Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after Cryologic solid surface vitrification or slow-rate freezing q Jan M.J. Aerts a, * , Janina B.P. De Clercq a , Silke Andries a , Jo L.M.R. Leroy a , Stefan Van Aelst b , Peter E.J. Bols a a Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Department of Veterinary Medicine, Laboratory of Veterinary Physiology, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium b Faculty of Sciences, Department of Applied Mathematics and Computer Science, Ghent University, Krijgslaan 281-S9, 9000 Gent, Belgium article info Article history: Received 9 June 2008 Accepted 29 July 2008 Available online 7 August 2008 Keywords: Cryopreservation Vitrification Mouse ovary Follicle survival Preantral follicles Transplantation abstract This study was designed to asses murine preantral follicle survival and growth, after cryopreservation of ovarian tissue by two different methodologies, solid-surface vitrification by the Cryologic vitrification method (CVM) and slow-rate freezing (SRF). Cryotreated tissue was stored in liquid nitrogen for 24 h, and upon warming follicle viability was assessed by live/dead fluorescent probes, and by 7-day autotransplantation of both cryotreated tissue types to the left and right kidney capsule of the donor animals (n = 16). The live/dead assay immediately upon tissue warming did not allow a distinction to be made in terms of follicle viability between the CVM and SRF cryoprocedure. In grafted tissue, follicular survival and growth was assessed by conventional histological examination and proliferating cell nuclear antigen immunohistochemistry. In each experimental group (control, CVM and SRF), follicles were classified according to developmental stage, and a comparison of the proportions of follicle stages between the three groups was executed by statistical analysis of variance. The fraction of primordial follicles in CVM and SRF grafts significantly decreased as compared to control tissue, whereas intermediary and pri- mary follicles significantly increased. The proportion of secondary and antral follicles after SRF was sig- nificantly larger than after CVM, but did not differ significantly between CVM and control tissue. The observed massive follicle activation is a typical transplantation effect, but testifies to the survival of cryopreserved follicles. In both types of cryotreated tissue, growing follicles, including antral stage, were present in grafts from all recipient animals. The significantly more abundant further developed stages in SRF treated tissue, however, suggest that CVM treated tissue may have suffered a growth disadvantage. To our knowledge, this is the first time that the CVM technique has been utilized to vitrify preantral follicles. Ó 2008 Elsevier Inc. All rights reserved. Introduction In recent years cryopreservation of ovarian cortical tissue has received a lot of attention, and murine as well as human ovarian tissue banking has become a conceivable prospect [36,46]. The ovarian cortex contains mostly primordial follicles, which repre- sent a resting stockpile of germ cells. Throughout reproductive life only a small fraction of these follicles produce oocytes competent to undergo successful maturation and ovulation. The rest of the oocytes (>99.9%) perish through a process called atresia. Cryopres- ervation of this follicular population, with the aim of future in vitro or in vivo development, is therefore an attractive concept to in- crease the yield of mature follicles for production of offspring, for research into early follicular dynamics, or for preservation of rare and endangered species. Several workers have investigated the feasibility of ovarian cor- tex cryopreservation by conventional slow-rate freezing (SRF) or by a variety of vitrification techniques. To determine the effective- ness of the cryotechnique or freezing protocol, or for comparison between different procedures, assessment of follicular survival after specimen warming is required. Follicle morphology of cooled–warmed ovarian cortex has been examined by histological analysis, and the developmental stages 0011-2240/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2008.07.011 q None of the authors of the manuscript received funding, grants, or in-kind support in support of the research or the preparation of the manuscript. None of the authors of the manuscript has an association or financial involvement (i.e. consultancies/advisory board, stock ownerships/options, equity interest, patents received or pending, royalties/honorary) with any organization or commercial entity having a financial interest in or financial conflict with the subject matter or research presented in the manuscript. On behalf of all co-authors, Jan Aerts, corresponding author of the manuscripts entitled: Follicle survival and growth to antral stages in short-term murine ovarian cortical transplants after vitrification by the Cryologic method or slow-rate freezing. * Corresponding author. Fax: +32 3 820 24 33. E-mail addresses: jan.aerts@ua.ac.be, jan_aerts@yahoo.com (J.M.J. Aerts). Cryobiology 57 (2008) 163–169 Contents lists available at ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo