Sperm motility and DNA fragmentation analysis in relation to EA.

<p>A) Sperm motility analysis in neat ejaculate (N = 76) (■) as well as in the processed fraction (N = 32) from four study intervals. Processed fraction was incubated at 37°C and motility analysis was performed at 0h (●), 6h (▲), and 24h (▼) time interval. Please note that differences in sperm motility with corresponding EA intervals were not statistically significant. B) Box plot depicting the DNA fragmentation level as measured by the sperm chromatin dispersion (SCD) assay in the neat ejaculate (N = 56) (□) and processed fraction (N = 32) (■). ^aP < 0.05 <i>Vs</i> corresponding group in EA-5; ^bP < 0.001 <i>Vs</i> corresponding group in EA-7; ^c P < 0.01 <i>Vs</i> corresponding group in EA-7; ^dP < 0.05 <i>Vs</i> corresponding group in EA-7. C) Sperm DNA longevity analysis by SCD assay in the processed fraction at 0h (■), 1h (●), 6h (▲), and 24h (▼) time interval.</p

Sperm Chromatin Immaturity Observed in Short Abstinence Ejaculates Affects DNA Integrity and Longevity <i>In Vitro</i>

<div><p>Background</p><p>The influence of ejaculatory abstinence (EA) on semen parameters and subsequent reproductive outcome is still debatable; hence understanding the impact of EA on sperm structural and functional integrity may provide a valuable information on predicting successful clinical outcome.</p><p>Objective</p><p>To understand the influence of EA on sperm chromatin maturity, integrity, longevity and global methylation status.</p><p>Methods</p><p>This experimental prospective study included 76 ejaculates from 19 healthy volunteers who provided ejaculates after observing 1, 3, 5 and 7 days of abstinence. Sperm chromatin maturity, DNA integrity and global methylation status were assessed in the neat ejaculate. Sperm motility, DNA integrity and longevity were assessed in the processed fraction of the fresh and frozen-thawed ejaculates to determine their association with the length of EA.</p><p>Results</p><p>Spermatozoa from 1 day ejaculatory abstinence (EA-1) displayed significantly higher level of sperm chromatin immaturity in comparison to EA-3 (P < 0.05) and EA-5 (P < 0.01) whereas; the number of 5-methyl cytosine immunostained spermatozoa did not vary significantly across groups. On the other hand, <i>in vitro</i> incubation of processed ejaculate from EA-1 resulted in approximately 20 and 40 fold increase in the DNA fragmented spermatozoa at the end of 6 and 24h respectively (P < 0.01–0.001).</p><p>Conclusion</p><p>Use of short-term EA for therapeutic fertilization would be a clinically valuable strategy to improve the DNA quality. However, use of such spermatozoa after prolonged incubation <i>in vitro</i> should be avoided as it can carry a substantial risk of transmitting DNA fragmentation to the oocytes.</p></div

Basic semen characteristics in relation to different EA intervals.

<p>Basic semen characteristics in relation to different EA intervals.</p

Sperm chromatin maturity and hypermethylation level at various EA periods.

<p><b>A)</b> Box plot demonstrating the percentage of aniline blue positive spermatozoa (suggestive of immature chromatin) in the neat ejaculate from four study intervals (N = 32) (^a P < 0.05 <i>Vs</i> EA-1, ^b P < 0.01 <i>Vs</i> EA-1). <b>B)</b> Box plot demonstrating the percentage of 5mC positive spermatozoa (suggestive of hypermethylation) across study intervals (N = 40). Please note that the differences were not statistically significant.</p

DNA fragmentation analysis in frozen thawed spermatozoa.

<p>DNA fragmentation level was measured by the sperm chromatin dispersion (SCD) assay in the processed fraction at 0 (N = 32) (□) and 6h (N = 32) (■). Please note that fold change in the sperm DNA fragmentation level with corresponding interval of EA were not statistically significant.</p