Comet assay: A prognostic tool for DNA integrity assessment in infertile men opting for assisted reproduction.

Background & objectives: The growing concern on transmission of genetic diseases in assisted reproduction technique (ART) and the lacunae in the conventional semen analysis to accurately predict the semen quality has led to the need for new techniques to identify the best quality sperm that can be used in assisted procreation techniques. This study analyzes the sperm parameters in the context of DNA damage in cytogenetically normal, AZF non deleted infertile men for DNA damage by comet assay. Methods: Seventy infertile men and 40 fertile controls were evaluated for the semen quality by conventional semen parameters and the sperms were also analyzed for DNA integrity by comet assay. The patients were classified into oligozoospermic (O), asthenozoospermic (A), teratozoospermic (T), oligoasthenoteratozoospermic (OAT) categories and infertile men with normal semen profile. The extent of DNA damage was assessed by visual scoring method of comets. Results: Idiopathic infertile men with normal semen profile (n=18) according to conventional method and patients with history of spontaneous abortions and normal semen profile (n=10) had high degree of DNA damage (29 and 47% respectively) as compared to fertile controls (7%). The O, A, T and OAT categories of patients had a variably higher DNA damage load as compared to fertile controls. Interpretation & conclusions: The normal range and threshold for DNA damage as a predictor of male fertility potential and technique which could assess the sperm DNA damage are necessary to lower the trauma of couples experiencing recurrent spontaneous abortion or failure in ART.

Antioxidant levels in blood and seminal plasma and their impact on sperm parameters in infertile men.

Excess reactive oxygen species (ROS) beyond the scavenging capacity of antioxidants leads to DNA damage and oxidation of lipoprotein components at the cellular and subcellular level. The oxidative stress (OS) adversely affects sperm function by altering membrane fluidity, permeability and impairs sperm functional competence. In the present study, the OS status in seminal plasma and blood serum in infertile men and its relationship with spermatozoa parameters have been investigated. Four groups of infertile men viz., oligozoospermic (n = 15), asthenozoospermic (n = 17), teratozoospermic (n = 19), and oligoasthenoteratozoospermic (n = 9), and healthy fertile controls (n = 40) have been analyzed for superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and malondialdehyde (MDA) in seminal plasma and blood serum. Significant correlation between blood serum SOD and sperm count has been observed in patients (p = 0.018) and controls (p = 0.021). Similarly, significant correlation between blood serum GSH and sperm progressive motility in patients (p = 0.036) and controls (p = 0.029) is observed. The low seminal MDA is associated with increase in sperm progressive motility in patients (p = 0.039) and controls (p = 0.028). Positive correlation is found between increased seminal MDA levels and abnormal sperm morphology in both patients and controls (r = 0.523, p = 0.029; r = 0.612, p = 0.034 respectively). Correlations between blood SOD and sperm count and between blood GSH levels and progressive motility suggest that these can be important biochemical markers in assaying the sperm count and motility. A negative correlation of motility with seminal MDA indicates that sperm membrane lipid peroxidation affects the fluidity and thus mobility of sperm axoneme. This affects functional competence of the sperm and acts like a biological safeguard. The results of the present study suggest the prospects of using the blood serum and seminal plasma antioxidants as a valuable tool to evaluate the sperm reproductive capacity and functional competence.

Role of reactive oxygen species in the pathogenesis of mitochondrial DNA (mtDNA) mutations in male infertility.

Infertility affects about 15 per cent married couples half of which may be attributed to men with low sperm motility (asthenozoospermia), low sperm count (oligozoospermia) or abnormal sperm morphology (teratozoospermia). As mitochondria are the energy source for initiation, differentiation and function of the germ cells, mutation in mitochondrial genome can impair the formation of mature spermatozoa. Mutations in mitochondrial genome are identified in patients with fertility problems. However, mitochondria are also both the source and target of reactive oxygen species (ROS). ROS are normally generated at low levels by human spermatozoa in order to perform its physiological function. However, if the generation of these reactive free radicals overwhelm the antioxidant defense system, this can lead to oxidative stress, which is characterized by mitochondrial and nuclear genome damage. So both ROS and mtDNA mutations are considered to be the major aetiological factors in a variety of human diseases including male infertility. Identification of novel mutations in mtDNA of infertile patients with supraphysiological levels of ROS are considered to be important to gain better understanding of the aetiology of idiopathic infertility. Early detection and prompt antioxidant therapy can prevent ROS induced DNA damage. This has far reaching impact if such men opt for assisted reproductive technology (ART)/in vitro fertilization.

Evaluation of nuclear DNA damage in human spermatozoa in men opting for assisted reproduction.

Diagnosis of sperm DNA integrity of semen sample is important for consistently high reproductive efficiency. The conventional parameters of semen analysis take into account morphology, motility, and concentration of spermatozoa in the sample, which are insufficient for evaluation of reproductive potential. Current studies have implicated abnormal organization of genomic material in sperms as a probable cause in 20 per cent cases of male infertility. This is especially important in the era of assisted reproduction technique (ART) when a majority of infertile couples opt for assisted reproduction and in where cases DNA integrity is a better diagnostic and prognostic marker as compared to routine semen analysis. This article reviews and discusses some of the current techniques employed for evaluating chromatin structure or DNA damage in spermatozoa. These different techniques include single cell gel electrophoresis (COMET assay), Terminal tranferase dUTP Nick End Labelling (TUNEL), sperm chromatin structure assay (SCSA), In situ nick translation (ISNT) and acridine orange test. These techniques are independent measure of sperm quality and assist in semen quality assessment by detecting defects in DNA integrity or chromatin structure. The discussed techniques vary in their level of accuracy, cost input, sophistication of analysis and their application depends upon the sensitivity required for analysis. The article also briefly outlines the DNA packaging and the causes of DNA damage in spermatozoa. During chromatin packing 85 per cent of the histones are replaced by protamine while the residual histones act as marker of genes which are expressed in early embryonic development. Among the different aetiological factors observed to be responsible for DNA damage in human spermatozoa increased reactive oxygen species (ROS), oxidative stress is highly correlated with greater DNA fragmentation index (DFI). Oxidative stress leads to single and double strand breaks in sperm DNA. Apoptosis and abnormal chromatin packing also contribute to DNA damage. The significance of chromatin structure studies is more stressed owing to the greater awareness to transmission of genetic diseases because of higher incidence of gene imprinting defects, increased cancer frequency and other congenital and non-congenital defects in children conceived through assisted reproduction techniques.

Necessity of nuclear and mitochondrial genome analysis prior to assisted reproductive techniques/intracytoplasmic sperm injection.

Assisted reproductive technique (ART) has revolutionized the management of severe male factor infertility and in some countries 5% babies are conceived through ART/intra cytoplasmic sperm injection (ICSI). However, the carry-home live birth rate after several ART cycles is low (18-25%) and this is financially, physically and emotionally crippling for the couples. Genetic factors could lead to pre or post-implantation failure and thus explain for low ART success rate. Thus, this study was planned to understand, if infertile men harbour genetic abnormalities which may be iatrogenically transmitted by ART and adversely affect growth potential of embryo. Ninety infertile men underwent semen, cytogenetic, Yq microdeletion and mitochondrial mutation analysis. Of these, 14.4% cases harboured cytogenetic abnormality, and 8.89% Yq microdeletions. A high frequency of mitochondrial mutations was found in 23 men with asthenospermia. It is important to understand that through ART genetic abnormalities are transmitted to offspring, resulting in impaired growth and development potential of embryo and poor take-home live birth rate. Thus, genetic analysis is strongly recommend in all men with idiopathic infertility who opt for ART to counsel couples and provide them with most adapted therapeutics.

Azoospermia factor deletions in varicocele cases with severe oligozoospermia.

BACKGROUND: Varicocele is the most common cause of male infertility. The etiology and pathophysiology of varicocele are multifactorial. When low sperm counts are associated with varicocele, varicocelectomy can partially restore spermatogenesis and fertility. Few recent studies have reported that in some varicocele cases, there may be an associated genetic etiology. Presence of a genetic factor like azoospermia factor microdeletions may lead to irreversible spermatogenic arrest in these cases, but very few reports support these findings. However, it is still not understood why some cases improve after varicocelectomy and why some cases show no improvement in semen parameters postoperatively. AIM: It is important to distinguish varicocele cases from Yq microdeletions as these cases have irreversible testicular damage and thus carry a poor prognosis after varicocelectomy. SETTINGS: Research and Referral tertiary care hospital. Design: Prospective study. MATERIALS AND METHODS: Seventy-two infertile men with varicocele were referred for Yq microdeletion analysis from the infertility clinic of AIIMS and Army Research and Referral Hospital. Genomic DNA was isolated from blood and polymerase chain reaction microdeletion screening was done in these cases to determine the presence or deletion of AZF loci. RESULTS: In this study 7 (9.7%) varicocele cases harbored Yq microdeletion. The sperm count in cases which harbored Yq microdeletion was significantly lower than in cases without Yq microdeletion. CONCLUSION: Varicocele cases with Yq microdeletion do not show improvement in semen parameters post-varicocelectomy. Detection of Yq microdeletion determines prognosis and future management in such cases.