| Cryopreservation of human sperm is currently widely used in Assisted Reproductive Technology(ART) in male fertility storage.With the increasing of male infertility patients, how to save the oligozoospermic sample,retrograde ejaculation sperm, and sperm retrieved from the epididymis or testis in patients with azoospermia has been attracting more attention.Permeating cryoprotectants(CPA) was used in conventional freezing will produce osmotic shock, negative effect on DNA in addition and removal,and lose sperm by washing and centrifugation to removal CPA. Vitrified without permeable cryoprotectants obtain high cooling rates,avoid the ice crystal in frozen and complex treatment program after thawing, improve the recovery of viability, motility and DNA integrity. Therefore, it is a hot spot to explore the use of suitable carrier vitrification small numbers of sperm in ART.Objective: The aim of our study was to use the self-made closed straw vitrify micro quantities human sperm with or without permeating cryoprotectants, and compare vitrified spermatozoa with conventional rapid freezing in terms of their post-thaw parameters(DNA integrity, mitochondrial membrane potential and 24 h motility), with the aim of evaluate a new type of cryopreservation carrier for small numbers of human spermatozoa vitrification.Methods:1 Normal sperm freezing group : Semen samples were collected from the department of reproductive medicine in the second hospital of Hebei Medical University from January 2015 to October 2015.Ejaculates were obtained from 19 patients by masturbation after 2~7d of sexual abstinence. All specimens had at least 40 million spermatozoa/ml,50% progressively motile, and 1.5ml semen volume. Semen analysis was performed according to the WHO laboratory manual for the examination and processing of human semen,5th ed. Each ejaculate was density gradients prepared and divided into five aliquots for: Group A, fresh control;Group B, conventional freezing + CPA;Group C,vitrification + CPA;Group D, vitrification + sucrose; Group E,vitrification without cryoprotectants. Group B used 1.8ml tube for conventional rapid freezing, Group C?Group D and Group E used self-made closed straw for vitrification. After storage at least 24 h, the samples were warmed, assessed and two-two comparisons the parameters:viability,motility,PR,MMP,DNA integrity,24 h motility,24 h PR,24 h motility/ motility and 24 h PR/PR.2 Epididymal sperm freezing group : Five epididymal sperm samples were collected from PESA-ICSI cycles performed at the department of reproductive medicine in the second hospital of Hebei Medical University from January 2015 to October 2015. Each sample was divided into four aliquots for: Group F,fresh control;Group G,vitrification of epididymal sperm + CPA;Group H,vitrification of epididymal sperm + sucrose;Group I, vitrification of epididymal sperm without cryoprotectants. After the storage at least 24 h, the samples were warmed, assessed and two-two comparisons the parameters(viability, motility, PR and DNA integrity).3 Testicular sperm freezing group : Six testicular sperm samples were collected from TESA-ICSI cycles performed at the department of reproductive medicine in the second hospital of Hebei Medical University from January 2015 to October 2015.After washing and incubating,each simple was divided into four aliquots for: Group J,fresh control;Group K,vitrification of testicular sperm + CPA;Group L, vitrification of testicular sperm + sucrose;Group M, vitrification of testicular without cryoprotectants. After the storage at least 24 h, the samples were warmed, assessed motility.Results:1 Normal sperm freezing group :(1)All of the parameters after frozen/thawed were lower than fresh control group.(2)No significant difference was observed in the sperm viability, motility, DNA integrity,MMP and 24 h motility between group B and group D [55.00(8.00) vs 55.00(8.00),34.00(7.80)vs26.80(11.20),94.00(3.00) vs 94.00(2.00), 49.10(6.80) vs 46.60(9.10),20.40(6.80) vs 18.50(8.2), P>0.05].(3)Comparing the viability, motility, PR and MMP of group C?group D and group E[44.00(5.00)vs 55.00(8.00)vs42.00(8.00),18.90(7.10)vs26.80(11.20)vs17.90(6.60), 13.90(6.70) vs 25.4(12.7) vs 13.20(5.60), 40.10(8.30) vs 46.60(9.10) vs 35.40(5.4) ],group D has a stronger cryoprotective effect than group C and group E(P<0.05).(4)Group C has a stronger cryoprotective effect on the DNA integrity compared with group E[95.00(2.00)vs92.00(3.00), P<0.05].2 Epididymal sperm freezing group :(1)The sperm viability was no significant difference after different methods vitrification epididymal sperm thawed[18.00(7.00), 26.00(2.00), 23.00(2.00)].(2)The DNA integrity of vitrification of group G was significantly lower among three cryopreservation groups[12.00(3.00)vs 26.00(2.00)vs 26.00(4.00), P<0.05].3 Testicular sperm freezing group :Motile spermatozoa were seen after all vitrification groups thawed.Conclusion: The parameters(viability, motility, PR, MMP and DNA integrity) of vitrified micro quantities human sperm with sourse was similar to that recorded for spermatozoa on the conventional rapid freezing.But the vitrification of spermatozoa requires smaller volume and absence of permeating cryoprotectants, the method is simple and avoid the sample in liquid nitrogen potential cross contamination by the closed straw. Vitrification of micro quantities human sperm can be recommended in the field of assisted reproduction, especially in the case of a very small amount of sperm. |
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