ICSI后人精子激活鼠卵细胞能力的研究及实验方法探讨
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摘要
自1992年Palermo等将胞浆内单精子显微注射(Intracytoplasmic sperm injection, ICSI)应用于人类并获成功分娩以来,该技术因其单精受精率高,多精受精率低以及不受精子浓度、形态影响等优点,而被广泛应用于临床。这是人类在人工辅助助孕技术(artificial reproduction technology, ART)方面的一个突破性进展。至今全世界采用ICSI技术已经超过35,000个治疗周期,超过10,319例妊娠,出生婴儿超过5,352名。该技术在治疗不孕症,尤其是男性不育中取得了令人瞩目的进展。ICSI为男性不育的治疗带来了革命性的突破,成为严重少精子症、阻塞性无精子症和一些非阻塞性无精子症的首选治疗方法。
ICSI是借助显微操作仪将精子直接注入卵母细胞胞浆的一项辅助受精技术,近十年来广泛应用于人类辅助生育。ICSI是一种旁路受精方式,丰富了经典的受精理论,也可作为研究受精机制的实验模型。
目的:探讨提高小鼠卵细胞浆内单精子注射受精率的方法及研究各种质量精子的鼠卵细胞激活能力。
方法:选用鼠龄8~10周的健康昆明雌鼠作为卵子的供体,通过HMG/HCG方案行超促排卵,断髓处死后,从鼠输卵管中取卵。通过ICSI技术,了解不同采卵时间、不同前培养时间对ICSI的影响。精液标本来源于大连市妇产医院生殖中心不孕症门诊患者。用SWIM-UP法分别处理正常组、少精组、弱精组的精液,选择优质的精子进行ICSI ,计算卵母细胞激活率、卵细胞形态正常率,并进行各参数间对比。
结果: 1、不同采卵时间的观察显示, hCG注射后18h获卵的成熟度高于其他各组,呈显著性差异(P<0.05)。2、卵细胞体外培养时间的选择:卵细胞体外培养2 h组卵细胞激活率、形态正常率均明显高于4 h组,差异具有统计学意义(P < 0. 05)。3、实验对照组、空白对照组卵细胞激活率远远低于实验组,差异有统计学意义(P < 0. 01)。4、空白对
Objective: To study methods raising average percentage of ferti- lization by intracytoplasmic sperm injection (ICSI) in mouse eggs. To study oocyte activation capability of different parameters human semen samples.
Methods: KunMing white mice (8~10 weeks of age) were used as donors of eggs. The influence of different phases of collecting eggs and different culture time on ICSI was investigated. Semen samples according to WHO (1994) recommendation were evaluated, and were treated by SWIM-UP.
Results: 1. The mature rate of mouse oocyte, which the collection of oocytes is done 18 hours after the injection of HCG, is significant higher than the other groups (P<0.05).2. The oocyte activation rate and normal morphy rate in the group which culture time is 2 hours is higher than 4 hours. The difference is significant (P<0.05). 3. The activation rate of the experimental controlled group and the opening controlled group are significant lower than the experiment group (P<0.01).4. The survival rate of the opening controlled group is higher than the other groups (P<0.05).5. No significant difference on activation rate occurred between different groups of sperm (P>0.05).
Conclusion:1. The proper time of the oocyte collecting and the oocyte culture is important for the result of ICSI.2. It might be suggested that the mouse oocyte activation capacity of human sperm do not directly relate to initial semen sample results.3. The fertilization rate of the mouse oocyte after ICSI is low. The volume and the ability of resistence of injury to mouse oocyte may result in the above results.4.The failure of oocyte
ICSI是借助显微操作仪将精子直接注入卵母细胞胞浆的一项辅助受精技术,近十年来广泛应用于人类辅助生育。ICSI是一种旁路受精方式,丰富了经典的受精理论,也可作为研究受精机制的实验模型。
目的:探讨提高小鼠卵细胞浆内单精子注射受精率的方法及研究各种质量精子的鼠卵细胞激活能力。
方法:选用鼠龄8~10周的健康昆明雌鼠作为卵子的供体,通过HMG/HCG方案行超促排卵,断髓处死后,从鼠输卵管中取卵。通过ICSI技术,了解不同采卵时间、不同前培养时间对ICSI的影响。精液标本来源于大连市妇产医院生殖中心不孕症门诊患者。用SWIM-UP法分别处理正常组、少精组、弱精组的精液,选择优质的精子进行ICSI ,计算卵母细胞激活率、卵细胞形态正常率,并进行各参数间对比。
结果: 1、不同采卵时间的观察显示, hCG注射后18h获卵的成熟度高于其他各组,呈显著性差异(P<0.05)。2、卵细胞体外培养时间的选择:卵细胞体外培养2 h组卵细胞激活率、形态正常率均明显高于4 h组,差异具有统计学意义(P < 0. 05)。3、实验对照组、空白对照组卵细胞激活率远远低于实验组,差异有统计学意义(P < 0. 01)。4、空白对
Objective: To study methods raising average percentage of ferti- lization by intracytoplasmic sperm injection (ICSI) in mouse eggs. To study oocyte activation capability of different parameters human semen samples.
Methods: KunMing white mice (8~10 weeks of age) were used as donors of eggs. The influence of different phases of collecting eggs and different culture time on ICSI was investigated. Semen samples according to WHO (1994) recommendation were evaluated, and were treated by SWIM-UP.
Results: 1. The mature rate of mouse oocyte, which the collection of oocytes is done 18 hours after the injection of HCG, is significant higher than the other groups (P<0.05).2. The oocyte activation rate and normal morphy rate in the group which culture time is 2 hours is higher than 4 hours. The difference is significant (P<0.05). 3. The activation rate of the experimental controlled group and the opening controlled group are significant lower than the experiment group (P<0.01).4. The survival rate of the opening controlled group is higher than the other groups (P<0.05).5. No significant difference on activation rate occurred between different groups of sperm (P>0.05).
Conclusion:1. The proper time of the oocyte collecting and the oocyte culture is important for the result of ICSI.2. It might be suggested that the mouse oocyte activation capacity of human sperm do not directly relate to initial semen sample results.3. The fertilization rate of the mouse oocyte after ICSI is low. The volume and the ability of resistence of injury to mouse oocyte may result in the above results.4.The failure of oocyte
引文
1 Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmatic injection of single spermatozoon into anoocyte. Lancet, 1992, 340: 17–18
2 Montag M, Isachenko E, vanderven K, et al. Clinical application of artificial oocyte activation. Human Reprod, 2005, 20: 355
3 Eliyahu E, Talmor-Cohen A, Shalgi R. Signaling through protein kinases during egg activation. J Reprod Immunol, 2002, 53: 161-169
4 黄天华, 高小平, 李丹. 人精子染色体研究. 遗传与疾病, 1987, 4
5 王长娟,周连富,徐桂香. 人精子激活卵细胞相关因子检测方法的建立. 吉林大学学报, 2002, vol 28 No.2
6 张立珠 临床生殖内分泌与不育症
7 Sousa M, Tesarik J. Ultrastructural analysis of fertilizatioin failure after intracyto- plasmic sperm injection. Hum Reprod, 1994, 9: 2374-2380
8 Perry AC, Wakayama T, Yanagimachi R. A novel trans-complementation assay suggests full mammalian oocye activation is coordinately initiated by multiple, submembrane sperm components. Biol Reprod, 1999, 60: 747-755
9 kimura Y,Yanagimachi R,Kuretake S,et al. Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material. Biol reprod,1998, 58:1407-1415
10 Ito J, Kawano N, Hirabayashi M, et al. The role of calcium/calmodulin dependent protein kinase Ⅱ on the inactivation of MAP kinase and p34cdc2 kinase during fertilization and activation in pig oocytes. Reproduction, 2004, 128: 409-415
11 Watanabe S. Frequent structural chromosome aberrations in immotile human sperm exposed to culture media. Human Reprod, 2004, 19: 940-947
12 Flaherty SP, Payne D, Swann NJ, et al. Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection(ICSI). Reprod Fertil Dev, 1995, 7: 197-210
13 Dozortsev D, Rybouchkin A, De Sutter P, et al. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Human Reprod, 1995, 10: 403-407
14 Reynier P, May-panloup P, Chretein MF, et al. Mitochondrial DNA content affects the fertilizability of human oocytes. Mol Hum Reprod, 2001, 7: 425-429
15 Miyara F, Aubriot FX, Glissant A, et al. Multiparameter analysis of human oocytesat metaphase Ⅱ stage after IVF failure in non-male infertility. Hum Reprod, 2003, 18: 1494-1503
16 Wiland E, Wojda A, Kamieniczna M, et al. Idiopathic infertility in married couples in the light of cytogenetic analysis and sperm penetration assay. Folia Histochem Cytobiol, 2001, 39: 35-41
17 Morris ID, Ilott S, Dixon L, et al. The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis(Comet assay)and its relationship to fertilization and embryo development. Hum Reprod, 2002, 17: 990-998
18 杨益寿, 熊素芳, 龙文,等. 汪昌介小鼠卵细胞质内显微注入单精子受精的实验研究. 解剖学报, 1998, Vo l129, No14
1 Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 1992, 340: 17–18
2 Land JA, Evers JL. Risks and complications in assisted reproduction techniques: Report of an ESHRE consensus meeting. Hum Reprod, 2003, 18(2): 455–457
3 Helmerhorst FM, Perquin DA, Donker D, et al. Perinatal outcome of singletons and twins after assisted conception: a systematic review of controlled studies. BMJ. 2004, 328: 261–266
4 Cook R, Bradley S, Golombok S. A preliminary study of parental stress and child behaviour in families with twins conceived by in-vitro fertilization. Hum Reprod 1998, 13: 3244–3246
5 De Sutter P, Van der Elst J, Coetsier T, et al. Single embryo transfer and multiple pregnancy rate reduction in IVF/ICSI: a 5-year appraisal. Reprod Biomed Online, 2003, 6: 464–469
6 ASRM/SARTR. Assisted reproductive technology in the United States: 1999 results generated from the American Society for Reproductive Medicine/ Society for Assisted Reproductive Technology Registry. Fertil Steril. 2002, 78: 918–931
7 Gray RH, Wu LY. Subfertility and risk of spontaneous abortion. Am J Public Health, 2000, 90: 1452–1454
8 Vernaeve V, Tournaye H, Osmanagaoglu K, et al. Intracytoplasmic sperm injection with testicular spermatozoa is less successful in men with nonobstructive azoosper- mia than in men with obstructive azoospermia. Fertil Steril, 2003, 79: 529–533
9 Vernaeve V, Bonduelle M, Tournaye H, et al. Pregnancy outcome and neonatal data of children born after ICSI using testicular sperm in obstructive and non-obstructive azoospermia.. Hum Reprod, 2003, 18: 2093–2097
10 Bonduelle M, Aytoz A, Van Assche E, et al. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum Reprod, 1998, 13 : 781–782
11 Govaerts I, Devreker F, Delbaere A,et al. Short-term medical complications of 1500 oocyte retrievals for in vitro fertilization and embryo transfer. Eur J Obstet Gynecol Reprod Biol, 1998, 77: 239–243
12 Klip H, Burger CW, Kenemans P, et al. Cancer risk associated with subfertility and ovulation induction: a review. Cancer Causes Control, 2000, 11:319–344
13 Wood S, Thomas K, Sephton V, et al. Postoperative pain, complications, and satisfaction rates in patients who undergo surgical sperm retrieval. Fertil Steril, 2003, 79: 56–62
14 Beutel M, Kupfer J, Kirchmeyer P, et al. Treatment-related stresses and depression in couples undergoing assisted reproductive treatment by IVF or ICSI. Andrologia, 1999, 31: 27-35
15 Harari O, Bourne H, Baker G, et al. High fertilization rate with intracytoplasmic sperm injection in mosaic Klinefelter’s syndrome. Fertil Steril, 1995, 63: 182-184
16 Palermo GD, Schlegel PN, Sills ES, et al. Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter’s syndrome. N Engl J Med, 1998, 338: 588-590
17 Testart J, Gautier E, Brami C, et al. Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Hum Reprod, 1996, 11: 2609-2012
18 Martin RH. The risk of chromosomal abnormalities following ICSI. Hum Reprod, 1996, 11: 924-925
19 Vogt PH, Edelmann A, Kirsch S , et al. Human Y chromosome azoospermia fact- ors(AZF)mapped to different subregions in Yq11. Hum Mol Genet, 1996, 5: 933-943
20 Van Steirteghem A, Bonduelle M, Devroey P, et al. Follow-up of children born after ICSI. Hum Reprod Update, 2002, 8: 111-116
21 Silber SJ, Repping S. Transmission of male infertility to future generations: lessons from the Y chromosome. Hum Repro Update, 2002, 8: 217-219
22 ean M, Rival JM, Mensier A, et al. Prenatal diagnosis of ring chromosome 14 after intracytoplasmic sperm injection. Fertil Steril, 1997, 67: 164-165
23 Tripp BM, Kolon TF, Bishop C, et al. Intracytoplasmic sperm injection and potential transmission of genetic disease. JAMA, 1997, 277: 963-964
24 Mulhall JP, Reijo R, Alagappan R, et al. azoospermic men with deletion of the DAZ gene cluster are capable of completing spermatogenesis: fertilization, normal embryonic development and pregnancy occur when retrieved testicular spermatozoa are used for intracytoplasmic sperm injection. Hum Reprod, 1997, 12: 503-508
25 Kostiner DR, Turek PJ, Reijo RA. Male infertility: analysis of the markers and genes on the human Y chromosome. Hum Reprod, 1998, 13: 3032-3028
2 Montag M, Isachenko E, vanderven K, et al. Clinical application of artificial oocyte activation. Human Reprod, 2005, 20: 355
3 Eliyahu E, Talmor-Cohen A, Shalgi R. Signaling through protein kinases during egg activation. J Reprod Immunol, 2002, 53: 161-169
4 黄天华, 高小平, 李丹. 人精子染色体研究. 遗传与疾病, 1987, 4
5 王长娟,周连富,徐桂香. 人精子激活卵细胞相关因子检测方法的建立. 吉林大学学报, 2002, vol 28 No.2
6 张立珠 临床生殖内分泌与不育症
7 Sousa M, Tesarik J. Ultrastructural analysis of fertilizatioin failure after intracyto- plasmic sperm injection. Hum Reprod, 1994, 9: 2374-2380
8 Perry AC, Wakayama T, Yanagimachi R. A novel trans-complementation assay suggests full mammalian oocye activation is coordinately initiated by multiple, submembrane sperm components. Biol Reprod, 1999, 60: 747-755
9 kimura Y,Yanagimachi R,Kuretake S,et al. Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material. Biol reprod,1998, 58:1407-1415
10 Ito J, Kawano N, Hirabayashi M, et al. The role of calcium/calmodulin dependent protein kinase Ⅱ on the inactivation of MAP kinase and p34cdc2 kinase during fertilization and activation in pig oocytes. Reproduction, 2004, 128: 409-415
11 Watanabe S. Frequent structural chromosome aberrations in immotile human sperm exposed to culture media. Human Reprod, 2004, 19: 940-947
12 Flaherty SP, Payne D, Swann NJ, et al. Assessment of fertilization failure and abnormal fertilization after intracytoplasmic sperm injection(ICSI). Reprod Fertil Dev, 1995, 7: 197-210
13 Dozortsev D, Rybouchkin A, De Sutter P, et al. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Human Reprod, 1995, 10: 403-407
14 Reynier P, May-panloup P, Chretein MF, et al. Mitochondrial DNA content affects the fertilizability of human oocytes. Mol Hum Reprod, 2001, 7: 425-429
15 Miyara F, Aubriot FX, Glissant A, et al. Multiparameter analysis of human oocytesat metaphase Ⅱ stage after IVF failure in non-male infertility. Hum Reprod, 2003, 18: 1494-1503
16 Wiland E, Wojda A, Kamieniczna M, et al. Idiopathic infertility in married couples in the light of cytogenetic analysis and sperm penetration assay. Folia Histochem Cytobiol, 2001, 39: 35-41
17 Morris ID, Ilott S, Dixon L, et al. The spectrum of DNA damage in human sperm assessed by single cell gel electrophoresis(Comet assay)and its relationship to fertilization and embryo development. Hum Reprod, 2002, 17: 990-998
18 杨益寿, 熊素芳, 龙文,等. 汪昌介小鼠卵细胞质内显微注入单精子受精的实验研究. 解剖学报, 1998, Vo l129, No14
1 Palermo G, Joris H, Devroey P, et al. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet, 1992, 340: 17–18
2 Land JA, Evers JL. Risks and complications in assisted reproduction techniques: Report of an ESHRE consensus meeting. Hum Reprod, 2003, 18(2): 455–457
3 Helmerhorst FM, Perquin DA, Donker D, et al. Perinatal outcome of singletons and twins after assisted conception: a systematic review of controlled studies. BMJ. 2004, 328: 261–266
4 Cook R, Bradley S, Golombok S. A preliminary study of parental stress and child behaviour in families with twins conceived by in-vitro fertilization. Hum Reprod 1998, 13: 3244–3246
5 De Sutter P, Van der Elst J, Coetsier T, et al. Single embryo transfer and multiple pregnancy rate reduction in IVF/ICSI: a 5-year appraisal. Reprod Biomed Online, 2003, 6: 464–469
6 ASRM/SARTR. Assisted reproductive technology in the United States: 1999 results generated from the American Society for Reproductive Medicine/ Society for Assisted Reproductive Technology Registry. Fertil Steril. 2002, 78: 918–931
7 Gray RH, Wu LY. Subfertility and risk of spontaneous abortion. Am J Public Health, 2000, 90: 1452–1454
8 Vernaeve V, Tournaye H, Osmanagaoglu K, et al. Intracytoplasmic sperm injection with testicular spermatozoa is less successful in men with nonobstructive azoosper- mia than in men with obstructive azoospermia. Fertil Steril, 2003, 79: 529–533
9 Vernaeve V, Bonduelle M, Tournaye H, et al. Pregnancy outcome and neonatal data of children born after ICSI using testicular sperm in obstructive and non-obstructive azoospermia.. Hum Reprod, 2003, 18: 2093–2097
10 Bonduelle M, Aytoz A, Van Assche E, et al. Incidence of chromosomal aberrations in children born after assisted reproduction through intracytoplasmic sperm injection. Hum Reprod, 1998, 13 : 781–782
11 Govaerts I, Devreker F, Delbaere A,et al. Short-term medical complications of 1500 oocyte retrievals for in vitro fertilization and embryo transfer. Eur J Obstet Gynecol Reprod Biol, 1998, 77: 239–243
12 Klip H, Burger CW, Kenemans P, et al. Cancer risk associated with subfertility and ovulation induction: a review. Cancer Causes Control, 2000, 11:319–344
13 Wood S, Thomas K, Sephton V, et al. Postoperative pain, complications, and satisfaction rates in patients who undergo surgical sperm retrieval. Fertil Steril, 2003, 79: 56–62
14 Beutel M, Kupfer J, Kirchmeyer P, et al. Treatment-related stresses and depression in couples undergoing assisted reproductive treatment by IVF or ICSI. Andrologia, 1999, 31: 27-35
15 Harari O, Bourne H, Baker G, et al. High fertilization rate with intracytoplasmic sperm injection in mosaic Klinefelter’s syndrome. Fertil Steril, 1995, 63: 182-184
16 Palermo GD, Schlegel PN, Sills ES, et al. Births after intracytoplasmic injection of sperm obtained by testicular extraction from men with nonmosaic Klinefelter’s syndrome. N Engl J Med, 1998, 338: 588-590
17 Testart J, Gautier E, Brami C, et al. Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Hum Reprod, 1996, 11: 2609-2012
18 Martin RH. The risk of chromosomal abnormalities following ICSI. Hum Reprod, 1996, 11: 924-925
19 Vogt PH, Edelmann A, Kirsch S , et al. Human Y chromosome azoospermia fact- ors(AZF)mapped to different subregions in Yq11. Hum Mol Genet, 1996, 5: 933-943
20 Van Steirteghem A, Bonduelle M, Devroey P, et al. Follow-up of children born after ICSI. Hum Reprod Update, 2002, 8: 111-116
21 Silber SJ, Repping S. Transmission of male infertility to future generations: lessons from the Y chromosome. Hum Repro Update, 2002, 8: 217-219
22 ean M, Rival JM, Mensier A, et al. Prenatal diagnosis of ring chromosome 14 after intracytoplasmic sperm injection. Fertil Steril, 1997, 67: 164-165
23 Tripp BM, Kolon TF, Bishop C, et al. Intracytoplasmic sperm injection and potential transmission of genetic disease. JAMA, 1997, 277: 963-964
24 Mulhall JP, Reijo R, Alagappan R, et al. azoospermic men with deletion of the DAZ gene cluster are capable of completing spermatogenesis: fertilization, normal embryonic development and pregnancy occur when retrieved testicular spermatozoa are used for intracytoplasmic sperm injection. Hum Reprod, 1997, 12: 503-508
25 Kostiner DR, Turek PJ, Reijo RA. Male infertility: analysis of the markers and genes on the human Y chromosome. Hum Reprod, 1998, 13: 3032-3028