Vitrification technique for female germinative tissue cryopreservation and banking

Abstract

Objective: To report on a device designed for the vitrification of germinative tissue, and a systematic vitrification/warming protocol.Methods: We 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).Results: There 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).Conclusions: There 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.

Investigating the impact of vitrification on bovine ovarian tissue morphology, follicle survival, and transcriptomic signature

“…It can be learned faster compared to slow freezing, requires significantly less freezing time, and does not demand expensive equipment, resulting in reduced cost [ 51 ]. Additionally, the equipment for vitrification is lightweight and portable, enabling practitioners to perform the procedure in clinics near patients, thereby significantly enhancing the accessibility of fertility preservation treatments [ 52 ]. It is worth noting that despite of these advantages, most live births resulting from the use of cryopreserved ovarian tissue have been achieved through slow freezing protocols, while only four live births have been reported using the vitrification protocol [ 13 , 14 ].…”

“…However, we partially addressed this limitation by utilising a sufficiently large number of animals, ovaries and tissue pieces. Moreover, numerous studies have employed different approaches concerning the size of the tissue pieces used in experiments, primarily varying in length (ranging from 2.5 to 10 mm) and width (from 1 to 10 mm), while, maintaining a thickness of less than 2 mm [ 16 , 17 , 52 , 83 – 86 ]. In our study, we followed these recommendations and used the tissue pieces of 2 × 5 mm in size with 1–1.5 mm in thickness.…”

Nanowarming improves survival of vitrified ovarian tissue and follicular development in a sheep model

“…In 2009 a vitrification procedure by Kagawa et al [ 20 ] was introduced that became widely accepted by the scientific community in this field [ 61 , 62 ]. While there are claims that ovarian cortex vitrification by conventional vitrification lacks adverse side effects [ 63 ], there are evidence that suggests otherwise [ 61 ]. More specifically, during the warming process of the cryopreserved tissue, lack of uniform and fast thermal distribution results in formation of ice particles that later leads to tissue damage [ 9 , 12 ].…”

Morphological study on the effects of sample size on ovarian tissue vitrification

“…Thus, as stated by Yavin & Arav (2007) , sample volume is an essential factor for successful vitrification, the smaller the tissue sample, the less liquid is necessary to cool it and the lower is the probability of ice-crystal formation and tissue damage. A recent report described the use of a dissecting tool for slicing ovarian tissue in fragments measuring 1 x 7 x 7 mm, prior to cryopreservation ( Almodin et al ., 2020 ). After vitrification using the Vitroequip procedure, the authors described no statistical differences in follicular morphology between fresh and cryopreserved tissue samples, showing that relatively large fragments shall be used for cryostorage of human ovarian cortex samples.…”