Additional file 1: of Global DNA methylation levels are altered by modifiable clinical manipulations in assisted reproductive technologies

Effect of parental age and sex of newborns. (DOCX 87 kb

Additional file 2: of Global DNA methylation levels are altered by modifiable clinical manipulations in assisted reproductive technologies

Distribution of global methylation fractions based on modifiable factors: mode of egg fertilization by LUMA (A) and LINE1 assay (B); oxygen tension by LUMA (C) and LINE1 assay (D); type of embryo transfer by LUMA (E) and LINE1 assay (F) and; embryo transfer day by LUMA (G) and LINE1 assay (H). (PPTX 385 kb

Pairwise LD Analysis and Pairwise FGT at the <i>H19</i>/<i>IGF2</i> DMR

<div><p>(A) Pairwise LD test between ten SNPs covering a 31-kb region containing <i>H19</i>/<i>IGF2</i> DMR shows a major breakdown of LD which corresponds to the LDU step shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-g003" target="_blank">Figure 3</a>B. Intensity of LD is coded in colors as shown.</p><p>(B) Pairwise FGT between the same ten SNPs. A “1” indicates recombination between that pair of loci (all four gametes) and “0” indicates only three types of gametes (recombination between the two loci is uncertain). Considering that a historical recombination would break the haplotype inside of which it appeared, at least eight haplotype blocks could be identified. The ten markers used for both analyses in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-g004" target="_blank">Figure 4</a>A and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-g004" target="_blank">4</a>B are the same as depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-g003" target="_blank">Figure 3</a>B (CEPH)</p></div

LD Analysis at Human Chromosomal Regions Containing Imprinted Genes

<div><p>(A) Comparison of LDU values at imprinted versus control regions. Each LDU/Mb value obtained for a given imprinted bin was plotted against the LDU/Mb value of the corresponding control region. Note that most of the imprinted regions reach higher LDU values compared with their corresponding control regions (dotted line with slope 1 corresponds to virtual positions in cases with equal LDU/Mb values at imprinted and control regions).</p><p>(B) The number of haplotype blocks/Mb is higher at imprinted genes compared with their corresponding control regions (dotted line indicates equal values).</p><p>(C) The mean sizes of haplotype blocks are significantly smaller at imprinted regions versus flanking control regions (dotted line indicates equal sizes).</p><p>(D) There is a significant excess in the number of hot-spots of recombination at imprinted versus control regions (see text), and the total length of the hot-spots appears greater in imprinted regions than in control regions. Each value corresponds to the total length of all hot-spots of recombination (in base pairs [bp]) for a given imprinted or control region.</p></div

Distribution of Recombination Events at Human 11p15.5 Imprinted Cluster

<div><p>Positions of markers used for mapping recombinants in this region are indicated in Mb from the telomeric end (Tel) of the short arm. Imprinted genes are shown on the left side of the figure. Arrows correspond to direction and parental-specific origin of transcription: blue are paternally transcribed genes, red are maternally expressed genes, and black are genes with biallelic expression or unknown imprinting status. The two known germline imprints at this locus are shown by colored oval shapes on the left side of the figure: the blue oval corresponds to the paternally methylated <i>IGF2</i>/<i>H19</i> DMR and the red oval corresponds to the maternally methylated <i>KCNQ1OT1</i> DMR. Each vertical bar on the right side of the figure corresponds to a meiotic recombination event, delimitated by the nearest informative markers: Labeled in blue are crossovers in paternal meiosis, and labeled in red are recombinations in maternal meiosis.</p><p>An asterisk (*) represents an unidentified polymorphism found at <i>MUC5B</i> locus, and double asterisks (**) indicate an unidentified TaqI polymorphism found at <i>TH</i> locus (genotypes available through CEPH database—see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#s4" target="_blank">Materials and Methods</a>).</p></div

LD Analysis at Human 11p15.5 Imprinted Cluster

<div><p>(A) Population-specific metric LD map for about 1-Mb region containing imprinted genes at human 11p15.5 chromosome. Positions along the chromosome are shown in bp on the <i>x-</i>axis. Straight lines are representing the genome-wide slopes (LDU/Mb) corresponding to each population, as extrapolated from De La Vega et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.0020101#pgen-0020101-b016" target="_blank">16</a>]. Note that LD extends less far in the region containing imprinted genes compared with a region of similar length from the rest of genome, in agreement with the interpretation of higher recombination in these areas (i.e., breakdown of LD has been converted to implied recombination rate and rendered graphically as red rectangular “hot spots”). Location of CpG islands in the region are depicted as shown in MapView; dark blue represents CpG islands larger than 500 bp, and light blue represents CpG islands over 200 bp. For both categories, G + C content is higher than 50% and the observed CpG/expected CpG content is higher than 0.6. The two black arrows correspond to the regions containing the primary germline imprints at <i>H19</i>/<i>IGF2</i> DMR (left arrow) and <i>KCNQ1OT1</i> DMR (right arrow), respectively. Both are located at regions exhibiting steps of LD and recombination hot-spots and are zoomed-in in (B) and (C). The red open arrows correspond to smaller steps, which are variable between populations and do not correspond with any hot-spot of recombination.</p><p>(B) The metric LD map for the region containing <i>H19</i>/<i>IGF2</i> DMR using data from the four populations (HapMap) and the set of CEPH individuals analyzed in this study. The three horizontal bars correspond to recombinants mapped at this region, one in maternal meiosis (red) and two in paternal meioses (blue). The blue oval shape corresponds to the <i>H19</i>/<i>IGF2</i> DMR.</p><p>(C) The metric LD map for the region containing <i>KCNQ1OT1</i> DMR using data from the four populations (HapMap). Two recombinants (horizontal blue bars) were mapped at this region in paternal meioses. The red oval shape corresponds to the <i>KCNQ1OT1</i> DMR.</p></div