Associations Between Endometriosis and Gut Microbiota

The gut microbiota has been associated with many diseases, including endometriosis. However, very few studies have been conducted on this topic in human. This study aimed to investigate the association between endometriosis and gut microbiota. Women with endometriosis (N=66) were identified at the Department of Gynaecology and each patient was matched with three controls (N=198) from the general population. All participants answered questionnaires about socioeconomic data, medical history, and gastrointestinal symptoms and passed stool samples. Gut bacteria were analyzed using 16S ribosomal RNA se- quencing, and in total, 58 bacteria were observed at genus level in both patients with endometriosis and controls. Comparisons of the microbiota between patients and controls and within the endometriosis cohort were performed. Both alpha and beta diversities were higher in controls than in patients. With the false discovery rate q<0.05, abundance of 12 bacteria belonging to the classes Bacilli, Bacteroidia, Clostridia, Coriobacteriia, and Gammaproteobacter differed significantly between patients and controls. Differences observed between patients with or without isolated ovarian endometriosis, involvement of the gastrointestinal tract, gastrointestinal symptoms, or hormonal treatment disappeared after calculation with false discovery rate. These findings indicate that the gut microbiota may be altered in endometriosis patients.

Endometriosis . Gastrointestinal symptoms . Gut microbiota . Pathophysiology

Endometriosis is an inflammatory, estrogen-dependent dis- ease defined by presence of endometrial tissue outside the uterine cavity, affecting approximately 6 –10% of reproduc- tive women [ 1, 2]. Besides gynecological symptoms [ 3, 4], gastrointestinal symptoms affect up to 90% of patients with endometriosis [5]. The most common gastrointestinal symp- tom is bloating, followed by nausea, constipation, diarrhea, and vomiting [5, 6]. The gastrointestinal tract is a complex ecosystem with a symbiosis of food molecules, gut mucosal cells, immune sys- tem cells, and microorganisms. It is a dynamic environment, and the commensal bacteria, or microbiota, are contently changing [7]. The gut microbiota is thought to play a major role in the maintenance of health and development of disease and has through inflammatory and metabolic changes been proven to affect conditions both inside and outside of the gastrointestinal tract [8–10]. Systemic levels of estrogen in post-menopausal women have been associated with fecal microbiome richness and levels of fecal Clostridia taxa [ 11]. Therefore, the gut micro- biota has been suggested to have an impact on estrogen levels in men and post-menopausal women and to be involved in estrogen-dependent diseases [11, 12]. Higher estrogen levels stimulate epithelial proliferation in the female reproductive tract and have been shown to drive diseases such as endome- triosis and endometrial cancer [ 13]. Recent studies have shown that the gut microbiota is a major regulator of inflam- matory processes outside the gastrointestinal tract [14], factors which may be involved in the pathogenesis of endometriosis [15]. The cytoplasmic protein AXIN1 is involved in the reg- ulation of apoptosis and has been reported to be a potential new biomarker for endometriosis [ 16], with correlations to clinical data such as gastrointestinal symptoms and hormone treatment [17]. Due to the impact of immunological and hormonal changes in patients with endometriosis, and the impact of gut microbi- ota on immune and estrogen responses, it has been * Bodil Ohlsson [email protected] 1 Department of Internal Medicine, Skåne University Hospital, Lund University, Jan Waldenströms street 15, floor 5, 20502 Malmö, Sweden 2 Department of Clinical Sciences in Malmö, Lund University, Malmö, Sweden https://doi.org/10.1007/s43032-021-00506-5 / Published online: 3 March 2021 Reproductive Sciences (2021) 28:2367–2377 hypothesized that the gut microbiota is involved in the patho- genesis of endometriosis [18]. The primary aim of the present study was to investigate the gut microbiota in endometriosis in comparison with healthy controls. The secondary aim was to examine any differences regarding microbiota abundance within the endometriosis cohort, dependent on disease locali- zation, symptoms, or treatment.

Study Design Patients with endometriosis ( N=66) were recruited from the Department of Gynaecology at Skåne University Hospital and were matched with three controls each from the Malmö Offspring Study (MOS). The study participants answered a questionnaire concerning sociodemographic data and medical history, completed the visual analog scale for irritable bowel syndrome (VAS-IBS), and passed stool samples. The gut mi- crobiota was identified at genus level using 16S rRNA se- quencing. Comparisons in gut microbiota were performed be- tween patients and controls, and within the patient cohort, using two-tailed Mann-Whitney U test, Fisher ’s exact test, Spearman’s correlations test, Shannon diversity index, and Bray Curtis dissimilarity index. Patients Patients were recruited from the Department of Gynaecology at Skåne University Hospital, Malmö, according to the ICD- 10 classification of endometriosis, N80. The main inclusion criteria were to have a diagnosis of endometriosis, confirmed by laparoscopy or laparotomy. General inclusion criteria were an age above 18 years and to comprehend the Swedish or English languages. Exclusion criteria were an uncertain diag- nosis of endometriosis, current pregnancy, diagnosed inflam- matory bowel disease (IBD), living far from the hospital, and multiple or severe somatic or psychiatric comorbidities. Between September 2016 and March 2017, 266 women who fulfilled the inclusion criteria were identified. Of these, 196 women were excluded because they were as follows: unwill- ing to participate (N=162), had moved too far from the hospi- tal (N=23), had a non-surgically confirmed diagnosis ( N=7), or an uncertain diagnosis of endometriosis ( N=2). This re- duced the number of women to 72. Out of these, 66 women passed stool samples and were thereby included in the present study. Controls The controls were recruited from 2644 individuals who had previously been extracted from the MOS [19], which consists of descendants to participants in the Malmö Diet and Cancer Cardiovascular Cohort (MDC-CC). The recruitment of partic- ipants to the MDC-CC and the MOS took place in the 1990s and 2010s, respectively [20, 21]. Each case was matched with three controls according to sex (only women), age (± 730 days), body mass index (BMI) (± 2 BMI units), and smoking. The participants from MOS who had not answered question- naires and passed stool samples or were diagnosed with celiac disease, Crohn ’s disease, ulcerative colitis, irritable bowel syndrome (IBS), or lactose intolerance were excluded from the matching process. Study Questionnaires The patients with endometriosis answered questions regarding their endometriosis-associated symptoms such as onset of symptoms, trigger factors, and treatment. They also answered a questionnaire regarding education, occupation, marital sta- tus, smoking habits, alcohol habits, physical activity, medical history, and pharmacological treatments. The participants in the MOS answered a similar questionnaire. They were also asked the following question: “Have you experienced bowel symptoms during the last 2 weeks?” All participants from MOS who had answered “yes” were excluded from the pres- ent study. VAS-IBS The VAS-IBS is a psychometrically validated questionnaire used to estimate gastrointestinal symptoms in patients with functional bowel disease [ 22]. With the VAS-IBS question- naire, the patients estimated the severity of symptoms over the last 2 weeks regarding abdominal pain; constipation; diarrhea; bloating and flatulence; vomiting and nausea; and intestinal symptom’s influence on daily life. The VAS-IBS question- naire measures each of the symptoms on a continuous scale from 0 to 100 mm where 0 represents no problems and 100 represents very severe problems. The scales were inverted from the original scales [ 22]. Reference values from 52 healthy women were used as controls [ 23]. Gut Microbiota and Laboratory Analyses Stool samples were collected by the patients and controls at home in sterile tubes (Sarstedt, Numbrecht, Germany) and put in the freezer until they were brought to the lab. The samples were kept at –80 °C until the extraction of microbial DNA. Microbial DNA was extracted from the stool samples using a QIAamp column Stool Kit. The V1 –V3 regions of the 16S ribosomal RNA gene were pairwise (300*2 base pairs) ampli- fied and sequenced using a HiSeq Illumina at GATC Biotech (Constance, Germany). The sequences were stored as fastq files which were aligned by FLASH and binned together to 2368 Reprod. Sci. (2021) 28:2367–2377 operational taxonomic units (OTUs) using QIIME [ 24, 25]. The sequences were then matched with the reference database Greengenes and classified at genus level. In total, 937,892,146 reads, with an average of 434,008 reads per sample, were included in the analysis. Finally, the data was normalized using the cumulative sum scaling with the R package metagenomSeq. In total, 64 bacteria at genus level occurred in the control group and 66 occurred in the group of patients with endome- triosis. Bacteria that only occurred in <10 of the samples were excluded, leaving 58 bacteria at genus level in the control group and 62 in the patient group, and thus, 58 bacteria were included in the statistical analyses between patients and con- trols, and 62 bacteria in calculations within the endometriosis cohort. Levels of plasma AXIN1 and fecal calprotectin were ana- lyzed by ELISA as previously described by Dihm et al. [ 17]. Data Categorization Smoking habits were divided into current smoking and no current smoking, regardless of previous smoking habits. Alcohol intake was divided into <1 or ≥1 standard glass per week. Physical activity was divided into <1 or ≥1hp e rw e e k of activity which lead to breathlessness. Hormone treatment was divided into current treatment or no current treatment, regardless of previous treatment. Hormonal treatment includ- ed estrogen, combined oral contraceptives, progestin, and gonadotropin-releasing hormone (GnRH) analogs. Localization of endometriosis lesions were divided into iso- lated ovarian lesions or spread to any other location and bowel involvement or no bowel involvement. Gastrointestinal symp- toms were divided into having symptoms or not having any symptoms specified on the VAS-IBS scales, i.e., abdominal pain; diarrhea; constipation; bloating and flatulence; vomiting and nausea; and influence of intestinal symptom’s on daily life the last 2 weeks, considered together as one value. If none of the values exceeded a predetermined value determined in a previous study of healthy female volunteers, the patient was categorized as having no symptoms [23]. Statistical Analyses Statistical analyses were performed using the software SPSS© statistical computer package version 26 for Windows. Since the distribution of the quantitative data was skewed, descrip- tive statistics were calculated by the Mann-WhitneyU test and Spearman’s correlation test. Fisher ’s exact test was used for dichotomous variables. A sensitivity analysis was performed where all patients and controls who had received antibiotic treatment in the last 6 months prior to inclusion in the study were removed. Beta diversity was calculated using the Bray-Curtis dissim- ilarity index to detect differences in microbiota composition among the groups. Beta diversity was calculated using vegdist; further statistical difference for dissimilarity index was tested with Adonis, all within the R package vegan. Alpha diversity was tested using the Shannon diversity index to analyze diversity of genus among the samples. Alpha di- versity was calculated usingdiversity. Furthermore, a variance test (ANOVA) was performed [26]. Values are presented as median and interquartile range or number and percentage. Q-values are thep-values adjusted for FDR set at 5% according to the Benjamini-Hochberg method, to adjust for multiple comparisons, and considered our main

[27].

Basal Characteristic A total of 66 women with endometriosis and 198 controls were included in the study, who showed similar basal characteristics (Table 1). The median age of the patients was 37.8 (32.8– 43.3) years and the median BMI was 25.0 (22.0–28.0) kg/m 2. The vast majority of the women had an education from secondary school or university (95.4%) and were either studying or working (83.4%). Most of the patients were non-smokers (84.8%) and drank less than 1 standard glass of alcohol/week (63.6%). Of all, 27 pa- tients (40.9%) had ≥ 1 h/week of physical activity which led to breathlessness. A minority of the patients (28.8%) were living alone. More patients (62.1 vs. 51.5%) than controls (8.1 vs. 17.2%) were currently treated with hor- mone therapy or analgesic drugs (non-steroidal anti- inflammatory drugs, opioids, paracetamol) (Table 1). Endometriosis Characteristics Twenty-seven patients (40.9%) had isolated ovarian endome- triosis and 18 patients (27.3%) had involvement of the gastro- intestinal tract (Supplementary Table 1). Of the 41 patients who were currently treated with hormone therapy, 20 patients (48.8%) were treated with estrogen or combined oral contra- ceptives, 19 patients (46.3%) were treated with progestin, and 8 patients (19.5%) were treated with GnRH analogs. The ma- jority of the patients (86.4%) had suffered from gastrointesti- nal symptoms over the last 2 weeks prior to inclusion in the study. There was no difference in gastrointestinal symptoms between patients with isolated ovarian lesions or spread le- sions ( p=0.71), gastrointestinal tract involvement or not (p=1.00), or with or without hormone treatment ( p=0.47). There was no difference in hormone treatment between pa- tients with isolated ovarian lesions or spread lesions (p=0.31) 2369Reprod. Sci. (2021) 28:2367–2377 or gastrointestinal tract involvement or not (p=0.57). The me- dian value of plasma AXIN1 was 390.0 (357.5 –420.0) pg/ml and the median value of feces calprotectin was 25.00 (25.00– 29.50) mg/kg. Of the patients, a total of 8 women (12.1%) had Table 1 Basal characteristics in endometriosis patients and controls Variables Controls N=198 Patients N=66 p-value Age, years 37.0 (32.0–44.0) 37.8 (32.8 –43.3) 0.88 BMI, kg/m2 24.7 (22.1–27.5) 25.0 (22.0 –28.0) 0.70 Education level, N (%) 1.00 Missing value 2 1 Graduated primary 8 (4.1) 2 (3.0) Graduated secondary 79 (39.9) 27 (40.9) Graduated university 109 (55.1) 36 (54.5) Occupation, N (%) 0.05 Missing value 14 1 Full time 105 (53.0) 33 (50.0) 51–99% 49 (24.7) 10 (15.2) 1–50% 15 (7.6) 7 (10.6) Sick or early retirement 3 (1.5) 5 (7.6) Unemployed 4 (2.0) 6 (9.1) Student 8 (4.0) 5 (7.6) Current smoking, N (%) 30 (15.2) 10 (15.2) 1.00 Alcohol intake ≥ 1 glass/week, N (%) 71 (35.9) 24 (36.4) 1.00 Physical activity ≥ 1 h/week, N (%) 93 (47.0) 27 (40.9) 0.48 Lives alone, N (%) 44 (22.2) 19 (28.8) 0.18 Hormone treatment, N (%) 16 (8.1) 41 (62.1) <0.001 Missing value 1 Antibiotic treatment last 6 months, N (%) 29 (14.6) 12 (18.2) 0.56 Missing value 1 Analgesic treatment, N (%) 34 (17.2%) 34 (51.5%) <0.001 Missing value 6 Visual analog scale for irritable bowel syndrome Missing value 1 Abdominal pain (mm) 47 (13–72)

values 5 (1–15) Constipation (mm) 28 (2–60)

values 9 (1–22) Diarrhea (mm) 17 (2–55)

values 3 (0–10) Bloating and flatulence 62 (20–76)

values 14 (1–29) Vomiting and nausea (mm) 15 (2–50)

values 2 (0–3) Psychological well-being (mm) 37 (13–62)

values 4 (0–16) Intestinal symptoms influence on daily life (mm) 52 (17–80)

values 2 (0–18) Gastrointestinal symptoms were assessed by the visual analog scale for irritable bowel syndrome, 0 –100mm, where 0 mm represents no symptoms and 100 mm maximal symptoms [ 22]. Reference values from healthy controls are shown [ 23]. Values are presented as median (interquartile range) or numbers (percentage). Mann- Whitney U te st or Fisher ’s exact test. p-values <0.05 were considered statistically significant BMI body mass index 2370 Reprod. Sci. (2021) 28:2367–2377 been born by caesarean section. There were no significant differences regarding endometriosis characteristics between the subgroups (data not shown). Bacterial Analysis The adonis test showed a significantly higher beta diversity in the control group compared to the endometriosis group. However, R2 was very small (0.02) (Fig. 1). The ANOVA test showed a significantly higher alpha diversity, p=4.9e−05, in the control group compared to the endometriosis group (Fig.2). Nineteen gut bacteria at genus level differed in abundance between endometriosis patients and controls. With the FDR set at 0.05, this number was reduced to 12 bacteria (Fig. 3, Table 2). These bacteria belonged to the classes Bacteroidia (N=4), Clostridia (N=4), Coriobacteriia (N=2), Bacilli (N=1), and Gammaproteobacter ( N=1). Two bacteria belonging to the Bacteroidia class ( Bacteroides and Parabacteroides)a n d two belonging to the Clostridia class ( Oscillospira and Coprococccus) were observed in higher abundance in pa- tients, and two other bacteria in the Bacteroidia (Paraprevotella and one unidentified) and Clostridia (Lachnospira and one unidentified) classes were observed in lower abundance, compared to controls. Genus belonging to the Bacilli (Turicibacter) and the Coriobacteriia (unidentified) classes were found in lower abundance, whereas an unidenti- fied genus in the class of Gammaproteobacter was found in higher abundance, compared to the controls (Fig. 3,T a b l e2). Patients with isolated ovarian endometriosis had a higher abundance of one unidentified genus and Lachnobacterium belonging to the Clostridia class and Adlercreutzia belonging to the Coriobacteriia class, com- pared to those with spread disease (Table 3). Patients with endometrial involvement of th e gastrointestinal tract had a higher abundance of Lactococcus belonging to the class Bacilli compared to them without involvement (Table 4). Endometriosis patients with gastrointestinal symptoms had a lower abundance of SMB53 in the Clostridia class, and lower and higher abundance of Odoribacter and Prevotella , respectively, belonging to the Bacteroidia class, compared to those without symptoms (Table 5). When the abundance of bacteria and the degree of symp- toms were compared on bacteria which differed between patients with and without gastrointestinal symptoms, there was a correlation between Prevotella and the symptoms constipation ( R=0.307, p=0.014); bloating and flatulence (R=0.297, p=0.016); and vomiting and nausea ( R=0.295, p=0.017). Patients with hormonal treatment had a higher abundance of Blautia and Ruminococcus belonging to the Clostridia class, and Butyricimonas in the Bacteroidia class, compared with those without treatment (Table 6). There was a correlation between levels of fecal calprotectin and the abundance of Ruminococcus (R=0.260, p=0.038), whereas pla sma AXIN1 levels did not correlate with any bacteria abundance (data not shown). With the FDR set at 0.05, these results lost sig- nificance, and there were no significant differences of microbiota abundance within the cohort depending on dis- ease localization, symptoms, or hormone treatment (Tables 3, 4, 5,a n d6 ). There was no difference in gut bacteria between patients with and without current anal- gesic treatment (data not shown). Fig. 1 Plot visualizing the beta diversity (Bray-Curtis dissimilarity index) of gut micro- biota, colored by healthy controls (1) and patients with endometri- osis (2) 2371Reprod. Sci. (2021) 28:2367–2377 Sensitivity Analysis After exclusion of all participants who had received antibiotic treatment in the last 6 months, 17 bacteria differed between the groups in the initial calculation. After FDR adjustment, only three bacteria with a significant difference in abundance be- tween patients and controls were detected, namely Lachnospira, Oscillospira , and a genus in the order Bacteroidales (Supplementary Table 2). In patients with sole ovarian involvement, difference in abundance of Prevotella Fig. 2 Boxplot of alpha diversity (Shannon diversity index) for gut microbiota in healthy controls (1) and patients with endometriosis (2). p-values <0.05 were consid- ered statistically significant Fig. 3 Stacked bar plots of the 19 genus (mean relative abundance) from Table2 that significantly differed between healthy (group 1) and patients with endometriosis (group 2) 2372 Reprod. Sci. (2021) 28:2367–2377 was gained whilst those of Lachnobacterium and Adlercreutzia were lost, compared to those with spread endo- metriosis (Supplementary Table 3 ), whereas calculations re- garding gastrointestinal i nvolvement were unaffected (Supplementary Table 4 ). In patients with gastrointestinal symptoms, difference in the abundance of Turicibacter was gained whilst those of Odoribacter and Prevotella were lost, compared to those without gastrointestinal symptoms (Supplementary Table 5 ). Patients with current hormonal treatment had a difference in abundance of a genus in the family S247, compared to those without treatment, whilst dif- ferences of Ruminococcus and Butyricimonas disappeared (Supplementary Table 6). The differences within the endome- triosis cohort lost significance after FDR adjustment (Supplementary Table 3–6).

Generally, the overall diversity of gut microbiota was signif- icantly higher among controls compared to patients with en- dometriosis. Especially, the alpha diversity differed consider- ably whilst the beta diversity was only marginally higher in controls than in endometriosis patients. There were differ- ences in abundance of 12 genus belonging to the classes Bacilli, Bacteroidia, Clostr idia, Coriobacteriia, and Gammaproteobacter between endometriosis patients and con- trols, without any significant differences within the endome- triosis cohort after FDR adjustments. A systematic review from 2020 identified 13 clinical stud- ies that investigated the connection between endometriosis and the microbiome [ 28]. Out of the 13 studies, only six had Table 2 Bacteria with significant difference between endometriosis patients and controls Bacteria Controls N=198 Patients N=66 p-value Q-value g__Paraprevotella; f__Paraprevotellaceae; o__Bacteroidales; c__Bacteroidia 0.71 (0.00 –4.70) 0.00 (0.00 –1.11) <0.001 0.00058 g__Adlercreutzia; f__Coriobacteriaceae; o__Coriobacteriales; c__Coriobacteriia 6.76 (4.91 –8.97) 5.15 (3.10 –7.31) <0.001 0.00029 g__f__o__Bacteroidales; c__Bacteroidia 0.63 (0.00–2.69) 0.00 (0.00 –0.50) <0.001 0.00019 g__Lachnospira; f__Lachnospiraceae; o__Clostridiales; c__Clostridia 12.43 (11.60–13.31) 3.47 (1.34 –4.88) <0.001 0.00015 g__Oscillospira; f__Ruminococcaceae; o__Clostridiales; c__Clostridia 10.67 (9.81–11.62) 11.79 (10.60 –12.53) <0.001 0.00012 g__f__Coriobacteriaceae; o__Coriobacteriales; c__Coriobacteriia 8.24 (6.72–9.45) 6.95 (5.25 –8.65) 0.001 0.0096 g__Bacteroides; f__Bacteroidaceae; o__Bacteroidales; c__Bacteroidia 15.29 (14.25–16.45) 16.08 (15.14 –17.26) 0.001 0.0083 g__Parabacteroides; f__Porphyromonadaceae; o__Bacteroidales; c__Bacteroidia 11.27 (9.98 –12.47) 11.92 (10.95 –13.20) 0.001 0.0073 g__f__o__SHA98; c__Clostridia 2.63 (0.00–5.70) 0.00 (0.00 –4.01) 0.004 0.026 g__f__Enterobacteriaceae; o__Enterobacteriales; c__Gammaproteobacter 3.28 (1.06 –5.56) 4.38 (2.30 –7.16) 0.007 0.041 g__Turicibacter; f__Turicibacteraceae; o__Turicibacterales; c__Bacilli 4.50 (2.57–6.75) 2.89 (0.00 –5.84) 0.008 0.042 g__Coprococcus; f__Lachnospiraceae; o__Clostridiales; c__Clostridia 10.31 (9.34–11.25) 10.81 (9.95 –11.76) 0.009 0.044 g__f__o__YS2; c__4C0d2 0.00 (0.00–3.89) 0.00 (0.00 –1.11) 0.012 0.054 g__f__o__RF32; c__Alphaproteobacteria 3.27 (0.00–6.72) 0.00 (0.00 –5.18) 0.016 0.066 g__f__Peptostreptococcaceae; o__Clostridiales; c__Clostridia 6.90 (5.10–8.60) 6.04 (3.74 –8.15) 0.024 0.093 g__f__Barnesiellaceae; o__Bacteroidales; c__Bacteroidia 11.53 (9.43–12.65) 10.43 (7.45 –12.55) 0.029 0.11 g__f__Halanaerobiaceae; o__Halanaerobiales; c__Clostridia 8.53 (7.13–9.56) 7.85 (6.12 –9.08) 0.033 0.011 g__f__o__RF39; c__Mollicutes 2.86 (0.19–7.70) 0.54 (0.00 –6.59) 0.040 0.13 g__f__Lachnospiraceae; o__Clostridiales; c__Clostridia 3.83 (2.37–5.11) 12.72 (12.08 –13 .57) 0.040 0.12 Values of operational taxonomic unit (OTU) are presented as median (interquartile range). Mann-Whitney U test. The q-value is the adjusted p-value with a false discovery rate (FDR) of 5% and considered the main result Table 3 Bacteria with significant difference between patients with isolated ovarian and spread endometriosis Bacteria Only ovarium N=27 Spread N=38 p-value Q-value g__f__Christensenellaceae; o__Clostridiales; c__Clostridia 5.76 (3.86–6.94) 3.77 (0.82 –6.10) 0.014 0.868 g__Lachnobacterium; f__Lachnospiraceae; o__Clostridiales; c__Clostridia 6.68 (5.99 –7.80) 6.62 (5.21 –7.70) 0.036 1.000 g__Adlercreutzia; f__Coriobacteriaceae; o__Coriobacteriales; c__Coriobacteriia 6.30 (4.61 –7.30) 4.64 (2.47 –6.76) 0.046 0.951 Values of operational taxonomic unit (OTU) are presented as median (interquartile range). Mann-Whitney U test. The q-value is the adjusted p-value with a false discovery rate (FDR) of 5% and our main results 2373Reprod. Sci. (2021) 28:2367–2377 studied the gut microbiota, and only a single study had been performed in human gut microbiota [28]. The main finding in Ata et al. 2019 [ 29] was that two women with stage 3 –4 endometriosis had an Escherichia/Shigella- dominant gut microbiome at genus level, whereas none of the endometriosis-free controls exhibited this dominance. Escherichia and Shigella belong to the family Enterobacteriaceae. We found a non-significant enrichment of Enterobacteriaceae in endometriosis patients, although we did not identifyEscherichia/Shigellaat genus level using 16 S rRNA sequencing. Since the previous study only included as few as 14 patients [29], and our findings lost significance due to correction for multiple testing, further studies are required to determine the true significance of Enterobacteriaceae be- tween subjects with and without endometriosis. No differ- ences between endometriosis patients and controls in micro- biota composition could be found in a recent study examining rectal swab samples [ 30]. A few studies have examined and found altered microbiota composition in the human reproduc- tive tract in endometriosis, but the low number of studies makes it impossible to estimate the associations between mi- crobiota alterations in the reproductive tract and the gastroin- testinal tract [28, 31]. Several animal studies support the hypothesis that endome- triosis has an impact on the gut microbiota. Rhesus monkeys with endometriosis had a significantly altered gut microbiota profile compared to healthy controls, and endometriosis was associated with higher concentrations of Gram-negative bac- teria and lower concentrations of Lactobacilli [32]. When en- dometriosis was induced in mice, a higher beta diversity of gut microbiota was developed first after 42 days, with similar alpha diversity, among the endometriosis mice compared with control mice [ 33]. In contrast, other mouse studies with endometriosis induction have described effects on the gut mi- crobiota already after 21 days; one study found decreased diversity, richness, and abundance of gut microbiota [ 34] and one found increased alpha and beta diversity [35], where- as a third study could not identify any effect at all [ 36]. Interestingly, antibiotic treatment to mice reduced the endo- metriosis lesions and inflammatory responses, changes which were restored after oral feces gavage [35]. Although different models were used to induce and evaluate endometriosis in mice, the results point to an association between endometri- osis and gut microbiota and suggest that gut bacteria promote endometriotic lesion progression [ 31, 34–36]. However, the microbiota alterations may also depend on other functions, e.g., subclinical infections [37]. Our results indicated that the beta diversity was slightly higher in the general population, whilst the alpha diversity was significantly higher, compared to the endometriosis patients. Even though present and previ- ous results are contradicting, which might be explained by different species, our results indicate that long-term exposure to endometriotic tissue may affect the gut microbiota in human. Endometriosis is an estrogen-dependent disease and high levels of estrogen have been linked to the pathogenesis of endometriosis [13]. The symptoms related to the disease are commonly treated with estrogen, combined oral contracep- tives, progestin, or GnRH analogs, which abolish ovulation by lowering of the systemic levels of estrogen [ 38]. Previous studies have shown that the gut microbiota has an impact on estrogen levels and a bi-directional relationship in estrogen- dependent diseases [ 11, 12]. Thus, gut microbiota could be in volved in the development and symptomology of endome- triosis, but endometriosis and its treatment may also affect the composition of gut microbiota. In the present study, we could Table 4 Bacteria with significant differences between endometriosis patients with and without involvement of the gastrointestinal (GI) tract Bacteria GI tract not involved N=47 GI tract involved N=18 p-value Q-value g__Lactococcus; f__Streptococcaceae; o__Lactobacillales; c__Bacilli 2.30 (1.05 –4.16) 3.90 (1.81–6.11) 0.034 1.000 Values of operational taxonomic unit (OTU) are presented as median (interquartile range). Mann-Whitney U test. The q-value is the adjusted p-value with a false discovery rate (FDR) of 5% and our main results Table 5 Bacteria with significant difference between patients with and without gastrointestinal (GI) symptoms Bacteria No GI symptoms N=8 GI symptoms N=57 p-value Q-value g__SMB53; f__Clostridiaceae; o__Clostridiales; c__Clostridia 6.96 (5.81–8.52) 4.72 (2.54 –6.92) 0.011 0.682 g__Odoribacter; f__Odoribacteraceae; o__Bacteroidales; c__Bacteroidia 3.06 (0.54 –6.08) 0.00 (0.00 –0.82) 0.028 0.868 g__Prevotella; f__Prevotellaceae; o__Bacteroidales; c__Bacteroidia 0.00 (0.00 –1.63) 4.96 (2.90 –10.44) 0.030 0.620 Values of operational taxonomic unit (OTU) are presented as median (interquartile range). Mann-Whitney U test. The q-value is the adjusted p-value with a false discovery rate (FDR) of 5% and our main results 2374 Reprod. Sci. (2021) 28:2367–2377 not prove that changes in gut microbiota would cause or con- tribute to gastrointestinal symptoms. A previous study has described an association between the protein AXIN1 and endometriosis in humans [17]. The study showed that plasma levels of AXIN1 were higher in patients with current hormone treatment, positively correlated with both duration and degree of gastrointestinal symptoms, and negatively correlated with levels of fecal calprotectin. However, we could not find any correlation between AXIN1 levels and abundance of bacteria, which differed between those with or without gastrointestinal symptoms or hormonal treatment. The correlation be tween fecal calprotectin and Ruminococcus could possibly reflect more inflammation in the group treated with hormones [ 17]. The results of the current study did not suggest that the localization of the endometriosis lesions is related to an altered profile of gut microbiota, in accordance with a recent study concerning potential plasma biomarkers for endometriosis, such as AXIN1, ST1A1, CXCL9, and OSM [16]. On the other hand, patients with pelvic endometriosis, with or without ovarian involvement, may have a higher prevalence of tenascin-C autoantibodies than patients with isolated ovarian endometriosis [39]. Antibiotic treatment has beenshown to affect the gut micro- biota for up to 6 months [40]. In the FDR-controlled sensitivity analysis, only three bacteria at genus level differed significantly between patients and controls. It is unknown whether the reduced number of significant differences in bacteria abundance was due to the lower number of participants in the sensitivity analysis, or if antibiotic treatment had an impact on the results. The strength of the present study is the examination of a human cohort and matched controls. However, some of the controls could theoretically also suffer from endometriosis. Since all participants from the MOS who suffered from gas- trointestinal symptoms were excluded, this risk was mini- mized. Another limitation is that only 16S rRNA has been examined, and not the whole microbiota genome. Furthermore, adjustments for food and several other con- founders were not possible to perform. Examination of biopsy samples instead of feces may be more representative. Also, large numbers of statistical calculations of several bacteria were performed. By using FDR, we have tried to reduce the effect of multiple testing. On the other hand, by using FDR, there is a risk to reduce the significance of a true association, which had been found if a smaller number of bacteria had been measured. Therefore, we have chosen to show also the

before FDR-adjustment. The large number of different bacteria in the gut in combi- nation with varying lifestyle habits and several confounders for the microbiota composition may lead to different results in different studies, maybe only by chance. One of the great challenges for the future is to standardize sample collection and analysis, with appropriate adjustments for confounders, to be able to compare different studies. Sample collection prior to any endometriosis treatment is important. In addition, asso- ciations between the microbiota composition of the gastroin- testinal tract and the reproductive tract and estrogen levels would be of interest to evaluate. Furthermore, the microbiota composition should be related to fecal metabolites, to better understand the functional role of the composition [ 34]. Our results indicate that the overall gut microbial diversity is significantly higher in controls compared to patients with endometriosis. Although the analyses showed significant re- sults for a number of bacteria at the genus level, the differ- ences could be a coincidence depending on multiple compar- isons. Based on the cross-sectional study design, it is not pos- sible to decide whether the gut microbiota has any major im- pact on endometriosis development and the related symptoms or whether endometriosis affects the gut microbiota. However, our study suggests that the gut microbiota may be altered to some extent in patients with endometriosis. The findings put a perspective on microbiota profiling in endome- triosis and provides a basis for further research on the patho- physiology, diagnosis, and treatment of endometriosis. Supplementary Information The online version contains supplementary

available at https://doi.org/10.1007/s43032-021-00506-5.

We would like to thank Malin Ek and the staff at the Clinical Research Unit at the Department of Internal Medicine, Skåne University Hospital, Malmö, for collecting all data, and Johan Hultman for setting up the microbiota pipeline. Table 6 Bacteria with significant difference between patients with and without current hormonal treatment Bacteria No treatment N=24 Treatment N=41 p-value Q-value g__Blautia; f__Lachnospiraceae; o__Clostridiales; c__Clostridia 10.80 (9.85–12.15) 12.12 (11.05 –13.30) 0.009 0.558 g__Ruminococcus; f__Lachnospiraceae; o__Clostridiales; c__Clostridia 8.54 (8.12 –10.33) 9.75 (6.82 –10.74) 0.019 0.589 g__Butyricimonas; f__Odoribacteraceae; o__Bacteroidales; c__Bacteroidia 8.91 (6.04 –10.62) 9.31 (4.27 –12.47) 0.034 0.703 Values of operational taxonomic unit (OTU) are presented as median (interquartile range). Mann-Whitney U test. The q-value is the adjusted p-value with a false discovery rate (FDR) of 5% and our main results 2375Reprod. Sci. (2021) 28:2367–2377 Code Availability Code is not available due to European laws. Author’s Contribution Conceptualization, B.R. and B.O.; methodology, A.S., B.R., and B.O.; software, L.B., A.S., and B.O.; validation, A.S., B.R., L.B., and B.O.; formal analysis, A.S., L.B., and B.O.; investigation, B.R.; resources, B.O.; data curation, A.S. and B.O.; writing —original draft preparation, A.S.; writing —review and editi ng, B.R., L.B., M.O.M., and B.O.; visualization, A.S. and L.B.; supervision, M.O.M and B.O.; project administration, B.O.; funding acquisition, B.R. and B.O. All authors have read and agreed to the published version of the manuscript. Funding Open access funding provided by Lund University. This re- search was funded by grants from Bengt Ihre Foundation, Dir Albert Påhlsson’s Foundation, and Development Foundation of Region Skåne. Data Availability Data can be provided from the authors upon request. Declarations Ethics Approval and Consent to Participate This study was ap- proved by the Ethics Review Board of Lund University, No 2012/594, 2012/564, and 2016/56. All subjects gave written, informed consent before inclusion in the study. Consent for Publication All subjects gave written, informed con- sent before inclusion in the study. Conflict of Interest The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adap- tation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, pro- vide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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