Measurement of biochemical indicators:

Venous blood was drawn from neonates within 30 min after birth, and neonatal insulin levels were measured by radioimmunoassay. Furthermore, blood glucose levels were measured by glucose oxidase method, whereas hemoglobin A1c (HbA1c) levels were measured by agarose gel electrophoresis with automatic glycated hemoglobin analyzer.

Measurement of PDX1 gene methylation rate: Measurement by methylation-specific PCR method: The DNA concentration was measured after DNA purification of peripheral blood mononuclear cell (PBMCs), and individual genomic DNA was transformed using the heavy sulfite method after the ideal concentration was reached. PDX1 gene fragments were amplified by hot-start PCR using the transformed DNA as a template. In terms of PDX1 primers, they were forward: 5’-GGCACAGCGGAGCCTAT-3’ and reverse: 5’-GCCACCC-CTGCAGCTT-3’ with a fragment length of 354 bp. With a total volume of 25μl of the reaction system, PCR was amplified under the conditions of pre-denaturation at 95°C for two minutes, denaturation at 94°C for 20s, annealing at 54°C for 30s, and extension at 67°C for 20s, for a total of 40 cycles.

Correlation analysis of neonatal blood glucose levels with maternal PDX1, NGN3 and Pax6 levels.

After the cycle, the PCR was extended at 72°C for five minutes. The purified fragments were connected with PMD18-T vector, and 10 single positive clones were selected from each group of samples after transformation and coating for sequencing, which was repeated for three times. If the cyclic threshold was 37 or below, the fluorescence intensity at the end point of amplification was 2-3 times that of the negative control group, and the dissolution curve was unimodal and the corresponding temperature was consistent with the so-called temperature of the positive control, it was determined to be positive, and methylation would be determined after three consecutive positive times. Methylation rate = number of methylated patients/total number of patients.

Measurement of mRNA expression levels of NGN3 and Pax6: RT-PCR was employed to measure the mRNA expression levels of NGN3 and Pax6, Trizol was utilized to extract total RNA from peripheral blood mononuclear cells for reverse transcription, and NGN3 and Pax6mRNA were amplified by PCR.