<i>In Vitro</i> Matured Oocytes Are More Susceptible than <i>In Vivo</i> Matured Oocytes to Mock ICSI Induced Functional and Genetic Changes

<div><p>Background</p><p>Concerns regarding the safety of ICSI have been intensified recently due to increased risk of birth defects in ICSI born children. Although fertilization rate is significantly higher in ICSI cycles, studies have failed to demonstrate the benefits of ICSI in improving the pregnancy rate. Poor technical skill, and suboptimal <i>in vitro</i> conditions may account for the ICSI results however, there is no report on the effects of oocyte manipulations on the ICSI outcome.</p><p>Objective</p><p>The present study elucidates the influence of mock ICSI on the functional and genetic integrity of the mouse oocytes.</p><p>Methods</p><p>Reactive Oxygen Species (ROS) level, mitochondrial status, and phosphorylation of H2AX were assessed in the <i>in vivo</i> matured and IVM oocytes subjected to mock ICSI.</p><p>Results</p><p>A significant increase in ROS level was observed in both <i>in vivo</i> matured and IVM oocytes subjected to mock ICSI (P<0.05-0.001) whereas unique mitochondrial distribution pattern was found only in IVM oocytes (P<0.01-0.001). Importantly, differential H2AX phosphorylation was observed in both <i>in vivo</i> matured and IVM oocytes subjected to mock ICSI (P <0.001).</p><p>Conclusion</p><p>The data from this study suggests that mock ICSI can alter genetic and functional integrity in oocytes and IVM oocytes are more vulnerable to mock ICSI induced changes.</p></div

Effect of mock injection on Reactive oxygen species level in <i>in vivo</i> and <i>in vitro</i> matured murine oocytes assessed using 2′,7′-dichlorodihydrofluorescein diacetate (DCHFDA) staining.

<p>A. Oocytes retrieved from superovulated mice, subjected to mock ICSI were assessed for ROS production. The relative ROS intensity in standard control (N = 19), ICSI control (N = 22) and ICSI group (N = 20), was determined. The error bars represent the corresponding SEM (Mean ± SEM). ^aP <0.05: Standard control Vs ICSI control, ^bP <0.01: Standard control Vs ICSI group, ^cP < 0.001: ICSI control Vs ICSI group. B. <i>In vitro</i> matured metaphase II oocytes, subjected to mock ICSI were assessed for ROS production. The relative ROS intensity in standard control (N = 28), ICSI control (N = 25) and ICSI group (N = 26), was determined (Mean ± SEM). ^dP < 0.001: Standard control Vs ICSI group and ICSI control Vs ICSI group.</p

Mitochondrial distribution and activity as measured by Rhodamine 123 and JC1 staining of oocytes.

<p>A. Oocytes retrieved from superovulated mice, subjected to mock ICSI were assessed for mitochondrial distribution. The percentage of oocytes displaying uniform (closed bar); and aggregated (grey bar) distribution in standard control (N = 28), ICSI control (N = 49) and ICSI group (N = 36), was determined. B. <i>In vitro</i> matured metaphase II oocytes, subjected to mock ICSI were evaluated for mitochondrial distribution. The percentage of oocytes displaying uniform (closed bar); aggregated (grey bar) and peripheral (open bar) distribution in standard control (N = 33), ICSI control (N = 33) and ICSI group (N = 32), was determined. ^aP <0.05: Uniform distribution pattern in standard control of figure A Vs Standard control in figure B. ^bP < 0.001: Standard control Vs ICSI control. ^cP < 0.01: Standard control Vs ICSI group. ^dP < 0.0001: percentage of oocytes displaying peripheral distribution in ICSI group with other two groups. C. Mitochondrial activity as measured by the JC1 ratio in IVM oocytes in standard control (N = 81); ICSI control (N = 69) and ICSI group (N = 68). Please note that difference were not significant.</p

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

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

Basic semen characteristics in relation to different EA intervals.

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

Incidence of sperm aneuploidy in the health workers exposed to ionizing radiation at workplace.

<p>Incidence of sperm aneuploidy in the health workers exposed to ionizing radiation at workplace.</p

Characteristics of the subjects included in the study.

<p>Characteristics of the subjects included in the study.</p