Systematic review and meta-analysis on patented and non-patented vitrification processes to ovarian tissue reported between 2000 and 2021.

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.

Use of green microalgae Chlorella as a nutritional supplement to support oocyte and embryo production in goats.

This study aimed to evaluate the use of green microalgae as a nutritional supplement for oocyte and embryo production in goats. Two experiments were performed on adult goats to obtain oocytes (EVO; n = 14) and in vivo embryos (IVD; n = 14). In both, the donors were divided into control (n = 7) and Chlorella (n = 7) groups. All goats received a base diet, and donors were orally supplemented with Chlorella pyrenoidosa (CH) in the Chlorella groups. For EVO, donors received 10 g CH for 14 days, and for IVD, 20 g CH was given for six days before embryo recovery. In EVO and IVD, food intake in the CH group was comparatively low, and it showed relatively high subcutaneous adipose deposition. In addition, the CH group exhibited an increase in triglyceride, cholesterol, and plasma glucose levels. In IVD, a significant increase in peripheral glutathione peroxidase levels was noticed. In EVO, the CH group showed relatively large follicular size and an increase in intrafollicular levels of triglycerides, glucose, and glutathione peroxidase. No differences were observed in the oocyte collected, and CH oocytes showed a low intensity of MitoTracker fluorescence (MT). In IVD, the CH group had a high proportion of transferable embryos, and these structures exhibited high fluorescence intensities for MT and H2DCFDA probes. We concluded that under these conditions, CH did not enhance the quality of the recovered oocytes. However, a daily dose of 20 g CH improved the quality of embryos and stimulated their mitochondrial functionality.

Dyslipidemia induced by lipid diet in late gestation donor impact on growth kinetics and in vitro potential differentiation of umbilical cord Wharton's Jelly mesenchymal stem cells in goats.

Mesenchymal stem cells (MSC) from the umbilical cord (UC) have several attractive properties for clinical use. This study aimed to verify the impact of a lipid-rich diet during late gestation of donor goats on the growth and differentiation of MSCs from UC. From the 100th day of pregnancy to delivery, 22 goats were grouped based on their diet into the donor-lipid (DLD; n = 11) and donor-baseline (DBD; n = 11) diet groups. Diets were isonitrogenous and isoenergetic, differing in fat content (2.8% vs. 6.3% on a dry matter basis). Wharton's jelly (WJ) fragments were cultured. After primary culture, samples of WJ-MSCs were characterized by the expression of CD90, CD73, CD34, CD45, CD105, and Fas genes, mitochondrial activity using MitoTracker (MT) fluorescence probe, and growth kinetics. Population doubling time (PDT) was also determined. WJ-MSCs were differentiated into chondrocytes, adipocytes and osteocytes, and the mineralized area and adipocytes were determined. The lipid diet significantly increased triglyceride and cholesterol levels during pregnancy. The DLD group showed sub-expression of the CD90 gene, a high MT intensity, and a low proliferation rate at the end of the subculture. The mean PDT was 83.9 ± 1.3 h. Mineralized area and lipid droplet stain intensity from osteogenic and adipogenic differentiations, respectively, were greater in DLD. We conclude that in donor goats, dietary dyslipidemia during late pregnancy affects the ability of UC-derived MSCs to express their developmental potential in vitro, thus limiting their possible use for therapeutic purposes.

Ultra-diluted Folliculinum 6 cH impairs ovine oocyte viability and maturation after in vitro culture.

This study investigated the effect of Folliculinum 6 cH on the oocyte meiosis resumption and viability rates, progesterone production and mitochondrial activity after in vitro maturation of cumulus-oocyte complexes (COCs) in sheep. Sheep ovaries were collected at a local slaughterhouse and COCs were recovered by slicing technique. The selected COCs were maturated in TCM199 (Control treatment), or control medium supplemented with 0.05% ethanol (v/v) (the vehicle of the homeopathic preparation - Ethanol treatment) or with Folliculinum 6 cH. After 24 h of in vitro maturation (IVM), oocytes were mechanically denuded and incubated with Hoechst 33342 and MitoTracker (0.5 µM) Orange CMTMRos for analysis of viability and chromatin configuration, and mitochondrial activity, respectively. The results showed that Folliculinum 6 cH addition increased oocyte degeneration and reduced meiotic resumption compared to the control (P < 0.05). Interestingly, the percentages meiotic resumption and oocyte maturation were lower in the Folliculinum 6 cH treatment compared to its vehicle (Ethanol treatment) (P < 0.05). On the other hand, when the treatments were compared, higher mitochondrial activity was observed in the Ethanol treatment (P < 0.05). In conclusion, contrary to its vehicle, the addition of Folliculinum 6 cH to the IVM medium promoted oocyte degeneration and affected negatively the mitochondrial distribution, impairing meiosis resumption.

In vitro study of Withanolide D toxicity on goat preantral follicles and its effects on the cell cycle.

The Withanolide D is a chemotherapeutic potential against the human tumor cell. However, there is no report on the effect of this compound on ovarian function, especially on preantral folliculogenesis. The aim of this study was to evaluate the toxicity of a new candidate to anticancer drug, Withanolide D (WD) on morphologic integrity, development (activation and granulosa cell proliferation) and gene expression of ABCB1 protein of caprine preantral follicles. Ovarian fragments were cultured in vitro for 2 or 6 days in α-MEM or α-MEM added with paclitaxel (PTX -0.1 µg/mL; negative control) and different concentrations of WD (WD1.5, WD3.0 or WD6.0). The higher dose of WD showed a toxic effect similar to PTX and higher (P < 0.05) than other treatments after 2 and 6 days. In addition, WD6.0 reduced the cell proliferating compared to PTX or mild dose. The expression of ABCB1 remained unchanged in the presence of the chemotherapeutic agents (PTX and WD) throughout the culture period. In conclusion, WD exerted a toxic effect observed by decreasing follicular survival and cell proliferation, on the preantral caprine follicles similar to PTX, whose negative effect on folliculogenesis is already widely known.