Genetics and epigenetics of endometriosis : analysis of microRNAs and family sequencing

L'endométriose est une pathologie gynécologique chronique, inflammatoire et hormono-dépendante, définie par la présence de tissu endométrial fonctionnel en dehors de la cavité utérine. Elle affecte environ 10% des femmes en âge de procréer et peut occasionner des douleurs pelviennes et une infertilité. C'est une maladie complexe dont l'étiologie et la physiopathologie demeurent mal connues. La première partie de ce travail concerne les bases génétiques de l'endométriose qui est une maladie répondant à une hérédité multigénique. À ce jour, les études d'association pangénomiques n'ont mis en évidence que des variants d'effets relativement modestes, dans des formes non familiales. Nous proposons ici une approche fondée sur l'étude de familles concentrant des cas sévères, dans le but de mettre en évidence de nouveaux polymorphismes affectant possiblement des gènes majeurs. Quatre-vingt cinq familles présentant au moins deux cas d'endométriose sévère ont été sélectionnées. Un prélèvement d'ADN a été réalisé pour chaque malade de la famille, ainsi que les parents des sujets atteints, puis les régions codantes (exomes) ont été séquencées. La mise en évidence d'un ou plusieurs variants dans ces formes familiales pourrait permettre de découvrir de nouveaux gènes impliqués dans la maladie et peut-être poser les bases d'un dépistage familial. La deuxième partie de ce travail s'intéresse aux mécanismes épigénétiques de l'endométriose et plus particulièrement aux micro-ARNs. S'il est clair que leur expression est dérégulée dans l'endométriose, l'identification des micro-ARNs en cause est loin d'être achevée, les études publiées à ce jour étant discordantes entre elles. À l'aide d'une approche bioinformatique originale et sans a priori, s'appuyant sur les données de la base miRWalk 2.0 couplées aux données d'expression de kystes ovariens endométriosiques (endométriomes), nous avons identifié 8 micro-ARNs potentiellement régulateurs. Étant donné la forte corrélation existant entre la concentration tissulaire et circulante des micro-ARNs, les 8 micro-ARNs sélectionnés ont été quantifiés par RT-qPCR dans le plasma de 93 patientes et 95 témoins indemnes de la maladie. Deux sur les huit testés étaient surexprimés chez les patientes endométriosiques : let-7b-5p et miR-92a-3p (p<0.005). Trois micro-ARNs parmi les 8 sélectionnés, incluant let7b-5p et miR-92a-3p, ciblaient potentiellement le gène KIAA1324, l'un des plus dérégulés dans l'endométriome et dont l'expression est modulée par les oestrogènes. Nous avons ensuite réalisé des cultures primaires de cellules endométriales à partir de biopsies d'endomètre de 10 femmes porteuses d'un endométriome. Ces cellules ont été transfectées avec des mimics de micro-ARNs (let-7b-5p et miR-92a-3p) afin d'étudier leur effet sur l'expression de KIAA1324. L'expression du gène KIAA1324 a été mesurée par RT-qPCR et une baisse d'expression allant jusqu'à 40% a été observée en présence des deux micro-ARNs testés. L'analyse immunohistochimique comparant la lésion ovarienne à l'endomètre eutopique d'une même patiente a révélé un marquage moins intense de KIAA1324 dans la lésion par rapport à l'endomètre, ce qui est en accord avec les données transcriptomiques. En conclusion, nous avons mis en évidence l'implication potentielle d'un nouveau gène dans la physiopathologie de l'endométriose, le gène KIAA1324 dont l'expression est diminuée par deux micro-ARNs circulants, let-7b-5p et miR-92a-3p. Ces deux micro-ARNs ont été retrouvés surexprimés dans la maladie; leur quantification dans le plasma pourrait constituer un outil de diagnostic précoce dans l'endométriose.Endometriosis is a chronic, inflammatory and hormono-dependent gynecological disease, defined by the presence of functional endometrial tissue outside the uterine cavity. It affects around 10% of women of childbearing age and possibly causes pelvic pain and infertility. It is a heterogeneous disease; its etiology and pathophysiology remain poorly understood. The first part of this work deals with the genetic basis of endometriosis, that is considered a complex trait. To date, genome-wide association studies have only revealed genomic variants with a slight effect, in non-familial forms. We propose an approach based on the analysis of families harboring multiple and severe cases, in order to highlight not ever described polymorphisms, possibly afflicting gene expression with a major effect. Eighty-five families with at least two cases of severe endometriosis were selected. A sample of genomic DNA was taken from each subject, including their parents, and submitted to exome sequencing. Identification of variants in these familial forms may allow to focus on new genes possibly involved in the disease and draw the basis of familial screening. The second part of this work focuses on the epigenetic mechanisms of endometriosis, particularly on micro-RNAs (miRNAs). While their expression is clearly deregulated in endometriosis, precise identification of the miRNAs involved in the disease is still to be achieved, as the studies published so far have produced discordant results. Using an original and unbiased bioinformatics approach based on miRWalk 2.0 database and transcriptomic data from endometriotic ovarian cysts (endometriomas) we identified 8 putative miRNAs. Given the strong correlation between the tissue and plasma concentration of the miRNAs, we quantified these miRNAs by RT-qPCR in the plasma of 93 patients and 95 healthy controls. Two miRNAs were upregulated inpatients: let-7b-5p and miR-92a-3p (p <0.005). Three miRNAs, including let7b-5p and miR-92a-3p, potentially targeted KIAA1324, an estrogen-regulated gene and one of the most deregulated genes in endometrioma. We then performed primary endometrial cells cultures from endometrial biopsies of 10 women with endometrioma. These cells were transfected with miRNAs mimics (let-7b-5p and miR-92a-3p) in order to study their effect on the expression of KIAA1324. KIAA1324 expression was measured by RT-qPCR and a strong decrease of about 40% was observed in the presence of these two miRNAs. Immunohistochemistry assay of KIA1324c in endometrioma as compared to eutopic endometrium of the same patient revealed a fainter staining of KIAA1324 in the lesion as compared to the endometrium, which is concordant with the transcriptomic data. In conclusion, we have revealed the putative implication of a new gene in the pathophysiology of endometriosis, KIAA1324 whose expression is reduced by two circulating microRNAs, let-7b-5p and miR-92a-3p. These two microRNAs were found to be upregulated in the disease; their quantification in plasma could provide a tool for early diagnosis in endometriosis

Subtotal Hysterectomy with Single Port Access Laparoscopy: Gadget or Progress?

International audienceThe strengths of surgical laparoscopy compared to laparotomy include shorter hospitalization, reduction in post-operative pain and adhesions, and better cosmetic outcomes. Since 2008, Single Port Access Laparoscopy (SPAL) has been used in order to offer additional cosmetic benefits and to further reduce post-operative morbidity. The aim of this study was to assess the feasibility of a subtotal hysterectomy using SPAL technique, as well as the benefits and the limitations of this technique

Subtotal Hysterectomy with Single Port Access Laparoscopy: Gadget or Progress?

International audienceThe strengths of surgical laparoscopy compared to laparotomy include shorter hospitalization, reduction in post-operative pain and adhesions, and better cosmetic outcomes. Since 2008, Single Port Access Laparoscopy (SPAL) has been used in order to offer additional cosmetic benefits and to further reduce post-operative morbidity. The aim of this study was to assess the feasibility of a subtotal hysterectomy using SPAL technique, as well as the benefits and the limitations of this technique

Deep shaving and transanal disc excision in large endometriosis of mid and lower rectum: the Rouen technique

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Continuous Amenorrhea May Be Insufficient to Stop the Progression of Colorectal Endometriosis

International audienceWe present the case of a patient in whom consecutive imaging assessment and surgery demonstrated the obvious progression of colorectal endometriosis under continuous medical therapy. A 26-year-old nullipara presented with secondary dysmenorrhea, deep dyspareunia, diarrhea, and constipation during menstruation. Magnetic resonance imaging (MRI) assessment revealed 2 right ovarian endometriomas, but no deep endometriosis lesion. Intraoperatively, we found a 2-cm length of thickened and congestive area of sigmoid colon, along with small superficial lesions arising in the small bowel and appendix. We performed ablation of ovarian endometriomas and appendectomy, and decided to not resect the bowel. Postoperative computed tomography-based virtual colonoscopy (CTC) revealed a slight abnormality of the sigmoid colon. Endorectal ultrasound identified a normal rectum and sigmoid colon. Despite long-term continuous medical treatment, the patient presented 4 years later with impaired digestion consisting in constipation alternating with diarrhea, bloating, dyschesia, and pelvic pain. MRI and CTC revealed an abnormal sigmoid colon from 42 to 50 cm above the anus, with digestive tract diameter reduced from 10 mm down to the virtual lumen, along with an overall rigid appearance. Laparoscopy revealed the extent of endometriosis lesions in the sigmoid colon and multiple implantations in the small bowel. We performed sigmoid and small bowel resection. This case demonstrates the obvious progression of deep rectal endometriosis despite 4 years of continuous hormonal therapy

Surgical Outcomes of Urinary Tract Deep Infiltrating Endometriosis

International audienceTo report the outcomes of surgical management of urinary tract endometriosis

Recurrent Hemoperitoneum During Pregnancy in Large Deep Endometriosis Infiltrating the Parametrium

International audienceWe present the case of a young woman at 16 weeks' gestation who presented to a peripheral hospital with severe recurrent hemoperitoneum related to severe deep endometriosis infiltrating the left parametrium. She underwent 2 surgical open procedures in emergency, followed by pregnancy loss. Deep endometriosis infiltrated the rectum, the vagina, and the left parametrium, leading to stenosis of the left ureter and advanced destruction of the left kidney. Ovarian reserve was low with an antimullerian hormone level at .6 ng/mL. To improve endometriosis-related symptoms and preserve fertility, a laparoscopic conservative rectal and ureteral management was proposed with an aim to relieve symptoms, avoid further destruction of the left kidney, preserve the right splanchnic nerves and inferior hypogastric plexus, and enhance spontaneous conception. We performed a combined vaginal-laparoscopic approach that consisted of vaginal infiltration resection, adhesiolysis, rectal shaving, ureterolysis, and restoration of the permeability of the fallopian tubes. Seven months after surgery the patient spontaneously conceived and is doing well

Unbiased In Silico Analysis of Gene Expression Pinpoints Circulating miRNAs Targeting KIAA1324, a New Gene Drastically Downregulated in Ovarian Endometriosis

Objective: To identify circulating miRNAs associated with ovarian endometriosis (OMA), and to analyze candidate genes targeted by these miRNAs. Methods: Putative regulating miRNAs were identified through an original bioinformatics approach. We first queried the miRWalk 2.0 database to collect putative miRNA targets. Then, we matched it to a transcriptomic dataset of OMA. Moving from gene expression in the tissue to possible alterations in the patient plasma, a selection of these miRNAs was quantified by qRT-PCR in plasma samples from 93 patients with isolated OMA and 95 patients surgically checked as free from endometriosis. Then, we characterized the genes regulated by more than one miRNA and validated them by immunohistochemistry and transfection experiments on endometrial cell primary cultures obtained from endometrial biopsies of 10 women with and without endometriosis with miRNA mimics. Stromal and epithelial cells were isolated and cultured separately and gene expression levels were measured by RT-qPCR. Results: Eight miRNAs were identified by bioinformatics analysis. Two of them were overexpressed in plasma from OMA patients: let-7b-5p and miR-92a-3p (p &lt; 0.005). Three miRNAs, let-7b and miR-92a-3p, and miR-93-5p potentially targeted KIAA1324, an estrogen-responsive gene and one of the most downregulated genes in OMA. Transfection experiments with mimics of these two miRNAs showed a strong decrease in KIAA1324 expression, up to 40%. Immunohistochemistry revealed a moderate-to-intense staining for KIAA1324 in the eutopic endometrium and a faint-to-moderate staining in the ectopic endometrium for half of the samples, which is concordant with the transcriptomic data. Discussion and Conclusion: Our results suggested that KIAA1324 might be involved in endometriosis through the downregulating action of two circulating miRNAs. As these miRNAs were found to be overexpressed, their quantification in plasma could provide a tool for an early diagnosis of endometriosis

Long-term functional outcomes following colorectal resection versus shaving for rectal endometriosis

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