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

J Marschalek, C Egarter, K Nouri, S Dekan, J Ott, M Frank, D Pietrowski, J Marschalek, C Egarter, K Nouri, S Dekan, J Ott, M Frank, D Pietrowski

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Background: Vitrification has superseded the slow freezing method for cryopreservation of oocytes, embryos, and sperm, but there are as yet no standard protocols for its use in ovarian tissue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.

Keywords: Cell viability; Fertility preservation; Ovarian tissue cryopreservation; Transgender; Vitrification.

Conflict of interest statement

JO received remuneration for lecturing from Lenus Pharma GesmbH outside the submitted work. All other authors declare that they have no conflict of interest.

© 2021. The Author(s).

Figures

Fig. 1
Fig. 1
Study flow chart
Fig. 2
Fig. 2
Representative example image of a Fluorescence Activated Cells Sorting (FACS) analysis of non-vitrified (left) and vitrified (right) ovarian cells after enzymatic digestion. Upper lane: Contour Plot of ovarian cells. Lower lane: Dot Plot of DAPI (4,6 Diamino-2-Phenylindole, Dihydrochloride) stained cells
Fig. 3
Fig. 3
A Rate of viable ovarian cells before and after vitrification/warming using the DMSO-containing protocol 1 and the DMSO-free protocol 2. B Rate of viable ovarian cells before and after vitrification/warming using the open and the closed vitrification system. *p < 0.05
Fig. 4
Fig. 4
Number of defective follicles with regard to different protocols
Fig. 5
Fig. 5
Example of morphologically normal and defective follicles. Example of HE stained ovarian histological tissue sections before (A) and after (B) vitrification showing normal (1) and atretic (2) follicles. Stroma cells (3) and Oocyte nucleus (4) were marked (Magnification 400X)

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