S. Venkatesh

Telomere length in reproduction

Telomeres, noncoding hexameric tandem repeats located at the ends of chromosomes, maintain chromosome stability and genome integrity. These guanine‐rich repeats are highly conserved during evolution, and their role is dependent on their length and structure. They have multiple functions, including regulating the reproductive lifespan by mediating synapsis and homologous recombination of the chromosomes. Short telomeres result in meiotic arrest, segregation abnormalities and dysjunction, which lead to an increased incidence of aneuploid germ cells. In addition, shortened telomeres in men result in apoptosis of germ cells, whereas, in women, they result in meiotic arrest. In somatic cells, telomere shortening occurs at each consecutive round of replication, which induces senescence in vitro and in vivo. However there is a 2‐fold elongation of telomeres during spermatogenesis. Spermatozoa, are terminally differentiated cells, have longer telomeres than spermatogonia and pachytene spermatocytes. In addition to genetic factors, lifestyle factors and psychological stress also play crucial role in modulating telomere length. Because not much is known about its role in reproduction, we focused this review on the function, structure and length dynamics of the telomere in the reproductive process.

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.

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.

An evolutionary insight into mutation of ATPase6 gene in primary ovarian insufficiency

In our earlier study [1], we reported the association of primary ovarian insufficiency (POI) with non-synonymous mutations in the ATPase6 gene. It has been claimed that these changes are phylogenetic dependent and are not associated with the POI [2]. It has also been quoted that out of the six mutations reported in the ATPase6 gene, five were attributable to distinct south Asian, West Eurasian and East Asian haplogroups’. However, it is not acceptable at this point of view as five different ethnicities or haplogroups cannot be classified based on only these five different mitochondrial substitutions. After the published comment on our findings, we did an intensive study of mtDNA haplogroups of each patient samples and found that on the basis of single mitochondrial substitution, the whole lineage cannot be defined and so their haplogroups. Every individual whether it is a case or control, falls under a specific haplogroup which is characterized by the series of homoplamsic mitochondrial mutations. If any non-synonymous mutation is found in mitochondrial genome which is not a part of individual specific phylogenetic lineage then it may alter the health profile of the same individual and hence included in our study as pathogenic [1]. Moreover, if the transitions occur in the coding region and interestingly if they are nonsynonymous mutations changing the chemical nature of the amino acid, the individual carrying this may be more susceptible for the disease. Thus, the severity of mutations to the health of any individual is not affected by whether they are phylogenetic dependent or not. Moreover, our study is strictly based on clinical association, where cases had high ROS levels, and was not intended for phylogenetic analysis. After an intensive study of mtDNA haplogroups (Table 1), importantly, the virtual absence of transitions at 8137 and 8584 suggests that the patients are neither falling in M8 nor in U7 haplogroup. Instead, the presence of 8502 transition in the sample containing 8684C mutation suggests that they fall in M2 haplogroup [3]. This mutation is present in 7 out of 20 patients. In addition to that the absence of 1811T mutation in the cases suggests yet another evidence contrary to comments, as the presence of 1811T is mandatory to define either M39 or U20304070809 haplogroup [4, 5]. Moreover, the frequently found 12705T with 9094C (which is present in 5 out of 20 patients) was claimed to be overwhelming Indian specific R haplogroup instead of U2b2, whereas the non-synonymous 9094C has never been reported as a part of macro haplogroup R. Hence, now the correlation is more positive between 8684C, 9094G and occurrence of POI. We have also not found any nine base pair deletion (represents B4 and B5 haplogroup) in the samples having 9123G transition which was claimed to fall in B4a haplogroup. At last, the nonsynonymous mutation 9064G was claimed to be present in four different haplogroups and intriguingly they all belong to macro haplogroup M, but surprisingly we have found 10398G instead of 10400T which shows that this sample may fall in any haplogroup but not in M. In addition to this, we have also initiated another study enrolling more cases on these aspects to come to a conclusion, because reactive oxygen species are by product of mitochondrial respiratory chain and may be produced in excess in cases with mitochondrial dysfunction due to mitochondrial nucleotide changes [6]. We further strongly state that the results of our preliminary pilot study [1] highlights the need for larger S. Venkatesh R. Dada (&) Laboratory for Molecular Reproduction and Genetics, Department of Anatomy, All India Institute of Medical Sciences, New Delhi 110029, India e-mail: rima_dada@rediffmail.com