体外受精与体外培养对小鼠囊胚发育潜能的影响
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摘要
目的评估体外受精和体外培养对囊胚期胚胎细胞数及发育潜能的影响。方法 (1)雌性ICR小鼠,自然交配获取囊胚为In vivo组,体内受精-体外培养获取囊胚为IVC组,体外受精获取囊胚为IVF组;(2)对三组囊胚行免疫杀伤法检测并在荧光显微镜下计数总细胞数、内细胞团细胞数、内细胞团与总细胞数的比值;(3)提取三组小鼠囊胚mRNA,采用实时荧光定量聚合酶链反应(qRT-PCR)方法检测Pou5f1等细胞因子的mRNA表达水平;(4)三组囊胚移植入D3.5假孕鼠,取D10.5胚胎进行形态学检测,计算胚胎植入率、成活率、流产率。结果 (1)IVF组和IVC组囊胚与In vivo组相比,细胞总数、滋养外胚层(TE)细胞数、内细胞团(ICM)细胞数均显著降低(P<0.01),TE/ICM细胞数的比值三组之间无显著差异(P>0.05);(2)IVF组和IVC组囊胚与In vivo组相比,多潜能基因OCT4、Nanog、Sox2表达水平相对上调,而早期细胞命运分化相关基因Fgf4、Gata4、Sox17、Eomes、Elf5、Lif表达水平下调,差异有统计学意义(P<0.05);(3)三组间胚胎植入率无显著性差异(P>0.05),而IVC组、IVF组活产率显著低于In vivo组,流产率显著高于In vivo组,差异有统计学意义(P<0.05)。结论体外受精会导致囊胚质量下降,并影响胚胎植入后的进一步发育,导致胚胎成活率降低。
Objective:To evaluate the effect of in vitro fertilization and in vitro culture on the development potential of blastocyst.Methods:The ICR mice were used for the study.The blastocysts from in vitro fertilization were named as IVF group,and the blastocysts from in vivo fertilization or in vitro culture were named as IVC group and the in vivo group respectively.The blastocysts mRNA were extracted and the mRNA expression of cytokines such as Pou5 f1 was detected by real-time quantitative polymerase chain reaction(qRT-PCR)in the three groups.The blastocysts were transplanted into Day 3.5 pseudo-pregnant rats,and the embryos on Day10.5 were taken for morphological examination.The embryo implantation rate,survival rate and abortion rate were compared among the three groups.Results:Compared with the in vivo group,the total cell number,the cell numbers of trophectoderm(TE)and the inner cell mass(ICM)were significantly decreased in IVF and IVC group(P<0.01).The ratio of TE/ICM cell number was not significantly different among the three groups(P>0.05).The expressions of OCT4,Nanog and Sox2 in IVF group and IVC group were significantly up-regulated than that in in vivo group,while the expression of Fgf4,Gata4,Sox17,Eomes,Elf5 and Lif were significantly down-regulated in early stage(P<0.05).There was no significant difference in embryo implantation ratesamong the three groups(P>0.05),but the live birth rate in IVC group and IVF group were significantly lower than the in vivo group(P<0.05),and the abortion rate was significantly higher than the in vivo group(P<0.05).Conclusions:In vitro fertilization leads to a decline in the quality of the blastocyst,which affects the further development of the embryo and leads to decline the live birth rate.
Objective:To evaluate the effect of in vitro fertilization and in vitro culture on the development potential of blastocyst.Methods:The ICR mice were used for the study.The blastocysts from in vitro fertilization were named as IVF group,and the blastocysts from in vivo fertilization or in vitro culture were named as IVC group and the in vivo group respectively.The blastocysts mRNA were extracted and the mRNA expression of cytokines such as Pou5 f1 was detected by real-time quantitative polymerase chain reaction(qRT-PCR)in the three groups.The blastocysts were transplanted into Day 3.5 pseudo-pregnant rats,and the embryos on Day10.5 were taken for morphological examination.The embryo implantation rate,survival rate and abortion rate were compared among the three groups.Results:Compared with the in vivo group,the total cell number,the cell numbers of trophectoderm(TE)and the inner cell mass(ICM)were significantly decreased in IVF and IVC group(P<0.01).The ratio of TE/ICM cell number was not significantly different among the three groups(P>0.05).The expressions of OCT4,Nanog and Sox2 in IVF group and IVC group were significantly up-regulated than that in in vivo group,while the expression of Fgf4,Gata4,Sox17,Eomes,Elf5 and Lif were significantly down-regulated in early stage(P<0.05).There was no significant difference in embryo implantation ratesamong the three groups(P>0.05),but the live birth rate in IVC group and IVF group were significantly lower than the in vivo group(P<0.05),and the abortion rate was significantly higher than the in vivo group(P<0.05).Conclusions:In vitro fertilization leads to a decline in the quality of the blastocyst,which affects the further development of the embryo and leads to decline the live birth rate.
引文
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[14]HosseinNia P,Hajian M,Tahmoorespur M,et al.Expression profile of developmentally important genes in pre and periimplantation goat embryos produced in vitro[J].Int J Fertil Steril,2016,10:310-319.
[15]Veraguas D,Gallegos PF,Velasquez AE,et al.FSH stimulation of anestrous cats improves oocyte quality and development of parthenogenetic embryos[J].Theriogenology,2017,87:25-35.
[16]Rodríguez-Alvarez L,Cox J,Tovar H,et al.Changes in the expression of pluripotency-associated genes during preimplantation and peri-implantation stages in bovine cloned and in vitro produced embryos[J].Zygote,2010,18:269-279.
[17]Majumdar G,Majumdar A,Verma IC,et al.Relationship between morphology,euploidy and implantation potential of cleavage and blastocyst stage embryos[J].J Hum Reprod Sci,2017,10:49-57.
[18]Pathak S,Kedia-Mokashi N,Saxena M,et al.Effect of tamoxifen treatment on global and insulin-like growth factor2-H19locus-specific DNA methylation in rat spermatozoa and its association with embryo loss[J].Fertil Steril,2009,91:2253-2263.
[19]Ankolkar M,Patil A,Warke H,et al.Methylation analysis of idiopathic recurrent spontaneous miscarriage cases reveals aberrant imprinting at H19 ICR in normozoospermic individuals[J].Fertil Steril,2012,98:1186-1192.
[20]Liu Y,Tang Y,Ye D,et al.Impact of abnormal DNA methylation of imprinted loci on human spontaneous abortion[J].Reprod Sci,2017,1:1933719117704906.
[21]Carrell DT.Aberrant methylation of the H19 imprinting control region may increase the risk of spontaneous abortion[J].Epigenomics,2013,5:23-24.
[2]Adashi EY,Gleicher N.Is a blanket elective single embryo transfer policy defensible?[J].Rambam Maimonides Med J,2017,8.
[3]Wang X,Du M,Guan Y,et al.Comparative neonatal outcomes in singleton births from blastocyst transfers or cleavage-stage embryo transfers:a systematic review and meta-analysis[J].Reprod Biol Endocrinol,2017,15:36.
[4]薛侠,施文浩,师娟子,等.D5选择性的单囊胚移植和双囊胚胎移植妊娠结局比较[J].生殖医学杂志,2014,23:276-279.
[5]Richard B,Marina G,Kristina VN,et al.Manipulating the mouse embryo[M].New York:Cold Spring Harbor Laboratory Press,2014:1-795.
[6]Lane M,Gardner DK.Nonessential amino acids and glutamine decrease the time of the first three cleavage divisions and increase compaction of mouse zygotes in vitro[J].J Assist Reprod Genet,1997,14:398-403.
[7]Cardellicchio L,Reschini M,Paffoni A,et al.Frozen-thawed blastocyst transfer in natural cycle:feasibility in everyday clinical practice[J].Arch Gynecol Obstet,2017,295:1509-1514.
[8]Qu P,Qing S,Liu R,et al.Effects of embryo-derived exosomes on the development of bovine cloned embryos[J/OL].PLoS One,2017,12:e174535.
[9]王保垒,赵禹,刘军,等.共培养对小鼠囊胚质量及其表观遗传修饰的影响[J].生物工程学报,2009,25:733-738.
[10]Maluf M,Perin PM,Foltran Januário DA,et al.In vitro fertilization,embryo development,and cell lineage segregation after pre-and/or postnatal exposure of female mice to ambient fine particulate matter[J].Fertil Steril,2009,92:1725-1735.
[11]Whitmill A,Liu Y,Timani KA,et al.Tip110 deletion impaired embryonic and stem cell development involving downregulation of stem cell factors Nanog,Oct4,and Sox2[J].Stem Cells,2017,35:1674-1686.
[12]Guo G,Huss M,Tong GQ,et al.Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst[J].Dev Cell,2010,18:675-685.
[13]Soma M,Iha M,Kihara Y,et al.Preferential emergence of cell types expressing markers for primitive endoderm lineages in mouse embryonic stem cells expressing exogenous EGAM1homeoprotein[J].J Biosci Bioeng,2012,114:342-346.
[14]HosseinNia P,Hajian M,Tahmoorespur M,et al.Expression profile of developmentally important genes in pre and periimplantation goat embryos produced in vitro[J].Int J Fertil Steril,2016,10:310-319.
[15]Veraguas D,Gallegos PF,Velasquez AE,et al.FSH stimulation of anestrous cats improves oocyte quality and development of parthenogenetic embryos[J].Theriogenology,2017,87:25-35.
[16]Rodríguez-Alvarez L,Cox J,Tovar H,et al.Changes in the expression of pluripotency-associated genes during preimplantation and peri-implantation stages in bovine cloned and in vitro produced embryos[J].Zygote,2010,18:269-279.
[17]Majumdar G,Majumdar A,Verma IC,et al.Relationship between morphology,euploidy and implantation potential of cleavage and blastocyst stage embryos[J].J Hum Reprod Sci,2017,10:49-57.
[18]Pathak S,Kedia-Mokashi N,Saxena M,et al.Effect of tamoxifen treatment on global and insulin-like growth factor2-H19locus-specific DNA methylation in rat spermatozoa and its association with embryo loss[J].Fertil Steril,2009,91:2253-2263.
[19]Ankolkar M,Patil A,Warke H,et al.Methylation analysis of idiopathic recurrent spontaneous miscarriage cases reveals aberrant imprinting at H19 ICR in normozoospermic individuals[J].Fertil Steril,2012,98:1186-1192.
[20]Liu Y,Tang Y,Ye D,et al.Impact of abnormal DNA methylation of imprinted loci on human spontaneous abortion[J].Reprod Sci,2017,1:1933719117704906.
[21]Carrell DT.Aberrant methylation of the H19 imprinting control region may increase the risk of spontaneous abortion[J].Epigenomics,2013,5:23-24.