Patients and study design
A total of 124 cycles of single vitrified-warmed blastocyst transfer performed between October 2020 and March 2023 in a single in vitro fertilization (IVF) center (Kinutani Women’s Clinic, Hiroshima, Japan) were included in the analysis. All transferred blastocysts from the PGT-A cycles were evaluated using the KAT-Score (v2, Varinos Inc, Tokyo, Japan). However, cases with pre-implantation genetic testing for structural chromosome rearrangements or multiple embryo transfer cycles including two-step embryo transfer were excluded from the analysis (Fig. 1). The main outcome measures were clinical pregnancy (presence or absence of gestational sac) and live birth (presence or absence of live birth).
This study was approved by the Ethics Committee of Kinutani Women’s Clinic, Hiroshima, Japan. Informed consent was obtained from all patients included in this study.
Ovarian stimulation and oocyte retrieval
Patients were treated with gonadotropin hormone-releasing hormone (GnRH) agonist using a short or long protocol, following a progestin-primed ovarian stimulation protocol, in accordance with each patient’s ovarian response and medical history of IVF treatment. Serum estradiol levels were monitored and oocyte growth evaluated by transvaginal ultrasound. In some cycles, the minimal stimulation protocol and natural cycle were used. When a follicle reached a mean diameter ≥ 18 mm, human chorionic gonadotropin (Aska Pharmaceutical, Tokyo, Japan), 250 µg of choriogonadotropin alpha (Ovidrel; Merck Serono, Darmstadt, Germany), or GnRH agonist was administered. Transvaginal oocyte retrieval was performed 35–37 h after administration.
Fertilization procedure, embryo culture, time-lapse monitoring
Prior to conventional IVF, collected cumulus–oocyte complexes were cultured for approximately 4 h in G-IVF medium (Vitrolife, Gothenburg, Sweden) at 37°C under an atmosphere of 6% CO2, 5% O2, and 89% N2. Cumulus–oocyte complexes were denuded by pipetting using 80 IU/ml hyaluronidase (Irvine Scientific, Santa Ana, CA, USA). The maturity of denuded oocytes was assessed by visualization of the first polar body. Only metaphase II oocytes were used for intracytoplasmic sperm injection (ICSI). Embryos fertilized by ICSI or conventional IVF were cultured in DNP dishes (Dai Nippon Printing Co., Ltd., Tokyo, Japan) with SAGE 1 Step (Origio, Målöv, Denmark) at 37°C under an atmosphere 6% CO2, 5% O2, and 89% N2 for 5–7 days and evaluated for development to the blastocyst stage. The oocytes were cultured individually in a time-lapse system (CCM-iBIS; Astec, Fukuoka, Japan). A total of 1–25 embryos were placed in a 100 µl drop of medium, and images of the embryos were recorded automatically at 15-min intervals.
Blastocyst biopsy protocols and NGS analysis
Trophectoderm (TE) biopsy was performed on expanded blastocysts on days 5–7 regardless of morphological grade. First, artificial shrinkage was performed using a laser system (Zilos-TK™; Hamilton Thorn Bioscience Inc., Beverly, MA, USA) to create a space between the zona pellucida and TE cells. While holding the blastocyst in an inner cell mass (ICM) at a position of approximately 8 to 9 o’clock, serial laser pulses were used to create a small hole (~ 10 µm) in the zona pellucida at a position of approximately 3 to 4 o’clock. The biopsy pipette was pushed into the hole created by the laser to aspirate the TE cells. Five to 10 TE cells were aspirated into the biopsy pipette and collected by flicking the holding pipette and biopsy pipette with the aid of several laser pulses. Biopsied samples were processed for whole-genome amplification and NGS at Varinos Laboratory. The VeriSeq PGS kit (Illumina K.K., San Diego, CA, USA) on the MiSeq system (Illumina K.K., San Diego, CA, USA) was used in 24-sample runs following the manufacturer’s protocol. The copy number of each sample was analyzed using BluFuse Multi Software (Illumina K.K., San Diego, CA, USA). A molecular karyotype profile consistent with mosaicism was detected when a whole chromosome or chromosomal segment resulted in intermediate copy number levels of 20–80% between whole numbers according to the guidelines of the Preimplantation Genetic Diagnosis International Society [21].
Vitrification and warming of blastocysts and embryo transfer
Blastocyst vitrification was performed using the method reported by Hiraoka et al. [22] within 1 h after embryo biopsy. The blastocysts were placed in equilibration solution containing 7.5% (v/v) ethylene glycol (Sigma-Aldrich, St. Louis, MO, USA) and 7.5% (v/v) dimethyl sulfoxide (Nacalai Tesque Inc., Kyoto, Japan) in mHTF (Irvine Scientific, Santa Ana, CA, USA) supplemented with 20% (v/v) serum protein substitute (Origio, Målöv, Denmark) at 37°C. Blastocysts were then transferred into vitrification solution containing 15% (v/v) ethylene glycol, 15% (v/v) dimethyl sulfoxide, and 0.5 M sucrose (Sigma-Aldrich, St. Louis, MO, USA) in mHTF supplemented with 20% (v/v) serum protein substitute for 1 min at 37°C. The blastocysts were loaded onto Cryotops (Kitazato Corporation, Fujinomiya, Japan) at a minimum volume and immediately immersed in liquid nitrogen at − 196°C. For the warming protocol, the tip of the Cryotop was immersed directly in 1.0 M sucrose solution for 1 min at 37°C. The blastocyst was transferred to 0.5 M sucrose solution for 3 min and washed twice in mHTF supplemented with 20% serum protein substitute for 5 min at 37°C. Prior to embryo transfer, the warmed blastocyst was cultured for approximately 3–4 h in EmbryoGlue (Vitrolife, Gothenburg, Sweden). Luteal phase support was started using intravaginal micronized progesterone or natural cycles. Embryo transfers were performed under ultrasound guidance using a soft catheter (Origio, Målöv, Denmark).
KAT-Score-based scoring of embryos
Embryos were scored 0 (complete euploidy) to 10 (complete aneuploidy) using the KAT-Score v2 (Table 1). First, a score from 1 to 8 was assigned for each type of mosaicism and a score of 9 for segmental aneuploidy. Next, a score of 0.2–0.8 was added to the total depending on the level of mosaicism in the range of 20–80%. When mosaicism was present on multiple chromosomes, the score of the chromosome with the highest level of mosaicism was assigned as the mosaicism score. Figure 2 shows an example of the scoring.
Table 1
Knowledge-based Aneuploidy Theoretical Score (KAT-Score) for pre-implantation genetic testing.
Mosaic type | Score |
Aneuploid | 10 |
Segmental aneuploid | 9 |
High chromosomal mosaic complex | 8 |
High chromosomal mosaic gain | 7 |
High chromosomal mosaic loss | 6 |
Low chromosomal mosaic complex | 5 |
Low chromosomal mosaic gain | 4 |
Low chromosomal mosaic loss | 3 |
High segmental mosaic gain/loss | 2 |
Low segmental mosaic gain/loss | 1 |
Euploid | 0 |
Mosaic level | Score |
80% | 0.8 |
70% | 0.7 |
60% | 0.6 |
50% | 0.5 |
40% | 0.4 |
30% | 0.3 |
20% | 0.2 |
Note: Low=low-level mosaicism >20% to <50%, High=high-level mosaicism ≥50% to <80%, Complex=mosaicism of monosomies and trisomies found on multiple chromosomes, Gain=trisomy, Loss=monosomy.
Statistical analysis
Statistical analysis was performed using JMP 14.0 software (SAS Institute, Inc., Cary, NC, USA) and GraphPad PRISM 6.03 software (GraphPad Inc., San Diego, CA, USA). Patient characteristics were presented as means and standard deviation (SD). The KAT-Score data were not normally distributed, so the Wilcoxon rank-sum test was used to compare the scores as non-parametric continuous variables. We calculated the area under the receiver operating characteristic curve (AUC) of the KAT-Score for predicting clinical pregnancy and ongoing pregnancy. Adjusted odds ratios (aORs) were calculated using multivariate logistic regression analysis. Differences were considered significant at P < 0.05.