Three group comparison (feature set = 5000) and gene ontology analysis.

<p>Bar graph showing gene ontology analysis on the basis of a) molecular function b) cellular location, and c) biological processes, when three groups (fertile control, asthenozoospermic and normozoospermic infertile) were compared simultaneously.</p

Differential Genes Expression between Fertile and Infertile Spermatozoa Revealed by Transcriptome Analysis

<div><p>Background</p><p>It was believed earlier that spermatozoa have no traces of RNA because of loss of most of the cytoplasm. Recent studies have revealed the presence of about 3000 different kinds of mRNAs in ejaculated spermatozoa. However, the correlation of transcriptome profile with infertility remains obscure.</p><p>Methods</p><p>Total RNA from sperm (after exclusion of somatic cells) of 60 men consisting of individuals with known fertility (n=20), idiopathic infertility (normozoospermic patients, n=20), and asthenozoospermia (n=20) was isolated. After RNA quality check on Bioanalyzer, AffymetrixGeneChip Human Gene 1.0 ST Array was used for expression profiling, which consisted of >30,000 coding transcripts and >11,000 long intergenic non-coding transcripts.</p><p>Results</p><p>Comparison between all three groups revealed that two thousand and eighty one transcripts were differentially expressed. Analysis of these transcripts showed that some transcripts [ribosomal proteins (<i>RPS25</i>, <i>RPS11</i>, <i>RPS13</i>, <i>RPL30</i>, <i>RPL34</i>, <i>RPL27</i>, <i>RPS5</i>), <i>HINT1</i>, <i>HSP90AB1</i>, <i>SRSF9</i>, <i>EIF4G2</i>, <i>ILF2</i>] were up-regulated in the normozoospermic group, but down-regulated in the asthenozoospermic group in comparison to the control group. Some transcripts were specific to the normozoospermic group (up-regulated: <i>CAPNS1</i>, <i>FAM153C</i>, <i>ARF1</i>, <i>CFL1</i>, <i>RPL19</i>, <i>USP22</i>; down-regulated: <i>ZNF90</i>, <i>SMNDC1</i>, <i>c14orf126</i>, <i>HNRNPK</i>), while some were specific to the asthenozoospermic group (up-regulated: <i>RPL24</i>, <i>HNRNPM</i>, <i>RPL4</i>, <i>PRPF8</i>, <i>HTN3</i>, <i>RPL11</i>, <i>RPL28</i>, <i>RPS16</i>, <i>SLC25A3</i>, <i>C2orf24</i>, <i>RHOA</i>, <i>GDI2</i>, <i>NONO</i>, <i>PARK7</i>; down-regulated: <i>HNRNPC</i>, <i>SMARCAD1</i>, <i>RPS24</i>, <i>RPS24</i>, <i>RPS27A</i>, <i>KIFAP3</i>). A number of differentially expressed transcripts in spermatozoa were related to reproduction (n = 58) and development (n= 210). Some of these transcripts were related to heat shock proteins (<i>DNAJB4</i>, <i>DNAJB14</i>), testis specific genes (<i>TCP11</i>, <i>TESK1</i>, <i>TSPYL1</i>, <i>ADAD1</i>), and Y-chromosome genes (<i>DAZ1</i>, <i>TSPYL1</i>).</p><p>Conclusion</p><p>A complex RNA population in spermatozoa consisted of coding and non-coding RNAs. A number of transcripts that participate in a host of cellular processes, including reproduction and development were differentially expressed between fertile and infertile individuals. Differences between comparison groups suggest that sperm RNA has strong potential of acting as markers for fertility evaluation.</p></div

Three group comparison and gene set analysis for comparison among normal fertile control, asthenozoospermic infertile, and idiopathic normozoospermic infertile groups.

<p>Only pathways that strongly correlated (r≥0.5) with expression of meta-gene set have been shown.</p><p>Three group comparison and gene set analysis for comparison among normal fertile control, asthenozoospermic infertile, and idiopathic normozoospermic infertile groups.</p

Three group comparison and gene set analysis.

<p>Bar graph showing correlation between expression of meta-gene set and the pathway (outcome) involved.</p

Three group comparison (feature set = 5000) and protein class analysis.

<p>PANTHER analysis of most significantly (top 5000) differentially expressed transcripts to classify them into protein classes.</p

Three group comparison and co-expression network analysis.

<p>Fruchterman-Reingold plot showing connections between co-expressing genes. Sample classes are colour-coded and probes are coloured according to the class with the highest median expression value across the corresponding samples. Colour-codes: class 1 (red: asthenozoospermic), class 2 (green: fertile control), and class 3 (blue: normozoospermic infertile).</p

Forest plot.

<p>Meta-analysis using allele frequency (a), cumulative allele meta-analysis using fixed effect (b) and random effect models (c).</p

Publication bias (Genotype meta-analysis).

<p>Funnel plot of standard error by log odds ratio using fixed model for observed set of studies (a) and after imputation (b). Funnel plot of precision by log odds ratio using fixed model for observed set of studies (c) and after imputation (d). Funnel plot of standard error by log odds ratio using random effect model for observed set of studies (e) and after imputation (f). Funnel plot of precision by log odds ratio using random effect model for observed set of studies (g) and after imputation (h).</p

Statistical analysis of mutant allele/genotype in our case-control study.

#<p>The calculations for mutant allele have been done with reference to allele ‘C’; for mutant genotypes with reference to ‘CC’ between cases and controls.</p>$<p>Azoo-azoospermia; Astheno-asthenozoospermia; Normo-normozoospermia; OAT-oligoasthenoteratozoospermia; Uncateg: uncategorized.</p><p>*Indicates statistically significant value.</p

Allele and genotype distribution.

<p>Bar diagram showing distribution of allele and genotypes in control and different groups of infertile individuals in our population.</p