Chromosome screening using culture medium of embryos fertilised in vitro: a pilot clinical study

Rui Fang, Weimin Yang, Xin Zhao, Fang Xiong, Caiqing Guo, Jianping Xiao, Li Chen, Xiaoqing Song, Honghua Wang, Jie Chen, Xiao Xiao, Bing Yao, Li-Yi Cai, Rui Fang, Weimin Yang, Xin Zhao, Fang Xiong, Caiqing Guo, Jianping Xiao, Li Chen, Xiaoqing Song, Honghua Wang, Jie Chen, Xiao Xiao, Bing Yao, Li-Yi Cai

Abstract

Background: Previous studies from this as well as other research groups suggested that non-invasive chromosome screening (NICS) with embryo culture medium can be used to identify chromosomal ploidy and chromosomal abnormalities. We here report a series of clinical cases utilizing the technology.

Methods: A total of 45 couples underwent in vitro fertilisation during a period between February 2016 and February 2017. Karyotyping revealed normal chromosomes in both partners in 23 couples, and chromosomal rearrangements in at least one partner in 22 couples. Intracytoplasmic sperm injection (ICSI) was used for fertilization. NICS was carried out using embryo culture medium at the blastocyst stage via multiple annealing and looping-based amplification cycles, whole-genome amplification and next-generation sequencing.

Results: A total of 413 embryos were obtained; 170 blastocysts were subjected to NICS. The screening showed euploidy in 79 embryos, aneuploidy in 52 embryos, and mosaic ploidy for 33 embryos. The rate of euploidy was comparable in couples with normal karyotype (50.7%; 38/75) vs. chromosomal rearrangement (43.2%; 41/95). A total of 52 euploid embryos (50 oocyte retrieval cycles) were transferred in 43 women. Biochemical pregnancy rate was 72.0% (36/50). Clinical pregnancy rate was 58.0% (29/50). The rate of spontaneous miscarriage was 3/29 (none with chromosomal aneuploidy). A total of 27 healthy babies were delivered.

Conclusions: NICS could identify embryo chromosomal abnormalities in couples either with or without chromosomal rearrangement, with satisfying clinical outcomes.

Keywords: Assisted reproductive technology; Chromosomal ploidy; Clinical outcome; Next-generation sequencing; Non-invasive chromosome screening.

Figures

Fig. 1
Fig. 1
Schematic representation of the study design and work flow

References

    1. Machtinger R, Racowsky C. Morphological systems of human embryo assessment and clinical evidence. Reprod Biomed Online. 2013;26:210–221. doi: 10.1016/j.rbmo.2012.10.021.
    1. Minasi MG, Colasante A, Riccio T, Ruberti A, Casciani V, Scarselli F, et al. Correlation between aneuploidy, standard morphology evaluation and morphokinetic development in 1730 biopsied blastocysts: a consecutive case series study. Hum Reprod. 2016;31:2245–2254. doi: 10.1093/humrep/dew183.
    1. Munne S, Fragouli E, Colls P, Katz-Jaffe M, Schoolcraft W, Wells D. Improved detection of aneuploid blastocysts using a new 12-chromosome FISH test. Reprod Biomed Online. 2010;20:92–97. doi: 10.1016/j.rbmo.2009.10.015.
    1. Dreesen J, Destouni A, Kourlaba G, Degn B, Mette WC, Carvalho F, et al. Evaluation of PCR-based preimplantation genetic diagnosis applied to monogenic diseases: a collaborative ESHRE PGD consortium study. Eur J Hum Genet. 2014;22:1012–1018. doi: 10.1038/ejhg.2013.277.
    1. 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. doi: 10.1016/j.fertnstert.2009.10.015.
    1. Scott RT, Jr, Upham KM, Forman EJ, Zhao T, Treff NR. Cleavage-stage biopsy significantly impairs human embryonic implantation potential while blastocyst biopsy does not: a randomized and paired clinical trial. Fertil Steril. 2013;100:624–630. doi: 10.1016/j.fertnstert.2013.04.039.
    1. Zhao HC, Zhao Y, Li M, Yan J, Li L, Li R, et al. Aberrant epigenetic modification in murine brain tissues of offspring from preimplantation genetic diagnosis blastomere biopsies. Biol Reprod. 2013;89:117. doi: 10.1095/biolreprod.113.109926.
    1. Wu Y, Lv Z, Yang Y, Dong G, Yu Y, Cui Y, et al. Blastomere biopsy influences epigenetic reprogramming during early embryo development, which impacts neural development and function in resulting mice. Cell Mol Life Sci. 2014;71:1761–1774. doi: 10.1007/s00018-013-1466-2.
    1. Stigliani S, Anserini P, Venturini PL, Scaruffi P. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation. Hum Reprod. 2013;28:2652–2660. doi: 10.1093/humrep/det314.
    1. Hammond ER, McGillivray BC, Wicker SM, Peek JC, Shelling AN, Stone P, et al. Characterizing nuclear and mitochondrial DNA in spent embryo culture media: genetic contamination identified. Fertil Steril. 2017;107:220–228. doi: 10.1016/j.fertnstert.2016.10.015.
    1. Palini S, Galluzzi L, De Stefani S, Bianchi M, Wells D, Magnani M, et al. Genomic DNA in human blastocoele fluid. Reprod Biomed Online. 2013;26:603–610. doi: 10.1016/j.rbmo.2013.02.012.
    1. Gianaroli L, Magli MC, Pomante A, Crivello AM, Cafueri G, Valerio M, et al. Blastocentesis: a source of DNA for preimplantation genetic testing. Results from a pilot study. Fertil Steril. 2014;102:1692–1696. doi: 10.1016/j.fertnstert.2014.08.021.
    1. Tobler KJ, Zhao Y, Ross R, Benner AT, Xu X, Du L, et al. Blastocoel fluid from differentiated blastocysts harbors embryonic genomic material capable of a whole-genome deoxyribonucleic acid amplification and comprehensive chromosome microarray analysis. Fertil Steril. 2015;104:418–425. doi: 10.1016/j.fertnstert.2015.04.028.
    1. Farra C, Choucair F, Awwad J. Non-invasive pre-implantation genetic testing of human embryos: an emerging concept. Hum Reprod. 2018;33:2162–2167. doi: 10.1093/humrep/dey314.
    1. Magli MC, Pomante A, Cafueri G, Valerio M, Crippa A, Ferraretti AP, et al. Preimplantation genetic testing: polar bodies, blastomeres, trophectoderm cells, or blastocoelic fluid? Fertil Steril. 2016;105:676–683. doi: 10.1016/j.fertnstert.2015.11.018.
    1. Perloe M, Welch C, Morton P, Venier W, Wells D, Palini S. Validation of blastocoele fluid aspiration for preimplantation genetic screening using array comparative genomic hybridization (aCGH) Fertil Steril. 2013;100:S208. doi: 10.1016/j.fertnstert.2013.07.1384.
    1. Poli M, Jaroudi S, Sarasa J, Spath K, Child T, Wells D. The blastocoel fluid as a source of DNA for preimplantation genetic diagnosis and screening. Fertil Steril. 2013;100:S37. doi: 10.1016/j.fertnstert.2013.07.1789.
    1. Shamonki MI, Jin H, Haimowitz Z, Liu L. Proof of concept: preimplantation genetic screening without embryo biopsy through analysis of cell-free DNA in spent embryo culture media. Fertil Steril. 2016;106:1312–1318. doi: 10.1016/j.fertnstert.2016.07.1112.
    1. Xu J, Fang R, Chen L, Chen D, Xiao JP, Yang W, et al. Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization. PNAS USA. 2016;113:11907. doi: 10.1073/pnas.1613294113.
    1. Balaban B, Brison D, Calderon G, Catt J, Conaghan J, Cowan L, et al. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26:1270. doi: 10.1093/humrep/der037.
    1. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114. doi: 10.1093/bioinformatics/btu170.
    1. Li H, Durbin R. Fast and accurate long-read alignment with Burrows–Wheeler transform. Bioinformatics. 2010;26:589. doi: 10.1093/bioinformatics/btp698.
    1. Greco E, Litwicka K, Arrivi C, Varricchio MT, Caragia A, Greco A, et al. The endometrial preparation for frozen–thawed euploid blastocyst transfer: a prospective randomized trial comparing clinical results from natural modified cycle and exogenous hormone stimulation with GnRH agonist. J Assist Reprod Genet. 2016;33:1–12. doi: 10.1007/s10815-016-0736-y.
    1. Li S, Jing W, Yan C, Zhou D, Yin T, Xu W, et al. Intrauterine administration of hCG-activated autologous human peripheral blood mononuclear cells (PBMC) promotes live birth rates in frozen/thawed embryo transfer cycles of patients with repeated implantation failure. J Reprod Immunol. 2017;119:15–22. doi: 10.1016/j.jri.2016.11.006.
    1. Lattes K, Checa MA, Vassena R, Brassesco M, Vernaeve V. There is no evidence that the time from egg retrieval to embryo transfer affects live birth rates in a freeze-all strategy. Human Reprod. 2016;32:368. doi: 10.1093/humrep/dew306.
    1. Minasi MG, Fiorentino F, Ruberti A, Biricik A, Cursio E, Cotroneo E, et al. Genetic diseases and aneuploidies can be detected with a single blastocyst biopsy: a successful clinical approach. Hum Reprod. 2017;32:1–8. doi: 10.1093/humrep/dex215.
    1. De Rycke M, Belva F, Goossens V, Moutou C, SenGupta SB, Traeger-Synodinos J, et al. ESHRE PGD Consortium data collection XIII: cycles from January to December 2010 with pregnancy follow-up to October 2011. Hum Reprod. 2015;30:1763–1789. doi: 10.1093/humrep/dev122.
    1. Capalbo A, Rienzi L, Cimadomo D, Maggiulli R, Elliott T, Wright G, et al. Correlation between standard blastocyst morphology, euploidy and implantation: an observational study in two centers involving 956 screened blastocysts. Hum Reprod. 2014;29:1173–1181. doi: 10.1093/humrep/deu033.
    1. Alfarawati S, Fragouli E, Colls P, Stevens J, Gutiérrezmateo C, Schoolcraft WB, et al. The relationship between blastocyst morphology, chromosomal abnormality, and embryo gender. Fertil Steril. 2011;95:520–524. doi: 10.1016/j.fertnstert.2010.04.003.
    1. Taylor TH, Gitlin SA, Patrick JL, Crain JL, Wilson JM, Griffin DK. The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans. Hum Reprod Update. 2014;20:571–581. doi: 10.1093/humupd/dmu016.
    1. Bolton H, Graham SJ, Van der Aa N, Kumar P, Theunis K, Fernandez Gallardo E, et al. Mouse model of chromosome mosaicism reveals lineage-specific depletion of aneuploid cells and normal developmental potential. Nat Commun. 2016;7:11165. doi: 10.1038/ncomms11165.
    1. Galluzzi L, Palini S, Stefani SD, Andreoni F, Primiterra M, Diotallevi A, et al. Extracellular embryo genomic DNA and its potential for genotyping applications. Future Sci OA. 2015;1:FSO62. doi: 10.4155/fso.15.62.

Source: PubMed

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