Cryopreservation Of Preantral Follicles Research Articles

Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments

Cryopreservation of preantral follicles is a promising technique to preserve female fertility. The aim of this study was to evaluate the effect of vitrification on the development of secondary follicles included in ovarian tissue or isolated after microdissection. An important end point included is the capacity of grown oocytes to resume meiosis. Sheep ovarian cortexes were cut into fragments and split into three different groups: (1) fresh (control): secondary follicles isolated without any previous vitrification; (2) follicle-vitrification (follicle-vit): secondary follicles vitrified in isolated form; and (3) tissue-vitrification (tissue-vit): secondary follicles vitrified within fragments of ovarian tissue (in situ former) and subsequently subjected to isolation. From the three groups, isolated secondary follicles were submitted to IVC for 18 days. After IVC, cumulus-oocyte complexes (COCs) were harvested from follicles. As an additional control group, in vivo grown, in vivo-grown COCs were collected from antral ovarian follicles. All, recovered COCs were matured and the chromatin configuration was evaluated. Data were analyzed by ANOVA, and the means were compared by Student-Newman-Keuls test, and by chi-square. Differences were considered to be significant when P < 0.05. Isolated preantral follicles from all treatments had normal morphology, antrum formation, and low follicle degeneration after IVC. The growth rate between control and follicle-vit did not differ (P > 0.05), and was higher (P < 0.05) than for tissue-vit. The percentage of follicles that decreased diameter during IVC was significantly higher in tissue-vit than the in follicle-vit. Recovery rate of oocytes from normal follicles was higher in follicle-vit than in tissue-vit. Furthermore, oocyte viability was lower in tissue-vit than other treatments, and follicle-vit did not differ from control and in vivo grown. The percentage of oocytes meiosis resuming was not different between treatments except for in vivo grown. After vitrification, only follicle-vit showed metaphase I oocyte. We conclude that secondary follicles vitrified after isolation displayed a better follicular growth rate, oocyte viability, percentage of oocytes reaching the metaphase I stage, and fewer follicles with decreased diameter after IVC.

Total oxidative status of mouse vitrified pre-antral follicles with pre-treatment of alpha lipoic acid.

Background: Cryopreservation of pre-antral follicles is a hopeful technique to preserve female fertility. The aim of the present study was to evaluate reactive oxygen species (ROS) and total antioxidant capacity (TAC) levels of mouse vitrified pre-antral follicles in the presence of alpha lipoic acid (ALA). Methods: Isolated pre-antral follicles (140–150 µm in diameter) were divided into vitrified–warmed and fresh groups. Each group was subjected to in vitro maturation with or without ALA for 12 days, followed by adding human chronic gonadotropin to induce ovulation. In vitro fertilization was performed to evaluate their developmental competence. In parallel, the amount of ROS and TAC were assessed after 0, 24, 48, 72, and 96 h of culture by 2',7'-dichlorofluorescin assay and ferric reducing/antioxidant power assay, respectively. Results: The respective rates of survival, antrum formation, and metaphase II oocytes were significantly higher in ALA-supplemented groups compared to the groups not treated with ALA. TAC and ROS levels were significantly decreased and increased, respectively during the culture period up to 96 h in the absence of ALA in both vitrified and non-vitrified samples. However, with pretreatment of ALA, TAC levels were increased significantly and remained constant up to 96 h in vitrified-warmed pre-antral follicles, while ROS levels completely returned to the level of starting point after 96 h of culture in the presence of ALA. Conclusion: Pretreatment of ALA positively influences development of pre-antral follicles in vitrified and non-vitrified samples through increasing follicular TAC level and decreasing ROS levels.

Effects of vitrification solutons and equilibration times on the morphology of cynomolgous ovarina tissues vitrified ultra-rapidly by direct plungging into liquid nitrogen

OBJECTIVE: Cryopreservation of preantral follicles in ovarian tissues has been expected to be an effective measure for preserving young women who need to undergo cytotoxic therapy. However, cryopreservation protocol has not yet been established. In this study, we assessed effects of vitrification solutions and equilibration times on morphologies of cynomolgus ovarian tissues vitrified ultra-rapidly using newly developed vitrification system which is consisted of 4 fine needles (Cryosupport). DESIGN: Experimental animal study. MATERIALS AND METHODS: Ovarian cortical sections (1 cm x 1cm x 0.1-0.2 cm) of 7 cynomolgus monkeys (85-137 months old) were randomly allocated to 6 groups. Ovarian sections were vitrified using 2 different vitrification solutions (35% (v/v) ethyleneglycol (EG) + 5% (w/v) PVP + 0.5 M sucrose (EG35) vs. 20% (v/v) EG + 20% (v/v) DMSO + 0.5 M sucrose (VSED)) after 3 different exposure times (5, 10 and 20 min). Ovarian sections put on the Cryosupport were vitrified ultra-rapidly by a direct application into liquid nitrogen. After warming, follicles were analyzed using light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: Five-minute exposure to EG35 brought in better follicle morphology (93%) compared with other conditions (77-89%) by LM and better mitochondrial morphology (88%) compared with other conditions by TEM (40-81%). Especially, the proportions of morphologically-normal follicles (77%) and of normal structure of mitochondria in oocytes (40%) of preantral follicles vitrified using VSED after 5 min were lower (P<0.01) than those achieved from follicles vitrified by other conditions (follicles: 87-94% and mitochondria: 78-88%, respectively). CONCLUSIONS: Results of the present study indicate that a combination of 35% EG with 5% PVP can support the morphology of vitrified preantral follicles and mitochondria in oocytes vitrified after the exposure for 5 min. Shorter exposure time to cryoprotectant might be expected to decrease the cytotoxic effect on the ovarian tissues.

Comparison of the developmental potential of 2-week-old preantral follicles derived from vitrified ovarian tissue slices, vitrified whole ovaries and vitrified/transplanted newborn mouse ovaries using the metal surface method

BackgroundCryopreservation of preantral follicles or ovarian tissues would enable the storage of large numbers of primordial follicles or preantral follicles and preserves the structural integrity of somatic and reproductive cells. In the present study, we compared the developmental potential of cryopreserved two-week-old mouse preantral follicles, ovarian tissue slices, two-week-old mouse ovaries and newborn mouse ovaries using a metal plate with a high cooling rate for cooling the droplet of vitrification solution.MethodsGroups of 2 to 4 samples (including of 14-day old preantral follicles, ovarian tissue slices, whole ovaries, and whole newborn ovaries) were exposed to 4% ethylene glycol (EG) in DPBS + 10% FBS for 15 min and then rinsed in a vitrification solution composed of 6 M ethylene glycol and 0.4 M trehalose in DPBS + 10% FBS. Equilibration in room temperature was performed for 20–30 seconds for preantral follicle and 5 min equilibration was performed in an ice bath for ovaries. The samples were dropped onto the surface of metal plate around -180°C in the volume of 2 μl and 6 μl. After thawing, the ovarian tissue was mechanically isolated for collecting the preantral follicles. The thawed newborn ovaries were transplanted under the renal capsule of recipient male mice for 14 days. Preantral follicles collected from each groups were cultured individually in 20-μl droplets of α-MEM culture medium in culture dish for 12 days. On the day 12 of culture, the cumulus-oocyte complexes (COCs) were collected for IVM and IVF. Fertilization and embryo cleavage were scored.ResultsAfter the vitrification of 14-day-old preantral follicles using 2 μl or 6 μl droplet onto surface of metal plate, the results indicated that no significant difference in survival rate, antral-like cavity formation, COCs collected, 2 cell embryo cleavage and blastocyst development was found in vitrification of the 2 μl and 6 μl droplet groups. As comparing 14-day old ovarian tissue (ovarian tissue slices and whole ovaries) and whole newborn ovaries vitrified in 6 μl droplet, lower success rates of antral-like cavity formation and COCs collection were found in the whole ovaries group.ConclusionOur results suggest that the metal plate surface vitrification method is an appropriate and convenient method for cryopreservation of mouse ovaries and preantral follicles. The droplet volume of vitrification solution in 2 μl and 6 μl can be an option.

Cryopreservation of caprine ovarian tissue using glycerol and ethylene glycol

Cryopreservation of ovarian tissue may be a potential alternative for the conservation of genetically superior animals, including high milk- and meat-producing goat breeds. However, until now, no information was available concerning the cryopreservation of preantral follicles (PF) enclosed in caprine ovarian tissue. The objective of the present study was to evaluate the structural and ultrastructural characteristics of caprine PF after exposure to and cryopreservation of ovarian tissue in 1.5 and 3 M glycerol (GLY) and ethylene glycol (EG). At the slaughterhouse, each ovarian pair from five adult mixed breed goats was divided into nine fragments and randomly distributed into treatment groups. One fragment was immediately fixed for histological examination and ultrastructural analysis, after slaughter (control). Four of the ovarian fragments were equilibrated at 20 °C for 20 min in 1.8 ml of MEM containing 1.5 or 3 M GLY or EG for a toxicity test and the final four fragments were slowly frozen using these cryoprotectants at the concentrations above. After toxicity testing and freezing/thawing, the ovarian fragments were fixed for histological examination. Histological analysis showed that after toxicity testing and cryopreservation of the ovarian tissue in GLY or EG at both concentrations, the percentage of normal PF was significantly lower than controls. Ultrastructural analysis of PF frozen in 1.5 and 3 M GLY, as well as 3 M EG demonstrated that these follicles remained morphologically normal. In conclusion, we demonstrated cryopreservation of caprine PF in ovarian tissue.