Slow Freezing Versus Vitrification of Mouse Ovaries: from Ex Vivo Analyses to Successful Pregnancies after Auto-Transplantation

Carmen Terren,Carine Munaut,MaïTé Fransolet,Michelle Nisolle,Marie Ancion

doi:10.1038/s41598-019-56182-8

Carmen Terren, Carine Munaut + Show 3 more

Open Access

https://doi.org/10.1038/s41598-019-56182-8

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Abstract

Slow freezing (SF) is the reference method for ovarian tissue cryopreservation. Vitrification (VT) constitutes an alternative but controversial method. This study compares SF and VT (open [VTo] and closed [VTc] systems) in terms of freezing damage and fertility restoration ability. In vitro analyses of C57Bl/6 SF or VTo-ovaries, immediately after thawing/warming or after culture (cult), revealed that event though follicular density was similar between all groups, nuclear density was decreased in VTo-ovaries compared to CT-ovaries (CT = 0.50 ± 0.012, SF = 0.41 ± 0.03 and VTo = 0.29 ± 0.044, p < 0.01). Apoptosis was higher in VTo-cult ovaries compared to SF-cult ovaries (p < 0.001) whereas follicular Bmp15 and Amh gene expression levels were decreased in the ovaries after culture, mostly after VTo (p < 0.001). Natural mating after auto-transplantation of SF, VTo and VTc-ovaries revealed that most mice recovered their oestrous cycle. Fertility was only restored with SF and VTo ovaries (SF: 68%; VTo: 63%; VTc: 0%; p < 0.001). Mice auto-transplanted with SF and VTo-ovaries achieved the highest number of pregnancies. In conclusion, in vitro, no differences between SF and VTo were evident immediately after thawing/warming but VTo ovaries displayed alterations in apoptosis and follicular specific proteins after culture. In vivo, SF and VTo ovary auto-transplantation fully restored fertility whereas with VTc-ovary auto-transplantation no pregnancies were achieved.

Highlights

In recent decades, considerable therapeutic advances have allowed notable improvements in the survival of cancer patients
Two successful deliveries have been reported after vitrification (VT)[12] and two after VT followed by in vitro activation of the human ovarian cortex (IVA-OCTP)[13,14]
The ovaries were removed through small dorsolateral skin incisions and were placed either in Leibovitz L-15 medium (Lonza, Verviers, Belgium) supplemented with 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific, Gibco, Waltham, Mass., USA) (transport solution for slow freezing (SF)) or in Dulbecco’s phosphate-buffered solution (DPBS; Thermo Fisher Scientific, Gibco, Waltham, Mass., USA) supplemented with 20% FBS (transport solution for vitrification (VT))

Summary

Introduction

Considerable therapeutic advances have allowed notable improvements in the survival of cancer patients. Lee et al performed a comparison between SF and VT for human ovarian tissue cryopreservation and transplantation onto the back muscle of ovariectomized female SCID mice[25]. Their results indicate that SF is superior to VT in terms of follicle survival and growth after transplantation. While the open system enables extremely high cooling rates, the direct contact with LN2 increases potential cross-contamination risks[26,27] To address these safety considerations, closed systems were developed, first and mainly for oocyte and embryo cryopreservation[28,29], and are currently the only ones used in laboratories. The second aim was to analyse the resumption of ovarian and reproductive functions in vivo after cryopreservation and auto-transplantation of ovaries into mice with ovarian failure

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