Sundararajan Venkatesh

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.

Analysis of sperm nuclear protein gene polymorphisms and DNA integrity in infertile men

Sperm nuclear proteins, the protamines (PRM) and transition nuclear proteins (TNP) play a crucial role in sperm nuclear condensation. The compact packaging of sperm DNA by protamines maintains sperm genome integrity, which is prerequisite for normal sperm function. However the effect of nucleotide variations in PRM and TNP genes on sperm DNA integrity and male fertility is not clear. This case-control study was planned to analyze PRM and TNP gene nucleotide variations and sperm DNA integrity in 100 oligozoospermic infertile men and 100 fertile controls. Protamine and TNP genes were amplified by polymerase chain reaction and sequenced. Flow cytometry-sperm chromatin structure assay (FC-SCSA) was applied to measure the DNA fragmentation index (DFI) in sperm. Semen analysis was performed as per WHO [1999] guidelines with slight modification. In total, 7 nucleotide variations including two novel changes, a non-synonymous mutation in the exon-2 of PRM2 gene (c.443C > A) and a novel insertion of T (c.396_397InsT) at the 3′ UTR region of TNP1 were detected. None of the nucleotide changes were observed with increased risk frequency in the oligozoospermic infertile men compared to the controls. Though overall DFI was significantly (p < 0.0001) higher in infertile men compared to controls (36.31 ± 7.25 vs. 26.49 ± 2.78) irrespective of nucleotide changes, no such difference was observed between 100 infertile men or pooled population of 200 with and without mutations. However it was observed that two cases with novel nucleotide changes PRM2 c.443C > A and TNP1 c.396_397InsT had higher DFI value of 34.82% and 43.85%, respectively. In conclusion, our pilot study for the first time in the Indian population revealed two rare novel mutations in sperm nuclear protein genes that are perhaps associated with higher sperm DNA fragmentation.

Chromosomal abnormalities & oxidative stress in women with premature ovarian failure (POF)

Background & objectives: Premature ovarian failure (POF) is defined as the cessation of ovarian function under the age of 40 yr and is characterized by amenorrhoea, hypoestrogenism and elevated serum gonadotrophin levels. The cause of POF remains undetermined in majority of the cases. This study was aimed to investigate the type and frequency of cytogenetic abnormalities in patients with idiopathic POF and also to study the role of oxidative stress in such cases. Methods: Seventy five women with idiopathic POF were included in this study. Chromosome analysis was done in peripheral blood lymphocytes by conventional GTG banding to identify numerical or structural abnormalities. Cytogenetically normal cases were investigated for reactive oxygen species (ROS) levels in their blood by luminol-chemiluminescence assay. Results: Eighteen chromosomal anomalies were identified in POF patients (24%). Majority of the cases were found to have X-chromosome abnormalities (28%). Overall median ROS range was found to be significantly higher (P<0.01) in POF patients [50480 (120,132966) RLU/min] compared to controls [340 (120,5094) RLU/min]. Among these, 50 per cent of the POF patients had higher ROS levels, 20 per cent had medium elevation and 30 per cent were found to have normal values comparable to controls. Interpretation & conclusions: X-chromosome anomalies were found to be the major contributor of POF. Oxidative stress may be the underlying aetiology in idiopathic premature ovarian failure. Thus the results of this study highlight the role of cytogenetic abnormalities and supraphysiological levels of ROS in causation of idiopathic POF. But the role of oxidative stress needs to be confirmed by other studies on patients from different geographical areas and from different ethnicities.

Genetic Testing in Male Infertility

Infertility is a major health problem which affects approximately 22% of married couples in reproductive age. The apparent increased incidence of male infertility, in parallel with the widespread use of in vitro fertilization (IVF), raises concern as to the impact of advanced assisted conception techniques in transmitting genetic anomalies to the offspring. Recent research has widely focused on genetic factors underlying male infertility and several genetic tests are now clinically available. Genetic testing plays an important role not only to identify the etiology of male infertility but also aids in counseling as well as in the prevention of the transmission of genetic defects to the offspring via assisted reproduction. This review is focused on the syndromic and non-syndromic causes of male infertility and the diagnostic tests use in their evaluation.

Segregation of sperm subpopulations in normozoospermic infertile men

Sperm function is essential for fertilization and embryogenesis yet semen contain a heterogeneous population of sperm. This study was designed to evaluate two different sperm populations separated by the density gradient method. Semen from 25 idiopathic normozoospermic infertile men was processed by double density gradient centrifugation and evaluated for sperm present in the 50% (upper) layer and the 90% (lower) layer for reactive oxygen species (ROS), sperm chromatin integrity, and morphology. The population of sperm in the 90% layer showed significantly lower ROS levels (22.90 (0.92, 85.32) vs. 382.03 (158.30, 1409.51) and lower DNA fragmentation index (DFI) (24.26 (22.54, 25.50) vs. 29.93 (28.48, 31.25) and higher number of sperm with normal morphology (55 (45.0, 60.0) vs. 32.5 (20, 40) compared to sperm in the 50% layer. However, in the original raw semen, sperm DFI (27.02 (26.19, 27.76)) and percentage high DNA stainability (% HDS) (3.1 (2.40, 3.78)) cells were significantly higher compared to the 90% layer population. Density gradient separation of the sperm subpopulation from the original semen favors the selection of sperm with genome integrity, low levels of ROS, and normal morphology. Therefore presence of pathological sperm in the semen may disrupt the function of normal spermatozoa, and hence the selection of the normal sperm subpopulation may be a better candidate for assisted conception. Further studies are required to evaluate the gradient separated sperm population in assisted reproductive techniques (ART).

Acridine orange binding to RNA interferes DNA fragmentation index calculation in sperm chromatin structure assay.

Sperm chromatin is a highly organized, condensed, and compact structure, which is considered to be an important factor for normal fertilization and pregnancy outcome (2). The sperm chromatin structure assay (SCSA) is an excellent and most predictable and clinically correlated test, widely used in estimation of DNA fragmentation index. Acridine orange, a metachromic dye, has the property of shifting fluorescence to red when it binds to single-stranded DNA (ssDNA) or RNA from green when it is bound to native doublestranded DNA (dsDNA). In our recent work on SCSA, we found decreased mean red fluorescence after treatment with RNase compared to the mean value without RNase treatment (unpublished data). This shows that the number of cells showing red fluorescence (indicating DNA damage) may be overestimated by SCSA due to presence of single stranded RNA, which also binds to acridine orange.