Advertisement

Identification of biparental and diploid blastocysts from monopronuclear zygotes with the use of a single-nucleotide polymorphism array

  • Ping-Yuan Xie
    Affiliations
    National Engineering and Research Center of Human Stem Cells, Changsha, People's Republic of China
    Search for articles by this author
  • Yi Tang
    Affiliations
    Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, People's Republic of China

    Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, People's Republic of China

    Institute of Reproductive and Stem Cell Engineering, Central South University, School of Basic Medical Science, Changsha, People's Republic of China
    Search for articles by this author
  • Liang Hu
    Affiliations
    Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, People's Republic of China

    Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, People's Republic of China

    Institute of Reproductive and Stem Cell Engineering, Central South University, School of Basic Medical Science, Changsha, People's Republic of China
    Search for articles by this author
  • Qi Ouyang
    Affiliations
    National Engineering and Research Center of Human Stem Cells, Changsha, People's Republic of China

    Institute of Reproductive and Stem Cell Engineering, Central South University, School of Basic Medical Science, Changsha, People's Republic of China
    Search for articles by this author
  • Yi-Fan Gu
    Affiliations
    Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, People's Republic of China

    Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, People's Republic of China

    Institute of Reproductive and Stem Cell Engineering, Central South University, School of Basic Medical Science, Changsha, People's Republic of China
    Search for articles by this author
  • Fei Gong
    Affiliations
    Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, People's Republic of China

    Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, People's Republic of China

    Institute of Reproductive and Stem Cell Engineering, Central South University, School of Basic Medical Science, Changsha, People's Republic of China
    Search for articles by this author
  • Li-Zhi Leng
    Affiliations
    Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, People's Republic of China

    Institute of Reproductive and Stem Cell Engineering, Central South University, School of Basic Medical Science, Changsha, People's Republic of China
    Search for articles by this author
  • Shuo-Ping Zhang
    Affiliations
    Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, People's Republic of China

    Institute of Reproductive and Stem Cell Engineering, Central South University, School of Basic Medical Science, Changsha, People's Republic of China
    Search for articles by this author
  • Bo Xiong
    Affiliations
    Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, People's Republic of China
    Search for articles by this author
  • Guang-Xiu Lu
    Affiliations
    National Engineering and Research Center of Human Stem Cells, Changsha, People's Republic of China

    Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, People's Republic of China

    Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, People's Republic of China

    Institute of Reproductive and Stem Cell Engineering, Central South University, School of Basic Medical Science, Changsha, People's Republic of China
    Search for articles by this author
  • Ge Lin
    Correspondence
    Reprint requests: Ge Lin, Ph.D., Institute of Reproductive and Stem Cell Engineering, School of Basic Medical Science, Central South University, Xiangya Road 84, Changsha 410078, People's Republic of China.
    Affiliations
    National Engineering and Research Center of Human Stem Cells, Changsha, People's Republic of China

    Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, People's Republic of China

    Key Laboratory of Reproductive and Stem Cell Engineering, Ministry of Health, Changsha, People's Republic of China

    Institute of Reproductive and Stem Cell Engineering, Central South University, School of Basic Medical Science, Changsha, People's Republic of China
    Search for articles by this author

      Objective

      To select normal fertilized diploid blastocysts in patients who had only monopronucleated (1PN) embryos for transfer.

      Design

      Experimental study.

      Setting

      University-affiliated center.

      Patient(s)

      Couples who were undergoing intracytoplasmic sperm injection treatment and had 1PN blastocysts.

      Intervention(s)

      In a preliminary test, limited cells of parthenogenetic human embryonic stem cells (phESCs) and normal fertilized blastocysts were analyzed with the use of a low-density single-nucleotide polymorphism (SNP) array to identify the distribution pattern and rate of heterozygosity. In the clinical application, 1PN blastocysts were analyzed with the use of the SNP array. Only diagnosed normal blastocysts were transferred. The diagnosed uniparental blastocysts were validated by imprinted gene expression.

      Main Outcome Measure(s)

      Distribution pattern and rate of heterozygosity between parthenogenesis and normal fertilization.

      Result(s)

      In the pretest, phESCs exhibited distinct distribution pattern and lower rate of heterozygosity, compared with normal fertilized blastocysts after SNP analysis. In particular, homozygous hESCs showed a panhomozygosity distribution pattern, hybrid phESCs showed a partial homozygosity distribution pattern, and normal fertilized blastocysts exhibited a panheterozygosity distribution pattern with an average of 20.21% heterozygosity rate; 13.6% was found to be the minimum cutoff to predict normal fertilized samples. In the clinical application, 24 1PN blastocysts were analyzed; 10/24 showed chromosomal abnormalities, 3/24 showed panhomozygosity with 0.45%–0.8% heterozygosity, and 1/24 showed partial homozygosity with 6.54% heterozygosity. The remaining 10 blastocysts, with a panheterozygosity distribution pattern and higher genomic heterozygosity rate, were diagnosed as normal-fertilization diploid embryos; three were transferred and resulted in two healthy newborns.

      Conclusion(s)

      The low-density SNP array might serve as a cost-effective method to identify biparental origin and diploid 1PN blastocysts for transfer.
      Identificación de blastocistos biparentales y diploides a partir de zigotos monopronucleares utilizando arrays de polimorfismos de nucleótido único

      Objetivo

      Seleccionar blastocistos diploides normales fecundados en pacientes que tenían solo embriones monopronucleares (1PN) para transferir.

      Diseño

      Estudio experimental.

      Localización

      Centro afiliado a la Universidad.

      Pacientes

      Parejas que acudieron a tratamientos con inyección intracitoplasmática de espermatozoides y tuvieron blastocistos con 1 PN.

      Intervención (es)

      En una prueba preliminar, se analizaron un número limitado de células madre embriogénicas partenogenéticas humanas (phESCs) y blastocistos fecundados normales con el uso de arrays de polimorfismos de nucleótido único (SNP) de baja densidad para identificar el patrón de distribución y la tasa de heterocigosidad. En la aplicación clínica, los blastocistos de 1PN se analizaron con el uso del array de SNP. Solo se transfirieron los blastocistos diagnosticados normales. Los blastocistos uniparentales fueron validados analizando expresión de genes con impronta.

      Medidas principales

      Patrón de distribución y tasa de heterocigosidad entre partenogénesis y fecundación normal.

      Resultado (s)

      En la prueba previa, las células phESC mostraron un patrón de distribución distinto y una menor tasa de heterocigosidad, en comparación con los blastocitos fecundados normales después del análisis de SNP.
      En particular, los homocigotos hESCs mostraron un patrón de distribución de panhomozigosidad, los híbridos phESCs mostraron un patrón de distribución de homocigosis parcial, y los blastocistos fecundados normales exhibieron un patrón de distribución panheterocigosidad con un promedio de 20.21% tasa de heterocigosidad; Se descubrió que 13.6% es el límite mínimo para predecir muestras fecundadas normales. En la aplicación clínica, se analizaron 24 blastocistos de 1PN; 10/24 mostraron anomalías cromosómicas, 3/24 mostraron panhomozigosidad con 0.45% -0.8% de heterocigosidad y 1/24 mostró homocigosidad parcial con 6.54% de heterocigosidad. Los 10 blastocistos restantes, con un patrón de distribución de panheterocigosidad y una mayor tasa de heterocigosidad genómica, se diagnosticaron como embriones diploides de fecundación normal; tres fueron transferidos y resultaron en dos recién nacidos sanos.

      Conclusión (es)

      El array de SNP de baja densidad podría servir como un método rentable para identificar el origen biparental y los blastocistos diploides 1PN para la transferencia.

      Key Words

      To read this article in full you will need to make a payment

      References

        • Balakier H.
        • Squire J.
        • Casper R.F.
        Characterization of abnormal one pronuclear human oocytes by morphology, cytogenetics and in-situ hybridization.
        Hum Reprod. 1993; 8: 402-408
        • Munne S.
        • Tang Y.X.
        • Grifo J.
        • Cohen J.
        Origin of single pronucleated human zygotes.
        J Assist Reprod Genet. 1993; 10: 276-279
        • Staessen C.
        • Janssenswillen C.
        • Devroey P.
        • van Steirteghem A.C.
        Cytogenetic and morphological observations of single pronucleated human oocytes after in-vitro fertilization.
        Hum Reprod. 1993; 8: 221-223
        • Nagy Z.P.
        • Janssenswillen C.
        • Janssens R.
        • de Vos A.
        • Staessen C.
        • van de Velde H.
        • et al.
        Timing of oocyte activation, pronucleus formation and cleavage in humans after intracytoplasmic sperm injection (ICSI) with testicular spermatozoa and after ICSI or in-vitro fertilization on sibling oocytes with ejaculated spermatozoa.
        Hum Reprod. 1998; 13: 1606-1612
        • van der Heijden G.W.
        • van den Berg I.M.
        • Baart E.B.
        • Derijck A.A.
        • Martini E.
        • de Boer P.
        Parental origin of chromatin in human monopronuclear zygotes revealed by asymmetric histone methylation patterns, differs between IVF and ICSI.
        Mol Reprod Dev. 2009; 76: 101-108
        • Azevedo A.R.
        • Pinho M.J.
        • Silva J.
        • Sa R.
        • Thorsteinsdottir S.
        • Barros A.
        • et al.
        Molecular cytogenetics of human single pronucleated zygotes.
        Reprod Sci. 2014; 21: 1472-1482
        • Levron J.
        • Munne S.
        • Willadsen S.
        • Rosenwaks Z.
        • Cohen J.
        Male and female genomes associated in a single pronucleus in human zygotes.
        Biol Reprod. 1995; 52: 653-657
        • Bradley C.K.
        • Traversa M.V.
        • Hobson N.
        • Gee A.J.
        • McArthur S.J.
        Clinical use of monopronucleated zygotes following blastocyst culture and preimplantation genetic screening, including verification of biparental chromosome inheritance.
        Reprod Biomed Online. 2017; 34: 567-574
        • Dasig D.
        • Lyon J.
        • Behr B.
        • Milki A.A.
        Monozygotic twin birth after the transfer of a cleavage stage embryo resulting from a single pronucleated oocyte.
        J Assist Reprod Genet. 2004; 21: 427-429
        • Gras L.
        • Trounson A.O.
        Pregnancy and birth resulting from transfer of a blastocyst observed to have one pronucleus at the time of examination for fertilization.
        Hum Reprod. 1999; 14: 1869-1871
        • Itoi F.
        • Asano Y.
        • Shimizu M.
        • Honnma H.
        • Murata Y.
        Birth of nine normal healthy babies following transfer of blastocysts derived from human single-pronucleate zygotes.
        J Assist Reprod Genet. 2015; 32: 1401-1407
        • Staessen C.
        • van Steirteghem A.C.
        The chromosomal constitution of embryos developing from abnormally fertilized oocytes after intracytoplasmic sperm injection and conventional in-vitro fertilization.
        Hum Reprod. 1997; 12: 321-327
        • Liao H.
        • Zhang S.
        • Cheng D.
        • Ouyang Q.
        • Lin G.
        • Gu Y.
        • et al.
        Cytogenetic analysis of human embryos and embryonic stem cells derived from monopronuclear zygotes.
        J Assist Reprod Genet. 2009; 26: 583-589
        • Reichman D.E.
        • Jackson K.V.
        • Racowsky C.
        Incidence and development of zygotes exhibiting abnormal pronuclear disposition after identification of two pronuclei at the fertilization check.
        Fertil Steril. 2010; 94: 965-970
        • Mateo S.
        • Parriego M.
        • Boada M.
        • Vidal F.
        • Coroleu B.
        • Veiga A.
        In vitro development and chromosome constitution of embryos derived from monopronucleated zygotes after intracytoplasmic sperm injection.
        Fertil Steril. 2013; 99: 897-902.e1
        • Yao G.
        • Xu J.
        • Xin Z.
        • Niu W.
        • Shi S.
        • Jin H.
        • et al.
        Developmental potential of clinically discarded human embryos and associated chromosomal analysis.
        Sci Rep. 2016; 6: 23995
        • Mateo S.
        • Vidal F.
        • Parriego M.
        • Rodriguez I.
        • Montalvo V.
        • Veiga A.
        • et al.
        Could monopronucleated ICSI zygotes be considered for transfer? Analysis through time-lapse monitoring and PGS.
        J Assist Reprod Genet. 2017; 34: 905-911
        • Lin G.
        • OuYang Q.
        • Zhou X.
        • Gu Y.
        • 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
        • Kim K.
        • Ng K.
        • Rugg-Gunn P.J.
        • Shieh J.H.
        • Kirak O.
        • Jaenisch R.
        • et al.
        Recombination signatures distinguish embryonic stem cells derived by parthenogenesis and somatic cell nuclear transfer.
        Cell Stem Cell. 2007; 1: 346-352
        • Kim K.
        • Lerou P.
        • Yabuuchi A.
        • Lengerke C.
        • Ng K.
        • West J.
        • et al.
        Histocompatible embryonic stem cells by parthenogenesis.
        Science. 2007; 315: 482-486
        • Lin G.
        • Xie Y.
        • Ouyang Q.
        • Qian X.
        • Xie P.
        • Zhou X.
        • et al.
        HLA-matching potential of an established human embryonic stem cell bank in China.
        Cell Stem Cell. 2009; 5: 461-465
        • Gardner D.K.
        • Surrey E.
        • Minjarez D.
        • Leitz A.
        • Stevens J.
        • Schoolcraft W.B.
        Single blastocyst transfer: a prospective randomized trial.
        Fertil Steril. 2004; 81: 551-555
        • Kuwayama M.
        Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method.
        Theriogenology. 2007; 67: 73-80
        • Tan Y.Q.
        • Tan K.
        • Zhang S.P.
        • Gong F.
        • Cheng D.H.
        • Xiong B.
        • et al.
        Single-nucleotide polymorphism microarray–based preimplantation genetic diagnosis is likely to improve the clinical outcome for translocation carriers.
        Hum Reprod. 2013; 28: 2581-2592
        • Northrop L.E.
        • Treff N.R.
        • Levy B.
        • Scott Jr., R.T.
        SNP microarray–based 24 chromosome aneuploidy screening demonstrates that cleavage-stage FISH poorly predicts aneuploidy in embryos that develop to morphologically normal blastocysts.
        Mol Hum Reprod. 2010; 16: 590-600
        • Picelli S.
        • Faridani O.R.
        • Bjorklund A.K.
        • Winberg G.
        • Sagasser S.
        • Sandberg R.
        Full-length RNA-seq from single cells using Smart-Seq2.
        Nat Protoc. 2014; 9: 171-181
        • Yan L.
        • Yang M.
        • Guo H.
        • Yang L.
        • Wu J.
        • Li R.
        • et al.
        Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells.
        Nat Struct Mol Biol. 2013; 20: 1131-1139
        • Rosenbusch B.
        The chromosomal constitution of embryos arising from monopronuclear oocytes in programmes of assisted reproduction.
        Int J Reprod Med. 2014; 2014: 418198
        • Yan J.
        • Li Y.
        • Shi Y.
        • Feng H.L.
        • Gao S.
        • Chen Z.J.
        Assessment of sex chromosomes of human embryos arising from monopronucleus zygotes in in vitro fertilization and intracytoplasmic sperm injection cycles of Chinese women.
        Gynecol Obstet Invest. 2010; 69: 20-23
        • Macas E.
        • Imthurn B.
        • Roselli M.
        • Keller P.J.
        Chromosome analysis of single- and multipronucleated human zygotes proceeded after the intracytoplasmic sperm injection procedure.
        J Assist Reprod Genet. 1996; 13: 345-350
        • Leng L.
        • Ouyang Q.
        • Kong X.
        • Gong F.
        • Lu C.
        • Zhao L.
        • et al.
        Self-diploidization of human haploid parthenogenetic embryos through the Rho pathway regulates endomitosis and failed cytokinesis.
        Sci Rep. 2017; 7: 4242
        • Ulbright T.M.
        Gonadal teratomas: a review and speculation.
        Adv Anat Pathol. 2004; 11: 10-23
        • Devriendt K.
        Hydatidiform mole and triploidy: the role of genomic imprinting in placental development.
        Hum Reprod Update. 2005; 11: 137-142
        • Strain L.
        • Warner J.P.
        • Johnston T.
        • Bonthron D.T.
        A human parthenogenetic chimaera.
        Nat Genet. 1995; 11: 164-169
        • Sultan K.M.
        • Munne S.
        • Palermo G.D.
        • Alikani M.
        • Cohen J.
        Chromosomal status of uni-pronuclear human zygotes following in-vitro fertilization and intracytoplasmic sperm injection.
        Hum Reprod. 1995; 10: 132-136
        • Harton G.L.
        • de Rycke M.
        • Fiorentino F.
        • Moutou C.
        • SenGupta S.
        • Traeger-Synodinos J.
        • et al.
        ESHRE PGD consortium best practice guidelines for amplification-based PGD.
        Hum Reprod. 2011; 26: 33-40
        • Huang L.
        • Ma F.
        • Chapman A.
        • Lu S.
        • Xie X.S.
        Single-cell whole-genome amplification and sequencing: methodology and applications.
        Annu Rev Genomics Hum Genet. 2015; 16: 79-102
        • Anderegg C.
        • Markert C.L.
        Successful rescue of microsurgically produced homozygous uniparental mouse embryos via production of aggregation chimeras.
        Proc Natl Acad U S A. 1986; 83: 6509-6513
        • Huan Q.
        • Gao X.
        • Wang Y.
        • Shen Y.
        • Ma W.
        • Chen Z.J.
        Comparative evaluation of human embryonic stem cell lines derived from zygotes with normal and abnormal pronuclei.
        Dev Dyn. 2010; 239: 425-438
        • Chen X.
        • Luo Y.
        • Fan Y.
        • Yue L.
        • Wu X.
        • Chen Y.
        • et al.
        Triploid and diploid embryonic stem cell lines derived from tripronuclear human zygotes.
        J Assist Reprod Genet. 2012; 29: 713-721
        • 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: 2016-2023
        • Liu J.
        • Wang X.L.
        • Zhang X.
        • Shen C.Y.
        • Zhang Z.
        Live births resulting from 0PN-derived embryos in conventional IVF cycles.
        J Assist Reprod Genet. 2016; 33: 373-378
        • Li M.
        • Lin S.
        • Chen Y.
        • Zhu J.
        • Liu P.
        • Qiao J.
        Value of transferring embryos that show no evidence of fertilization at the time of fertilization assessment.
        Fertil Steril. 2015; 104: 607-611.e2
        • Matt D.W.
        • Ingram A.R.
        • Graff D.P.
        • Edelstein M.C.
        Normal birth after single-embryo transfer in a patient with excessive polypronuclear zygote formation following in vitro fertilization and intracytoplasmic sperm injection.
        Fertil Steril. 2004; 82: 1662-1665