Cryopreservation of Ovarian and Testicular Tissue and the Influence on Epigenetic Pattern

Abstract

Ovarian tissue cryopreservation (OTC) or testicular tissue cryopreservation (TTC) are effective and often the only options for fertility preservation in female or male patients due to oncological, medical, or social aspects. While TTC and resumption of spermatogenesis, either in vivo or in vitro, has still be considered an experimental approach in humans, OTC and autotransplantation has been applied increasingly to preserve fertility, with more than 200 live births worldwide. However, the cryopreservation of reproductive cells followed by the resumption of gametogenesis, either in vivo or in vitro, may interfere with sensitive and highly regulated cellular processes. In particular, the epigenetic profile, which includes not just reversible modifications of the DNA itself but also post-translational histone modifications, small non-coding RNAs, gene expression and availability, and storage of related proteins or transcripts, have to be considered in this context. Due to complex reprogramming and maintenance mechanisms of the epigenome in germ cells, growing embryos, and offspring, OTC and TTC are carried out at very critical moments early in the life cycle. Given this background, the safety of OTC and TTC, taking into account the epigenetic profile, has to be clarified. Cryopreservation of mature germ cells (including metaphase II oocytes and mature spermatozoa collected via ejaculation or more invasively after testicular biopsy) or embryos has been used successfully for many years in medically assisted reproduction (MAR). However, tissue freezing followed by in vitro or in vivo gametogenesis has become more attractive in the past, while few human studies have analysed the epigenetic effects, with most data deriving from animal studies. In this review, we highlight the potential influence of the cryopreservation of immature germ cells and subsequent in vivo or in vitro growth and differentiation on the epigenetic profile (including DNA methylation, post-translational histone modifications, and the abundance and availability of relevant transcripts and proteins) in humans and animals.

Keywords: epigenetic; genomic imprinting; medically assisted reproduction; ovarian tissue cryopreservation; testicular tissue cryopreservation.

Figure 1

Scheme of epigenetic control. The epigenome includes reversible DNA methylation, post-translational histone modifications (PTMs), and the abundance and availability of relevant transcripts and proteins to establish or maintain DNA methylation. Methylation (Me), acetylation (Ac), and ubiquitylation (Ub).

Figure 2

DNA methylation pattern in gametogenesis and early embryogenesis. The DNA methylation pattern is erased in primordial germ cells (PGC) followed by subsequent de novo establishment of the germ cell-specific DNA methylation profile for imprinted genes and gDNA. During early embryogenesis, global DNA demethylation occurs in the zygote and growing embryo. During embryogenesis, cell- and tissue-specific de novo gDNA reprogramming is established. Methylation pattern of imprinted genes is maintained after fertilisation and embryogenesis.

Figure 3

Strategies for fertility preservation in female patients. Conventional oocyte/zygote/embryo cryopreservation (left pathway) and ovarian tissue cryopreservation and in vivo/in vitro oogenesis (right pathway). Left pathway: Cryopreservation of mature oocytes or embryos after controlled ovarian stimulation and medically assisted reproduction techniques. Right pathway: Ovarian tissue cryopreservation followed by in vivo or in vitro folliculogenesis and oogenesis. After orthotopic or heterotopic transplantation, fertilisation can occur spontaneously or via medically assisted reproduction. Techniques to avoid the risk of remission of malignant cells including in vitro follicle culture (IVC), in vitro maturation of immature oocytes (IVM), and artificial ovaries (3D AO) followed by medically assisted reproduction or autotransplantation.

Figure 4

Strategies for fertility preservation in male patients. Cryopreservation of mature spermatozoa (left pathway) and (experimental) testicular tissue cryopreservation and in vivo/in vitro spermatogenesis (right pathway). Left pathway: Cryopreservation of mature spermatozoa or testicular tissue followed by medically assisted reproduction (MAR) techniques (including IVF, ICSI, TESE-ICSI). Right pathway: Testicular tissue cryopreservation followed by in vivo or in vitro spermatogenesis. Resumption of gametogenesis can occur after tissue grafting or, to avoid the risk of remission of malignant cells, after isolation of spermatogonial stem cells (SSC), in vitro proliferation/spermatogenesis, SSC transplantation, and/or MAR techniques.