Noninvasive chromosome screening of human embryos by genome sequencing of embryo culture medium for in vitro fertilization

Abstract

Preimplantation genetic screening (PGS) is widely used to select in vitro-fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of in vitro fertilization (IVF). A disadvantage of PGS is that it requires biopsy of the preimplantation human embryo, which can limit the clinical applicability of PGS due to the invasiveness and complexity of the process. Here, we present and validate a noninvasive chromosome screening (NICS) method based on sequencing the genomic DNA secreted into the culture medium from the human blastocyst. By using multiple annealing and looping-based amplification cycles (MALBAC) for whole-genome amplification (WGA), we performed next-generation sequencing (NGS) on the spent culture medium used to culture human blastocysts (n = 42) and obtained the ploidy information of all 24 chromosomes. We validated these results by comparing each with their corresponding whole donated embryo and obtained a high correlation for identification of chromosomal abnormalities (sensitivity, 0.882, and specificity, 0.840). With this validated NICS method, we performed chromosome screening on IVF embryos from seven couples with balanced translocation, azoospermia, or recurrent pregnancy loss. Six of them achieved successful clinical pregnancies, and five have already achieved healthy live births thus far. The NICS method avoids the need for embryo biopsy and therefore substantially increases the safety of its use. The method has the potential of much wider chromosome screening applicability in clinical IVF, due to its high accuracy and noninvasiveness.

Keywords: IVF; MALBAC; PGS; WGA; chromosomal abnormalities.

Conflict of interest statement

X.S.X. and S.L. are cofounders of Yikon Genomics Company, Ltd.

Figures

Fig. 1.

Diagram of the validation procedure of the noninvasive chromosome screening (NICS) assay. Briefly, D3 embryos achieved via intracytoplasmic sperm injection (ICSI) were placed in blastocyst culture medium. Both the D3–D5 culture medium and the corresponding whole embryos were collected and used for WGA by multiple annealing and looping-based amplification cycles (MALBAC). Whole-genome–amplified products from both the D3–D5 culture medium and the embryo were sequenced using an Illumina HiSeq 2500 platform. The chromosome ploidy information was obtained from both the culture medium and the corresponding embryo.

Fig. 2.

Examples of validation of results from the comparison of NICS versus the whole-blastocyst embryos. (A and B) Equivalent karyotype results obtained from NICS and the corresponding blastocyst embryo. Fig. 2A shows consistent results from a normal/transferrable embryo, and Fig. 2B shows consistent results from an embryo with a chromosome loss in chr18. (C) An example of inconsistent results obtained from NICS and the blastocyst embryo, with the embryo showing balanced chromosomal composition and the NICS assay identifying chromosome gains of chr10 and chr17.

Fig. S1.

CNVs of embryos EM23, EM42, and EM38 determined by high-sequencing depth (30×). Top graph corresponds to spent culture media, and bottom graph is for the corresponding biopsy of embryo at blastocyst stage. (A) NICS and trophectoderm (TE) biopsy results for EM23 with a NICS coverage rate of 24.1%. (B) NICS and TE biopsy results for EM42 with a NICS coverage rate of 32.9%. (C) NICS and TE biopsy results of EM38 with a NICS coverage of 65.0%. Note the consistency of the results in all NICS cases and the corresponding embryo biopsy. As shown in B, the copy number for chromosome 1 was one and not two, and therefore embryo EM42 could not be used for transfer.

Fig. 3.

Embryo screening and selection using NICS from a patient carrying a balanced translocation of chr14/15. A total of three embryos successfully developed to the blastocyst stage, and D3–D5 culture medium from each embryo was collected for the NICS assay to screen for chromosomal abnormalities. (A and B) Embryos showed chromosomal abnormalities with chr14/15 and therefore could not be transferred. (C) An embryo showed balanced chromosomal composition and was therefore transferred into the uterus of the patient, resulting in a successful pregnancy and a healthy live birth.

Fig. S2.

Chromosomal screen results from whole embryos (A and C), and their corresponding culture media (B and D). E corresponds to a whole live embryo with normal chromosome results using NICS. (B, D, and E) The NICS results for the three blastocysts. E shows a balanced chromosomal composition, and the embryo was therefore transferred into the uterus of the patient. B and D show chromosome gain/loss from chr14/chr15. Therefore, the corresponding embryos were not transferred but were lysed for chromosome screening to confirm the NICS results (A and C). NICS results for both embryos were consistent with chromosomal ploidy information from the blastocyst embryo.