TITLE

OPTIMUM FOR CRYOPRESERVATION OF HUMAN OVARIAN TISSUE: GENE EXPRESSION AS A DETECTOR OF EFFECTIVENESS

AUTHOR(S)
Isachenko, V.; Lapidus, I.; Isachenko, E.; Hancke, K.; Nawroth, F.; Wiegratz, I.; Kreienberg, R.
PUB. DATE
October 2010
SOURCE
Reproductive BioMedicine Online (Reproductive Healthcare Limited;Oct2010 S3 Supplemen, Vol. 20, pS54
SOURCE TYPE
Academic Journal
DOC. TYPE
Article
ABSTRACT
Objective: Cryopreservation, the most important stage of the cryobanking of ovarian tissue, can be carried out using one of two methods: conventional ("slow") freezing, and vitrification (direct plunging into liquid nitrogen). For human oocytes vitrification is more effective compared to conventional freezing (Isachenko et al., 2005a,b; 2006a). However, these comparative data are limited for human ovarian tissue. It was previously shown that for human ovarian tissue the effectiveness of conventional freezing is higher compared to vitrification (Isachenko et al., 2007, 2009a, b). However, this question (what is better, traditional freezing or modern vitrification?) is topical and at present the respective discussions in the field of reproductive medicine are more than intensive. Particularly, the results of our comparative investigations (freezing vs vitrification) (Isachenko et al., 2009a) were agitated a criticism of some colleges, by opinion of which it is not correct to compare the protocol of vitrification developed by us (Isachenko et al., 2008) with standard conventional freezing. By opinion of some colleges, there are effective protocols of vitrification of human ovarian tissue. Taking into account this critical opinion, the aim of our investigations was the comparison of effectiveness of recently published and, by analyse of literature, most effective protocol of vitrification of human ovarian tissue (Keros et al., 2009c, traditionally experienced group from Sweden) and conventional freezing with our modifications (Isachenko et al., 2009a). Materials and Methods: Ovarian tissue from 5 patients was transported to the laboratory within 20 min at 32 to 34°C, divided into smaller pieces (1x1 to 1.5x0.7 to 1mm) and randomly distributed into three groups: Group 1: control (fresh tissue), Group 2: pieces after vitrification/warming, Group 3: pieces after conventional freezing/thawing. All pieces were cultured in vitro for 12 days (Isachenko et al., 2006b). The viability of the tissue was evaluated by the development of the follicles and GAPDH gene expression after in vitro culture. Total RNA preparation, RT, PCR, and real-time PCR were performed by the routine methodology using GenElute Total RNA purification-, DNase I treatment-, Superscript II reverse transcriptase-, PCR RedTaq Ready Master Mix- and SYBR Master Mix- Kits.
ACCESSION #
59358144

 

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