P-725 Role of Non-Invasive Preimplantation Genetic Testing- Aneuploidy (NIPGT-A) using spent culture media (SCM) and its concordance with Trophoectoderm (TE) biopsy: A Prospective Cohort Study
Authors: Sethi A , Singh N , Gupta R , Rani L , Saini M , Malhotra N , Mahey R , Patel G 
Published: June 2023

Abstract

Study question
What is the ploidy concordance rate between spent culture media (SCM) and trophectoderm biopsy (TE) using Next Generation Sequencing (NGS) and correlation with clinical outcome?

Summary answer
DNA could be isolated from SCM with successful NGS library sequencing. Ploidy and per-chromosome concordance rate of TE biopsy vs SCM was 68.38% and 85.13%.

What is known already
Many challenges are associated with TE biopsy, and the possibility of NiPGT-A using cell free DNA (cfDNA) from SCM is very intriguing, as it will be simpler, safer and cheaper alternative to the gold standard TE biopsy. The bottlenecks associated with TE biopsy like additional equipment and trained embryologist, can be substantially reduced. The disparities in reported results in various studies comparing SCM vs TE, could be attributed to culture media contamination (maternal, paternal or environment), different laboratory workflow methodologies used for embryo culture, different whole genome amplification methods, library sequencing methods and different algorithms for analysis.

Study design, size, duration
Prospective cohort study was conducted in tertiary care centre from August 2021-June 2022. Ethical Approval was taken from Institute Ethics Committee. Couples with female age ≥ 35 years, one or more implantation failures, male factor infertility requiring ICSI, opting for elective single euploid blastocyst transfer, were included in study. Forty four blastocysts were obtained from 14 patients who underwent IVF/ICSI cycles, gave consent for TE biopsy and SCM collection for PGT-A and NiPGT-A respectively.

Participants/materials, setting, methods
Individualised ovarian stimulation was carried out with GnRH antagonist protocol. ICSI was done for fertilization. Embryos were cultured in sequential medium. On day 3, after thorough washing embryo was cultured separately in 10μl micro-droplets. SCM was collected on day-5, before biopsy. DNA extraction, amplification and library preparation from TE biopsy and SCM was done using Veriseq PGS kit (Illumina) and sequencing was done on Illumina MiSeq system. Euploid embryo transfer was done in FET cycle

Main results and the role of chance
Out of 44 blastocysts, 31 were day 5 and 13 were day 6. WGA-DNA from TE biopsy and SCM was 100% successful. Average concentration of amplified DNA obtained from TE biopsy and SCM was 34.88 ± 9.56 ng/µl, 32.3 ± 6.84 ng/µl respectively. Quality control parameters for library preparation and sequencing for samples were as per recommended standards. CNV visualization and analysis was done using BlueFuse Multi-Software (Illumina). Ploidy concordance rate could be analysed in 26 embryos, which was 68.38%, per chromosome concordance was 85.13% and sex-chromosome concordance was 73.0%. The sensitivity, specificity, PPV, NPV and diagnostic accuracy of NiPGT-A was 66.6%, 60%, 87.5%, 30% and 65.38% respectively. On comparing day 5 and day 6 blastocysts, day 6 had better concordance rate 72.7% vs 60%, p &gt; 0.05. On comparing good (&gt;BB) and poor morphology (&lt;BB)embryos, &lt;BB had better concordance rate 83.33% vs 50%, p = 0.16. Per chromosome concordance rate was higher for &lt;BB embryos, 90.5% vs 80.5%,p= 0.001. Full maternal cell contamination was suspected when TE was aneuploid/mosaic, XY and SCM was euploid, XX (n = 4/14), assessed only in XY embryos. Single euploid blastocyst transfer had 66.6%clinical pregnancy rate. One resulted in healthy live-birth, two on-going pregnancies, 30weeks and 12weeks period of gestation.

Limitations, reasons for caution
Before considering NiPGT-A in routine clinical practice, each embryology lab needs standardization, to implement necessary modifications in routine embryology protocol. Labs should ensure that embryo viability is not affected and should try to minimize chances of maternal or external contamination in SCM to reliably predict concordance rates with high confidence.

Wider implications of the findings
NiPGT-A is useful technique to assess ploidy but presently, NiPGT-A in combination with PGT-A, can help in prioritizing embryos according to their implantation potential. Further studies are required to find out whether NiPGT-A may serve as an alternative to PGT-A and implemented alone in routine clinical practice in future.

Trial registration number
CTRI/2021/04/033121