Genexpression und Morphologie der Follikel nach konventionellem Einfrieren und Vitrifikation von humanem Ovarialgewebe

 

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Zusammenfassung

Fragestellung: Bei der Kryokonservierung, dem wichtigsten Verfahren für die Lagerung von Ovarialgewebe, können 2 Methoden unterschieden werden: das konventionelle („langsame“) Einfrieren und die Vitrifikation (direktes Eintauchen in flüssigen Stickstoff). In früheren Studien wurde gezeigt, dass die Effektivität der Vitrifikation im Vergleich zum konventionellen Einfrieren von menschlichen Eizellen und Embryonen höher ist. Allerdings sind die Erfahrungen mit beiden Methoden für humanes Ovarialgewebe begrenzt. Das Ziel dieser Studie war daher der Vergleich des konventionellen Einfrierens mit der Vitrifikation bei menschlichem Ovarialgewebe. Material und Methodik: Ovarialgewebefragmente von 5 Patientinnen wurden innerhalb von 20 Minuten bei 32 bis 34 °C in das Labor transportiert. Die Fragmente wurde in kleinere Stücke aufgeteilt (1 × 1 bis 1,5 × 0,7 bis 1 mm) und zufällig in die 3 folgenden Gruppen verteilt: Gruppe 1: Kontrollgruppe (natives Gewebe), Gruppe 2: Vitrifikation/Erwärmung, Gruppe 3: konventionelles Einfrieren/Auftauen. Alle Fragmente wurden in vitro für 12 Tage kultiviert. Die Lebensfähigkeit des Gewebes wurde durch die Entwicklung der Follikel und die GAPDH-Genexpression nach der Kultivierung bewertet. Ergebnisse: Für die Gruppen 1, 2 und 3 waren 93, 74 und 78 % der Follikel morphologisch normal. Die molekularbiologische Analyse zeigte jedoch, dass die Intensität der GAPDH-Genexpression im Gewebe nach konventionellem Einfrieren im Gegensatz zur Vitrifikation stark erhöht war. Schlussfolgerung: Somit ist anzunehmen, dass für die Kryokonservierung von humanem Ovarialgewebe das konventionelle Einfrieren besser geeignet ist als die Vitrifikation.

Abstract

Purpose: 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 and immediate immersion into liquid nitrogen). For human oocytes and embryos vitrification is more effective compared to conventional freezing. However, these comparative data are limited for human ovarian tissue. The aim of this study was to compare conventional freezing of ovarian tissue with vitrification. Material and Methods: Ovarian tissue from 5 patients was transported to the laboratory within 20 min at 32 to 34 °C, divided into smaller pieces (1 × 1 to 1,5 × 0,7 to 1 mm) 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. The viability of the tissue was evaluated by the development of the follicles and GAPDH gene expression after in vitro culture. Results: 93, 74 and 78 % of the follicles of groups 1, 2 or 3 were morphologically normal. Molecular analysis showed that the intensity of GAPDH gene expression in the tissue after conventional freezing was greatly increased compared to after vitrification. Conclusion: It was concluded that for the cryopreservation of human ovarian tissue conventional freezing is more suitable than vitrification.

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Vladimir Isachenko

Universitätsfrauenklinik Ulm

Prittwitzstraße 43

89075 Ulm

Email: v.isachenko@yahoo.com