短时常规体外受精及补救性卵胞浆内单精子注射胚胎染色体非整倍体分析
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摘要
在体外受精—胚胎移植(in vitro fertilization-embryo transfer, IVF-ET)周期中有10%～20%的几率会出现完全的受精失败,其原因可能是由于卵母细胞和精子内在的结构或功能缺陷、精卵结合障碍等而引起,具体到每一个受精失败周期中是不同而又很难确定的。然而预测IVF受精失败亦非常困难,国内外也多有报道认为选择合适的卵子及时补救卵胞浆内单精子注射(intracytoplasmic sperm injection, ICSI)可获得妊娠和分娩。为有效降低周期的取消率,我们尝试行短时IVF结合短时IVF补救ICSI的受精方式。但这两种技术本身是否会影响到胚胎染色体非整倍体情况,尚未见相关报道。
目的
本研究拟运用二轮荧光原位杂交(fluorescence in situ hybridization, FISH)技术分析常规IVF、短时IVF和短时IVF后行补救ICSI胚胎非整倍体情况,更为科学的评价短时IVF及短时IVF后行补救ICSI的安全性,为临床提供更有力的支持依据。
方法
选取2009年7月～2010年1月郑州大学第一附属医院生殖医学中心314个IVF-ET周期、465个短时IVF-ET周期和82个短时IVF后补救ICSI周期资料。经患者知情同意后收集同期各组废弃胚胎。采用Tween-20/HCl消化透明带后吹打法获得卵裂球,Tween-20/HCl+甲醇/冰醋酸法裂解固定卵裂球,应用荧光杂交探针LSI13/LSI21和CEPX/Y进行二轮FISH, Imstar荧光分析软件拍摄、记录、存档后进行分析。
所有数据采用SPSS11.0统计软件包处理,数据采用百分率表示。计数资料采用x2检验。检验标准取α=0.05,P＜0.05为有显著性差异。
结果
1.统计分析结果
回顾性分析314个IVF-ET周期中4082枚卵母细胞,465个短时IVF-ET周期中6030枚卵母细胞和82个短时IVF后补救ICSI周期中749枚卵母细胞受精情况。短时IVF组总受精率、2PN受精率都不低于常规IVF组,短时IVF组优胚率显著高于常规IVF组；短时后补救ICSI组总受精率85.2%显著高于其他两组(P＜0.05)；短时后补救ICSI组多PN受精率8.0%显著高于常规IVF组(P<0.05)。
2.卵裂球固定和杂交情况
共收集废弃胚胎152只,固定率(固定卵裂球数/获得卵裂球数)为91.9%(352/383)；出信号率(出信号卵裂球数/固定卵裂球数)为96.0%(338/352)。信号反应143只胚胎染色体情况。
3.卵裂球FISH结果
短时IVF周期的胚胎中胚胎染色体异常率不高于常规IVF周期的胚胎,短时IVF后补救ICSI组与常规IVF组相比胚胎染色体异常率也无显著增高。各组中2PN胚胎的染色体二体率恒定较高,但也存在非整倍体及嵌合体现象。1PN组的胚胎也表现较高的染色体二体率。而≥3PN组中,染色体非整倍体率为81.0%,显著高于1PN组、2PN组(P＜0.05)。数据中可观察到随年龄增加胚胎的染色体异常率呈增高趋势,2PN胚胎≥35岁组染色体异常率显著高于＜30岁组和30～34岁组(P＜0.05)。按平均获卵数13枚为界分组对比染色体异常率差异无显著性。
结论
1.短时IVF不降低受精率,不增加胚胎染色体异常几率,是一种安全有效的受精方式。
2.短时IVF失败后补救ICSI可及时挽救受精失败,有效减少周期取消率,且不增加正常胚胎的染色体异常风险。
3.2PN胚胎是移植的首选对象。但也存在染色体异常几率。可能为体外受精妊娠率低下原因之一。IVF中1PN胚胎染色体具有较高的二体型比率,说明IVF中1PN胚胎很大可能为正常受精。3PN胚胎染色体异常高发,不具有移植价值。
4.胚胎染色体异常几率随年龄增高而增高,尤其是≥35岁女性,染色体异常几率明显升高。获卵数目对胚胎染色体异常形成关系不大。
Failed fertilization after IVF occurs in 10-20% of cycles.The reason is different and difficult to determine in each IVF-ET cycle.Sperm defect,disturbances in sperm-oocyte interaction and oocyte abnormality have all been proposed as possible causes of failed fertilization after IVF.It is difficult to forecast the result of failed fertilization. As it report, we will obtain the good pregnancy result if choose the appropriate oocyte to rescue ICSI in time. We advise using short-time IVF and rescue ICSI in order to effectively reduce the rate of cycle cancellation.But whether these two kind of technologies itself will affect chromosomal aneuploid of embryo,it is unknown yet.
Objectives
To discuss whether short-time IVF and rescue ICSI will affect chromosomal aneuploid of embryo,we use two round FISH to analyze embryo's chromosomal aneuploid.We will appraisal scientific two kind of technology's security and provide the theoritical evidence for supporting in clinical practice.
Methods
Abandoned embryos for research were recruited from patients who were performed with IVF/rescue ICSI treatment in Reproductive Medical Center in the First Affiliated Hospital of Zhengzhou University from July 2009 to January 2010. All of the patient have given informed consent for their abandoned embryos. To use aspirate-pull method to obtain blastomere form embryo whose zone lysed by Tween-20/HCl+Methanol/glacial acetic acid. To use Tween-20/HCl+Methanol/ glacial acetic acid methods to fix blastomeres. To compare aneuploid rate and differences of each group after two round FISH with probe LSI13,LSI21,CEPX and CEPY.
All data were dealed with SPSS 11.0 statistical package byx2test.α=0.05 is considered as test criterion for statistical analyse. We take P<0.05 as the significance difference standard.
Results
1. Statistical analysis result
We have retrospective analysis 4082 ovocytes from 314 IVF-ET cycles,6030 ovocytes from 465 short-time IVF-ET cycles and 749 ovocytes from 82 rescue ICSI cycles. The total fertilization rate and the 2PN fertilization rate of short-time IVF group are not lower than the conventional IVF group.The good embryo rate of short-time IVF group is significance higher than the conventional IVF group.The total fertilization rate of rescue ICSI group is significance higher than other two groups. Its multi-PN fertilization rate is significance higher than conventional IVF group.
2. Fix and hybridization outcomes
The total number of collecting abandoned embryo is 152. The fixing rate was 91.9%,while signals rate was 96.0%. The signal responds 143 embryois chromosome.
3. FISH outcomes of blastomeres
The chromosomal abnormality rate of short-time IVF group is similar with the conventional IVF cycle. The rescue ICSI group has the same result. The diploidy rate every groups' 2PN embryo is stable, but it also has the aneuploid and gomphosis phenomenon. The 1PN embryo also displays the high chromosome two bodies rate. But the chromosomal aneuploid rate of≥3PN embryo in every group is significance higher than 1PN group and 2PN group. Advanced maternal age, more elder may result in higer abnormality rate. In the≥35 year old group,2PN embryo chromosomal abnormality rate is significance higher than the younger group. But number of oocytes retrieved may have no effect on embryonic chromosomal abnormality.
Conclusions
1. Short-time IVF does not decrease the fertilization rate, and does not increase the embryo chromosomal abnormality. It is a security effective fertilizing method.
2. Rescue ICSI after short-time IVF could be useful to save the failed fertilization. It will effectively reduce cycle cancellation rate, and could not increase the 2PN embryos' chromosomal abnormality risk.
3. The 2PN embryos are the best choice to transfer. But they also has the chromosomal abnormality rate. Maybe this is the key factor on lower clinical pregnant rate. The 1PN embryos' chromosome from IVF cycles has a high level diploidy rate. Maybe it declares that the 1PN embryos from IVF cycles would be fertilized normally. Embryos derived from≥3PN are not suitable for transplantation, because of the higher chromosomal abnormality rate.
4. Advanced maternal age, especially≥35 year; more elder may result in higer abnormality rate. The number of oocytes retrieved may have no effect on embryonic' chromosomal abnormality.
目的
本研究拟运用二轮荧光原位杂交(fluorescence in situ hybridization, FISH)技术分析常规IVF、短时IVF和短时IVF后行补救ICSI胚胎非整倍体情况,更为科学的评价短时IVF及短时IVF后行补救ICSI的安全性,为临床提供更有力的支持依据。
方法
选取2009年7月～2010年1月郑州大学第一附属医院生殖医学中心314个IVF-ET周期、465个短时IVF-ET周期和82个短时IVF后补救ICSI周期资料。经患者知情同意后收集同期各组废弃胚胎。采用Tween-20/HCl消化透明带后吹打法获得卵裂球,Tween-20/HCl+甲醇/冰醋酸法裂解固定卵裂球,应用荧光杂交探针LSI13/LSI21和CEPX/Y进行二轮FISH, Imstar荧光分析软件拍摄、记录、存档后进行分析。
所有数据采用SPSS11.0统计软件包处理,数据采用百分率表示。计数资料采用x2检验。检验标准取α=0.05,P＜0.05为有显著性差异。
结果
1.统计分析结果
回顾性分析314个IVF-ET周期中4082枚卵母细胞,465个短时IVF-ET周期中6030枚卵母细胞和82个短时IVF后补救ICSI周期中749枚卵母细胞受精情况。短时IVF组总受精率、2PN受精率都不低于常规IVF组,短时IVF组优胚率显著高于常规IVF组；短时后补救ICSI组总受精率85.2%显著高于其他两组(P＜0.05)；短时后补救ICSI组多PN受精率8.0%显著高于常规IVF组(P<0.05)。
2.卵裂球固定和杂交情况
共收集废弃胚胎152只,固定率(固定卵裂球数/获得卵裂球数)为91.9%(352/383)；出信号率(出信号卵裂球数/固定卵裂球数)为96.0%(338/352)。信号反应143只胚胎染色体情况。
3.卵裂球FISH结果
短时IVF周期的胚胎中胚胎染色体异常率不高于常规IVF周期的胚胎,短时IVF后补救ICSI组与常规IVF组相比胚胎染色体异常率也无显著增高。各组中2PN胚胎的染色体二体率恒定较高,但也存在非整倍体及嵌合体现象。1PN组的胚胎也表现较高的染色体二体率。而≥3PN组中,染色体非整倍体率为81.0%,显著高于1PN组、2PN组(P＜0.05)。数据中可观察到随年龄增加胚胎的染色体异常率呈增高趋势,2PN胚胎≥35岁组染色体异常率显著高于＜30岁组和30～34岁组(P＜0.05)。按平均获卵数13枚为界分组对比染色体异常率差异无显著性。
结论
1.短时IVF不降低受精率,不增加胚胎染色体异常几率,是一种安全有效的受精方式。
2.短时IVF失败后补救ICSI可及时挽救受精失败,有效减少周期取消率,且不增加正常胚胎的染色体异常风险。
3.2PN胚胎是移植的首选对象。但也存在染色体异常几率。可能为体外受精妊娠率低下原因之一。IVF中1PN胚胎染色体具有较高的二体型比率,说明IVF中1PN胚胎很大可能为正常受精。3PN胚胎染色体异常高发,不具有移植价值。
4.胚胎染色体异常几率随年龄增高而增高,尤其是≥35岁女性,染色体异常几率明显升高。获卵数目对胚胎染色体异常形成关系不大。
Failed fertilization after IVF occurs in 10-20% of cycles.The reason is different and difficult to determine in each IVF-ET cycle.Sperm defect,disturbances in sperm-oocyte interaction and oocyte abnormality have all been proposed as possible causes of failed fertilization after IVF.It is difficult to forecast the result of failed fertilization. As it report, we will obtain the good pregnancy result if choose the appropriate oocyte to rescue ICSI in time. We advise using short-time IVF and rescue ICSI in order to effectively reduce the rate of cycle cancellation.But whether these two kind of technologies itself will affect chromosomal aneuploid of embryo,it is unknown yet.
Objectives
To discuss whether short-time IVF and rescue ICSI will affect chromosomal aneuploid of embryo,we use two round FISH to analyze embryo's chromosomal aneuploid.We will appraisal scientific two kind of technology's security and provide the theoritical evidence for supporting in clinical practice.
Methods
Abandoned embryos for research were recruited from patients who were performed with IVF/rescue ICSI treatment in Reproductive Medical Center in the First Affiliated Hospital of Zhengzhou University from July 2009 to January 2010. All of the patient have given informed consent for their abandoned embryos. To use aspirate-pull method to obtain blastomere form embryo whose zone lysed by Tween-20/HCl+Methanol/glacial acetic acid. To use Tween-20/HCl+Methanol/ glacial acetic acid methods to fix blastomeres. To compare aneuploid rate and differences of each group after two round FISH with probe LSI13,LSI21,CEPX and CEPY.
All data were dealed with SPSS 11.0 statistical package byx2test.α=0.05 is considered as test criterion for statistical analyse. We take P<0.05 as the significance difference standard.
Results
1. Statistical analysis result
We have retrospective analysis 4082 ovocytes from 314 IVF-ET cycles,6030 ovocytes from 465 short-time IVF-ET cycles and 749 ovocytes from 82 rescue ICSI cycles. The total fertilization rate and the 2PN fertilization rate of short-time IVF group are not lower than the conventional IVF group.The good embryo rate of short-time IVF group is significance higher than the conventional IVF group.The total fertilization rate of rescue ICSI group is significance higher than other two groups. Its multi-PN fertilization rate is significance higher than conventional IVF group.
2. Fix and hybridization outcomes
The total number of collecting abandoned embryo is 152. The fixing rate was 91.9%,while signals rate was 96.0%. The signal responds 143 embryois chromosome.
3. FISH outcomes of blastomeres
The chromosomal abnormality rate of short-time IVF group is similar with the conventional IVF cycle. The rescue ICSI group has the same result. The diploidy rate every groups' 2PN embryo is stable, but it also has the aneuploid and gomphosis phenomenon. The 1PN embryo also displays the high chromosome two bodies rate. But the chromosomal aneuploid rate of≥3PN embryo in every group is significance higher than 1PN group and 2PN group. Advanced maternal age, more elder may result in higer abnormality rate. In the≥35 year old group,2PN embryo chromosomal abnormality rate is significance higher than the younger group. But number of oocytes retrieved may have no effect on embryonic chromosomal abnormality.
Conclusions
1. Short-time IVF does not decrease the fertilization rate, and does not increase the embryo chromosomal abnormality. It is a security effective fertilizing method.
2. Rescue ICSI after short-time IVF could be useful to save the failed fertilization. It will effectively reduce cycle cancellation rate, and could not increase the 2PN embryos' chromosomal abnormality risk.
3. The 2PN embryos are the best choice to transfer. But they also has the chromosomal abnormality rate. Maybe this is the key factor on lower clinical pregnant rate. The 1PN embryos' chromosome from IVF cycles has a high level diploidy rate. Maybe it declares that the 1PN embryos from IVF cycles would be fertilized normally. Embryos derived from≥3PN are not suitable for transplantation, because of the higher chromosomal abnormality rate.
4. Advanced maternal age, especially≥35 year; more elder may result in higer abnormality rate. The number of oocytes retrieved may have no effect on embryonic' chromosomal abnormality.
引文
[1]Lizardi PM, Huang X, Zhu Z, et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification[J]. Nat Genet.1998 Jul;19(3):225～232
[2]Corneveaux JJ, Kruer MC, Hu-Line D, et al. SNP-based chromosomal copy number ascertainment following multiple displacement whole-genome amplification [J]. Biotechniques. 2007 Jul;42(1):77～83
[3]Jasmine F, Ahsan H, Andrulis IL, et al. Whole-genome amplification enables accurate genotyping for microarray-based high-density single nucleotide polymorphism array [J]. Cancer Epidemiol Biomarkers Prev.2008 Dec; 17(12):3499～3508
[4]Spits C, Le Caignec C, De Rycke M, et al. Optimization and evaluation of single-cell whole-genome multiple displacement amplification [J]. Hum Mutat.2006 May;27(5):496-503
[5]Dean FB, Hosono S, Fang L, et al. Comprehensive human genome amplification using multiple displacement amplification[J]. Proc Natl Acad Sci U S A.2002 Apr 16;99(8):5261～ 5266
[6]Jiang Z, Zhang X, Deka R, et al. Genome amplification of single sperm using multiple displacement amplification [J]. Nucleic Acids Res.2005 Jul 7;33(10):e91
[7]Ivanov PL, Fomichev AA. Whole-genome amplification by MDA to improve sensitivity of forensic expert examination of chromosomal DNA[J]. Sud Med Ekspert.2008 Jul-Aug;51(4):16-18.
[8]Hosono S, Faruqi AF, Dean FB, et al. Unbiased whole-genome amplification directly from clinical samples[J]. Genome Res.2003 May;13(5):954-964.
[9]Ling J, Zhuang G, Zhang CH,et al. Evaluation of genome coverage and fidelity of multiple displacement amplification from single cells by SNP array[J]. Mol Hum Reprod.2009 Nov;15(11):739～747
[10]Hellani A, Coskun S, Tbakhi A,et al. Clinical application of multiple displacement amplification in preimplantation genetic diagnosis[J]. Reprod Biomed Online.2005 Mar;10(3):376～380.
[11]Kennett JY, Watson SK, Saprunoff H, et al. Technical demonstration of whole genome array comparative genomic hybridization [J]. J Vis Exp.2008 Aug 5;(18).pii:870.doi:10.3791/870.
[12]Panelli S, Damiani G, Espen L, et al. Ligation overcomes terminal underrepresentation in multiple displacement amplification of linear DNA[J]. Biotechniques.2005 Aug;39(2):174,176,178 passim
[13]Sun G, Kaushal R, Pal P, et al. Whole-genome amplification:relative efficiencies of the current methods[J]. Leg Med (Tokyo).2005 Oct;7(5):279-286
[14]Hellani A, Coskun S, Benkhalifa M, et al. Multiple displacement amplification on single cell and possible PGD applications[J]. Mol Hum Reprod.2004 Nov;10(11):847～852
[15]Renwick PJ, Lewis CM, Abbs S, et al. Determination of the genetic status of cleavage-stage human embryos by microsatellite marker analysis following multiple displacement amplification[J]. Prenat Diagn.2007 Mar;27(3):206～215
[16]Wells D, Sherlock JK, Handyside AH, et al. Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation[J]. Nucleic Acids Res.1999 Feb 15;27(4):1214～1218.
[17]Grewal SS, Kahn JP, MacMillan ML, et al. Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotype-identical sibling selected using preimplantation genetic diagnosis[J]. Blood.2004 Feb 1; 103(3):1147～1151
[18]Lledo B, Ten J, Galan FM, et al. Preimplantation genetic diagnosis of Marfan syndrome using multiple displacement amplification[J]. Fertil Steril.2006 Oct;86(4):949-955.
[19]Renwick PJ, Trussler J, Ostad-Saffari E, et al. Proof of principle and first cases using preimplantation genetic haplotyping--a paradigm shift for embryo diagnosis[J]. Reprod Biomed Online.2006 Jul;13(1):110～119.
[20]Renwick P, Ogilvie CM. Preimplantation genetic diagnosis for monogenic diseases: overview and emerging issues[J]. Expert Rev Mol Diagn.2007 Jan;7(1):33～43.
[2]Corneveaux JJ, Kruer MC, Hu-Line D, et al. SNP-based chromosomal copy number ascertainment following multiple displacement whole-genome amplification [J]. Biotechniques. 2007 Jul;42(1):77～83
[3]Jasmine F, Ahsan H, Andrulis IL, et al. Whole-genome amplification enables accurate genotyping for microarray-based high-density single nucleotide polymorphism array [J]. Cancer Epidemiol Biomarkers Prev.2008 Dec; 17(12):3499～3508
[4]Spits C, Le Caignec C, De Rycke M, et al. Optimization and evaluation of single-cell whole-genome multiple displacement amplification [J]. Hum Mutat.2006 May;27(5):496-503
[5]Dean FB, Hosono S, Fang L, et al. Comprehensive human genome amplification using multiple displacement amplification[J]. Proc Natl Acad Sci U S A.2002 Apr 16;99(8):5261～ 5266
[6]Jiang Z, Zhang X, Deka R, et al. Genome amplification of single sperm using multiple displacement amplification [J]. Nucleic Acids Res.2005 Jul 7;33(10):e91
[7]Ivanov PL, Fomichev AA. Whole-genome amplification by MDA to improve sensitivity of forensic expert examination of chromosomal DNA[J]. Sud Med Ekspert.2008 Jul-Aug;51(4):16-18.
[8]Hosono S, Faruqi AF, Dean FB, et al. Unbiased whole-genome amplification directly from clinical samples[J]. Genome Res.2003 May;13(5):954-964.
[9]Ling J, Zhuang G, Zhang CH,et al. Evaluation of genome coverage and fidelity of multiple displacement amplification from single cells by SNP array[J]. Mol Hum Reprod.2009 Nov;15(11):739～747
[10]Hellani A, Coskun S, Tbakhi A,et al. Clinical application of multiple displacement amplification in preimplantation genetic diagnosis[J]. Reprod Biomed Online.2005 Mar;10(3):376～380.
[11]Kennett JY, Watson SK, Saprunoff H, et al. Technical demonstration of whole genome array comparative genomic hybridization [J]. J Vis Exp.2008 Aug 5;(18).pii:870.doi:10.3791/870.
[12]Panelli S, Damiani G, Espen L, et al. Ligation overcomes terminal underrepresentation in multiple displacement amplification of linear DNA[J]. Biotechniques.2005 Aug;39(2):174,176,178 passim
[13]Sun G, Kaushal R, Pal P, et al. Whole-genome amplification:relative efficiencies of the current methods[J]. Leg Med (Tokyo).2005 Oct;7(5):279-286
[14]Hellani A, Coskun S, Benkhalifa M, et al. Multiple displacement amplification on single cell and possible PGD applications[J]. Mol Hum Reprod.2004 Nov;10(11):847～852
[15]Renwick PJ, Lewis CM, Abbs S, et al. Determination of the genetic status of cleavage-stage human embryos by microsatellite marker analysis following multiple displacement amplification[J]. Prenat Diagn.2007 Mar;27(3):206～215
[16]Wells D, Sherlock JK, Handyside AH, et al. Detailed chromosomal and molecular genetic analysis of single cells by whole genome amplification and comparative genomic hybridisation[J]. Nucleic Acids Res.1999 Feb 15;27(4):1214～1218.
[17]Grewal SS, Kahn JP, MacMillan ML, et al. Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotype-identical sibling selected using preimplantation genetic diagnosis[J]. Blood.2004 Feb 1; 103(3):1147～1151
[18]Lledo B, Ten J, Galan FM, et al. Preimplantation genetic diagnosis of Marfan syndrome using multiple displacement amplification[J]. Fertil Steril.2006 Oct;86(4):949-955.
[19]Renwick PJ, Trussler J, Ostad-Saffari E, et al. Proof of principle and first cases using preimplantation genetic haplotyping--a paradigm shift for embryo diagnosis[J]. Reprod Biomed Online.2006 Jul;13(1):110～119.
[20]Renwick P, Ogilvie CM. Preimplantation genetic diagnosis for monogenic diseases: overview and emerging issues[J]. Expert Rev Mol Diagn.2007 Jan;7(1):33～43.