Effect of Cooling, Freezing and Thawing Rates and Storage Conditions on Preservation of Human Spermatozoa

Abstract

Human spermatozoa was relatively resistant to cooling shock. However, when diluted semen was cooled faster than 10" C per minute from room temperature (RT) to 5" C and rewarmed to RT, percentage motility and percentage alive of spermatozoa decreased when compared to the slower cooling rates (< 5" Clmin). The optimum cooling rate from RT to 5" C resulting in maximum survival of human spermatozoa was found to be 0.5 to 1" C per minute when cooled from RT to 5" C and subsequently frozen-thawed inliquid nitrogen(LN,). The optimal freezing rate of 10" C perminute,from 5" to -80" C, resulted in higher survival of human spermatozoa than slower (1.1 " C/min) or faster (87.1 " C/ min) freezing rates. Slow thawing in 20 or 35" C air, on a dry bench, resulted in better survival than the other slower or faster thawing methods used. The temperature at which human semen samples were transferred to LN2 significantly influenced spermatozoa survival. Survival was higher when transferred at -30" C or lower when compared with samples transferred at -1 5" C or higher. However, maximal spermatozoa survival was obtained when the samples were transferred at -80" C or lower. Transfer of human semen from LN2 to -25 to -30" C and storage for 24 hours significantly reduced spermatozoa viability when compared with storage at 196" C or -80 to -85" C. No significant differences were found between storage temperatures of -80 to -85" C and -196" C in the maintenance of spermatozoa viability for up to 90 days. EinfluR der Kiihlungs-, Gefrier-und Auftau-Raten und der Lagerungsbedingungen auf die Erhaltung menschlicher SpermatozoenZusammenfassung : Menschliche Spermatozoen waren relativ resistent gegeniiber dem Kiihlungs-Schock. Wenn das Sperma jedoch schneller gekihlt wurde, und zwar um 10" C pro Minute von der Raumtemperatur (RT) auf 5" C und wiedererwarmt wurde, fie1 der Prozentsatz der Motilitiit und der unbeweglichen Spermatozoen gegeniiber der langsameren Kiihlungsmte (unter 5" C/min.) ab. Die optimale Kiihlungsrate von der RT auf 5" C, die in einer maximalen Uberlebensrate der menschlichen Spermatozoen resultierte, wurde gefunden bei 9,5-1" Clmin., wenn von der RT auf 5" C gekiihlt wurde und spater gefroren-aufgetaut in fliissigem Stickstoff (LN,). Die optimale Gefrierrate von 10" C/min., von 5" auf -80" C resultierte in einer hoheren Uberlebensrate der menschlichen Spermatozoen gegenuber langsameren Gefrierraten (1 .lo C/min.) oder schneller (87.1" Clmin.). Langsames Auftauen in 20 oder 35" C Luft, auf einer Trockenbank, f~r t e zu einer besseren Cooling and Thawing 53 iiberlebensrate, als es mit den anderen Auftaumethoden erreicht werden konnte. Die Temperatur, bei welcher die menschlichen Spermaproben in LN2 uberfirt wurden, beeinfldte signifkant das tiberleben der Spermatozoen; diese Rate war hoher, wenn auf -30" C oder niedriger iiberfirt wurde, verglichen mit Proben, die auf -15" C oder hoher uberfirt wurden. Die maximale Spermatozoen-Uberlebensrate wurde erreicht, wenn die Spermaproben auf -80" C oder niedriger uberfiirt wurd...

Human Sperm Cryopreservation: Update on Techniques, Effect on DNA Integrity, and Implications for ART

“…It has been shown that the optimal initial cooling rate of the specimen from room temperature to 5°C is 0.5–1°C/min [10]. The sample is then frozen from 5°C to −80°C at a rate of 1–10°C/min.…”

“…Mitochondria are placed along the midpiece between the plasma membrane and the nine fibrous columns, to form a coating that provides energy necessary for sperm motility [27]. The greatest amount of energy is provided by molecules of ATP synthesized either by glycolysis in the cytoplasm [28] or through oxidative phosphorylation (oxphos) in the mitochondria [10]. …”

Survival of Human Sperm Following Controlled- and Noncontrolled-Rate Cryopreservation/Die Überlebensrate menschlicher Spermatozoen nach einer kontrollierten und nicht kontrollierten Kryokonservierung

“…Two factors which are important for successful freezing of semen are (1) slow cooling to + 5 O C (Mahadevan and Trounson -1984) and (2) adequate circulation of liquid nitrogen vapors around semen to produce uniform and fast rates of freezing from + 5 O C to -80 "C (Mahadevan and Trounson -1984;Hammitt and Martin -1988).…”

“…A second objective was to examine whether sperm survival following NCR freezing could be improved by thawing at a faster rate, in 40 O C water, than with thawing at the standard slower rate of room temperature (Mahadeven and Trounson -1984;Serafini and Marrs -1986). It was hypothesized that a faster rate of thawing might be required with NCR freezing because the rate of semen freezing from + 5 O C to -180°C was faster for the NCR than for the CR method.…”

Options thérapeutiques

“…la congélation et la décongélation (Mahadevan et Trounson 1984). Ces méthodes, basées sur un refroidissement lent dans le glycérol, ont été largement adoptées pour la conservation du sperme pour la FIV et l'IAD ).…”