Vitrification of caprine secondary and early antral follicles as a perspective to preserve fertility function.

The present study aimed to evaluate the structure, survival and development of isolated caprine (secondary-SEC and early antral-EANT) follicles, after vitrification in the presence of synthetic polymers and in vitro culture. Additionally, transzonal projections (TZPs) and p450 aromatase enzyme were evaluated. After isolation, SEC and EANT follicles were in vitro cultured for six days or vitrified. After one week, SEC and EANT follicles were warmed and also in vitro cultured for six days. Data revealed that the percentage of morphologically normal follicles was similar between fresh and vitrified follicles in both follicular categories and antrum formation rate was similar between fresh and vitrified SEC follicles. Fluorescence by calcein-AM did not show difference between fresh and vitrified (SEC and EANT) follicles, however, the trypan blue test showed low viability for vitrified follicles. The integrity of TZPs was not affected between fresh and vitrified SEC follicles, however, in vitrified EANT follicles, there were signs of TZPs loss. Regarding steroidogenic function, it was observed a positive staining for p450 aromatase enzyme in fresh and vitrified SEC and EANT follicles. It was concluded that SEC follicles seem to be more resistant to vitrification than EANT follicles, as shown by the trypan blue test and TZPs assay. Future studies may confirm this hypothesis, in order to consolidate the use of SEC and EANT follicles as an alternative to ovary cryopreservation.

In situ cultured preantral follicles is a useful model to evaluate the effect of anticancer drugs on caprine folliculogenesis.

Despite the increase in the incidence of cancer, the number of women who survive cancer treatment is growing. However, one of the principal results of chemotherapy is premature ovarian failure (POF). The aim of this study was to use the in situ culture preantral follicles as an in vitro model to evaluate the toxicity of two anticancer drugs, doxorubicin (DXR) and paclitaxel (PTX), on the integrity and development of ovarian follicles. Fragments of the ovarian cortex of goats were cultured in vitro for 1 or 7 days in α-MEM(+) supplemented with different concentrations of DXR (0.003, 0.03, or 0.3 µg/mL) and PTX (0.001, 0.01, or 0.1 µg/mL). Analyses were performed before and after culture to evaluate tissue integrity by classical histology, apoptosis by TUNEL assay, DNA laddering kit and the detection of activated caspase 3, and DNA damage by the immune detection of phosphorylated histone H2A.x (H2AXph139). Both DXR and PTX reduced the number of morphologically normal primordial and developing follicles. Positive staining for TUNEL and active caspase 3 was detected in all the samples (P < 0.05). Therefore, we propose the in situ culture of caprine preantral follicles as a useful experimental model for assessing the toxic effects of the chemotherapeutic agents on ovarian folliculogenesis. Microsc. Res. Tech. 79:773-781, 2016. © 2016 Wiley Periodicals, Inc.

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.

Impacto dos agentes antineoplásicos sobre os folículos ovarianos e importância das biotécnicas reprodutivas na preservação da fertilidade humana / Impact of antineoplastic agents on the ovarian follicles and importance of reproductive biotechnologies in the preservation of human fertility

A preservação da fertilidade de jovens mulheres em idade reprodutiva tem se tornado um dos grandes desafios da medicina, já que a maioria dos tratamentos contra o câncer pode causar insuficiência ovariana prematura, devido à toxicidade ovariana dos agentes quimioterápicos.Com base nessa sensibilidade e com vistas a reverter ou minimizar os danos às células reprodutivas decorrentes da quimioterapia, cada vez se tornam mais frequentes os estudos e a busca tanto por opções para preservar a fertilidade feminina quanto por tratamentos para células cancerosas que sejam mais seletivos. Este artigo tem como objetivo apresentar uma visão geral sobre os danos causados pelos quimioterápicos à função ovariana e as possíveis opções para a preservação da fertilidade feminina em pacientes com câncer e as perspectivas em oncofertilidade. Foi feita uma pesquisa no banco de dados National Library of Medicine (PubMed), em que foram recuperados artigos publicados entre 1967 e 2015 sobre agentes quimioterápicos e seus danos à fertilidade feminina.

The preservation of fertility in young women of reproductive age has become a major challenge in medicine, since most cancer treatments can cause premature ovarian failure due to ovarian toxicity of chemotherapeutic agents. Based on this sensitivity it is necessary to revert or minimize damage to reproductive cells resulting from chemotherapy. It have become more frequent studies and researches for alternatives to preserve female fertility and treatments that are more selective for cancer cells. Currently there are several options for preservation of fertility in women undergoing chemotherapy treatments.This article aims to present an overview of the damage caused by chemotherapy ovarian function and possible options for preserving fertility in female cancer patients and prospects in oncofertilidade. A search on databases were searched for relevant articles: the National Library of Medicine (PubMed) and the Virtual Health Library. Articles published between 1967 and 2015 on chemotherapeutic agents and their damage on female fertility was held.

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.