Made available in DSpace on 2015-04-14T13:36:02Z (GMT). No. of bitstreams: 1 464257.pdf: 2419406 bytes, checksum: 7e6915df6695417911cc50d9c2a4ea00 (MD5) Previous issue date: 2014-10-14 / Endometriosis is a well know estrogen dependent disease and it?s most common symptoms are severe pelvic pain and infertility. It affects up to 15% of patients on reproductive age and up to 50% of infertile patients. Its pathogenesis still unclear and there is evidence for a role of genetic components. The microRNA135a and 135b (miR135) silence gene expression and increased miR135 down-regulated HOXA 10, a key mediator of endometrial receptivity and implantation. MiRNA are aberrantly regulated in the endometrium of women with endometriosis when compared to the endometrium of disease free women. Considering that several genes are known to be differentially expressed in eutopic and ectopic endometrium of women with endometriosis, we analyzed the expression of miR135 in the ectopic endometrium, compared with the expression in the eutopic from the same patients, and also evaluate if there is different levels of expression during the menstrual cycle. We evaluated thirty one subjects who underwent surgery from March 2013 through May 2014 for diagnosis or treatment of endometriosis, they had endometrium and endometriosis lesions biopsies taken. Approval was obtained from the PUCRS and Santa Casa Hospital Investigations Committee. Eight subjects were excluded due to low levels of mRNA. The samples were divided according to the menstrual cycle as follows: proliferative, day 1-14 (n=11) and secretory, day 15-28 (n=12). For miRNA detection, we used the poly (A) RT-PCR method using Invitrogen NCode miRNA first-strand cDNA synthesis MIRC-50 kit (Invitrogen, California, USA). Gene transcripts were amplified by real-time PCR using the AB 7500 (Applied Biosystems, California, USA) with the forward specific primers to miR135a and miR135b and the universal reverse primer complementary to the anchor primer. U6 small nuclear RNA was used as a control to determine relative miRNA expression. Relative mRNA level was presented using the formula 2&#8722;&#916;&#916;Ct. Statistical analysis was performed using unpaired Mann Whitney test for the ectopic vs. eutopic endometrium samples and for comparison between different phases of the menstrual cycle. All the analyses considered a p< 0.05 as significant. Tweenty three patients submitted to laparoscopic surgery for diagnosis or treatment of endometriosis had endometrium biopsy taken and excision of endometriosis lesions. All endometriosis lesions samples expressed miR135a and miR135b. Comparing with the eutopic endometrium, there weren?t difference on its expression. When the subjects were divided by the menstrual cycle phase, during the secretory phase the expression of mir135a and 135b was lower in the ectopic endometrium comparing to the proliferative phase. MicroRNA is involved in endometrial receptivity, and there is evidence of a relation between miR135a and miR135b with HOXA10, a well know gene that is down regulated in women with endometriosis and has a strong influence on embryo implantation. Here we showed similar expression levels of miR135a and miR135b in the ectopic endometrium when compared with eutopic endometrium. However, we detected a lower expression of miR135 during the secretory phase that is likely due to physiological lower levels of estrogen and higher levels of progesterone during this phase. / Endometriose ? uma doen?a estrog?nio dependente que, entre seus sintomas mais comuns, est?o dor p?lvica e infertilidade. Afeta at? 15% das pacientes em idade reprodutiva e at? 50% das pacientes inf?rteis. A etiopatogenia ainda n?o ? bem clara, mas h? evid?ncias do envolvimento de componentes gen?ticos. O microRNA 135a e 135b (miR135) silencia a express?o g?nica e o aumento na express?o do miR135 diminui a express?o do HOXA 10, um importante mediador da receptividade endometrial e implanta??o. MicroRNAs t?m sua express?o alterada no endom?trio de mulheres com endometriose quando comparado com o endom?trio de mulheres sem a doen?a. Considerando que v?rios genes s?o conhecidos por terem sua express?o alterada no endom?trio t?pico quando comparado ao endom?trio ect?pico das pacientes com endometriose, foi analisado a express?o do miR135 neste dois tecidos endometriais na mesma paciente em diferentes fases do ciclo menstrual. Ap?s aprova??o pelos Comit?s de ?tica em Pesquisa do Hospital S?o Lucas da PUCRS e da Santa Casa de Porto Alegre, foram realizadas biopsias endometriais e ex?rese de les?es de endometriose de trinta e uma pacientes submetidas ? cirurgia no per?odo de mar?o de 2013 a maio 2014 para diagn?stico ou tratamento de endometriose Oito pacientes foram exclu?das devido a n?veis de mRNA muito baixos. As amostras foram divididas de acordo com o ciclo menstrual, fase proliferativa, dia 1 a 14 (n=11) e fase secretora, dia 15 a 28 (n=12). Para a detec??o de miRNA foi utilizado o m?todo poly (A) RT-PCR utilizando o kit Invitrogen NCode miRNA first-strand cDNA synthesis MIRC-50 kit (Invitrogen, California, USA). A transcri??o g?nica foi amplificada por PCR em tempo real, utilizando o aparelho AB 7500 (Applied Biosystems, Calif?rnia, USA). Foram utilizados oligonucleot?deos iniciadores espec?ficos para o miR135a e 135b e oligonucleot?deo iniciador universal. Para determinar a express?o relativa, foi utilizado o gene U6. N?veis relativos de mRNA foram apresentados utilizando a formula 2&#8722;&#916;&#916;Ct. An?lise estat?stica foi realizada utilizando o teste de Mann Whitney, considerando como significativo um p<0,05. Vinte e tr?s pacientes tiveram suas amostras analisadas. Todas as amostras expressavam n?veis de miR135a e miR135b. Comparando o endom?trio ect?pico com o endom?trio t?pico n?o houve diferen?a na express?o do microRNA. Quando as pacientes foram divididas nas diferentes fases do ciclo menstrual, observou-se que durante a fase secretora, a express?o do miR135a e miR135b foi menor do que na fase proliferativa. Em conclus?o, microRNAs est?o envolvidos na receptividade endometrial e h? evid?ncia da rela??o entre o miR135a e miR135b com HOXA10, um gene sabidamente diminu?do na endometriose e relacionado ? implanta??o embrion?ria. Neste trabalho, foi demonstrado uma express?o semelhante do miR135 no endom?trio ect?pico em compara??o com o endom?trio t?pico e uma diminui??o nesta express?o quando comparado a fase secretora com a proliferativa, provavelmente devido a baixos n?veis de estrog?nio e altos n?veis de progesterona presentes nesta fase.