Clinical and Experimental Reproductive Medicine

The Korean Society for Reproductive Medicine (대한생식의학회)
권호 표지

Optimized Methods to Maintain Motility and Viability in Normozoospermic Males

정자 운동성 및 수명 보존을 위한 최적 배양에 관한 연구

  • You, Young-Ah (Department of Animal Science & Technology, Chung-Ang University) ;
  • Mohamed, E.A. (Department of Animal Science & Technology, Chung-Ang University) ;
  • Oh, Shin-Ae (Department of Animal Science & Technology, Chung-Ang University) ;
  • Pang, Myung-Geol (Department of Animal Science & Technology, Chung-Ang University)
  • 유영아 (중앙대학교 산업과학대학 동물자원과학과) ;
  • ;
  • 오신애 (중앙대학교 산업과학대학 동물자원과학과) ;
  • 방명걸 (중앙대학교 산업과학대학 동물자원과학과)
  • Published : 2009.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

Objectives: To determinate the optimal culture condition to maintain lifespan in human sperm, we evaluated the effect of different temperature on sperm motility and viability up to 5 days in normal specimens. Methods: Ejaculated semen samples with normal semen parameters were gently washed in HEPES buffered Tyrod's-Albumin-Lactate-Pyruvate (HTALP) media. Each 5 ml of HTALP + 0.3% albumin with $1{\times}10^6$ sperm/ml was incubated for 5 days in $37^{\circ}C$, $22^{\circ}C$, and $4^{\circ}C$. The sperm motility and kinematics were analyzed using computer assisted sperm analysis (CASA), membrane integrity was assessed by hypoosmotic swelling test (HOST), and capacitation status was evaluated by chlorotetracycline (CTC) fluorescence pattern. Each parameter was measured on day 1, 3, and 5, respectively. Results: The motility, viability and live/uncapacitated pattern were demonstrated significantly in temperature- and time-dependent difference (p<0.05). While the sperm cultured for 1 day in each temperature was not significantly different, the sperm cell kept in $22^{\circ}C$ after 3 days were preserved sperm motility, viability, membrane integrity, and F pattern better than in other culture temperatures. Conclusions: HTALP can be used a basic medium for culture and longevity preservation, and sperm cell kept at $22^{\circ}C$ is beneficial for assisted reproductive techniques.

목 적: 본 연구는 정상 정액을 배양액으로 세척한 후 $4^{\circ}C$, $22^{\circ}C$, $37^{\circ}C$에서 5일 동안 보존하면서 정자의 운동성, 생존성을 관찰하여 정자의 운동성과 수명 유지를 위한 적정 배양 환경을 분석하고자 하였다. 연구방법: 정액검사 시 정상으로 판정된 남성의 정자를 HTALP 배양액으로 세척하여 정자의 최종 농도 $1{\times}10^6/ml$로 각 5 ml을 배양온도 $4^{\circ}C$, $22^{\circ}C$, $37^{\circ}C$에서 5일 동안 배양하였다. 1일, 3일 5일째에 CASA에 의해 운동성을 측정하였고, HOST로 정자막의 온전성을 분석하였으며, CTC pattern으로 수정능획득 상태 분석하여 최적의 배양 환경을 분석하였다. 결 과: 정자의 운동성, 생존성 및 수정능획득이 야기되지 않은 정자는 배양일에 따라 유의하게 감소하였다 (p<0.05). 또한 정자 배양 후 1일에는 정자의 운동성, 생존성 및 정자막의 온전성과 CTC pattern은 온도에 따라 차이가 없었으나, 배양 후 3일과 5일에서는 $22^{\circ}C$에서 배양된 정자가 다른 배양온도에 비해 가장 잘 보존되었다 (p<0.05). 결 론: HTALP로 세척된 정자를 $22^{\circ}C$에서 보존 시 5일까지 정자의 운동성과 수명을 보조생식술에 적합한 수준으로 유지시킬 수 있는 최적 배양 환경으로 제시할 수 있다.

Keywords

References

  1. Esfandiari N, Saleh RA, Blaut AP, Sharma RK, Nelson DR, Thomas AJ Jr, Falcone T, Agarwal A. Effects of temperature on sperm motion characteristics and reactive oxygen species. Int J Fertil Womens Med 2002; 47: 227-33
  2. Appell RA, Evans PR. The effect of temperature on sperm motility and viability. 1977; 28: 1329-32
  3. Grizard G, Chevalier V, Griveau JF, Le Lannou D, Boucher D. Infuence of seminal plasma on cryopreservation of human spermatozoa in a biological material-free medium: study of normal and low-quality semen. J Androl 1999; 22: 190-6 https://doi.org/10.1046/j.1365-2605.1999.00170.x
  4. Saito K, Kinoshita Y, Kanno H, Iwasaki A, Hosaka M. A new method of the electrolyte-free long-term preservation of human sperm at 4 degree C. Fertil Steril 1996; 65: 1210-3 https://doi.org/10.1016/S0015-0282(16)58340-X
  5. Cohen J, Fehilly CB, Walters DE. Prolonged storage of human spermatozoa at room temperature or in a refrigerator. Fertil Steril 1985; 44: 254-62 https://doi.org/10.1016/S0015-0282(16)48747-9
  6. Hossain A, Osumamkpe C, Nagamani, M. Extended culture of human spermatozoa in the laboratory may have practical value in the assisted reproductive procedures. Fertil Steril 2008; 89: 237-9 https://doi.org/10.1016/j.fertnstert.2007.01.170
  7. Petrrlla et al. Optimizing incubation conditions for the preservation of sperm motility in processed semen samples. Fertil Steril 2005; 84: 513-5 https://doi.org/10.1016/j.fertnstert.2005.01.138
  8. 문신용, 류범용, 방명걸, 오선경, 이재훈, 서창석, 김석현, 최영민, 김정구, 이진용. 남성 불임의 진단 및 체외수정의 예후인자로서 정자형태의 정밀 분석과 정자 첨체반응 및 햄스터 난자 침투 분석의 비교 연구. 대한불임학회지 2002; 29: 57-66
  9. 최두석, 문신용, 장윤석. 남성 불임검사 중 정자형태와 정자 운동성 검사 및 저장성용액 내 정자 팽창검사의 상관관계 및 가임능력 예측에 관한 연구. 대한산부인과학회잡지 1993; 36: 2497-509
  10. DasGupta S, Mills CL, Fraser LR. Ca(2+)-regulatd cnanges in the capacitation state of human spermatozoa assessed by a chlorocycline fluorescence assay. J Reprod Fertil 1993; 99:135-43 https://doi.org/10.1530/jrf.0.0990135
  11. Fraser LR, Abeydeera LR, Niwa K. Ca(2+)-regulating mechanisms that modulate bull sperm capacitation and acrosomal exocytosis as determined by chlorotetracycline analysis. Mol Reprod Dev 1995; 40: 233-41 https://doi.org/10.1002/mrd.1080400213
  12. Perez LJ, Valcarcel A, de Las Heras MA, Moses DF, Baldassarre H. In vitro capacitation and induction of acrosomal exocytosis in ram spermatozoa as assessed by the chlorocycline assay. Theriogenology 1996; 45: 1037-46 https://doi.org/10.1016/0093-691X(96)00031-3
  13. Jeyendran, RS, Van der Ven HH, Prez-Pelaez M, et al. Development of an assay to assess the functional integrity of the human sperm membranes and its relationship to other semen characteristics. J Reprod Fert 1984; 70: 219-28 https://doi.org/10.1530/jrf.0.0700219
  14. Hirano Y, Shibahara H, Obara H, Suzuki T, Takamizawa S, Yamaguchi C, Tsunoda H, Sato I. Relationships between sperm motility characteristics assessed by the omputer-aided sperm analysis (CASA) and fertilization rates in vitro. Journal of Assisted Reproduction and Genetics 2001; 18: 213-8
  15. Shibahara H, Obara H, Ayustawati, Hirano Y, Suzuki T, Ohno A, Takamizawa S, Suzuki M. Prediction of pregnancy by intrauterine insemination using CASA estimates and strict and criteria in patients with male factor infertility. International Journal of Andrology 2004; 27: 63-8 https://doi.org/10.1111/j.0105-6263.2004.00437.x
  16. Hatasaka HH, Holmgren WJ, Chatterton RT, et al. The differential hypoosmotic swelling pattern predicts the sperm penetration outcome in an 'infetile' population. 46th annual meeting of American Fertility Society, 1990; Abstract 199
  17. Verheyen G, Joris H, Crits K, Nagy Z, Tournaye H, Van Steirteghem A. Comparison of different hypo-osmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection. Hum Reprod Update 1997; 3: 195-203 https://doi.org/10.1093/humupd/3.3.195
  18. Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, Crowe JH. Cold shock damage is due to lipid phase transitions in cell membranes: A demonstration using sperm as a model. J Exp Zool 1993; 265: 432-7 https://doi.org/10.1002/jez.1402650413
  19. Ladha, S. Lipid heterogeneity and membrane fluidity in a highly polarized cell, the mammalian spermatozoon. J Membr Biol 1998; 165: 1-10 https://doi.org/10.1007/s002329900415
  20. Saito K, Kinoshita Y, Kanno H, Iwasaki A. The role of potassium ion and extracellular alkalization in reinitiation of human spermatozoa preserved in electrolyte-free solution at 4 degrees C. Fertil Steril 1996; 56: 1214-8
  21. Chantler E, Abraham-Peskir JV, Little S, McCann C, Medenwaldt R. Effect of cooling on the motility and function of human spermatozoa. Cryobiology 2000; 41: 125-34 https://doi.org/10.1006/cryo.2000.2274
  22. Calamera JC, Fernandez PJ, Buffone MG, Acosta AA, Doncel GF. Effects of long-term in vitro incubation of human spermatozoa:functional parameters and catalase effect Andpologia 2001; 33: 79-86
  23. Marin-Brigglier C, Tezon JG, Miranda PV, Vazquez-Levin MH. Effect of incubation human sperm at room temperature on capacitation-related events. Fert Steril 2002; 77: 252-9 https://doi.org/10.1016/S0015-0282(01)02982-X