Solid-surface vitrification is an appropriate and convenient method for cryopreservation of isolated rat follicles.
BACKGROUND
Cryopreservation of isolated follicles may be a potential option to restore fertility in young women with cancer, because it can prevent the risks of cancer transmission. Several freezing protocols are available, including slow-rate freezing, open-pulled straws vitrification (OPS) and solid-surface vitrification (SSV, a new freezing technique). The purpose of our study was to investigate the effects of these freezing procedures on viability, ultrastructure and developmental capacity of isolated rat follicles.


METHODS
Isolated follicles from female Sprague-Dawley rats were randomly assigned to SSV, OPS and slow-rate freezing groups for cryopreservation. Follicle viability assessment and ultrastructural examination were performed after thawing. In order to study the developmental capacity of thawed follicles, we performed in vitro culture with a three-dimensional (3D) system by alginate hydrogels.


RESULTS
Our results showed that the totally viable rate of follicles vitrified by SSV (64.76%) was slightly higher than that of the OPS group (62.38%) and significantly higher than that of the slow-rate freezing group (52.65%; P < 0.05). The ultrastructural examination revealed that morphological alterations were relatively low in the SSV group compared to the OPS and slow-rate freezing groups. After in vitro culture within a 3D system using alginate hydrogels, we found the highest increase (28.90 +/- 2.21 microm) in follicle diameter in follicles from the SSV group. The estradiol level in the SSV group was significantly higher than those in the OPS and slow-rate freezing groups at the end of a 72-hr culture period (P < 0.05).


CONCLUSIONS
Our results suggest that the SSV method is an appropriate and convenient method for cryopreservation of isolated rat follicles compared with the conventional slow-rate freezing method and the OPS method.
Xing
Weijie
W
Reproductive Medicine Center, the First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China.
Zhou
Canquan
C
Bian
Jiang
J
Montag
Markus
M
Xu
Yanwen
Y
Li
Yubin
Y
Li
Tao
T
D000464:Alginates / Q000494:pharmacology; D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival / Q000502:physiology; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods*; D005260:Female; D005615:Freezing; D020100:Hydrogels / Q000494:pharmacology; D006080:Ovarian Follicle / Q000166:cytology / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D010053:Ovary / Q000187:drug effects / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley
D000464:Alginates; D020100:Hydrogels
Anim Reprod Sci. 2000 Jul 2;60-61:357-64
10844207
J Reprod Dev. 2008 Jun;54(3):149-55
18296868
Cryobiology. 2008 Oct;57(2):137-41
18680737
Reprod Fertil Dev. 2007;19(2):383-91
17257525
Reprod Biomed Online. 2006 Aug;13(2):228-34
16895638
Mol Reprod Dev. 2004 Feb;67(2):186-92
14694434
Theriogenology. 2000 Sep 15;54(5):651-8
11101028
Acta Vet Scand. 1997;38(4):349-52
9503677
Diabetes. 1997 Jul;46(7):1120-3
9200645
Cell Tissue Res. 2007 Jan;327(1):167-76
16937112
Biol Reprod. 1996 May;54(5):1059-69
8722627
Fertil Steril. 1997 Mar;67(3):481-6
9091334
Reprod Domest Anim. 2008 Jun;43(3):360-366
18086252
Cryobiology. 2001 Mar;42(2):139-44
11448116
Cryobiology. 2002 Feb;44(1):5-13
12061843
Hum Reprod. 2006 Sep;21(9):2223-7
16720622
Fertil Steril. 2001 Mar;75(3):588-93
11239546
Biol Reprod. 2000 Aug;63(2):513-8
10906058
Nat Biotechnol. 1999 Dec;17(12):1234-6
10585728
Hum Reprod. 2009 Jan;24(1):92-9
18815120
Hum Reprod. 2006 Feb;21(2):413-20
16199426
Biomaterials. 2007 Oct;28(30):4439-48
17643486
Arch Med Res. 2001 Nov-Dec;32(6):587-94
11750734
J Reprod Dev. 2010 Feb;56(1):176-81
19815985
Theriogenology. 1997 Jan 15;47(2):501-9
16728002
Theriogenology. 2003 Apr 15;59(8):1839-50
12566156
Cryobiology. 1984 Aug;21(4):407-26
6467964
BMC Biotechnol. 2008 Apr 04;8:38
18394156