Reliability of the comet assay in cryopreserved human sperm

被引:71
作者
Duty, SM
Singh, NP
Ryan, L
Chen, Z
Lewis, C
Huang, T
Hauser, R
机构
[1] Harvard Univ, Sch Publ Hlth, Dept Environm Hlth, Occupat Hlth Program, Boston, MA 02115 USA
[2] Harvard Univ, Sch Publ Hlth, Dept Biostat, Boston, MA 02115 USA
[3] Univ Washington, Dept Bioengn, Seattle, WA 98195 USA
[4] Dana Farber Canc Inst, Dept Biostat Sci, Boston, MA 02115 USA
[5] Massachusetts Gen Hosp, Vincent Obstet & Gynecol Serv Androl Lab, Boston, MA 02114 USA
[6] Massachusetts Gen Hosp, IVF Unit, Boston, MA 02114 USA
关键词
comet assay; cryopreservation; human sperm; intra-class correlation; reliability;
D O I
10.1093/humrep/17.5.1274
中图分类号
R71 [妇产科学];
学科分类号
100211 ;
摘要
BACKGROUND: Although the comet assay has potential value for measuring DNA damage in large epidemiological human sperm studies, it is impractical to perform the assay daily on fresh semen samples. Therefore, before its use in epidemiological studies, the reliability of the comet assay in measuring DNA damage in cryopreserved sperm should be compared with that in fresh human sperm. METHODS: Semen samples from 16 men were cryopreserved in liquid nitrogen (LN) using four methods: flash freezing with and without cryopreservative, and programmable freezing with and without cryopreservative. Neutral microgel electrophoresis was performed and comets were stained with YOYO-1. Comet length was measured using an eyepiece micrometer at x400 magnification. RESULTS: The highest correlation was between comet assay results obtained from fresh human semen compared with semen flash frozen without cryopreservative (R = 0.88). However, the method of cryopreservation, as compared with other sources of variability, accounted for only 6% of the variability. Inter-individual variability accounted for 20%, and individual sperm-to-sperm variability within an ejaculate accounted for 65%. CONCLUSIONS: Flash-freezing in LN without cryopreservative most closely reproduced the results obtained using fresh human semen samples, and thereby represents the most appropriate cryopreservation method for human semen in epidemiological studies utilizing the neutral comet assay.
引用
收藏
页码:1274 / 1280
页数:7
相关论文
共 27 条
  • [1] Future research directions to study genetic damage in germ cells and estimate genetic risk
    Adler, ID
    [J]. ENVIRONMENTAL HEALTH PERSPECTIVES, 1996, 104 : 619 - 624
  • [2] Anderson D, 1997, TERATOGEN CARCIN MUT, V17, P97, DOI 10.1002/(SICI)1520-6866(1997)17:3<97::AID-TCM1>3.0.CO
  • [3] 2-8
  • [4] BOX GEP, 1978, STAT EXPT, P556
  • [5] CRYOPRESERVATION OF BOAR SEMEN IN MINI-STRAWS AND MAXI-STRAWS
    BWANGA, CO
    DEBRAGANCA, MM
    EINARSSON, S
    RODRIGUEZMARTINEZ, H
    [J]. JOURNAL OF VETERINARY MEDICINE SERIES A-PHYSIOLOGY PATHOLOGY CLINICAL MEDICINE, 1990, 37 (09): : 651 - 658
  • [6] Assessment of DNA integrity and morphology of ejaculated spermatozoa from fertile and infertile men before and after cryopreservation
    Donnelly, ET
    Steele, EK
    McClure, N
    Lewis, SEM
    [J]. HUMAN REPRODUCTION, 2001, 16 (06) : 1191 - 1199
  • [7] INDIVIDUALITY OF DNA DENATURATION PATTERNS IN HUMAN SPERM AS MEASURED BY THE SPERM CHROMATIN STRUCTURE ASSAY
    EVENSON, DP
    JOST, LK
    BAER, RK
    TURNER, TW
    SCHRADER, SM
    [J]. REPRODUCTIVE TOXICOLOGY, 1991, 5 (02) : 115 - 125
  • [8] Fleiss JL, 1999, DESIGN ANAL CLIN EXP, DOI DOI 10.1002/9781118032923
  • [9] Cryoprotective agent and temperature effects on human sperm membrane permeabilities: convergence of theoretical and empirical approaches for optimal cryopreservation methods
    Gilmore, JA
    Liu, J
    Woods, EJ
    Peter, AT
    Critser, JK
    [J]. HUMAN REPRODUCTION, 2000, 15 (02) : 335 - 343
  • [10] Hallak J, 2000, INT J FERTIL WOMEN M, V45, P38