A polymorphism in a let-7 microRNA binding site of KRAS in women with endometriosis.

Endometriosis is found in 5-15% of women of reproductive age and is more frequent in relatives of women with the disease. Activation of KRAS results in de novo endometriosis in mice, however, activating KRAS mutations have not been identified in women. We screened 150 women with endometriosis for a polymorphism in a let-7 microRNA (miRNA) binding site in the 3'-UTR of KRAS and detected a KRAS variant allele in 31% of women with endometriosis as opposed to 5% of a large diverse control population. KRAS mRNA and protein expression were increased in cultured endometrial stromal cells of women with the KRAS variant. Increased KRAS protein was due to altered miRNA binding as demonstrated in reporter assays. Endometrial stromal cells from women with the KRAS variant showed increased proliferation and invasion. In a murine model, endometrial xenografts containing the KRAS variant demonstrated increased proliferation and decreased progesterone receptor levels. These findings suggest that an inherited polymorphism of a let-7 miRNA binding site in KRAS leads to abnormal endometrial growth and endometriosis. The LCS6 polymorphism is the first described genetic marker of endometriosis risk

Diminuição da expressão do miRNA 135 na fase secretora do ciclo menstrual em pacientes com endometriose

Endometriose é uma doença estrogênio dependente que, entre seus sintomas mais comuns, estão dor pélvica e infertilidade. Afeta até 15% das pacientes em idade reprodutiva e até 50% das pacientes inférteis. A etiopatogenia ainda não é bem clara, mas há evidências do envolvimento de componentes genéticos. O microRNA 135a e 135b (miR135) silencia a expressão gênica e o aumento na expressão do miR135 diminui a expressão do HOXA 10, um importante mediador da receptividade endometrial e implantação. MicroRNAs têm sua expressão alterada no endométrio de mulheres com endometriose quando comparado com o endométrio de mulheres sem a doença. Considerando que vários genes são conhecidos por terem sua expressão alterada no endométrio tópico quando comparado ao endométrio ectópico das pacientes com endometriose, foi analisado a expressão do miR135 neste dois tecidos endometriais na mesma paciente em diferentes fases do ciclo menstrual.Após aprovação pelos Comitês de Ética em Pesquisa do Hospital São Lucas da PUCRS e da Santa Casa de Porto Alegre, foram realizadas biopsias endometriais e exérese de lesões de endometriose de trinta e uma pacientes submetidas à cirurgia no período de março de 2013 a maio 2014 para diagnóstico ou tratamento de endometriose Oito pacientes foram excluídas devido a níveis 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 detecção de miRNA foi utilizado o método poly (A) RT-PCR utilizando o kit Invitrogen NCode miRNA first-strand cDNA synthesis MIRC-50 kit (Invitrogen, California, USA). A transcrição gênica foi amplificada por PCR em tempo real, utilizando o aparelho AB 7500 (Applied Biosystems, Califórnia, USA). Foram utilizados oligonucleotídeos iniciadores específicos para o miR135a e 135b e oligonucleotídeo iniciador universal. Para determinar a expressão relativa, foi utilizado o gene U6. Níveis relativos de mRNA foram apresentados utilizando a formula 2−ΔΔCt. Análise estatística foi realizada utilizando o teste de Mann Whitney, considerando como significativo um p<0,05.Vinte e três pacientes tiveram suas amostras analisadas. Todas as amostras expressavam níveis de miR135a e miR135b. Comparando o endométrio ectópico com o endométrio tópico não houve diferença na expressão do microRNA. Quando as pacientes foram divididas nas diferentes fases do ciclo menstrual, observou-se que durante a fase secretora, a expressão do miR135a e miR135b foi menor do que na fase proliferativa.Em conclusão, microRNAs estão envolvidos na receptividade endometrial e há evidência da relação entre o miR135a e miR135b com HOXA10, um gene sabidamente diminuído na endometriose e relacionado à implantação embrionária. Neste trabalho, foi demonstrado uma expressão semelhante do miR135 no endométrio ectópico em comparação com o endométrio tópico e uma diminuição nesta expressão quando comparado a fase secretora com a proliferativa, provavelmente devido a baixos níveis de estrogênio e altos níveis de progesterona presentes nesta fase.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−ΔΔCt. 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

Falência ovariana precoce associada a deleção no braço longo do cromossomo: relato de dois casos e revisão da literatura Premature ovarian failure with a deletion in the long arm of chromosome: report of two cases and review of the literature

Falência ovariana prematura pode ser idiopática ou estar associada a várias distúrbios auto-imunes ou genéticos, como as deleções do cromossomo X. Relatamos dois novos casos de deleções do braço longo do cromossomo X, em pacientes nuligrávidas apresentando amenorréia secundária e infertilidade. Nenhuma paciente referia história familiar de falência ovariana prematura e relatavam desenvolvimento puberal normal. A avaliação genética mostrou deleção distal no braço longo do cromossomo X, sendo os resultados 46,X,del(Xq22) e 46,X,del(Xq13q28), respectivamente. Após o diagnóstico as pacientes optaram por fertilização in vitro com óvulos doados.<br>Premature ovarian failure may be idiopathic or associated with several autoimmune and genetic disorders as X chromosome deletions. We report two cases of preamture ovarian failure associated with a deletion in the long arm of X chromosome. Both patients were nulligravidas presenting secondary amenorrhea and complaints of infertility, without family history of premature ovarian failure and reporting normal puberal development. Their karyotypes showed deletions of the distal long arm of all X chromosomes and were 46,X, del(Xq22) and 46,X, del(Xq13q28), respectively. After the diagnosis the patients decided to be submitted to an in vitro fertilization with egg donation

Injeção intracitoplasmática de espermatozóides na azoospermia não-obstrutiva: comparação com histopatologia testicular prévia Resultados de la inyección intracitoplasmática de espermatozoides en hombres con azoospermia no obstructiva: utilidad de la biopsia testicular previa Outcome of intracytoplasmic sperm injection in non-obstructive azoospermia according to previous testicular histology

Objetivo: avaliar o resultado de injeção intacitoplasmática de espermatozóides (ICSI) em homens com azoospermia não obstrutiva de acordo com a histologia testicular prévia. Desenho: estudo retrospectivo e transversal. Materiais e métodos: foram estudados os resultados laboratoriais e clínicos em 59 casais (79 ciclos) submetidos a ICSI com uso de espermatozoide testicular. Foram divididos três grupos de acordo com a histologia testicular obtida em biópsia a fertilização (hipoespermatogênese, parada de maturação espermática e aplasia de células germinativas) e os resultados da ICSI foram comparados entre os grupos. Resultados: o achado histoplatológico mais frequante foi hipoespermatogenese (61%), seguido por parada de maturação (22%) e aplasia de células germinativas (17%). A recuperação espermática e a taxa de fertilização oocitária foram superiores no grupo com hipoespermatogênese (p Objetivo: evaluar el resultado de la inyección intracitoplasmática de espermatozoides (ICSI) en parejas cuyos hombres mostraron azoospermia no obstructiva en conformidad con el hallazgo histológico del testículo. Diseño: estudio retrospectivo con análisis transversal. Materiales y métodos: han sido estudiados los resultados de laboratorio y clínicos en 59 parejas (79 ciclos) sometidas a la ICSI. Los hombres han sido divididos en 3 grupos de acuerdo con el reporte histológico obtenido en biopsia previa a la fertilización (hipoespermatogénesis, detención de la maduración espermática y aplasia de las células germinativas) y los resultados han sido comparados entre los grupos. Resultados: el hallazgo principal fue la hipoespermatogénesis (61%), seguido por la detención de la maduración espermática (22%) y la aplasia de las células germinativas (17%). Los espermatozoides estuvieron presentes en 87,7% y la tasa de fertilización (58,8%) en los casos de hipoespematogenesis fue significativamente más grande (p Objective: evaluating ICSI outcome using testicular spermatozoa in patients having non-obstructive azoospermia according to the histological finding of a previous testicular biopsy. Design: retrospective and transversal study. Patients and methods: we evaluated the laboratory outcome and clinical results of 59 couples undergoing 79 ICSI cycles with testicular sperm retrieval. These patients were divided into three groups according to testicular histology (hypospermatogenesis, maturation arrest and germ cell aplasia) revealed in biopsy prior to ICSI. The ICSI was compared to the other groups. Results : the most frequent testicular histological finding was hypospermatogenesis (61%), followed by maturation arrest (22%) and germ cell aplasia (17%). Sperm recovery and oocyte fertilisation were higher in the hypospermatogenesis group (p < 0,01) than in maturation arrest (50% and 40.7%) and germ cell aplasia (21.4% and 36.8%). Embryo cleavage was higher in patients having hypospermatogenesis (95.9%) followed by maturation arrest (87.5%) and germ cell aplasia (71.4%) (p = 0.001). The groups presented no difference in embryo development. Total clinical pregnancy rate per ICSI cycles and per cycles with embryo transfer were 25.3% and 37.7%, respectively. Conclusions: testicular biopsy has clinical value when counselling infertile couples. Although patients with hypospermatogenesis returned the best results, sperm recovery and oocyte fertilization are possible, even in cases where no spermatozoa were found in testicular biopsy

Septo uterino, duplicação cervical e septo vaginal: relato de uma malformação incomum Utero septado, duplicación del cuello y septo vaginal: informe de una rara malformación Septate uterus, cervical duplication and vaginal septum: a report of an uncommon malformation

Objetivo: describir una rara alteración de la fusión de los canales de Müller. Diseño: presentación de caso Reporte del caso: una paciente asiste a la consulta con un examen de ultrasonido en cuya descripción se plantea la sospecha de útero septado. El examen ginecológico reveló un septo vaginal longitudinal, que llegaba hasta la región del himen y la presencia de dos cuellos. La ecografía pélvica tridimensional mostró duplicación del cuello, útero septado que comprometía desde el istmo hasta la cavidad uterina pero sin división del cuerpo uterino, compatible con útero septado y cuello doble. Conclusión: este caso representa una rara malformación no incluida dentro de la clasificación habitual de las malformaciones Müllerianas y que no tiene explicación embriogénica, que se apoya en la teoría de la fusión unidireccional de los canales de Müller. La ecografía tridimensional, es un examen simple y de bajo costo y demostró ser una buena opción para el diagnóstico definitivo de esta malformación, por lo que debe ser considerada dentro del arsenal de exámenes complementarios.Objective: to describe a case of an unusual müllerian anomaly. Design: case report. Case report: a 34 years old white nulligravida presented with complains of suspected uterine septum observed during a routine ultrasonographic examination. Gynecological examination revealed a longitudinal vaginal septum which arrived in hymeneal region and two uterine cervixes. Three dimensional pelvic ultrasonography showed cervix duplication, uterine septum from isthmus to endometrial cavity and absence of uterine body division, compatible with complete uterine septum and true dual cervices. Conclusion: this case represents a rare malformation that is not included in usual classification of müllerian malformations and is not explained by the traditional embryologic vision that supports the unidirectional müllerian fusion. The three-dimensional ultrasonography, a non-invasive and lower cost exam, is a diagnostic option that must be considered in diagnosis arsenal of müllerian malformations

Septo uterino, duplicação cervical e septo vaginal: relato de rara malformação mülleriana com gestação a termo Septate uterus, cervical duplication and vaginal septum: a report of an uncommon malformation with normal term pregnancy

Apresentamos um caso de gestação espontânea em uma paciente com útero septado completo e duplicação cervical. Paciente com 34 anos, branca, nuligesta, ciclos regulares, com suspeita de septo uterino em exame ecográfico. Ao exame, apresentava septo vaginal longitudinal até a região himenal e dois colos uterinos. Solicitada ecografia pélvica tridimensional que evidenciou duplicação cervical, septo uterino do istmo à cavidade endometrial e ausência de divisão do corpo uterino, compatível com útero septado completo e duplicação cervical verdadeira. Um mês após, relatou relação sexual desprotegida e atraso menstrual. Ao exame ecográfico foi visualizado saco gestacional único na cavidade uterina direita. Apresentou gestação sem intercorrências. A cesariana ocorreu com 37 semanas, com recém-nascido do sexo feminino saudável e puerpério normal. Esse caso ilustra uma gestação espontânea, sem intercorrências, em uma rara anomalia, cujo impacto reprodutivo ainda não está totalmente elucidado.<br>This report describes an unusual case of spontaneous pregnancy in a patient with Müllerian anomaly. The patient was a 34-years old, white, nulligravida, with regular menstrual cycles, and suspected uterine septum observed during a routine ultrasonographic examination. The gynecological examination revealed a complete longitudinal vaginal septum and two uterine cervices. Three-dimensional pelvic ultrasonography showed cervix duplication, uterine septum from isthmus to endometrial cavity and absence of uterine body division, compatible with complete uterine septum and true dual cervices. She returned after one month and reported unprotected sexual intercourse and delayed menstrual period. She was pregnant, had a good pregnancy evolution, and delivered a healthy term baby girl, by cesarean section, at 37 weeks of pregnancy. This report describes a case of normal-term pregnancy in a patient with a rare anomaly (vaginal septum and two cervices) who became spontaneously pregnant without treatment

Atelosteogenesis Type 2/Diastrophic Dysplasia Phenotypic Spectrum: From Prenatal to Preimplantation Genetic Diagnosis

Atelosteogenesis type II (AO2) and diastrophic dysplasia (DTD) are two recessively inherited, severe skeletal dysplasias caused by mutations in the SLC26A2 gene. AO2 is an invariably lethal condition, while DTD patients may reach adult life, although both diseases have overlapping diagnostic features. Here we report a patient with an intermediate phenotype between AO2 and DTD and present the successful application of preimplantation genetic diagnosis (PGD) in this situation. Sequencing of SLC26A2 alleles in the infant identified two compound heterozygous mutations, p.Arg178Ter and p.Arg279Trp, of paternal and maternal origin, respectively. At request from the parents, PGD was developed by haplotype mapping of parental SLC26A2 alleles in eleven five-day embryos. Transference to the mother was attempted twice, finally resulting in pregnancy and delivery of a healthy baby. This exemplifies the utility of PGD for inherited lethal conditions with a significant risk of recurrence, and highlights the importance of accurate diagnosis of skeletal dysplasias with prenatal manifestation

Atelosteogenesis Type 2/Diastrophic Dysplasia Phenotypic Spectrum: From Prenatal to Preimplantation Genetic Diagnosis

Atelosteogenesis type II (AO2) and diastrophic dysplasia (DTD) are two recessively inherited, severe skeletal dysplasias caused by mutations in the SLC26A2 gene. AO2 is an invariably lethal condition, while DTD patients may reach adult life, although both diseases have overlapping diagnostic features. Here we report a patient with an intermediate phenotype between AO2 and DTD and present the successful application of preimplantation genetic diagnosis (PGD) in this situation. Sequencing of SLC26A2 alleles in the infant identified two compound heterozygous mutations, p.Arg178Ter and p.Arg279Trp, of paternal and maternal origin, respectively. At request from the parents, PGD was developed by haplotype mapping of parental SLC26A2 alleles in eleven five-day embryos. Transference to the mother was attempted twice, finally resulting in pregnancy and delivery of a healthy baby. This exemplifies the utility of PGD for inherited lethal conditions with a significant risk of recurrence, and highlights the importance of accurate diagnosis of skeletal dysplasias with prenatal manifestation