Skip to main content
Top
Published in: Journal of Assisted Reproduction and Genetics 2/2016

01-02-2016 | Stem Cell Biology

Pronuclear removal of tripronuclear zygotes can establish heteroparental normal karyotypic human embryonic stem cells

Authors: Hong-Qing Liao, Qi OuYang, Shuo-Ping Zhang, De-Hua Cheng, Guang-Xiu Lu, Ge Lin

Published in: Journal of Assisted Reproduction and Genetics | Issue 2/2016

Login to get access

Abstract

Purpose

This study aimed to derive heteroparental normal karyotypic human embryonic stem cells (hESCs) from microsurgically corrected tripronuclear (3PN) zygotes.

Methods

After sequential culture for 5–6 days, embryos developed from microsurgically corrected 3PN zygotes were analyzed by fluorescence in situ hybridization (FISH) using probes for chromosomes 17, X and Y. Intact 3PN zygotes from clinical in vitro fertilization (IVF) cycles were cultured as the control group. The inner cell mass (ICM) of blastocysts that developed from microsurgically corrected 3PN zygotes was used to derive hESC lines, and the stem cell characteristics of these lines were evaluated. G-banding analysis was adopted to identify the karyotype of the hESC line, and the heteroparental inheritance of the hESC line was analyzed by DNA fingerprinting analysis.

Results

The blastocyst formation rate (13.5 %) of the microsurgically corrected 3PN zygotes was significantly higher (P < 0.05) than that of intact 3PN zygotes (8.7 %). The diploid rate of the blastocysts (55.0 %) was significantly higher (P < 0.05) than that of the arrested cleavage-stage embryos (18.4 %) in microsurgically corrected 3PN zygotes. The triploid rate of the microsurgically corrected 3PN zygotes (5.7 %) was significantly lower (P < 0.01) than that of intact 3PN zygotes (19.4 %). Furthermore, we established one heteroparental normal karyotypic hESC line from the microsurgically corrected tripronuclear zygotes.

Conclusions

Pronuclear removal can effectively remove the surplus chromosome set of 3PN zygotes. A combination of pronuclear removal and blastocyst culture enables the selection of diploidized blastocysts from which heteroparental normal karyotypic hESC lines can be derived.
Literature
1.
go back to reference Pieters MH, Dumoulin JC, Ignoul-Vanvuchelen RC, Bras M, Evers JL, Geraedts JP. Triploidy after in vitro fertilization: cytogenetic analysis of human zygotes and embryos. J Assist Reprod Genet. 1992;9:68–76.CrossRefPubMed Pieters MH, Dumoulin JC, Ignoul-Vanvuchelen RC, Bras M, Evers JL, Geraedts JP. Triploidy after in vitro fertilization: cytogenetic analysis of human zygotes and embryos. J Assist Reprod Genet. 1992;9:68–76.CrossRefPubMed
3.
go back to reference Porter R, Han T, Tucker MJ, Graham J, Liebermann J, Sills ES. Estimation of second polar body retention rate after conventional insemination and intracytoplasmic sperm injection: in vitro observations from more than 5000 human oocytes. J Assist Reprod Genet. 2003;20:371–6.PubMedCentralCrossRefPubMed Porter R, Han T, Tucker MJ, Graham J, Liebermann J, Sills ES. Estimation of second polar body retention rate after conventional insemination and intracytoplasmic sperm injection: in vitro observations from more than 5000 human oocytes. J Assist Reprod Genet. 2003;20:371–6.PubMedCentralCrossRefPubMed
5.
go back to reference Plachot M, Crozet N. Fertilization abnormalities in human in-vitro fertilization. Hum Reprod. 1992;7 Suppl 1:89–94.CrossRefPubMed Plachot M, Crozet N. Fertilization abnormalities in human in-vitro fertilization. Hum Reprod. 1992;7 Suppl 1:89–94.CrossRefPubMed
6.
go back to reference Staessen C, Van Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum Reprod. 1997;12:321–7.CrossRefPubMed Staessen C, Van Steirteghem AC. The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization. Hum Reprod. 1997;12:321–7.CrossRefPubMed
7.
go back to reference Grossmann M, Calafell JM, Brandy N, Vanrell JA, Rubio C, Pellicer A, et al. Origin of tripronucleate zygotes after intracytoplasmic sperm injection. Hum Reprod. 1997;12:2762–5.CrossRefPubMed Grossmann M, Calafell JM, Brandy N, Vanrell JA, Rubio C, Pellicer A, et al. Origin of tripronucleate zygotes after intracytoplasmic sperm injection. Hum Reprod. 1997;12:2762–5.CrossRefPubMed
8.
go back to reference Golubovsky MD. Postzygotic diploidization of triploids as a source of unusual cases of mosaicism, chimerism and twinning. Hum Reprod. 2003;18:236–42.CrossRefPubMed Golubovsky MD. Postzygotic diploidization of triploids as a source of unusual cases of mosaicism, chimerism and twinning. Hum Reprod. 2003;18:236–42.CrossRefPubMed
10.
go back to reference Zaragoza MV, Surti U, Redline RW, Millie E, Chakravarti A, Hassold TJ. Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatidiform mole. Am J Hum Genet. 2000;66:1807–20. doi:10.1086/302951.PubMedCentralCrossRefPubMed Zaragoza MV, Surti U, Redline RW, Millie E, Chakravarti A, Hassold TJ. Parental origin and phenotype of triploidy in spontaneous abortions: predominance of diandry and association with the partial hydatidiform mole. Am J Hum Genet. 2000;66:1807–20. doi:10.​1086/​302951.PubMedCentralCrossRefPubMed
13.
go back to reference Rawlins RG, Binor Z, Radwanska E, Dmowski WP. Microsurgical enucleation of tripronuclear human zygotes. Fertil Steril. 1988;50:266–72.PubMed Rawlins RG, Binor Z, Radwanska E, Dmowski WP. Microsurgical enucleation of tripronuclear human zygotes. Fertil Steril. 1988;50:266–72.PubMed
14.
go back to reference Gordon JW, Grunfeld L, Garrisi GJ, Navot D, Laufer N. Successful microsurgical removal of a pronucleus from tripronuclear human zygotes. Fertil Steril. 1989;52:367–72.PubMed Gordon JW, Grunfeld L, Garrisi GJ, Navot D, Laufer N. Successful microsurgical removal of a pronucleus from tripronuclear human zygotes. Fertil Steril. 1989;52:367–72.PubMed
15.
go back to reference Malter HE, Cohen J. Embryonic development after microsurgical repair of polyspermic human zygotes. Fertil Steril. 1989;52:373–80.PubMed Malter HE, Cohen J. Embryonic development after microsurgical repair of polyspermic human zygotes. Fertil Steril. 1989;52:373–80.PubMed
16.
go back to reference Palermo G, Munne S, Cohen J. The human zygote inherits its mitotic potential from the male gamete. Hum Reprod. 1994;9:1220–5.PubMed Palermo G, Munne S, Cohen J. The human zygote inherits its mitotic potential from the male gamete. Hum Reprod. 1994;9:1220–5.PubMed
17.
go back to reference Ivakhnenko V, Cieslak J, Verlinsky Y. A microsurgical technique for enucleation of multipronuclear human zygotes. Hum Reprod. 2000;15:911–6.CrossRefPubMed Ivakhnenko V, Cieslak J, Verlinsky Y. A microsurgical technique for enucleation of multipronuclear human zygotes. Hum Reprod. 2000;15:911–6.CrossRefPubMed
19.
go back to reference Gu Y-F, Lin G, Lu C-F, Lu G-X. Analysis of the first mitotic spindles in human in vitro fertilized tripronuclear zygotes after pronuclear removal. Reprod BioMed Online. 2009;19:745–54.CrossRefPubMed Gu Y-F, Lin G, Lu C-F, Lu G-X. Analysis of the first mitotic spindles in human in vitro fertilized tripronuclear zygotes after pronuclear removal. Reprod BioMed Online. 2009;19:745–54.CrossRefPubMed
20.
go back to reference Kattera S, Chen C. Normal birth after microsurgical enucleation of tripronuclear human zygotes: case report. Hum Reprod. 2003;18:1319–22.CrossRefPubMed Kattera S, Chen C. Normal birth after microsurgical enucleation of tripronuclear human zygotes: case report. Hum Reprod. 2003;18:1319–22.CrossRefPubMed
21.
go back to reference Gardner DK, Schoolcraft WB. No longer neglected: the human blastocyst. Hum Reprod. 1998;13:3289–92.CrossRefPubMed Gardner DK, Schoolcraft WB. No longer neglected: the human blastocyst. Hum Reprod. 1998;13:3289–92.CrossRefPubMed
22.
go back to reference Jones GM, Trounson AO, Gardner DK, Kausche A, Lolatgis N, Wood C. Evolution of a culture protocol for successful blastocyst development and pregnancy. Hum Reprod. 1998;13:169–77.CrossRefPubMed Jones GM, Trounson AO, Gardner DK, Kausche A, Lolatgis N, Wood C. Evolution of a culture protocol for successful blastocyst development and pregnancy. Hum Reprod. 1998;13:169–77.CrossRefPubMed
23.
go back to reference Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–7.CrossRefPubMed Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, et al. Embryonic stem cell lines derived from human blastocysts. Science. 1998;282:1145–7.CrossRefPubMed
24.
go back to reference Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 2000;18:399–404. doi:10.1038/74447.CrossRefPubMed Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol. 2000;18:399–404. doi:10.​1038/​74447.CrossRefPubMed
27.
go back to reference Lin G, OuYang Q, Zhou XY, Gu YF, Yuan D, Li W, et al. A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro fertilization procedure. Cell Res. 2007;17:999–1007. doi:10.1038/cr.2007.97.CrossRefPubMed Lin G, OuYang Q, Zhou XY, Gu YF, Yuan D, Li W, et al. A highly homozygous and parthenogenetic human embryonic stem cell line derived from a one-pronuclear oocyte following in vitro fertilization procedure. Cell Res. 2007;17:999–1007. doi:10.​1038/​cr.​2007.​97.CrossRefPubMed
29.
go back to reference Angell RR, Templeton AA, Messinis IE. Consequences of polyspermy in man. Cytogenet Cell Genet. 1986;42:1–7.CrossRefPubMed Angell RR, Templeton AA, Messinis IE. Consequences of polyspermy in man. Cytogenet Cell Genet. 1986;42:1–7.CrossRefPubMed
30.
go back to reference Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod. 1987;37:395–401.CrossRefPubMed Kola I, Trounson A, Dawson G, Rogers P. Tripronuclear human oocytes: altered cleavage patterns and subsequent karyotypic analysis of embryos. Biol Reprod. 1987;37:395–401.CrossRefPubMed
31.
go back to reference Rosenbusch B, Schneider M, Sterzik K. The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization. Hum Reprod. 1997;12:2257–62.CrossRefPubMed Rosenbusch B, Schneider M, Sterzik K. The chromosomal constitution of multipronuclear zygotes resulting from in-vitro fertilization. Hum Reprod. 1997;12:2257–62.CrossRefPubMed
32.
go back to reference Iliopoulos D, Vassiliou G, Sekerli E, Sidiropoulou V, Tsiga A, Dimopoulou D, et al. Long survival in a 69, XXX triploid infant in Greece. Genet Mol Res. 2005;4:755–9.PubMed Iliopoulos D, Vassiliou G, Sekerli E, Sidiropoulou V, Tsiga A, Dimopoulou D, et al. Long survival in a 69, XXX triploid infant in Greece. Genet Mol Res. 2005;4:755–9.PubMed
33.
go back to reference Clouston HJ, Fenwick J, Webb AL, Herbert M, Murdoch A, Wolstenholme J. Detection of mosaic and non-mosaic chromosome abnormalities in 6- to 8-day old human blastocysts. Hum Genet. 1997;101:30–6.CrossRefPubMed Clouston HJ, Fenwick J, Webb AL, Herbert M, Murdoch A, Wolstenholme J. Detection of mosaic and non-mosaic chromosome abnormalities in 6- to 8-day old human blastocysts. Hum Genet. 1997;101:30–6.CrossRefPubMed
34.
go back to reference Evsikov S, Verlinsky Y. Mosaicism in the inner cell mass of human blastocysts. Hum Reprod. 1998;13:3151–5.CrossRefPubMed Evsikov S, Verlinsky Y. Mosaicism in the inner cell mass of human blastocysts. Hum Reprod. 1998;13:3151–5.CrossRefPubMed
35.
go back to reference Magli MC, Jones GM, Gras L, Gianaroli L, Korman I, Trounson AO. Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum Reprod. 2000;15:1781–6.CrossRefPubMed Magli MC, Jones GM, Gras L, Gianaroli L, Korman I, Trounson AO. Chromosome mosaicism in day 3 aneuploid embryos that develop to morphologically normal blastocysts in vitro. Hum Reprod. 2000;15:1781–6.CrossRefPubMed
36.
go back to reference Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munne S. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum Reprod. 2001;16:1954–8.CrossRefPubMed Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munne S. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Hum Reprod. 2001;16:1954–8.CrossRefPubMed
39.
go back to reference Janny L, Menezo YJ. Maternal age effect on early human embryonic development and blastocyst formation. Mol Reprod Dev. 1996;45:31–7.CrossRefPubMed Janny L, Menezo YJ. Maternal age effect on early human embryonic development and blastocyst formation. Mol Reprod Dev. 1996;45:31–7.CrossRefPubMed
41.
go back to reference Jiang C, Cai L, Huang B, Dong J, Chen A, Ning S, et al. Normal human embryonic stem cell lines were derived from microsurgical enucleated tripronuclear zygotes. J Cell Biochem. 2013;114:2016–23. doi:10.1002/jcb.24547.CrossRefPubMed Jiang C, Cai L, Huang B, Dong J, Chen A, Ning S, et al. Normal human embryonic stem cell lines were derived from microsurgical enucleated tripronuclear zygotes. J Cell Biochem. 2013;114:2016–23. doi:10.​1002/​jcb.​24547.CrossRefPubMed
42.
go back to reference Fan Y, Li R, Huang J, Zhao HC, Ding T, Sun X, et al. Improved efficiency of microsurgical enucleated tripronuclear zygotes development and embryonic stem cell derivation by supplementing epidermal growth factor, brain-derived neurotrophic factor, and insulin-like growth factor-1. Stem Cells Dev. 2014;23:563–75. doi:10.1089/scd.2013.0420.PubMedCentralCrossRefPubMed Fan Y, Li R, Huang J, Zhao HC, Ding T, Sun X, et al. Improved efficiency of microsurgical enucleated tripronuclear zygotes development and embryonic stem cell derivation by supplementing epidermal growth factor, brain-derived neurotrophic factor, and insulin-like growth factor-1. Stem Cells Dev. 2014;23:563–75. doi:10.​1089/​scd.​2013.​0420.PubMedCentralCrossRefPubMed
Metadata
Title
Pronuclear removal of tripronuclear zygotes can establish heteroparental normal karyotypic human embryonic stem cells
Authors
Hong-Qing Liao
Qi OuYang
Shuo-Ping Zhang
De-Hua Cheng
Guang-Xiu Lu
Ge Lin
Publication date
01-02-2016
Publisher
Springer US
Published in
Journal of Assisted Reproduction and Genetics / Issue 2/2016
Print ISSN: 1058-0468
Electronic ISSN: 1573-7330
DOI
https://doi.org/10.1007/s10815-015-0634-8

Other articles of this Issue 2/2016

Journal of Assisted Reproduction and Genetics 2/2016 Go to the issue