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J Med Genet. 2007 August; 44(8): 498–508.
Published online 2007 May 11. doi:  10.1136/jmg.2007.049650
PMCID: PMC2597934

Coordinated transcriptional regulation patterns associated with infertility phenotypes in men

Abstract

Introduction

Microarray gene‐expression profiling is a powerful tool for global analysis of the transcriptional consequences of disease phenotypes. Understanding the genetic correlates of particular pathological states is important for more accurate diagnosis and screening of patients, and thus for suggesting appropriate avenues of treatment. As yet, there has been little research describing gene‐expression profiling of infertile and subfertile men, and thus the underlying transcriptional events involved in loss of spermatogenesis remain unclear. Here we present the results of an initial screen of 33 patients with differing spermatogenic phenotypes.

Methods

Oligonucleotide array expression profiling was performed on testis biopsies for 33 patients presenting for testicular sperm extraction. Significantly regulated genes were selected using a mixed model analysis of variance. Principle components analysis and hierarchical clustering were used to interpret the resulting dataset with reference to the patient history, clinical findings and histological composition of the biopsies.

Results

Striking patterns of coordinated gene expression were found. The most significant contains multiple germ cell‐specific genes and corresponds to the degree of successful spermatogenesis in each patient, whereas a second pattern corresponds to inflammatory activity within the testis. Smaller‐scale patterns were also observed, relating to unique features of the individual biopsies.

Keywords: testis, infertility, microarray, spermatogenesis, germ cell

Spermatogenesis is one of the most fundamental developmental processes, partly responsible for the perpetuation of all animal species including humans. It is regulated by highly coordinated waves of gene expression; unsurprisingly, therefore, when transcription is compromised infertility can ensue. The transition from spermatogonial germ cell to mature spermatozoon involves mitosis, meiosis, quality control of the germ cell population via apoptosis, and extensive morphological differentiation. This differentiation necessitates restructuring every organelle within the developing germ cell, as well as the genesis of novel organelles (flagellum, acrosome) found in no other cell type.1 It has been estimated that up to 4% of the entire genome is expressed specifically in developing germ cells.2 The impact of this vast transcriptional complexity is only now beginning to be unravelled, and is as yet poorly understood. In particular, while there is a growing body of transcriptional data covering normal testis development in mice,2,3,4,5 there are comparatively few data for human spermatogenesis, and in particular for the transcriptional consequences of subfertility phenotypes.

Previous studies that have addressed this issue include Sha et al, who compared normal adult (n = 2) and fetal (n = 3) testis to detect transcripts specific to germ cells,6 Fox et al,7 who compared patients with Sertoli‐cell only (SCO) (n = 3) and obstructive azoospermia (n = 3) for the same purpose, and Rockett et al8 who compared patients with obstructive (n = 4) and nonobstructive (n = 4) azoospermia. A significant drawback of both the latter studies is that they treat obstructive patients with azoospermia as physiologically normal, despite the fact that secondary effects of obstruction can lead to marked changes in testis cell composition.9 Furthermore, the grouping together of different pathological phenotypes under a common heading such as non‐obstructive or obstructive azoospermia leads to confounding of the observed effects, and difficulty in interpretation.

The more recent study of Feig et al (n = 28) addresses these issues by stringently filtering the patient cohort chosen for analysis to include only biopsies with a highly homogeneous cellular composition and well‐defined spermatogenesis status.10 This, however, will have the effect of excluding from the analysis all forms of subfertility that do not give rise to a highly uniform testicular pathology. Their targeted selection of the patient set was performed according to four predefined categories: SCO, meiotic arrest, hypospermatogenesis and full spermatogenesis, thus allowing analysis of the genes expressed in different cell types.

The approach used in the current study was to carry out an unbiased assessment of testicular transcriptional activity for all patients for whom biopsy material was available. Biopsies from patients undergoing testicular sperm extraction (TESE) for the purposes of assisted reproduction were used for microarray expression analysis using the Illumina RefSet oligonucleotide clone set. Each individual patient was treated as a separate sample for the purpose of one way analysis of variance, allowing us to investigate expression signatures specific to selected biopsies. To date, no other study has examined individual data from subfertile males in this way.

Methods

Patients

Testicular biopsies were taken from patients attending for treatment the Assisted Conception Unit at Birmingham Women's Healthcare NHS Trust. All patients were undergoing testicular biopsies in the course of their treatment and gave their informed consent to donate a portion of the biopsy material for this research project (ethics approval “Genes controlling spermatogenesis”, South Birmingham REC, LREC #0374).

Patients fell into six categories; idiopathic azoospermia (10 patients; AZ1–AZ10), cryptozoospermia (e patients, CR1–CR8), obstructive azoospermia due to congenital bilateral absence of the vas deferens (3 patients, AVD1–AVD3), obstructive azoospermia due to vasectomy (8 patients, VAS1–VAS8), non‐obstructive azoospermic following cancer treatment (3 patients, CA1–CA3), and a single patient who was unable to produce a semen sample in clinic (O1). Vasectomy reversal had been attempted for five patients (table 11)) but had failed. “Cryptozoospermia” denotes patients with very low sperm counts, and encompasses two related categories:

Table thumbnail
Table 1 Patient phenotypes
  1. Sperm not detected in normal ejaculate, small numbers of sperm found after centrifugation to pellet cells.
  2. Most ejaculates completely devoid of sperm, occasional sperm found in repeat samples from the same patient.

All patients had clinical investigations including a physical examination and hormonal assessment (follicle‐stimulating hormone (FSH) and testosterone). Karyotype analysis, assessment for Yq microdeletion and analysis for the most frequent cystic fibrosis transmembrane regulator (CFTR) gene mutations were performed in selected patients.

Testicular sperm recovery

Open testicular biopsies were carried out either under general anaesthesia or under local anaesthesia (a mixture of 0.5% bupivacaine and 2% lignocaine). Both testes were palpated and a decision made as to which testis was to be incised according to the size and consistency of the testis. The scrotal skin was stretched, and an incision of approximately 20 mm was made through the skin, the dartos muscle and the tunica albuginea. Careful haemostasis was secured using diathermy. Gentle pressure was then applied to the testis, and a small specimen of the protruding testicular mass was removed with fine scissors. Usually two specimens were taken from a single incision, and up to three incisions were made in a single testis. The tunica albuginea was sutured with continuous 3‐0 polyglactin suture, and the same suture used for subcuticular closure of the skin, but with interrupted sutures. Pressure was then applied over the incision suture for 5 minutes to secure haemostasis.

The testicular specimens were passed to the embryologist for identification of sperm. Following disruption of the tissue and microscopic observation, the sample was frozen for later use if sperm were observed. A randomly taken portion of the biopsy material was removed for the research project and placed in storage media (RNALater; Ambion Inc., Austin, Texas, USA (patients AZ1‐AZ6, CR1‐CR6 and VAS1‐VAS4) or Cook Sperm Media; Cook IVF, Spencer, Indiana, USA (remaining patients)). The sample was then snap‐frozen in liquid nitrogen and transferred to Cambridge University for processing. Where sufficient tissue was available, a further random piece of the biopsy was sent for histological examination using H&E.

Clinical findings and histological analysis

Histological analysis was performed on some patient tissue to identify the extent of spermatogenesis present, tubule condition and tissue types present. Representative photographs were taken of each available sample (see supplementary file 1; available online at http://jmg.bmj.com/supplemental). Results presented are based on histology reports from pathology labs at Birmingham Women's Hospital supplemented by our own independent analysis on further sections. Given the focal nature of some forms of testicular pathology, this histological analysis might introduce misclassification in some patients; however, owing to limited material available form testis biopsy procedures it is not possible to send more samples for histological analysis to obtain a more reliable classification.

Patients were followed through treatments that used sperm obtained from the testicular biopsy and outcomes of treatment were recorded (number of eggs collected, fertilisation rate, number and quality of embryos transferred, pregnancy rate (biochemical and clinical) and live birth rate).

RNA preparation and cDNA amplification

Total RNA was isolated from the biopsies using TRI reagent (Sigma‐Aldrich, St Louis, Missouri, USA) and cleaned using RNEasy MinElute columns (Qiagen, Valencia, California, USA), according to the manufacturers' protocols. Reference human total testis RNA was purchased from Ambion (Foster City, California, USA) and RNA quality assessed on an Agilent BioAnalyzer (Agilent Biotechnology, Santa Clara, California, USA). RNA samples of good quality were obtained from 33 patients. Two independent cDNAs were generated for each RNA sample, according to the manufacturer's protocols (SMART; Clontech, Mountain View, California, USA) as modified by Petalidis et al.11 Briefly, 50 ng of total testis RNA was reverse‐transcribed in a total volume of 10 μl, then 2 μl of first‐strand cDNA was used as the template in a 40 μl PCR amplification (19 cycles at 95° for 5 s, 65° for 5 s, and 68° for 6 min). Amplified cDNAs were purified using MinElute columns (Qiagen). In total, 192 amplifications were performed for the reference sample and pooled, to give a large stock of uniform reference cDNA.

Array analysis and data extraction

Dual‐colour hybridisations were performed as previously described,3 comparing the labelled cDNAs derived from the biopsy samples to the common reference cDNA pool. Two hybridisations were performed for each amplified cDNA, incorporating a dye reversal, thus giving four technical replicate experiments for each biopsy (two independent amplifications for each biopsy, and two dye‐swapped hybridisations for each amplified sample). For each hybridisation, 250 ng each of control/reference cDNA was labelled using Cy3‐tagged or Cy5‐tagged dCTP (BioPrime kit; Invitrogen Corp., Carlsbad, California, USA). Arrays were fabricated by the Centre for Microarray Resources at Cambridge University Department of Pathology. The probe set used was the Illumina RefSet oligonucleotide collection. This oligo array is built with an open‐source probe collection designed to yield information about ~21 000 human genes. The collection includes ~16 000 probes designed against ~12 600 single‐isoform and 1300 multiple‐isoform curated RefSeq genes, and a further ~6800 probes for validated RefSeq genome annotations. Data were collected using a GenePix scanner (Axon Instruments), and quantified with BlueFuse software (BlueGnome, Cambridge, UK). Expression data was log2 transformed prior to normalisation and statistical analysis. We found that the optimum normalisation for this dataset was Lowess normalisation, followed by per‐slide normalisation to the mode of the fluorescence ratio distribution. All data obtained in this study are available through the public ArrayExpress database (http://www.ebi.ac.uk/arrayexpress; accession number E‐MEXP‐1019).

Data analysis

Normalised data was analysed using the R library FSPMA,12 which is based on the mixed‐model analysis of variance library YASMA.13 The analysis of variance model used a nested design with on‐slide replicated spots as the innermost effect, nested inside technical replicate amplifications, and then dye‐swap replicate hybridisations, with patient phenotype as the outermost effect. The inner two effects were considered to be random effects, and dye‐swap and patient phenotype considered to be fixed effects. The p values were calculated using the FSPMA analysis of variance model and a false discovery rate (FDR) correction for multiple comparisons.14 Principal components analysis (PCA) and hierarchical clustering were performed using Knowledge Discovery Environment software (InforSense). Prior to PCA, a row‐based normalisation was applied, normalising the expression ratio data for each gene by subtracting the mean log2 value across all samples. Thus, for the purposes of the PCA, the expression level of each gene was measured relative to the average expression across all patients. This normalisation focuses the PCA on the differences between the patients, rather than the differences between each patient and the reference sample. Pearson correlation was used as the distance metric for the hierarchical clustering, and the unweighted pair‐group centroid clustering algorithm used.

Gene ontology analysis

Onto‐Express software (http://vortex.cs.wayne.edu/onto express/) was used to analyse the distribution of gene ontology (GO) categories in selected groups of genes. A binomial distribution was used in assessing the statistical significance of GO category enrichment, with Benjamini–Hochberg FDR correction for multiple testing.14

Results

Phenotypic analysis of the patient group

Patient history, Assisted Conception Unit (ACU) findings and histological data are summarised in Table 11.. The table shows patient number, significant aspects of patient history relating to the subfertility, FSH levels, histological findings, and whether or not mature/motile sperm were found following TESE at the ACU (see supplementary table 11 for details of intracytoplasmic sperm injection cycles and success rates; available online at http://jmg.bmj.com/supplemental). The patient judged to have the most normal testis histology was patient AZ6. In most cases where histological analysis was performed, seminiferous tubules could be clearly identified; however, three of the biopsies contained tissue that could not be clearly identified, resembling either efferent ducts (patients AZ8, CA1) or unidentified muscular structures (patient AZ1).

Patient O1 and his partner achieved a spontaneous pregnancy; however, the histology showed clear spermatogenic deficiency. This is consistent with a diagnosis of oligozoospermia. No karyotypic abnormalities or Y chromosome microdeletions were found. One of the CBAVD patients (patient AVD2) was found to have a CFTR mutation. Patient CA1 was a carrier for a CFTR mutation, but did not lack vasa deferentia.

Analysis of variance selection of significantly varying genes

A mixed model analysis of variance was used to select genes that varied significantly between patients. Using a threshold p value of 0.05, 19 423 probes (~85% of the gene set) were found to vary significantly across the dataset. In total, 11 048 probes (~48.5% of the gene set) were significant at a threshold of p<1×10−6. These numbers, while high, are not unexpected given the large number of samples and the vast physiological difference between a functioning testis and a germ cell‐deficient testis. In order to select a tractable number of genes for downstream analysis, an extremely stringent threshold of p<1×10−16 was used, yielding 3278 significant genes. Data presented below are based on this set of very highly significant genes. Similar results were obtained for both the PCA and hierarchical clustering analyses when using less stringent thresholds for significance (data not shown).

Principal components analysis

Testis tissue is composed of a large number of different cell types, all of which contribute in varying proportions to the aggregate expression profile. Because germ cell number and differentiation state varies widely between the biopsies, the expression data presented here represents the net result of a large number of interacting processes. PCA is a statistical method of disentangling the various effects contributing to the overall dataset variance, and yields a number of principle components or “axes”, which together add up to the observed expression values. If a given gene A scores highly on any given axis and gene B scores low on the same axis, then patients with a high A:B ratio will also score highly on the same axis. In contrast, a low A:B ratio would lead to that patient scoring lower on that axis. Thus, PCA components represent patterns of correlated gene‐expression changes. If a given patient's expression profile is a good fit to the pattern defined by a given axis, that patient will score highly on that axis.

For this dataset, most of the variation in the dataset was captured by the first two principal components: PCA 1, explaining 53% of the total variance in the dataset, and PCA 2, explaining 18% of the total variance (full PCA variance breakdown is available as supplementary table 22;; available online at http://jmg.bmj.com/supplemental). For these two principal components, the 500 genes with the most positive or most negative correlations with the component were selected, and the distribution of gene ontologies assessed using Onto‐Express. This analysis indicates the functional gene categories associated with these patterns of coordinated gene expression. A GO category was considered significantly enriched in any given group of genes if the FDR‐adjusted p value was <0.05 and there were >3 genes contained within the category. Table 22 shows which categories were enriched at the extremes of each PCA axis. Figure 1A1A is a PCA biplot indicating the mapping of the complete gene set onto the PCA1 and PCA2 axes.

figure mg49650.f1
Figure 1 PCA biplots indicating significant aspects of the dataset. (A) Gene biplot showing distribution of the gene set on the PCA axes. Highlighted genes illustrate the functional correlates of each axis. PCA1, germ‐cell content; PCA2, ...
Table thumbnail
Table 2 GO categories found to be over‐represented in the genes at the extremes of each PCA axis

PCA1: germ cell/somatic cell distinction

Genes mapping to the negative end of the PCA1 axis were enriched for multiple GO categories associated with germ cell progression and sperm function. These genes included several well‐known germ cell specific genes, e.g. Prm1, Prm2, Tnp1, Ldh3 and Sycp3 (see Matzuk and Lamb15 for a review of testis pathways). In contrast, the genes mapping to the positive end of the PCA1 axis were enriched for multiple housekeeping functions such as ribosomal components, electron transport chain components and extracellular matrix proteins. Genes specifically expressed in somatic cell types (and not expressed in germ cells) also scored highly positive on the PCA1 axis. This axis thus distinguishes between germ cell‐specific gene expression and ubiquitous or somatic cell gene expression.

PCA2: inflammatory mediators

Genes scoring highly on the PCA2 axis were enriched for GO categories relating to inflammatory processes. Furthermore, the four genes most strongly associated with this axis were the known pro‐inflammatory cytokines (C–C motif) ligand 2 (CCL2), interleukins 6 and 8, and (C–X–C motif) ligand 1 (CXCL1). Other pro‐inflammatory cytokines/chemokines (colony‐stimulating factor 3, leukaemia inhibitory factor)16 and immediate early response genes (epidermal growth factor 1 (EGR1) and FOS) also scored highly on this axis. On this basis, we therefore deduced that this axis relates to the degree of inflammatory processes occurring in each biopsy. Interestingly, the genes scoring lowest on this axis (correlated negatively with inflammatory gene expression) include known Leydig cell genes such as Insl317 and sterol carrier protein 2.18 Genes scoring highly on the PCA2 axis also scored highly on the PCA1 axis, indicating that increased inflammatory gene expression correlates with absence of germ‐cell gene expression and thus with the severity of the testicular phenotype.

Comparison of PCA data with patient phenotype

Table 33 shows patient scores on the PCA1 and PCA2 axes. These scores represent how closely each patient correlates with the prototypical expression pattern represented by each principle component. Patients scoring highly on the PCA1 axis have a high ratio of somatic‐cell to germ‐cell transcripts (i.e. pronounced germ‐cell deficiency), whereas patients scoring high on the PCA2 axis have a high ratio of inflammatory gene to other gene transcripts (i.e. raised levels of inflammation in the tissue sample).

Table thumbnail
Table 3 Patient scores for PCA1 and PCA2

In general, the level of germ‐cell gene expression indicated by the PCA1 data is consistent with the histological and ACU findings summarised in table 11.. Notably, patient AZ6, with the testis histology profile closest to normal, scored at the low extreme of the PCA1 axis (ie high germ‐cell gene expression), and near zero on the PCA2 axis (no overexpression of inflammatory mediators). Patient O1, despite achieving a spontaneous pregnancy with his partner, showed a moderate reduction in germ‐cell mRNAs and also showed the highest score of any patient on the inflammatory axis represented by PCA2.

The patient distribution on the PCA1 axis was non‐normally distributed (Kolmogorov–Smirnov goodness‐of‐fit test p = 0.03). Inspection showed a bimodal distribution, with patients tending to score either high or low on this axis, with few intermediate scores. This suggests that most patients in this study have either no germ cells (complete SCO) or significant numbers of late–stage germ cells. This is consistent with the results of Feig et al,10 who found that 26.7% of patients had a Johnsen score19 of [less-than-or-eq, slant]2, and 57.3% a score of [gt-or-equal, slanted]8. In contrast, the patient distribution on the PCA2 axis was consistent with a normal distribution (K‐S test, p = 0.88), indicating a continuous range of inflammatory phenotypes among patients.

Figure 1B1B shows a PCA biplot for all patients, coded according to the ACU findings. There was a highly significant difference (t test, p<1×10−4) in PCA1 score between the azoospermic patients and those with motile sperm, whereas those that had mature but nonmotile sperm were more widely distributed on the PCA1 and 2 axes. A minority (n = 5) of the patients with motile sperm had PCA1 scores >0, and thus grouped more closely with the azoospermic patients than the others with motile sperm. These five patients (AZ10, CR4, CR7, AVD1 and CA1) are highlighted in figure 1B1B.. In each case, the more detailed histological analysis was in agreement with the expression profiles. No mature sperm was seen at histology for patients AZ10, CR7, AVD1 and CA1, and patient CR4 showed mature but abnormally shaped sperm.

FSH levels

FSH is raised in SCO syndrome, due to a lack of inhibin feedback.20 Figure 1C1C shows a PCA biplot for all patients where FSH data was available; it can be seen that raised FSH is strongly correlated with the PCA1 axis (t test comparison of normal/elevated p = 6×10−4), indicating almost complete loss of germ‐cell transcripts in these patients. There was no correlation with the PCA2 axis (t test p = 0.6).

Secondary effects of obstructive azoospermia

In most cases where sperm was found in the biopsy, the cause of the subfertility was obstructive rather than being due to an innate failure of germ‐cell production. Obstruction of the normal male ductal system often leads to substantial secondary change in the testis, including rupture of the rete testis and pressure atrophy of the seminiferous epithelium.21,22,23,24 This can be clearly seen in the histological results in table 11 for vasectomised patients and those with congenital absence of the vas deferens. We were interested to see whether these changes were observable in the expression data. Figure 1D1D indicates the locations of these patients in the PCA biplot. The majority of these patients scored strongly negative for PCA1, indicating high levels of germ‐cell transcripts relative to the patient set as a whole. These levels of germ‐cell transcripts presumably indicate a normally functioning spermatogenic process. However, some showed a reduction in germ cell activity (less strongly negative PCA1). This was correlated (Pearson coefficient  = 0.670, p<0.05) with an increase in PCA2, indicating increased inflammatory activity associated with the loss of germ‐cell transcripts This is consistent with tissue destruction following pressure‐induced rupture of the testicular ductal system.

Hierarchical cluster analysis

We used hierarchical clustering to seek out smaller‐scale patterns in the dataset, as a complement to the large‐scale trends seen in the PCA analysis. Figure 2A2A shows a heat map for all selected genes across all patients. Five major zones can be seen, and a smaller sixth zone can be defined based on upregulation of a group of genes across multiple patients.

figure mg49650.f2
Figure 2 Heat maps showing gene‐expression levels across patients. (A) All genes. Six major “zones” can be defined, representing groups of coregulated genes. (B) Genes specifically upregulated in the three highly abnormal ...

Gene ontology data was used to examine the functional distribution of transcripts within each of the heat map zones (table 44).). Zone 1 is enriched for functional categories associated with late spermatid development and motility, but also for categories associated with meiotic cells, e.g. synaptonemal complex genes (note: the mitosis‐associated categories relating to this zone are a result of misannotation in the Gene Ontology database, which records some protamines and transition proteins as mitotic genes). Zone 2 is enriched for functional categories associated with DNA synthesis, repair and cell division. Zones 3 and 4 are enriched for functional categories associated with energy production, protein synthesis and other metabolic functions. Zone 5 is enriched for cholesterol and lipid metabolism categories. Zone 6 contained too few genes to show specific category enrichment; however, inspection showed it to contain many of the inflammatory mediators outlined above in association with PCA2.

Table thumbnail
Table 4 GO categories found to be over‐represented (p<0.05 following FDR correction, bolded p<0.01) in each of the major zones of the heatmap shown in figure 2</figref>

Figure 1E1E shows the PCA biplot of the gene‐expression data, coloured according to the zone assigned by the hierarchical cluster analysis. It can be seen that zones 1–4 divide the PCA1 axis into more specific subregions. Zone 6 highlights the inflammatory genes at the upper extreme of PCA2. Zone 5 is not significantly separated from other zones on a PCA1/PCA2 biplot, however a PCA1/PCA3 biplot (supplementary figure 11;; available online at http://jmg.bmj.com/supplemental) shows that zone 5 genes are associated with this third principle component. Zone 5 genes and PCA3 genes were enriched for GO categories relating to lipid metabolism, and also included the known Leydig cell gene Insl3 (see supplementary files; available online at http://jmg.bmj.com/supplemental), thus it may be that this zone and principal component relate to a shift in the proportions of interstitial tissue to seminiferous tubule tissue.

This analysis also reveals clusters of genes specifically upregulated in the unclassifiable biopsies AZ1, AZ8 and CA1 (Figure 2B2B).). These biopsies were enriched for ontology categories relating to the cytoskeleton (including smooth muscle actin and myosin isoforms) and to defence mechanisms. Patients AZ1 and CA1 (but not AZ8) showed upregulation of lactoferrin, a known epididymal transport gene.25,26 In addition, known prostate genes such as secretoglobin 2a127 and PAGE428 were also overexpressed in these patients. For all three samples, the enrichment for defence‐related GO categories was due to beta‐defensin genes, which are known epididymal antigens.29 However, the specific family members involved were different for the three samples. Figure 33 shows expression data for all defensin genes present in the gene set.

figure mg49650.f3
Figure 3 Expression of defensin genes present in the gene set. Values plotted are mean (SEM) for patients AZ1, AZ8 and CA1, and the aggregate mean (SEM) for the complete dataset.

Strikingly, the defensins appear to be regulated in chromosome‐specific groups. There was upregulation of β‐defensins mapping to 20p13 in patients AZ1 and CA1 but not in patient 1004. Three of the four β‐defensins mapping to 20q11 are downregulated in all three patients. Four of the six β‐defensins mapping to 8p23 are upregulated in patients AZ1 and AZ8, but not in patient CA1. These changes in β‐defensin expression are specific to these three patients; across the dataset as a whole there is very little change in expression of these genes. There was no change in expression of α‐ defensins across the dataset.

Discussion

Understanding the transcriptional control of fertility, and how perturbed transcription can lead to loss or reduction of fertility, will require many studies correlating clinical phenotypes with expression data. Although this study does not purport to be encyclopaedic, it is nevertheless the largest investigation to date of expression changes associated with male infertility. The patient group analysed in this study was not specifically targeted based on known causes of azoospermia, thus this constitutes an unbiased survey of the range of pathological phenotypes present among patients TESE via open biopsy.

PCA was used to define functional axes of gene expression, the most significant (PCA1) relating to germ‐cell/somatic‐cell gene expression, and the next most significant (PCA2) relating to inflammatory gene expression. Together, these accounted for most of the variance in the dataset. Known Leydig cell genes scored low on the PCA2 axis, indicating a negative correlation with inflammatory gene activity. This is in agreement with previous reports that pro‐inflammatory cytokines suppress steroidogenic activity.30,31

The levels of germ‐cell transcription indicated by the PCA1 score for each patient was in good agreement with the ACU findings relating to the presence of sperm in the biopsies in the majority of cases. In a minority of cases, sperm was found by the ACU despite an expression profile suggestive of low germ‐cell gene expression. In each case, the histological analysis of biopsy material was in agreement with our expression data. In these patients, the motile sperm detected by the ACU presumably derive from small foci of normal spermatogenesis, possibly outside the area of biopsy used for this study, within a largely degenerate testis. This illustrates the difficulty of classifying testes with a heterogeneous pathological phenotype. Interestingly, three of these patients (AZ10, CR7, CA1) had a clinical history suggestive of testicular dysgenesis syndrome, with features such as undescended testis and inguinal hernia (AZ10), varicocoele (CR7), and testicular carcinoma (CA1).

Our hierarchical cluster analysis defined groups of coregulated transcripts, which proved to fractionate the PCA1 axis into distinct subclusters. These subclusters were found to represent functionally distinct groups of genes, with specific GO categories being enriched within each zone. This functional subdivision agrees well with our overall interpretation of PCA1 as representing germ‐cell specificity. Genes expressed exclusively in germ cells, such as spermatid antigens and meiosis‐specific proteins (eg, synaptonemal complex proteins) were most strongly associated with the negative extreme of PCA1, and fell into zone 1. Genes relating to DNA synthesis and repair were expressed preferentially, but not exclusively in germ cells, and thus fell into zone 2, strongly negative for PCA1, but not as extreme as zone 1. Zones 3 and 4 fractionate the remaining genes still further, zone 3 being enriched for GO categories related to energy production, and zone 4 for protein synthesis and other metabolic functions. The slight distinction between zones 3 and 4 on the PCA1 axis may reflect greater energy demands in developing germ cells, and greater protein synthesis demands in the supporting somatic lineages. The results of Feig et al10 form a complementary classification to ours. The combination of PCA and hierarchical clustering in our analysis fractionates genes based on the degree of germ‐cell specificity, whereas their analysis of different patient groups fractionates genes based on germ‐cell subtype, assigning different groups of genes to different stages of germ‐cell differentiation. Thus, their analysis shows in which germ‐cell stage a given gene is expressed, whereas ours provides a measurement of the extent to which any given gene is preferentially expressed in germ‐cell or somatic‐cell lineages.

In addition to this germ‐cell axis (PCA1), our analysis revealed a second dimension of variability (PCA2) among patients in terms of inflammatory gene activity. Genes scoring high on PCA2 also scored high on PCA1, indicating that in general, increase in inflammatory‐gene activity correlates with loss of germ‐cell transcripts. We further analysed this effect specifically within the obstructive azoospermia category (vasectomised patients and patients with CBAVD), and again found that loss of germ cells correlates with increasing inflammatory mediators, suggesting pressure atrophy of the seminiferous epithelium and consequent inflammatory tissue destruction. The analysis of Feig et al10 did not reveal this source of variability between patients. We speculate that this is because tissue destruction via testicular inflammation may lead to heterogeneity across the testis, and that patients with inflammatory pathology were therefore excluded from their dataset.

Three biopsies showed histology that was not seminiferous, with unidentified tubular structures present (patients AZ1, AZ8, CA1). In two cases (AZ8, CA1), the tubules had some characteristics of efferent duct tissue, while the third contained muscular tubes of unknown origin. This most likely reflects the difficulty of accurately obtaining tubule material from the very small testes often associated with defective spermatogenesis; however, it may also indicate abnormal differentiation of testis structures in patients with testicular dysgenesis. These biopsies showed no expression of germ‐cell genes. We found expression of muscle genes, and also of known epididymal antigens such as lactoferrin and β‐defensins. Interestingly, the defensin genes followed a coordinated pattern of upregulation and downregulation of linked gene clusters. This suggests that regulation of these genes occurs via chromatin modification affecting whole chromosomal domains. The coordinated differences in defensin expression observed between patients AZ1, AZ8 and CA1 may indicate a spatial element to defensin regulation—that is, different segments of the male gonad may selectively express different combinations of defensins. This is consistent with other work showing spatial regulation of other defensin genes along the length of the epididymis.32,33 More intriguingly, there was also expression of prostate genes such as PAGE4 and secretoglobin 2a1 in these biopsies. These have not previously been shown to be expressed in any testicular or epididymal cell type. It may be that very severely dysgenetic testes show ectopic expression of varying subsets of genes associated with non‐testis tissue types. Expansion of the study to include expression profiles of normal epididymal tissue and/or prostate tissue, as well as more patients with testicular dysgenesis, would help resolve this issue.

This study is the largest expression analysis survey to date of subfertile human men and demonstrates that robust, reproducible expression data can be generated from very small amounts of biopsy material. This represents a significant milestone in our ability to garner expression data from such patients. In agreement with Feig et al,10 we found the dataset to be bimodal, with most patients either showing either comparatively high levels of germ‐cell gene transcription or none. This is unsurprising given the current clinical practice of only taking biopsies from azoospermic or cryptozoospermic patients. This means that testicular expression data cannot be obtained for the much larger number of patients presenting with varying degrees of hypospermatogenesis. Many unanswered questions about male fertility can only be answered by studying these patients directly. For example, oligozoospermia is known to be linked to high rates of sperm aneuploidy, with clear implications for the risk of generating aneuploid embryos.34 A better understanding of the transcriptional abnormalities seen in oligozoospermic patients may reveal markers associated with the success or failure of various assisted‐reproduction techniques, and the risks associated with each for parent and child. Given that the technology is now in place to address these questions, we therefore urge a debate on the merits of further expression profiling of the much larger population of oligozoospermic patients.

Another general principle that emerges from this study is the vast amount still to be discovered about the genetic networks necessary for normal testis function. Up to 4% of the entire genome is expressed specifically in developing male germ cells,2 and as yet the function of many of these genes is unknown. Examining PCA1 (representing germ cell genes), we found enrichment for the “unknown” category in all three major GO trees: molecular function, biological process and cellular component (table 11).). Indeed, together with “spermatogenesis”, these constituted the four most significant GO categories enriched in germ cell genes. This dramatically indicates the current lack of knowledge concerning the detailed transcriptional mechanisms of sperm development. Transcriptional array studies, including the body of data generated here, will be a vital component of the research involved in unravelling the complexity of a functional spermatozoon.

Supplementary material is available on the JMG website at http://jmg.bmj.com/supplemental

Acknowledgements

The study was funded by the Wellcome Trust. We are grateful to Birmingham Women's Healthcare NHS Trust for sponsorship of this project and to the staff of the Assisted Conception Unit, Birmingham Women's Hospital.

Abbreviations

ACU - Assisted Conception Unit

CTFR - cystic fibrosis transmembrane regulator

FDR - false discovery rate

FSH - follicle‐stimulating hormone

GO - gene ontology

PDA - principal components analysis

SCO - Sertoli‐cell only

TESE - testicular sperm extraction

Footnotes

Competing interests: None declared.

The last two authors should be considered joint last authors.

Supplementary material is available on the JMG website at http://jmg.bmj.com/supplemental

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