DNA methylation associated with polycystic ovary syndrome: a systematic review.

PURPOSE: Polycystic ovary syndrome (PCOS) is an endocrine metabolic disease that affects women of reproductive age and is one of the main causes of anovulatory infertility. However, the cause of PCOS is yet fully understood, and genetic factors play an important role in its etiology. In this study, we reviewed the main genes involved in the etiology of PCOS and the influence of DNA methylation, aiming to answer the study´s guiding question: 'What is the influence of DNA methylation on the main genes involved in PCOS?'. METHODS: We used the MEDLINE database, and inclusion criteria (primary and original articles, written in English, found through our entry terms) and exclusion criteria (literature reviews and articles that used animals to perform the experiments and that focused in other epigenetics mechanism without being DNA methylation) were applied. RESULTS: Twenty-three scientific articles, from a total of 43 articles read in full, were chosen for this study. Eighteen studies confirmed DNA methylation associated with PCOS. CONCLUSION: The most relevant genes related to PCOS were INSR, LHCGR, and RAB5B, which may be epigenetically altered in DNA, with the first two genes hypomethylated and the last hypermethylated. The epigenetic changes presented in the genes related to PCOS or their promoters were only at the CpG sites.

Educação remota na continuidade da formação médica em tempos de pandemia: viabilidade e percepções / Distance Learning in continued medical training during the pandemic: feasibility and perceptions

Resumo: Introdução: Com a interrupção das atividades educacionais das universidades por causa da pandemia da Covid-19, formas de ensino virtuais, como a educação remota (ER), passaram a ser debatidas no meio acadêmico. No entanto, faz-se necessária uma avaliação criteriosa da ER antes de sua aplicação visando à qualidade do ensino. Objetivo: Assim, o objetivo deste estudo foi avaliar a viabilidade da implantação da ER para discentes de um curso de Medicina. Método: Aplicou-se um formulário on-line no qual constavam questões sobre aspectos demográficos e socioeconômicos, sobre acesso às tecnologias digitais e uma pergunta subjetiva relacionada ao uso da ER. Os dados quantitativos foram comparados pelos testes Kruskal-Wallis e Mann-Whitney com p < 0,05. Analisaram-se os dados qualitativos por meio do método de Classificação Hierárquica Descendente, e houve ainda uma análise pós-fatorial. Resultados: O perfil socioeconômico de 266 participantes foi variado, sendo a maioria dos discentes brasileiros do sexo masculino, adultos jovens, cor da pele parda e renda familiar média elevada. Todos relataram ter acesso à energia elétrica, mas não à água encanada, à coleta pública de lixo e ao esgotamento sanitário. Todos possuíam pelo menos um equipamento eletrônico para acesso à internet, mas com variação no tipo e na velocidade da conexão. Um total de 80,8% dos discentes avaliou como viável a implantação da ER para o seguimento do curso. No entanto, 8,65% dos discentes afirmaram que os equipamentos e a internet disponíveis não permitiriam o acompanhamento das atividades on-line. Foi observada uma correlação significativa entre a velocidade de acesso à internet e a renda familiar média, a cor da pele e o local da residência (p < 0,05), bem como entre possuir equipamentos adequados à ER e a renda familiar média (p < 0,05). A análise da pergunta objetiva revelou seis categorias: a necessidade de organização dos procedimentos e o anseio pela volta à normalidade; custos e benefícios da ER; planejamento e garantia da acessibilidade de todos os estudantes à internet de qualidade; acreditar que a realização da ER não é a solução para o problema; capacitação de professores e acadêmicos para o uso das plataformas digitais; e o querer realizar a ER; surgindo uma variedade de opiniões, visões e realidades pelos discentes. Conclusão: Apenas o seguimento da ER, sem garantia de acesso para todos os discentes, é insuficiente e necessita da intervenção dos gestores para não haver prejuízo na aprendizagem daqueles menos favorecidos.

Abstract: Introduction: With the interruption of University teaching activities due to the COVID-19 pandemic, various virtual learning strategies, such as Distance Learning (DL), have be discussed in the academic environment. However, before applying such strategies, a careful evaluation is required in order to ensure high quality learning. Objective: The aim of this study was to evaluate the feasibility of implementing DL for students at a medical school. Method: An online form was applied containing a demographic questionnaire, socioeconomic questionnaire, a questionnaire on access to digital technologies and a subjective question related to the use of DL. Quantitative data were compared using the Kruskal-Wallis or Mann-Whitney test, with p<0.05. Qualitative data were analyzed using the Descending Hierarchical Classification method and post-factorial analysis. Results: The socioeconomic profile of 266 participants was varied, with most of the students being young adults, Brazilian and male with brown skin color and a high average family income. All students reported having access to electricity, but not running water, public garbage collection and sanitation. All had at least one electronic equipment for internet access, but with variations in the type and speed of internet connection. 80.8% of the students considered the implementation of DL feasible for following the course. However, 8.65% stated that the equipment and the available internet equipment would not allow them to follow online activities. A significant correlation was observed between internet access speed and the average family income, skin color and the place of residence (p<0.05); as well as between having equipment suitable for DL and the average family income (p<0.05). Analysis of the objective question revealed six categories: the need to organize procedures and the desire to return to normality; DL costs and benefits; planning and ensuring the accessibility of all students to quality internet; to believe that the realization of DL is not the solution to the problem; training teachers and academics to use digital platforms; and wanting to perform the DL; giving rise to a variety of opinions, views and realities from the students. Conclusion: Merely carrying on with DL, without any guaranteed access for all students, is insufficient and requires the intervention of managers to avoid detrimental consequences for the learning of those less favored.

In vitro culture systems as an alternative for female reproductive toxicology studies.

SummaryStudies have shown that daily exposure to different products, whether chemical or natural, can cause irreversible damage to women's reproductive health. Therefore it is necessary to use tests that evaluate the safety and efficacy of these products. Most reproductive toxicology tests are performed in vivo. However, in recent years, various cell culture methods, including embryonic stem cells and tissues have been developed with the aim of reducing the use of animals in toxicological tests. This is a major advance in the area of toxicology, as these systems have the potential to become a widely used tool compared with in vivo tests routinely used in reproductive biology and toxicology. The present review describes and highlights data on in vitro culture processes used to evaluate reproductive toxicity as an alternative to traditional methods using in vivo tests.

ATP-binding cassette (ABC) transporters in caprine preantral follicles: gene and protein expression.

The multidrug resistance proteins ABCB1, ABCC2 and ABCG2 are an energy-dependent efflux pump that functions in systemic detoxification processes. Physiologically expressed in a variety of tissues, most abundantly in the liver and intestinal epithelia, placenta, blood-brain barrier and various stem cells, until now, these pumps were not identified in goat ovarian tissue. Therefore, the aim of this study is to analyze ABCB1, ABCC2, and ABCG2 mRNA and protein expression in goat preantral follicles. Fragments (3 × 3 × 1 mm) from five pairs of ovary (n = 10) obtained from five goat were collected and immediately submitted to qPCR, Western blot, and immunofluorescence assay for mRNA detection and identification and localization of the ABC transporters, respectively. mRNA for ABCB1, ABCC2, and ABCG2 and the presence of their proteins were observed on ovarian tissue samples. Positive marks were observed for the three transport proteins in all follicular categories studied. However, the marks were primarily localized in the oocyte of primordial, transition and primary follicle categories. In conclusion, goat ovarian tissue expresses mRNA for the ABCB1, ABCC2 and ABCG2 transporters and the expression of these proteins in the preantral follicles is a follicle-dependent stage.

Activin-A promotes the development of goat isolated secondary follicles in vitro.

The role of activin-A in follicular development and on the mRNA expression levels of different genes in goat secondary follicles was evaluated. Goat secondary follicles (≥ 150 µm) were cultured for 18 days under control conditions or with the addition of either 50 or 100 ng/ml activin-A (Experiment 1). The mRNA levels for the genes that code for activin-A, ActR-IA, ActR-IB, ActR-IIA, ActR-IIB, follicle stimulating hormone receptor (FSH-R) and P450 aromatase were measured in each condition (Experiment 2). We observed that after 6 days of culture, the antrum formation rate was higher in cultures with added activin-A than in the cultured control (P < 0.05). The addition of 50 ng/ml activin-A increased the follicular growth rate in the final third of the culture (days 12-18), resulting in a higher percentage of meiosis resumption (P < 0.05). On day 6, the addition of activin-A (50 ng/ml) increased the levels of ActR-IA mRNA compared with the cultured control (P < 0.05). After 18 days, the addition of 50 ng/ml activin-A significantly increased the levels of its own mRNA compared with the non-cultured control. Moreover, this treatment reduced the mRNA levels of P450 aromatase in comparison with the cultured control (P < 0.05). Higher levels of P450 aromatase mRNA were found for both activin-A treatments compared with the non-cultured control (P < 0.05). No difference in estradiol levels was detected among any of the tested treatments. In conclusion, the addition of activin-A to culture medium stimulated early antrum formation as well as an increase in the daily follicular growth rate and the percentage of meiosis resumption.

Effect of medium composition on the in vitro culture of bovine pre-antral follicles: morphology and viability do not guarantee functionality.

Summary This study investigated the effect of three different culture media (α minimum essential medium (α-MEM), McCoy or TCM199 during the in vitro culture (IVC) of bovine isolated pre-antral follicles. Pre-antral follicles greater than 150 µm in size were isolated and cultured for 0 (control), 8 or 16 days in one of the abovementioned culture media. Follicles were evaluated for survival, growth and antrum formation at days 8 and 16. The results showed that TCM199 was the most suitable medium to preserve follicular viability and ultrastructure, resulting in the highest rates of antrum formation. In conclusion, TCM199 promotes the in vitro development of isolated pre-antral follicles without hampering follicular functionality by sustaining in vitro growth and antrum formation.

Ultrastructural features of agouti (Dasyprocta aguti) preantral follicles cryopreserved using dimethyl sulfoxide, ethylene glycol and propanediol.

The objective was to develop an efficient protocol for cryopreservation of agouti (Dasyprocta aguti) ovarian tissue. Agouti ovarian fragments were placed, for 10 min, in a solution containing MEM and fetal bovine serum plus 1.5 M dimethyl sulfoxide (DMSO), ethylene glycol (EG) or propanediol (PROH); some of those fragments were subsequently cryopreserved in a programmable freezer. After exposure and/or thawing, all samples were fixed in Carnoy prior to histological analysis. To evaluate ultrastructure, follicles from the control and all cryopreserved treatments were fixed in Karnovsky and processed for transmission electron microscopy. After exposure and freezing, there was a significant decrease in the percentage of morphologically normal preantral follicles in all treatments when compared to the control (92.67 ± 2.79, mean ± SD). However, there were no significant difference when the exposure and freezing procedures were compared using the same cryoprotectant. Moreover, there was no significant difference among cryoprotectants at the time of exposure (DMSO: 64.7 ± 3.8; EG: 70.7 ± 11.2, PROH: 63.3 ± 8.5) or after freezing (DMSO: 60.6 ± 3.6, EG: 64.0 ± 11.9; PROH: 62.0 ± 6.9). However, only follicles frozen with PROH had normal ultrastructure. In conclusion, preantral follicles enclosed in agouti ovarian tissue were successfully cryopreserved using 1.5 M PROH, with satisfactory maintenance of follicle morphology and ultrastructure.

Freezing solution containing dimethylsulfoxide and fetal calf serum maintains survival and ultrastructure of goat preantral follicles after cryopreservation and in vitro culture of ovarian tissue.

Goat ovarian cortex fragments were subjected to slow freezing in the presence of various solutions containing intracellular cryoprotectants, including 1.0 M ethylene glycol (EG), propanediol (PROH), or dimethyl sulfoxide (DMSO), with or without sucrose and/or fetal calf serum (FCS). Histological examination revealed that only the DMSO-containing solutions were able to maintain a follicular ultrastructure similar to the morphology observed in the fresh control. Therefore, fragments previously cryopreserved in DMSO solutions (with and without sucrose and/or FCS) were cultured in vitro for 48 h and then subjected to viability, histological, and ultrastructural analysis. No significant differences were observed among the percentages of morphologically normal follicles in cryopreserved ovarian tissue before in vitro culture (DMSO: 62.5%; DMSO + sucrose: 68.3%; DMSO + FCS: 60.0%; DMSO + sucrose + FCS: 60.0%) and after culture (DMSO: 60.8%; DMSO + sucrose: 64.2%; DMSO + FCS: 70.8%; DMSO + sucrose + FCS: 55.0%). Following in vitro culture, the viability analysis showed that only the freezing solution containing DMSO and FCS (75.6%) maintained a percentage of viable follicles similar to that observed after culture without cryopreservation (89.3%). As determined by ultrastructural analysis, morphologically normal preantral follicles were detected in the fresh control and in fragments cultured before and after cryopreservation with DMSO and FCS. Thus, a freezing solution containing DMSO and FCS, under the experimental conditions tested here, guaranteed the maintenance of viability and follicular ultrastructure after short-term in vitro culture.