Arch Gynecol Obstet (2010) 282:695–705 DOI 10.1007/s00404-010-1623-x
REPRODUCTIVE MEDICINE
Nucleotide variations in mitochondrial DNA and supra-physiological ROS levels in cytogenetically normal cases of premature ovarian insufficiency Manoj Kumar • Dhananjay Pathak • Alka Kriplani • A. C. Ammini • Pankaj Talwar Rima Dada
•
Received: 6 June 2010 / Accepted: 20 July 2010 / Published online: 1 August 2010 Ó Springer-Verlag 2010
Abstract Premature ovarian insufficiency (POI) is defined as the cessation of ovarian function under the age of 40 years and is characterized by amenorrhea, hypoestrogenism, and elevated serum gonadotrophin concentration (FSH). It is a heterogeneous disorder with a multicausal pathogenesis; however, majority of cases are idiopathic. In idiopathic POI, involvement of unknown mechanisms may increase rate of oocyte apoptosis. Studies have shown that elevated reactive oxygen species (ROS) levels affect the quality of gametes. Mitochondrial mutations in different complexes of electron transport chain have been reported to disrupt the electron flow which lead to formation of more superoxide ions or increased levels of ROS. This study was aimed to screen the mitochondrial genome for variations in idiopathic POI (n = 25) and occult ovarian insufficiency (OI) (n = 5) patients. 30 patients diagnosed with POI and occult OI were enrolled in this study. Blood samples were collected from the patients and controls. DNA was extracted using phenol chloroform M. Kumar D. Pathak R. Dada (&) Laboratory for Molecular Reproduction and Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi 110029, India e-mail:
[email protected] A. Kriplani Department of Obstetrics and Gynaecology, All India Institute of Medical Sciences, New Delhi 110029, India A. C. Ammini Department of Endocrinology and Metabolism, All India Institute of Medical Sciences, New Delhi 110029, India P. Talwar Assisted Reproduction Technology Centre, Army Hospital Research and Referral, Delhi Cantonment, Delhi 110010, India
method. A total of 102 nucleotide variations were observed in patients as compared with 58 nucleotide variations in controls. 24% variations were found to be non-synonymous and 76% were synonymous. It was found that 48% variations were in complex I, 8% in complex III, 24% in complex IV, and 20% in complex V of electron transport chain. We found most of the non-synonymous mitochondrial variations in complex I (48%) of the respiratory chain which is the largest enzyme complex and is associated with oxidative stress. Some non-synonymous pathogenic alterations (p.M31T, p.W239C, p.L128Q) and non pathogenic alterations (ATPase6:p.T53I, ATPase6:p.L190F, ATPase6:p.L199L) were found to be significantly higher in cases as compared with controls. The preliminary data suggest that the mitochondrial mutations and subsequent decline in ATP levels may accelerate follicular atresia and lead to POI. The results of this preliminary study highlight the need to extend this study by analyzing large number of samples in different ethnic populations and analyze for ROS levels and mitochondrial mutations in oocytes as they are of different embryonic origin and develop in a different microenvironment. Keywords Premature ovarian insufficiency Amenorrhea Oxidative stress Mitochondrial mutations ETC Premature ovarian failure
Introduction Premature ovarian insufficiency (POI) or premature menopause is a condition characterized by amenorrhea, hypoestrogenism, and elevated follicle-stimulating hormone in women aged below 40 years. It is a common condition occurring in 1% of all women, and in 0.1% of women aged
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below 30 years [1]. POI is a heterogeneous disorder with a multifactorial pathogenesis such as chromosomal, genetic, mitochondrial aberrations, enzymatic, iatrogenic, immunological, or infections [2, 3]. These factors may influence the ovary at any stage of life, including prepubertal, pubertal, or reproductive stages [4]. But majority of POI cases are idiopathic, and the underlying mechanisms are largely unknown. In the embryo, germ cells migrate from the urogenital ridge to the primitive ovary where they proliferate to form 3.5 9 106 oocytes in each ovary by about 20 weeks of intrauterine life. But as compared with number of oocytes formed in the ovaries, only \500 oocytes are released in the entire reproductive life span of a woman which means that most of these germ cells are destroyed through apoptosis [5, 6]. Either decreased number of follicles (formed during ovarian development) or an increased rate of follicle loss may result in POI. In idiopathic POI, there may be involvement of several mechanisms affecting the rate of oogenesis and oocyte apoptosis or leading to accelerated apoptosis of germ cells [7]. Recent study by Venkatesh et al. [3] reported increased nucleotide variations in ATPase6 gene and increased reactive oxygen species (ROS) levels in POI. Thus, this study planned to analyze the whole mitochondrial genome for nucleotide variations in cytogenetically normal cases of POI and occult OI. Oxidative stress (OS) is underlying mechanism in several female reproductive disorders like endometriosis, polycystic ovaries, and POI [3, 8]. OS induces lipid peroxidation, structurally and functionally alters protein and DNA, and also promotes apoptosis [9, 10]. OS occurs when the generation of ROS and other free radical species exceeds the antioxidant defense mechanism. Baisong Lu et al. [11] suggested that the high superoxide ion levels lead to decrease in the bioavailability of nitric oxide and increase in ROS levels and oxidative stress, which provide a novel link between mitochondrial dysfunction and infertility. Another study [12] suggested that increased production of ROS contributes to oophoritis associated with POI. In living cells the main sources of ROS are mitochondrial respiratory chain and lymphocytes, and external factors like smoking, air pollution, exposure to xenobiotics, and electromagnetic radiation [13]. It was also reported that increased level of ROS produce increased mitochondrial DNA nucleotide variations [14] and also cause nuclear DNA damage, but mitochondrial DNA damage persists longer and is more extensive than nuclear DNA damage. Mitochondrial dysfunction has been associated with a wide range of human conditions including atherosclerosis and cardiovascular disease [15], insulin resistance, age-related neurodegenerative diseases [16], human aging [17], and male infertility [10].
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Mitochondrial alterations in different complexes of electron transport chain have been reported to disrupt the electron flow which leads to formation of more superoxide ions and thus increased levels of ROS and low ATP levels [3, 14]. Various studies have shown that oxidative stress affects the quality of gametes. As compared with spermatozoa, female germ cells develop under hypoxic condition in the ovarian cortex; however, exposure to supraphysiological levels of ROS are detrimental to developing oogonia [3]. The control of ovarian stromal cells and germ cell function is a diverse paradigm and increased ROS levels may be one of the modulators of ovarian germ cell and stromal cell physiology [18]. Oxidative stress may lead to a decreased complement of oocytes in the ovaries at birth or their accelerated atresia. So, it can be hypothesized that POI occurs either due to accelerated follicular atresia or impaired differentiation of follicles due to mitochondrial nucleotide alterations which lead to ATP depletion and raised ROS levels. Mitochondria are the power house of the cell and are both source and target of free radicals. Oocytes contain highest number of mitochondrial DNA copies and thus it is possible that supraphysiological ROS levels induce mitochondrial DNA damage, and mitochondrial DNA with nucleotide alterations further produce more free radicals by Ozawa effect [14]. The role of mitochondrial nucleotide variations and subsequently oxidative stress in female infertility deserves further investigation, and larger studies on different ethnic populations are warranted. In a previous study we reported the role of mitochondrial variation in a single mitochondrial gene ATPase6 and oxidative stress in POI [3]. To further establish a strong correlation between mitochondrial nucleotide variations and POI, in this study we analyzed the complete mitochondrial genome in cytogenetically normal idiopathic cases of POI and occult OI patients. In cases with elevated ROS levels and mitochondrial nucleotide variations, early diagnosis and prompt antioxidant administration may prevent or delay germ cell apoptosis and prevent premature cessation of ovarian function.
Materials and methods Clinical examination and selection of cases After approval from the institutional review board (IRB#IRB00006862; All India Institute of Medical Sciences, Delhi, India), 30 cases (25 POI and 5 occult OI) referred for cytogenetic and molecular analysis from Department of Obstetrics and Gynaecology, Endocrinology (AIIMS, New Delhi, India) and Army Research and
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Referral hospital (New Delhi), were enrolled in this study. Patients were eligible for inclusion only if they met standard clinical criteria for POI. POI was defined as the absence of menstruation along with elevated FSH levels ([40 mIU/ml) before 35 years of age. The patients were enrolled if they were not on any drugs or did not have any history of recent infection or inflammation and were nonsmokers and also had normal weight. Subject’s height, weight, age, complete gynecological, occupational, medical, and family history were collected from each patient. Physical and clinical examination was done to identify secondary sexual characteristics or any syndromic features. 30 ethnically and age-matched fertile females with normal menstrual cycle and no other medical problem were enrolled as controls. Peripheral blood sample (5 ml) was collected from patients and controls after informed consent. Sample collection and DNA isolation Only cases with idiopathic POI and occult OI were enrolled for this study. These cases were confirmed to be cytogenetically normal. 5 ml of peripheral blood samples were collected in EDTA tubes from all participating individuals after obtaining their written consent. DNA was extracted from whole blood samples of all POI patients and controls using phenol chloroform method. ROS measurement One milliliter blood was used for measurement of ROS. Blood taken from patient/control was centrifuged at 3009g for 7 min and the plasma was removed and transferred into a microcentrifuge tube. To 400 ll of 5 M luminol (5-amino-2,3-dihydro-1,4-phthalazinedione: sigma) was added to the mixture and served as a probe. ROS levels were assessed by measuring the luminol-dependant chemiluminescence with the luminometer (Sirus, Berthold) for 15 min. The results were expressed in relative light unit per minute (RLU/min) per 400 ll of blood plasma. PCR amplification, sequence, and analysis of the mitochondrial DNA coding region The entire coding region of the mitochondrial genome was amplified in all patients and controls using 24 separate sets of primers [19]. Primers were used to amplify the entire coding region of the mitochondrial genome. Complete mtDNA genome was sequenced except for the D-loop as it is a hyper-variable region. All fragments were sequenced in both forward and reverse directions for confirmation of any detected variant. The mitochondrial DNA sequence of patients and controls were aligned with the revised Cambridge sequence (rCRS) (NC_012920). All the mismatched
697
nucleotide sequences from patients were noted carefully and compared with controls. All these variations were searched in the Human mitochondrial genome database such as Mitomap (http://www.mitomap.org), mtDB (http://www.genpat.uu.se/mtDB/) and Ensembl Database (http://www.ensembl.org/) for their significance. Computational assessment of missense mutations Two homology-based programs polymorphism phenotyping (Polyphen) available at http://genetics.bwh.harvard. edu/pph/ and sorting intolerant from tolerant (SIFT) analysis tool available at http://sift.jcvi.org/ were used to predict the functional impact of missense changes identified in this study. Polyphen structurally analyzes an amino acid polymorphism and predicts whether that amino acid change is likely to be deleterious to protein function [20, 21]. Polyphen scores of [2.0 indicate the polymorphism is probably damaging to protein function. Scores of 1.5–2.0 are possibly damaging, and scores of \1.5 are likely benign. SIFT is a sequence homology-based tool that sorts intolerant from tolerant amino acid substitutions and predicts whether an amino acid substitution in a protein will have a phenotypic effect [22]. SIFT is based on the premise that protein evolution is correlated with protein function. Positions with normalized probabilities\0.05 are predicted to be deleterious, and those C0.05 are predicted to be tolerated in case of SIFT. The substitution may occur at a specific site, e.g. Active or binding, or in a non-globular, e.g., transmembrane region. Polyphen uses the predicted hydrophobic and transmembrane (PHAT) matrix score to evaluate possible functional effect of a substitution in the transmembrane region. At this step, Polyphen memorises all positions that are annotated in the query protein as BINDING, ACT_SITE, LIPID, or METAL. At a later stage, if the search for a homologous protein with known 3D structure is successful, it is checked whether the substitution site is in spatial contact with these critical residues. Positions important for function should be conserved in an alignment of the protein family, whereas unimportant positions should appear diverse in an alignment.
Results A total of 102 mitochondrial nucleotide variations were observed in patients as compared with 58 nucleotide variations in controls. Out of 102 variations, 78 were found in POI and 24 in patients of occult ovarian insufficiency. Thus, we found increased number of mitochondrial sequence variations in patients as compared with controls. Table 1 displays all mtDNA sequence variants (n = 102) detected in patients.
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Table 1 Mitochondrial variations in patients S. no.
Complex
Nucleotide change
Locus
Codon change
Amino acid change
Type of mutation
Polyphen/ SIFT score
Pathogenic (yes/no)
Accession number
Frequency of variation
1
I
3398T[C
ND1
ATA[ACA
p.M31T
NS
2.231/0.00
Yes
Reported
12/30
2
I
3438G[A
ND1
GGG[GGA
p.G44G
SYN
NA
NA
Reported
2/30
3
I
3507C[G
ND1
ACC[ACG
p.T67T
SYN
NA
NA
GU397546
1/30
4
I
3741C[T
ND1
ACC[ACT
p.T145T
SYN
NA
NA
Reported
3/30
5
I
3834G[A
ND1
CTG[CTA
p.L176L
SYN
NA
NA
Reported
2/30
6
I
*3970C[T
ND1
CTA[TTA
p.L222L
SYN
NA
NA
Reported
1/30
7 8
I I
4113G[A *4852T[A
ND1 ND2
CTG[CTA CTG[CAG
p.L269L p.L128Q
SYN NS
NA 1.95/0.00
NA Yes
Reported GU397533
1/30 7/30
9
I
4883C[T
ND2
CCC[CCT
p.P138P
SYN
NA
NA
Reported
1/30
10
I
4916A[G
ND2
CTA[CTG
p.L149L
SYN
NA
NA
Reported
2/30
11
I
4944A[G
ND2
ATC[GTC
p.I159V
NS
0.468/0.29
No
Reported
1/30
12
I
5004T[C
ND2
TTA[CTA
p.L179L
SYN
NA
NA
Reported
1/30
13
I
5178C[A
ND2
CTA[ATA
p.L237I
NS
0.147/0.11
No
Reported
1/30
14
I
*5186A[T
ND2
TGA[TGT
p.W239C
NS
1.982/0.00
Yes
Reported
9/30
15
I
10142C[T
ND3
AAC[AAT
p.N28N
SYN
NA
NA
Reported
2/30
16
I
10188A[G
ND3
ATA[GTA
p.M44V
NS
1.389/0.08
No
GU397531
1/30
17
I
10265T[C
ND3
ATT[ATC
p.I69I
SYN
NA
NA
Reported
1/30
18
I
10310G[A
ND3
CTG[CTA
p.L84L
SYN
NA
NA
Reported
1/30
19
I
10217A[G
ND3
ATA[ATG
p.M53M
SYN
NA
NA
Reported
1/30
20
I
10400C[T
ND3
ACC[ACT
p.T114T
SYN
NA
NA
Reported
3/30
21
I
10535T[C
ND4L
TAT[TAC
p.Y22Y
SYN
NA
NA
Reported
1/30
22 23
I I
10586G[A 10667T[C
ND4L ND4L
TCG[TCA TTT[TTC
p.S39S p.F66F
SYN SYN
NA NA
NA NA
Reported Reported
1/30 1/30
24
I
10727C[T
ND4L
GGC[GGT
p.G86G
SYN
NA
NA
GU397527
1/30
25
I
10556C[T
ND4L
TCC[TCT
p.S29S
SYN
NA
NA
GU397528
1/30
26
I
*11467A[G
ND4
TTA[TTG
p.L236L
SYN
NA
NA
Reported
6/30
27
I
11887G[A
ND4
CTG[CTA
p.L376L
SYN
NA
NA
Reported
2/30
28
I
*11914G[A
ND4
ACG[ACA
p.L385L
SYN
NA
NA
Reported
3/30
29
I
12106C[T
ND4
CTC[CTT
p.L449L
SYN
NA
NA
Reported
1/30
30
I
*12007G[A
ND4
TGG[TGA
p.W416W
SYN
NA
NA
Reported
4/30
31
I
12107C[T
ND4
CTC[CTT
p.T449T
SYN
NA
NA
GU397543
2/30
32
I
*12372G[A
ND5
CTG[CTA
p.L12L
SYN
NA
NA
Reported
6/30
33
I
*12561G[A
ND5
CAG[CAA
p.Q75Q
SYN
NA
NA
Reported
1/30
34
I
12618G[A
ND5
TTG[TTA
p.L94L
SYN
NA
NA
Reported
2/30
35
I
12793T[C
ND5
TTG[CTG
p.L153L
SYN
NA
NA
Reported
1/30
36
I
13194G[A
ND5
CTG[CTA
p.L286L
SYN
NA
NA
Reported
1/30
37
I
13368G[A
ND5
GGG[GGA
p.G344G
SYN
NA
NA
Reported
5/30
38 39
I I
13500T[C 13656T[C
ND5 ND5
GGT[GGC CTT[CTC
p.G338G p.L440L
SYN SYN
NA NA
NA NA
Reported Reported
1/30 2/30
40
I
13812T[C
ND5
GCT[GCC
p.A492A
SYN
NA
NA
Reported
1/30
41
I
13635T[C
ND5
GGT[GGC
p.G433G
SYN
NA
NA
Reported
1/30
42
I
13674T[C
ND5
AAT[AAC
p.N446N
SYN
NA
NA
Reported
4/30
43
I
12477T[C
ND5
AGT[AGC
p.S47S
SYN
NA
NA
Reported
1/30
44
I
14034T[C
ND5
ATT[ATC
p.N566N
SYN
NA
NA
Reported
1/30
45
I
13194G[A
ND5
CTG[CTA
p.T286T
SYN
NA
NA
Reported
1/30
46
I
12338T[C
ND5
ATA[ACA
p.M1T
NS
Benign/0.00
No
Reported
4/30
47
I
13708G[A
ND5
GCA[ACA
p.A458T
NS
0.610/0.34
No
Reported
5/30
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Table 1 continued S. no.
Complex
Nucleotide change
Locus
Codon change
Amino acid change
Type of mutation
Polyphen/ SIFT score
Pathogenic (yes/no)
Accession number
Frequency of variation
48
I
13780A[G
ND5
ATC[GTC
p.I482V
NS
0.775/0.07
No
Reported
1/30
49
I
13928G[C
ND5
AGC[ACC
p.S531T
NS
0.342/1.00
No
Reported
1/30
50
I
14002A[G
ND5
ACC[GCC
p.T556A
NS
0.532/0.51
No
Reported
1/30
51
I
12735C[A
ND5
ACC[ACA
p.T133T
SYN
NA
NA
GU397537
1/30
52
I
13317G[A
ND5
CTG[CTA
p.L327L
SYN
NA
NA
GU397539
1/30
53
I
*12498C[T
ND5
TTC[TTT
p.F54F
SYN
NA
NA
GU397538
1/30
54
I
*13676A[G
ND5
AAC[AGC
p.N447S
NS
1.023/0.14
No
GU397540
4/30
55
I
13860C[T
ND5
ACC[ACT
p.T508T
SYN
NA
NA
GU397541
1/30
56 57
III III
15458T[C 15287T[C
CYB CYB
TCC[CCC TTT[CTT
p.S238P p.F181L
NS NS
0.049/0.05 0.967/0.01
No No
Reported Reported
1/30 6/30
58
III
15629T[C
CYB
TTA[CTA
p.L295L
SYN
NA
NA
Reported
1/30
59
III
*14783T[C
CYB
TTA[CTA
p.L13L
SYN
NA
NA
Reported
11/30
60
III
15043G[A
CYB
GGG[GGA
p.G99G
SYN
NA
NA
Reported
5/30
61
III
15049C[T
CYB
GGC[GGT
p.G101G
SYN
NA
NA
Reported
1/30
62
III
15286C[T
CYB
ACC[ACT
p.T180T
SYN
NA
NA
Reported
1/30
63
III
15457C[T
CYB
CTC[CTT
p.L237L
SYN
NA
NA
GU397548
1/30
64
IV
6150G[A
CO1
GTT[ATT
p.V83I
NS
0.114/0.42
No
Reported
1/30
65
IV
6261G[A
CO1
GCC[ACC
p.A120T
NS
0.182/0.65
No
Reported
1/30
66
IV
6164C[T
CO1
ATT[ATC
p.I87I
SYN
NA
NA
Reported
2/30
67
IV
6176T[C
CO1
GAT[GAC
p.D91D
SYN
NA
NA
Reported
1/30
68
IV
6179G[A
CO1
ATG[ATA
p.M92M
SYN
NA
NA
Reported
2/30
69
IV
7106A[G
CO1
TCA[TCG
p.S401S
SYN
NA
NA
GU397532
1/30
70
IV
6182G[A
CO1
GCG[GCA
p.A93A
SYN
NA
NA
Reported
1/30
71 72
IV IV
6392T[C 8027G[A
CO1 CO2
AAT[AAV GCC[ACC
p.N163N p.A148T
SYN NS
NA 0.386/1.00
NA No
Reported Reported
1/30 1/30
73
IV
7645T[C
CO2
CTT[CTC
p.L20L
SYN
NA
NA
Reported
1/30
74
IV
7729A[G
CO2
ACA[ACG
p.T45T
SYN
NA
NA
Reported
1/30
75
IV
8023T[C
CO2
ATT[ATC
p.I146I
SYN
NA
NA
Reported
1/30
76
IV
8137C[T
CO2
TTC[TTT
p.F184F
SYN
NA
NA
Reported
1/30
77
IV
*8251G[A
CO2
GGG[GGA
p.G222G
SYN
NA
NA
Reported
6/30
78
IV
8116A[G
CO2
GGA[GGG
p.G177G
SYN
NA
NA
GU397550
1/30
79
IV
7702G[A
CO2
CTG[CTA
p.L39L
SYN
NA
NA
Reported
1/30
80
IV
7759T[C
CO2
GCT[GCC
p.A58A
SYN
NA
NA
Reported
1/30
81
IV
7828A[G
CO2
CTA[CTG
p.L81L
SYN
NA
NA
Reported
1/30
82
IV
7967C[T
CO2
CTA[TTA
p.L128L
SYN
NA
NA
Reported
1/30
83
IV
9374A[G
CO3
CAA[CAG
p.Q56Q
SYN
NA
NA
GU397530
2/30
84
IV
9752C[A
CO3
TTC[TTA
p.F182L
NS
0.792/0.54
No
GU397529
1/30
85
IV
9614A[G
CO3
GTA[GTG
p.V136V
SYN
NA
NA
Reported
2/30
86 87
IV IV
9512C[T 9767C[T
CO3 CO3
TAC[TAT ACC[ACT
p.Y102Y p.T187T
SYN SYN
NA NA
NA NA
Reported Reported
1/30 2/30
88
IV
9852A[G
CO3
ACT[GCT
p.T216A
NS
0.090/1.00
No
Reported
1/30
89
IV
9861T[C
CO3
TTC[CTC
p.F219L
NS
0.027/0.35
No
Reported
1/30
90
V
8410C[T
ATP8
CCC[CCT
p.P15P
SYN
NA
NA
Reported
1/30
91
V
8537A[G
ATP8
ATC[GTC
p.I58V
NS
0.758/0.09
No
Reported
1/30
92
V
8414C[T
ATP8
CTC[TTC
p.L17F
NS
0.112/0.44
No
Reported
1/30
93
V
8679A[G
ATP6
AAA[AAG
p.K51K
SYN
NA
NA
Reported
1/30
94
V
8790G[A
ATP6
CTG[CTA
p.L88L
SYN
NA
NA
Reported
1/30
123
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Table 1 continued S. no.
Complex
Nucleotide change
Locus
Codon change
Amino acid change
Type of mutation
Polyphen/ SIFT score
Pathogenic (yes/no)
Accession number
Frequency of variation
95
V
*8865G[A
ATP6
GTG[GTA
p.V113V
SYN
NA
NA
Reported
1/30
96
V
9123G[A
ATP6
CTG[CTA
p.L199L
SYN
NA
NA
Reported
5/30
97
V
*8684C[T
ATP6
ACC[ATC
p.T53I
NS
0.219/1.00
No
Reported
8/30
98
V
9094C[T
ATP6
CTT[TTT
p.L190F
NS
0.002/0.73
No
Reported
6/30
99
V
9064G[A
ATP6
GCA[ACA
p.A180T
NS
0.012/0.37
No
Reported
1/30
100
V
8779C[T
ATP6
CTC[TTC
p.T85T
SYN
NA
NA
GU397553
1/30
101
V
9189C[T
ATP6
TAC[TAT
p.Y221Y
SYN
NA
NA
GU397554
1/30
102
V
8676C[T
ATP6
ATC[ATT
p.I50I
SYN
NA
NA
GU397555
2/30
* Mitochondrial variations in patients and controls, Reported http://www.mitomap.org, SYN synonymous, NS not synonymous, NA not applicable, ND1 NADH dehydrogenase subunit 1; ND2 NADH dehydrogenase subunit 2; ND3 NADH dehydrogenase subunit 3; ND4 NADH dehydrogenase subunit 4; ND5 NADH dehydrogenase subunit 5; CO1 cytochrome c oxidase I; CO2 cytochrome c oxidase II; ATPase6 ATP synthase subunit a (F-ATPase protein 6); ATPase8 ATP synthase protein 8; CYB cytochrome
All novel variations (19 in patients, Table 1) (12 in controls, Table 2) were submitted to the GenBank database and accession numbers were obtained (GU397527–GU597555). Out of 102 variations observed in patients 25 (24.50%) were non-synonymous and 77 (75.50%) were synonymous (Fig. 1a), whereas in controls 43 (76.79%) variations were synonymous and 13 (23.21%) were non-synonymous (Fig. 1b; Table 3). Thus, we found higher number of pathogenic non-synonymous variations in patients. 54% (55 of 102) nucleotide variations were observed in mitochondrial complex I in POI and occult OI cases. Figure 2 compares the distribution of mitochondrial nucleotide variations between patients and controls within the coding regions of the mitochondrial genome. Table 4 compares the number of mitochondrial variations found in the mitochondrial respiratory complexes between POI, occult OI, and controls. Thus, the mitochondrial respiratory chain complex I, IV, and V had significantly higher number of mitochondrial variations in patients as compared with controls. In complex III, the number of mitochondrial variations in patients was not significantly higher as compared with controls (Fig. 3). FSH levels were significantly (p \ 0.0001) higher in patients with POI [105.76 mIU/ml (55.76–194.76 mIU/ ml)] and occult OI [40.56 mIU/ml (25.20–62.64 mIU/ml)] as compared with controls (Table 3). Also the levels of FSH were significantly higher in POI cases than in occult OI cases. The overall median range of ROS was found to be significantly (p \ 0.005) higher in patients [48,160 RLU/min (120–130,122 RLU/min)] as compared with controls [340 RLU/min (120–5,096 RLU/min)]. Among the patients, 64% had very high levels of ROS, 24% have moderately elevated levels, and 12% had normal values comparable to controls. It was found that cases with occult OI had moderate elevation of ROS levels [15,120 RLU/min (1,225–20,106 RLU/min)].
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Computational assessment of non-synonymous variations was done using online tools (Polyphen and SIFT). Nucleotide changes ND1:g.3398T[C, ND2:g.5186A[T, ND2:g.4852T[A were predicted to be pathogenic, whereas all other non-synonymous variations were benign. Table 5 describes the frequency of pathogenic mutations in patients and controls. All pathogenic mutations were found to be significantly higher in POI and occult OI cases as compared with controls. ND1:g.3398T[C and ND2:4852T[A were observed in 12 and 7 patients, respectively, whereas ND2:g.5186A[T was found in nine patients, significantly higher as compared with controls (Table 5). Three nonpathogenic nucleotide variations (ATPase6:g.8684C[T, ATPase6:g.9094C[T, ATPase6:g.9123G[A) were found to be significantly higher in cases as compared with controls. Three nucleotide alterations (CO2:g.8251G[A, ND5:g.13368G[A and ND5:g.13708G[A) previously reported to be associated with increased ROS levels (oxidative stress) were found in 16 cases.
Discussion The precise patho-physiological mechanism responsible for POI is unknown. This study identified significantly higher nonsynonymous pathogenic nucleotide variations in POI. Thus, ovarian insufficiency may be associated with more mtDNA damage and dysfunction which indicates abnormal mitochondrial biogenesis during oocyte growth. These mitochondrial nucleotide alterations lead to increased production of free radicals with simultaneous decrease in production of ATP. Adequate ATP levels are required for optimal oogenesis germ cell growth, development, and differentiation. Mitochondrial dysfunction due to nucleotide alterations may result in impaired oogenesis or accelerated germ cell apoptosis and POI.
Arch Gynecol Obstet (2010) 282:695–705
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Table 2 Mitochondrial DNA variations in controls S. no.
Complex
Nucleotide change
Locus
Codon change
Change in protein
Type of mutation
Polyphen/ SIFT score
Pathogenic (yes/no)
Accession number
Frequency of variation
1
I
3591G[A
ND1
CTG[CTA
p.T95T
SYN
NA
NA
Reported
1/30
2
I
3915G[A
ND1
GGG[GGA
p.G203G
SYN
NA
NA
Reported
1/30
3
I
3918G[A
ND1
GAG[GAA
p.E204E
SYN
NA
NA
Reported
1/30
4
I
3933A[G
ND1
TCA[TCG
p.S209S
SYN
NA
NA
GU397544
1/30
5
I
*3970C[T
ND1
CTA[TTA
p.L222L
SYN
NA
NA
Reported
2/30
6
I
3996C[T
ND1
AAC[AAT
p.N230N
SYN
NA
NA
Reported
1/30
7 8
I I
4029C[A 3424G[A
ND1 ND1
ATC[ATA GGC[GGT
p.I241M p.G41G
NS SYN
Benign/0.07 NA
NA NA
GU397545 Reported
1/30 1/30
9
I
4793A[G
ND2
ATA[ATG
p.M108M
SYN
NA
NA
Reported
1/30
10
I
*4852T[A
ND2
CTG[CAG
p.L128Q
NS
1.951/0.00
Yes
GU397533
2/30
11
I
*5186A[T
ND2
TGA[TGT
p.W239C
NS
1.982/0.03
Yes
Reported
1/30
12
I
5348C[T
ND2
TAC[TAT
p.Y293Y
SYN
NA
NA
Reported
1/30
13
I
5351A[G
ND2
CTA[CTG
p.L294L
SYN
NA
NA
Reported
1/30
14
I
*10310G[A
ND3
CTG[CTA
p.T84T
SYN
NA
NA
Reported
1/30
15
I
10609T[C
ND4L
ATA[ACA
p.M47T
NS
Benign/0.23
No
Reported
1/30
16
I
*11467A[G
ND4
TTA[TTG
p.L236L
SYN
NA
NA
Reported
1/30
17
I
*11914G[A
ND4
ACG[ACA
p.T385T
SYN
NA
NA
Reported
1/30
18
I
*12007G[A
ND4
TGG[TGA
p.W416W
SYN
NA
NA
Reported
1/30
19
I
12073C[T
ND4
TTC[TTT
p.F438F
SYN
NA
NA
GU397542
1/30
20
I
*12107C[T
ND4
CTC[CTT
p.T449T
SYN
NA
NA
GU397543
1/30
21
I
12133C[T
ND4
TCC[TCT
p.S458S
SYN
NA
NA
Reported
1/30
22 23
I I
*12372G[A 12373A[G
ND5 ND5
CTG[CTA ACT[GCT
p.T12T p.T13A
SYN NS
NA Benign/0.00
NA No
Reported Reported
1/30 1/30
24
I
12406G[A
ND5
GTT[ATT
p.V24I
NS
0.299/0.72
No
Reported
1/30
25
I
12486C[T
ND5
CCC[CCT
p.P50P
SYN
NA
NA
GU397534
1/30
26
I
*12561G[A
ND5
CAG[CAA
p.Q75Q
SYN
NA
NA
Reported
1/30
27
I
13204G[A
ND5
GTC[ATC
p.V290I
NS
0.710/1.00
No
GU397535
1/30
28
I
12477T[C
ND5
AGT[AGC
p.S47S
SYN
NA
NA
Reported
1/30
29
I
12681T[C
ND5
AAT[AAC
p.N115N
SYN
NA
NA
Reported
1/30
30
I
13806C[T
ND5
GCC[GCT
p.A490A
SYN
NA
NA
GU397536
1/30
31
I
14058C[T
ND5
TCC[TCT
p.S574S
SYN
NA
NA
Reported
1/30
32
III
*14783T[C
CYB
TTA[CTA
p.L13L
SYN
NA
NA
Reported
2/30
33
III
14872C[T
CYB
ATC[ATT
p.I42I
SYN
NA
NA
Reported
1/30
34
III
15119G[A
CYB
GCA[ACA
p.A125T
NS
0.760/0.00
No
Reported
1/30
35
III
15172G[A
CYB
GGG[GGA
p.G142G
SYN
NA
NA
Reported
1/30
36
III
15217G[A
CYB
GGG[GGA
p.G157G
SYN
NA
NA
Reported
1/30
37
III
15385C[T
CYB
TCC[TCT
p.S213S
SYN
NA
NA
Reported
1/30
38 39
III III
15431G[A 15484A[G
CYB CYB
GCC[ACC TCA[TCG
p.A229T p.S246S
NS SYN
0.033/0.03 NA
No NA
Reported GU397547
1/30 1/30
40
III
15670T[C
CYB
CAT[CAC
p.H308H
SYN
NA
NA
Reported
1/30
41
IV
6032G[A
CO1
CAG[CAA
p.Q43Q
SYN
NA
NA
Reported
1/30
42
IV
6320T[C
CO1
CCT[CCC
p.P139P
SYN
NA
NA
Reported
1/30
43
IV
6734G[A
CO1
ATG[ATA
p.M277M
SYN
NA
NA
Reported
1/30
44
IV
7316G[A
CO1
ATG[ATA
p.M471M
SYN
NA
NA
Reported
1/30
45
IV
7738T[C
CO2
ACT[ACC
p.T51T
SYN
NA
NA
Reported
1/30
46
IV
7762G[A
CO2
CAG[CAA
p.Q59Q
SYN
NA
NA
Reported
1/30
47
IV
8143T[C
CO2
GCT[GCC
p.A186A
SYN
NA
NA
GU397549
1/30
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Table 2 continued S. no.
Complex
Nucleotide change
Locus
Codon change
Change in protein
Type of mutation
Polyphen/ SIFT score
Pathogenic (yes/no)
Accession number
Frequency of variation
48
IV
*8251G[A
CO2
GGG[GGA
p.G222G
SYN
NA
NA
Reported
1/30
49
V
8503T[G
ATP8
AAT[AAG
p.N46K
NS
0.090/1.00
No
GU397551
1/30
50
V
8584G[A
ATP6
GCA[ACA
p.A20T
NS
0.362/0.19
No
Reported
1/30
51
V
8594T[C
ATP6
ATC[ACC
p.I23T
NS
1.579/0.27
No
Reported
2/30
52
V
8650C[T
ATP6
CTA[TTA
p.L42L
SYN
NA
NA
Reported
1/30
53
V
*8684C[T
ATP6
ACC[ATC
p.T53I
NS
0.219/1.00
No
Reported
1/30
54
V
8718A[G
ATP6
AAA[AAG
p.K64K
SYN
NA
NA
Reported
1/30
55
V
8812A[G
ATP6
ACC[GCC
p.T96A
NS
0.908/0.03
No
Reported
1/30
56 57
V V
*8865G[A 8886G[A
ATP6 ATP6
GTG[GTA AAG[AAA
p.V113V p.K120K
SYN SYN
NA NA
NA NA
Reported GU397552
1/30 1/30
58
V
8925A[G
ATP6
ACA[ACG
p.T133T
SYN
NA
NA
Reported
1/30
* Mitochondrial variations found both in patients and controls, Reported http://www.mitomap.org, SYN synonymous, NS not synonymous, NA not applicable, ND1 NADH dehydrogenase subunit 1, ND2 NADH dehydrogenase subunit 2, ND3 NADH dehydrogenase subunit 3, ND4 NADH dehydrogenase subunit 4, ND5 NADH dehydrogenase subunit 5, CO1 cytochrome c oxidase I; CO2 cytochrome c oxidase II, ATPase6 ATP synthase subunit a (F-ATPase protein 6), ATPase8 ATP synthase protein 8, CYB cytochrome
Fig. 1 a Pie chart distribution of mitochondrial mutations (Reported) found in POF patients. b Pie chart distribution of novel mitochondrial mutation found in POF patients Table 3 Comparison of ROS and FSH levels of POF, occult OI, and controls Subjects
ROS (RLU/min)
Age (years)
FSH (mIU/ml)
POI (n = 25)
48,160 (120–130,122)
23.96 ± 2.87
105.76 (55.76–194.76)
Occult OI (n = 5)
15,120 (1,225–20,106)
21.44 ± 2.5
40.56 (25.20–62.64)
Controls (n = 30)
340 (120–5,096)
25.95 ± 3.25
2.86 (1.80–6.13)
Significance
\0.005
\0.0001
In this study majority of mitochondrial nucleotide changes were predominantly transitions rather than transversions, similar to the mtDNA changes previously reported in association with other diseases. In this study we found 76% (120 of 158) synonymous nucleotide changes. Synonymous variations have been routinely classified as innocuous polymorphisms and are assumed to be functionally neutral. However, synonymous mutations may result in skipping of certain exons as reported in CFTR gene [23]. Carlini and Stephan [24] reported that when synonymous changes were introduced in the fruit fly
123
Fig. 2 Gene-wise distribution of mitochondrial mutations in different mitochondrial genes
alcohol dehydrogenase gene, they changed several codons to sub-optimal synonyms due to which production of the encoded enzyme was reduced. This may be the mechanism by which the synonymous changes in different genes adversely affect various enzymes in ETC and result in lower ATP production. In this study we found 77 synonymous mitochondrial alterations in patients which may have similar effect due to codon bias. It was recently thought that variations in synonymous sites were selectively neutral, but codon bias has now been recognized as an important evolutionary force, and there is a positive correlation between degree, codon bias, and level of gene expression [25]. Different codons coding for same amino acid not only have a different secondary structure which
Arch Gynecol Obstet (2010) 282:695–705
703
Table 4 Number of mitochondrial variations (complex wise) between patients and controls Mitochondrial respiratory chain complexes
POF (n = 25)
Complex I
Complex III
Complex IV
Complex V
43
07
21
09
Occult OI (n = 5)
12
02
06
04
Controls (n = 30)
31
09
08
10
Table 5 Frequency of pathogenic mitochondrial variations found in patients and controls Subjects
Pathogenic mutations ND1:g.3398T[C ND2:g.5186A[T ND2:g.4852T[A
POI 10 (n = 25)
8
6
Occult OI (n = 5)
1
1
1
2
2
Controls Nil (n = 30)
Fig. 3 Distribution of mitochondrial mutations in different complexes of mitochondrial respiratory chain or electron transport chain
may affect both rate and accuracy of translation [26] but also reduce translation efficacy by 2.13% [24]. In this study we found 24.50% nucleotide changes were non-synonymous which could adversely affect electron transport chain (ETC) and thus impair ATP production and low ATP levels may lead to impaired folliculogenesis/oogenesis. We observed three mitochondrial variations CO2:g.8251G[A, ND5:g.13368G[A and ND5:g.13708G[A in 6, 5, and 5 patients as compared with 1, 0 and 0 controls, respectively, and these mitochondrial alterations have been reported to produce high ROS levels [27] and are associated with oxidative stress. Three nucleotide changes ND1:g.3398T[C, ND2:g.5186A[T and ND2:g.4852T[A were predicted pathogenic and may adversely affect OXPHOS efficiency and reduce ATP production. ND1:g.3398T[C and ND2:g.5186A[T were found in significantly higher number of patients (12:9) as compared with controls (0:1) (Table 5). This may impair growth and development of oogonia/oogenesis and also may accelerate apoptosis of germ cells. In POI and occult OI, 22 of 25 mitochondrial alterations were between dissimilar amino acid residues and were non-pathogenic. Two nucleotide changes (ND5:g.12338T[C and CYB:g.15287T[C) were found to be intolerant using SIFT tool whereas
predicted to be benign by Polyphen (Table 1). Nucleotide changes g.12338T[C and g.15287T[C were found in four and six patients, respectively. Although the ND5:g. 12338T[C and CYB:g.15287T[C replacement do not seem to be deleterious mutations, these replacements might alter the function of the complex I and CYB subunit. Protein tertiary structure is usually driven by the burial of hydrophobic residues, and it has been shown that energetics of DNA tertiary structure assembly were determined to be primarily driven by the hydrophobic effect [28]. So, the replacement of non-polar/hydrophobic amino acid to polar/ hydrophilic or vice versa could disrupt the structure of mitochondrial complexes and thus affect OXPHOS efficiency and reduce ATP production. It has also been reported that mtDNA mutations induce insufficient amounts of active mitochondria and decline in OXPHOS efficiency, all of which could contribute to poor oocyte quality [3, 29]. The decline in OXPHOS efficiency can result in leakage of electrons from the electron transport chain at the inner mitochondrial membrane, and these electrons get transferred to the oxygen molecule, resulting in an unpaired electron in the orbit. This may further damage mitochondrial DNA [8], thus leading to higher levels of ROS and increases the number of mitochondrial nucleotide alterations. In this study, ROS levels showed a positive correlation with FSH levels in cases (POI and occult OI) (Table 3). Study inclusion criteria were strict to eliminate all confounding factors which could lead to high ROS levels like smoking, intake of drugs, and history of infection or inflammation, etc. The respiratory chain embedded in the mitochondrial inner membrane is composed of five multiheteromeric enzyme complexes (I, II, III, IV, and V), but the structural components of complex II are encoded exclusively by nuclear genes. In this study majority of the patients harboring non-synonymous and pathogenic mitochondrial variations were detected in complex I and V of the respiratory chain. MTND is the largest enzyme complex of mitochondrial respiratory chain complexes, and complex V plays an important role in production of ATP. MTND genes harbor most mtDNA mutations that arise in
123
704
protein-encoding genes and deficiencies of this complex cause a wide range of clinical phenotypes (mitochondrial encephalomyopathies, PCG, infertility, LHON and sensorineural hearing impairment) [30]. Due to deficiencies or disruption of these complexes electron shuttle is blocked, resulting in elevated ROS levels as found in this study. Sugioka et al. [31] reported that complex I and III are the major sites for ROS production in respiratory chain and are associated with elevated production of ROS. In this study three non-synonymous mutations were detected in complex III. Excessive ROS production causes the opening of permeability transition pore and subsequently the release of cytochrome c, which activate the caspase cascade pathway that can induce cellular degeneration [32] and may be underlying cause of POI due to accelerated apoptosis of germ cells. More than 99% of the follicles from the initial pool undergo atresia or apoptosis [33]. In a woman’s life, only 400 follicles will undergo recruitment, final selection, and ovulation. According to Gougeon and Busso [34] the different steps from the primordial stage to ovulation take at least 3 months. In theory, we can hypothesize that POI can occur: (a) because of an initial decrease in the primordial follicle pool; (b) because of accelerated follicular atresia; (c) because of impaired maturation of follicle. Recent studies [35] have shown that low-quality oocytes have lower mitochondrial membrane potential, increase in mitochondrial DNA damage, mitochondria with abnormal morphology, and changes in mitochondrial gene expression. These mitochondrial changes arise from excessive ROS production [30]. In previous studies from our laboratory we have already reported increased ROS levels in POI cases which harbored ATPase mutations [3]. Low ATP levels may lead to impaired oogenesis and low primordial follicle production or may also lead to accelerated depletion by activation of the caspase apoptotic pathway [3, 32]. It has been shown that regulated generation of ROS by the pre-ovulatory follicle is an important promoter of oogenesis. Behrman et al. [12] showed that granulosa and luteal cells respond negatively to high ROS levels, and supra-physiological ROS levels adversely affect second meiotic division progression, leading to diminished gonadotrophin levels and anti-steroidogenic actions, DNA damage, and inhibited ATP production. Oxygen limitation or deficiency is known to stimulate follicular angiogenesis, which is important for follicular growth and development. Impairment of angiogenesis within ovarian follicles contributes to follicular atresia [36]. Free radicals may act as signal transducers [37] or intracellular messengers [38] of the angiogenic response. Mitochondria are the major consumers of cellular oxygen, thereby providing support to the hypothesis that ROS are involved in intracellular signaling between tissue hypoxia and angiogenic response [39].
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Chao et al. [40] investigated ovarian mitochondrial DNA (mtDNA) mutation and oxidative damage and found that oxidative damage to the ovaries increased with cycles of stimulation, with a statistically significant increase in lipid peroxides. Venkatesh et al. [3] reported significantly high ROS levels and ATPase mutations in POI cases as compared with controls. In present study we found significantly (p \ 0.005) increased ROS levels in POI cases [48,160 RLU/min (120–130,122 RLU/min)] as compared with controls [340 RLU/min (120–5,096 RLU/min)]. The role of OS in female fertility and sub-fertility is an area deserving continued research. Such preliminary studies on analysis of complete mitochondrial genome and oxidative stress should be followed up by larger studies and in ethnically different populations. It would also be worth analyzing ova for mitochondrial nucleotide alterations as they develop in a different microenvironment in the ovary and are of different embryological origin; however, due to ethical constraints such studies are not feasible. But in a study in male infertility from our laboratory we found seminal ROS levels correlated with systemic blood ROS levels [10]. Thus, this study on blood ROS and mitochondrial nucleotide alterations in POI is important, though it would be ideal to conduct further, similar studies on oocytes. The therapeutic tools currently available for the treatment of mitochondrial diseases due to mtDNA mutations are few, and their efficacy is not yet well established [41, 42]. In the treatment of mitochondrial disorders in which ROS play important pathogenic role, antioxidants may be considered as a potentially useful therapeutic tool in association with other therapeutic approaches. One study [43] showed CoQ10 supplement yielded better quality oocytes and embryos and both showed increased ATP levels following CoQ10 administration. Thus, large studies on role of mitochondrial mutations and OS on idiopathic POI cases are required. In cases which harbor mitochondrial nucleotide variations and have high ROS levels, early detection and prompt antioxidant administration may delay/prevent oxidative stress-induced mitochondrial mutations and damage to developing germ cells. Conflict of interest
None.
References 1. Coulam CB, Adamson SC, Annegers JF (1986) Incidence of premature ovarian insufficiency. Obstet Gynecol 67:604–606 2. Goswami D, Conway GS (2005) Premature ovarian failure. Hum Reprod Update 11(4):391–410 3. Venkatesh S, Kumar M, Sharma A, Kriplani A, Ammini AC, Talwar P, Agarwal A, Dada R (2010) Oxidative stress and ATPase6 mutation is associated with primary ovarian insufficiency. Arch Gynecol Obstet. doi:10.1007/s00404-010-1444-y
Arch Gynecol Obstet (2010) 282:695–705 4. Coulam CB (1982) Premature gonadal failure. Fertil Steril 38(6):645–655 5. Hsueh AJ, Billig H, Tsafriri A (1994) Ovarian follicle atresia: a hormonally controlled apoptotic process. Endocr Rev 15(6):707–724 6. Hsueh AJ, Eisenhauer K, Chun SY, Hsu SY, Billig H (1996) Gonadal cell apoptosis. Recent Prog Horm Res 51:433–455 7. Morita Y, Tilly JL (1999) Oocyte apoptosis: like sand through an hourglass. Dev Biol 213:1–17 8. Agarwal A, Gupta S, Sharma RK (2005) Role of oxidative stress in female reproduction. Reprod Biol Endocrinol 3:28 9. Ames BN, Shigenaga MK, Hagen TM (1993) Oxidants, antioxidants, and the degenerative diseases of aging. Proc Natl Acad Sci USA 90:7915–7922 10. Shamsi MB, Venkatesh S, Tanwar M, Talwar P, Sharma RK, Dhawan A, Kumar R, Gupta NP, Malhotra N, Singh N, Mittal S, Dada R (2009) DNA integrity and semen quality in men with low seminal antioxidant levels. Mutat Res/Fundam Mol Mech Mutagen 665:29–36 11. Lu B, Christophe P, Tamas G, Christian G, Wilbur H, David B, Martin MM, Karl-Erik A, Paul AO, Colin EB (2008) A mutation in the inner mitochondrial membrane peptidase 2-like gene (Immp2l) affects mitochondrial function and impairs fertility in mice. Biol Reprod 78:601–610 12. Behrman HR, Kodaman PH, Preston SL, Gao S (2001) Oxidative stress and the ovary. J Soc Gynecol Investig 8:S40–S42 13. Agarwal A, Desai NR, Makker K, Varghese A, Mouradi R, Sabanegh E, Sharma R (2009) Effects of radiofrequency electromagnetic waves (RF-EMW) from cellular phones on human ejaculated semen: an in vitro pilot study. Fertil Steril 92(4):1318–1325 14. St John JC, Cooke ID, Barratt CL (1997) Mitochondrial mutations and male infertility. Nat Med 3:124–125 15. Madamanchi NR, Runge MS (2007) Mitochondrial dysfunction in atherosclerosis. Circ Res 100:460–473 16. Mancuso M, Coppede F, Migliore L, Siciliano G, Murri L (2006) Mitochondrial dysfunction, oxidative stress and neurodegeneration. J Alzheimer Dis 10:59–73 17. Sedensky MM, Morgan PG (2006) Mitochondrial respiration and reactive oxygen species in mitochondrial aging mutants. Exp Gerontol 41:237–245 18. Bedaiwy MA, Falcone T (2003) Peritoneal fluid environment in endometriosis: clinicopathological implications. Minerva Ginecol 55:333–345 19. Kumar M, Tanwar M, Saxena R, Sharma P, Dada R (2010) Identification of novel mitochondrial mutations in Leber’s hereditary optic neuropathy. Mol Vis 16:782–792 20. Sunyaev S, Ramensky V, Koch I, Lathe W 3rd, Kondrashov AS, Bork P (2001) Prediction of deleterious human alleles. Hum Mol Genet 10(6):591–597 21. Ramensky V, Bork P, Sunyaev S (2002) Human non-synonymous SNPs: server and survey. Nucleic Acids Res 30(17):3894–3900 22. Ng PC, Henikoff S (2003) SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res 31(13):3812–3814 23. Pagani F, Raponi M, Baralle FR (2005) Synonymous mutations in CFTR exon 12 affect splicing and are not neutral in evolution. PNAS 102:6368–6372 24. Carlini DB, Stephan W (2003) In vivo introduction of unpreferred synonymous codons into the Drosophila Adh gene results in reduced levels of ADH protein. Genetics 163(1):239–243 25. Carlini DB, Chen Y, Stephan W (2001) The relationship between third-codon position nucleotide content, codon bias, mRNA secondary structure and gene expression in the drosophilid alcohol dehydrogenase genes Adh and Adhr. Genetics 159(2):623–633
705 26. Parsch J, Braverman JM, Stephan W (2000) Comparative sequence analysis and patterns of covariation in RNA secondary structures. Genetics 154(2):909–921 27. van der Walt JM, Nicodemus KK, Martin ER, Scott WK, Nance MA, Watts RL, Hubble JP, Haines JL, Koller WC, Lyons K, Pahwa R, Stern MB, Colcher A, Hiner BC, Jankovic J, Ondo WG, Allen FH Jr, Goetz CG, Small GW, Mastaglia F, Stajich JM, McLaurin AC, Middleton LT, Scott BL, Schmechel DE, PericakVance MA, Vance JM (2003) Mitochondrial polymorphisms significantly reduce the risk of Parkinson disease. Am J Hum Genet 72(4):804–811 28. Sideraki V, Huang W, Palzkill T, Gilbert HF (2001) A secondary drug resistance mutation of TEM-1 beta-lactamase that suppresses misfolding and aggregation. Proc Natl Acad Sci USA 98(1):283–288 29. Hsieh RH, Au HK, Yeh TS, Chang SJ, Cheng YF, Tzeng CR (2004) Decreased expression of mitochondrial genes in human unfertilized oocytes and arrested embryos. Fertil Steril 81:912–918 30. Wong LJ (2007) Pathogenic mitochondrial DNA mutations in protein-coding genes. Muscle Nerve 36:279–293 31. Sugioka K, Nakano M, Totsune NH, Minakami H, Tero KS, Ikegami Y (1988) Mechanism of O2-generation in reduction and oxidation cycle of ubiquinones in a model of mitochondrial electron transport systems. Biochim Biophys Acta 936:377–385 32. Thouas GA, Trounson AO, Wolvetang EJ, Jones GM (2004) Mitochondrial dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro. Biol Reprod 71:1936–1942 33. Richardson SJ, Senikas V, Nelson JF (1987) Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion. J Clin Endocrinol Metab 65:1231–1237 34. Gougeon A, Busso D (2000) Morphologic and functional determinants of primordial and primary follicles in the monkey ovary. Mol Cell Endocrinol 25:33–42 35. Wang LY, Wang DH, Zou XY, Xu CM (2009) Mitochondrial functions on oocytes and preimplantation embryos. J Zhejiang Univ Sci B 10:483–492 36. Greenwald GS, Terranova PF (1988) Follicular selection and its control. In: Knobil E, Neill J (eds) The physiology of reproduction. Raven Press, New York, pp 387–445 37. Schroedl C, McClintock DS, Budinger GR, Chandel NS (2002) Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species. Am J Physiol Lung Cell Mol Physiol 283:L922–L931 38. Pearlstein DP, Ali MH, Mungai PT, Hynes KL, Gewertz BL, Schumacker PT (2002) Role of mitochondrial oxidant generation in endothelial cell responses to hypoxia. Arterioscler Thromb Vasc Biol 22:566–573 39. Basini G, Grasselli F, Bianco F, Tirelli M, Tamanini C (2004) Effect of reduced oxygen tension on reactive oxygen species production and activity of antioxidant enzymes in swine granulosa cells. Biofactors 20:61–69 40. Chao HT, Lee SY, Lee HM, Liao TL, Wei YH, Kao SH (2005) Repeated ovarian stimulations induce oxidative damage and mitochondrial DNA mutations in mouse ovaries. Ann N Y Acad Sci 1042:148–156 41. Marriage B, Clandinin MT, Glerum DM (2003) Nutritional cofactor treatment in mitochondrial disorders. J Am Diet Assoc 103(8):1029–1038 42. Tanaka M, Nishigaki Y, Fuku N, Ibi T, Sahashi K, Koga Y (2007) Therapeutic potential of pyruvate therapy for mitochondrial diseases. Mitochondrion 7(6):399–401 43. Bentov Y, Esfandiari N, Burstein E, Casper RF (2010) The use of mitochondrial nutrients to improve the outcome of infertility treatment in older patients. Fertil Steril 93(1):272–275
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