Bar graph illustrating the percentage of hypermethylated and hypomethylated CpGs in swim-up sperm samples and B cells.

<p>The number of detected CpGs varies according to the data extrapolation performed separately for each tested group: i) total CpGs, ii) autosomal CpGs, iii) X chromosome-linked CpGs and iv) Y chromosome-linked CpGs. * corresponds to p values <0.05 (the whiskers show the SD; n = 7).</p

Heatmap displaying the methylation status of CpG loci (n = 2386) mapping in the promoters of 45 imprinted genes in relation to quality-fractioned sperm populations (i.e. swim-up “up” and swim- “dn” sperm fractions).

<p>A) The dendrograms above the heatmap show hierarchical clustering based on the methylation data alone. Sperm populations and CpG loci are represented by columns and rows, respectively. Each cell indicates the CpG methylation level for one site in each sample. Methylation levels are represented in the scale on the right side of the heatmap and are referred lowest to highest as green (0.0) to red (1.0). (B) List of the 45 imprinted gene available in the array.</p

Novel Insights into DNA Methylation Features in Spermatozoa: Stability and Peculiarities

<div><p>Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic and cancer cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now extended also to hypomethylated piRNAs-linked genes, provides solid basis for future basic and clinical research.</p> </div

Description of CpGs in terms of number and methylation status in the swim-up sperm fraction and in B cells.

<p>The number of hypomethylated and hypermethylated loci are indicated as total, autosomic, X chromosome and Y chromosome-linked. A) Sperm CpG numbers refer singularly to the eight normozoospermic men, while B cells CpG numbers belong to two different subjects. The mean CpG number ± DS and the percentage calculated in respect to the mean total CpG number for each group are reported in the middle panel; B) Number of CpGs conserved among individuals: the first raw reports the number of CpGs shared by individuals, while the second raw reports the percentage of conserved CpGs compared to the mean CpG number reported in panel A.</p

Spermatozoa versus B cell: a 450K DNA methylation portrait.

<p>(A) Graph showing percentages of equally and differentially methylated CpG sites in swim-up sperm samples compared to B cells. (B) Graph showing percentages of hypermethylation and hypomethylation in spermatozoa relating to the differentially methylated CpGs proportion (4,3%). Graphs describing the hypermethylated (C) and hypomethylated (D) sites according to their i) functional genomic distribution; ii) CpG content/neighborhood context and iii) association with RNA transcripts.</p

Scatter plots reporting CpGs methylation levels between different samples deriving from the swim-up sperm selection procedure in the same individual EC01: (A) swim-up (Up) sperm fraction versus swim-down (Dn) sperm fraction; (B) whole sperm population at1h (Ws 1 h) versus whole sperm population at 2 h (Ws 2h); (C) whole sperm population at 1h versus swim-down sperm fraction; (D) whole sperm population at1h versus swim-up sperm fraction.

<p>R²  = Pearson coefficient.</p

Representative scatter plots reporting CpG methylation levels between different individuals EC01 and EC10.

<p>(A) swim-up (Up) sperm samples; (B) swim-down (Dn) sperm samples; (C) whole sperm population at 1 h (Ws 1 h) samples; (D) Box plot representing the inter-individual variability of DNA methylation levels in total CpGs from the swim-up, swim-down and whole sperm population at1h samples. The median value is shown. * corresponds to p value = 0.0213; R2  = Pearson coefficient. The boxes describe the lower quartile (Q1, 25%), median (Q2, 50%) and the upper quartile (Q3, 75%); the whiskers extend 1.5 times the IQR from the box.</p

Intra-group epigenetic distances for the promoters of <i>BRCA1, BRCA2, HTT, DMPK1, PSEN1</i> and <i>PSEN2</i> genes.

<p>This distance represents the net dissimilarity of DNA methylation profiles between two sequences: the higher the distance, the more dissimilar are the compared samples. Different individuals were crossed with each other and Euclidean distances were calculated for beta-values of CpG sites as a marker of inter-individual variability in three different sperm subpopulations: A) Whole sperm population; B) swim-down and C) swim-up fractions. Numbers on the X axis indicate the identity of the pair-wise comparisons inside the experimental group: individuals EC01, EC07, EC10, EC12, EC14, EC16, EC18 and EC10 are numbered 1 to 8. Distance values are displayed on the Y axis. The top and bottom blue guidelines represent the 0.025 and 0.975 quartiles, while the red guideline represents the mean distance value.</p

Heatmap displaying the methylation status of CpG loci (n = 297) mapping in 10 selected genes in relation to quality-fractioned sperm populations (i.e. swim-up “up” and swim-down “dn” sperm fractions).

<p>A) The dendrograms above the heatmap show hierarchical clustering based on the methylation data alone. Sperm populations and CpG loci are represented by columns and rows, respectively. Each cell indicates the CpG methylation level for one site in each sample. Methylation levels are represented in the scale on the right side of the heatmap and are referred lowest to highest as green (0.0) to red (1.0). B) Scatter plot reporting CpGs methylation levels between quality-fractioned sperm populations (Up vs Dn) among different individuals. R² = Pearson coefficient. C) List of the 10 analyzed genes, selected because previously reported as differently methylated in infertile men compared to normozoospermic controls.</p

High Resolution X Chromosome-Specific Array-CGH Detects New CNVs in Infertile Males

<div><h3>Context</h3><p>The role of CNVs in male infertility is poorly defined, and only those linked to the Y chromosome have been the object of extensive research. Although it has been predicted that the X chromosome is also enriched in spermatogenesis genes, no clinically relevant gene mutations have been identified so far.</p> <h3>Objectives</h3><p>In order to advance our understanding of the role of X-linked genetic factors in male infertility, we applied high resolution X chromosome specific array-CGH in 199 men with different sperm count followed by the analysis of selected, patient-specific deletions in large groups of cases and normozoospermic controls.</p> <h3>Results</h3><p>We identified 73 CNVs, among which 55 are novel, providing the largest collection of X-linked CNVs in relation to spermatogenesis. We found 12 patient-specific deletions with potential clinical implication. Cancer Testis Antigen gene family members were the most frequently affected genes, and represent new genetic targets in relationship with altered spermatogenesis. One of the most relevant findings of our study is the significantly higher global burden of deletions in patients compared to controls due to an excessive rate of deletions/person (0.57 versus 0.21, respectively; p = 8.785×10⁻⁶) and to a higher mean sequence loss/person (11.79 Kb and 8.13 Kb, respectively; p = 3.435×10⁻⁴).</p> <h3>Conclusions</h3><p>By the analysis of the X chromosome at the highest resolution available to date, in a large group of subjects with known sperm count we observed a deletion burden in relation to spermatogenic impairment and the lack of highly recurrent deletions on the X chromosome. We identified a number of potentially important patient-specific CNVs and candidate spermatogenesis genes, which represent novel targets for future investigations.</p> </div