The effect of different vitrification protocols on cell survival in human ovarian tissue: a pilot study

Abstract

Background Vitrification has superseded the slow freezing method for cryopreservation of oocytes, embryos, and sperm, but there are as yet no standard protocols for its use in ovarian tissue cryopreservation (OTC). Published protocols diverge mainly with regard to the extent of supplementation of dimethyl sulfoxide (DMSO) to the vitrification medium, and to the use of an open or closed vitrification system. We investigated the viability of cells after vitrification/warming, using ovarian tissue of transgender patients, by means of Fluorescence Activated Cells Sorting (FACS), and histomorphological analyses using a DMSO-containing (P1) and a DMSO-free protocol (P2) in an open or closed vitrification setting. Results Twelve ovarian samples were donated from female-to-male transgender patients: 6 were vitrified according to protocol 1, the other 6 according to protocol 2. The amount of viable cells was 90.1% (P1) and 88.4% (P2) before vitrification. After vitrification and subsequent warming, viable cells were reduced to 82.9% (P1, p = 0.093) and 72.4% (P2, p = 0.019). When comparing the closed and the open systems, the decline in cell viability from pre- to post-vitrification was significant only for the latter (p = 0.037). Histological examination reveals no significant differences with respect to degenerated follicles before or after vitrification. Conclusion These results led us to conclude that a protocol containing DMSO results in a higher viability of ovarian cells than a protocol that uses ethylene glycol as cryoprotective agent in vitrification. The use of an open vitrification system led to significant decline in the rate of viable cells. Trial registration NCT03649087, retrospectively registered 28.08.2018.

Different Impacts of Cryopreservation in Endothelial and Epithelial Ovarian Cells

“…Moreover, the use of DMSO as a cryoprotective agent seems to be a contributing factor for cell survival, as reported earlier [26,28].…”

“…We hypothesize that this might protect KGN cells from apoptosis, as the finding that estrogen can suppress apoptosis and promote tumor cell proliferation has already been demonstrated for several estrogen-dependent cancer cell types [27]. Moreover, the use of DMSO as a cryoprotective agent seems to be a contributing factor for cell survival, as reported earlier [26,28].…”

DMSO derives Trophectoderm and Clonal Blastoid from Single Human Pluripotent Stem Cell

“…In cryopreservation protocols, particularly vitrification, higher concentrations of DMSO (∼15% vs. 0.5-2% in this study) are used. Interestingly, several comparative studies showed better survival of human embryos or cells with DMSO-containing cryoprotectants than in DMSO-free ones 43,47,48 . It is tempting to speculate that remnants of or exposures to DMSO could have contributed to the health of human embryos, similar to what is observed in blastoids.…”

Impact of repeated cryopreservation on embryo health and implantation potential

“…Moreover, the type of cryoprotectants used for vitrification may also impact embryo implantation potential. While sucrose, dimethyl sulfoxide (DMSO) and 1,2-propanediol (PROH), are ubiquitously used and have been reported to have no detrimental impact on the viability and morphology of oocytes and embryos [32][33][34] , there are contradictory studies that suggest that DMSO and PROH lead to premature centromere separation in murine oocytes and consequent embryo aneuploidy [35][36][37][38] . Furthermore, the concentration of cryoprotectants employed in clinical settings varies depending on the commercial vitrification kits used, such as: Vitrification Kit (Kitazato BioPharma, Shizuoka, Japan) 39 , Sage Vitrification Kit (Origio, Denmark) 40 and Rapid-I devices (Vitrolife) 41 .…”