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Abstract
Objective
Many clinical trials have proven the close relationship between the loss of human mitochondrial DNA and aging process. The purpose of this study was to evaluate the different types of mitochondrial DNA deletion and its frequency in luteinized granulosa cells in different aged groups of women undergoing in vitro fertilization (IVF).
Methods
The ovum pick up was done in 51 women who participated in Konyang University IVF program, and mitochondrial DNAs extracted from luteinized granulosa cells, were screened to search for different types of deletion and its frequency. The deleted mitochondrial DNA were analyzed by polymerase chain reaction method. DNA sequencing was performed to reveal exact deletion point.
Results
Three different types of deletions (4,977 bp, 7,150 bp, and 5,777 bp) were confirmed. To find the difference between the aged groups, we have divided women into groups younger than 32 years, between 32 to 37 years, and older than 37 years. The deletion of 4,977 bp was 60.9% (14/23) in younger than 32 years, 46.2% (6/13) in 32 to 37 years, 46.7% (7/15) in older than 37 years. There was no statistical significance between aged groups and the incidence of the deletion. The deletion of 7150 bp was 34.8% (8/23), in younger than 32 years, 30.8% (4/13) in 32 to 37 years, 40% (6/15) in older than 37 years. We investigated relationship between the frequency of deletion and the aging, but there was no statistical significance. In case of 5,777 bp, the deletion was 43.5% (10/23) in younger than 32 years, 30.8% (4/13) in 32 to 37 years, 53.3% (8/15) in older than 37 years. It showed no statistical significance as well as other types.
Conclusion
In this study we have found three different types of deletion of mitochondrial DNA obtained from luteinized granulosa cells in women with infertility. There were no significant differnces of each type of deletion in 3 different aged groups of infantile women. The limitation of this study is that the analyze were done qualitatively. If we could provide the quantitative analyze it could be applied clinically.
Figures and Tables
Figure 1
The existence of a sufficient amount of mitochondrial DNA for the PCR reaction. Mitochondrial DNA of cells in follicular fluid was subjected to PCR using primer L625 and H726. Amplified 1,030 bp bands that indicated similar density were detected in all samples. Marker, DNA molecular weight marker.
*Group A: women under 32 years of age.
†Group B: women between 32 and 37 years of age.
‡Group C: women over 37 years of age.
Figure 2
Detection of deleted mitochondrial DNA in human follicular fluid cells by PCR using primer L729 and H1390. Mitochondrial DNA of three groups was sujected to PCR, separated on a 1% agarose gel, and stained with ethidium bromide. Amplification of the 6.6-kb region resulted in a 0.77-kb band (arrow A) with 5-kb deletion of mitochondrial DNA. Marker, DNA molecular weight marker.
*Group A: women under 32 years of age.
†Group B: women between 32 and 37 years of age.
‡Group C: women over 37 years of age.
Figure 3
Detection of deleted mitochondrial DNA in human follicular fluid cells by polymerase chain reaction (PCR) using primer L820, H1619 and nested-PCR using primer L853, H1587, L820 and H1436. mitochondrial DNA of three groups was subjected to PCR, separated on a 1% agarose gel, and stained with ethidium bromide. Amplification of the 6.2 kb region resulted in a 0.4 kb band (arrow B) with 5.8 kb deletion of mitochondrial DNA. And amplification of the 7.4 kb region resulted in a 0.22 kb band (arrow C) with 7.2 kb deletion of mitochondrial DNA. Marker, DNA molecular weight marker.
*Group A: women under 32 years of age.
†Group B: women between 32 and 37 years of age.
‡Group C: women over 37 years of age.
Table 1
Synthesized primers used for polymerase chain reaction, cloning and sequencing
*Primers L625, L729, L790, L820 and L853 were used for amplification of the light strand of mitochondrial DNA. And primers H726, H1363, H1390, H1436, H1587, and H1619 were used for amplification of the heavy strand of mitochondrial DNA.
†Numbering of mitochondrial DNA is according to that of Anderson et al27.
Table 2
Primers, sequences and length of amplified product of wild type and deleted mitochondrial DNA
Table 3
Nucleotide sequences of the deletion breakpoint regions of mitochondrial DNA
Direct repeat sequences are underlined. The numbering of nucleotides is according to that of Anderson et al.27 The breakpoints are indicated by parentheses and the nucleotides shown inside the parentheses (indicated by italics) were deleted.
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