Effects of Oxygen Free Radical on the In-vitro Development of Human Embryos

Hwang, Kyung Joo; Yoo, Jung Hyun; Kwon, Hyuck Chan; Yang, Hyun Won; Ryu, Hee Sug; Chang, Ki Hong; Sohn, Sung Hyang; Oh, Kie Suk

Abstract

INTRODUCTION: In human IVF-ET only a few oocytes develop to be good quality embryos depending on the incubation conditions and the quality of ovum and sperm, while the rest show abnormal morphology due to unequal cell division or fragmentation of the cell. Fragmented embryos limit developmental potential and rarely result in implantation. Such abnormal embryo development have been reported to be due to genetic defects or inadequate culture environment. This in-vitro environment differs from in-vivo conditions in that the oxygen concentration is higher(20 %) and in such conditions, the mouse embryos show a higher ROS in simple culture media. ROS is believed to cause damage to the cell membrane and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of ROS within embryos and morphological features of cell damage induced by ROS.
MATERIALS AND METHODS
A total of 53 human oocytes and embryos(31 fragmented and 10 nonfragmented embryos, 12 unfertilized oocytes) were obtained in IVF-ET programs. The relative intensity of ROS within embryos was reassured using 2,7-dichlorodihydrofluorescein diacetate (DCHFDA ; Molecular Probes, USA) by Quanti cell 500(Imaging Co. UK), and DNA fragmentation was observed via TEM(Zeiss EM 902A, Germany) and ApopTag kit(Oncor Co. USA).
RESULTS
The ROS levels was significantly higher in fragmented embryos(72.21 +/- 9.62) compared to nonfragmented embryos(31.30 +/- 3.50, p<0.05) and unfertilized oocytes(30.75 +/- 2.67, p<0.05). Apoptosis was only observed in fragmented embryos, and was absent in nonfragmented embryos. Also the DAPI staining showed that the nuclei of fragmented embryos were broken up into irregular shapes, and the EM showed confirmation of apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres.
CONCLUSION
We conclude that there is a direct relationship between increased ROS and apoptosis, and that further studies should be undertaken.