1. Genome-wide screen of ovary-specific DNA methylation in polycystic ovary syndrome 
Ying-Ying Yu, Ph.D., Cui-Xiang Sun, Ph.D., Yin-Kun Liu, Ph.D., Yan Li, Ph.D., Li Wang, Ph.D., Wei Zhang, Ph.D. 
Fertility and Sterility 
Volume 104, Issue 1, Pages e6 (July 2015)
DOI: /j.fertnstert
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
DNA methylation profiles from polycystic ovary syndrome (PCOS) and control ovaries. (A) Comparison of immunoprecipitation (IP) enrichment of methylated DNAs from PCOS and control. The scatter plot is a comparison of methylation levels as represented by log2 ratios in PCOS and control ovarian tissues. Each dot represents the log2 ratio calculated for a probe from PCOS and control. The correlation of the IP enrichment between PCOS and control is R = (B) Venn diagram for in CpG islands (CGIs) and methylated genes in PCOS versus control data sets. The genes closest to the detected methylated peaks were annotated using the HG18 (NCBI36 version) human genome assembly. The PCOS-specific peaks were excluded that contained the common annotated gene surrounding the other specific peaks in the control. (C) Distribution of methylated CpGs in different genomic subregions in PCOS and control. All methylated CGIs were categorized into several subtypes according to the different subregions of methylated CGIs in the genome, including promoter, intragenic, downstream, degenerate promoter, degenerate intragenic, degenerate downstream, and unknown. The number and percentage of methylated CGIs in the different genomic subregions were compared between PCOS and control.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
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3. Figure 2
Methylation levels vary with respect to distance relative to the transcription start site (TSS). The region pertaining to −1,000 base pair (bp) to +1,000 bp upstream or downstream of the TSS were separated into bins representing every window of 200 bp. The marginal regions (−2,000 ∼ −1,000 and +1,000 ∼ +2,000) were analyzed as complete units rather than 200-bp windows because of sparse distribution of methylated peaks within these regions (peak score > 2). The box plots represent the distribution of peak scores in these bins of equal bp width with the x-axis representing the distance to the potential TSS. The black line marks the median percentiles, and the lower and upper limits of the box represent the 25th and 75th percentiles. Red-colored bars indicate PCOS, and green-colored bars indicate control samples.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Promoter classes are differentially methylated. (A) Comparison of DNA methylation levels of promoters in women with polycystic ovary syndrome (PCOS) and controls. In each class, promoters were grouped into one of three bins based on their DNA methylation level in PCOS (log2 ratio < 0, 0 ≤ log2 ratio ≤ 0.4, and log2 ratio > 0.4). For promoters in each bin, the methylation measurement in control was subtracted from that in PCOS. A positive value reflects higher methylation in PCOS than in controls. The box plots illustrate that methylation of LCPs (low CpG promoters) and ICPs (intermediate CpG promoters) is very similar between PCOS and controls, whereas the hypermethylation of HCPs (high CpG promoters) detected in PCOS (log2 ratio > 0.4) is mostly absent in control. (B) The number of promoters that are hypermethylated in both PCOS and controls (PCOS & control), or in PCOS only (PCOS specific). In each class, PCOS hypermethylated promoters were defined as having a log2 ratio > 0.4.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
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5. Supplemental Figure 1
Seven candidate genes validated by methylation specific polymerase chain reaction (PCR) (MSP). Regions of interest were amplified using primers specific for methylated and unmethylated versions of sites. The U represents unmethylated PCR products, and M represents methylated products. Lymphocyte represents negative control (unmethylated), and M.SssI represents the positive control (methylated). P1 and P2 represent two randomly selected samples from the polycystic ovary syndrome (PCOS) group. C1 and C2 represent two randomly selected samples from the control group. Methylation of these gene promoters was semiquantitatively scored as the methylation level (ML). (A) SLC2A8, (B) NRIP1, (C) IGF2BP2, (D) INSR, (E) CYP19A1, (F) AMH, and (G) AMHR2. ∗P<.05, ∗∗P<.01, ∗∗∗P<.001.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 2
Top 10 molecular function, process, and pathway maps identified from the microarray data through the MetaCore tool. Genes associated with the methylation profiles were uploaded into GeneGo MetaCore software. The resultant list of molecular function, process, and pathway maps is arranged by descending P values.
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7. Supplemental Figure 3
Mechanism of the disruption of gene transcription by DNA methylation in normal and disease states. A schematic representation of the regulation of gene transcription by DNA methylation patterns derived from the results presented. The epigenetic modification of DNA by methylation targets different regions of the genome. Alterations in DNA methylation patterns lead to changes in gene transcription that ultimately drive the disease state. The normal and disease scenarios are depicted in the left and right columns, respectively.
Fertility and Sterility  , e6DOI: ( /j.fertnstert )
Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions