The role of gut dysbiosis in endometriosis' diagnosis and treatment approaches - case report

We report a case of a 33-year-old nulliparous woman that presented to Prof. Dr. Panait Sîrbu Obstetrics and Gynecology Hospital (Bucharest, Romania) with painful symptoms, dysmenorrhea visual analog scale (VAS) 10, dyspareunia, menorrhagia, and GI symptoms (constipation, dyschezia, abdominal flatulence), with little relief from non-steroidal anti-inflammatory medication. The patient has been under clinical monitoring by our multidisciplinary medical team since July 2022. This is a case reported in line with the Surgical CAse REport (SCARE) criteria. Medical history The patient was diagnosed with EM in 2012. Her medical and surgical history allowed us to recollect two laparoscopic surgeries for EM in 2012 and 2018, both of which were pathologically confirmed. The patient underwent laparoscopic right ovarian cystectomy (endometrioma 5 cm in diameter) in 2012 and laparoscopic left ovarian cystectomy (endometrioma 7 cm in diameter) and hysteroscopic polypectomy in 2018. We have no data available regarding the deep infiltrative EM lesions back in 2012 and 2018. Between the two procedures, she received numerous combined oral contraceptive treatments (Ethinylestradiol 0.02 mg and Gestoden 0.075 mg, then Ethinylestradiol 0.03 mg and Drospirenone 3 mg) that didn’t improve her painful symptomatology. After three months since the 2018 surgery, she presented for recurrent pelvic pain and secondary amenorrhea with a negative pregnancy test. The US evaluation revealed an enlarged pelvic mass measuring 1.69 cm in diameter, with a Doppler echo free lumen due to blood accumulation, the patient being diagnosed with hematometra (a collection of blood inside the uterine cavity) and cervical stenosis (Figure 2 ). The cause of the hematometra was the obliteration of the cervical ostium. She underwent a surgical dilation to drain the blood from the uterus and a Levonorgestrel-releasing intrauterine system was inserted. After two months, the intrauterine device was removed due to menorrhagia and pelvic pain. A cervical Pap smear performed in February 2022 reported atypical squamous cells of undetermined significance (ASCUS), with the following colposcopy features of low-grade cervical intraepithelial neoplasia (CIN). She underwent a loop electrosurgical excision procedure (LEEP) with the histopathological result of low-grade CIN. The follow-up Pap smear and colposcopy in 2021 was normal, with no features of cervical dysplasia (Figure 3 ). Medical evaluation Medical evaluation in July 2022 revealed the following: Clinical examination Physical examination revealed limited uterine mobility and rebound discomfort in the pelvic region. The uterus and adnexa were both within usual limits, while the Douglas’ pouch was utterly obliterated. Ultrasound aspect of hematometra – yellow arrow (A–C) Normal colposcopy aspect after the loop electrosurgical excision procedure (LEEP) Transvaginal ultrasound The General Electric (GE) Voluson E8 machine was used to do the TVUS examination. We requested the patient to lie on her back while we put the sterile US probe into the vagina. To determine the presence of EM cysts and lesions, we examined the uterus, both adnexa, as well as the anterior and posterior pelvic compartments. The TVUS identified two small left endometriomas, each measuring 1.5 cm and 1 cm in diameter, the ovaries being adherent to the posterior uterine wall. The sliding sign was negative in the posterior compartment, suggesting a lack of mobility of the rectum against the uterus, indicating adhesion and the presence of EM lesions. A 2.63 cm-diameter hypoechogenic intestinal nodule was also identified in the retrocervical area (Figure 4 ). The irregular contour of the uterosacral ligaments suggested EM lesions. Ultrasound aspect of the retrocervical endometriosis nodule Magnetic resonance imaging with endometriosis protocol The MRI examination was performed using EM protocol: US gel instillation inside the vagina and rectum for a better expansion and visualization of the pelvic organs. The MRI described the “kissing-ovaries” sign and the appearance of the rectosigmoid EM nodule, which supported the US diagnosis of deep infiltrative EM. Additionally, according to both imaging methods, adenomyosis is diagnosed by describing an asymmetrical thickening of the myometrium, particularly in the anterior uterine wall (Figure 5 ). We emphasize the importance of a trained US gynecologist that can perform a correct preoperative assessment of the lesions which can aid in the correct planning of the surgical intervention. The diagnosis of deep EM is based on imaging investigations – TVUS and pelvic MRI with EM protocol: #Enzian P0 O2/0 T2/0 A2 B2/2 C2 FA (left endometrioma, left parameter nodule, left and right uterosacral ligaments nodules, retrocervical nodule, rectal nodule, adenomyosis). MRI with EM protocol results: (A) Rectal EM nodule; (B) Left endometrioma; (C) Recto-cervical nodule; (D) Adenomyosis lesion. In all images, the structures mentioned above are indicated by yellow arrows. EM: Endometriosis; MRI: Magnetic resonance imaging Paraclinical examination Further investigations revealed a mild anemia [hemoglobin (Hb) 10.6 mg/dL, mean corpuscular volume (MCV) 76 fL with a normal interval of 80–96 fL, and iron deficit at 22 mg/dL]. The patient received oral and intravenous treatment with iron and folic acid. Cancer antigen 125 (CA125) 43.1 IU/mL (normal range 0–35 IU/mL). In some neoplasia (ovarian, endometrial, and Fallopian tube cancers) or gynecological diseases including EM, uterine fibroids, or ovarian masses, this tumor marker is elevated in a non-specific manner. Mild hypovitaminosis D (15 ng/mL, with a normal interval 20 and 40 ng/mL) for which received supplements with vitamin D 5000 IU per day. The vaginal smear revealed an infection with Ureaplasma urealyticum and Mycoplasma hominis, as well as vaginal candidiasis, for which she was prescribed oral Doxycycline 100 mg for 14 days, oral Fluconazole 150 mg for three days and intravaginal cream with Nystatin. Urinary infection with Enterobacter aerogenes, for which she was prescribed oral antibiotics (Cefuroxime 1 g for seven days). Anti-Müllerian hormone (AMH) 0.44 ng/dL (normal range 2–6.8 ng/mL). Anti-thyroid peroxidase (TPO) antibodies 54 IU/mL (normal range less than 35 IU/mL). The presence of anti-TPO antibodies with a normal thyroid function [thyroid stimulating hormone (TSH), triiodothyronine (T3), thyroxine (T4) in normal ranges]. This may increase the risk of developing future thyroid disorders, especially autoimmune. Stool sample The stool samples were analyzed to assess microbiome status with the GI Effects® Comprehensive Stool Profile. GI Effects® is a stool test panel containing information regarding several inflammatory markers – calprotectin, eosinophil protein X (EPX), immunoglobulin A (IgA), maldigestion markers – pancreatic elastase-1 (PE-1), putrefactive short-chain fatty acids (SCFAs), long-chain fatty acids (LCFAs), triglycerides, phospholipids, cholesterol, bacterial metabolic products – butyrate, acetate, propionate, β-glucuronidase, and 24 commensal microbes [measured by polymerase chain reaction (PCR)], microscopic and PCR detected parasites and yeasts. We identified a pattern of inflammation-associated dysbiosis. The test compares the seven major bacterial phyla abundance and diversity in commensal species to a healthy cohort which might point to more significant differences in the gut microbiota (GM) composition of the patient. The intestinal microbiota test confirms the presence of a commensal intestinal flora that is rich in the species reported in the literature of specialty and contains Bacteroidetes and Fusobacteria phylum more than 25% than the healthy cohort, Actinobacteria phylum less than 50% than the healthy cohort. The presence of Proteobacteria and Firmicutes phylum was relatable to healthy cohorts, and we also observed a total absence of Euryarchaeota and Verrucomicrobia phylum. The fecal test shows a high increase in calprotectin and fecal secretory IgA, depicting permeability and increased immune defense of the intestinal mucosa. β-Glucuronidase was also detected in our patient test, having a value of 518 U/g (normal range 368–6266 U/g). Analyzing the results in the test regarding commensal bacteria deoxyribonucleic acid (DNA) determined through 16S ribosomal ribonucleic acid (rRNA) gene PCR amplification technique, we observed the high values of the following species: Bacteroides – Prevotella group (Bacteroides vulgatus), Barnesiella spp. and Odoribacter spp. and a barely detectable amount of Prevotella spp. In the Firmicutes phylum, we identified an abundance of Butyrivibrio crossotus, Lactobacillus spp., Pseudoflavonifractor spp. and Veillonella spp. The Firmicutes/Bacteroidetes ratio in our patient was low: 8 (normal range 12–620). The GI microbiome culture identified high levels of non-pathogen bacteria Escherichia coli, Bifidobacterium, and potential pathogens: Hafnia alvei and Enterobacter cloacae. The mycology culture performed by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI–TOF–MS) technology identified the presence of potential pathogens: Candida albicans and C. glabrata. Moreover, in the stool sample, moderate amount of Giardia lamblia was detected through microscopic examination and confirmed by PCR analysis. The patient received staged oral treatment for giardiasis (Metronidazole 500–750 mg/day × 7 days), for H. alvei and E. cloacae (800 mg of Sulfamethoxazole and 160 mg of Trimethoprim every 12 hours for five days) and for C. albicans and C. glabrata (Nystatin 500 000 IU per os three times daily seven days). The patient was also subjected to the administration of probiotics for three months (June–August 2022) to restore healthy flora – Multi Strain 100 Billion colony-forming units (CFUs) [Lactobacillus acidophilus (CUL 60), L. acidophilus (CUL 21), Bifidobacterium bifidum (CUL 20), B. animalis subsp. lactis (CUL 34), L. salivarius (CUL 61), L. acidophilus (NCFM®), B. lactis HNO19™, L. rhamnosus HN001™, and Saccharomyces Boulardii] to prevent Candida spp. overgrowth. In parallel, personalized diet was instituted containing natural sources of Lactobacillus spp. and avoiding high-sugar foods. The evolution was good, bloating, and abdominal cramping was reduced by 80%. In September 2022, the patient underwent laparoscopic surgery for the excision of EM implants, left salpingectomy, rectosigmoid resection with mechanical termino-terminal anastomosis, ileo-cecal and appendicular resection with latero-lateral anastomosis. The tubal permeability test was positive for the right Fallopian tube. The complete diagnosis after the surgery was: deep EM #Enzian P3 01/0 T3m/2+ A3 B3/3 C3 FI FA FO (left endometrioma, rectovaginal septum nodule, left parameter nodule, right parameter nodule, left uterosacral ligament nodule, right uterosacral ligament nodule, rectosigmoid nodule, ileal nodules, appendicular nodule, left round ligament nodule, left lumbo-ovarian nodule, diaphragm nodule, adenomyosis). The endometriosis fertility index (EFI) was 3. The subsequent histopathology exam confirmed the diagnosis of EM for all the excised lesions. The immediate postoperative evolution of the patient was favorable, with early resumption of intestinal transit and oral nutrition. The follow-up in November 2022 with improved symptomatology (lack of pelvic pain, dysmenorrhea, and GI symptoms), no anemia and normal blood tests. The fertility prognostic was discussed. Because of her low AMH value (0.44 ng/dL) and poor EFI score (3), she was advised to use assisted reproductive technology (ART) to conceive for improved outcomes, particularly in vitro fertilization (IVF). After EM surgery staging, pregnancy rates can be predicted using the EFI score, which is a reliable and validated clinical method.

Endometriosis (EM) is an estrogen-dependent, chronic inflammatory disease caused by the spread of eutopic endometrial tissue to ectopic locations [ 1 ]. EM was first addressed in literature in the 1800s, but it did not receive serious consideration until the 20th century [ 2 ]. Even though substantial research is being done to identify the causes, the physiopathogenesis of this illness is still not fully understood, and it is still referred to as “enigmatic and missed disease” [ 3 ]. About 6–10% of women of reproductive age are affected by EM, and roughly 50% of infertile women are suffering from EM and sometimes the presence of this disease can be easily overlooked due to the scarcity/diversity of symptoms [ 4 , 5 ]. Sometimes a lot of time passes from the initial symptoms to a conclusive diagnosis, the average period being estimated to be between 5–8 and even 12 years due to the lack of simple tests or markers [ 2 ]. Patients that present themselves with dysmenorrhea (painful periods), non-cyclical chronic pelvic pain, cyclical or perimenstrual symptoms, with or without abnormal bleeding, dyspareunia (painful intercourse), infertility, dysuria (painful urination) or hematuria should be investigated for an EM diagnosis [ 6 , 7 , 8 , 9 ]. A suggestive diagnosis can be obtained using a variety of noninvasive imaging techniques including magnetic resonance imaging (MRI), transvaginal ultrasound (TVUS), transrectal ultrasound (TRUS), and three-dimensional (3D) ultrasound (US) [ 10 ]. All these modalities rely on the experience and expertise of the interpreter [ 11 ]. Up to 90% of EM patients also experience gastrointestinal (GI) issues in combination with these gynecological symptoms [ 2 ]. The most common GI symptoms are abdominal bloating, nausea, vomiting, transit disorders – constipation, diarrhea, dyschezia (painful defecation), rectal bleeding [ 7 ]. However, these symptoms may also be very suggestive for an imbalance in the intestinal microbiota known under the term of intestinal dysbiosis. It was shown that patients with EM may exhibit intestinal, vaginal, or uterine dysbiosis, which can affect estrogen metabolism, modulate the immune system, and worsen the condition [ 12 , 13 , 14 ]. There are several theories that support the emergence of EM, with some promoting an origin in uterine tissues and others suggesting an origin in extrauterine tissues [ 15 ]. As a result, EM etiology is multifaceted, involving various theories and processes, including inadequate immunity, uncontrolled cell growth, poor cell signaling and genetic predisposition [ 16 ]. According to studies, it is largely accepted that the initial stage in the emergence of EM is the presence of viable endometrial cells outside the endometrial cavity [ 15 ]. EM is an estrogen-dependent disorder with a chronic inflammatory pattern. A persistent inflammatory state is maintained, thus the growth of endometriotic implants is accelerated by abnormal immune cell activation and the release of proinflammatory cytokines [ 16 ]. Because of their unique character, macrophages are less able to phagocytose ectopic endometrial tissue [ 17 ]. Intraperitoneally produced cytokines reduce intestinal motility and gastric acid secretion, which significantly lowers lactobacilli species and encourages the emergence of Gram-negative bacterial species [ 18 ]. The three main ways in which bacteria enter the peritoneal fluid are: (i) bacterial efflux happens when intestinal permeability and epithelial junctions are reduced due to dysbiosis, which affects the stability of the GI epithelial barrier [ 19 ]; (ii) the bacteria pass into the lymphatic circulation by stimulating the dendritic follicular cells at the level of the lymph nodes in the intestinal wall [ 4 , 19 ]; (iii) directly through the bloodstream [ 20 ]. Between EM and the disbalanced microbiota a bidirectional connection exists (Figure 1 ). The immune system is suppressed, so the intestinal barrier becomes more permeable and pathogen defense mechanisms are reduced [ 19 ]. The normal roles of the intestinal microbiota – nutrient absorption and synthesis, preservation of the intestinal mucosal integrity, defense against pathogens, maturation of the immune system, and regulation of inflammatory disorders – are dysregulated when there is an imbalance in the main bacterial phyla [ 19 , 20 ]. Endometriosis and dysbiosis: bidirectional relationship

The literature related to a possible correlation between EM and intestinal dysbiosis support the theory of the altered immune response by dysbiosis changes, which promotes and maintains a chronic inflammatory response, altering the population of immune cells and promoting the growth of EM implants. There is scientific evidence to support the idea that the immune system response may be regulated by the microbiome [ 21 , 22 ]. It is evident that an inefficient immune response appears to play a significant part in the pathogenesis of EM [ 21 ]. Inflammation caused by dysbiosis’ activation of the body immune response might further encourage and exacerbate retrograde menstruation and the associated endometrial cell adhesion in the peritoneum [ 22 ]. The relationship between EM and GM is bidirectional, EM implants promote the maintenance of bacterial pathogens through alterations in estrogenic metabolism. Microbiota contributes to the development of EM by promoting a long-lasting inflammatory and hyperestrogenic environment [ 23 , 24 ]. There are various microbial ecosystems to consider while researching the relationship between EM and intestinal microbiome (IM). EM appears to be associated with elevated levels of different microorganisms across the IM: Bacteroides, Clostridia, Prevotella, Proteobacteria, Firmicutes, Bacteroidetes, Enterobacteriaceae, Brucellacea, Klebsiella, Gardnerella, Shigella, Streptococcus, Cyanobacteria, Saccharibacteria, Actinobacteria, Fusobacteria, Paraprevotella, E. coli, Odoribacter, Veillonella, Ruminococcus [ 25 , 26 , 27 , 28 , 29 , 30 , 31 ]. The study of Shan et al. (2020) identified an increased Firmicutes/Bacteroidetes ratio in the EM group. This ratio has been cited in several studies as a dysbiosis indicator [ 28 ]. Their study identified in the EM group an abundance of Actinobacteria, Cyanobacteria, Saccharibacteria, Fusobacteria, Bifidobacterium, Blautia, Dorea, Streptococcus and Acidobacteria and a decrease in Tenericutes, Lachnospira and Eubacterium [ 28 ]. The study proposed a correlation between intestinal dysbiosis in EM patients and elevated levels of estrogen and inflammatory factors [serum interleukin-8 (IL-8)] [ 28 ]. The intestinal microbiota of our patient showed the presence of Bacteroidetes phylum (more than 25% than the healthy cohort) supported by literature studies [ 26 , 28 , 29 , 31 ]. The presence of Fusobacteria is supported by the findings of Shan et al. [ 28 ]. A total absence of Euryarchaeota and Verrucomicrobia phylum was observed in our patient GM. Shan et al. also identified the presence of Verrucomicrobia phylum in the stool samples of EM patients [ 28 ], whereas there is no evidence of Euryarchaeota in the microbiome of EM patients. There is no correlation in the literature between the absence of these species and the development of EM. The presence of Proteobacteria and Firmicutes in the stool probe of our patient is supported by the findings of Yuan et al. [ 30 ] and Shan et al. [ 28 ]. We did not detect increased levels of Actinobacteria, as opposed to the data in literature [ 28 , 29 , 30 ]. Another interesting relationship, between the IM and chronic stress in EM patients, was researched by Xu et al. in 2017 [ 31 ]. In the chronically stressed EM group compared to the control group, Xu et al. found that the inflammatory cytokines nuclear factor-kappa B (NF-κB), p65 and cyclooxygenase-2 (COX-2) were present, along with enhanced levels of catecholamines and cortisol. The imbalance of Paraprevotella, Odoribacter, Veillonella, and Ruminococcus was also discovered to be connected to the stress levels of the host via the inflammatory gut–brain axis [ 31 ]. The study conducted by Yuan et al. in 2018 on a group of laboratory mice (22 EM group and 20 control group) whose endometrial tissue was implanted in the peritoneal cavity demonstrated the appearance of intestinal dysbiosis 42 days after the persistence of EM lesions. The species identified in the intestinal flora were Firmicutes and Actinobacteria, whereas Bacteroidetes was present in the control group [ 30 ]. The Firmicutes/Bacteroidetes ratio, which is significant as a marker of dysbiosis, was higher in the EM group. This study supports the existence of a two-way relationship between EM and microbiota. The Firmicutes/Bacteroidetes ratio in our patient was lower than the normal values of control groups. An increase or a decrease in the ratio indicates the existence of gut dysbiosis and it is influenced by many factors: age, weight, diet, antibiotics, supplements, physical activity [ 24 ]. There are few data in the literature regarding the Firmicutes/Bacteroidetes ratio in EM, the most relevant study being performed on mice [ 30 ]. The relevant studies showed an increase in the Firmicutes/Bacteroidetes ratio in EM patients [ 30 , 32 ], yet not ruling out interpreting a decrease in the ratio as a dysbiosis marker. β-Glucuronidase was also measured in the stool sample of our patient. Nine out of 40 different bacterial strains, which are part of the dominant bacterial groups from intestinal flora, were shown to have β-glucuronidase activity, according to Dabek et al.: Bacteroides ovatus, representatives of the Lachnospiraceae family within the Firmicutes phylum: Roseburia intestinalis and R. hominis and Faecalibacterium prausnitzii (Oscillospiraceae family within the Bacillota phylum) [ 33 ]. The significant impact on β-glucuronidase activity seen in R. hominis suggests that some components of the GM may have different levels of enzyme activity depending on the colonic exposure to glycosides, which is influenced by the dietary pattern. The Firmicutes phylum appeared to be the most associated with a high level of β-glucuronidase. The prevalence of Firmicutes phylum was consistent with that of healthy cohorts in our patient. Even though we detected a β-glucuronidase activity, lower than that reported in the literature, this can correlate with the lower presence of Firmicutes phylum. By releasing β-glucuronidase, accelerating estrogen deconjugation, and increasing the amount of free circulating estrogen, dysbiosis also affects the metabolism of estrogen [ 28 , 34 ]. Despite being in the early phase, EM patients have responded well to antibiotic and probiotic therapy trials. Studies have shown the effectiveness of antibiotics in the progression of the disease and the reduction of chronic inflammation, especially broad-spectrum antibiotics, or Metronidazole [ 30 ]. Using probiotics to increase the Lactobacillus spp. one can reestablish the intestinal and vaginal flora. It has also been shown that supplying probiotics to EM patients causes a reduction in their painful symptoms [ 31 ].

The composition of the intestinal microbiota can be used as a screening test in the diagnosis of EM, combined with clinical and imaging examination. For women that are not yet diagnosed with EM and present themselves with chronic pain and GI symptoms and do not respond to anti-inflammatory treatment, the determination of the intestinal microbiota can be an excellent screening marker to guide the patient to the gynecologist specializing in EM. The two practical applications of the association between dysbiosis and EM can be split into a diagnosis/prevention strategy and a therapeutic method. It will be advantageous to examine how the microbiota can be employed as a non-invasive EM diagnostic tool. The treatment methods may be additional to surgical treatment, with the possibility of improving chronic painful symptoms and quality of life. The main strength of our article is the collection of microbiota data from the intestinal probe of our patient, that was comparable to the findings in the literature of specialty. This case supports the evidence found in the studies mentioned above and further research and data analyses on a larger group of patients are needed to deepen our knowledge regarding the connection between microbiota and EM.

The authors declare that they have no conflict of interests.