The goals of this international multicenter cross-sectional study are:

1. To provide patients with a comprehensive PGT solution capable of simultaneously detecting embryonic chromosomal aneuploidy, mosaicism, microdeletions/ microduplications, heteroploidy, and heterozygosity (LOH) in a single assay, thereby reducing miscarriage and birth defects;
2. To perform PGT analysis on abnormally fertilized embryos, select euploid embryos with normal ploidy, and calculate embryo utilization rates;
3. To reduce the false-positive rate through confirmation of mosaic embryos and subsequent analysis of its origin, thereby minimizing embryo wastage;
4. To provide molecular genetic evidence for expert consensus on clinical management of atypically fertilized embryos, of pathogenic/likely pathogenic small CNVs, optimize mosaic embryo transfer strategies, and inform preconception intervention;
5. To enhance international PGT testing standards through international multi-center collaboration.

The study will enroll patients undergoing PGT-A from seven domestic and international centers, with patient enrollment expected to be completed within one year. PGT-A upgrade testing will be performed on embryos from enrolled patients, and the incidence rates of Incidence of microdeletions/microduplications, heteroploidy, LOH will be statistically analyzed. All patients who undergo embryo transfer will be followed up for clinical outcomes and birth defects.

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.

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.

This study is a cross-sectional study with a two-sided test and α = 0.05. Based on the analysis of existing data from previous PGT-A upgrade studies performed in 2PN embryos, the total incidence rate (p) of microdeletion/microduplication, heteroploidy, and UPD/LOH in euploid embryos is estimated to be 2.0%, with a margin of error (δ) of 0.005. The required number of euploid embryos is 3,012, and the calculation formula is provided below. Given that the euploidy rate is approximately 50%, a total of 6,024 embryos tested by PGT-A upgrade needs to be enrolled. Considering the potential dropout risk (10%), 6,694 embryos from two pronuclei (PN) are included. All embryos from 0PN/1PN/3PN are included.

Considering regulatory requirements, the IPD will not be shared.