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

Paula Barros Terraciano,Cristiana Palma Kuhl,Emily Pilar,Jaquelline Festa,Isabel Durli,Charles Ferreira,Tuane Alves Garcez,Markus Berger,Raquel De Almeida Schneider,Vitória De Oliveira Batista,Eduardo Pandolfi Passos,Elizabeth Cirne Lima

doi:10.5935/1518-0557.20190057

Abstract

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.

Highlights

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
Ovarian transplantation has been used for many years in animal models to study ovarian endocrine function (Bagwell et al, 1976), and it was later adapted for ovarian function studies after cryopreservation (Sugimoto et al, 2000)

Summary

Introduction

Ovarian transplantation has been used for many years in animal models to study ovarian endocrine function (Bagwell et al, 1976), and it was later adapted for ovarian function studies after cryopreservation (Sugimoto et al, 2000). Most of the follicular loss in cryopreserved tissue does not occur during the cryopreservation/thawing processes, but rather during the warm ischemic time after retransplantation (Liu et al, 2002; Gosden, 2000). Interventions such as transplantation to granulation tissue (Israely et al, 2006) or incubation of the pretransplantation tissue with growth factors (Schnorr et al, 2002), vitamin E (Nugent et al, 1998), or other antioxidants (Weissman et al, 1999; Kim et al, 2004; Sapmaz et al, 2003) have shown moderate or no effect to increase follicular survival. Live births have been demonstrated after whole ovary cryopreservation and vascular retransplantation, both in sheep (Imhof et al, 2006) and in rat models (Silber et al, 2008), the procedure as a whole has low effectiveness, leading to low live-birth rates

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Conclusion