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Mol. Hum. Reprod. Advance Access originally published online on January 18, 2006
Molecular Human Reproduction 2005 11(12):843-846; doi:10.1093/molehr/gah243
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© The Author 2006. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes

C.C.W. Chan1, V.W.S. Liu, E.Y.L. Lau, W.S.B. Yeung, E.H.Y. Ng and P.C. Ho

Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Hong Kong SAR, China

1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, 102 Pokfulam Road, Hong Kong SAR, China. E-mail: cwcchan{at}graduate.hku.hk


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies analysing the incidences of mitochondrial DNA (mtDNA) deletions and mtDNA content in unfertilized oocytes in relation to donors’ age have been controversial. The objective of the study was to compare these two parameters in unfertilized oocytes and relate them to the donors’ age. Fifty-two women donated 155 unfertilized metaphase II (MII) oocytes. The incidence of 4977 bp deletion was 34.6%, and the mtDNA copy number was 598 350 ± 265 862. Women ≥35 years of age had a significantly higher incidence of 4977 bp deletion, lower mtDNA copy number, higher FSH level and poorer ovarian response when compared with younger women. The mtDNA copy number was negatively correlated with the donor’s age. The higher incidence of mtDNA deletion and lower mtDNA copy number in older women suggested that these two parameters may reflect ovarian ageing.

Key words: age/deletion/mitochondrial DNA copy number/unfertilized oocyte


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Mitochondria are maternally inherited organelles that use oxidative phosphorylation to supply ATP to the cell. Each mitochondrion carries its own multicopy genome, which is a circular, double-stranded DNA molecule with 16.6 kb (Anderson et al., 1981Go). Mitochondrial DNA (mtDNA) codes for 13 essential subunits of the respiratory chain complexes that provide the main ATP supply of the cell (Wallace, 1992Go). There is an increase in the rate of accumulation of somatic mtDNA mutations concomitant with a decrease in mitochondrial respiratory function during the aging process (Beckman and Ames, 1998Go; Wallace, 1999Go). Accumulation of mtDNA mutations in a specific tissue is inversely correlated to its replication potential and directly related to its metabolic state.

The 4977 bp deletion, also known as the common deletion, is one of the most frequently observed mtDNA mutations. This mutation accumulates with age in various human tissues and has been proposed as a marker of natural aging (Meissner et al., 1997Go; Meissner and von Wurmb, 1998Go; von Wurmb et al., 1998Go). Although Keefe et al. (1995)Go suggested that the common deletion is positively correlated with patients’ age and may, therefore, serve as a marker of oocyte senescence, other studies have failed to confirm this observation (Chen et al., 1995Go; Brenner et al., 1998Go; Barritt et al., 1999Go; Hsieh et al., 2002Go).

The total mitochondrial mass determines the functional competency of the cell, and it is highly correlated with the total mtDNA copy number (Wu et al., 2002Go). Using various methods, the mtDNA content of unfertilized human oocytes has been quantified (Chen et al., 1995Go; Reynier et al., 2001Go; Barritt et al., 2002Go; May-Panloup et al., 2005Go). None of these studies related the presence of mtDNA deletion to the mtDNA content.

In this study, we investigated the incidence of 4977 bp deletion and the total mtDNA copy number in a group of unfertilized oocytes and related the incidence of the deletion to the mtDNA content and the donors’ age.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Human oocyte collection
The study was approved by the Institutional Review Board of the hospital. Written consents were obtained from all women donating their oocytes for research. Oocytes were obtained from women undergoing IVF treatment at Queen Mary Hospital. The long protocol for pituitary down-regulation, ovarian stimulation and oocyte retrieval used in this study has been published previously (Chan et al., 2005Go). Unfertilized oocytes were obtained from patients undergoing IVF treatment, but the oocytes were unfertilized after conventional insemination or ICSI. Oocytes were considered to be unfertilized if no pronucleus or second polar body could be seen 48 h following insemination.

Only oocytes at metaphase II (MII) were subjected to analysis. To ensure uniformity of laboratory conditions and eliminate the possibility of contamination by sperm mtDNA, the zona pellucida of all oocytes were removed using acidified Tyrode’s solution. Each oocyte was individually washed in phosphate-buffered saline (PBS), then transferred to a labelled Eppendorf tube and frozen in liquid nitrogen until analysis.

DNA extraction
The oocytes were thawed at room temperature and then incubated in 10 µl lysis buffer (50 mM Tris–HCl, pH 8.5, 0.1 mM EDTA, 0.5% Tween-20, 200 µg/ml proteinase K) at 55°C for 2 h. Finally, proteinase K was heat inactivated at 95°C for 10 min.

PCR
The presence of mtDNA in a single human oocyte was confirmed by standard PCR with a set of primers (forward primer L467 and reverse primer H725) for amplifying a fragment outside the hotspot of deletion area. Each PCR mixture was made to a final volume of 20 µl containing 5 pmol of each primer, 0.2 mM of dNTP, 0.5 U of Taq polymerase and 2.5 mM magnesium chloride. The initial denaturation step was performed at 94°C for 2 min. This was followed by 45 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, elongation at 72°C for 30 s and a final extension at 72°C for 2 min. Negative control was performed by substituting the oocyte DNA sample with an equivalent amount of water in the PCR mixture. A positive and a negative control were included in each run.

Nested PCR
The nested PCR was employed to detect the presence of 4977 bp deletion. Two pairs of primers used (outer primers L8282 + H13650 [GenBank] and inner primers L8334 + H13578 [GenBank] ) flanked the region of the 4977 bp deletion site. If the deletion was present, the nested PCR product was 268 bp long. In the absence of the deletion, there was no DNA amplification (Figure 1). The PCR mixture was the same as described. The following amplification profile was used: 1 cycle at 94°C for 2 min; 30 cycles at 94°C for 30 s, 50°C for 30 s, 72°C for 30 s and 1 cycle at 72°C for 2 min. All experiments included a water-only negative control and a positive control with the 4977 bp deletion. The PCR products were separated on 2% agarose gel stained with ethidium bromide. PCR products were confirmed by DNA sequencing.


Figure 1
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  		Figure 1. Nested PCR to detect the presence of {Delta}mtDNA4977 in mtDNA. The PCR primers used for amplification are actually over 5 kb apart as indicated by the numbers (nucleotide positions of the 5" end of the primers) shown in the figure, and the elongation time is only 30 s. (A) Therefore, no PCR product could be generated in the absence of the deletion because the product would be too long (>5 kb) to be amplified under such condition. (B) In the presence of {Delta}mtDNA4977, a PCR product would be generated because the 4977 bp deletion brought the primers closer to each other, and the size of the product is only 268 bp long.

 

Real-time quantitative PCR
Real-time quantitative PCR (qPCR) was performed using the ABI PRISM 7700 Sequence Detector (Perkin-Elmer/Applied Biosystems, Foster City, CA, USA). The conditions used were as described in Wang et al. (2005)Go with modifications. Briefly, 1 µl of DNA sample was added to a final 10 µl reaction mixture containing 1 x PCR buffer, 5 mM magnesium chloride, 0.2 mM of dNTP, 5 pmol of each primer, 1 pmol of dually labelled (5" Fam and 3" Tamra) TaqMan probe and 0.5 U Taq polymerase. The following quantification cycling protocol was used: 95°C for 4 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 50°C for 15 s and elongation at 72°C for 15 s. The measurement of mtDNA copy number was performed in duplicates for each sample in separate qPCR runs. Recombinant plasmid containing mtDNA fragment [nucleotide position from 3182 to 4216, spanning from 16S rRNA, tRNA (Leu) and ND1 genes coding region] was used as standard DNA. No mtDNA deletion was reported before in this particular segment of mtDNA. This makes the segment suitable to be used for measuring mtDNA copy number in DNA samples. The interrun coefficient of variations of standard DNA in seven separate runs were less than 5%, which indicated an acceptable degree of precision (Wang et al., 2005Go). Figure 2 shows a typical example of our qPCR with good linearity.


Figure 2
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  		Figure 2. Real-time quantitative PCR to show the amplification curve and regression curve for the measurement of mtDNA copy number in oocyte. (A) Amplification plots obtained from mtDNA standard at six orders of magnitude (from 103 to 108 copies) range. Fluorescence signal was measured at the end of each elongation step and was plotted against the cycle number. (B) Log-linear regression curve was then obtained from threshold cycle number against the mtDNA standards with copy number ranging from 103 to 108. The correlation coefficient is 0.997 in this particular run. The mtDNA copy number in a sample could then be determined from the linear regression line.

 

Statistical analysis
The primary outcome was the percentage of oocytes with the 4977 bp deletion in the unfertilized oocytes. Categorical and continuous data were compared using chi-square test and Student’s t-test, respectively. A two-sided P value of <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
One hundred and fifty-five unfertilized MII oocytes were donated for research from 52 patients. The mean age of the donors was 32.7 ± 4.2 years (range 25–42 years). The mean duration of subfertility was 4.4 ± 2.1 years. The causes of subfertility are listed below: male factor (28), tuboperitoneal factor (14), endometriosis (6) and unexplained cause (4). The overall fertilization rate was 57 ± 20%. In 19 (36.5%) patients, the oocytes were fertilized with ICSI while in 33 (65.5%) patients the oocytes were fertilized with conventional insemination. The fertilization rate was similar between those fertilized by ICSI (58 ± 25%) and by conventional insemination (56 ± 18%) (P = 0.550). The incidence of 4977 bp deletion was 34.6%, and the mtDNA copy number was 598 350 ± 265 862. Women ≥35 years of age had a significantly higher incidence of 4977 bp deletion and a lower mtDNA copy number when compared with younger women (Table I). Women in the younger age group had significantly lower early follicular phase FSH level, higher estradiol (E2) on day of HCG, required less HMG dose and had more oocytes retrieved. When logistic regression was applied, age was the only significant predictor of the presence of the 4977 bp deletion, while mtDNA copy number, early follicular phase FSH and smoking were not (Table II). The mtDNA copy number was also negatively correlated with the donors’ age (Pearson’s correlation coefficient = –0.185, P = 0.041).


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  		Table I. Comparison of various parameters in donors of unfertilized oocytes according to their age

 

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  		Table II. Logistic regression table showing the various factors in predicting the presence of {Delta}mtDNA4977 in unfertilized oocytes

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Previous studies have demonstrated that the incidence of 4977 bp deletion in unfertilized oocytes varied from 32 to 66% with various PCR strategies. In our study, using nested PCR, the incidence of 4977 bp deletion in unfertilized MII oocytes was 34.6%, in accordance with the reported range. In contrast to most of the previous studies, we have shown that the incidence of 4977 bp deletion was significantly higher in older women. In fact, an association between donors’ age and the deletion has been described in the initial study by Keefe et al. (1995)Go. This observation is in line with the hypothesis that there is an age-related accumulation of mtDNA rearrangements in human oocytes. Some previous studies that failed to show such an association did not restrict their analysis to MII oocytes (Barritt et al., 1999Go; Hsieh et al., 2002Go). It has been shown that the incidences of mtDNA rearrangements are inversely related to oocyte development, and hence, the incidences were highest in germinal vesicle, followed by MI and MII oocytes (Barritt et al., 1999Go), representing a possible selection process such that immature oocytes with mitochondrial deletion could not develop further. Analysing mtDNA deletion in a mixture of oocytes in different stages of development, the incidence in the MII oocytes would be over-estimated. If most of the donors were young, the age-related effect would not be apparent. This helps to explain in part the different observation in the incidence of 4977 bp deletion in relation to donors’ age.

The absolute number of mtDNA in unfertilized human oocytes varied from 50 000 to 1 550 000 (Chen et al., 1995Go; Reynier et al., 2001Go; Barritt et al., 2002Go; May-Panloup et al., 2005Go). The total mtDNA copy number of our samples fell within the reported range. In this study, we showed a negative correlation between the mtDNA content and the donors’ age. This contrasted with the findings from Barritt et al. (2002)Go. However, another group of investigators also reported lower mtDNA content in women with high FSH who were also older (May-Panloup et al., 2005Go). The explanation for the discrepancy is not clear. Nevertheless, we believe that our findings are genuine, as we also showed a lower mtDNA copy number in women who were older than 35 years of age.

In conclusion, the higher incidence of mtDNA deletion and lower mtDNA copy number in older women suggests that these two parameters may reflect ovarian aging.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
This study was supported by the Hong Kong Obstetrical and Gynaecological Trust Fund.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F et al. (1981) Sequence and organization of the human mitochondrial genome. Nature 290,457–465.[CrossRef][Medline]

Barritt JA, Brenner CA, Cohen J and Matt DW (1999) Mitochondrial DNA rearrangements in human oocytes and embryos. Mol Hum Reprod 5,927–933.[Abstract/Free Full Text]

Barritt JA, Kokot M, Cohen J, Steuerwald N and Brenner CA (2002) Quantification of human ooplasmic mitochondria. Reprod Biomed Online 4,243–247.[Medline]

Beckman KB and Ames BN (1998) Mitochondrial aging: open questions. Ann N Y Acad Sci 854,118–127.[CrossRef][Web of Science][Medline]

Brenner CA, Wolny YM, Barritt JA, Matt DW, Munne S and Cohen J (1998) Mitochondrial DNA deletion in human oocytes and embryos. Mol Hum Reprod 4,887–892.[Abstract/Free Full Text]

Chan CCW, Ng EHY, Tang OS, Yeung WSB, Lau EYL and Ho PC (2005) A prospective randomized double-blind study to compare two doses of recombinant human chorionic gonadotrophin in inducing final oocyte maturity and the hormonal profile during the luteal phase. J Clin Endocrinol Metab 90,3933–3938.[Abstract/Free Full Text]

Chen X, Prosser R, Simonetti S, Sadlock J, Jagiello G and Schon EA (1995) Rearranged mitochondrial genomes are present in human oocytes. Am J Hum Genet 57,239–247.[Web of Science][Medline]

Hsieh RH, Tsai NM, Au HK, Chang SJ, Wei YH and Tzeng CR (2002) Multiple rearrangements of mitochondrial DNA in unfertilized human oocytes. Fertil Steril 77,1012–1017.[CrossRef][Web of Science][Medline]

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May-Panloup P, Chretien MF, Jacques C, Vasseur C, Malthiery Y and Reynier P (2005) Low oocyte mitochondrial DNA content in ovarian insufficiency. Hum Reprod 20,593–597.[Abstract/Free Full Text]

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Reynier P, May-Panloup P, Chretien MF, Morgan CJ, Jean M, Savagner F, Barriere P and Malthiery Y (2001) Mitochondrial DNA content affects the fertilizability of human oocytes. Mol Hum Reprod 7,425–429.[Abstract/Free Full Text]

von Wurmb N, Oehmichen M and Meissner C (1998) Demonstration of the 4977 bp deletion in human mitochondrial DNA from intravital and postmortem blood. Mutat Res 422,247–254.[Medline]

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Submitted on August 13, 2005; revised on October 20, 2005; accepted on October 26, 2005


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