Repeated vitrification/warming of human sperm gives better results than repeated slow programmable freezing

Abstract

In this study, we compared the effects of repeated freezing/thawing of human sperm by our in-house method of rapid freezing with slow programmable freezing. Sperm samples from 11 normozoospermic subjects were processed through density gradients and divided into three aliquots: non-frozen, rapid freezing and slow programmable freezing. Sperm in the rapid freezing group had better motility and viability than those in the slow freezing group (P<0.01) after the first, second and third cycles of freezing/thawing, but there was no difference in morphology. In the second experiment, rapid freezing was repeated three times in 20 subjects. The samples from each thawing cycle were evaluated for DNA fragmentation using the alkaline comet assay. DNA fragmentation began to increase considerably after the second cycle of freezing/thawing, but to a level that was not clinically important. In the third experiment, rapid freezing was done repeatedly in 10 subjects, until no motile sperm were observed after thawing. The median number of repeated freezing/thawing that yielded no motile sperm was seven (range: 5-8, mean: 6.8). In conclusion, we demonstrated that repeated freezing/thawing of processed semen using our rapid freezing method gave better results than standard slow programmable freezing. This method can help maximize the usage of precious cryopreserved sperm samples in assisted reproduction technology.

Effect of sperm storage and selection techniques on sperm parameters

“…In cases where repeated cryopreservation is done on the same sample, vitrification resulted in better post-thaw sperm parameters than standard slow programmable freezing [Vutyavanich et al 2012]. This study was performed using high-quality semen samples, so further studies are needed using patients with low-quality sperm [Vutyavanich et al 2012]. Case reports of live birth using vitrified spermatozoa and ICSI or IUI suggests it may be widely used in the near future [Sánchez et al 2012b].…”

“…Vitrification, on the other hand, does not require any post-thaw processing. In cases where repeated cryopreservation is done on the same sample, vitrification resulted in better post-thaw sperm parameters than standard slow programmable freezing [Vutyavanich et al 2012]. This study was performed using high-quality semen samples, so further studies are needed using patients with low-quality sperm [Vutyavanich et al 2012].…”

Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage

“…Successful vitrification of small volume of semen and the use of vitrification devices were reported previously [10, 20] by aliquoting a suspension of washed semen aliquoted by a single channel pipette into liquid nitrogen and collected by a strainer, also demonstrated superiority of such method over conventional freezing. Rapid cooling rate in liquid nitrogen also greatly shortens the process of change of state of cellular contents [21, 22], effectively avoiding any damage resulting from crystallisation action compared with the 2-stage cooling process in conventional slow freezing [23]. The immersion and complete cooling of the vitrification device in LN2 prior to the start of the procedure minimises the heat insulation effect of film boiling during plunging of the sample into LN2.…”

Density Gradient Centrifugation Pra-freezing Mengoptimalkan Persentase Morfologi Normal Spermatozoa Pasca-thawing

“…2 Kriopreservasi spermatozoa pada umumnya digunakan sebelum dilakukan kemoterapi atau radioterapi, pada kasus oligoasthenoteratozoospermia (OAT) yang berat, gangguan ejakulasi serta pada kasus donor spermatozoa. 3 Prosedur kriopreservasi yang terdiri atas prosedur freezing dan thawing akan memberikan efek merugikan terhadap struktur dan fungsi spermatozoa, 4 yakni meningkatkan persentase spermatozoa dengan motilitas yang buruk dan morfologi abnormal, 5,6 menginduksi dekondensasi kromatin, serta meningkatkan fragmentasi DNA spermatozoa. 7 Teknik freezing yang digunakan saat ini dapat dibedakan menjadi teknik freezing konvensional dan teknik freezing mutakhir.…”