WCSS analysis using raw counts from RNA-seq data at E18.5, 12 weeks, and 39 weeks. (A) Nine temporal expression patterns (k = 9) across mouse ovarian development, using Natural as the training group. (B) Top 20 enriched biological processes associated with ovarian function and their respective temporal expression cluster. (C) Changes in temporal expression patterns for all genes from Natural to IVF groups. (D) Top 5 enriched biological processes using the genes that change temporal expression patterns (clusters). (E–J) Differential expression analysis was performed using DeSeq2. (E) Volcano plot (E18.5 gonads). (F) Heatmap with the most DEGs upregulated (Up) or downregulated (Down) and (G) corresponding volcano plot (12-week ovaries). (H) Heatmap with the most DEGs up- or downregulated and (I) corresponding volcano plot (39-week ovaries). (J) Heatmap with the most DEGs up- or downregulated. Color boxes at the top denote the experimental group. (K) Immunofluorescence staining for CD31 in 12-week ovaries. Original magnification, ×400. (L) Representative Western blot for CD31, LINE1 ORF1p, cleaved caspase 3, TOM20, and GAPDH. (M) Immunofluorescence staining for cleaved caspase 3 expression in 12-week ovaries. (N) Quantification of normalized protein expression for cleaved caspase 3, CD31, LINE1 ORF1p, and TOM20 in 12-week ovaries. Western blot band intensities were normalized to GAPDH and subsequently normalized within each blot to the mean intensity of the Natural group to account for inter-blot variability. Each data point represents 1 biological replicate. Sample sizes are indicated above each panel as the number of animals (litters). Data are presented as the mean ± SEM. Normality was assessed using the Shapiro-Wilk test. Data that did not meet the normality assumption were analyzed using the nonparametric Mann-Whitney U test, whereas normally distributed data were analyzed using an unpaired 2-tailed t test with Welch’s correction. *P < 0.05 and *P < 0.01 compared with Naturals.