Progestins Related to Progesterone and Testosterone Elicit Divergent Human Endometrial Transcriptomes and Biofunctions.

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use

Lgr5 Does Not Vary Throughout the Menstrual Cycle in Endometriotic Human Eutopic Endometrium

Endometriosis is characterized by the abnormal presence of endometrium outside of the uterus, resulting in pelvic pain and infertility. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) has been postulated to be a marker of stem cells in the endometrium. However, LGR5 + cells have a macrophage-like phenotype in this tissue, so it is unclear what role LGR5 + cells actually play in the endometrium. Macrophages serve an important function in the endometrium to maintain fertility, while LGR5 + cells generally have a role in tumor progression and are involved in invasion in some cancers. We sought to determine whether LGR5 + cells vary across the menstrual cycle in women with endometriosis and whether there are implications for LGR5 in the aggressiveness of endometriosis and reproductive outcomes. We performed immunofluorescence, flow cytometry, and primary culture in vitro experiments on eutopic and ectopic endometrium from healthy and endometriosis patients and observed that neither LGR5 + cells nor LGR5 expression varied throughout the cycle. Interestingly, we observed that LGR5 + cell percentage overexpressing CD163 (anti-inflammatory marker) was higher in healthy endometrium, suggesting that in endometriosis, endometrium presents a more pro-inflammatory phenotype that likely leads to poor obstetric outcomes. We also observed higher levels of LGR5 + cells in ectopic lesions compared to eutopic endometrium and specifically in deep infiltrating endometriosis, indicating that LGR5 could be involved in progression and aggressiveness of the disease

Molecular alterations in eutopic endometrium of women with endometriosis and implications in its diagnostic

La endometriosi és una malaltia benigna que afecta a un 10-15% de les dones en edat reproductiva I els seus símptomes principals són dolor i infertilitat. El seu diagnòstic pot ser retardat fins a 6,7 anys de mitjana, i el diagnòstic principal és per laparoscòpia. L’objectiu principal d’aquesta tesi era trobar una nova eina diagnòstica menys invasiva que la cirurgia. Per aconseguir-ho, es van dur a terme dos objectius; trobar nous biomarcadors per la malaltia a l’endometri eutòpic i estudiar el marcador Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) com a possible biomarcador per a l’endometriosi. Per al primer objectiu, es va fer un estudi de Discovery de biomarcadors a partir de RNA-High-Sequencing i es va desenvolupar un classificador utilitzant el leave one out cross validation model de Partial Least Squares (PLS) a partir de mostres fixades i incloses en parafina de tots els tipus d’endometriosi i controls. No vam ser capaços de discernir entre tipus d’endometriosi però es va trobar una elevada sensitivitat i especificitat per a diagnosticar la malaltia en global. No obstant, al validar el model amb un increment del nombre de mostres, només el 60% de les pacients amb endometriosi van poder ser diagnosticades com a endometriosi. En conclusió, el model validat no es pot traslladar a la pràctica clínica. En el segon objectiu, vam estudiar LGR5 a l’endometri eutòpic amb RNA-High-Sequencing i vam descobrir que aquestes cèl·lules tenen un fenotip de macròfags. No vam observar diferències significants entre dones sanes i malaltes, per tant, LGR5 no és un bon biomarcador per a la malaltia. No obstant, vam trobar que l’endometriosi profunda, el tipus més agressiu de la malaltia, sobre-expressava gens únics relacionats amb infertilitat. Així doncs, concloem que LGR5 podria estar relacionat amb l’agressivitat de la malaltia així com en els resultats reproductius. Sabent que les cèl·lules LGR5 positives tenien un fenotip de macròfag, vam estudiar els macròfags a l’endometri eutòpic de dones amb la malaltia i vam trobar que presenten un fenotip més pro-inflamatori que les de l’endometri sa, el que podria influir en la fisiopatologia de la malaltia i en els resultats reproductius en aquestes dones.Endometriosis is a benign disease that affects 10-15% of reproductive age women and the main symptoms are pain and infertility. Its diagnostic can be delayed for 6.7 years in average, and the final diagnostic is by laparoscopy. The main objective of this thesis was to find a new and less invasive diagnostic approach for the disease than surgery. For this purpose, two objectives were performed; to find new biomarkers for the disease in eutopic endometrium and to study Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) as a putative biomarker for endometriosis. For the first objective, a discover study of biomarkers was done by RNA-High-Sequencing and a classifier was developed by using Partial Least Squares (PLS) leave one out cross validation model using formalin-fixed paraffin embedded samples from all types of endometriosis and controls. We were not able to distinguish between types of disease but there was a high sensitivity and specificity to diagnose the disease in global. However, when we validated the model by increasing the sample size, only the 60% of patients could be diagnosed as endometriosis. In conclusion, the validated model cannot be translated to the clinics. In the second objective, we studied LGR5 in eutopic endometrium by RNA-High-Sequencing and we discovered that these cells have a macrophage-like phenotype. We did not observe significant differences between healthy and diseased women and thus, LGR5 is not a good biomarker for the disease. However, we found that deep infiltrating endometriosis (DIE), the most aggressive type of the disease, overexpressed unique genes related to infertility. Hence, we concluded that LGR5 could be related to the aggressiveness of the disease as well as in reproductive outcomes. Knowing that LGR5 positive cells phenotype was macrophage-like, we studied macrophages in eutopic endometrium of diseased women and we found that they present a more pro-inflammatory phenotype than healthy endometrium which could also influence to the pathophysiology of the disease and to the reproductive outcomes in this women

Molecular alterations in eutopic endometrium of women with endometriosis and implications in its diagnostic

La endometriosi és una malaltia benigna que afecta a un 10-15% de les dones en edat reproductiva I els seus símptomes principals són dolor i infertilitat. El seu diagnòstic pot ser retardat fins a 6,7 anys de mitjana, i el diagnòstic principal és per laparoscòpia. L’objectiu principal d’aquesta tesi era trobar una nova eina diagnòstica menys invasiva que la cirurgia. Per aconseguir-ho, es van dur a terme dos objectius; trobar nous biomarcadors per la malaltia a l’endometri eutòpic i estudiar el marcador Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) com a possible biomarcador per a l’endometriosi. Per al primer objectiu, es va fer un estudi de Discovery de biomarcadors a partir de RNA-High-Sequencing i es va desenvolupar un classificador utilitzant el leave one out cross validation model de Partial Least Squares (PLS) a partir de mostres fixades i incloses en parafina de tots els tipus d’endometriosi i controls. No vam ser capaços de discernir entre tipus d’endometriosi però es va trobar una elevada sensitivitat i especificitat per a diagnosticar la malaltia en global. No obstant, al validar el model amb un increment del nombre de mostres, només el 60% de les pacients amb endometriosi van poder ser diagnosticades com a endometriosi. En conclusió, el model validat no es pot traslladar a la pràctica clínica. En el segon objectiu, vam estudiar LGR5 a l’endometri eutòpic amb RNA-High-Sequencing i vam descobrir que aquestes cèl·lules tenen un fenotip de macròfags. No vam observar diferències significants entre dones sanes i malaltes, per tant, LGR5 no és un bon biomarcador per a la malaltia. No obstant, vam trobar que l’endometriosi profunda, el tipus més agressiu de la malaltia, sobre-expressava gens únics relacionats amb infertilitat. Així doncs, concloem que LGR5 podria estar relacionat amb l’agressivitat de la malaltia així com en els resultats reproductius. Sabent que les cèl·lules LGR5 positives tenien un fenotip de macròfag, vam estudiar els macròfags a l’endometri eutòpic de dones amb la malaltia i vam trobar que presenten un fenotip més pro-inflamatori que les de l’endometri sa, el que podria influir en la fisiopatologia de la malaltia i en els resultats reproductius en aquestes dones.Endometriosis is a benign disease that affects 10-15% of reproductive age women and the main symptoms are pain and infertility. Its diagnostic can be delayed for 6.7 years in average, and the final diagnostic is by laparoscopy. The main objective of this thesis was to find a new and less invasive diagnostic approach for the disease than surgery. For this purpose, two objectives were performed; to find new biomarkers for the disease in eutopic endometrium and to study Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) as a putative biomarker for endometriosis. For the first objective, a discover study of biomarkers was done by RNA-High-Sequencing and a classifier was developed by using Partial Least Squares (PLS) leave one out cross validation model using formalin-fixed paraffin embedded samples from all types of endometriosis and controls. We were not able to distinguish between types of disease but there was a high sensitivity and specificity to diagnose the disease in global. However, when we validated the model by increasing the sample size, only the 60% of patients could be diagnosed as endometriosis. In conclusion, the validated model cannot be translated to the clinics. In the second objective, we studied LGR5 in eutopic endometrium by RNA-High-Sequencing and we discovered that these cells have a macrophage-like phenotype. We did not observe significant differences between healthy and diseased women and thus, LGR5 is not a good biomarker for the disease. However, we found that deep infiltrating endometriosis (DIE), the most aggressive type of the disease, overexpressed unique genes related to infertility. Hence, we concluded that LGR5 could be related to the aggressiveness of the disease as well as in reproductive outcomes. Knowing that LGR5 positive cells phenotype was macrophage-like, we studied macrophages in eutopic endometrium of diseased women and we found that they present a more pro-inflammatory phenotype than healthy endometrium which could also influence to the pathophysiology of the disease and to the reproductive outcomes in this women

The endometrial immune environment of women with endometriosis

BackgroundEndometriosis, a common oestrogen-dependent inflammatory disorder in women of reproductive age, is characterized by endometrial-like tissue outside its normal location in the uterus, which causes pelvic scarring, pain and infertility. While its pathogenesis is poorly understood, the immune system (systemically and locally in endometrium, pelvic endometriotic lesions and peritoneal fluid) is believed to play a central role in its aetiology, pathophysiology and associated morbidities of pain, infertility and poor pregnancy outcomes. However, immune cell populations within the endometrium of women with the disease have had incomplete phenotyping, thereby limiting insight into their roles in this disorder.Objective and rationaleThe objective herein was to determine reproducible and consistent findings regarding specific immune cell populations and their abundance, steroid hormone responsiveness, functionality, activation states, and markers, locally and systemically in women with and without endometriosis.Search methodsA comprehensive English language PubMed, Medline and Google Scholar search was conducted with key search terms that included endometriosis, inflammation, human eutopic/ectopic endometrium, immune cells, immune population, immune system, macrophages, dendritic cells (DC), natural killer cells, mast cells, eosinophils, neutrophils, B cells and T cells.OutcomesIn women with endometriosis compared to those without endometriosis, some endometrial immune cells display similar cycle-phase variation, whereas macrophages (Mø), immature DC and regulatory T cells behave differently. A pro-inflammatory Mø1 phenotype versus anti-inflammatory Mø2 phenotype predominates and natural killer cells display abnormal activity in endometrium of women with the disease. Conflicting data largely derive from small studies, variably defined hormonal milieu and different experimental approaches and technologies.Wider implicationsPhenotyping immune cell subtypes is essential to determine the role of the endometrial immune niche in pregnancy and endometrial homeostasis normally and in women with poor reproductive history and can facilitate development of innovative diagnostics and therapeutics for associated symptoms and compromised reproductive outcomes

Molecular alterations in eutopic endometrium of women with endometriosis and implications in its diagnostic /

La endometriosi és una malaltia benigna que afecta a un 10-15% de les dones en edat reproductiva I els seus símptomes principals són dolor i infertilitat. El seu diagnòstic pot ser retardat fins a 6,7 anys de mitjana, i el diagnòstic principal és per laparoscòpia. L'objectiu principal d'aquesta tesi era trobar una nova eina diagnòstica menys invasiva que la cirurgia. Per aconseguir-ho, es van dur a terme dos objectius; trobar nous biomarcadors per la malaltia a l'endometri eutòpic i estudiar el marcador Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) com a possible biomarcador per a l'endometriosi. Per al primer objectiu, es va fer un estudi de Discovery de biomarcadors a partir de RNA-High-Sequencing i es va desenvolupar un classificador utilitzant el leave one out cross validation model de Partial Least Squares (PLS) a partir de mostres fixades i incloses en parafina de tots els tipus d'endometriosi i controls. No vam ser capaços de discernir entre tipus d'endometriosi però es va trobar una elevada sensitivitat i especificitat per a diagnosticar la malaltia en global. No obstant, al validar el model amb un increment del nombre de mostres, només el 60% de les pacients amb endometriosi van poder ser diagnosticades com a endometriosi. En conclusió, el model validat no es pot traslladar a la pràctica clínica. En el segon objectiu, vam estudiar LGR5 a l'endometri eutòpic amb RNA-High-Sequencing i vam descobrir que aquestes cèl·lules tenen un fenotip de macròfags. No vam observar diferències significants entre dones sanes i malaltes, per tant, LGR5 no és un bon biomarcador per a la malaltia. No obstant, vam trobar que l'endometriosi profunda, el tipus més agressiu de la malaltia, sobre-expressava gens únics relacionats amb infertilitat. Així doncs, concloem que LGR5 podria estar relacionat amb l'agressivitat de la malaltia així com en els resultats reproductius. Sabent que les cèl·lules LGR5 positives tenien un fenotip de macròfag, vam estudiar els macròfags a l'endometri eutòpic de dones amb la malaltia i vam trobar que presenten un fenotip més pro-inflamatori que les de l'endometri sa, el que podria influir en la fisiopatologia de la malaltia i en els resultats reproductius en aquestes dones.Endometriosis is a benign disease that affects 10-15% of reproductive age women and the main symptoms are pain and infertility. Its diagnostic can be delayed for 6.7 years in average, and the final diagnostic is by laparoscopy. The main objective of this thesis was to find a new and less invasive diagnostic approach for the disease than surgery. For this purpose, two objectives were performed; to find new biomarkers for the disease in eutopic endometrium and to study Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) as a putative biomarker for endometriosis. For the first objective, a discover study of biomarkers was done by RNA-High-Sequencing and a classifier was developed by using Partial Least Squares (PLS) leave one out cross validation model using formalin-fixed paraffin embedded samples from all types of endometriosis and controls. We were not able to distinguish between types of disease but there was a high sensitivity and specificity to diagnose the disease in global. However, when we validated the model by increasing the sample size, only the 60% of patients could be diagnosed as endometriosis. In conclusion, the validated model cannot be translated to the clinics. In the second objective, we studied LGR5 in eutopic endometrium by RNA-High-Sequencing and we discovered that these cells have a macrophage-like phenotype. We did not observe significant differences between healthy and diseased women and thus, LGR5 is not a good biomarker for the disease. However, we found that deep infiltrating endometriosis (DIE), the most aggressive type of the disease, overexpressed unique genes related to infertility. Hence, we concluded that LGR5 could be related to the aggressiveness of the disease as well as in reproductive outcomes. Knowing that LGR5 positive cells phenotype was macrophage-like, we studied macrophages in eutopic endometrium of diseased women and we found that they present a more pro-inflammatory phenotype than healthy endometrium which could also influence to the pathophysiology of the disease and to the reproductive outcomes in this women

Macrophages display proinflammatory phenotypes in the eutopic endometrium of women with endometriosis with relevance to an infectious etiology of the disease

ObjectiveTo phenotype transcriptomically M1 macrophages (Mϕ1) and M2 macrophages (Mϕ2) in the endometrium of women with endometriosis.DesignProspective experimental study.SettingUniversity research laboratory.Patient(s)Six women with endometriosis and five controls without disease, in the secretory phase of the menstrual cycle.Intervention(s)Mϕ1, Mϕ2, uterine natural killer, and T regulatory cells were isolated from human endometrium using a uniquely designed cell-specific fluorescence activating cell sorting panel. Transcriptome profiles were assessed by RNA high sequencing, bioinformatics, and biological pathway analyses.Main outcomes measure(s)Differential gene expression between Mϕ1 and Mϕ2 in women with and without endometriosis and in Mϕ1 versus Mϕ2 in each group was determined and involved different biologic and signaling pathways.Result(s)Flow cytometry analysis showed no significant differences in total numbers of leukocytes between control and endometriosis groups, although Mϕ1 were higher in the endometriosis group versus controls. Statistical transcriptomic analysis was performed only in Mϕ1 and Mϕ2 populations due to larger sample sizes. Bioinformatic analyses revealed that in women with endometriosis, endometrial Mϕ1 are more proinflammatory than controls and that Mϕ2 paradoxically have a proinflammatory phenotype.Conclusion(s)As Mϕ are phenotypically plastic and their polarization state depends on their microenvironment, the altered endometrial environment in women with endometriosis may promote endometrial Mϕ2 polarization and an Mϕ1 proinflammatory phenotype. Moreover, aberrant phenotypes of Mϕ may contribute to abnormal gene expression of the eutopic endometrium and a proinflammatory environment in women with endometriosis relevant to the pathophysiology of the disease and compromised reproductive outcomes