Methylation analysis of idiopathic recurrent spontaneous miscarriage cases reveals aberrant imprinting at H19 ICR in normozoospermic individuals Mandar.

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Methylation analysis of idiopathic recurrent spontaneous miscarriage cases reveals aberrant imprinting at H19 ICR in normozoospermic individuals Mandar Ankolkar, M.Sc., Anushree Patil, M.D., Himangi Warke, M.D., Vinita Salvi, M.D., Neelam Kedia Mokashi, M.Sc., Shilpa Pathak, Ph.D., N.H. Balasinor, Ph.D. Fertility and Sterility Volume 98, Issue 5, Pages 1186-1192 (November 2012) DOI: 10.1016/j.fertnstert.2012.07.1143 Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 1 A schematic representation of the H19 DMR methylation status obtained by the QUMA sequence analysis software. Each black circle denotes a methylated CpG and each white circle represents an unmethylated CpG. Each vertical column of circles (1–18) represents a CpG locus. CpG 4–8 span the CTCF-binding site 6. CpG 7 corresponding to nucleotide 7966 in GenBank accession no. AF125183 is a known polymorphic site (C/T). Each horizontal row of circles containing a sequence of 18 CpGs represents a single clone analyzed. Fertility and Sterility 2012 98, 1186-1192DOI: (10.1016/j.fertnstert.2012.07.1143) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Semen analysis. All the values have been plotted as box and whisker graphs with median and interquartile range (5%–95%) between the Control and RSM groups. Values outside the interquartile range (outliers) have been indicated as dots. (A) Sperm concentration of Control and RSM is expressed in millions/mL (n = 26 in both groups). (B) Sperm motility of Control and RSM groups is expressed as percentage of cells with normal motility (n = 26 in both groups). (C) Sperm chromatin compaction status analyzed by the chromomycin A3 (CMA3) staining in Control (n = 23) and RSM (n = 26) participants. It is expressed as percentage of cells stained positive with CMA3. (D) Sperm morphology analyzed by the Papanicolaou staining protocol in Control (n = 24) vs. RSM (n = 26) groups is expressed as percentage of spermatozoa with normal morphology. Fertility and Sterility 2012 98, 1186-1192DOI: (10.1016/j.fertnstert.2012.07.1143) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions

Figure 3 H19 ICR analysis. All the values have been plotted as box and whisker graphs with median and interquartile range (5%–95%) between Control and RSM groups (n = 26, in each group). Values outside the interquartile range (outliers) have been indicated as dots. (A) Complete H19 ICR methylation is expressed as percentage of methylated CpGs in the entire region of the H19 ICR analyzed. (B) Percentage of clones with 100% methylated CpGs, indicates sperms with normal methylation. (C) CTCF methylation levels expressed as a percentage of CpGs methylated in the sixth CTCF-binding region analyzed (CpG 4–8). *Significance calculated at 95% confidence interval. Fertility and Sterility 2012 98, 1186-1192DOI: (10.1016/j.fertnstert.2012.07.1143) Copyright © 2012 American Society for Reproductive Medicine Terms and Conditions