FISH技术检测流产组织染色体异常的临床应用研究
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摘要
【目的】建立标准化的自然流产或死胎组织样本处理、质量控制及荧光原位杂交(fluorescence in situ hybridization, FISH)操作程序,为临床推广应用奠定基础；探讨本地区孕妇早、中、晚孕自然流产或死胎病例中染色体异常的发生情况,为临床完善产前诊断与遗传咨询提供理论依据。
【方法】建立标准化荧光原位杂交技术(FISH)操作平台,完成156例自然流产或死胎组织的染色体异常检测。
【结果】本研究共收集临床病例156例,受检样本杂交成功率100%。其中早孕132例,中孕18例,晚孕6例。共检出染色体异常病例82例,占52.6%,以三体型最为常见。高龄组孕妇(年龄≥5岁)与非高龄组(<35岁)的染色体异常胚胎比例分别为63.2%(24/38)和49.2%(58/118),差异不具有统计学意义；反复自然流产组染色体异常胚胎为15例(48.4%),单次自然流产组胚胎染色体异常67例(53.6%),差异不具有统计学意义。自然流产或死胎病例中,女性胚胎占60.9%(95/156),男性胚胎占39.1%(61/156),两组染色体异常的发生率分别为45.3%和63.9%,可认为两组间差异有统计学意义(P=0.023)。
【结论】胚胎染色体异常是早期自然流产的主要原因,FISH技术能够快速、准确、高效地检测自然流产或死胎染色体异常情况,有利于判断流产的原因,并达到针对性地指导预防及治疗的目的,具有良好的临床应用前景。
【Objective】To explore an optimal method of sample pretreatment, operating sequence and quality control of chorionic villus cells or fetal tissue by fluorescence in situ hybridization(FISH).To discuss the incidence of chromosomal abnormalities of miscarriage tissue collected from pregnant women in the first, second and last trimester in cases of spontaneous abortion or fetal death.
【Methods】Establish a standardized operating platform, detection the chromosomal abnormalities of 156 cases of complete miscarriage fetal tissue by FISH.
[Results] Of the 156 cases, FISH was successfully tested in all cases(100%).There were 132 cases of the first trimester,18 cases of the second trimester and 6 cases of the last trimester. In which abnormal embryo karyotypes were identified in 82 cases (52.6%). Trisomy was the most frequent embryo karyotype abnormalities in these cases. The proportion of fetal chromosomal abnormalities in the advanced maternal age group was 63.2%(24/38),which in the non-advanced maternal age group was 49.2%(58/118). There was not statistically significant difference between the two groups. Chromosomal aberration occurred in 67 cases (53.6%) of the first spontaneous abortion and in 15 cases (48.4%) of recurrent abortions. Female fetuses accounted for 60.9%(95/156)in the spontaneously aborted fetuses, while male fetuses accounted for 39.1%(61/156). The incidence of chromosomal abnormalities in the two groups were 45.3% and 63.9%, there were significant differences between the two groups (P=0.023).
【Conclusion】Embryo chromosomal abnormality is the most important reason of spontaneous abortion, and fluorescence in situ hybridization is a rapid, accurate and efficient way helps identify the causes of abortion and ensure the success of the next pregnancy. It has a good prospect of clinical application.
【方法】建立标准化荧光原位杂交技术(FISH)操作平台,完成156例自然流产或死胎组织的染色体异常检测。
【结果】本研究共收集临床病例156例,受检样本杂交成功率100%。其中早孕132例,中孕18例,晚孕6例。共检出染色体异常病例82例,占52.6%,以三体型最为常见。高龄组孕妇(年龄≥5岁)与非高龄组(<35岁)的染色体异常胚胎比例分别为63.2%(24/38)和49.2%(58/118),差异不具有统计学意义；反复自然流产组染色体异常胚胎为15例(48.4%),单次自然流产组胚胎染色体异常67例(53.6%),差异不具有统计学意义。自然流产或死胎病例中,女性胚胎占60.9%(95/156),男性胚胎占39.1%(61/156),两组染色体异常的发生率分别为45.3%和63.9%,可认为两组间差异有统计学意义(P=0.023)。
【结论】胚胎染色体异常是早期自然流产的主要原因,FISH技术能够快速、准确、高效地检测自然流产或死胎染色体异常情况,有利于判断流产的原因,并达到针对性地指导预防及治疗的目的,具有良好的临床应用前景。
【Objective】To explore an optimal method of sample pretreatment, operating sequence and quality control of chorionic villus cells or fetal tissue by fluorescence in situ hybridization(FISH).To discuss the incidence of chromosomal abnormalities of miscarriage tissue collected from pregnant women in the first, second and last trimester in cases of spontaneous abortion or fetal death.
【Methods】Establish a standardized operating platform, detection the chromosomal abnormalities of 156 cases of complete miscarriage fetal tissue by FISH.
[Results] Of the 156 cases, FISH was successfully tested in all cases(100%).There were 132 cases of the first trimester,18 cases of the second trimester and 6 cases of the last trimester. In which abnormal embryo karyotypes were identified in 82 cases (52.6%). Trisomy was the most frequent embryo karyotype abnormalities in these cases. The proportion of fetal chromosomal abnormalities in the advanced maternal age group was 63.2%(24/38),which in the non-advanced maternal age group was 49.2%(58/118). There was not statistically significant difference between the two groups. Chromosomal aberration occurred in 67 cases (53.6%) of the first spontaneous abortion and in 15 cases (48.4%) of recurrent abortions. Female fetuses accounted for 60.9%(95/156)in the spontaneously aborted fetuses, while male fetuses accounted for 39.1%(61/156). The incidence of chromosomal abnormalities in the two groups were 45.3% and 63.9%, there were significant differences between the two groups (P=0.023).
【Conclusion】Embryo chromosomal abnormality is the most important reason of spontaneous abortion, and fluorescence in situ hybridization is a rapid, accurate and efficient way helps identify the causes of abortion and ensure the success of the next pregnancy. It has a good prospect of clinical application.
引文
[1]乐杰.妇产科学[M].第7版,北京：人民卫生出版社,2008,83.
[2]Bagislar S, Ustuner I, Cengiz B, et al. Extremely skewed X-chromosome inactivation paterns in women with recurrent spontaneous abortion[J]. Aust N Z J Obstet Gynecol,2006,46(5):384-387.
[3]林其德.原因不明复发性流产的基础与临床研究进展.中华妇产科志,2003,38(8)：481.
[4]Adolfsson A, P.G Larsson. Cumulative incidence of previous spontaneous abortion in Sweden in 1983-2003:a register study[J]. Acta Obstetricia Gynecologica,2006,85: 741-774.
[5]Modvig J, L. Schmidt, M. Damsgaard. Measurement of total risk of spontaneous abortion: the virtue of conditional risk estimation [J]. American Journal of Epidemiology 1990.132 (6):1021-1038.
[6]Simon C, Rubio C, Vidal F et al. Increased chromosome abnormalities in human preimplantation embryos after in-vitro fertilization in patients with recurrent miscarriage. Reprod Fertil Dev,1998,10:87-92.
[7]Gall J, Pardue M.Formationand detection of RNA-DNA hybridmolecules incytological preparations[J].Proc Natl Acad Sci USA,1969,63:378-383.
[8]Maning J, E Hersheynd, Brokertr, et al. A new method of in situ hybridizato[J] Chromosoma,1975,53(2):107-117.
[9]Rudkin GT. High resolution detection of DNA-RNA hybrids in situ by indirect immuno-fluorescence[J]. Nature,1977,265:472-473.
[10]Langer PR, Waldrop AA, Ward C. Enzymatic synthesis of biotinlabeled polynucleotides Novel nucleic acid affinity probes[J]. Proc Natl Acad Sci, USA,1981,78:6633-6637.
[11]卢建,章钧,何蕴韶.荧光原位杂交技术及其临床应用[J].分子诊断与治疗杂志,2009,1(1)：38-42.
[12]Griffin DK, Wilton LJ, Handyside AH, et al. Fluorescent in situ hybridization for simultaneous detection of X and Y chromosome specific probes for sexing of human preimplantation embryonic nuclei[J].Hum Genet,1992,89:18-22.
[13]Tepperberg J, Pettenati MJ, Rao PN, et al. Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH):2-years multicenter retrospective study and review of the literature [J]. Prenat Diagn,2001,21 (4):293-301.
[14]Bianchi DW, Simpson J L, Jackson L G, et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood:Analysis of NIFTY data[J]. PrenatDiagn,2002,22: 609-615.
[15]Baart EB, Martini E, Van Opstal D. Screening for aneuploidies of ten different chromosomes in two rounds of FISH:A short and reliable protoco[J]. PrenatDiagn,2004, 24(12):955-961.
[16]Wyandt HE, Tonk VS, Huang XL, et al. Correlation of abnormal rapid FISH and chromosome results from amniocytes for prenatal diagnosis [J]. Fetal Diagn Ther,2006, 21:235-240.
[17]Caine A, Maltby AE, Parkin CA, et al. Prenatal detection of Down's syndrome by rapid aneuploidy testing for chromosomes 13,18,and 21 by FISH or PCR without a full karotype:a cytogenetic risk assessment[J].Lancet,2005,366(9480):123-128.
[18]Gouas L, Goumy C, Veronese L, et al. Gene dosage methods as diagnostic tools for the identification of chromosome abnormalities[J].Pathol Biol,2008,56(6):345-353.
[19]Phupong, Praphanphoj, Shotelersuk. Prenatal exclusion of subtelomeric deletion 1p by fluorescent in situ hybridization[J].Arch Gynecol Obstet,2007,275(4):237-240.
[20]Kucheria K, Jobanputra V, Talwar R, et al. Human molecular cytogenetics:diagnosis, and disease management[J]. Teratog Carcing Mutagen,2003:235-240.
[21]Carrera M. Screening prenatal de aneuploidias:QF-PCR vs FISH[J]. Prog Diag Prenat, 2001,13:262-266.
[22]Stephenson MD, Awartani KA, Robinson WP. Cytogenetic analysis of miscarriages from couples with recurrent miscarriage:a case-control study[J].Hum Repord,2002,17 446-451.
[23]Lewin P, KeinfingerP, Bazin Aetal. Difining the efficiency of fluorescence in situ hybridization on uncultured amniocytes on a retrospective cohort of 27 407 prenatal diagnosis[J]. Prenat Diagn,2000,20:1.
[24]Stumm M, Wegner RD, Bloechle M, et al. Interphase M-FISH applications using comer cialprobes in prenataland PGD diagnostics[J]. Cytogenet Genome Res,2006,114(3-4): 296-301.
[25]Choolani M, Ho SS, Razvi K, et al. Fast FISH:technique for ultrarapid fluorescence in situ hybridization on uncultured amniocytes yielding results within 2h of amniocentesis [J]. Mol Hum Reprod,2007,13(6):355-359.
[26]Shaffer LG, Bui TH. Molecular cytogenetic and rapid aneuploidy detection methods in prenatal diagnosis[J]. Am J Med Genet C Semin Med Genet,2007,145C(1):87-98.
[27]Lebedev IN, Ostroverkhova NV, Nikitina TV, et al. Features of chromosomal abnormalities in spontaneous abortion cell culure failures detected by interphase FISH analysis. Eur J Hum Genet,2004,12(7):513-520.
[28]郎景和,边旭明,向阳.妇产科遗传学[M].北京：人民卫生出版社,2005：109-113.
[29]Wilmink FA, Van Opstal D, Papatsonis DNM, et al. False positive FISH diagnosis of chromonosomy X in uncultured amniotic fluid cells due to a chromosome Y deletion [J].Prent Diagn,2008,28 (9):871-873.
[30]Klinger K, Landes G, Shook D, et al. Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH)[J]. Am J Hum Genet,1992,51(1):55-65.
[31]Wauters J, Assche EV, AntsaklisA, et al. Fully automated FISH examination of amniotic fluid cells[J]. Prenat Diagn,2007 Oct,27(10):951-955.
[32]Sullivan AE, Silver RM, LaCoursiere DY, et al. Recurrent fetal aneuploidy and recurrent miscarriage[J]. Obstetri Gynecol,2004,104(4):784-788.
[33]顾莹,张旸,祝建疆等.107例稽留流产胎儿绒毛染色体分析[J].中国优生与遗传杂志,2010,18(4)：59.
[34]Carrera M, Ribas I, Torrents M, et al. Spontaneous repeteat abortions and numerical chromosome anomalies:genetic diagnosis before preimplantation alternative diagnosis[J]. Prog Diagn Prenat,1996,8:342-347.
[35]Diego-Alvarezl D, Garcia-Hoyos M, Trujillo MJ, et al. Application of quantitative fluorescent PCR with short tandem repeat markers to the study of aneuploodies in spontaneous miscarriages[J]. Hum Repord,2005,20(5):1235-1243.
[36]Carp H, Toder V, Aviram A. Karyotype of the abortus in recurrent miscarriage[J]. Fertil & Ster,2001,75(4):678-682.
[37]Simpson J L. Causes of fetal wastage[J]. Clin Obstet Gynecol,2007,50(1):10-30.
[38]徐两蒲,林元,彭淑华等.112例早期绒毛细胞染色体检查及其结果分析[J].中国优生与遗传杂志,2010,06(25)：813-815.
[39]夏家辉,李麓芸等.染色体病[M],第1版.北京：科学出版,1989：226.
[40]李璞.医学遗传学[M],第2版.北京：中国协和医科大学出版社,2008.
[41]周焕庚,夏家辉,李麓芸等.人类染色体[M],第1版.北京：科学出版社,1987.249.
[42]熊丽,刘洁,邓康等.早期自然流产绒毛细胞培养及染色体核型分析110例[J].南方医科大学学报,2009,29(1)：64-67.
[43]Angela TA Thein, SherifA Abdel-Fattah, et al. An assessment of the use of interphase FISH with chromosome specific probes as an alternative to cytogenetics in prenatal diagnosis[J]. Prenat Diagn,2000,20:275-280.
[44]伍学淼,张化冰.嵌合型染色体致性别分化异常[J].中国实用内科杂志,2004,24(11)：635-655.
[45]Vorsanova SG. Evidence for high frequency of chromosomal mosaicism in spontaneous abortions revealed by interphase FISH analysis [J]. Histochem Cytochem,2005,53(3): 375-380.
[46]Nybo Andersen, A. M.J. Wohlfahrt, P. Christens, et al. Maternal age and fetal loss: population based register linkage study[J]. British MedicaJournal2000,320:1708-1712.
[47]刘宝,高尔生.中国已婚育龄妇女自然流产的危险因素分析[J].中国公共卫生。2002,18(7)：890-892.
[48]史庆华,许波,杨庆岭等.非整倍体和人类生殖健康[J].中国科学技术大学学报.2008,38(8)：883-889.
[49]Homer H A. Mad 2 and spindle assembly checkpoint function during meiosis I in mammalian oocytes[J]. Histol Histopathol,2006,21(8):873-886.
[50]Lebedev I. Molecular cytogenetics of recurrent missed abortions[J]. Med Res,2006, 124(1):9-10.
[51]何国平,刘雨生,童先宏等.30例体外受精-胚胎移植后流产绒毛原代培养及染色体核型分析[J].中国妇幼保健.2011,26：400-402.
[52]刘丽娟.自然流产性别特异性胚胎淘汰及其发生机制的初步研究[D].南方医科大学,2010.
[2]Bagislar S, Ustuner I, Cengiz B, et al. Extremely skewed X-chromosome inactivation paterns in women with recurrent spontaneous abortion[J]. Aust N Z J Obstet Gynecol,2006,46(5):384-387.
[3]林其德.原因不明复发性流产的基础与临床研究进展.中华妇产科志,2003,38(8)：481.
[4]Adolfsson A, P.G Larsson. Cumulative incidence of previous spontaneous abortion in Sweden in 1983-2003:a register study[J]. Acta Obstetricia Gynecologica,2006,85: 741-774.
[5]Modvig J, L. Schmidt, M. Damsgaard. Measurement of total risk of spontaneous abortion: the virtue of conditional risk estimation [J]. American Journal of Epidemiology 1990.132 (6):1021-1038.
[6]Simon C, Rubio C, Vidal F et al. Increased chromosome abnormalities in human preimplantation embryos after in-vitro fertilization in patients with recurrent miscarriage. Reprod Fertil Dev,1998,10:87-92.
[7]Gall J, Pardue M.Formationand detection of RNA-DNA hybridmolecules incytological preparations[J].Proc Natl Acad Sci USA,1969,63:378-383.
[8]Maning J, E Hersheynd, Brokertr, et al. A new method of in situ hybridizato[J] Chromosoma,1975,53(2):107-117.
[9]Rudkin GT. High resolution detection of DNA-RNA hybrids in situ by indirect immuno-fluorescence[J]. Nature,1977,265:472-473.
[10]Langer PR, Waldrop AA, Ward C. Enzymatic synthesis of biotinlabeled polynucleotides Novel nucleic acid affinity probes[J]. Proc Natl Acad Sci, USA,1981,78:6633-6637.
[11]卢建,章钧,何蕴韶.荧光原位杂交技术及其临床应用[J].分子诊断与治疗杂志,2009,1(1)：38-42.
[12]Griffin DK, Wilton LJ, Handyside AH, et al. Fluorescent in situ hybridization for simultaneous detection of X and Y chromosome specific probes for sexing of human preimplantation embryonic nuclei[J].Hum Genet,1992,89:18-22.
[13]Tepperberg J, Pettenati MJ, Rao PN, et al. Prenatal diagnosis using interphase fluorescence in situ hybridization (FISH):2-years multicenter retrospective study and review of the literature [J]. Prenat Diagn,2001,21 (4):293-301.
[14]Bianchi DW, Simpson J L, Jackson L G, et al. Fetal gender and aneuploidy detection using fetal cells in maternal blood:Analysis of NIFTY data[J]. PrenatDiagn,2002,22: 609-615.
[15]Baart EB, Martini E, Van Opstal D. Screening for aneuploidies of ten different chromosomes in two rounds of FISH:A short and reliable protoco[J]. PrenatDiagn,2004, 24(12):955-961.
[16]Wyandt HE, Tonk VS, Huang XL, et al. Correlation of abnormal rapid FISH and chromosome results from amniocytes for prenatal diagnosis [J]. Fetal Diagn Ther,2006, 21:235-240.
[17]Caine A, Maltby AE, Parkin CA, et al. Prenatal detection of Down's syndrome by rapid aneuploidy testing for chromosomes 13,18,and 21 by FISH or PCR without a full karotype:a cytogenetic risk assessment[J].Lancet,2005,366(9480):123-128.
[18]Gouas L, Goumy C, Veronese L, et al. Gene dosage methods as diagnostic tools for the identification of chromosome abnormalities[J].Pathol Biol,2008,56(6):345-353.
[19]Phupong, Praphanphoj, Shotelersuk. Prenatal exclusion of subtelomeric deletion 1p by fluorescent in situ hybridization[J].Arch Gynecol Obstet,2007,275(4):237-240.
[20]Kucheria K, Jobanputra V, Talwar R, et al. Human molecular cytogenetics:diagnosis, and disease management[J]. Teratog Carcing Mutagen,2003:235-240.
[21]Carrera M. Screening prenatal de aneuploidias:QF-PCR vs FISH[J]. Prog Diag Prenat, 2001,13:262-266.
[22]Stephenson MD, Awartani KA, Robinson WP. Cytogenetic analysis of miscarriages from couples with recurrent miscarriage:a case-control study[J].Hum Repord,2002,17 446-451.
[23]Lewin P, KeinfingerP, Bazin Aetal. Difining the efficiency of fluorescence in situ hybridization on uncultured amniocytes on a retrospective cohort of 27 407 prenatal diagnosis[J]. Prenat Diagn,2000,20:1.
[24]Stumm M, Wegner RD, Bloechle M, et al. Interphase M-FISH applications using comer cialprobes in prenataland PGD diagnostics[J]. Cytogenet Genome Res,2006,114(3-4): 296-301.
[25]Choolani M, Ho SS, Razvi K, et al. Fast FISH:technique for ultrarapid fluorescence in situ hybridization on uncultured amniocytes yielding results within 2h of amniocentesis [J]. Mol Hum Reprod,2007,13(6):355-359.
[26]Shaffer LG, Bui TH. Molecular cytogenetic and rapid aneuploidy detection methods in prenatal diagnosis[J]. Am J Med Genet C Semin Med Genet,2007,145C(1):87-98.
[27]Lebedev IN, Ostroverkhova NV, Nikitina TV, et al. Features of chromosomal abnormalities in spontaneous abortion cell culure failures detected by interphase FISH analysis. Eur J Hum Genet,2004,12(7):513-520.
[28]郎景和,边旭明,向阳.妇产科遗传学[M].北京：人民卫生出版社,2005：109-113.
[29]Wilmink FA, Van Opstal D, Papatsonis DNM, et al. False positive FISH diagnosis of chromonosomy X in uncultured amniotic fluid cells due to a chromosome Y deletion [J].Prent Diagn,2008,28 (9):871-873.
[30]Klinger K, Landes G, Shook D, et al. Rapid detection of chromosome aneuploidies in uncultured amniocytes by using fluorescence in situ hybridization (FISH)[J]. Am J Hum Genet,1992,51(1):55-65.
[31]Wauters J, Assche EV, AntsaklisA, et al. Fully automated FISH examination of amniotic fluid cells[J]. Prenat Diagn,2007 Oct,27(10):951-955.
[32]Sullivan AE, Silver RM, LaCoursiere DY, et al. Recurrent fetal aneuploidy and recurrent miscarriage[J]. Obstetri Gynecol,2004,104(4):784-788.
[33]顾莹,张旸,祝建疆等.107例稽留流产胎儿绒毛染色体分析[J].中国优生与遗传杂志,2010,18(4)：59.
[34]Carrera M, Ribas I, Torrents M, et al. Spontaneous repeteat abortions and numerical chromosome anomalies:genetic diagnosis before preimplantation alternative diagnosis[J]. Prog Diagn Prenat,1996,8:342-347.
[35]Diego-Alvarezl D, Garcia-Hoyos M, Trujillo MJ, et al. Application of quantitative fluorescent PCR with short tandem repeat markers to the study of aneuploodies in spontaneous miscarriages[J]. Hum Repord,2005,20(5):1235-1243.
[36]Carp H, Toder V, Aviram A. Karyotype of the abortus in recurrent miscarriage[J]. Fertil & Ster,2001,75(4):678-682.
[37]Simpson J L. Causes of fetal wastage[J]. Clin Obstet Gynecol,2007,50(1):10-30.
[38]徐两蒲,林元,彭淑华等.112例早期绒毛细胞染色体检查及其结果分析[J].中国优生与遗传杂志,2010,06(25)：813-815.
[39]夏家辉,李麓芸等.染色体病[M],第1版.北京：科学出版,1989：226.
[40]李璞.医学遗传学[M],第2版.北京：中国协和医科大学出版社,2008.
[41]周焕庚,夏家辉,李麓芸等.人类染色体[M],第1版.北京：科学出版社,1987.249.
[42]熊丽,刘洁,邓康等.早期自然流产绒毛细胞培养及染色体核型分析110例[J].南方医科大学学报,2009,29(1)：64-67.
[43]Angela TA Thein, SherifA Abdel-Fattah, et al. An assessment of the use of interphase FISH with chromosome specific probes as an alternative to cytogenetics in prenatal diagnosis[J]. Prenat Diagn,2000,20:275-280.
[44]伍学淼,张化冰.嵌合型染色体致性别分化异常[J].中国实用内科杂志,2004,24(11)：635-655.
[45]Vorsanova SG. Evidence for high frequency of chromosomal mosaicism in spontaneous abortions revealed by interphase FISH analysis [J]. Histochem Cytochem,2005,53(3): 375-380.
[46]Nybo Andersen, A. M.J. Wohlfahrt, P. Christens, et al. Maternal age and fetal loss: population based register linkage study[J]. British MedicaJournal2000,320:1708-1712.
[47]刘宝,高尔生.中国已婚育龄妇女自然流产的危险因素分析[J].中国公共卫生。2002,18(7)：890-892.
[48]史庆华,许波,杨庆岭等.非整倍体和人类生殖健康[J].中国科学技术大学学报.2008,38(8)：883-889.
[49]Homer H A. Mad 2 and spindle assembly checkpoint function during meiosis I in mammalian oocytes[J]. Histol Histopathol,2006,21(8):873-886.
[50]Lebedev I. Molecular cytogenetics of recurrent missed abortions[J]. Med Res,2006, 124(1):9-10.
[51]何国平,刘雨生,童先宏等.30例体外受精-胚胎移植后流产绒毛原代培养及染色体核型分析[J].中国妇幼保健.2011,26：400-402.
[52]刘丽娟.自然流产性别特异性胚胎淘汰及其发生机制的初步研究[D].南方医科大学,2010.
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