<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

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

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