Experimental and Molecular Pathology

DROSHA rs10719 and DICER1 rs3742330 polymorphisms in endometriosis and different diseases: Case-control and review studies.

Jéssica Vilarinho Cardoso, Rui Medeiros, Francisca Dias, Isabelle Alves Costa, Renato Ferrari, Plinio Tostes Berardo, Jamila Alessandra Perini


Objective: DROSHA and DICER1 enzymes participate in the main stages of microRNA synthesis. Polymorphisms can influence mRNAs stability and genes expression, and hence affect the binding of miRNAs. Thus, the present study evaluated the association of DROSHA and DICER1 polymorphisms in the development of endometriosis and other diseases.
Methods: A total of 240 endometriosis cases and 242 controls were genotyped for the DROSHA rs10719 G > A and DICER1 rs3742330 A > G polymorphisms using the TaqMan system. The association between polymorphisms and endometriosis was estimated by binary logistic regression. A literature review was also performed including all published articles (PubMed database) until December 2020, regarding the association of the studied polymorphisms and different diseases.
Results: DICER1 rs3742330GG was only found in endometriosis cases (2.1%) and deep infiltrative endometriosis (DIE) (2.5%). The DICER1 rs3742330GG genotype was significantly associated with endometriosis (P < 0.05), suggesting a tendency to present an increased risk for disease. DROSHA rs10719A and DICER1 rs3742330G allele frequencies varied among populations (6%–79% and 10.2%–55.1%, respectively). In the Brazilian population, the frequencies of these alleles were 42.3% and 7.3%, respectively. Both polymorphisms were risk factors for nonsyndromic orofacial clefts, tuberculosis, stroke ischemia and mortality after stroke, recurrent idiopathic pregnancy loss, and some types of cancer. Moreover, the DICER1 rs3742330 polymorphism was a protective factor for precancerous cervical lesions, different types of cancer and tuberculosis.
Conclusions: The results suggest that only the DICER1 rs3742330 A > G polymorphism may be associated with susceptibility to endometriosis. The frequencies of both polymorphisms were significantly different among populations, and there were discrepancies in the risk associations with the development of diseases.

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Rev. Bras. Saúde Mater. Infant.

Epidemiological profile of women with endometriosis: a retrospective descriptive study

Jéssica Vilarinho Cardoso, Daniel Escorsim Machado, Mayara Calixto da Silva, Plinio Tostes Berardo, Renato Ferrari, Maurício Simões Abrão, Jamila Alessandra Perini


Objectives: to describe the epidemiological and clinical profile of women with endometriosis and to determine the association with the prognostic characteristics of the disease.
Methods: retrospective descriptive study involving 237 women attended at two referral hospitals for endometriosis, between 2011 and 2017. Associations between groups were estimated using logistic regression models
Results: most women (65.4%) were of reproductive age (29-39 years), with a body mass index in the range of 18.5-24.9 kg/m2 and a high prevalence (23-81%) of symptoms of the disease, with 49.5% being infertile. The average time of diagnosis was 5 years. Ovarian endometrioma and/or deep infiltrative endometriosis (DIE) were the most frequent type of endometriosis (87%), and 59% of patients were in the III/IV stage of the disease. Approximately 87% of women with surgical diagnosis were aged over 30, married (70%) and had lower parity. Dyspareunia was negatively associated with superficial endometriosis. Infertility was positively associated with age (30-39 years) and DIE in the uterine tubes; dysmenorrhea with DIE in the uterosacral ligament; cyclic intestinal complaints with DIE in the rectosigmoid and intestine, and with DIE classification and III/IV stage.
Conclusions: knowing the epidemiological and clinical profile of Brazilian women with endometriosis can help in diagnosis and treatment planning.

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European Journal of Obstetrics & Gynecology and Reproductive Biology

Combined effect of vascular endothelial growth factor and its receptor polymorphisms in endometriosis: a case-control study

Jéssica Vilarinho Cardoso, Maurício Simões Abrão, Rosane Vianna-Jorge, Renato Ferrari, Plínio Tostes Berardo, Daniel Escorsim Machado, Jamila Alessandra Perini


Objective: Endometriosis is a multifactorial gynecological disease, whose pathogenesis is crucially dependent on angiogenesis, which is signaled via vascular endothelial growth factor (VEGF) and its receptor (VEGFR2). We hypothesize that single nucleotide polymorphisms (SNPs) in VEGF and VEGFR2 genes may influence the onset and/or the progression of endometriosis. The main aim of this study was to investigate the contribution of VEGF and VEGFR2 SNPs as risk factors for endometriosis, as well as their association with endometriosis symptoms.
Study design: A case-control study was conducted, involving 293 endometriosis patients and 223 controls, who were submitted to laparoscopic or laparotomy surgery at hospitals from the Brazilian public health system. Genotyping of VEGF (2578C > A, 460T > C, 1154G > A, +405G > C and +936C > T) and VEGFR2 (604T > C, 1192C > T) SNPs was performed by TaqMan real-time polymerase chain reaction. The association between SNPs and endometriosis, deep infiltrating endometriosis (DIE) or endometriosis symptoms was estimated by odds ratios (OR) with their 95% confidence intervals (CI), which were calculated using multivariate logistic regression models. Results: VEGF variant alleles 2578A and 1154A were associated with increased endometriosis risk (OR: 1.39, 95% CI: 1.04–1.87 and OR: 1.63, 95% CI: 1.12–2.37, respectively), whereas VEGF 405C and VEGFR2 1192T were associated with lower risk of endometriosis (OR: 0.66, 95% CI: 0.43–1.00 and OR: 0.58, 95% CI: 0.40–0.84, respectively). The combination of wild-type genotypes of both VEGF 2578C > A and 1154G > A with variant genotypes of both VEGF +405G > C and VEGFR2 1192C > T showed the best protective effect against the development of endometriosis, either considering all cases (OR: 0.33, 95% CI: 0.12–0.89) or only DIE (OR: 0.30, 95% CI: 0.10–0.87). The combination of variant genotypes of VEGF 2578C > A, 1154G > A, +405G > C and VEGFR2 1192C > T was also protective against DIE (OR: 0.67, 95% CI: 0.46–0.96). VEGFR2 1192C > T were associated with reduced cyclical urinary complaints (OR: 0.40, 95% CI: 0.18–0.88).
Conclusions: Our results indicate that VEGF SNPs 2578C > A and 1154G > A increase endometriosis risk, whereas VEGF +405G > C and VEGFR2 1192C > T are protective against disease development, with VEGFR2 1192C > T also reducing cyclical urinary symptoms. The combined analysis of VEGF–VEGFR2 genotypes suggests a gene–gene interaction in endometriosis susceptibility.

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BMC Women's Health

Role of vascular endothelial growth factor polymorphisms (-2578C > A, -460 T > C, -1154G > A, +405G > C and +936C > T) in endometriosis: a case–control study with Brazilians

Jamila Alessandra Perini1, Jessica Vilarinho Cardoso, Plínio Tostes Berardo, Rosane Vianna-Jorge, Luiz Eurico Nasciutti, Marta Bellodi-Privato, Daniel Escorsim Machado and Mauricio Simões Abrão


Background: Endometriosis is regarded as a complex and heterogeneous disease in which genetic and environmental factors contribute to the phenotype. The Vascular Endothelial Growth Factor (VEGF) plays important roles in the pathogenesis of endometriosis. The present study was aimed at investigating the contribution of VEGF polymorphisms as risk factors for the development of endometriosis. This is the first study to evaluate the combined influence of the five most common VEGF polymorphisms.
Methods: This study was conducted at two hospitals from the Brazilian public health system, and comprised 294 women submitted to laparoscopic or laparotomy surgery: 182 patients had a histologically confirmed diagnosis of endometriosis (cases), whereas 112 had no evidence of the disease (controls). The VEGF polymorphisms were determined by TaqMan real-time polymerase chain reaction. The odds ratio (OR) with their 95% confidence intervals (CI) were calculated using an unconditional logistic regression model.
Results: Endometriosis patients and controls did not differ regarding age distribution, whereas the body mass index was significantly lower in endometriosis patients, when compared with controls (23.1 ± 3.9 versus 27.3 ± 5.9, P < 0.001). The evaluation of gynecological symptoms, including dysmenorrhea, non-cyclic chronic pelvic pain, dyspareunia and infertility, indicates significantly higher prevalences among endometriosis cases. The variant allele -1154A was significantly associated with endometriosis, either considering all cases (OR: 1.90, 95% CI: 1.23–2.97), deep infiltrating endometriosis (DIE) (OR: 1.83, 95% CI: 1.16-2.90) or moderate and severe endometriosis (stages III-IV) (OR: 1.97, 95% CI: 1.21-3.19). No significant differences were found in allele or genotype distributions of the –2578C > A, -460 T > C, +405G > C and +936C > T polymorphisms between endometriosis cases and controls. A total of six haplotypes were inferred derived from four polymorphisms (-2578C > A, -460 T > C, -1154G > A and +405G > C). There was a protective association between CCGG haplotype and endometriosis, either considering all cases (OR: 0.36, 95% CI: 0.15–0.86), DIE (OR: 0.37 95% CI: 0.15 – 0.90) or stages III-IV (OR: 0.35 95% CI: 0.13 – 0.95).
Conclusions: The present results indicate a positive association between VEGF -1154G > A and the risk of developing endometriosis, whereas the CCGG haplotype may be protective against the development of disease.

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Journal of Experimental & Clinical Cancer Research

Higher expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) and metalloproteinase-9 (MMP-9) in a rat model of peritoneal endometriosis is similar to cancer diseases

Daniel E Machado, Plínio T Berardo, Celia Y Palmero, Luiz E Nasciutti


Background: Endometriosis is a common disease characterized by the presence of a functional endometrium outside the uterine cavity, causing pelvic pain, dysmenorrheal, and infertility. This disease has been associated to development of different types of malignancies; therefore new blood vessels are essential for the survival of the endometrial implant. Our previous observations on humans showed that angiogenesis is predominantly found in rectosigmoid endometriosis, a deeply infiltrating disease. In this study, we have established the experimental model of rat peritoneal endometriosis to evaluate the process of angiogenesis and to compare with eutopic endometrium.
Methods: We have investigated the morphological characteristics of these lesions and the vascular density, VEGF and its receptor Flk-1 and MMP-9 expression, and activated macrophage distribution, using immunohistochemistry and RT-PCR.
Results: As expected, the auto-transplantation of endometrium pieces into the peritoneal cavity is a well-established method for endometriosis induction in rats. The lesions were cystic and vascularized, and demonstrated histological hallmarks of human pathology, such as endometrial glands and stroma. The vascular density and the presence of VEGF and Flk-1 and MMP-9 were significantly higher in endometriotic lesions than in eutopic endometrium, and confirmed the angiogenic potential of these lesions. We also observed an increase in the number of activated macrophages (ED-1 positive cells) in the endometriotic lesions, showing a positive correlation with VEGF.
Conclusion: The present endometriosis model would be useful for investigation of the mechanisms of angiogenesis process involved in the peritoneal attachment of endometrial cells, as well as of the effects of therapeutic drugs, particularly with antiangiogenic activity.

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Composition of sulfated glycosaminoglycans and immunodistribution of chondroitin sulfate in deeply infiltrating endometriosis affecting the rectosigmoid

Plínio T. Berardo, Maurício S. Abrao, Maísa L.S. Souza , Daniel E. Machado, Luiz-Cláudio F. Silva, Luiz E. Nasciutti


The composition of sulfated glycosaminoglycans (GAGs) and the tissue distribution of chondroitin sulfate (CS) were analyzed in deeply infiltrating endometriosis (DIE) of rectosigmoid, using metachromatic staining, and biochemical analysis employing electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against CS. The sulfated GAGs were characterized as dermatan sulfate (DS), heparan sulfate (HS) and CS; and DS strongly predominated compared to HS and CS. Immunostaining procedures showed that CS was concentrated in the endometriosis foci, distributed throughout the stroma around the glands. This is the first report describing the composition of sulfated GAGs and the tissue location of CS in DIE by means o histochemical, biochemical and immunohistochemical analyses. These results confirmed that in DIE o rectosigmoid, as in eutopic endometrium [Nasciutti, L.E., Ferrari, R., Berardo, P.T., Souza, M.L.S., Takiya C.M., Borojevic, R., Abrao, M.S., Silva, L.C.F., 2006. Distribution of chondroitin sulfate in human endometrium. Micron 37, 544–550], CS was the dominant sulfated GAG in stroma of the lesion foci.

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Fertility and Sterility

Vascular density and distribution of vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 (Flk-1) are significantly higher in patients with deeply infiltrating endometriosis affecting the rectum

Daniel Escorsim Machado, M.Sc., Mauricio Simoes Abrao, M.D., Ph.D., Plinio Tostes Berardo, M.D., Ph.D., Christina Maeda Takiya, M.D., Ph.D., and Luiz Eurico Nasciutti, Ph.D.


Objective: To analyze vascular density and immunolocalization of angiogenic vascular endothelial growth factor (VEGF) and its receptor Flk-1 in the proliferative and secretory eutopic human endometrium and in three different sites of endometriosis: the ovary, bladder, and rectum.
Design: Prospective study.
Setting: University hospital.
Patient(s): Thirty women with endometriosis (10 ovarian, 10 bladder, 10 rectal) and 32 control women (10 proliferative endometrium, 10 secretory endometrium, 4 normal ovary, 4 normal bladder, 4 normal rectum).
Intervention(s): Normal endometrial samples were obtained from women during laparoscopic ablation of subserous myoma, and biopsy specimens of endometriosis were obtained from patients undergoing surgery for the diagnosis and treatment of endometriosis. Normal tissues of ovary, bladder, and rectum were obtained from these organs beside the lesions of endometriosis.
Main Outcome Measure(s): Blood vessels were quantified according to the number of von Willebrand factor–positive endothelial cells. The VEGF and Flk-1 distribution were evaluated semiquantitatively by immunohistochemical staining.
Result(s): More blood vessels were found in cases of endometriosis, particularly rectal endometriosis, compared with the respective control samples and with the eutopic endometrium, and they were localized in endometrial stroma around the glands. The VEGF and Flk-1 expression levels were also higher in cases of endometriosis, especially rectal endometriosis.
Conclusion(s): Vascularization and VEGF and Flk-1 expression are significantly higher in deeply infiltrating endometriosis affecting the rectum, reinforcing the hypothesis that antiangiogenesis therapy may constitute a new modality of treatment, especially in cases of deep endometriosis involving the rectum. (Fertil Steril 2008; 90:148–55. 2008 by American Society for Reproductive Medicine.)

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Distribution of chondroitin sulfate in human endometrium

Luiz E. Nasciutti, Renato Ferrari, Plínio T. Berardo, Maísa L. S. Souza, Christina M. Takiya, Radovan Borojevic, Maurício S. Abrão, Luiz-Claudio F. Silva


Sulfated glycosaminoglycan (GAG) composition was characterized in the human endometrium during proliferative and secretory phases of the menstrual cycle. Sulfated GAGs were analyzed in endometrium tissue using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed that CS was the main sulfated GAG species detected, accompanied by small amounts of heparan sulfate and dermatan sulfate. CS was distributed overall the connective stroma, around arteriole vessels and glands, and there was no important difference in the immunostaining between the proliferative and secretory endometrium phases. Our findings extend previous observations on the GAG composition in the human endometrium providing new information regarding the tissue distribution and location of endometrial CS.

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