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[P-181] A COMPARATIVE STUDY OF THREE COMMERCIALLY AVAILABLE CRYOPROTECTANTS FOR CRYOPRESERVATION OF HUMAN SPERM.

S. Verza Junior, C. Manzzi Feijó, D. Telles Schneider, S. C. Esteves. ANDROFERT - Centro de Referência em Infertilidade Masculina, Campinas, Brazil

Objective: Indications for cryopreservation of human spermatozoa have spread out since the introduction and developments of assisted reproduction techniques. Refinements in freezing protocols and cryoprotectants are clinical useful specially for poor semen quality and for freezing spermatozoa retrieved from the testis and epidydimis. The aim of this study is to compare 3 commercially available cryoprotectants for cryopreservation of human spermatozoa.

Design: Prospective, comparative laboratory study.

Material and methods: IRB was obtained for this study. Semen specimens from 23 infertile men were obtained by masturbation after 2-3 days of sexual abstinence. After liquefaction, a small aliquot was removed for semen analysis for determination of total (%MOT) and progressive motility (%PROG). Samples were then divided into three equal aliquots, each of them diluted (1:1, v/v) with a different cryoprotectant, as follows: group 1: TEST yolk buffer with glycerol (TYB, Irvine Scientific); group 2: Sperm Freezing Medium (SFM, Medicult); and group 3: Sperm Freezing Medium (SFM, LifeGlobal). Specimens were then cryopreserved by using the standard liquid nitrogen vapor technique, and stored in liquid nitrogen for at least 48 h before thawing. Thawing was carried out at room temperature for 5 minutes and at 37°C for 20 minutes. After removing cryoprotectants, %MOT, %PROG and cryosurvival rates were determined. Student´s t test was used to analyze %MOT and % PROG from before freezing to after thawing. Repeated measures analysis of variance was used to compare differences in sperm parameters among the 3 groups after thawing.

Results: There was a significant decrease in the total (%MOT) and progressive (%PROG) motility from before freezing to after thawing for all cryoprotectants tested. However, we observed better post-thaw sperm survival and motility when cryoprotectants 2 and 3 were used as compared to cryoprotectant 1.

Sperm parameters before freezing and after thawing by using 3 different cryoprotectants
	Group 1: TYB (Irvine)	Group 2: SFM (MediCult)	Group 3: SFM (GlobalLife)	p2
% motility before freezing (%MOT)	62.2±16.2	62.7±14.4	63.3±15.6	NS
% motility after thawing (%MOT)	30.7±16.5	35.4±14.6	35.6±17.2	0.041x2;0.011x3
progressive motility before freezing (%PROG)	55.2±9.9	56.8±8.1	55.1±11.4	NS
progressive motility after thawing (%PROG)	27.6±16.2	33.8±8.3	34.6±15.7	0.011x2; 0.011x3
cryosurvival rate (%CRYO)	48.3±21.8	55.9±17.7	56.1±22.3	0.041x2;0.021x3
p1	<0.01	<0.01	<0.01
NS= Not significant; p1=comparison from before freezing to after thawing; p2= comparison among cryoprotectants tested; P<0.05 considered significantCryosurvival rate: %MOT after thawing/%MOT before freezing

Conclusion: All cryoprotectants proved to be efficient for cryopreservation of human sperm. However, we observed better cryosurvival rates when using cryoprotectants manufactured by Medicult and LifeGlobal as compared to Irvine. These findings may be useful for the selection of cryoprotectants that can optimize cryosurvival rates. Finally, we suggest that cryoprotectans that yields better sperm recovery should be the choice specially when the laboratory is dealing with poor sperm quality samples.

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