Relationship between age and blastocyst chromosomal ploidy analyzed by noninvasive preimplantation genetic testing for aneuploidies (niPGT-A)

Abstract

Objective: To assess the relationship between human blastocyst chromosomal ploidy established by niPGT-A and increasing age. Methods: This is a prospective multicenter study carried out by ten assisted reproduction centers after their embryologists acquired training and validated their results with the previous use of niPGT-A. A total of 94 couples with indication for niPGT-A due to increase maternal age, male factor, repeated implantation failures, recurrent abortion or because they requested niPGT-A were included in this study. The couples had no karyotype abnormalities. After ICSI, the embryos were cultured until blastocyst stage using one or two step culture systems, single or sequential media respectively, at 37ºC in an atmosphere of 6-7% CO2 and 5-20% O2 incubators. On day 3, we re-evaluated cleavage embryos to complete cumulus cells removal. The embryos were then cultured in individual well, with 20µl of medium under oil until they reached blastocyst stage. The blastocysts were vitrified and stored in liquid nitrogen. After that, the spent blastocyst culture medium (20µl) was transferred to a PCR tube and sent for analysis in the genetic laboratory, where it was stored at -80ºC until sequencing. A total of 243 samples of spent blastocyst culture medium were collected on the 5th/6th day. Cell-free DNA secreted on culture medium was amplified using NICS Sample Preparation Kit (Yikon Genomics), based on the MALBAC technology. After whole genome amplification, the DNA was measured using a Qubit 2.0 fluorometer and subjected to next generation sequencing (NGS) using Illumina MiSeq ® platform. The data were analyzed using the ChromGo ® software (Yikon Genomics). Results: The mean age of the patients was 38±4.08 years with an interval of 20-44 years. The euploid was diagnosed in 36.4% (80/220) of cases, aneuploidy in 31.3% (69/220), and mosaicism in 32.3% (71/220; with ≥60% aneuploidy) of blastocysts. Mosaic values ranged from 29.8% to 33.8% in different age groups. Individually, the most frequent chromosomal abnormality was XXY (Klinefelter Syndrome) occurring in 18 cases, followed by chromosome 21 (trisomy/monosomy) in 8 cases. The niPGT-A data showed a ≥60% incidence of aneuploid cells in all cases of chromosomal mosaicism (n=71). Conclusion: A high degree of mosaicism with aneuploidy cells was detected, and some hypotheses were suggested for this data (niPGT-A sensitivity in detecting the self-correction of chromosomal abnormalities phenomenon). However, it did not vary remarkably with age. On the other hand, euploidy levels had a negative correlation with age and aneuploidy levels had a positive relationship. This is the first report in the literature to relate chromosomal ploidy in blastocysts using niPGT-A and increasing patient age.

Are tri‐pronuclear embryos that show two normal‐sized pronuclei and additional smaller pronuclei useful for embryo transfer?

“…Nevertheless, in this study, patients’ ages in the cycles with 2.1PN zygotes were higher than those without 2.1PN zygotes, and the two patients with mosaic embryos were relatively young (30 and 32 years). Although it has been reported that chromosomal aneuploidy is associated with age, 20 , 21 , 22 , 23 further research is required to gain a more complete understanding of whether the aneuploidy of 2.1PN embryos is associated with patient age.…”

Prevalencia de aneuploidías en blastocistos de día 5 vs. día 6: desfase de 24 horas como factor determinante

“…Vagini et al (2020) usando PGT-A relacionaron la edad con aneuploidías, viendo incrementada su prevalencia y reportando el síndrome de Klinefelter como la cromosomopatía más frecuente (18/71 [25%]) 16 . Kroener et al (2012) analizando blastocistos D5 y todos aquellos con desfase de al menos 24 horas (mórulas D5 y D6, blastocistos D6), evaluaron el grado morfológico, reportaron que los que tuvieron un desfase de 24 horas no mostraron una diferencia significativa en presentar aneuploidías, sin embargo, le dieron mayor importancia al grado morfológico; es decir, un embrión óptimo, independientemente del día de desarrollo, tiene altas posibilidades de ser euploide 17 .…”

An Update on Non-invasive Approaches for Genetic Testing of the Preimplantation Embryo

“…The majority of “mosaic” results indicated high-level mosaicism, which may relate to the process and purpose of embryonic self-correction mechanisms. Similar to invasive PGT-A, mosaicism did not vary depending on maternal age, while aneuploidy levels had a positive correlation with maternal age [ 109 ]. In another recent study, SCM niPGT-A results were also investigated relevant to pregnancy outcome, and live births were associated with low embryonic cfDNA in SCM [ 70 ].…”