Prospective International Multi-center Clinical Trial of PGT-A Upgrade

The study will enroll 6,694 embryos derived from typical fertilization (2PN) that meet the inclusion and exclusion criteria in patients undergoing PGT-A, as well as all embryos derived from atypical fertilization (0PN/1PN/3PN). In contrast to conventional PGT-A testing, PGT-A upgrade testing will be performed on the embryos to comprehensively analyze multiple embryonic abnormalities in a single detection, including aneuploidy, mosaicism, microdeletion/microduplication, heteroploidy, and loss of heterozygosity (LOH), and to calculate their respective incidence rates.

In addition to embryos derived from 2PN, embryos from 0PN/1PN/3PN will also be cultured to the blastocyst stage for trophectoderm (TE) cell biopsy. Euploid embryos identified by PGT-A upgrade testing will be recorded, and the utilization rate of atypically fertilized embryos will be evaluated.

For mosaic embryos, a previously established parental haplotype origin algorithm will be applied to distinguish true versus false mosaicism and identify the origin of abnormalities, thereby recognizing "false-positive" mosaic embryos and further increasing the number of transferable embryos.

Patients will receive euploid embryo transfer (from 2PN) in accordance with routine clinical practice. In cases where no 2PN-derived euploid embryos are available, transfer of 0PN/1PN/3PN-derived euploid embryos and embryos classified as "false-positive" mosaic may be considered after the patient has been fully informed of the risks and provided informed consent.

All transfer cycles will be followed up for prenatal diagnosis results and birth defects. The primary outcome measures are the incidence rates of microdeletion/microduplication, heteroploidy, and LOH. The secondary outcome measures include embryo utilization rate, clinical pregnancy rate, ongoing pregnancy rate, live birth rate, miscarriage rate, concordance rate between prenatal diagnosis results and PGT-A results, and birth defect rate.

The maximum follow-up duration will be 1 year after embryo transfer. Clinical and embryology laboratory procedures during the study will not be altered and will be performed in accordance with each center's routine practice.

• PGT-A upgrade (Other): A comprehensive PGT solution capable of simultaneously detecting embryonic chromosomal aneuploidy, mosaicism, microdeletions/ microduplications, heteroploidy, and LOH in a single assay.

Primary outcomes

• Incidence of microdeletions/microduplications [Two months after oocyte retrieval]: Trophectoderm biopsy samples undergo whole genome amplification followed by NGS. Microdeletions and microduplications are identified according to Human Genome Assembly GRCh19 (hg19) or updated versions. The incidence will be calculated as the number of embryos with pathogenic or likely pathogenic microdeletions/microduplications (1-4M) divided by the total number of embryos.
• Incidence of heteroploidy [Two months after oocyte retrieval]: Trophectoderm biopsy samples undergo whole genome amplification followed by NGS. Heteroploidy is identified according to Human Genome Assembly GRCh19 (hg19) or updated versions. The incidence will be calculated as the number of embryos with heteroploidy divided by the total number of embryos.
• Incidence of loss of heterozygosity [Two months after oocyte retrieval]: Trophectoderm biopsy samples underwent whole genome amplification followed by NGS. Loss of heterozygosity were identified according to Human Genome Assembly GRCh19 (hg19) or updated versions. The incidence will be calculated as the number of embryos with loss of heterozygosity divided by the total number of embryos

Secondary outcomes

• Transferable embryo rate [Two months after oocyte retrieval]: Trophectoderm biopsy samples undergo whole genome amplification followed by NGS. Normal karyotype is identified according to Human Genome Assembly GRCh19 (hg19) or updated versions. The rate will be calculated as the number of embryos with normal karyotype divided by the total number of embryos.
• Clinical pregnancy rate [28-30 days after embryo transfer]: Transvaginal ultrasonography will be performed. Clinical pregnancy will be diagnosed with detection of an intrauterine gestational sac
• Ongoing pregnancy rate [12 weeks after the embryo transfer]: Transvaginal ultrasonography will be performed. Ongoing pregnancy will be diagnosed with detection of an intrauterine gestational sac
• Live birth rate [Two weeks after the newborn's birth]: Live birth rate is defined as delivery of any viable infant at 28 weeks or more of gestation, after embryo transfer
• Early miscarriage rate [12 weeks of after the embryo transfer]: Number of pregnancy losses / number of clinical pregnancies after transfer
• Concordance between prenatal diagnosis results and PGT-A results [16-24 weeks of gestation]: Prenatal diagnosis result: Chromosomal analysis performed on fetal samples obtained through chorionic villus sampling or amniocentesis, including karyotyping, chromosomal microarray analysis or next-generation sequencing. PGT-A result: Chromosomal analysis performed by PGT-A upgrade.
• Birth defect rate [At 1 year postpartum]: Physical examination, echocardiography, X-ray/MRI, ophthalmologic examination, hearing screening

Sex: Female, ages: 20 Years to 46 Years

Inclusion Criteria:

(1) Any one of the following conditions being met is sufficient:

1. advanced maternal age (AMA, age ≥35 years),
2. recurrent implantation failure (RIF),
3. recurrent miscarriage (RM),
4. severe male factor (SMF). (2) And at least one blastocyst is available.

Exclusion Criteria:

1. Couples undergoing PGT-SR due to chromosomal structural abnormalities carried by either one or both members, such as balanced translocations, Robertsonian translocations, inversions, complex chromosomal rearrangements, and pathogenic microdeletions or microduplications;
2. Couples undergoing PGT-M;
3. Conditions with established impact on uterine morphology or endometrial receptivity, including untreated uterine malformations (septate uterus, unicornuate uterus, didelphic uterus, etc.) and untreated hydrosalpinx;
4. Embryos coming from oocyte or sperm (gametes) donation;
5. Individuals with contraindications to pregnancy or assisted reproduction technology.

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• Ge Lin, Principal investigator, CITIC-Xiangya Reproductive & Genetic Hospital