Ploidy Testing of Blastocoel Fluid for Screening May Be Technically Challenging and More Invasive Than That of Spent Cell Culture Media

Abstract

Background: Recent studies have demonstrated that both blastocoel fluid (BF) and spent cell culture media (SCM) have potential as materials for non-invasive or less-invasive pre-implantation genetic analysis. BF may allow more opportunity to obtain cell-free DNA from the inner cell mass (ICM), and it has a lower risk of containing contaminant DNA from cumulus cells, sperm and culture media. There are no data regarding the ICM as a gold standard to evaluate the chromosome constitution of BF or SCM for embryo liquid biopsy.

Methods: Two hundred eighteen donated human blastocysts were warmed and cultured in blastocyst culture media for 18-24 h. The corresponding SCM was collected, and only clear ICM was biopsied in blastocysts; otherwise, the whole blastocyst (WB) was biopsied. Quantitative PCR was performed to determine the DNA levels in the SCM and BF before and after amplification. ChromInst was used to amplify BF/SCM and blastocyst DNA before sequencing. Chromosomal copy number variation (CNV) was investigated to evaluate the chromosome constitution.

Results: In total, 212 blastocysts were available for SCM and BF collection. The technical success rates (next-generation sequencing data) were 100 and 69.8% (148/212) for SCM and BF, respectively. Among the 148 blastocysts with both SCM and BF data, 101 were euploid and 47 were aneuploid based on ICM (n = 89) or WB (n = 59) analysis as the gold standard. Among all blastocysts, SCM was comparable to BF [specificity: 80.2 versus 61.4% (P = 0.005, χ² test); sensitivity: 91.5 versus 87.2% (P = 0.738, χ² test); negative predictive value (NPV): 95.3 versus 91.2% (P = 0.487, χ² test); positive predictive value (PPV): 68.3% versus 51.3% (P = 0.042, χ² test)]. The SCM and BF samples were 83.8% (124/148) and 69.6% (103/148) concordant with the corresponding ICM/WB samples when only two categories, euploid or aneuploid/mosaic, were grouped to calculate the concordance.

Conclusions: Compared with BF, SCM has superior diagnostic performance, and it is non-invasive for embryos.

Clinical trial registration: [http://www.chictr.org.cn], identifier [ChiCTR-BPD-17014087].

Keywords: blastocoel fluid; embryos; inner cell mass; pre-implantation genetic testing of aneuploidies; spent culture medium.

FIGURE 1

The validation procedure of the study design. Human blastocysts were donated by in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) patients with preserved surplus embryos after giving birth to live healthy babies. We then validated non-invasive chromosome screening (NICS) using spent culture medium (SCM) versus blastocoel fluid (BF) as a source of DNA. In each embryo, we obtained the chromosome ploidy results from the inner cell mass (ICM), trophectoderm biopsy (TE), SCM, and BF after whole-genome amplification (WGA) and library preparation by NICSInst. Copy number variation (CNV) was determined by ChromGo Analysis Software.

FIGURE 2

Flowchart of sample processing success and loss. In total, 212 of 218 blastocysts were fully re-expanded. Blastocentesis failed in 38 full-expanded blastocysts. No valid sequencing data (low reads number) were generated in an additional 26 blastocysts. Inner cell mass was available as a reference for 89 blastocysts, and whole embryo was used as a reference for the remaining 59 blastocysts. Among the 148 blastocysts with both SCM and BF data, 101 were euploid, and the remaining 47 were aneuploid.