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.

Morphological and ultrastructural changes in seminal coagulum of the squirrel monkey (Saimiri collinsi Osgood, 1916) before and after liquefaction.

Studies with squirrel monkey semen are of special interest due to the large amount of coagulation that is a component of the semen, which is a problem that has to be overcome when the objective is harvesting of gametes. In the present study, there was characterization of the seminal coagulum of captive S. collinsi. Four samples of ejaculates were collected using electroejaculation procedures from four animals. The aim in conducting this study was to evaluate seminal coagulum of S. collinsi using histological and scanning electron microscopy (SEM) procedures before and after semen liquefaction in an ACP-118® extender. Seminal coagulum of S. collinsi was composed of a superficial plate (external), which coats the spongy seminal plasma matrix of S. collinsi. Additionally, there were sperm in the external and internal components of the coagulum with these gametes being isolated or grouped and with there being a heterogeneous distribution of gametes. The supplementation of semen with ACP-118® resulted in a partial dissolution of the seminal plate and spongy matrix portions of the seminal coagulum within the first hour of incubation.

Equol: A Microbiota Metabolite Able to Alleviate the Negative Effects of Zearalenone during In Vitro Culture of Ovine Preantral Follicles.

The impact of zearalenone (ZEN) on female reproduction remains an issue, since its effects may differ among exposed cell types. Besides the use of decontaminants in animal diet, other approaches should be considered to minimise ZEN effects after exposure. Since the first organ in contact with ZEN is the gastrointestinal tract, we hypothesise that products of microbiota metabolism may play a role in ZEN detoxification. We aimed to evaluate the effect of 1 µmol/L ZEN and 1 µmol/L equol (a microbial metabolite), alone or in combination, on the survival and morphology of in vitro cultured ovarian preantral follicles. Ovaries from 12 sheep were collected at a local abattoir and fragmented, and the ovarian pieces were submitted to in vitro culture for three days in the presence or absence of the test compounds. The follicular morphology was impaired by ZEN, but equol could alleviate the observed degeneration rates. While ZEN decreased cell proliferation in primary and secondary follicles, as well as induced DNA double-strand breaks in primordial follicles, all these observations disappeared when equol was added to a culture medium containing ZEN. In the present culture conditions, equol was able to counteract the negative effects of ZEN on ovarian preantral follicles.

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification.

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.

Stroma cell-derived factor 1 and connexins (37 and 43) are preserved after vitrification and in vitro culture of goat ovarian cortex.

This study aimed to evaluate the follicular morphology and development (follicular activation, cell proliferation, and hormone production), as well as the distribution pattern of Connexins 37 and 43 and SDF-1α after vitrification and in vitro culture of goat ovarian tissue. The study involved four experimental groups: fresh control, vitrified control, fresh culture and vitrified culture. The ovarian fragments were vitrified by a solid surface technique using the Ovarian Tissue Cryosystem and subsequently in vitro cultured for 7 days. The percentage of normal preantral follicles was similar between vitrified control and vitrified culture. However, both vitrified control and vitrified culture treatments showed a significant reduction of morphologically normal follicles in comparison to fresh control. A higher percentage of developing follicles (transition, primary and secondary) was observed in both fresh culture and vitrified culture treatments. Progesterone and estradiol production decreased (P < 0.05) during in vitro culture. SDF-1α and Cx37 proteins were detected in oocytes and granulosa cells from all the treatments. However, in vitrified cultured tissue, only granulosa cells were labeled with Cx37. Connexin 43 was detected in the granulosa, theca cells and zona pellucida in all the treatments. In conclusion, in vitro culture of vitrified goat ovarian cortex was able to promote follicle survival and did not alter the expression of SDF-1α and 43. However, the expression of Cx 37 was modified after in vitro culture of vitrified tissue.

Effects of long-term in vitro exposure of ejaculated boar sperm to zearalenone and α-zearalenol in sperm liquid storage medium.

The effects of in vitro exposure of porcine spermatozoa to zearalenone (ZEN) and α-zearalenol (α-ZOL) were studied by evaluating several parameters of an in vitro fertilization (IVF) system. For this purpose, boar spermatozoa cultured with semen storage medium containing 0 (control), 10 and 1000 µg/L of ZEN and α-ZOL for 1 week at 5°C were used for IVF of in vitro matured oocytes. Overall, there were no significant differences in the rates of total penetration, monospermic fertilization, and polyspermic fertilization of oocytes inseminated with spermatozoa from the different groups. Similarly, ZEN and α-ZOL at 10 and 1000 µg/L did not have detrimental effects on the cleavage and development to blastocysts of oocytes after in vitro fertilization. Although the motility, viability, and plasma membrane integrity of spermatozoa significantly decreased after 3 weeks of storage compared to non-stored spermatozoa (P < 0.05), ZEN and α-ZOL at the evaluated concentrations did not exert detrimental effects on the above parameters, even after 3 weeks of storage. These results indicate that prolonged exposure of boar spermatozoa to ZEN and α-ZOL up to 1000 µg/L under reduced metabolic conditions does not affect their in vitro function.

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.

Short-term preservation of canine preantral follicles: Effects of temperature, medium and time.

The use of the large pool of preantral follicles is a promising alternative to provide high numbers of fertilizable oocytes to reproductive biotechnology. This issue is particularly important to canids, since current rates of success of in vitro techniques using oocytes are very limited, and many species within this family are threatened by extinction. The aim of this study was to evaluate effects of temperature, medium and time on morphology and viability of canine preantral follicles during short-term preservation. Canine ovaries were cut into fragments which were incubated in 0.9% NaCl solution or in minimum essential medium (MEM) at 4, 20 or 38 degrees C for 2, 6, 12 or 24 h. Afterwards, preantral follicles were analyzed by histology, transmission electron microscopy and viability testing using trypan blue, calcein-AM and ethidium homodimer-1. Percentages of morphological normal and viable follicles were maintained similar to control (time 0 h) after incubation in 0.9% NaCl at 4 or 20 degrees C for up to 6h and at 38 degrees C for 2 h. Using MEM, such preservation was possible for 12h at 4 or 20 degrees C, and for 6h at 38 degrees C. These results indicate that preservation of canine preantral follicles might be better accomplished through hypothermic (4 or 20 degrees C) storage in MEM, which ensures maintenance of morphology and viability for up to 12h.

Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue.

Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 degrees C for 1h (protocol 1) or at 4 degrees C for 24h (protocol 2) in 0.9% saline solution. At the end of each treatment, preantral follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. To confirm viability staining, ultra-structure of the follicles was evaluated by transmission electronic microscopy (TEM). Data were compared by Chi-square test (P<0.05). The storage of the ovaries at 20 degrees C for 1h (78%) and 4 degrees C for 24h (80%) did not reduce significantly the percentage of viable preantral follicles when compared to the control (75%). Similar results were obtained when ovarian fragments, respectively, for protocols 1 and 2, were exposed to MEM (78 and 77%), 1.5M EG (78 and 71%), as well as frozen in 1.5M EG (74 and 77%). Percentages of viable follicles in control were similar to those observed after exposure (75%) and freezing (76%) in presence of 1.5M DMSO only when protocol 1 was used. The increase of the concentration from 1.5 to 3.0M, for all cryoprotectants, reduced significantly the percentage of viable preantral follicles after freezing. Ultra-structural analysis has confirmed trypan blue results, showing that not only basement membrane, but also organelles, were intact in viable preantral follicles. In conclusion, ovarian tissue cooling at 4 degrees C for 24h before cryopreservation (protocol 2) does not affect the viability of bovine preantral follicles when 1.5M EG is present in the cryopreservation medium.