an overweight (BMI 25-29) or an obese BMI (Table 1).

Discussion
The aim of our retrospective study was to assess the prevalence of pathogenic microbial communities in the endometrium relative to the body weight among women undergoing fertility treatment.Our analysis of 132 women revealed disparities in endometrial microbiota composition across varying BMI categories.Obese individuals exhibited a significantly higher prevalence of pathogenic organisms within the endometrium, with 36.2% of this group demonstrating such microbial profiles compared to 17% in the normal-weight group.These findings align with previous studies suggesting that obesity may be associated with endometrial microbiota changes.For instance, one study reported increased microbial diversity in endometrial tumors of obese women [23].Another study another identified a higher prevalence of microbial community types with elevated levels of Protobacteria in obese individuals [23].
While the relationship between obesity and the endometrial microbiome is not fully understood, several factors associated with obesity could potentially increase the risk of dysbiosis and chronic endometritis.First, obesity is known to cause chronic, low-grade inflammation throughout the body [27].This inflammation extends to the endometrium, as shown in a recent prospective cohort study.They found that obese patients had enhanced recruitment of protective, anti-inflammatory immune cell types and lower concentrations of endometrial tissue inflammatory markers after achieving weight loss with bariatric surgery [28].This is relevant to our study because endometrial dysbiosis, characterized by an increased relative abundance of pathogenic microorganisms among lactobacillus species, is associated with significantly higher tissue concentrations of inflammatory markers (IL-6, IL-1β, HIF-1α, and COX-2) [29].Second, obesity dysregulates immune Comparison of the prevalence microbial subgroups between obese and normal-weight patients.Normal BMI is <25, obese BMI is ≥30.A total of 17% (normal BMI) vs. 36.2%(obese BMI) had pathogenic endometrium.Results are statistically significant (p = 0.029).SPSS Fisher's exact test was used for statistical analysis.
Table 1 shows the demographic information of the participants and their microbial panel results according to their body mass index.Normal includes Lactobacillus predominant without pathogens.Dysbiotic is the absence of any Lactobacillus spp.without pathogens.Pathogenic includes the presence of one or more of the listed pathogens (Table 2) outside of the normal range.Table 2 lists the bacteria that were analyzed in the EMMA/ALICE panels and their reference ranges.The presence of ≥1 of the Lactobacillus spp.(left) above the reference range was required to make the designation of "Normal".If no LB spp.were detected above the reference range the endometrial tissue was "Dysbiotic".If any of the listed pathogens were present above their reference range, of LB, the tissue was "Pathogenic".

Discussion
The aim of our retrospective study was to assess the prevalence of pathogenic microbial communities in the endometrium relative to the body weight among women undergoing fertility treatment.Our analysis of 132 women revealed disparities in endometrial microbiota composition across varying BMI categories.Obese individuals exhibited a significantly higher prevalence of pathogenic organisms within the endometrium, with 36.2% of this group demonstrating such microbial profiles compared to 17% in the normalweight group.These findings align with previous studies suggesting that obesity may be associated with endometrial microbiota changes.For instance, one study reported increased microbial diversity in endometrial tumors of obese women [23].Another study another identified a higher prevalence of microbial community types with elevated levels of Protobacteria in obese individuals [23].
While the relationship between obesity and the endometrial microbiome is not fully understood, several factors associated with obesity could potentially increase the risk of dysbiosis and chronic endometritis.First, obesity is known to cause chronic, low-grade inflammation throughout the body [27].This inflammation extends to the endometrium, as shown in a recent prospective cohort study.They found that obese patients had enhanced recruitment of protective, anti-inflammatory immune cell types and lower concentrations of endometrial tissue inflammatory markers after achieving weight loss with bariatric surgery [28].This is relevant to our study because endometrial dysbiosis, characterized by an increased relative abundance of pathogenic microorganisms among lactobacillus species, is associated with significantly higher tissue concentrations of inflammatory markers (IL-6, IL-1β, HIF-1α, and COX-2) [29].Second, obesity dysregulates immune function, which may impair the body's ability to clear infections, including those that may contribute to chronic endometritis [30].
Analyzing the relationship between the uterine microenvironment and obesity is relevant because of the role they both play in the complex process of implantation and pregnancy maintenance [31,32].The presence of pathogenic microorganisms within the endometrium impairs early pregnancy development [19].Our findings suggest a potential relationship between the prevalence of these pathogenic organisms within the endometrium and an obese BMI.
It is crucial to acknowledge the limitations of our study.Our study's retrospective nature and the small sample sizes in the pathogenic groups introduce inherent biases which limit our ability to establish causation.Prospective studies with larger cohorts are warranted to validate and expand upon our observations.An additional limitation of our study was that our normal weight controls were from patients with infertility and thus, samples were not necessarily from a normal endometrium.Furthermore, endometrial sample collection limitations, including potential contamination with lower reproductive tract organisms, must be acknowledged.
In conclusion, our retrospective study highlights a potential association between obesity and the prevalence of pathogenic microbial communities in the endometrium among women undergoing fertility treatment.While our findings suggest a potential link between body weight and the composition of the endometrial microbiome, causality cannot be definitively established based on our observational data.Further prospective studies with larger cohorts are warranted to elucidate the complex relationship between obesity and the endometrial microbiome and to explore potential underlying mechanisms.These insights are crucial for advancing our understanding of the interplay between obesity, the endometrial microbiome, and reproductive health outcomes.

Figure 1 .
Figure 1.Comparison of the prevalence microbial subgroups between obese and normal-weight patients.Normal BMI is <25, obese BMI is ≥30.A total of 17% (normal BMI) vs. 36.2%(obese BMI) had pathogenic endometrium.Results are statistically significant (p = 0.029).SPSS Fisher's exact test was used for statistical analysis.

Table 1 .
Demographic information and results.
Figure 1.Comparison of the prevalence microbial subgroups between obese and normal-weight patients.Normal BMI is <25, obese BMI is ≥30.A total of 17% (normal BMI) vs 36.2% (obese BMI) had pathogenic endometrium.Results are statistically significant (p = 0.029).SPSS Fisher's exact test was used for statistical analysis.

Table 2 .
Bacteria analyzed in EMMA and ALICE panels with corresponding reference ranges.