Immature Cryopreserved Ovary Restores Puberty and Fertility in Mice without Alteration of Epigenetic Marks

Abstract

BackgroundProgress in oncology could improve survival rate in children, but would probably lead to impaired fertility and puberty. In pre-pubertal girls, the only therapeutic option is the cryopreservation of one ovary. Three births have been reported after reimplantation of cryopreserved mature ovary. Conversely, reimplantation of ovary preserved before puberty (defined as immature ovary) has never been performed in humans.Methodology/Principal FindingsIn order to analyze ovarian function, we performed transplantation using fresh or cryopreserved immature grafts in pre-pubertal or adult mice. Puberty as well as cyclic hormonal activity was restored. All follicle populations were present although a significant reduction in follicle density was observed with or without cryopreservation. Although fertility was restored, the graft is of limited life span. Because ex vivo ovary manipulation and cryopreservation procedure, the status of genomic imprinting was investigated. Methylation status of the H19 and Lit1 Imprinting Control Regions in kidney, muscle and tongue of offsprings from grafted mice does not show significant alteration when compared to those of unoperated mice.Conclusions/SignificanceThese results demonstrate that immature ovarian grafting can restore spontaneous puberty and fertility. However, these data suggest that follicle depletion leads to premature ovarian failure. This study addresses the very important epigenetics issue, and provides valuable information to the study of ovarian transplantation suggesting that these procedures do not perturb normal epigenetics marks. These results are highly relevant to the reimplantation question of immature cortex in women.

Genomic imprints as a model for the analysis of epigenetic stability during assisted reproductive technologies

“…Ultra-rapid vitrification of mouse preantral follicles followed by long-term IVM to GV oocytes led to Snrpn LOM (1/50 strands) but not Igf2r LOM (0/15 strands) or H19 GOM (0/58 strands) in pooled vitrified oocytes (Trapphoff et al 2010). In the second study, mice produced following whole ovary cryopreservation maintained normal H19 and Kcnq1ot1 methylation ratios (Sauvat et al 2008). Unfortunately, averaging methylation levels from three tissues from 5 to 36 mice may have obscured imprinting defects in individual mice.…”

Preserving Fertility in Prepubertal Children

“…The increased frequency of developmental impairment, as seen in Beckwith-Wiedemann or Angelman syndromes or in tumors such as retinoblastoma in children born after assisted reproductive technologies (e.g., ovarian stimulation, in vitro fertilization or in vitromaturation of oocytes), has led us to explore the status of genomic imprinting in pups born after ovarian cryopreservation in our experimental model. We found the absence of a significant difference compared with nonmanipulated mice, suggesting that cryopreservation or ovarian transplantation does not interfere with proper establishment of genomic imprinting [19]. This finding should, however, be confirmed by additional studies in other animal models.…”

“…Until recently, only two experiments in animal models using fetal or neonatal ovarian cortex had been performed. We addressed this issue by performing orthotopic ovarian transplantation using fresh or cryopreserved whole ovaries from nonpubescent mice and demonstrated the efficiency of these immature (defined as preserved before puberty) cryopreserved ovaries in restoring both endocrine function and fertility, even in immature recipients [19]. However, we observed a dramatic reduction in follicle density after transplantation independent of the cryopreservation process or experimental group as previously reported in human and other experimental models.…”

“…In our experimental mouse model, we observed smaller litter sizes in grafted groups compared with controls. Since reimplantation in this model was done with only one ovary, we then performed unilateral ovariectomy in a new control group of mice, and this resulted in a litter size similar to that in unmanipulated control mice [19]. …”

Surgery of Ovarian Tumors in Children

“…Until recently, this was not performed in animal models either, except in two experiments using fetal or neonatal ovarian cortex. We addressed this question by performing orthotopic ovarian transplantation using fresh and/or cryopreserved whole ovaries from nonpubescent mice, and demonstrated the efficiency of these immature (defined as preserved before puberty) cryopreserved ovaries to restore both endocrine function and fertility even in immature recipients [9]. However, we observed, as previously reported in human and other experimental models, a dramatic reduction in follicle density after transplantation independent of cryopreservation process and experimental group.…”