Mitochondrial Genetic Variation in Iranian Infertile Men
Several recent studies have shown that mitochondrial DNA mutations lead
to major disabilities and premature death in carriers. More than 150 mutations in
human mitochondrial DNA (mtDNA) genes have been associated with a wide spectrum of
disorders. Varicocele, one of the causes of infertility in men wherein abnormal inflexion
and distension of veins of the pampiniform plexus is observed within spermatic cord, can
increase reactive oxygen species (ROS) production in semen and cause oxidative stress
and sperm dysfunction in patients. Given that mitochondria are the source of ROS
production in cells, the aim of this study was to scan nine mitochondrial genes (
Materials and Methods:
In this cross-sectional study, polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing were used to detect and identify point mutations respectively in 9 mitochondrial genes in 72 infertile men with varicocele and 159 fertile men. In brief, the samples showing altered electrophoretic patterns of DNA in the SSCP gel were sent for DNA sequencing to identify the exact nucleotide variation.
Ten type nucleotide variants were detected exclusively in mitochondrial DNA of infertile men. These include six novel nucleotide changes and four variants previously reported for other disorders.
Mutations in mitochondrial genes may affect respiratory complexes in combination with environmental risk factors. Therefore these nucleotide variants probably lead to impaired ATP synthesis and mitochondrial function ultimately interfering with sperm motility and infertility.
The major concern among married couples when they are unsuccessful to conceive after one year of regular unprotected intercourse is that they may be infertile. Male factors can be attributed to half of these cases (1,2). The most common surgically reversible cause of infertility is varicocele. Its prevalence is about 4.4-22.6% in the general population, 21-41% in men with primary infertility and 75-80% in men with secondary infertility (3,4). Despite the advances in molecular medicine, the pathophysiology of varicocele induced infertility remains unknown. Several proposed mechanisms include venous pressure changes and increased testicular temperature due to dilation and tortuosity of the pampiniform plexus of veins, oxidative stress, retrograde flow of renal or adrenal products, Leydig cell dysfunction and hyperthermia (5,6). In addition, a number of patients with varicocele have genetic abnormalities like Yq-microdeletions (7). Among them, oxidative stress-induced DNA damage appears to be a more likely cause which may severely affect sperm quality leading to infertility (8). This damage is one of the potential etiological factors in varicocele. A major source of partially reduced derivatives of molecular oxygen (O2) is mitochondria (9). The variety of reactive oxygen species (ROS) that mitochondria produce principally include hydrogen peroxide (H2O2), superoxide anion (O2.-) and the hydroxyl radical (OH.) (10,11). In normal physiology, ROS perform several roles in regulating cellular functions by interacting with cellular components (12). In fertile men, physiological levels of ROS play important roles in sperm function, acrosome reaction, capacitation, hyper-activation and the penetration of oocyte by spermatozoa. However, in varicocele patients ROS generation is abnormally enhanced (13,14).
Specific point mutations and deletions of mitochondrial DNA (mtDNA) have been associated with poor sperm motility and semen quality in several studies. Sperm mtDNA is highly sensitive to mutations due to increased ROS by-products generated during oxidative respiration (15). When large amounts of mutant mtDNA accumulate in the testes, reduction in ATP production, mitochondrial respiratory dysfunction and meiotic arrest are induced in spermatogonia cells (16). Each mitochondrion has 2-10 mitochondrial genomes responsible for coding the subunits of the OXPHOS complex. The OXPHOS machinery is made up of over 80 different polypeptides, of which the mtDNA encodes 13 polypeptides including complex I, III, IV and V subunits (17).
In this study, for first time, we further analyzed
nine genes (
Materials and Methods
This study was a cross sectional study. Seventy two Iranian infertile men with clinical varicocele were recruited in the study. The varicocele diagnosis was made by the urologists for the patients by physical examination in standing position and via scrotal palpation in a temperature controlled room (23°C). Semen analysis was performed according to the WHO laboratory manual (18). Patients with varicocele were in 3 grades: i. Grade I (n=12), ii. Grade II (n=27) and iii. Grade III (n=33). The control group (healthy volunteers) consisted of 159 fertile and normospermic men from the Yazd Infertility Center who fathered at least one child. The ethnic and geographical origin of all patients and controls was the same. All participants were fully informed of the objectives of the study and those that signed the consent form were assigned to the study. All infertile men in the age group ranging from 22 to 36 years (mean, 29 years) were referred for evaluation of their infertility (1 year of unprotected intercourse and not leading to conception). The Yazd University Ethics Committee approved recruitment of patients and laboratory protocols in this study.
DNA extraction and mutation analysis
Peripheral blood samples were obtained from
varicocele patients and the DNA was extracted
using a standard salting-out procedure. Purified DNA
samples from leukocytes were used for the PCR
reactions. To amplify
Tm; Temperature melting.
For the SSCP assay, PCR products were heatdenatured at 95 ºC for 5 minutes and chilled on ice for 5 minutes, and then loaded onto an 8% nondenaturing polyacrylamide/TBE 0.5x gel. Gels were stained with silver nitrate to reveal the bands of single strand DNA. Various band patterns of the amplified PCR products were marked and scored. The typical gene variants got sequenced using a commercial company (Macrogen, South Korea). All the data obtained from automated sequencing was checked with Sequencher. The online multiple sequence alignment software ClustalW2 (
Software and databases
We used the tool PolyPhen-2 (
The GraphPad Prism software (GraphPad Software, Inc. USA) was used for statistical analysis. Distributions of continuous variables in groups were expressed as mean ± SD, and compared with unpaired Student’s t test. P<0.05 were regarded as statistically significant.
The age difference between the 72 Iranian infertile men with varicocele (mean age of 30.76 ± 6.47) and 159 normal controls (mean age: 28.8 ± 6.01) was not significant (P=0.785). Mutation analyses for the mitochondrial
The m.9911C>A mutation, an aromatic amino acid phenylalanine codon (TTC), changes to leucine codon (TTA), a hydrophobic amino acid at position 235 (designated F235L) in 1 patient (Fig .2,) and the novel 9929C>A mutation changes a polar tyrosine to threonine.
Two novel mutations were detected in 5 patients with one (9929C>A) being a nonsense mutation and changes tyrosine to stop codon (Y241X) and the other (10141C>G) being a missense mutation that changes Asparagine to Lysine (N27K). Also, three synonymous polymorphisms were found that were not reported previously (Table 2,). The other variation was the m.10463T>C substitution (homoplasmic state) in the tRNAArggene that was found in 3 patients.
|- Protein alignment of m.9911C>A missense mutation MT-COX3 and the arrow indicate the site of the F235L mutation.|
|Locus||Position||Nucleotide change||Amino acid position||No. of individuals||Hetero/Homo||Previously reported|
|MT-COX3||9932||G→A||W242W||1||Homo||Yes (21, 22)|
|MT-TR||10463||T→C||tRNAArg||6||Homo||Yes (23, 24)|
One of the most frequent causes of male infertility is varicocele, however, the pathogenic mechanisms by which it leads to changes in spermatogenesis are not clear (25). Some of these mechanisms may be related to mutations in mitochondrial complexes that affect flagellar movement and cause sperm dysmotility.
DNA alterations including point mutations and deletions of mtDNA have been reported in infertile patients with low sperm motility (26). The effect of mtDNA mutations on male infertility has also been studied. Shamsi et al. (27) reported that generation of ROS and mtDNA mutations are associated with pathogenic molecular mechanisms. Agarwal et al. (28) showed an increased oxidative stress in varicocele patients. Thangaraj et al. (29) demonstrated that sperm mitochondrial mutations is one of the causes of low sperm motility which is strongly dependent on ATP biosynthesis which is carried out by the mitochondrial OXPHOS system. Furthermore, it has been demonstrated that cells with some base substitutions in mtDNA can greatly influence semen quality (9,30,31).
It has been established that mitochondrial dysfunction caused by mtDNA mutations and oxidative damages is one of the important reasons for most types of infertility such as Varicocele (32). The mtDNAs alterations may accumulate in the spermatids or during gametogenesis and thereby impair the respiratory function and motility of spermatozoa (33).
We observed three heteroplasmic variations in 4 patients. A nine base pair heteroplasmic insertion in the non-coding MT-NC7 locus were found in 1 patient. Although this insertion (5ˊ-CCCCCTCTA-3ˊ) has been found in a noncoding region, it may cause mitochondrial rearrangements and DNA strand break affected by topoisomerases or DNA recombinase (34).
The heteroplasmic m.9911C>A and m.9929C>A transversions in
Here, we describe seven homoplasmic variants in 25 patients: two missense mutations in
Because sperms require an optimal energy to reach the oviduct during fertilization, the appropriate bioenergetic function of mitochondria is critical for male infertility. Therefore, any changes in mitochondrial genome can cause improper functioning of respiratory chain that in combination with environmental risk factors lead to infertility in men. This first Iranian study revealed that some Iranian infertile men carry variants in the nine mitochondrial genes and suggests that variants in these genes may be associated with varicocele.
This work was financially supported by Yazd University. We thank all the patients for providing blood samples for this study. The authors hereby declare that there is no conflict of interests.