RESUMO

Due to the great interest in ovarian cryopreservation and, consequently conservation and restoration of female fertility in the last decades, different vitrification procedures (vitrification devices or solutions) have been developed, patented, and used both for academic research purposes and for clinical use. Therefore, the present study aimed to provide a systematic review and meta-analysis of data obtained from the application of different patented and non-patented vitrification devices and solutions in different countries. For this purpose, relevant observational studies published between the years 2000 to 2021 were selected to verify the efficiency of ovarian vitrification processes on parameters such as morphology, viability, and apoptosis in preantral ovarian follicles after transplantation or in vitro culture. Our research revealed that, although several countries were considered in the study, the United States and Japan were the countries that registered the most processes, and 22 and 16 vitrification devices and solutions out of a total of 51, respectively were patented. Sixty-two non-patented processes were also considered in the study in all countries. We also observed that transplantation and in vitro ovarian culture were the techniques predominantly used to evaluate the efficiency of the devices and vitrification solutions, respectively. In conclusion, this review showed that patented or non-patented protocols available in the literature are able to successfully preserve preantral follicles present in ovarian tissue. Despite the satisfactory results reported so far, adjustments in ovarian vitrification protocols in order to minimize cryoinjuries to the follicles remain one of the goals of cryopreservation and preservation of the female reproductive function. We found that vitrification alters the morphology and viability, and offers risks leading in some cases to follicular apoptosis. However, adjustments to current protocols to develop an optimal procedure can minimize damage by not compromising follicular development after vitrification/warming.

RESUMO

Abstract This study aimed to investigate the effect of growth and differentiation factor 9 (GDF-9) during the in vitro culture of isolated caprine early antral follicles. The isolated and selected early antral follicles were individually cultured for 18 days, and the following treatments were tested: α-MEM+ (control treatment) or α-MEM+ supplemented with 200 ng/mL GDF-9. The following endpoints were evaluated: follicular growth and morphology, estradiol production, oocyte nuclear maturation, and relative expression of key genes related to steroidogenesis (CYP19A1, CYP17, and insulin receptor) and basement membrane remodeling (MMP-9 and TIMP-2). In both treatments, a decrease was observed in the percentage of morphologically intact follicles with a concomitant increase in the rates of extruded and degenerated follicles (P < 0.05). The GDF-9 treatment showed higher rates of extruded follicles only on day 6 of culture (P < 0.05). Follicle diameter increased progressively throughout the culture period (P < 0.05) with similar diameters between treatments at all culture times (P > 0.05). Growth and differentiation factor 9 increased the daily growth rate from the first to the second third of culture, with higher values (P < 0.05) than control in the second third. Oocyte maturation rate as well as estradiol levels and relative mRNA expression for CYP19A1, CYP17, MMP-9, TIMP-2, and insulin receptor genes were similar between treatments (P > 0.05). This study shows for the first time that GDF-9 added to a culture medium increased the follicle growth rate of goat early antral follicles cultured in vitro.

Resumo Este estudo teve como objetivo investigar o efeito do GDF-9 durante o cultivo in vitro de folículos antrais iniciais caprinos isolados. Os folículos antrais iniciais isolados e selecionados foram cultivados individualmente por 18 dias, e os seguintes tratamentos foram testados: α MEM+ (tratamento controle) ou α-MEM+ suplementado com 200 ng/mL de GDF-9 (tratamento GDF-9). Os seguintes parâmetros foram avaliados: crescimento e morfologia folicular, produção de estradiol, maturação nuclear do oócito e expressão relativa de genes-chave relacionados a esteroidogênese (CYP19A1, CYP17 e receptor de insulina) e remodelamento da membrana basal (MMP-9 e TIMP-2). Em ambos os tratamentos, observou-se diminuição na porcentagem de folículos morfologicamente intactos com aumento concomitante nas taxas de folículos extrusos e degenerados (P < 0,05). O tratamento GDF-9 apresentou maiores taxas de folículos extrusos apenas no 6º dia de cultivo (P < 0,05). O diâmetro do folículo aumentou progressivamente ao longo do período de cultivo (P < 0,05) com diâmetros semelhantes entre os tratamentos em todos os tempos de cultivo (P > 0,05). O GDF-9 aumentou a taxa de crescimento diário do primeiro para o segundo terço de cultivo, sendo maior (P < 0,05) que o controle no segundo terço. A taxa de maturação oocitária assim como os níveis de estradiol e a expressão relativa de RNAm para os genes CYP19A1, CYP17, MMP-9, TIMP-2 e receptor de insulina foram similares entre os tratamentos (P > 0,05). Em conclusão, este estudo mostra pela primeira vez que GDF-9 adicionado a um meio de cultivo aumentou a taxa de crescimento de folículos antrais iniciais caprinos cultivados in vitro.

RESUMO

The cryopreservation of secondary follicles (SF) is a promising alternative to preserve the reproductive potential both in humans and animals in situations in which the transplantation of ovarian tissue is not possible. The objective of the present study was cryopreserved SF isolated sheep. Beyond follicular morphology, viability and development, we investigated proteins related to steroidogenic function and basement membrane remodeling [metalloproteinases 2 (MMP-2) and 9 (MMP-9)] in fresh SF (FSF) and vitrified SF (VSF) followed by in vitro culture for 6 (D6) or 12 days (D12). The percentage of intact follicles, follicular and oocyte diameter of the VSF were lower than FSF on both days of culture (P < 0.05). The VSF viability was statistically reduced from D6 (95.5%) to D12 (77.3%) but did not differ from the FSF on both days (D6:96.2% to D12:86.5%). Antrum formation in the VSF (D6: 59.13%; D12: 79.56%) was significantly lower than the FSF (D6: 79.61%; D12: 92.23%). However, an increase in this percentage was observed from D6 to D12 in both groups. Aromatase showed stronger labeling on FSF D6 and VSF D12 compared to other treatments (P < 0.05). MMP-2 showed a similar pattern of labeling in FSF D6 and VSF D12, similarly to that observed in FSF D12 and VSF D6. MMP-9 was similar in FSF and VSF cultivated for 6 and 12 days. In conclusion, VSF are able to grow and develop during 12 days of in vitro culture and showed evidence of preservation of steroidogenic function and remodeling of the basement membrane.

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RESUMO

Withanolide D (WD) has been investigated as an antineoplastic drug. This study aimed to evaluate whether melatonin (MT) could attenuate toxic effects on preantral follicles enclosed in the ovarian cortex (experiment 1 - E1) or on isolated secondary follicles (experiment 2 - E2) exposed to WD. For E1, ovarian cortex was incubated for 48 h to: (1) α-MEM+; (2) α-MEM+ plus 6 µM WD; (3) α-MEM+ plus 3 mmol/L MT or (4) α-MEM+ plus WD and MT. For E2, secondary follicles were exposed for until 96 h in. (1) only to basic medium (α-MEM++/α-MEM++); (2) α-MEM++ plus 3 mmol/L MT (MT/MT); (3) α-MEM++ until 48 h, followed by more 48 h in 6 µM WD (α-MEM++/WD) or (4) a pre-exposure to MT for until 48 h, followed by more 48 h of exposure to WD plus MT (MT/MT + WD). The main results obtained showed that exposure to drugs caused damage to follicular morphology (WD or WD + MT) and diameter (WD) in the ovarian cortex or in isolated follicles. In pre-antral follicles in situ, ATM expression increased in the presence of WD, MT or association. As for the secondary follicles, ATM and γH2AX were immunostained in the granulosa and theca cells and oocytes in all treatments. TAp63α was immunostained in follicles included in the ovarian cortex and in isolated follicles. We conclude that melatonin did not provide protection and could have enhanced the toxic effect of WD to follicles surrounded or not by the ovarian cortex.

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RESUMO

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.

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