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A viable spermatozoon is a prerequisite for fertilization in intracytoplasmic sperm injection (ICSI). Thus, it is crucial to select viable but immotile spermatozoa on the day of ICSI. We report conflicting results in the identification of viable but immotile spermatozoa between the eosin-nigrosin staining and the laser test, which resulted in confusion for embryologists during assisted reproductive technology (ART). Three patients' semen samples that showed no motile spermatozoa are described in this report. To identify viable spermatozoa, we used both the eosin-nigrosin test and the laser test for each sample, and repeated the semen analysis twice in each patient. Viable but immotile spermatozoa selected by the laser test were used for ICSI. Viable spermatozoa were detected by both the eosin-nigrosin and laser tests in two patients (case 1, 95.00% vs. 24.21% and 92.68% vs. 22.22%; case 2, 41.18% vs. 23.48% and 39.81% vs. 22.52%), indicating consistent results between the two methods. In the third patient, the eosin-nigrosin test yielded viability rates of 20.75% and 19.14%, while the result of the laser test was 0%. Thus, testicular aspiration was performed to collect viable sperm from this patient. Normal fertilization was achieved after the injection of viable but immotile spermatozoa selected from these patients by the laser test, resulting in the birth of two healthy babies. Our study documents a case where the eosin-nigrosin test showed a limitation in identifying viable but immotile spermatozoa for ART, while the laser test may overcome this limitation. Larger samples may be required to corroborate the clinical value of the laser test.

A point of confusion for embryologists in the identification of viable spermatozoa by the eosin-nigrosin test