By Dr.Burde Kaustubh Girish DNB Narayana Hrudayalaya

Transcription

1 By Dr.Burde Kaustubh Girish DNB Narayana Hrudayalaya

2 The DNA is packed in sperm in a volume that is less than 10% somatic cell nucleus During spermatogenesis, DNA is initially packaged by histones Following meiosis, at the secondary spermatocyte stage of spermiogenesis, histones are replaced first by transition proteins and then by protamines. The solenoid structure is replaced by torroids (doughnut shapes), which are in turn supercoiled into torroidal loops. This highly compacted structure shuts down transcription during spermiogenesis.

3 DNA fragmentation is the separation or breaking of DNA strands into pieces. Mechanisms 1. Aberrant packaging during spermatogenesis 2. Apoptosis before ejaculation 3. Excessive production of reactive oxygen species in the ejaculate 4. Genetics 5. Exposure to environmental factors 6. Oxidative stress

4 Oxidative Stress caused by Aberrant chromatin packing during spermatogenesis Infection,Pyrexia, Elevated testicular temperature Recreational drugs use, Smoking, Alcohol Stress Diet Environmental and occupational pollutants Advanced age, Genetics Varicocoele, Leucocytospermia Sperm cytoplasmic droplets

5 Ratio of Assay-positive (damaged) sperm to all sperm and expressed as percentage Measured by Sperm DNA integrity Assay Tertiary investigation test

6 Direct (study actual breakage in DNA) 1. Comet 2. TUNEL Indirect (measure the relative proportions of single/abnormal and double/normal DNA within the sperm following acid treatment) 1. Acridine orange assay 2. Sperm chromatin structure assay

7 Terminal deoxynucleotidyl transferase dutp nick end labeling Assess DNA fragmentation Quantifies the incorporation of dutp at double stranded DNA breaks in reaction catalyzed by template independent enzyme Tdt. The positive and negative predictive values of the 20% sperm DNA fragmentation threshold were high: 92.8% (95% CI ) and 95.5% (95% CI ), respectively.

8 COMET assay uses the electro gel electrophoresis principle to assess the DNA damage. After DNA extraction and gel electrophoresis, damaged DNA looks like a comet. The level of DNA fragmentation is measured using special software after transferring a live video image of the microscopic field into the computer. One of the indices used is tail moment, which is a relation between the size and density (DNA percent) of the comet head and tail [= TL (tail length) 3 TD (tail density)]. The tail moment lies in one of three levels: mild, moderate, and severe, and their percentage in the sample are given.

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10 Measures in-situ DNA susceptibility to the acid-induced conformational helix-coil transition by AO fluorescence staining Advantage of acid and heat treatment Accurately etimated the percentage of DNA damaged sperms Has a cutoff point DFI >30%

11 DNA Fragmentation Index (%DFI: % sperm cells containing damaged DNA) Results are reported showing 4 statistical categories of fertility potential: 15% DFI = excellent to good sperm DNA integrity > 15 to < 25% DFI = good to fair sperm DNA integrity > 25 to < 50% DFI = fair to poor sperm DNA integrity 50% DFI = very poor sperm DNA integrity

12 Unexplained Infertility Arrested Embryo Development Poor Blastocyst Development Multiple Failed IVF/ICSI treatment Recurrent miscarriage in partner Advanced age Varicocoele Poor semen parameters Exposure to harmful substances

13 Semen samples in which more than one third of DNA is fragmented have a reduced chance of resulting in pregnancy. Sperm DNA fragmentation is significantly higher in infertile men and while men with poor semen parameters are more likely to have high sperm DNA fragmentation, High sperm DNA fragmentation is also found in men with normal semen parameters who may be diagnosed with unexplained infertility. Embryos derived from sperm whose DNA is highly fragmented have a poor prognosis. High sperm DNA fragmentation is more likely to affect

14 DNA damage in the embryo could result in cell degeneration and gene mutations, leading to arrested embryo development, miscarriage, abnormalities in the offspring and an increased susceptibility to childhood cancers In couples where the male partner has a high percentage of sperm with fragmented DNA, chances of a successful pregnancy are significantly reduced.

15 Provides a reliable analysis, beyond the routine semen sample assessment, of sperm DNA integrity that may help to identify men who are at risk of failing to initiate a healthy pregnancy Information helps in the clinical diagnosis, management and treatment of male infertility Prognostic value in assessing outcome of assisted conception treatment

16 If the damage is caused by free radicals, a change in lifestyle and diet designed to protect against oxidative stress may help reduce the levels of DNA fragmentation Antibiotics in the co-existence of an infection Life style changes drugs, smoking and occupation Diet fresh foods, particularly those containing antioxidants or vitamin C & E Varicocoele repair Testicular aspiration of sperm (DNA damage occurs at the post-testicular level, hence testicular sperm may have a better DNA integrity than ejaculated sperm) ICSI rather than IVF Initiatives to reduce the levels of fragmentation can be assessed by undertaking a second test three months later

17 If DNA fragmentation index is more than 30%, it is advisable to go for Intracytoplasmic Sperm Injection (ICSI) than In Vitro Fertilisation(IVF). Another modality is magnetic cell separation which separates apoptotic and normal cells No statistical difference between the outcomes of ICSI versus IVF in the group with DFI less than 30% was seen

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