Messenger RNA transcripts of the meiotic regulator BOULE in the testis of azoospermic men and their application in predicting the success of sperm retrieval

Yung Ming Lin1,5, Pao Lin Kuo2, Ying Hung Lin3, Yen Ni Teng4 and Johnny Shinn Nan Lin1

Departments of 1 Urology, 2 Obstetrics & Gynecology and 3 Institute of Basic Medical Science, National Cheng Kung University, College of Medicine, Tainan, Taiwan and Department of 4 Early Childhood Education and Nursery, Chia Nan University of Pharmacy and Science, Tainan, Taiwan

5 To whom correspondence should be addressed at: Department of Urology, National Cheng Kung University Hospital, 138 Sheng-Li Road, Tainan, Taiwan 704. Email: linym{at}mail.ncku.edu.tw


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Testicular sperm retrieval can lead to paternity for azoospermic patients with spermatogenic failure. The human BOULE gene, a meiotic regulator of germ cells, is a gene whose altered expression may be associated with sterility. We determined the levels of BOULE transcripts in the testes of azoospermic patients, and evaluated the relationship between BOULE transcript levels and patients' testicular phenotypes, clinical parameters and sperm retrieval results. METHODS and RESULTS: BOULE transcript levels in the testes of 41 azoospermic patients were examined by quantitative competitive-reverse transcription–polymerase chain reaction. A significant decrease in BOULE transcript levels was detected in patients with spermatogenic failure, and BOULE transcript levels progressively decreased with increasing severity of testicular failure. BOULE transcript levels did not correlate with the serum hormone parameters measured. Significantly higher BOULE transcript levels were detected in 19 patients with successful sperm retrieval than in 12 patients with failed sperm retrieval. When using a cut-off value of 0.5 for BOULE transcript ratio to predict the success of sperm retrieval, both the sensitivity and specificity value were 100%. CONCLUSIONS: We suggest the BOULE transcript plays an important role in human spermatogenesis and that the levels may predict the presence of testicular sperm in patients with spermatogenic failure.

Key words: azoospermia/BOULE/testis


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Since the advent of intracytoplasmic sperm injection (ICSI), many infertile men have been able to father offspring with a very limited number of sperm. In patients with azoospermia caused by testicular failure (non-obstructive azoospermia), testicular sperm extraction (TESE) plus ICSI is now considered the only way to fertilize oocytes and achieve a pregnancy. However, the presence of mature sperm at TESE in patients with non-obstructive azoospermia is variable, it was estimated that only 40–60% of patients would have successful TESE (Silber et al., 1995Go; Tournaye et al., 1997Go). Given that TESE and ICSI are invasive procedures for both the man and the woman, the identification of a parameter to predict the presence of sperm production in those patients would be of considerable value.

The human BOULE gene, a germ cell-specific cell cycle regulator, was recently identified and mapped to chromosome 2 (Xu et al., 2001Go). BOULE belongs to the DAZ (Deleted in AZoospermia) gene family, which consists of DAZ on the Y chromosome and DAZL (DAZ-Like) on chromosome 3. It is believed that the BOULE gene is highly conserved from flies to humans. Human BOULE and fly boule share 80% similarity in RNA binding domains and 42% similarity in protein sequence (Xu et al., 2001Go). In the fly, boule is required for completion of meiosis in male germ cells (Castrillon et al., 1993Go; Eberhart et al., 1996Go). Mutation of boule resulted in germ cell meiotic arrest and infertility; in addition, human BOULE could rescue this meiotic defect in infertile flies (Xu et al., 2003Go). Therefore, the human BOULE is considered to have a similar function to the fly boule, as a meiosis regulator.

In humans, BOULE is expressed exclusively in the male germline, and the BOULE protein can be detected mainly in the cytoplasm of the meiotic spermatocytes (Xu et al., 2001Go; Luetjens et al., 2004Go). The unique BOULE protein expression pattern strongly suggests that human BOULE is involved in the progression of cell cycles during meiosis. As meiosis is the key process for haploid germ cell production, it is tempting to speculate that the amount of BOULE transcripts correlates with the amount of sperm production as well as testicular phenotypes. To gain a better understanding of the role of BOULE in human spermatogenesis, we determined the BOULE mRNA transcript levels in the testes of azoospermic patients, and evaluated the relationship between BOULE mRNA transcript levels and patients' testicular phenotypes, clinical parameters as well as the results of sperm retrieval.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Patients
This study recruited 31 patients with non-obstructive azoospermia and 10 patients with obstructive azoospermia and normal spermatogenesis. Informed consent was obtained from all patients enrolled in the study. The study was approved by The National Scientific Council of Taiwan and the Institutional Review Board of National Cheng Kung University Medical Center. All patients had normal chromosome karyotypes and intact Y chromosome genes. The diagnosis of azoospermia was based on at least two separate semen analyses, two separate centrifuged semen sample analyses (3000 g, 15 min), detailed physical examination, endocrine profile testing, including luteinizing hormone (LH), follicular-stimulating hormone (FSH), prolactin and testosterone, and testicular biopsy and/or vaso-vesiculography. Serum levels of FSH, LH, prolactin and testosterone were measured using the commercial radioimmunoassay kits: Coat-A-Count FSH IRMA, Coat-A-Count LH IRMA, Coat-A-Count PRL IRMA and IMMULITE Total Testosterone (Diagnostic Products Corp., Los Angeles, CA). The intra-assay and inter-assay precision coefficients of variation were 2.4% and 4% for FSH, 1.2% and 2.2% for LH, 1.9% and 2.4% for PRL, and 6.7% and 7.7% for testosterone.

Testicular samples
For paternity purposes, the patients underwent sperm retrieval, either by microsurgical epididymal sperm aspiration (MESA) or TESE, and ICSI. Before sperm retrieval, these patients were asked to provide a small piece of testicular tissue (diameter 5 mm) for further study. For men undergoing MESA, an additional testicular incision was performed for sampling on the same side as the MESA procedure. For men undergoing TESE, a tiny sample was obtained from the pooling of testicular tissues. One third of the testicular tissue volume obtained was immersed in Bouin's solution and sent for histopathological diagnosis. This process allowed us to examine more than 100 cross-sections of seminiferous tubule. The diagnosis of the testicular histopathology was confirmed by two specialists, and was categorized according to the most advanced pattern of spermatogenesis present. The remaining two thirds of the tissue volume was cryopreserved for RNA extraction. Following sperm retrieval, positive sperm retrieval was defined as the presence of spermatozoa on wet preparation of the testicular tissues under an inverted microscopic examination (400x magnification).

Primer design
Primers specific for the human BOULE gene and the housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were designed. The nucleotide sequences spanning different exons were identified in GenBank using accession number BC023632 for GAPDH and accession number AF272858 for BOULE (National Center for Biotechnology Information). Another two sets of competitive reverse primers for BOULE and GAPDH were designed, based on the same nucleotide sequences and an additional short sequence upstream of the target cDNA incorporated into the 5' ends of the original reverse primers. The primer sequences, positions on the cDNAs, and expected sizes of the PCR products are listed in Table I.


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Table I. Oligonucleotide primers for BOULE mRNA amplification

 
RNA extraction and synthesis of cDNA by reverse transcription
The testicular tissue samples were stored under liquid nitrogen, using 2-methylbutane as cryoprotectant, until use. Before isolation of total cellular RNA, each specimen was sliced into 10 µm-thick pieces. Total cellular RNA was extracted from the tissue using standard methods (High PureTM RNA Tissue Kit, Boehringer Mannheim, Indianapolis, IN), and quantified by measuring total absorbance at 260 nm. One microgram of RNA was treated with RNase-free DNase (Qiagen, Valencia, CA) to remove contaminating genomic DNA. For the synthesis of complementary DNA (cDNA), 12 µl aliquots of master mixture containing 2 µl of RNA, 1 µl of 500 ng/µl oligo(dT)12–18 primer (Gibco/BRL, Grand Island, NY) and 9 µl of diethylpyrocarbonate-treated water were heated to 70°C for 10 min and put on ice. Reverse transcription (RT) reactions were performed in 20-µl aliquots containing master mixture, 4 µl of 5x first strand synthesis buffer, 0.1 M dithiothreitol, 10 mM of each dNTP, and 200 Units of SuperscriptTM II RNase H reverse transcriptase (Gibco/BRL). The RT temperature profile used was 42°C for 1 h, 75°C for 15 min, and final cooling to 4°C. Aliquots of the cDNA were stored at –20°C until use.

Quantitative competitive polymerase chain reaction (QC–PCR)
For QC–PCR, we synthesized wild and competitor RNA templates for internal standards. For the synthesis of internal standards, RNA extracted from the testicular tissue with normal spermatogenesis was subjected to RT–PCR. The products were separated by polyacrylamide gel electrophoresis using 1x TBE buffer (90 mM Tris–HCl, 90 mM boric acid, 1 mM EDTA, pH 8) at 110 V for 30–50 min. After initial confirmation by gel electrophoresis, competitor and wild PCR products were purified using the ConcertTM Rapid PCR Purification System, (Gibco/BRL) and subcloned into a pT7Blue T-vector (Novagen, Madison, WI). The inserts were confirmed by DNA sequencing using an automatic sequencer (ABI 377, Applied Biosystems/PE, Foster City, CA). The cDNA products were quantified by measuring the absorbance at 260 nm. To determine the equivalence of testicular cDNA and competitor cDNA, serial dilutions of competitor cDNA were co-amplified with testicular cDNA from tissue exhibiting normal spermatogenesis. The point at which the two lines crossed indicated the amount of competitor cDNA that should be added for subsequent standard curve construction and QC–PCR reaction. To produce the standard curve, a fixed amount of competitor cDNA was co-amplified with serial dilutions of wild cDNA, and the logarithm of the ratio of wild to competitor cDNA was plotted against the logarithm of the initial amounts of wild cDNA. The standard curves were highly reproducible and the R2 values for the GAPDH and BOULE standard curves were 0.9966 and 0.993, respectively (Figure 1).



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Figure 1. Standard QC–PCR curves generated for GAPDH (A) and BOULE (B). Increasing amounts (0.5, 1, 2, 4, 8, 16, 32 and 64 amol) of wild cDNA were coamplified with 2 amol of competitive cDNA for 30 cycles and separated on a polyacrylamide gel (upper panels). The intensities of the ratios of wild cDNA to competitor cDNA generated from these reaction mixtures were determined. The logs of the ratios of wild to competitor product density versus the logs of the amounts of initial wild cDNA added to the PCR reactions are shown in the graphs (lower panels).

 
The QC-PCR reaction consisted of 1 µl of competitor cDNA, 1 µl of cDNA from testicular samples and 23 µl of PCR-Mastermix containing 10x PCR buffer minus Mg+2, 1.5 mM MgCl2 solution, 0.2 mM of dNTP, 2.5 U Taq polymerase, corresponding paired primers, and water. The PCR temperature profile was 30 cycles of amplification (94°C for 1 min, 57°C for 1 min, and 72°C for 1.5 min), and a final extension at 72°C for 15 min. The PCR products were separated by polyacrylamide gel electrophoresis (5% for GAPDH, 8% for BOULE), stained with ethidium bromide, and analyzed by alpha-image (Alpha Innotech, San Leandro, CA). Ratios were calculated for the intensity of wild versus competitor bands for each lane of the gels. Amounts of transcripts were calculated by interpolation as previously described (Tsai and Wiltbank, 1996Go). The mRNA levels for GAPDH were expressed as the number of copies per nanogram of total RNA. The steady-state concentrations of mRNA for BOULE in each testicular sample were normalized to the amount of GAPDH gene (BOULE/GAPDH, transcript ratio).

Data analysis
We used GraphPad Prism 4 statistical software (GraphPad Software, San Diego, CA) for data analysis. The copy numbers of GAPDH and transcript ratios of BOULE in five histological groups (normal spermatogenesis, hypospermatogenesis, maturation arrest at spermatid stage, maturation arrest at spermatocyte stage and Sertoli cell-only syndrome) were presented as the mean±SEM. Transcript differences in patients with normal spermatogenesis and patients with spermatogenic failure (i.e. hypospermatogenesis, maturation arrest and Sertoli cell-only syndrome) were analyzed using an unpaired Student's t-test. Transcript differences or transcript ratios in patients with varying degrees of spermatogenic failure were analyzed using the Kruskal–Wallis test, and multiple pairwise comparisons were performed using Dunnett's test. Pearson product moment correlation coefficients were calculated to determine the correlation between BOULE transcript ratios and clinical parameters, i.e. serum FSH, LH, prolactin and testosterone level. Receiver operating characteristic (ROC) curve analysis of BOULE transcript ratios was used to determine the cut-off value that maximized the sensitivity and specificity for distinguishing between patients with successful sperm retrieval from those without. A P-value <0.05 was considered significant. Where appropriate, Bonferroni's method was used to correct significance levels for multiple testing of data.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Clinical variables of the patients
Of 10 patients with obstructive azoospermia and normal spermatogenesis, there were four patients with failed vasectomy reversal, one patient with failed vaso-epididymostomy, three patients with bilateral epididymal obstruction, and two patients with bilateral hypoplasia of the seminal vesicle. Neither of these two patients had congenital bilateral absence of vas deferens, nor genetic disorders. Testicular tissues were obtained by testicular biopsy (coinciding with MESA) in six of these 10 patients and by TESE in the other four patients. Sperm were successfully retrieved from all 10 patients. Of the 31 patients with non-obstructive azoospermia, the histopathological data indicated hypospermatogenesis in 13 patients, maturation arrest at the spermatocyte stage in two patients, maturation arrest at the spermatid stage in two patients, and Sertoli cell-only syndrome in 14 patients. Sperm were successfully retrieved from all 13 patients with hypospermatogenesis, two patients with maturation arrest at the spermatid stage and four of the 14 patients with Sertoli cell-only syndrome. One urologist, Y.M.Lin, performed all testicular biopsy, MESA and TESE procedures. The clinical characteristics of the study populations are summarized for each histopathological group in Table II.


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Table II. Clinical characteristics of the histopathological groups

 
Comparison of GAPDH mRNA transcript levels between patients with normal spermatogenesis and spermatogenic failure
The GAPDH mRNA transcript levels ranged from 11.8 to 45.6 x 103 copies/ng RNA (mean±SEM: 21.7±3.2 x 103 copies/ng RNA) for men with normal spermatogenesis, and from 6.2 to 67.9 x 103 copies/ng RNA (mean±SEM: 22.6±2.5 x 103 copies/ng RNA) for men with spermatogenic failure. No significant difference was detected between these two groups (P=0.83, unpaired Student's t-test). No significant difference in GAPDH mRNA transcript levels was detected between the groups when the patients were sub-divided into five histopathological groups (P=0.7201, Kruskal–Wallis test; Figure 2A).



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Figure 2. Transcripts of GAPDH mRNA (A) and mRNA transcript ratios of BOULE/GAPDH (B) in different testicular histological groups. For GAPDH, no significant difference was found among the groups (P=0.7201). For BOULE, significant differences were found between the five groups (P=0.0004). After multiple pairwise comparisons, significant differences were noted between the normal spermatogenesis group and the Sertoli cell-only group (P<0.01), and between the hypospermatogenesis group and the Sertoli cell-only group (P<0.01). Histology abbreviations: NR, normal spermatogenesis; HS, hypospermatogenesis; MA, maturation arrest; SCOS, Sertoli cell-only syndrome.

 
Comparison of BOULE mRNA transcript ratios between patients with normal spermatogenesis and spermatogenic failure
The BOULE mRNA transcript ratios ranged from 0.72 to 3.48 (mean±SEM: 1.85 x 0.3) for men with normal spermatogenesis, and from 0.09 to 3.2 (mean±SEM: 1.0 x 0.2) for men with spermatogenic failure. A significant decrease in BOULE mRNA transcript ratios was detected in the spermatogenic failure group (P=0.008, unpaired Student's t-test). Sub-dividing our patients into five groups according to the severity of testicular histopathology produced a progressive decrease in the BOULE mRNA transcript ratios (P=0.0004, Kruskal–Wallis test; Figure 2B). Pairwise comparisons of BOULE mRNA transcript ratios among the five groups revealed that there were significant differences between the normal spermatogenesis group and the Sertoli cell-only group (P<0.01), and between the hypospermatogenesis group and the Sertoli cell-only group (P<0.01).

BOULE mRNA transcript ratios and hormonal parameters
Figure 3 shows the correlation between BOULE mRNA transcript ratios and hormonal parameters. The correlation between BOULE mRNA transcript ratios and FSH levels (r=–0.267), BOULE mRNA transcript ratios and LH levels (r=–0.014), BOULE mRNA transcript ratios and prolactin levels (r=–0.203), and BOULE mRNA transcript ratios and testosterone levels (r=0.155) were low and categorized as not significant (P=0.104, 0.475, 0.341, and 0.234, respectively).



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Figure 3. Correlation between the serum levels of FSH (A), LH (B), testosterone (C), prolactin (D) and the BOULE mRNA transcript ratios. The r-value and significance value are shown on each graph.

 
BOULE mRNA transcript ratios and the results of sperm retrieval in patients with spermatogenic failure
We divided the 31 patients with spermatogenic failure into two groups based on the presence or absence of mature sperm recovered from TESE. The BOULE mRNA transcript ratios ranged from 0.69 to 3.2 (mean±SEM: 1.54 x 0.7) for 19 patients with successful sperm retrieval, and 0.09 to 0.3 (mean±SEM: 0.18 x 0.02) for 12 patients with failed sperm retrieval. A significant difference was noted between these two groups (P<0.0001, unpaired Student's t-test). We hypothesize that there may be a threshold level of BOULE mRNA transcripts required for completion of meiosis and that this level must be exceeded to produce intra-testicular sperm. The ROC curve analysis (Figure 4A) of BOULE mRNA transcript ratios indicates that the threshold that gave the maximum true-positive fraction (sensitivity) and false-positive fraction (1–specificity) was 0.5. At this cut-off value, the sensitivity and specificity for predicting the presence of sperm in patients with spermatogenic failure are 100% (Figure 4B). The calculated area under the curve is 1, thus demonstrating that the predictive value is perfect.



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Figure 4. ROC curve of BOULE mRNA transcript ratios (A) The fraction of true-positive results and false-positive results are shown. This analysis showed that successful sperm retrieval could be predicted with a sensitivity of 100% and specificity of 100% using a BOULE mRNA transcript ratio cutoff of 0.5 (B) The calculated area under the curve was 1, and the 95% confidence interval was 0.8235–1.0. Sperm (+) represents presence of sperm during sperm retrieval; sperm (–) represents absence of sperm during sperm retrieval.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, we report that BOULE mRNA transcript ratios were decreased in patients with spermatogenic failure, and these transcript ratios did not have a significant correlation with hormonal parameters. These results imply that BOULE, indeed, plays a role in sperm production but is not involved in the hormonal regulation pathway of spermatogenesis. A recent study indicated that BOULE protein expression was lacking in patients with complete meiotic arrest. This loss of BOULE protein expression resulted in the inactivation of maturation promoting factor, a factor required for meiosis completion, by down-regulation of CDC25A phosphatase. This halted the spermatogenic process at the spermatocyte stage (Luetjens et al., 2004Go). In this study, BOULE mRNA expression has been shown to be in agreement with BOULE protein expression. We demonstrated that two patients displayed meiotic arrest at the spermatocyte stage; both patients had low BOULE mRNA transcript ratios (0.09 and 0.29, respectively) and failed sperm retrieval. In contrast, the two patients who had successful sperm retrieval displayed meiotic arrest at the spermatid stage and had BOULE mRNA transcript ratios of 1.11 and 2.29, respectively. Although we do not have anti-BOULE antibody to demonstrate the loss of BOULE protein expression in those two patients with low BOULE transcript levels, their testicular phenotype and negative sperm retrieval results strengthen our assertion that the BOULE gene plays a key role in meiosis completion.

It is also possible that meiosis can be regulated by other factors acting upstream or downstream of BOULE. In Drosophia, Cdc25 is considered to be a general target for BOULE protein (Xu et al., 2003Go), and sa (spermatocyte arrest) and mia (meiosis I arrest) might act upstream of boule (Maines and Wasserman, 1999Go). A mutation in Cdc25 completely blocks meiosis in the male fly (Courtot et al., 1992Go), and mutations in sa and mia have been shown to result in the failure of BOULE protein to accumulate normally in the fly (Maines and Wasserman, 1999Go). Although, to date, human homologs of sa and mia have not been identified, further studies will be performed to identify the molecules upstream of the human BOULE–CDC25–CDC2/cyclin A (B) pathway, and to determine whether defects in components upstream or downstream of BOULE are associated with specific patient phenotypes.

In this study, BOULE mRNA transcripts were significantly decreased in patients with spermatogenic failure, especially in the Sertoli cell-only group. This result is not surprising given the germ-line specificity of BOULE. Higher BOULE mRNA transcript levels reflect increased numbers of germ cells entering the meiosis processes, and lower transcript levels may simply reflect a general loss of germ cells. However, it is interesting to note that BOULE mRNA transcripts were also detectable in all Sertoli cell-only specimens. Possible explanations for this include the expression of BOULE in the nucleus or cytoplasm of some type of non-germ lineage in the testis. It is possible that the BOULE protein level is too low to be detected by immunostaining or it is difficult to identify due to its nuclear location. A similar phenomenon occurred with other members of DAZ gene family. DAZ and DAZL mRNAs have been detected by RT–PCR in patients with a diagnosis of Sertoli cell-only syndrome (Kuo et al., 2004Go). To resolve this question, laser-capture microdissection, in combination with RT–PCR or western blotting analysis, may be useful. A second explanation for the detection of BOULE mRNA transcripts in Sertoli cell-only specimens is that these specimens may contain some germ cell foci, i.e. ‘incomplete’ Sertoli cell-only (Devroey et al., 1995Go; Silber et al., 1996Go). There have been many reports of patients with a Sertoli cell-only diagnosis who have undergone successful TESE (Silber et al., 1995Go; Tournaye et al., 1997Go). We believe that our histological diagnosis of Sertoli cell-only is correct because there were generally >100 seminiferous tubule cross-sections available for examination in each patient, the advanced category system was applied, and the histological diagnoses were confirmed by two experienced specialists. Our results also indicate the limited predictability of histological diagnosis at TESE. A third explanation for the detectable BOULE expression in these patients might be due to sample contamination. However, we believe that the presence of BOULE mRNA transcripts is not due to tissue contamination because BOULE mRNA transcripts were invariably detected in all 14 Sertoli cell-only specimens and DNase was routinely added before each cDNA synthesis.

In the present study, BOULE mRNA transcripts have been shown to correlate with the results of TESE. Similar predictive value tests have been evaluated for the other members of the DAZ gene family (DAZ and DAZL), but less predictive value was noted. Using a certain mRNA transcript level as a cut-off value, the sensitivity and specificity were 82.8% and 100%, respectively, for DAZ (Kuo et al., 2004Go), and 78.1% and 94%, respectively, for DAZL (our unpublished data). These divergent predictive values for the DAZ family might be explained by their distinct expression patterns. DAZL and DAZ are expressed mainly from germ-line stem cells to meiotic spermatocytes, whereas BOULE protein exists in the cytoplasm of pachytene spermatocytes, and persists through meiosis to round spermatids (Xu et al., 2001Go). The timeline for BOULE expression may be the reason for its higher predictive value for sperm production.

Although a number of studies have attempted to test the predictive powers of variable parameters, clinically, testicular samples for quantitative and/or qualitative evaluations are currently used to predict the presence of sperm at TESE. Qualitative measurement based on the most advanced pattern of spermatogenesis was 52–58% accurate (Su et al., 1999Go; Seo and Ko, 2001Go), and a positive result for quantitative measurement of mature spermatids predicted an 85% chance for subsequent success with TESE (Silber et al., 1997Go). In this study, BOULE mRNA transcripts have been shown to yield a predictive power of 100%, therefore, we believe that the measurement of BOULE mRNA transcripts may potentially be useful for predicting the presence of sperm in testis. Conversely, it is also noted that the sample size is limited in the present study, and the possibility exists that more samples may reduce the 100% predictive value reported.

Given that diagnostic testicular biopsy is less invasive than TESE, we recommend diagnostic testicular biopsy before attempting TESE for patients with non-obstructive azoospermia. A tiny piece of testicular tissue is sufficient for both histopathological examination and QC–PCR to detect BOULE mRNA transcripts. If we fail to identify spermatozoa, despite detecting high BOULE transcript levels from a tiny piece of biopsied sample, we hypothesize that it may be worthwhile to try TESE for sperm retrieval. Alternatively, if we fail to identify spermatozoa and the BOULE transcript levels are low in the biopsied sample, it may not be worthwhile to try more invasive TESE procedures. The BOULE transcript levels would also provide technicians in the ART laboratories with useful information to guide further attempts to search for sperm. This information will be highly valuable for counselling infertile couples before TESE–ICSI procedures. It would help avoid unnecessary, invasive TESE for men, and decrease the risks of potential complications from assisted reproductive techniques for women. Furthermore, we believe that a less invasive, more cost-effective, and outpatient-based method of diagnostic testicular sampling is necessary. A study is now ongoing to test the feasibility of applying this BOULE mRNA transcript assay to the testicular sample from a fine needle aspirate.

Because the testicular histology may be highly variable throughout the testis, it is possible that the biopsied sample may contain foci without germ cells. In this case, the BOULE transcript levels would be low. The same tissue may also contain other foci with many germ cells, which would give rise to high BOULE transcript levels. Although our QC–PCR assay has been shown to be highly predictable for successful sperm retrieval, the reliability of BOULE transcript levels obtained from diagnostic testicular biopsy should be further evaluated in future.

In summary, we have determined that BOULE mRNA transcript levels are significantly decreased in patients with spermatogenic failure and these mRNA transcript levels correlate with the success of sperm retrieval. Because BOULE is an important regulator of the human meiotic cell cycle, we believe that the level of BOULE mRNA transcript in the testis of azoospermic patients is informative. Using a cut-off value of 0.5 for the BOULE mRNA transcript ratio, the predictability for the success of sperm retrieval is ideal. Therefore, we recommend a prior diagnostic testicular biopsy, evaluating both testicular histology and BOULE mRNA transcript levels, for all couples attempting TESE–ICSI.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was sponsored by research grants NSC 91-2314-B-006-149 and NSC 91-3112-B-006-008, NSC 92-3112-B-006-002, and NSC 93-3112-B-006-004 from the National Science Council of Taiwan.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on July 28, 2004; accepted on November 11, 2004.