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

Abstract

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.

Laser-assisted selection of immotile spermatozoa has no effect on obstetric and neonatal outcomes of TESA-ICSI pregnancies

“…The male patients were placed in the supine position and disinfected following routine procedures [ 14 ]. Anesthesia was performed by using 2% lidocaine to block the spermatic cord.…”

“…Many approaches have been developed for detecting the viability of immotile sperm, and these include hypo-osmotic swelling (HOS) tests [ 9 ], use of chemicals for induction of tail movement [ 3 ] and laser [ 10 , 11 ]. Studies have shown that the use of laser-assisted selection of viable but immotile spermatozoa for ICSI can provide better merits than conventional ICSI using other immotile sperm selection methods [ 12 – 14 ]. ICSI with viable but immotile spermatozoa selected by laser assessment could result in similar fertilization and embryo cleavage rates when compared with use of motile testicular spermatozoa [ 15 ].…”

Pengaruh penambahan ekstrak bawang merah dalam pengencer Ringer’s Dextrose terhadap kualitas sperma Gallus domesticus pada suhu 5°C

“…shown dead sperm cells were able to absorb the eosin-nigrosine staining while live sperm cells could not to absorb the eosin-nigrosine (Kondracki et al, 2017) stated that sperm cells did not absorb the staining because their plasma membranes were fully reliable, meanwhile, the dead sperm cells' plasma membranes have been damaged so that they were able to absorb the stain. (Chen et al, 2019) reported that the evaluation of spermatozoa viability using the eosin-nigrosine staining test was recommended by the fifth edition of the World Health Organization (WHO) Laboratory Manual for examining human semen. By using this staining method, the immotile sperm could be known.…”

Laser-assisted selection of immotile spermatozoa has no effect on obstetric and neonatal outcomes of TESA-ICSI pregnancies

“…Many approaches have been developed for detecting the viability of immotile sperm, and these include hypo-osmotic swelling (HOS) tests [9], use of chemicals for induction of tail movement [3] and laser [10,11]. Studies have shown that the use of laser-assisted selection of viable but immotile spermatozoa for ICSI can provide better merits than conventional ICSI using immotile sperm methods [12][13][14]. ICSI with viable but immotile spermatozoa selected by laser assessment could result in similar fertilization and embryo cleavage rates when compared with use of motile testicular spermatozoa [15].…”