The Role of Peritoneal Macrophages in Endometriosis.

Endometriosis is an estrogen-dependent gynecological disorder, defined as the growth of endometrial stromal cells and glands at extrauterine sites. Endometriotic lesions are more frequently located into the abdominal cavity, although they can also be implanted in distant places. Among its etiological factors, the presence of immune dysregulation occupies a prominent place, pointing out the beneficial and harmful outcomes of macrophages in the pathogenesis of this disease. Macrophages are tissue-resident cells that connect innate and adaptive immunity, playing a key role in maintaining local homeostasis in healthy conditions and being critical in the development and sustainment of many inflammatory diseases. Macrophages accumulate in the peritoneal cavity of women with endometriosis, but their ability to clear migrated endometrial fragments seems to be inefficient. Hence, the characteristics of the peritoneal immune system in endometriosis must be further studied to facilitate the search for new diagnostic and therapeutic tools. In this review, we summarize recent relevant advances obtained in both mouse, as the main animal model used to study endometriosis, and human, focusing on peritoneal macrophages obtained from endometriotic patients and healthy donors, under the perspective of its future clinical translation to the role that these cells play on this pathology.

Isolation of functional mature peritoneal macrophages from healthy humans.

Macrophages play an important role in the inflammatory response. Their various biological functions are induced by different membrane receptors, including Toll-like receptors, which trigger several intracellular signaling cascades and activate the inflammasomes, which in turn elicit the release of inflammatory mediators such as cytokines. In this study, we present a novel method for the isolation of human mature peritoneal macrophages. This method can be easily implemented by gynecologists who routinely perform laparoscopy for sterilization by tubal ligation or surgically intervene in benign gynecological pathologies. Our method confirms that macrophages are the main peritoneal leukocyte subpopulation isolated from the human peritoneum in homeostasis. We showed that primary human peritoneal macrophages present phagocytic and oxidative activities, and respond to activation of the main proinflammatory pathways such as Toll-like receptors and inflammasomes, resulting in the secretion of different proinflammatory cytokines. Therefore, this method provides a useful tool for characterizing primary human macrophages as control cells for studies of molecular inflammatory pathways in steady-state conditions and for comparing them with those obtained from pathologies involving the peritoneal cavity. Furthermore, it will facilitate advances in the screening of anti-inflammatory compounds in the human system.

Investigation of anogenital distance as a diagnostic tool in endometriosis.

An association between anogenital distance (AGD) and endometriosis has been reported, suggesting that AGD may be a useful clinical tool in endometriosis. The predictive ability of AGD of women in discriminating presence and type of endometriosis was examined. A case-control study was conducted at the University Hospital 'Virgen de la Arrixaca', Murcia, Spain, between 2014 and 2015. A total of 114 participants diagnosed with endometriosis using ultrasound findings and 105 controls were recruited. Two AGD measurements were obtained: one from the anterior clitoral surface to the upper verge of the anus (AGDAC), and another one from the posterior fourchette to the upper verge of the anus (AGDAF). Parametric and non-parametric tests andreceiver operator characterstic analyses were used to determine relationships between AGD and presence of endometriosis and subgroups (ovarian endometriomas or deep infiltrating endometriosis [DIE]). The AGDAF, but not AGDAC, was associated with presence of endometriomas, DIE (P-values, <0.001-0.02), or both. The highest area under curve (0.91; 95% CI 0.84 to 0.97) was obtained for the DIE subgroup with the AGDAF measurement, with a sensitivity and specificity of 84.4% and 91.4%, respectively. AGDAF can therefore efficiently discriminate the presence of DIE and may be a useful clinical tool.

Vesicouterine fistula: teaching video on diagnosis and surgical treatment.

INTRODUCTION: A 42-year-old woman presented with urinary incontinence 9 years after the last of four vaginal deliveries. She had also had one Caesarean section. Immediately after the last delivery, she presented with haematuria, which resolved within a few hours, but the drain remained prophylactically for 7 days. Nine years later, she was referred to a specialist hospital. METHOD: The patient presented with continuous urinary incontinence, and physical examination revealed a loss of urine from the vagina, the latter confirmed by a methylene blue test showing loss of urine from the uterine cervix. Other diagnostic techniques used were cystography, cystoscopy and uro-CT. Based on a literature review of the management options for such patients and the relevant clinical details of our patient, a decision was made to perform a total abdominal hysterectomy and fistula repair. RESULTS AND DISCUSSION: Six months following surgery, the results were entirely satisfactory, with full urinary continence and significant improvement in the patient's quality of life. A discussion about controversial approaches to diagnosis and management is included.

Central pontine myelinolysis during pregnancy: Pathogenesis, diagnosis and management.

Central pontine myelinolysis (CPM) is a rare condition usually caused by rapid sodium correction in hyponatraemia after a severe neurological syndrome. Only few cases have been reported during pregnancy, most of which were reported in patients with hyperemesis. We describe the successful management of the first case of twin pregnancy in a patient who presented with CPM after treatment for premature labour and then review the literature on CPM in pregnancy (aetiology, diagnosis and management). Our patient required emergency delivery to achieve electrolyte and fluid balance. At six months, the twins remained asymptomatic and the mother had minor sequelae. The aetiology is not clear, and there is no evidence regarding the optimal treatment or prognosis of CPM. In our patient, desmopressin-contaminated atosiban showed a certain probability in the Karch-Lasagne algorithm of a causality relationship between hyponatraemia and the drug. To our knowledge, this is the first case of myelinolysis reported in a twin pregnancy possibly related to desmopressin-contaminated atosiban.

Uterine rupture in twin pregnancy with normal fetus and complete hydatidiform mole.

We describe a rare case of complete hydatidiform mole with twin live fetus (CHMTF) confirmed by histopathology, flow cytometry and polymerase chain reaction techniques. No malformations were observed, fetal karyotype was normal and ß-human chorionic gonadotropin levels were high (>100,000 IU/ml). The patient was informed of the risks and decided to continue with the pregnancy, but at week 15, she had to undergo hysterectomy due to uterine rupture. She subsequently developed persistent trophoblastic disease (PTD) with pulmonary metastases that required treatment with polychemotherapy. Patients with CHMTF should be informed of all known risks, including the considerable risk of PTD, which is similar to or, even higher than that associated with a singleton complete mole. The risk does not appear to be increased by continuing the pregnancy. Because so few series have been published, there is a lack of evidence-based clinical management guidelines. To our knowledge, this is the first report of uterine rupture in CHMTF.

Management of a dichorionic twin pregnancy with a normal fetus and an androgenetic diploid complete hydatidiform mole.

We describe a rare case of complete hydatidiform mole with twin live fetus (CHMTF) confirmed by histopathology, flow cytometry, and polymerase chain reaction techniques. No malformations were observed, fetal karyotype was normal and ß-human chorionic gonadotropin levels were increased (>100,000 IU/ml). Once the patient had been informed of the risks, it was decided to continue the pregnancy, but termination of pregnancy was necessary at week 13 + 5 due to maternal complications consisting of hyperthyroidism, hypertension and vaginal bleeding, followed by persistent trophoblastic disease (PTD). Patients diagnosed with CHMTF should be informed of all known risks, including the considerable risk of PTD, which is similar to - or according to some reports - even higher than that associated with a singleton complete mole and is not increased by continuing pregnancy. Due to the low number of series published, evidence-based clinical management guidelines are lacking.

Optimization of peritoneal fluid and leukocyte collection in patients with endometriosis.

OBJECTIVE: To propose a standardized protocol for peritoneal free fluid and leukocyte sample collection in women with endometriosis suitable for biomedical research on the basis of the surgical procedure, the clinical and technical conditions, and the quality of the samples obtained. DESIGN: Video showing the step-by-step collection procedure and the suitability of samples obtained for biomedical research. SUBJECTS: This study included 103 women with confirmed endometriosis by pathology analysis, who signed informed consent and were recruited from the Hospital "Virgen de la Arrixaca", Murcia, Spain. The study was approved by the Ethics Committee of University of Murcia (CEI 3156/2020). MAIN OUTCOME MEASURES: We analyzed the presence of free fluid in the peritoneal cavity and its relationship with hormonal treatment intake. In addition, the presence of blood contamination, the number of viable leukocytes and macrophages in free peritoneal fluid and lavages as well as their relationship with the lavage volume used, the body mass index, and the age of patients were analyzed. RESULTS: The presence of free peritoneal fluid, in which cells and molecules could be quantified, was scarce in the patients (21%), and it was not significantly related to hormonal treatment intake. The cell viability was higher than 98% in all collected samples; although 54% showed good quality and enough cellularity to be used in biomedical research, 40% were contaminated with blood and 6% had low cellularity. The number of leukocytes and macrophages recovered from the peritoneal lavages correlated positively with the lavage volume used and negatively with the body mass index and was independent of the age of the patients. CONCLUSION: We describe a standardized step-by-step procedure for peritoneal fluid and leukocyte collection in women with endometriosis, suitable for biomedical research, taking into account that not all women present free fluid in the peritoneal cavity. We propose to increase the lavage volume recommended by the World Endometriosis Research Foundation from 10 mL to at least 40 mL of sterile saline solution and its mobilization for at least 30 seconds within the peritoneal cavity, especially in patients with higher body mass index, to improve the efficiency of the procedure.

Fertility preservation in heterotopic cervical pregnancy: what is the best procedure?

We describe a patient who underwent assisted reproduction techniques and was diagnosed with heterotopic cervical pregnancy, and then discuss the management of this entity, which is rare and has no standard protocols. Treatment consisted of intra-arterial methotrexate (50 mg/m(2) body surface area) and simultaneous selective embolization of uterine arteries. The literature is also reviewed to identify other approaches and outcomes.

Hypothetical roadmap towards endometriosis: prenatal endocrine-disrupting chemical pollutant exposure, anogenital distance, gut-genital microbiota and subclinical infections.

BACKGROUND: Endometriosis is a gynaecological hormone-dependent disorder that is defined by histological lesions generated by the growth of endometrial-like tissue out of the uterus cavity, most commonly engrafted within the peritoneal cavity, although these lesions can also be located in distant organs. Endometriosis affects ~10% of women of reproductive age, frequently producing severe and, sometimes, incapacitating symptoms, including chronic pelvic pain, dysmenorrhea and dyspareunia, among others. Furthermore, endometriosis causes infertility in ~30% of affected women. Despite intense research on the mechanisms involved in the initial development and later progression of endometriosis, many questions remain unanswered and its aetiology remains unknown. Recent studies have demonstrated the critical role played by the relationship between the microbiome and mucosal immunology in preventing sexually transmitted diseases (HIV), infertility and several gynaecologic diseases. OBJECTIVE AND RATIONALE: In this review, we sought to respond to the main research question related to the aetiology of endometriosis. We provide a model pointing out several risk factors that could explain the development of endometriosis. The hypothesis arises from bringing together current findings from large distinct areas, linking high prenatal exposure to environmental endocrine-disrupting chemicals with a short anogenital distance, female genital tract contamination with the faecal microbiota and the active role of genital subclinical microbial infections in the development and clinical progression of endometriosis. SEARCH METHODS: We performed a search of the scientific literature published until 2019 in the PubMed database. The search strategy included the following keywords in various combinations: endometriosis, anogenital distance, chemical pollutants, endocrine-disrupting chemicals, prenatal exposure to endocrine-disrupting chemicals, the microbiome of the female reproductive tract, microbiota and genital tract, bacterial vaginosis, endometritis, oestrogens and microbiota and microbiota-immune system interactions. OUTCOMES: On searching the corresponding bibliography, we found frequent associations between environmental endocrine-disrupting chemicals and endometriosis risk. Likewise, recent evidence and hypotheses have suggested the active role of genital subclinical microbial infections in the development and clinical progression of endometriosis. Hence, we can envisage a direct relationship between higher prenatal exposure to oestrogens or estrogenic endocrine-disrupting compounds (phthalates, bisphenols, organochlorine pesticides and others) and a shorter anogenital distance, which could favour frequent postnatal episodes of faecal microbiota contamination of the vulva and vagina, producing cervicovaginal microbiota dysbiosis. This relationship would disrupt local antimicrobial defences, subverting the homeostasis state and inducing a subclinical inflammatory response that could evolve into a sustained immune dysregulation, closing the vicious cycle responsible for the development of endometriosis. WIDER IMPLICATIONS: Determining the aetiology of endometriosis is a challenging issue. Posing a new hypothesis on this subject provides the initial tool necessary to design future experimental, clinical and epidemiological research that could allow for a better understanding of the origin of this disease. Furthermore, advances in the understanding of its aetiology would allow the identification of new therapeutics and preventive actions.

Prognostic importance of atypical endometriosis with architectural hyperplasia versus cytologic atypia in endometriosis-associated ovarian cancer.

OBJECTIVE: Patients with endometriosis are at increased risk of ovarian cancer. It has been suggested that atypical endometriosis is a precursor lesion of endometriosis-associated ovarian cancer (EAOC). The aim of this study is to evaluate if cytologic (cellular) atypia and architectural atypia (hyperplasia), histologic findings described as atypical endometriosis, play a different role in patients with EAOC. METHODS: A prospective study was conducted between January 2014 and April 2017 at our institution with patients undergoing surgery with a histologic diagnosis of endometriosis, ovarian cancer, or EAOC. The prevalence and immunohistologic study (Ki-67, BAF250a, COX-2) of cases of cellular and architectural atypia in endometriosis were analyzed. RESULTS: Two hundred and sixty-six patients were included: the diagnosis was endometriosis alone in 159 cases, ovarian cancer in 81, and EAOC in 26. Atypical endometriosis was reported in 23 cases (12.43%), 39.13% of them found in patients with EAOC. Endometriosis with cellular atypia was found mainly in patients without neoplasm (71.4%), and endometriosis with architectural atypia was seen in patients with ovarian cancer (88.9%) (p=0.009). Ki-67 was significantly higher in endometriosis patients with architectural atypia than those with cellular atypia. CONCLUSION: The diagnosis of endometriosis with architectural atypia is important because it may be a precursor lesion of ovarian cancer; therefore, pathologists finding endometriosis should carefully examine the surgical specimen to identify any patients with hyperplasia-type endometriosis, as they may be at higher risk of developing EAOC.

Characterization of human peritoneal monocyte/macrophage subsets in homeostasis: Phenotype, GATA6, phagocytic/oxidative activities and cytokines expression.

Peritoneal macrophages play a critical role in the control of infectious and inflammatory diseases. Although recent progress on murine peritoneal macrophages has revealed multiple aspects on their origin and mechanisms involved in their maintenance in this compartment, little is known on the characteristics of human peritoneal macrophages in homeostasis. Here, we have studied by flow cytometry several features of human peritoneal macrophages obtained from the peritoneal cavity of healthy women. Three peritoneal monocyte/macrophage subsets were established on the basis of CD14/CD16 expression (CD14++CD16-, CD14++CD16+ and CD14highCD16high), and analysis of CD11b, CD11c, CD40, CD62L, CD64, CD80, CD86, CD116, CD119, CD206, HLA-DR and Slan was carried out in each subpopulation. Intracellular expression of GATA6 and cytokines (pro-inflammatory IL-6 and TNF-α, anti-inflammatory IL-10) as well as their phagocytic/oxidative activities were also analyzed, in an attempt to identify genuine resident peritoneal macrophages. Results showed that human peritoneal macrophages are heterogeneous regarding their phenotype, cell complexity and functional abilities. A direct relationship of CD14/CD16 expression, intracellular content of GATA6, and activation/maturation markers like CD206 and HLA-DR, support that the CD14highCD16high subset represents the mature phenotype of steady-state human resident peritoneal macrophages. Furthermore, increased expression of CD14/CD16 is also related to the phagocytic activity.

2-methoxyestradiol in the pathophysiology of endometriosis: focus on angiogenesis and therapeutic potential.

Endometriosis is a common condition among women of childbearing potential in which ectopic endometrial tissue is found outside the uterine cavity. Neoangiogenesis plays a major role in the development of endometriotic implants. Some evidence suggests that a disorder in the balance of proangiogenic and antiangiogenic factors that favors the former is induced by local hypoxia and is mediated by the hypoxia-inducible factor-vascular endothelium growth factor pathway could partially explain the development of this condition in some women. 2-methoxyestradiol is a biologically active metabolite of estradiol having antiangiogenic action. Changes in estradiol homeostasis have been locally observed in endometriosis. In this review, we summarize current knowledge of endometriosis pathophysiology, in particular, the balance between local 2-methoxyestradiol production and angiogenesis, which could promote the development of endometriotic lesions. 2-Methoxyestradiol emerges as a promising new candidate for the treatment of endometriosis.