Study subjects
This study is a single-centre prospective interventional clinical study. Patients who visited Hebei Maternity Hospital between October 2018 and December 2021 were recruited. Follow up regarding the primary outcome was completed in April 2023.
Inclusion criteria: the female patient was 22–40 years old, underwent intracytoplasmic sperm injection (ICSI) with ≥ 2 blastocysts, agreed to the first transfer as a single blastocyst transfer, and the transferred embryos were retrieved from a single cryopreservation.
Exclusion criteria: patients with chromosomal abnormalities, uterine anomalies, and endometrial thickness < 7 mm. Patients were recruited in the NICS group and the control group in the ratio of 1:2 numbers. This study was carefully reviewed and approved by the Medical Ethics Committee of Hebei Maternity Hospital (Number: 20180001). All patients provided informed consent to participate in this study. The number of Chinese Clinical Trial Registry was ChiCTR2300072566.
Oocytes retrieval and granulosa cell removal
According to the patient’s case, ovulation was stimulated by standard antagonist and progestin primed ovarian stimulation (PPOS), and the dose of gonadotropin was adjusted according to the patient’s ovarian response, hormone level and follicle size, and when the follicle diameter and hormone level reached the standard of triggering, the dose of 5,000 to 8,000 IU human chorionic gonadotrophin (HCG) or 0.1 mg gonadotropin-releasing hormone agonist (GnRHa) combined with 4,000 IU HCG was given, and the triggering was performed egg retrieval was performed 37 h later under the guidance of vaginal ultrasound. One to two hours after egg retrieval, oocytes were treated with hyaluronidase and blown and washed three times to remove granulosa cells.
Embryo culture and sample collection
After ICSI, fertilisation was checked at 16–18 h and two pronuclei and two polar bodies were clearly observed. On the afternoon of the second day after confirmation of fertilization, the granulosa cells of the embryos were again removed, blastocyst culture medium was replaced and the embryos continued to be cultured in the new drops. On the afternoon of the fourth day after fertilization, the blastocyst culture medium was replaced again and the embryos were washed three times, the volume of the culture medium was about 25 µl. These operations were effective in removing the contamination of maternal DNA. When the blastocysts developed to the standard of freezing, the blastocysts were cryopreserved as single blastocysts using the vitrification freezing method, and 20 µl culture medium of the corresponding blastocysts was collected into the RNase/DNAase-free PCR tubes, which containing 5 µl of preservation solution.
Morphologic grading of blastocysts
Before the blastocysts were frozen, the blastocysts were graded according to the Gardner and Schoolcraft morphological grading system [16, 17]. , which assessed the three components of blastocyst expansion, inner cell mass and trophectoderm. For the control group, the embryos were selected based on morphological grading and were transferred with single blastocysts. According to current expert consensus in China [18], D5 or D6 blastocysts graded as AA, AB, BA, or BB are considered High-quality blastocysts. Those graded as AC, BC, CA, or CB, as well as high-quality blastocysts, can be defined as Usable blastocysts. High-quality blastocysts such as AA, AB, BA, and BB were prioritized for transfer, followed by AC, BC, CA, and CB blastocysts.
Whole genome amplification, library prep and sequencing
Whole genome amplification (WGA) and next-generation sequencing (NGS) library preparation was performed on the collected blastocyst cultures using the NICSInst™ (Xukang Medical Technology (suzhou) Co., Ltd) library kit according to the instructions [11, 13]. A 10ul spent culture medium was pipetted from the sample preservation tube for WGA, and then the library was built for the amplified products. Quality control of NGS libraries was performed using Qubit 3.0 (Qubit® dsDNA HS Assay Kit, Thermo Fisher Scientific) and 1.5% agarose gel electrophoresis. After mixing the samples according to the aliquots, sequencing was performed using the Illumina platform, and approximately 2 M sequencing reads were obtained for each library.
Copy number variation (CNV) analysis and a.i. grading system
Data were analysed and visualised using ChromGo™ analysis software (Xukang Medical Technology (suzhou) Co., Ltd) [19]. High-quality reads were counted along the entire genome with a bin size of 1 Mb, and reads were normalized by GC and reference datasets, and the binary segmentation algorithm (CBS) was used for the detection of CNV fragments.
As described in the previous paper [15], the NICS-AI model has been established. The NICS-AI model was an artificial intelligence algorithm using R package caret 6.0–86. The model utilized the Random Forest (RF) machine learning algorithm, with whole embryo CNV results as the gold standard, to develop a copy number pattern in the blastocyst culture media associated with chromosomal euploidy or aneuploidy.
The machine learning methods trained on the following 11 features: 10 M-resolution CNV result, 10 M-resolution CNV result redefined by 50% mosaicism threshold, arm-resolution CNV result, arm-resolution CNV result redefined by 50% mosaicism threshold, whole chromosome-resolution CNV result, whole chromosome-resolution CNV result redefined by 50% mosaicism threshold, euploidy number with different resolution result, abnormal chromosome number, highest abnormal mosaicism proportion, largest abnormal fragment size corresponding to the highest mosaicism proportion, and presence of sex chromosome abnormality or not [15].
The NICS-AI model analysed these features from the sequencing results of the culture media to predict embryo euploidy probabilities, categorising them into ≥ 0.94, 0.7–0.94, and ≤ 0.7 as grades A, B, and C. The recommended embryos to be transferred were A and B-grade. The patients in the NICS group were transferred with A-grade blastocysts in preference to B-grade.
Embryo thawing and transfer
The embryos were thawed using the commercial thawing kit (Kitazato, Japan), and after thawing, the embryos were transferred according to the clinical routine.
Follow-up on clinical outcomes
The main clinical outcome was live birth rate of the first single blastocyst transfer. At 14 days after blastocyst transfer, the patient’s hCG value was measured. Clinical pregnancy was the presence of at least one gestational sac in the uterine cavity as determined by ultrasound at 28–30 days after transfer. An ongoing pregnancy was defined as a detectable fetal heart at week 12 of gestation. Live birth was defined as delivery of 1 live infant with a gestational age greater than 28 weeks. The following information was collected within 2 weeks of delivery: birth sex, birth weight, and neonatal score.
Statistical analysis
Data were analysed by normal distribution. Values that matched normal distribution were shown as mean and standard deviation, and t-tests were used for comparison between groups, while non-normally distributed data were shown as median (Q1-Q3), and Mann-Whitney tests were used between groups. The chi-square test was used to assess the comparison between groups of the component ratios or rates (%). Multiple logistic regression analysis was used to test clinical pregnancy rates, ongoing pregnancy rates, and live birth rates between groups. The demographic data, including female age, male age, number of previous early spontaneous abortions, indication, types of infertility, gonadotropins (Gn) dosage, and Gn days were incorporated into the model and used to analyze the odds ratio (OR) for clinical outcomes.
Statistical analysis was performed using SPSS, V.25.0 (SPSS, Chicago, Illinois, USA), and statistical differences were considered statistically significant at p < 0.05.
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