Samples

This study was approved by the Reproductive Medicine Ethics Committee of Jiangmen Central Hospital (Ethical Approval No.: Jiangmen Central Hospital Reproductive Ethics Review [2024] No. 034). From January 2024 to August 2025, a total of 2,229 patients underwent assisted reproductive treatment at this center. All embryos used in this study were discarded embryos donated for scientific research after obtaining informed consent from the patients. These cryopreserved discarded embryos were thawed in batches and subjected to trophectoderm biopsy, with the biopsy products undergoing PGT-A testing. Based on the PGT-A results, the donated embryos were classified as euploid, aneuploid, and mosaic embryos. Only euploid and aneuploid embryos proceeded to subsequent research, while mosaic embryos were not included in the scope of this study.

Controlled Ovarian Hyperstimulation and Embryo Culture

Controlled ovarian hyperstimulation was conducted in accordance with the standard protocols established by our center [6]. Embryos were sequentially cultured in G-1 and G-2 media (Vitrolife, Sweden) at 37 °C with 6% CO2. The embryos utilized in this study were surplus embryos, donated by patients for scientific research purposes, with ethical approval granted under Jiangmen Central Hospital Ethics Review [2024] 034A.

Embryo Biopsy and Transcriptome SequencingEmbryo Biopsy and Catheterization

After thawing the embryos, laser biopsy was performed, and 5–10 trophoblast cells were taken from each embryo and placed in tubes under a microscope.

Nuclear Cytoplasmic Separation

After placing the cells in the sample preservation solution (5 μ L), centrifuge for 30 s and let them stand at 4 ℃ for 5 min. Add 2.5 μ L of magnetic bead premix (2.3 μ L lysis buffer + 0.2 μ L magnetic beads), incubate at room temperature for 1 min after transient dissociation, and place on a magnetic rack for adsorption for 5 min. Take 4 μ L of supernatant as cytoplasm for subsequent RNA seq related experiments. In the remaining liquid, magnetic beads adsorb cell nuclei for subsequent PGT-A related experiments.

PGT-A Detection and RNA Seq Sequencing

PGT-A testing is used to distinguish between normal diploid embryos (n = 3) and non diploid embryos (n = 3). In this study, diploid embryos were defined as those with a non diploid rate between 0–20%; Non diploid embryos are defined as those with a non diploid rate ranging from 80 to 100%. Embryos with aneuploidy rates ranging from 20 to 80% are chimeric embryos and were not included in the scope of this study. The RNA Sequencing between diploid and aneuploid were completed by Yikon Genomics Company, Ltd..

Bioinformatics AnalysisPrincipal Component Analysis

Perform principal component analysis on transcriptome data using the R software package DUBStepR, with the following parameters: min.cells = 0.01 × ncol (input. data).

Identify Differentially Expressed Genes

Firstly, batch effect correction of gene expression values was performed using the R software package sva [7] on the transcriptome data of human normal diploid and non diploid embryos collected in this study, as well as on published transcriptome datasets of human normal embryos [8, 9]. The negative values of the expression matrix after batch effect correction were set to 0 [10]. Next, bilateral t-tests were used to identify differentially expressed genes (DEGs), and multiple t-tests corrected for false discovery rate (FDR) were used to more accurately identify DEGs. Genes that satisfy FDR < 0.05 and have an absolute value of log (fold change) ≥ 1 are identified as differentially expressed genes.

Gene Ontology Analysis

In order to investigate the functions of DEGs, we used the R software package GOstats [11] to perform Gene Ontology (GO) functional enrichment analysis on DEGs to identify dysregulated biological processes.

Pathway Analysis

In order to explore the pathways involved in DEGs, we used Metascapesoftware [12] to perform KEGG pathway enrichment analysis on the pathways involved in DEGs. A p-value < 0.05 was considered as representative GO and KEGG functions, and then visualized using the R software package ggplot2 [13].

Detection of Endocytosis

According to whether it depends on clathrin, endocytosis can be divided into clathrindependent endocytosis (CDE) and non clathrin dependent endocytosis (CIE). To verify these two endocytic pathways separately, we used two specific fluorescent molecular probes. Red fluorescent labeled Human Dil Low Density Lipoprotein (LDL) was used to detect CDE, and the probe was purchased from Shanghai Yisheng Biotechnology Co., Ltd. (Cat. No: 20614ES76). Fluorescein isothiocyanate labeled folate (FITC-PEG2K-Folic Acid, FA) was used to detect CIE, and the probe was purchased from Xi'an Qiyue Biotechnology Co., Ltd. (Cat. No: R-PF-0822). The working concentration of the above probe is 20 μ g/μ L, incubated at 37 ℃ in the dark for 3.5 h, and then incubated with hocheste33342 staining solution (Cat. No: BL1145A, biosharp) for another 0.5 h. Embryos were rinsed with live cell imaging solution (Cat. No: A596888DJ, Gibco, USA) and examined under a microscope. The microscopy equipment is an Olympus IX73 fluorescence inverted microscope.

Quantitative Real-Time PCRSample Collection

The sample sources for fluorescence quantitative PCR are divided into two parts: one part comes from blastocysts that have developed to the fifth to sixth day, and these blastocysts undergo PGT-A detection after biopsy. According to the test results, they were divided into diploid blastocysts (n = 3) and non diploid blastocysts (n = 3), and chimeric blastocysts were excluded from the group. The other part of the fluorescent quantitative PCR samples were obtained from patients' miscarriage products, which were divided into normal group (n = 3) and non diploid group (n = 3, T13, T18, and T22, respectively) based on the results of chromosome karyotyping examination. Chimeric miscarriage products were not included in the group.

RNA Extraction

After removing the aborted tissue from the -20 ℃ refrigerator, 300 μ L of RNA extraction solution (Lot No. 2504F048, Servicebio, Wuhan) was added and ground until no large tissue was visible to the naked eye. Then, 1 mL of extraction solution was added and centrifuged at 12,000 rpm at 4 ℃ for 15 min. Take 1 mL of supernatant, add 200 μ L of chloroform and shake vigorously. Let it stand at room temperature for 5 min, centrifuge at 12000 rpm at 4 ℃ for 15 min. Take 350–400 μ L of supernatant and add 500 μ L of isopropanol. Mix well and place in a -20° C refrigerator for 1.5 h. Centrifuge at 12000 rpm at 4° C for 15 min and discard the supernatant. Add 500 μ L of 70% ethanol to the precipitate and wash it. After centrifugation, open the lid to evaporate the alcohol, add 20 μ L of DEPC water to dissolve, measure the concentration, and store at -20 ℃ for later use.

cDNAsynthesis

TheRevertAid First Strand cDNA Synthesis Kit was purchased from Guangzhou Fengshuo Biotechnology Co., Ltd. (Lot.No 3183752, Thermo Scientific, USA). The reaction system is shown in Table 1.

Table 1 Reverse transcription reaction systemQuantitative Real-Time PCR

The fluorescent quantitative PCR kit was purchased from Shanghai Baisai Biotechnology Co., Ltd. (Lot No…, Takara, Japan). The fluorescence quantitative PCR instrument is Roche LightCycler 96. All primers were synthesized by Shanghai Shenggong Bioengineering Co., Ltd. (Guangzhou Branch). Pre denature at 95 ℃ for 30 s, cycle 40 times at 95 ℃ for 5 s and 60 ℃ for 30 s. At 60 ℃, measure the dissolution curve by increasing the temperature by 0.5 ℃ every 5 s until it reaches 95 ℃. The amplification system is shown in Table 2, and the primer sequences are shown in Table 3.

Table 2 Fluorescence quantitative PCR reaction systemDetection of Free Amino Acids in Cells

The levels of free amino acids in cells were detected separately in embryos and aborted tissues, and both samples underwent chromosome ploidy testing and were divided into diploid (normal diploid) and aneuploid groups. The sample size for both diploid and non diploid embryos is 8.The detection was completed by Beijing Qingxiang Research Institute using HPLC method [14].

Statistical Analysis

Each experiment should be repeated at least three times. Data analysis was conducted using GraphPad Prism 8 software, and statistical results were expressed as x ± SD (unless otherwise specified); Use Student's t-test or one-way ANOVA for significance analysis.