Monis Bilal Shamsi

Sperm DNA integrity assays: diagnostic and prognostic challenges and implications in management of infertility

Sperm is not a simple carrier of paternal genetic information but its role extends clearly beyond fertilization. Integrity of sperm genome is an essential pre-requisite for birth of healthy offspring and evaluation of sperm should entail DNA integrity analysis. DNA integrity analysis is a better diagnostic and prognostic marker of sperm reproductive potential. Conventional semen analysis emphasizes on sperm concentration, viability, motility and morphology and has been proven to be a poor indicator of reproductive potential and pregnancy outcome. To overcome the drawbacks associated with conventional semen analysis more useful fertility tests and molecular biomarkers have been explored. Among the different tests which have evolved for assessing the sperm reproductive potential, tests for sperm DNA quality are most promising. Sperm DNA damage has been closely associated with numerous indicators of reproductive health including fertilization, embryo quality, implantation, spontaneous abortion, congenital malformations and childhood diseases. It therefore has great potential as a prognostic test for both in vitro and in vivo conception. This review presents an updated account of tests that have better diagnostic and prognostic implications in the evaluation of sperm DNA damage. The basic principles, outline of methodology, advantage, disadvantage, clinical significance of each technique and implications of these tests have been discussed. The logistics of each test with respect to available resources and equipment in an andrology laboratory, the feasibility of performing these tests in routine diagnostic workup of infertile men and the opportunities and challenges provided by DNA testing in male fertility determination are also presented.

Clinical significance of sperm DNA damage threshold value in the assessment of male infertility

Sperm DNA integrity is a prerequisite for normal spermatozoal function. The aim of the study was to evaluate the role of sperm chromatin damage, its cut-off level and its effect on sperm parameters in men with idiopathic infertility by analyzing 100 idiopathic infertile men and 50 fertile controls. Semen samples were analyzed as per WHO 1999 guidelines and sperm chromatin structure assay (SCSA) was applied to measure DNA fragmentation index (DFI) in sperm. The mean DFI of infertile men (35.75) was significantly (P < .0001) higher as compared to controls (26.22). The threshold level of 30.28% was obtained as cut-off value to discriminate infertile men from fertile controls. Sperm count, forward motility, and normal morphology found to be negatively associated with DFI in overall study subjects. Infertile men with severe oligozoospermia had higher mean DFI (40.01 ± 11.31) than infertile men with oligozoospermia (35.11 ± 10.05) and normal sperm count (33.99 ± 9.96). Moreover 64% of infertile men have DFI > 30 against 6% of fertile controls (P < .0001). Higher sperm DNA fragmentation may be the underlying cause for poor semen quality in idiopathic infertile men and the threshold value of 30.28% is a clear discriminator to distinguish infertile men from fertile men of Indian population. Thus, DFI is a good prognostic marker as cases with higher sperm DFI may have poor success rate even after assisted conception and may experience recurrent pregnancy loss (RPL) and should be counseled accordingly.

Clinical significance of reactive oxygen species in semen of infertile Indian men

Reactive oxygen species (ROS) levels in semen are believed to play both physiological and pathological roles in male fertility. The study was aimed to find the clinical significance of ROS levels in infertile Indian men. This pilot study included 33 idiopathic infertile men and 18 proven fertile controls. ROS levels in the washed sperm were measured using chemiluminescence assay and expressed as 106 cpm per 20 million spermatozoa. Sperm count, percent sperm motility, and percent normal sperm morphology were found to be significantly (P < 0.0001) reduced in infertile men compared with the controls. Median (minimum, maximum range) ROS levels of the infertile group [24.90 (6.89, 44.71)] were found to be significantly (P < 0.0001) elevated compared with the fertile controls [0.167(0.15, 2.78)]. No significant correlation was seen between ROS levels and semen parameters. Elevated ROS levels in the idiopathic Indian infertile men may be one of the underlying reasons for impaired fertility. Therefore measurement of seminal ROS levels may be used in Indian infertile men for better understanding of the aetiology and selection of antioxidant regimen in the treatment of male infertility. However, large studies may be urgently warranted to find out the role of antioxidants in ROS elevated Indian infertile men through randomised, controlled clinical study.

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.

Genetic and epigenetic factors: Role in male infertility

Genetic factors contribute upto 15%–30% cases of male infertility. Formation of spermatozoa occurs in a sequential manner with mitotic, meiotic, and postmeiotic differentiation phases each of which is controlled by an intricate genetic program. Genes control a variety of physiologic processes, such as hypothalamus–pituitary–gonadal axis, germ cell development, and differentiation. In the era of assisted reproduction technology, it is important to understand the genetic basis of infertility to provide maximum adapted therapeutics and counseling to the couple.

Mitochondrial DNA variations in ova and blastocyst: Implications in assisted reproduction

Mitochondrial DNA (mtDNA) of oocyte is critical for its function, embryo quality and development. Analysis of complete mtDNA of 49 oocytes and 18 blastocysts from 67 females opting for IVF revealed 437 nucleotide variations. 40.29% samples had either disease associated or non-synonymous novel or pathogenic mutation in evolutionarily conserved regions. Samples with disease associated mtDNA mutations had low fertilization rate and poor embryo quality, however no difference in implantation or clinical pregnancy rate was observed. Screening mtDNA from oocyte/blastocyst is a simple, clinically reliable method for diagnostic evaluation of female infertility and may reduce risk of mtDNA disease transmission.

A COMPREHENSIVE WORK UP FOR AN ASTHENOZOOSPERMIC MAN WITH REPEATED INTRACYTOPLASMIC SPERM INJECTION (ICSI) FAILURE

Infertility affects about 15–20% couples attempting pregnancy and in about half cases the problem lies in the male. Among the sperm parameters, linear progressive motility is one of the most important predictors of fertility potential. Though genetic and chromosomal abnormalities are important aetiological factors in the pathogenesis of male infertility, the mechanism involved in impaired sperm motility is poorly understood. Here we report mitochondrial DNA (mtDNA) mutations with increased seminal reactive oxygen species (ROS) levels and higher DNA fragmentation level in the sperm resulting in decreased ATP production which plays an important role in sperm motility defect. Thus it is important to understand the aetiology of asthenozoospermia and to distinguish if infertile men harbour nuclear or mtDNA mutation as they are very important prognostic markers. This case study also highlights that routine semen parameters are very modest predictors of fertility outcome but ROS estimation and DNA integrity analysis by Comet assay have better diagnostic and prognostic capabilities. Thus this study is a detailed and comprehensive workup of an infertile asthenozoospermic male.

Herbo-mineral supplementation in men with idiopathic oligoasthenoteratospermia : A double blind randomized placebo-controlled trial

Introduction: There is insufficient scientific data on the medical management options for idiopathic oligoasthenoteratospermia (iOATs). We conducted a double blind, randomized, placebo-controlled trial to assess the efficacy and safety of the herbo-mineral supplement, Addyzoa®, in infertile men with iOATs. We also evaluated its effect on semen reactive oxygen species (ROS) levels, total antioxidant capacity (TAC) and DNA fragmentation index. Materials and Methods: Fifty infertile men with iOATS were recruited into an institutional ethics committee approved protocol from April to August 2009. Randomization was done using numbered, identical containers. Baseline semen samples were evaluated for routine parameters, ROS level, DNA fragmentation index and TAC. Drug/placebo was administered at a dose of two capsules twice a day for 3 months. All parameters were reassessed at 3 months and clinical side-effects were recorded. The study was registered with the Clinical Trials Registry of India and is available at www.ctri.in as study protocol number CTRI/2009/091/000551. Results: Forty-four subjects completed the study, 21 in the drug arm and 23 in the placebo arm. There was no difference in baseline parameters between the two groups. Men in the drug group had significant improvement in mean total motility from 23.2 ± 17.3% to 33.4 ± 23.2% (P-value: 0.008) and mean progressive (Type A+B) motility from 15.7 ± 12.6% to 22.6 ± 18.0% (P-value: 0.024). ROS, TAC and DFI did not change significantly in either group and did not show any correlation with other semen parameters. Conclusions: Treatment with Addyzoa resulted in a significant improvement in total and progressive motility in the semen of men with iOATs after 3 months of therapy. There was no change in the sperm concentration, ROS, DFI or TAC levels.

Chromosomal aberrations, Yq microdeletion, and sperm DNA fragmentation in infertile men opting for assisted reproduction

Male infertility is a multi‐factorial disorder, and identification of its etiology in an individual is critical for treatment. Systematically elucidating the underlying genetic causes (chromosomal and Yq microdeletion) and factors, such as reactive oxygen species (ROS) levels and total antioxidant capacity (TAC), which contribute to sperm DNA damage, may help to reduce the number of men with idiopathic infertility and provide them with the most suitable therapeutics and counseling. This study was done to comprehensively investigate genetic and oxidative stress factors that might be the etiology of a large percentage of men with idiopathic infertility. One hundred twelve infertile men and 76 fertile controls were screened for chromosomal aberrations and Yq microdeletions. ROS, TAC, and sperm DNA damage were assessed in cytogenetically normal, non‐azoospermic men with intact Y chromosome (n = 93). ROS was assessed in neat and washed semen by chemiluminescence; seminal TAC with a commercially available kit; and sperm DNA damage by the comet assay. Two men had cytogenetic abnormalities and seven men harbored Yq microdeletions. ROS levels in neat and washed semen of infertile men were significantly higher (P < 0.01) than controls. Infertile men had significantly lower (P < 0.01) TAC levels (1.79 mM), whereas sperm DNA fragmentation in infertile men was significantly higher (P < 0.01) than controls. Genetic factors and oxidative stress cumulatively account for large number of idiopathic infertile cases. Unlike, genetic causes, which cannot be cured, timely identification and management of oxidative stress may help to reverse/reduce the effects on induced DNA damage, and improve the outcomes for infertile males. Mol. Reprod. Dev. 79: 637–650, 2012.

Mosaic Status of Lymphocytes in Infertile Men with Klinefelter Syndrome

Abstract Chromosomal aneuploidies such as Klinefelter syndrome (47,XXY) are the most frequent chromosomal aberrations in infertile men. Normally the chromosomal status of patients is detected by karyotyping of up to 20 metaphase spreads, where low-grade mosaicism may be missed. This study was carried out to test whether Klinefelter patients with 47, XXY karyotype with few characteristic features of KFS may harbour normal 46,XY cell line. We analysed 150 well-spread G-banded metaphases in each Klinefelter patient(n-20). RESULTS: Of 20 patients, 4 patients had (5-12%) normal 46,XY cell line. This had not been identified on analyzing 20 metaphase spreads. CONCLUSIONS: These result suggests that 47,XXY patients with presence of XY cell line may have a higher probability of finding germ cells and isolated foci of spermatogenesis in the semeniferous tubules. This is a good diagnostic and prognostic marker for ART. Therefore infertile Klinefelter patients with low-grade mosaicism with 46, XY normal cell line, are good candidates for for ART/ICSI but in such cases if fertilization occurs it should be followed by preimplantation genetic diagnosis (PGD).