*2.6. Genotyping*

Genotyping was performed via IPLEX MassARRAY PCR using the Agena platform (Agena Bioscience, San Diego, CA, USA). IPLEX MassARRAY PCR and extension primers were designed from sequences containing each target SNP and 150 upstream and downstream bases with Assay Designer 4.0.0.2 (Agena Bioscience, San Diego, CA, USA) using the default settings. Single base extension reactions were performed on the PCR reactions with the iPLEX Gold Kit (Agena Bioscience) and 0.8 μL of the custom UEP pool. PCR reactions were dispensed onto SpectroChipArrays with a Nanodispenser (Agena Bioscience). An Agena Bioscience Compact MassArray Spectrometer was used to perform MALDI–TOF

mass spectrometry according to the iPLEX Gold Application Guide. The Typer 4 software package (Agena Bioscience) was used to analyse the resulting spectra, and the composition of the target bases was determined from the mass of each extended oligo. These panels were designed in collaboration with PATIA, and genotyping was performed using the Agena platform located at the Epigenetics and Genotyping laboratory, Central Unit for Research in Medicine (UCIM), Faculty of Medicine, University of Valencia, Valencia, Spain.

### *2.7. Statistics Analysis*

For the statistical analysis, 136 of the 149 patients were included, for whom genetic data were available. Different statistical tests were applied to examine the association between polymorphism in CUBN, MTHFR, MTR, and SLC19A1 and maternal and infant phenotypes and delivery complications. Maternal complications included preeclampsia, hypertension, and a family history of type 2 diabetes. Delivery complications included caesarean birth and induced birth. Neonatal phenotypes included macrosomia, admission to the intensive care unit, and PTB. The SNP information was decoded to numerical type depending on the presence of a reference or an alternative nucleotide of each sample. *p* values were computed using the chi-square test to determine whether the prevalence of genotype risk groups varied significantly depending on the phenotype or complication. *p* ≤ 0.05 was considered statistically significant. All of the aforementioned statistical analyses were performed using R software (version 3.2.0).
