多级梯度离心后X/Y精子浓度分析

罗熙; 李永刚; 李明颖; 曾鹏; 晏家骢

多级梯度离心后X/Y精子浓度分析

昆明理工大学附属云南省第一人民医院生殖医学科

基金项目:

基金：云南省卫生科技计划基金资助项目 (2012WS0073);

计量
- 文章访问数: 1872
- HTML全文浏览量: 670
- PDF下载量: 57
- 被引次数: 2
出版历程
- 收稿日期: 2017-03-16

Analysis of X/Y Sperm Concentration after Multistage Gradient Centrifugation

Funds:

基金：云南省卫生科技计划基金资助项目 (2012WS0073);

摘要

摘要: 目的在辅助生殖中, 利用多层密度梯度离心的方法来分选X/Y精子, 以检验常规IVF-ET中, X/Y精子比例是否失衡.方法使用Sperm Grad TM (Vitrolife, Sweden) 与Sperm Rinse TM (Vitrolife, Sweden) 配制10层梯度液, 分别为100%、95%、90%、85%、80%、75%、70%、65%、60%、55%.在离心管中制成多层梯度后, 每层梯度间分界明显.选择液化正常、浓度约50×106/mL、活动率约50%, 畸形率正常的5人精液混合后, 去除个体差异取3 mL进行梯度离心.分别收集每层梯度底部精子, 利用双色X/Y FISH探针 (Vysis) 进行FISH检测, 统计X/Y精子比例.结果 100%、95%、90%、85%、80%五层梯度中X精子含量比例分别为:51.41%、51.41%、52.19%、50.99%、54.10%.结论每层梯度液中X/Y精子含量比例虽有不同, 但差异并不明显.现在辅助生殖技术中, 利用商品化精子梯度分离液, 即使使用多层梯度进行离心分离, 也不会造成性别比例失衡.
- 梯度离心法 /
- X/Y精子分选 /
- FISH /
- 荧光原位杂交
Abstract: Objective In assisted reproduction, the X/Y sperm was sorted by multi density gradient centrifugation to examine whether the proportion of X/Y sperm was unbalanced in conventional IVF-ET.Method The use of Sperm Grad (Vitrolife, Sweden) and Sperm Rinse (Vitrolife, Sweden) to prepare 10 gradient liquid layer, were 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%.After the multilayer gradient was made in the centrifuge tube, the boundary between each gradient was obvious.Choose liquefaction normal, concentration of about 50×106/mL, activity rate of about 50%, low deformity rate of 5 people, after mixing semen, removing individual differences, and take 3 mL mixture gradient centrifugation.Collect each gradient bottom sperm, using two-color X/Y FISH probe (Vysis) for detection, and statistics X/Y sperm percentage.Results 100%, 95%, 90%, 85%, 80%, The proportion of X/Y sperm in each gradient was: 51.41%, 51.41%, 52.19%, 50.99%, 54.10%.Conclusions Although the proportion of X/Y sperm in each gradient was different, the difference was not obvious.In assisted reproductive technology, the use of commercialized sperm washing fluid, even if the use of multi-layer gradient centrifugation, will not cause gender imbalance.
- Gradient centrifugation /
- X/Y sperm sorting /
- FISH /
- Fluorescence in situ hybridization

HTML全文

参考文献(16)

[1]	[1]HENKEL R.Sperm preparation:state of the art physiological aspects and application of advanced sperm preparation methods[J].Asian J Androl, 2012, 14 (2) :260-269.[1]HENKEL R.Sperm preparation:state of the art physiological aspects and application of advanced sperm preparation methods[J].Asian J Androl, 2012, 14 (2) :260-269.
[2]	[2]PAASCH U, GRUNEWALD S, GLANDER H J.Sperm selection in assisted reproductive techniques[J].Soc Reprod Fertil Suppl, 2007, 65 (65) :515-525.[2]PAASCH U, GRUNEWALD S, GLANDER H J.Sperm selection in assisted reproductive techniques[J].Soc Reprod Fertil Suppl, 2007, 65 (65) :515-525.
[3]	[3]HENKEL R R, SCHILL W B.Sperm preparation for ART[J].Reprod Biol Endocrinol, 2003, 1 (1) :1-22.[3]HENKEL R R, SCHILL W B.Sperm preparation for ART[J].Reprod Biol Endocrinol, 2003, 1 (1) :1-22.
[4]	[4]MALVEZZI H, SHARMA R, AGARWAL A, et al.Sperm quality after density gradient centrifugation with three commercially available media:a controlled trial[J].Reprod Biol Endocrinol, 2014, 12 (1) :121.[4]MALVEZZI H, SHARMA R, AGARWAL A, et al.Sperm quality after density gradient centrifugation with three commercially available media:a controlled trial[J].Reprod Biol Endocrinol, 2014, 12 (1) :121.
[5]	[5]STAP J, HOEBE R A, MERTON J S, et al.Improving the resolution of cryopreserved X-and Y-sperm during DNA flow cytometric analysis with the addition of Percoll to quench the fluorescence of dead sperm[J].J Anim Sci, 1998, 76 (7) :1896-1902.[5]STAP J, HOEBE R A, MERTON J S, et al.Improving the resolution of cryopreserved X-and Y-sperm during DNA flow cytometric analysis with the addition of Percoll to quench the fluorescence of dead sperm[J].J Anim Sci, 1998, 76 (7) :1896-1902.
[6]	[6]CASSUTO N G, LE FOLL N, CHANTOT-BASTARAUDS, et al.Sperm fluorescence in situ hybridization study in nine men carrying a Robertsonian or a reciprocal translocation:relationship between segregation modes and high-magnification sperm morphology examination[J].Fertil Steril, 2011, 96 (4) :826-832.[6]CASSUTO N G, LE FOLL N, CHANTOT-BASTARAUDS, et al.Sperm fluorescence in situ hybridization study in nine men carrying a Robertsonian or a reciprocal translocation:relationship between segregation modes and high-magnification sperm morphology examination[J].Fertil Steril, 2011, 96 (4) :826-832.
[7]	[7]DURAKBASI-DURSUN H G, ZAMANI A G, KUTLU R, et al.A new approach to chromosomal abnormalities in sperm from patients with oligoasthenoteratozoospermia:detection of double aneuploidy in addition to single aneuploidy and diploidy by five-color fluorescence in situ hybridization using one probe set[J].Fertil Steril, 2008, 89 (6) :1709-1717.[7]DURAKBASI-DURSUN H G, ZAMANI A G, KUTLU R, et al.A new approach to chromosomal abnormalities in sperm from patients with oligoasthenoteratozoospermia:detection of double aneuploidy in addition to single aneuploidy and diploidy by five-color fluorescence in situ hybridization using one probe set[J].Fertil Steril, 2008, 89 (6) :1709-1717.
[8]	[8]RAMASAMY R, BESADA S, LAMB D J.Fluorescent in situ hybridization of human sperm:diagnostics, indications, and therapeutic implications[J].Fertil Steril, 2014, 102 (6) :1534-1539.[8]RAMASAMY R, BESADA S, LAMB D J.Fluorescent in situ hybridization of human sperm:diagnostics, indications, and therapeutic implications[J].Fertil Steril, 2014, 102 (6) :1534-1539.
[9]	[9]GUTHAUSER B, BOITRELLE F, ALBERT M, et al.Contraindication of ART following a sperm FISH analysis, even though only 12%of the spermatozoa had enlarged heads[J].Syst Biol Reprod Med, 2013, 59 (4) :214-217.[9]GUTHAUSER B, BOITRELLE F, ALBERT M, et al.Contraindication of ART following a sperm FISH analysis, even though only 12%of the spermatozoa had enlarged heads[J].Syst Biol Reprod Med, 2013, 59 (4) :214-217.
[10]	[10]MARTINEZ G, GILLOIS P, LE MITOUARD M, et al.FISH and tips:a large scale analysis of automated versus manual scoring for sperm aneuploidy detection[J].Basic Clin Androl, 2013, 23 (1) :13.[10]MARTINEZ G, GILLOIS P, LE MITOUARD M, et al.FISH and tips:a large scale analysis of automated versus manual scoring for sperm aneuploidy detection[J].Basic Clin Androl, 2013, 23 (1) :13.
[11]	[11]CITTADINI E, ALBANO C, CIMINO C, et al.Assisted reproductive technologies in severe male infertility[J].Acta Eur Fertil, 1992, 23 (3) :123.[11]CITTADINI E, ALBANO C, CIMINO C, et al.Assisted reproductive technologies in severe male infertility[J].Acta Eur Fertil, 1992, 23 (3) :123.
[12]	[12]FONG C Y, PEH G, SUBRAMANIAN A, et al.The use of discontinuous density gradients in stem cell research and application[J].Stem Cell Rev, 2009, 5 (4) :428-434.[12]FONG C Y, PEH G, SUBRAMANIAN A, et al.The use of discontinuous density gradients in stem cell research and application[J].Stem Cell Rev, 2009, 5 (4) :428-434.
[13]	[13]ANDERSEN C Y, JRGENSEN N.Improvement of sperm motility by the addition of progesterone to the Percoll medium during sperm purification[J].Hum Reprod, 1995, 10 (12) :3183-3185.[13]ANDERSEN C Y, JRGENSEN N.Improvement of sperm motility by the addition of progesterone to the Percoll medium during sperm purification[J].Hum Reprod, 1995, 10 (12) :3183-3185.
[14]李磊, 姚亚超, 刘寒艳, 等.ART中影响新生儿性别比的相关因素分析[J].生命科学研究, 2017, 21 (3) :228-232.[14]李磊, 姚亚超, 刘寒艳, 等.ART中影响新生儿性别比的相关因素分析[J].生命科学研究, 2017, 21 (3) :228-232.
[15]张力佳, 全松.辅助生殖技术中影响出生婴儿性别比的相关因素分析[J].南方医科大学学报, 2015, 53 (7) :977-984.[15]张力佳, 全松.辅助生殖技术中影响出生婴儿性别比的相关因素分析[J].南方医科大学学报, 2015, 53 (7) :977-984.
[16]	[]

相关文章(20)

[1]	何光勇, 唐健, 杨芳, 钱忠义, 杨榆玲, 罗兰. 人工驯养树鼩精子发生过程中MCM7蛋白的表达, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20210112
[2]	吕松琴, 孙溪若, 黄山, 徐丽萍, 张娟, 王义娜, 李晓非, 杨永锐. 干式荧光发光法在HBV感染诊疗中应用价值, 昆明医科大学学报. doi: 10.12259/j.issn.2095-610X.S20210812
[3]	刘洋, 黄娅娟, 廖婧, 孙春意, 翟淑娟, 周红林. 宫颈病变中应用FISH技术检测C-MYC基因及HC2技术、SPR技术检测HPV结果的相关性, 昆明医科大学学报.
[4]	刘洋, 李岱株, 孙春意, 卿清, 杨莹莹, 周红林. hTERC基因和C-MYC基因在宫颈病变筛查和诊断中的应用, 昆明医科大学学报.
[5]	相立峰, 王青青, 李明颖, 曾鹏, 李永刚. 印记基因H19在不同质量精子和胚胎中的甲基化状态, 昆明医科大学学报.
[6]	张危, 杨国际, 陈卫文, 李宏伟, 李秀萍, 曾晓燕. 不同荧光抗体剂量对HLA-B27的流式细胞结果的影响, 昆明医科大学学报.
[7]	姚金勇, 胡利平, 聂爱婷, 黄仁武, 邱建波, 张秀峰, 饶旼, 聂胜洁. MAOA μ-VNTR多态性荧光标记自动分型方法的建立及应用, 昆明医科大学学报.
[8]	李秀江. 混合斑中精子细胞的分离方法研究进展, 昆明医科大学学报.
[9]	吴建萍. 乳腺错构瘤X线、超声表现与病理11例病例并对照分析, 昆明医科大学学报.
[10]	曹宇. 环磷酰胺和白消安构建Wistar大鼠非梗阻性无精子症模型的比较, 昆明医科大学学报.
[11]	舒杰. “Y”型吻合预防吻合口瘘3例报告, 昆明医科大学学报.
[12]	熊伟. 奥沙利铂化疗对荧光HepG2肝癌细胞裸鼠原位移植瘤侵袭转移潜能的影响, 昆明医科大学学报.
[13]	王慧明. P2X4受体在大鼠脊髓小胶质细胞中的表达, 昆明医科大学学报.
[14]	左铁. 乳腺癌细胞MDA-MB-435s中侧群细胞的分选及其与非侧群细胞的生物学特点比较, 昆明医科大学学报.
[15]	张书林. 牵牛花综合症的荧光素眼底血管造影分析, 昆明医科大学学报.
[16]	王宇刚. 龟芪生精胶囊治疗男性少弱精子症临床观察, 昆明医科大学学报.
[17]	王芳. 原位心脏移植手术的麻醉管理, 昆明医科大学学报.
[18]	张璐. 细菌双杂交验证古菌UDG与TRX相互作用的研究, 昆明医科大学学报.
[19]	黄思宏. 肺泡蛋白沉积症的X线及CT表现, 昆明医科大学学报.
[20]	蔡吉芬. Ⅰ型主动脉夹层“杂交术”后合并急性冠脉综合症1例报道, 昆明医科大学学报.