Abstract
Non-obstructive azoospermia (NOA) is currently evaluated by the use of conventional histopathological methods. In some cases, focal spermatogenesis is present in the testes of patients with NOA which may be almost undetectable by routine histopathological examinations. Application of molecular markers in semen to predict the spermatogenesis status in the testis will emphasize the probability of finding sperm in NOA testis through further search using TESE or mTESE. Detection of germ cell-specific transcripts in semen is a signal of germ cells present in the testis. In this study, we used molecular methods to evaluate spermatogenesis status in azoospermic men. Semen samples were collected from 203 men with azoospermia. Total RNA was extracted from the semen precipitates. First-strand complementary deoxyribonucleic acid (cDNA) was synthesized by reverse transcriptase then, (RT)-PCRs were carried out using primers for testis stage-specific genes (DAZ, AKAP4, PRM1, and PRM2). Testicular tissue biopsies were used for evaluating spermatogenesis status in testis. Histopathological examination and LH, FSH, and testosterone level measurements (chemiluminescence assay) were performed. The presence of DAZ and PRM2 transcripts in semen significantly indicated the presence of spermatogonia and spermatids in the testicular tissues. Absence of all four markers in semen confirmed the histopathological results corresponding to sertoli cell only syndrome (SCO). Although TESE should not be excluded solely on this criteria, using PRM1, PRM2, AKAP4, and DAZ transcripts in semen would provide a non-invasive molecular diagnostic tool to better counsel patients before undergoing TESE.
Introduction
Infertility affects 10 − 15% of all individuals world-wide, half of which is due to male infertility [Matzuk and Lamb Citation2002]. Non-obstructive azoospermia (NOA) which is due to impaired spermatogenesis in the testis, occurs in 5% of infertile male subjects [Marcelli et al. Citation2008; Van Peperstraten et al. Citation2008]. Assisted reproductive techniques (ART) have helped azoospermic men to conceive a child from their own gametes, since focal spermatogenesis might still be present in small parts of the testes [Silber et al. Citation1997]. Mature sperm can be successfully retrieved by microdissection testicular sperm extraction (mTESE) or TESE (testicular sperm extraction) for intracytoplasmic sperm injection (ICSI) in approximately 30-70% of patients with NOA [Devroey et al. Citation1995; Houwen et al. Citation2008; Jezek et al. Citation1998; Sobek et al. Citation1998; Su et al. Citation1999; Tournaye et al. Citation1995].
At present, different methods such as the evaluation of endocrine profiles (FSH, LH, inhibin, and anti-müllerian hormone), histological examinations, and assessment of testicular volume and consistency are used to predict the presence of mature sperm in the testis. The predictive value of these methods are relatively low [Goulis et al. Citation2008; Sobek et al. Citation1998]. In addition, testicular biopsy is an invasive procedure with a small coverage of the entire testis [Schlegel and Su Citation1997]. More accurate methods are required to predict the presence of mature sperm in the entire testes of men with NOA.
The presence of immature germ cells in semen at different stages of spermatogenesis, has been previously documented [Bassol et al. Citation1990; Huang and Agarwal Citation2004; Yeung et al. Citation2007]. The presence of different stages of spermatogenic cells in the testis can be confirmed by the detection of certain mRNAs, exclusively expressed at a relevant maturation level [Matzuk and Lamb Citation2008]. Recently, highly specific cellular and molecular methods such as flow cytometry [Koscinski et al. Citation2005] and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) [Bauer and Patzelt Citation2003; Ferras et al. Citation2004; Patrizio et al. Citation2000; Song et al. Citation2000] have been used to detect germ cells in the testis. However, molecular methods are mainly based on detecting testis specific transcripts in testicular biopsies rather than seminal fluid.
Identification of molecular markers in semen samples as a non-invasive diagnostic tool, gives us a thorough image of the events which are in process in the testis [Platts et al. Citation2007; Yatsenko et al. Citation2006]. This method would provide prediction of even minute foci of spermatogenesis and small numbers of spermatozoa in NOA testis. In this study, we evaluated the spermatogenesis status of patients with NOA by testing for the presence of stage-specific gene transcripts in semen samples in conjunction with histopathological examination and endocrine profiles.
Results
Expression of testis-specific genes in semen was used as an indicator for the presence of different spermatogenic cells in the testis. The presence of testis-specific gene transcripts in semen was compared with testicular histopathological examination of NOA patients and also with the presence or absence of sperm in testicular tissue samples (TESE results). In addition, the endocrine profiles of the men with azoospermia were compared with molecular findings on their semen. On the whole, 93 semen samples were discarded due to patients' refusal to undergo testicular biopsy and the consequent absence of histopathological studies.
Table 1. The primer sequences.
The mean age of patients was 35.51 ± 7.1 (ranging from 23 to 63) years. The mean duration of infertility was 8.8 ± 6.9 years. Expression of β-actin, DAZ, AKAP4, PRM1, and PRM2 transcripts were analyzed in semen samples by RT-PCR as shown in using the primers listed in . The expression of the aforementioned genes was proven in the testicular tissue by normal spermatogenesis as positive controls.
Figure 1. RT-PCR results of DAZ, AKAP4, PRM1, and PRM2 genes in semen samples of NOA patients. The reference gene, β-actin, was used as the positive control. Lanes 1, 2, and 3: three NOA patients. BS: the band size for each gene.
Table 2. Serum hormone levels in NOA men that have been categorized as low, normal, and high according to reference ranges of hormones.
Histological and histopathological evaluations of the testis from 110 azoospermic men were as follows (a, b, c, d, and e): 65 men with sertoli cell only-syndrome (germ cell aplasia), 4 men with spermatogonial maturation arrest (MA), 20 men with spermatocyte MA, 6 men with spermatid MA, and 15 men with hypospermatogenesis. Sperm retrieval rate in our study was 15.74 %. The RT-PCR results for the five groups are summarized in . compares the presence of seminal molecular markers with successful sperm retrieval following TESE in NOA patients.
Figure 2. The agreement level between the relative frequency of patients expressing the genes in semen samples and testicular histopathological results. Testicular histopathology status in NOA patients are sertoli-cell only syndrom (a), spermatogonial MA (b), spermatocyte MA (c), spermatid MA (d), and hypospermatogenesis (e). β-actin was used as the positive control.
Figure 3. Seminiferous tubular histology patterns from TESE samples in NOA patients: a) sertoli-cell only syndrom, b) spermatogonial MA, c) spermatocyte MA, d) spermatid MA and, e) hypospermatogenesis. Tissues were stained by haematoxylin.eosin.
Table 3. Positive molecular results in NOA patients' semen sample compared with successful sperm retrieval after TESE.
According to the Kappa measure of agreement, there was a significant correlation between the expression of DAZ (Kappa value = 0.197, p = 0.035) and PRM2 (Kappa value = 0.305, p = 0.002) and the results from histopathological examinations. The presence of the PRM2 transcripts in the semen had a 70% sensitivity and a 77.5% specificity to predict the presence of mature spermatozoa in the testis. The presence of AKAP4 and PRM1 transcripts in semen were not compatible with histopathological findings. However, the specificity for both genes was sufficient to confirm the absence of spermatids or spermatozoa in the testis (85.5% and 81.6%, respectively). Molecular results for the relevant genes were not significantly correlated with the presence of sperm in testicular tissue samples (successful sperm retrieval following TESE).
Serum LH, FSH, and testosterone concentrations are summarized in . The probable relationship between the presence of gene transcripts in semen and hormonal results were monitored using the independent-sample T-test. However, findings show no significant correlation between the molecular findings and hormonal levels in the NOA men.
Discussion
At present, NOA patients can have a successful sperm retrieval using TESE or mTESE followed by ICSI [Devroey et al. Citation1995; Silber et al. Citation1995b]. Retrieving mature sperm with normal morphology from the testis of NOA patients plays an important role in these treatment approaches [Silber et al. Citation1995a; Tournaye et al. Citation1995].
Currently, determining the presence or absence of mature sperm in the testis of NOA patients is based on the traditional histopathological methods, which make use of testicular biopsies. Due to the limitations of these methods, application of seminal-based methods as non-invasive diagnostic tools are receiving more attention [Yatsenko et al. Citation2006]. Immature seminal germ-cells [Bassol et al. Citation1990; Huang and Agarwal Citation2004] may provide valuable information about spermatogenesis status in the testis. Koscinski et al. [2005] used flow cytometry to detect low numbers of immature germ-cells in the semen of NOA patients. More specifically, the pattern of germ cell-specific gene transcripts in semen can be used as a molecular marker for representing spermatogenesis status and may be a more suitable alternative for invasive diagnostic methods, such as testicular biopsy. Song et al. introduced the PRM2 transcript as a molecular marker to predict the presence of spermatids/mature sperms in the testis of NOA patients [Lee et al. Citation1998; Song et al. Citation2000]. However, this method was based on invasive testicular biopsy with limited diagnostic value.
In the present study, more than 50% of the participants were negative for germ-cells in histopathological evaluations of the biopsies (germ cell aplasia). Hence, the establishment of a non-invasive and accurate screening test with a high predictive value could prevent more than 50% of these patients from undergoing testicular biopsy. A significant correlation was observed between the presence of spermatogonia spermatids spermatozoa and expression of DAZ and PRM2 in the semen of the NOA patients. Expression of the DAZ gene begins in the premeiotic phase and continues until final stages of spermatogenesis [Lee et al. Citation1998; Menke et al. Citation1997; Szczerba et al. Citation2004]. Consequently, DAZ might be a useful germ-cell-specific internal control which confirms the presence of immature germ-cells in testis [Aslani et al. Citation2007]. The absence of DAZ transcripts and presence of β-actin transcripts in semen is consistent with germ cell aplasia in the testis.
Song et al. [2000] showed that deletion of the DAZ gene in NOA patients that express PRM2 correlates with the production of spermatozoa with poor morphology. Such genes can provide useful information about the pathophysiology of spermatogenesis [Lee et al. Citation1998]. Furthermore it may provide more understanding of the molecular basis of spermatogenesis and causes of male infertility.
The pattern of expression of AKAP4 was not compatible with the histopathological findings. This may be due to the small area of the testis which could be screened using this technique. However, AKAP4 was present in the semen of patients with positive TESE and control. This was sufficient to confirm the absence of spermatids and mature spermatozoa in semen, and subsequently the presence of spermatids or sperm in the testis.
A discrepancy was also observed between the molecular PRM1 transcripts in semen and histopathological results. This may reflect the independent expression of the PRM1 and PRM2 genes [Viguie et al. Citation1990; Wykes et al. Citation1995] even though they are both located on chromosome 16q13.3 (Gene Bank, Z46940) [Domenjoud et al. Citation1991]. Furthermore as Steger et al. have demonstrated, the PRM2 gene is expressed twice as much as the PRM1 gene in human testis using non-radioactive in-situ hybridization [Steger et al. Citation2000] . A similar result has been shown using Northern blot analysis [Wykes et al. Citation1995]. Therefore, PRM1 and PRM2 should be evaluated independently. Interestingly, AKAP4 and PRM1 correlated significantly with PRM2 transcripts. Our results show that absence of AKAP4, PRM1, and PRM2 transcripts in semen suggests their absence of mature sperm in the testis.
Comparison of the molecular and TESE results (successful sperm retrieval) did not show any significant correlation. The lack of correlation may be due to the association of the sampling of relatively small amounts of tissue from many negative TESE patients yet positive gene expression that surveys the entire tissue (). Friedler et al. showed that the first TESE results in mature sperm retrieval in 39% of patients (61% were negative TESE) [Friedler et al. Citation2002]. However a second TESE was positive in 25% of the first TESE negative group. Therefore, we propose that it may be beneficial to repeat TESE in the patients with negative first TESE but positive germ cell markers. This needs to be independently evaluated by additional studies.
In conclusion, by comparing histopathological findings and molecular results we showed that presence of germ cell-specific DAZ and PRM2 transcripts in the semen of NOA patients can be used as specific non-invasive markers to predict the presence of mature spermatids and/or sperm in the testis. However, it must be noted that histopathological findings are not always compatible with this spermatogenesis status assessment, and therefore multiple TESE may be required. The combination of molecular methods using testis-specific genes (PRM1, PRM2, AKAP4, DAZ, etc) and highly sensitive flow-cytometry may be able to predict spermatogenesis status in the testis of NOA men. These markers may provide better counsel before undergoing TESE or even multiple TESE. In future studies, analysis of other abundantly expressed germ-cell specific genes in larger population sizes by real-time PCR, and in combination with flow-cytometric methods, would further confirm the results of this preliminary study.
Materials and Methods
Semen Sample Collection
Semen samples were collected from 203 men with azoospermia attending Avicenna Infertility Clinic for infertility treatment. The semen samples were analyzed in accordance with the WHO 1999 guidelines [World Health Organization Citation1999]. As some couples refused to fulfill their infertility treatment, testicular biopsy was done only in 110 out of 203 individuals and their semen samples were kept for further studies. Written informed consent was taken from all participants for the use of their semen in the study. The Avicenna Research Institute's (ARI) Medical Ethics Committee approved the study. Testicular tissues of azoospermic men with all stages of spermatogenesis including mature spermatozoa were used as positive controls for the study.
Semen Sample Preparation
Following liquefaction of the semen at 37°C, each sample was centrifuged at 200 x g for 15 min. The pellets were washed 3 − 4 times by sterile phosphate buffered saline (PBS, 0.5 M) at 300 x g for 5 min. The final pellets were kept in liquid nitrogen (−196°C) prior to RNA extraction.
RNA Extraction
Frozen samples were thawed and washed twice by sterile PBS. The total RNA was extracted using RNA Bee kit (Biosite, Sweden). Lysis buffer (600 µl/106 sperm) was added to the samples. The lysates were homogenized by a pellet pestle (Sigma, USA) in a phenol-chloroform-isoamylic acid solution. Isopropanol was added to isolate and precipitate RNA from the aqueous phase. The isolated RNA was washed twice by ethanol (75%) and was left to be air-dried. The pellets were dissolved in diethylpyrocarbonate (DEPC)-treated water and stored at 70°C [Aarabi et al. Citation2008; Chomczynski and Sacchi Citation2006; Goodrich R 2007]. The purity of the extracted RNA was checked spectrophotometrically (Biophotometer, Eppendrof, Germany) at 260 and 280 nm.
RT-PCR Amplification
First-strand cDNA was synthesized by using 150 µg of the total RNA. The cDNA reactions occurred at 42°C in 60 min. The first-strand cDNA synthesis reaction mixture was prepared by using 10 µl of the isolated mRNA, 2 µl of 20 mM random hexamer (Roche, Germany), 2 µl of 10 mM dNTPs (Roche, Germany), 4 µl of 5X M-MLV reverse transcriptase buffer (Fermentase, EU), and 200 IU M-MLV-RT (Fermentase, EU) in a final volume of 20 µl.
PCR amplifications were carried out in a 25 µl reaction mixture, composed of 1 µl of the prepared cDNA, 1 µl of each primer (10 pM) (Bioneer, South Korea), 1 µl of 10 mM dNTPs (Roche, Germany), 1 IU Taq polymerase (Roche, Germany), 3 µl of 25 mM MgCl2 (Roche, Germany), and 2.5 µl of 10X PCR buffer (Roche, Germany).
PCR Amplification
Testis-specific genes were selected according to their expression stage. As shown earlier, deleted in azoospermia (DAZ) gene expression starts at the spermatogonial level [Huang et al. Citation2008; Lee et al. Citation1998; Menke et al. Citation1997; Szczerba et al. Citation2006; Warchol et al. Citation2001] and A-kinase anchoring protein-4 (AKAP4) gene is expressed only in the spermatid stage [Brown et al. Citation2003]. PRM1 and PRM2 expression were regarded as markers indicative of the presence of early spermatid and mature sperm [Matzuk and Lamb Citation2008]. β-actin was used as a housekeeping gene. All primers were chosen from different exons to eliminate possible contamination by genomic DNA (). For positive control reactions, cDNA was synthesized from testicular tissues of the azoospermic men containing mature sperm and all stages of spermatogenic cells. Positive and negative (water) controls were included in all PCR amplifications. PCR products were run on a 1.5% agarose gel, subsequently stained by ethidium bromide and visualized by UV transillumination [Lambard et al. Citation2004; Richter et al. Citation1999].
TESE and Histopathological Examination
TESE was performed [Friedler et al. Citation1997] and biopsied testicular tissues of azoospermic men were prepared for histopathological examination. Paraffin-embedded sections of the testicular tissues were dewaxed, rehydrated, and stained with hematoxylin and eosin [Aarabi et al. Citation2006; Song et al. Citation2000]. Results from histopathological evaluation fell into five categories: (1) sertoli cell only-syndrome (germ cell aplasia), (2) spermatogonial maturation arrest (MA), (3) spermatocyte MA, (4) spermatid MA, and (5) hypospermatogenesis.
Hormonal Analysis
All NOA patients underwent hormonal (LH, FSH, testosterone) assessment using a two-site immunoluminometric method (LIAISON, Italy) according to the manufacturer's manual. The patients hormone concentrations were categorized into high, normal, and low levels according to the hormone's reference ranges (Reference ranges for LH and FSH are 2-10 and 2-15 mIU/ml and for testosterone, 2-10 ng/ml).
Statistical Analysis
The data were statistically analyzed, using SPSS v.13.0. Kappa coefficient was used for agreement analysis between molecular and pathological results. Sensitivity and specificity for each molecular marker (gene transcript) was determined according to corresponding pathological results (Gold standard test). Chi-square test was used for the analysis of correlation between molecular results and presence or absence of sperm in testicular tissue samples. Correlation of molecular and hormonal results was analyzed in each sample using the independent-sample T test.
Abbreviations
| NOA: | = | non-obstructive azoospermia |
| RNA: | = | ribonucleic acid |
| cDNA: | = | complementary deoxyribonucleic acid |
| RT-PCR: | = | reverse transcriptase polymerase chain reaction |
| ART: | = | assisted reproductive technology |
| ICSI: | = | intracytoplasmic sperm injection |
| mTESE: | = | microdissection testicular sperm extraction |
| ARI: | = | Avicenna Research Institute |
| PBS: | = | phosphate buffered saline |
| DAZ: | = | deleted in azoospermia |
| AKAP4: | = | A-kinase anchoring protein-4 |
| PRM1: | = | protamine 1 |
| PRM2: | = | protamine 2 |
| MA: | = | maturation arrest |
| OA: | = | obstructive azoospermia. |
Acknowledgments
The authors would like to thank Dr. Hojjat Zeraati (Ph.D.) for his assistance in the statistical analysis of the paper. They are also grateful to Dr. S. Behnam Hashemi (M.D., M.P.H.) for editing the manuscript.
Declaration of Interest: This study was financially supported by grant No. 1424-33 from the Academic Center for Education, Culture and Research (ACECR). The authors had no conflict of interest in doing the research.There are no conflicts of interest.
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