Skip to main content

Advertisement

Comprehensive chromosome analysis of blastocysts before implantation using array CGH

Article metrics

Abstract

Background

Chromosomal abnormalities are common in embryos produced in vitro and cause implantation failure, miscarriage, and serious medical problems in infants. Because preimplantation genetic screening (PGS) is increasingly being used to detect aneuploidy in embryos with the purpose of improving implantation rates after IVF (in vitro fertilization), we aimed to validate the usefulness of array CGH for the preimplantation genetic screening (PGS) of embryos at the blastocyst stage of development.

Results

A total of 150 blastocysts were biopsied from couples undergoing IVF and analyzed using array CGH. We found that 54.5% (73/134) of the blastocysts were euploid embryos, whereas 45.5% of the embryos (61/134) had chromosomal abnormalities. Multiple chromosome abnormality was most frequently observed (34.4%), and dual aneuploidy was observed in 26.2% of the embryos. Monosomy (21.3%) appeared more frequently than trisomy (18%).

Conclusion

Chromosomal microarray analysis provided clinically significant cytogenetic information regarding the frequency and variety of chromosomal abnormalities observed in embryos at the blastocyst stage, suggesting that this is a useful tool for comprehensive aneuploidy screening in IVF.

Background

During IVF procedures, a preimplantation genetic diagnosis (PGD) is used to eliminate embryos carrying genetic diseases prior to implantation. The first application of PGD was successfully performed for couples at risk for transmitting recessive X-linked diseases to male offspring [1], whereby polymerase chain reaction (PCR) was used to determine the sex of the embryos.

Unlike PCR methods, preimplantation genetic screening (PGS) aims to provide a means for identifying potentially viable euploid embryos i.e., screening that may improve pregnancy rates. PGS was first described by Verlinsky et al. [2] and Munne et al. [3]. Although previous methods for embryo screening used fluorescence in situ hybridization (FISH) to analyze chromosomes [4, 5], the FISH approach is limited because the technique is unable to screen all chromosomes simultaneously. Conventional comparative genomic hybridization (CGH) has been used to comprehensively screen for aneuploidy in oocytes and embryos [6, 7]. However, although useful for selecting euploid embryos, the CGH protocol is not generally used because it is time consuming and complicated. At present, both array CGH (aCGH) and single nucleotide polymorphism (SNP) arrays have been validated as accurate methods for producing comprehensive analyses of chromosome in embryos that are compatible with day-3 biopsies and day-5 replacements in a fresh cycle [812]. The difference in mosaicism between embryos at days 3 and 5 has led to a preference for biopsies at the blastocyst stage [13, 14].

Here, we describe the results of an embryo analysis and the details of the chromosomal abnormalities found.

Results

In total, we analyzed 150 blastocysts from 49 couples undergoing IVF (Table 1). Amplification was not detected in 11 (7.3%) embryos, and noisy profile results were obtained for 3.6% (5/139) of the embryos. Euploidy was found in 54.5% of the embryos (73/134), whereas chromosomal abnormalities were found in 45.5% (61/134) of the embryos.

Table 1 Array-CGH results

The details of the array CGH results derived from aneuploid embryos (n=61) are summarized in Table 2. The type of chromosomal abnormality that was most frequently observed was multiple chromosomal abnormality (34.4%), and the second most frequent was dual chromosomal abnormality (26.2%). Monosomy (21.3%) appeared more frequently than trisomy (18%). Examples of array CGH profiles are shown in Figure 1.

Table 2 Chromosome abnormality analyzed
Figure 1
figure1

Examples of array CGH results are shown. (A) Array result displaying a female cell with the loss of chromosome 22. (B) A male cell with trisomy 21. (C) An XY cell showing two chromosomal abnormalities i.e., gain of chromosome 8 and loss of chromosome 14. (D) A male cell with multiple chromosomal abnormalities - gain of chromosomes 3 and 15 and loss of chromosome 22. (E) A normal female cell. (F) A cultured cell with a 2p duplication was used as a positive control.

The chromosomes that were most frequently detected to have aneuploidy were, in order, 15, 22, 21, 16, and 18. Chromosomes 4 and 12 were the least frequently found to have aneuploidy.

Discussion

Preimplantation genetic screening for aneuploidy is increasingly used to examine the chromosomes of embryos from couples undergoing IVF [810, 15, 16]. The purpose of PGS is to identify embryos that are free from chromosomal abnormalities. The main indications for PGS are maternal age, repeated implantation failure, and repeated miscarriage. We examined 150 embryos from 49 couples with these indications.

As shown in Table 1, we successfully analyzed 89.3% (134/150) of the embryos and found that 45.5% (61/134) of the embryos contained abnormal chromosomes. Although the array CGH method is robust and specific, we observed some failure in amplification and a noisy profile. Some cells containing degraded DNA or samples of low quality resulting from apoptosis can be obtained during the biopsy procedure, causing experimental error.

The results showed excessive single chromosome loss versus single chromosome gain (Table 2); frequent abnormalities in chromosomes 15, 22, 21, 16, and 18; and rare aneuploidy in chromosomes 4 and 12, which are similar to the results of previous reports [1719]. Although the frequency of chromosomal abnormalities varied, aneuploidy occurred in all of the 24 chromosomes (data not shown), suggesting that PGS is necessary for selecting healthy embryos during IVF procedures. Previous studies have shown the importance of screening embryos with improved pregnancy success as a result [19].

In conclusion, array CGH is a useful technique for the detection of chromosomal abnormalities during IVF procedures, as previously described [18]. However, embryo cultures up to days 5 or 6 should be established before performing array CGH experiments on blastocysts, and further evidence is required to determine whether PGS results in enhanced delivery rates[14].

Methods

Patient materials

A total of 150 blastocysts were collected from 49 couples who visited the clinic center to undergo IVF between September 2011 and December 2012. All patient materials were obtained and evaluated with informed patient consent and under approval from the Ethics Committees of MGMED clinic center and Seoul Rachel Fertility Center. All patients were provided with counseling regarding PGS using array CGH and signed an informed consent prior to entering the study.

Experimental procedures

The biopsied cells were washed in PBS and collected into PCR tubes. Whole-genome amplification was performed using a kit and following the manufacturer’s instructions (Sigma-Aldrich, Saint Louis, MO).

Approximately 3 μg of amplified DNA was used in the array CGH experiments, as described, with slight modifications [20]. Briefly, the amplified DNA was labeled with Cy-3 and Cy-5 dCTP for 3 h using a random priming method. The labeled DNA was purified, dissolved in hybridization buffer, and hybridized overnight. The slides were washed several times and dried as described [21]. Images of the slides were acquired with a GenePix4000B dual-laser scanner (Axon Instruments, Union City, CA) and analyzed with MacViewer software [21].

References

  1. 1.

    Handyside AH, Kontogianni EH, Hardy K, Winston RM: Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990, 344: 768–770. 10.1038/344768a0

  2. 2.

    Verlinsky Y, Cieslak J, Freidine M, Ivakhnenko V, Wolf G, Kovalinskaya L, White M, Lifchez A, Kaplan B, Moise J, et al.: Pregnancies following pre-conception diagnosis of common aneuploidies by fluorescent in-situ hybridization. Hum Reprod 1995, 10: 1923–1927.

  3. 3.

    Munne S, Dailey T, Sultan KM, Grifo J, Cohen J: The use of first polar bodies for preimplantation diagnosis of aneuploidy. Hum Reprod 1995, 10: 1014–1020.

  4. 4.

    Colls P, Goodall N, Zheng X, Munne S: Increased efficiency of preimplantation genetic diagnosis for aneuploidy by testing 12 chromosomes. Reprod Biomed Online 2009, 19: 532–538. 10.1016/j.rbmo.2009.05.002

  5. 5.

    Jansen RP, Bowman MC, de Boer KA, Leigh DA, Lieberman DB, McArthur SJ: What next for preimplantation genetic screening (PGS)? Experience with blastocyst biopsy and testing for aneuploidy. Hum Reprod 2008, 23: 1476–1478. 10.1093/humrep/den129

  6. 6.

    Voullaire L, Wilton L, Slater H, Williamson R: Detection of aneuploidy in single cells using comparative genomic hybridization. Prenat Diagn 1999, 19: 846–851. 10.1002/(SICI)1097-0223(199909)19:9<846::AID-PD657>3.0.CO;2-#

  7. 7.

    Wells D, Delhanty JD: Comprehensive chromosomal analysis of human preimplantation embryos using whole genome amplification and single cell comparative genomic hybridization. Mol Hum Reprod 2000, 6: 1055–1062. 10.1093/molehr/6.11.1055

  8. 8.

    Hellani A, Abu-Amero K, Azouri J, El-Akoum S: Successful pregnancies after application of array-comparative genomic hybridization in PGS-aneuploidy screening. Reprod Biomed Online 2008, 17: 841–847. 10.1016/S1472-6483(10)60413-0

  9. 9.

    Hu DG, Webb G, Hussey N: Aneuploidy detection in single cells using DNA array-based comparative genomic hybridization. Mol Hum Reprod 2004, 10: 283–289. 10.1093/humrep/gah038

  10. 10.

    Le Caignec C, Spits C, Sermon K, De Rycke M, Thienpont B, Debrock S, Staessen C, Moreau Y, Fryns JP, Van Steirteghem A, et al.: Single-cell chromosomal imbalances detection by array CGH. Nucleic Acids Res 2006, 34: e68. 10.1093/nar/gkl336

  11. 11.

    Johnson DS, Gemelos G, Baner J, Ryan A, Cinnioglu C, Banjevic M, Ross R, Alper M, Barrett B, Frederick J, et al.: Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol. Hum Reprod 2010, 25: 1066–1075. 10.1093/humrep/dep452

  12. 12.

    Treff NR, Su J, Tao X, Miller KA, Levy B, Scott RT Jr: A novel single-cell DNA fingerprinting method successfully distinguishes sibling human embryos. Fertil Steril 2010, 94: 477–484. 10.1016/j.fertnstert.2009.03.067

  13. 13.

    Schoolcraft WB, Fragouli E, Stevens J, Munne S, Katz-Jaffe MG, Wells D: Clinical application of comprehensive chromosomal screening at the blastocyst stage. Fertil Steril 2010, 94: 1700–1706. 10.1016/j.fertnstert.2009.10.015

  14. 14.

    Ly KD, Agarwal A, Nagy ZP: Preimplantation genetic screening: does it help or hinder IVF treatment and what is the role of the embryo? J Assist Reprod Genet 2011, 28: 833–849. 10.1007/s10815-011-9608-7

  15. 15.

    Hodes-Wertz B, Grifo J, Ghadir S, Kaplan B, Laskin CA, Glassner M, Munne S: Idiopathic recurrent miscarriage is caused mostly by aneuploid embryos. Fertil Steril 2012, 98: 675–680. 10.1016/j.fertnstert.2012.05.025

  16. 16.

    Ata B, Kaplan B, Danzer H, Glassner M, Opsahl M, Tan SL, Munne S: Array CGH analysis shows that aneuploidy is not related to the number of embryos generated. Reprod Biomed Online 2012, 24: 614–620. 10.1016/j.rbmo.2012.02.009

  17. 17.

    Munne S, Bahce M, Sandalinas M, Escudero T, Marquez C, Velilla E, Colls P, Oter M, Alikani M, Cohen J: Differences in chromosome susceptibility to aneuploidy and survival to first trimester. Reprod Biomed Online 2004, 8: 81–90. 10.1016/S1472-6483(10)60501-9

  18. 18.

    Gutierrez-Mateo C, Colls P, Sanchez-Garcia J, Escudero T, Prates R, Ketterson K, Wells D, Munne S: Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos. Fertil Steril 2011, 95: 953–958. 10.1016/j.fertnstert.2010.09.010

  19. 19.

    Yang Z, Liu J, Collins GS, Salem SA, Liu X, Lyle SS, Peck AC, Sills ES, Salem RD: Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet 2012, 5: 24. 10.1186/1755-8166-5-24

  20. 20.

    Park SJ, Jung EH, Ryu RS, Kang HW, Ko JM, Kim HJ, Cheon CK, Hwang SH, Kang HY: Clinical implementation of whole-genome array CGH as a first-tier test in 5080 pre and postnatal cases. Mol Cytogenet 2011, 4: 12. 10.1186/1755-8166-4-12

  21. 21.

    Choe J, Kang JK, Bae CJ, Lee DS, Hwang D, Kim KC, Park WY, Lee JH, Seo JS: Identification of origin of unknown derivative chromosomes by array-based comparative genomic hybridization using pre- and postnatal clinical samples. J Hum Genet 2007, 52: 934–942. 10.1007/s10038-007-0199-1

Download references

Acknowledgements

We wish to thank Eun Hye Jung, Jeong Eun Park and Kyung Yeon Moon for their contribution to the data summary. We are also grateful to other laboratory members for their enthusiastic participation.

Author information

Correspondence to Ho-Young Kang.

Additional information

Competing interest

The authors declare that they have no competing interests.

Authors’ contributions

MKC and SJP analyzed the data for the paper. HDJ and HJJ helped with the discussion and data summary. JHL performed various experiments. HYK drafted the manuscript, conceived of the study, and also approved the manuscript. All authors read and approved the final manuscript.

Mi Kyung Chung, Sang-Jin Park contributed equally to this work.

Authors’ original submitted files for images

Below are the links to the authors’ original submitted files for images.

Authors’ original file for figure 1

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chung, M., Jeong, H., Lee, J. et al. Comprehensive chromosome analysis of blastocysts before implantation using array CGH. Mol Cytogenet 6, 22 (2013) doi:10.1186/1755-8166-6-22

Download citation

Keywords

  • Chromosomal Abnormality
  • Preimplantation Genetic Diagnosis
  • Array Comparative Genomic Hybridization
  • Preimplantation Genetic Screening
  • Chromosomal Microarray Analysis