Influence of sperm immobilization on onset of Ca2+ oscillations after ICSI

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Abstract
Sperm immobilization prior to intracytoplasmic sperm injection (ICSI) is thought to be necessary for efficient fertilization. A variety of methods of sperm immobilization (pipetting, squeezing and piezo application) are currently employed in ICSI. The effect of differences in immobilization method on the timing of initial Ca2+ oscillations of oocytes in ICSI was investigated. Motile spermatozoa were immobilized in eosin Y solution using pipetting, squeezing and piezo application. Complete staining of the sperm head was achieved after 220.7, 42.2 and 5.0 s respectively. Oscillations after ICSI were measured fluorometrically for each method. The onset of Ca2+ oscillations was observed at 4.8 to 80.4 min after ICSI. Ca2+ oscillations developed earlier with the piezo method (14.4 ± 6.4 min) than other methods (pipetting, 43.1 ± 20.2 min, P < 0.01; squeezing, 18.4 ± 3.8 min, P = NS). The piezo method produced the earliest staining of the sperm head and may have caused the most damage to the sperm membrane. A more rapid onset of Ca2+ oscillations was also observed with the piezo method. The method of sperm immobilization may be important for the rapid release of sperm factors that initiate oocyte activation. This study also showed that Ca2+ oscillations develop earlier in human oocytes treated by ICSI than indicated in previous reports.