Tissue Vitrification Research Articles

Impacts of different synthetic polymers on vitrification of ovarian tissue

Type and concentration of cryoprotective agents (CPAs) are important factors which influence the likelihood of a successful ovarian tissue vitrification outcome. In an attempt to address this factor, the present study was conducted to evaluate the impacts of different synthetic polymers (Supercool X-1000, Supercool Z-1000 and PVP K-12) on vitrification of bovine ovarian tissue. From each ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control) or submitted to vitrification, either or not followed by in vitro culture for one or five days. Vitrification was performed using the ovarian tissue cryosystem (OTC) system. The ovarian tissues were intended for histological and viability analysis [Reactive oxygen species (ROS) production and degenerate cells assay (Ethidium homodimer-1)], as well as immunolocalization of AQP3 and AQP9 were measured. The results showed that during almost all the periods after warming, in treatment groups which contain polymer (X-1000, Z-1000 and PVP), the percentage of morphologically normal follicles was the highest in the X-1000 samples. Furthermore, post-thawed X-1000 group revealed stronger labeling for AQP9 in primordial and transitional follicles, when compared with others. However, morphology after cryopreservation did not correlate with follicle viability and function where the levels of degeneration and tissue damage of PVP K-12 group were lower in comparison with X-1000 group and only in PVP K-12 group, ROS level was similar to that of the fresh control group. We believe that in addition to permeating CPAs, the addition of one (Supercool X-1000) or maybe a combination (Supercool X-1000 and PVP K-12) of non-permeating polymers could be useful to improve the outcome for vitrified bovine ovarian tissue.

Effects of Different Vitrification Solutions and Protocol on Follicular Ultrastructure and Revascularization of Autografted Mouse Ovarian Tissue.

Objective Many attempts have been made to preserve fertility by improving the cryopreservation of the ovarian tissue. This current studyaimed to improve of direct cover vitrification (DCV) protocol on follicular preservation and angiogenesis in autografted ovarian tissue. Materials and Methods In this experimental study, sixty five female Balb/c mice (5-6 week-old) were anesthetized and their ovaries were dissected. The left ovaries were vitrified by DCV solution, thawed by descending concentrations of sucrose, and then autografted subcutaneously. The right ovaries were autografted with no vitrification procedure prior to transplantation. The animals were sacrificed under anesthesia on the 7thday after transplantation to obtain ovarian tissue. Follicular quality was assessed by histological and ultrastructure observations, and angiogenesis was examined by immunohistochemical staining and real-time polymerase chain reaction (PCR) analysis. Results The histological and ultrastructure features of the follicles preserved well after vitrification of the ovarian tissue by 10% ethylene glycol (EG) and 10% dimethyl sulfoxide (DMSO). Revascularizationwas manifested prominently in the DCV1-vitrified/grafted ovaries by von Willebrand factor (vWF) and alpha smooth muscle actin (α-SMA) immunostaining. The ovarian tissue vitrified in DCV1 protocol had higher expression levels of angiopoietin-2 (Ang-2) and vascular endothelial growth factor (VEGF) 7 days after autotransplantation (P<0.01). ConclusionThese findings suggest that DCV with 10% of both EG and DMSO, is an effective cryopreservation solution for preservation of good quality follicles as well an upregulation of angiogenic factors after ovarian tissue transplantation.

Assessment of DNA Repair Gene Expressions in Vitrified Mouse Preantral Follicles.

Objective Vitrification of the ovarian tissue is one of the techniques recommended for preserving the fertility of women who are dealing with infertility. Despite its benefits, our information about the molecular aspects of ovarian follicles vitrification is somehow ambiguous. Therefore, the aim of this study was to evaluate the expression pattern of DNA repair genes in vitrified preantral follicles. Materials and Methods In this experimental study, the isolated preantral follicles (n=906) from 14-16 days old mice (n=12) were divided into three groups: fresh, toxic and vitrified which were cultured in vitro for 12 days. Preantral follicles were vitrified using cryotop followed by exposure to equilibration solution for five minutes and vitrification solution (VS) for 30 seconds. In the toxic group, preantral follicles were only placed in equilibration and vitrification media and they were then placed in the warming solutions without exposure to liquid nitrogen. On the second and sixth days of the culture period, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to evaluate expression of the selected genes involved in DNA repair, including Msh6 (MutS homolog 6), Mre11 (Meiotic recombination 11), Brca1 (Breast cancer type 1), Rad51 (RAD51 recombinase), Pcna (Proliferating cell nuclear antigen) and Atm (ATM serine/threonine kinase). In addition, developmental parameters including growth, survival rate, antrum cavity formation and ovulation were analyzed. Results The relative mRNA expression of Msh6, Mre11, Brca1, Rad51, Pcna and Atm on the second and sixth days of the culture period in vitrified group was significantly higher than those of the control and toxic groups, but there was no significant difference between the toxic and control groups. In addition, developmental parameters of follicles were similar in both toxic and control groups, while both were significantly higher than that of vitrified group. ConclusionVitrification changes the expression pattern of DNA repair genes of the mouse preantral follicles.

Vitrification technique for female germinative tissue cryopreservation and banking.

ObjectiveTo report on a device designed for the vitrification of germinative tissue, and a systematic vitrification/warming protocol.MethodsWe obtained six fragments of cortical germinative tissue from a human ovary. We randomly chose two fragments and sent them to histological analysis. We vitrified four test samples and stored them for one week in liquid nitrogen (LN), and warmed one week later. We sent the vitrified/warmed fragments to the pathology laboratory, where they analyzed them morphologically under an optical microscope (10-40X). They analyzed the nuclear and cytoplasmic characteristics of the follicular cells, luteal layer, and stroma. The primordial and primary follicles in the fresh and vitrified/warmed fragments were counted and compared with the Mann-Whitney test (p<0.05).ResultsThere were ovarian follicles in different phases of maturation in both fresh and vitrified/warmed fragments, with a predominance of healthy-looking primordial and primary follicles. In the test fragments, the fusocellular architecture supporting the stromal cells exhibited some foci of edema, and were associated with cells with hydropic degeneration, with cytoplasmic fragmentation and eosinophilia. However, there were no signs of tissue necrosis or autolysis. There was no statistically significant difference between the number of follicles found in the control and test tissue fragments (p>0.05).ConclusionsThere were no significant morphological changes between fresh and vitrified/warmed germinative tissue. The vitrification device and protocol tested were effective in the preservation of human follicles, and should be considered for the banking germinative tissue for the restoration of fertility of women who are submitted to life-saving sterilizing treatments.

A conscious rethink: Why is brain tissue commonly preserved in the archaeological record? Commentary on: Petrone P, Pucci P, Niola M, et al. Heat-induced brain vitrification from the Vesuvius eruption in C.E. 79. N Engl J Med 2020;382:383-4. DOI: 10.1056/NEJMc1909867

ABSTRACT Brain tissue is ubiquitous in the archaeological record. Multiple, independent studies report the finding of black, resinous or shiny brain tissue, and Petrone et al. [2020 “Heat-induced Brain Vitrification from the Vesuvius Eruption in C.E. 79.” N Engl J Med. 382: 383–384; doi:10.1056/NEJMc1909867] raise the intriguing prospect of a role for vitrification in the preservation of ancient biomolecules. However, Petrone et al. (2020) have not made their raw data available, and no detailed laboratory or analytical methodology is offered. Issues of contamination and misinterpretation hampered a decade of research in biomolecular archaeology, such that addressing these sources of bias and facilitating validation of specious findings has become both routine and of paramount importance in the discipline. We argue that the evidence they present does not support their conclusion of heat-induced vitrification of human brain tissue, and that future studies should share palaeoproteomic data in an open access repository to facilitate comparative analysis of the recovery of ancient proteins and patterns of their degradation.

Ovarian tissue vitrification is more efficient than slow freezing to preserve ovarian stem cells in CF-1 mice.

Objective:The aim of this study was to investigate the efficacy of protocols for mice ovary cryopreservation to compare the differences in Mouse Vasa Homologue expression (a germline cell marker) and ovarian viability after vitrification or slow freezing.Methods: Female CF1 mice aged 40-45 days were randomly divided into three groups: Control, vitrification or slow freezing. Their ovaries were surgically removed, rinsed in saline solution and cryopreserved. For vitrification, we used a commercial protocol and for slow freeze, we used 1.5 M ethylene glycol (EG) as cryoprotectant. After that, the ovaries were processed for histological an immunohistochemical analysis, and counting of primordial, primary, pre-antral and antral follicles.Results: No significant difference was found in the proportion of high-quality primordial, primary and pre-antral follicles after thawing/warming in the slow freezing and vitrification groups. The immunohistochemistry for MVH antibody demonstrated that the slow freeze group had a higher number of unmarked cells (p=0.012), indicating a harmful effect on the MVH expression in the ovarian tissue, where the cell structure is complex.Conclusion: Although both protocols indicated similar results in the histological analysis of follicular counts, the vitrification protocol was significantly better to preserve ovarian stem cells, an immature germ cell population. These cells are able to self-renew having regeneration potential, and may be effective for the treatment of ovarian failure and consequently infertility.

Angiogenesis and follicular development in ovarian tissue of cattle following vitrification and post-warming culture on chicken chorioallantoic membrane

The purpose of this study was to assess the viability and growth of follicles in ovarian tissues of cattle vitrified using two non-permeating cryoprotectants (sucrose and trehalose) and two cryodevices (cryovial and cryotop). Cortical slices (1−2 mm3) from cattle ovaries (n = 5) were assigned to one of the 14 treatment groups. Cortical slices were vitrified in a TCM199 medium supplemented with ethylene glycol, DMSO, calf serum and either 0.5 M sucrose or trehalose, in cryovials or on cryotops. After warming, cortical slices were either fixed immediately for histology or grafted on a chorioallantoic membrane (CAM) of 10-day old chick embryos. Angiogenesis in ovarian tissues was determined. Viable and atretic preantral (primordial, primary and secondary) follicle densities were examined histologically. There was angiogenesis (chicken) in cortical slices grafted on the CAM by day 5 of culture, however, there was no difference for blood vessel densities when there was use of non-permeating cryoprotectants or cryodevices. Total, viable and atretic follicle densities did not differ (P > 0.05) with use of non-permeating cryoprotectants or cryodevices. The proportion of viable follicles was greater (P < 0.001) in fresh-control than CAM culture-control or vitrification groups. The inclusion of sucrose in the vitrification solution resulted in a larger number of atretic follicles than in the fresh-control group (P < 0.05). In summary, sucrose and trehalose, and cryotop and cryovial were equally suitable for vitrification of ovarian tissues of cattle. Vitrification of ovarian tissues of cattle with subsequent use of CAM culture adversely affected follicular development.

Impact Of Sodium Alginate On The Viability Of Zebrafish Oocytes After Ovarian Tissue Vitrification

Impact Of Sodium Alginate On The Viability Of Zebrafish Oocytes After Ovarian Tissue Vitrification

Vitrified Wharton’s jelly tissue as a biomaterial for multiple tissue engineering applications

Wharton’s Jelly (WJ) tissue is a promising biomaterial, for tissue engineering applications. However, its preservation over a long period in order to be readily available needs further optimization. A possible solution could be the vitrification and storage of WJ tissue at low temperatures. The aim of this study is to evaluate the effect of low temperature in the WJ tissue, which was stored at −196 °C, either with the vitrification or conventional cryopreservation methods. WJ tissues were isolated from human umbilical cords, cryopreserved with the above methods and remained for 1 year at −196 °C. Histological analysis of tissue’s extracellular matrix (ECM), isolation, and characterization of mesenchymal stromal cells (MSCs) were performed. Histological analysis revealed the presence of ECM components such as collagen, sulfated glycosaminoglycans (sGAGs), and the presence of cell nuclei only in vitrified samples. Furthermore, MSCs were isolated and expanded successfully from vitrified WJ tissues, whereas a few viable cells were obtained from conventionally cryopreserved tissues that were not further expanded. In conclusion, this study indicated the proper preservation of vitrified WJ tissues after 1 year of storage, which eventually could be used in tissue engineering and regenerative medicine approaches.

Effect of cryoprotectant type and concentration on the vitrification of collared peccary (Pecari tajacu) ovarian tissue

The aim of the present study was to establish a protocol for solid surface vitrification of peccary ovarian tissue by using different cryoprotectants. Ovarian pairs from five adult females were fragmented and two fragments (fresh control group) were immediately subjected to morphological evaluation using classical histology, transmission electron microscopy, and viability analysis using fluorescent probes. The remaining fragments (n = 18) were vitrified using a solid surface method with different concentrations (3 or 6 M) of ethylene glycol (EG), dimethyl sulfoxide (DMSO) or dimethyl formamide (DMF). After 2 weeks, samples were re-warmed and evaluated. A decrease in the percentage of morphologically normal preantral follicles (PFs) was verified for all the groups in comparison to the fresh control (92.0 ± 2.8%); however, if only the primordial follicles are considered, the most effective preservation (P < 0.05) was achieved with the use of EG at 3 M (74.2±7.3%) or DMSO at 6 M (75.0 ± 4.2%). Ultrastructural analysis indicated there were well-preserved PFs in all the groups evaluated, having well-defined membranes, a few vacuoles, and organelles that were uniformly distributed throughout the cytoplasm, mainly round and elongated mitochondria in close association with lipid droplets. Viability was preserved (P < 0.05) with the use of EG at 3 (97%) or 6 (97%) M, DMSO at 3 (100%), and DMF at 6 (97%) M. Solid surface vitrification, therefore, is an effective method for conservation of peccary female germplasm, especially with the use of EG at 3 M, which was highly effective for preservation of both the morphology and viability of PFs.

Negative air pressure treatment accelerates the penetration of permeable cryoprotectants into bovine ovarian tissue in vitrification protocol and improves cell density after vitrification

Effects of additional physical treatments during vitrification of the bovine ovarian tissue were examined for increasing of permeability of ethylene glycol (EG) and dimethyl sulfoxide (Me2SO). The concentrations of EG and Me2SO and histological changes in the ovarian tissue were evaluated. In the first equilibration step (7.5% EG and 7.5% Me2SO), all the 10-min physical treatments, i.e., negative (679 hPa) or positive (1347 hPa) air pressure applied with a disposable syringe, and shaking (60 rpm) applied with a laboratory shaker, were comparable to 25-min non-physical treatment (plain) vitrification. When effects of the negative air pressure were examined in the second equilibration step (20% EG and 20% Me2SO), its 10-min treatment was equivalent to 15-min plain vitrification (140–170 mg/g tissue). It was thus indicated that the negative air pressure treatment accelerates the penetration of permeable cryoprotectants into the ovarian tissue slices. Histological examination showed that the cell density and the amount of pan-cadherin in the tunica albuginea of the ovary was reduced by the vitrification, but was improved by the negative air pressure treatment. The amount of pan-cadherin in the tunica albuginea was recommended as a biomarker for evaluation of effectiveness of protocol for cryopreservation of bovine ovarian tissue and considered to be a candidate biomarker for human ovarian tissue cryopreservation.

Vitrification of canine ovarian tissues with polyvinylpyrrolidone preserves the survival and developmental capacity of primordial follicles

Ovarian tissue cryopreservation combined with immature follicle development can preserve female fertility in wildlife, regardless of age or reproductive timing. To investigate the effects of different cryopreservation methods and cryoprotectants on follicular survival and developmental capacity, ovarian cortical pieces from 15 dogs were cryopreserved by slow freezing or vitrification with different additional cryoprotectants as follows: dimethyl sulfoxide (DMSO), polyvinylpyrrolidone (PVP), combined DMSO and PVP (each at half the concentration of when used independently), or none (control). Cryopreserved ovarian tissues were evaluated by neutral red staining, histology, and xenotransplantation assays. Among cryopreservation conditions tested, vitrification with combined DMSO and PVP significantly improved the maintenance of follicular morphology compared to that in control. Furthermore, ovarian tissues vitrified using this condition maintained follicle morphology and developmental capacity 9 weeks after grafting, as shown by an increased percentage of primary and secondary follicles and a significant decrease in the transition stage from primordial to primary stage follicles 5 and 9 weeks after grafting. In contrast, slow freezing and control groups lost intact follicles by 5 weeks after grafting. The described cryopreservation techniques, which preserve canine follicle development, will build the foundation of ovarian tissue cryopreservation to preserve female fertility in wild canids.

Dietary Fish Oil and Vitamin E Reduce Cryoinjuries and Apoptosis in Vitrified Laying Hens' Ovarian Tissue.

The occurrence of cryoinjuries during ovarian tissue freezing necessitates development of methods that can overcome these challenges in cell and tissue cryopreservation. It has been hypothesized that omega-3 and vitamin E diet supplementation to the animal could be an appropriate strategy to preserve post-thaw ovarian quality. The laying hen is an appropriate animal model that can be used to study the effects of providing dietary supplements on the outcomes of ovary cryopreservation. The purpose of the study was to evaluate the dietary effects of fish oil, sunflower oil, and vitamin E on 68 laying hens according to the following treatments: basal diet + 1.5% sunflower oil (control; C); basal diet + 1.5% sunflower oil + 1.1 IU alpha-tocopherol/hen/day (E); basal diet + 1.5% fish oil + 1.1 IU alphatocopherol/hen/day (n-3+E); and basal diet + 1.5% fish oil (n-3). The effects on ovarian structure and preservation, apoptosis-related gene expression, and the fatty acid profiles in ovarian laying hen (n = 7 in each group) were studied. The number of intact primordial follicles in n-3+E group was significantly higher than other groups (85% vs. 71%, 72%, and 77% for n-3+E, C, E, and n-3, respectively; p < 0.01). There was a significant reduction in expression of Cas3, as well as Cas8, in n-3 and n-3+E than C and E (p ≤ 0.05). A trend to decrease in Bak (p = 0.089) and Bak/Bcl2 ratio (p = 0.095) in the mRNA was observed in n-3+E. Ovarian eicosapentaenoic acid (C20:5n3) concentration in n-3 was the highest among C, E, and n-3+E (p < 0.01). Docosahexaenoic acid (C22:6 n-3) concentrations in ovaries of the n-3 group were elevated five times more than control. The n-3: n-6 ratio in groups receiving omega-3 (n-3+E and n-3) was higher than other groups (p < 0.01). In conclusion, consumption of dietary omega-3 fatty acids with vitamin E improves the results of vitrification of ovarian tissues in laying hens.

Effects of new synthetic cryoprotectant agents on histological characteristics of various classes of vitrified bovine pre-antral follicles.

Previous studies have reported many discrepancies about the best type and concentration of cryoprotective agents (CPAs) and biological variability among various pre-antral follicle classes after cryopreservation of ovarian tissue. The aim of this study was to investigate the impacts of some synthetic polymers on histological characteristics of different types of pre-antral follicles after bovine ovarian tissue vitrification. From each bovine ovarian pair, fragments were recovered and immediately fixed for analysis (fresh control group) or submitted to vitrification (sucrose, X-1000, Z-1000 and polyvinylpyrrolidone groups), either followed by in vitro culture for 1 or 5 days. In this case, although, the addition of X-1000 resulted in greater percentages of normal follicles for almost all pre-antral follicle classes compared to those of other groups, there are some exceptions. These results indicate that the inclusion of polyvinylpyrrolidone in the freezing media can improve the morphology of the post-warmed transitional follicles and cultured primordial follicles on day five more than other CPAs. According to the results of this study, it can be concluded that although ovarian tissue cryopreservation is often performed to preserve the primordial follicles, by choosing the best combination of permeating and non-permeating CPAs (synthetic polymers), more advanced stages of bovine pre-antral follicles, transitional, primary and secondary follicles, may also survive the cryopreservation process.

Long-term follow-up of vitrified and autografted baboon (Papio anubis) ovarian tissue.

Can vitrified baboon ovarian tissue survive >5 months after autotransplantation and subsequently restore fertility? Our results show that ovarian tissue grafts can survive at least 18 months, but fertility restoration could not be confirmed due to lack of pregnancy. Ovarian function in baboons can be re-established after autografting of vitrified-warmed ovarian tissue fragments. Ovaries from five adult female baboons were used for this study. Unilateral ovariectomy was performed in each animal, followed by vitrification of ovarian fragments. Orthotopic autotransplantation of the vitrified-warmed ovarian fragments was carried out 1 day after ovariectomy. One month later, the other ovary was removed from each animal and vitrified. The next day, the ovarian samples were warmed and orthotopically autografted. Biopsies of grafted tissues were taken after 12 and 18 months for stromal tissue and follicle evaluation. Control samples were collected before vitrification. After grafting, follicle survival, growth and function and also the quality of stromal tissue were assessed histologically and by immunohistochemistry. Estrogen levels were measured, and cyclicity was monitored. All five animals were mated several times. Male baboons used in the mating experiments were not of proven fertility. After vitrification, warming and long-term grafting, follicles were able to grow. However, their function may have been negatively affected by vitrification and/or transplantation, as expression of kit ligand and c-kit differed from fresh ungrafted tissue (P < 0.05). Corpora lutea and/or ovulation stigma were observed in grafts, indicating successful ovulation in all the baboons, with estrogen levels comparable to those in adult female baboons. Not applicable. Despite our promising findings on ovarian function restoration, the vitrification procedure could not be validated. Moreover, male baboons used for mating were not of proven fertility. Our protocol of ovarian tissue vitrification successfully re-established ovarian function in a baboon model of autotransplantation. While more studies are required to determine whether this approach can indeed restore fertility, it may prove an easy way of cryopreserving ovarian tissue with a view to recovering ovarian function. This study was supported by grants from the Fonds National de la Recherche Scientifique (FNRS) (C.A.A. is an FRS-FNRS Research Associate; grant 5/4/150/5 awarded to M.M.D.), Fonds Spéciaux de Recherche, Fondation Saint Luc, Stichting tegen Kanker (Fondation contre le Cancer), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil; grant # 013/14 CAPES/WBI awarded to C.M.L.), Wallonie-Bruxelles International (awarded to C.A.A.), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq; grant awarded to Sarah R. Scalercio) and donations from the Ferrero family. None of the authors have any competing interests to declare.

Development of ovarian tissue vitrification method by using closed device

Development of ovarian tissue vitrification method by using closed device

Investigation of effects of cryoprotectant agents’ temperature on dog ovarian tissue vitrification

Investigation of effects of cryoprotectant agents’ temperature on dog ovarian tissue vitrification

Slush nitrogen vitrification of human ovarian tissue does not alter gene expression and improves follicle health and progression in long-term in vitro culture

To study whether slush nitrogen (SN) vs. liquid nitrogen (LN) vitrification affects human ovarian tissue gene expression and preserves follicle health during extended invitro culture. Randomized experimental study. University research laboratory. Ovarian biopsies collected by laparoscopic surgery from patients with benign gynaecologic conditions. None. Ovarian strips were vitrified with LN or SN, warmed, and analyzed before or after culture for 9days (d9) in gas-permeable dishes. Expression of genes involved in stress and toxicity pathways was analyzed in fresh and warmed strips by polymerase chain reaction (PCR) array and quantitative real-time-PCR. Fresh and vitrified/warmed strips were analyzed for follicle quality, progression, and viability before or after culture. The SN vitrification preserved follicle quality better than LN (% grade 1 follicles: fresh control, 54.2; LN, 29.3; SN, 48.8). Quantitative reverse transcription-PCR demonstrated a noticeable up-regulation of 13 genes in LN samples (range, 10-35) and a markedly lower up-regulation of only 5 genes (range, 3.6-7.8) in SN samples. Long-term invitro culture evidenced worse follicle quality and viability in LN samples than in both fresh and SN samples (% grade 1 follicle: fresh d0, 51.5; fresh d9, 41; LN d9, 16.4; SN d9, 55) and a highly significant reduction of primordial follicles and a concomitant increase of primary and secondary follicles in all samples. Follicle growth to the secondary stage was significantly higher in vitrified tissue than in fresh tissue, being better in SN than in LN vitrified tissue. Follicle quality, gene expression, viability, and progression are better preserved after SN vitrification.

Vitrification of cat ovarian tissue: Does fragment size matters?

The aim of the present study was to investigate whether or not the size of the ovarian fragment influences its resistance to cryostorage. For that purpose, ovaries were collected from 34 queens (various breeds, age 1-5year) by routine ovariectomy, transported to the laboratory and then sectioned in different sizes (3mm×3mm×3mm, 5mm×3mm×3mm and 7mm×3mm×3mm) and randomly assigned to a control (GC3, GC5 and GC7, respectively) or vitrified (GV3, GV5 and GV7, respectively) groups. Vitrified-warmed fragments were evaluated by histomorphology and immunohistochemistry (for apoptotic rates by using cleaved caspase-3). Histological examination reveals that 72.97% of the follicles in GV3 and 72.58% in GV5 were normal while only 42.86% of the follicles in GV7. The main morphological alteration presented in all groups was a detachment of the epithelial cells. Similarly, immunohistochemistry evaluation using caspase 3 revealed a small proportion of apoptotic cells in GV3 (8.43%) while in GV7 30.43% of the cells expressed cleaved caspase-3. These findings indicate that fragments sectioned in 3mm×3mm×3mm (27mm3 ) seem more adequate for perfusion of the cryoprotectant, causing less damage to the cell after vitrification-warming.

Ovarian tissue vitrification using open and closed devices, and thawing procedure

Ovarian tissue vitrification using open and closed devices, and thawing procedure