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- Original Article -

The prevalence of azoospermia factor microdeletion on the Y chromosome of Chinese infertile men detected by multi-analyte suspension array technology

Yi-Jian Zhu, Si-Yao Liu, Huan Wang, Ping Wei, Xian-Ping Ding

Institute of Medical Genetics, Key Laboratory of Bio-resources and Eco-environment, Ministry of Education, College of Life Science, Sichuan University, Chengdu 610064, China

Abstract

Aim: To develop a high-throughput multiplex, fast and simple assay to scan azoospermia factor (AZF) region microdeletions on the Y chromosome and establish the prevalence of Y chromosomal microdeletions in Chinese infertile males with azoospermia or oligozoospermia. Methods: In total, 178 infertile patients with azoospermia (non-obstructed), 134 infertile patients with oligozoospermia as well as 40 fertile man controls were included in the present study. The samples were screened for AZF microdeletion using optimized multi-analyte suspension array (MASA) technology. Results: Of the 312 patients, 36 (11.5%) were found to have deletions in the AZF region. The microdeletion frequency was 14% (25/178) in the azoospermia group and 8.2% (11/134) in the oligospermia group. Among 36 patients with microdeletions, 19 had deletions in the AZFc region, seven had deletions in AZFa and six had deletions in AZFb. In addition, four patients had both AZFb and AZFc deletions. No deletion in the AZF region was found in the 40 fertile controls. Conclusion: There is a high prevalence of Y chromosomal microdeletions in Chinese infertile males with azoospermia or oligozoospermia. The MASA technology, which has been established in the present study, provides a sensitive and high-throughput method for detecting the deletion of the Y chromosome. And the results suggest that genetic screening should be advised to infertile men before starting assisted reproductive treatments. (Asian J Androl 2008 Nov; 10: 873_881)

Keywords: Y chromosome microdeletion; azoospermia factor; male infertility; multi-analyte suspension array (MASA)

Correspondence to: Prof. Xian-Ping Ding, Institute of Medical Genetics, College of Life Science, Sichuan University, Chengdu 610064, China.
Tel: +86-28-8541-3096 Fax: +86-28-8541-5895
E-mail: brainding@263.net
Received 2008-03-16 Accepted 2008-06-01

DOI: 10.1111/j.1745-7262.2008.00436.x

1 Introduction

Infertility, which is defined as an inability to conceive or produce an offspring, is affecting about 15% of all the couples attempting to generate pregnancy. In the case of infertility, approximately 50% of the cases can be attributed to male factors [1]. It is reported that nearly 20% of all infertile men have idiopathic azoospermia or oligozoospermia (< 20 × 10⁶ spermatozoa/mL). Deletions of the azoospermia factor (AZF) of the Y chromosome are associated with severe spermatogenic failure and represent the most frequent molecular genetic cause of azoospermia and severe oligozoospermia [2]. These microdeletions were mapped to three non-overlapping regions, named as AZFa, AZFb, and AZFc, at Yq11.22-23 of the Y chromosome. Different candidate genes had been identified in AZF loci. In these cases, the molecular extensions of the most commen AZF microdeletions are already known as they result from intrachromosomal recombinant mechanisms and the gene content is also established, but the function of these genes is poorly understood.

Recently, infertility treatment has been performed by intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) techniques. However, deletions on the Y chromosome might be spread to the male offspring, causing the persistence of infertility problems in the next generations. Therefore, it is necessary for infertile men with azoospermia and severe oligozoospermia to have been screened for Y chromosome microdeletions before ICSI/IVF.

Currently, the most common method used to detect microdeletions of AZF on the Y chromosome is based on multiplex polymerase chain reaction (PCR) and conventional agarose electrophoresis. However, there are some limits to the application of the conventional methods, mainly owing to their low specificity, inaccuracy and time-consuming procedure [3]. So a more precise methodology is necessary to efficiently pursue diagnostic studies for male infertility. Here we present a novel and rapid method to scan for AZF region microdeletions of the Y chromosome in Chinese infertile males with azoospermia or oligozoospermia using multi-analyte suspension array (MASA) technology.

The molecular test developed uses Luminex (Luminex Corporation, San Diego, CA, USA) xMAP technology, a flow cytometer that allows simultaneous identification of the AZF microdeletion by mixing different sets of microspheres that contain specific capture probes derived from target sequence-tagged site (STS) markers in the AZF region. This technology permits the simultaneous detection of 100 analytes by combining 100 different sets of microspheres in a single reaction. Since each microsphere set is internally dyed with two spectral fluorochromes of different intensities, their unique spectral emissions are recognized by a red laser. On hybridization, the biotinylated amplicon bound to the surface of the microsphere is recognized by a green laser that quantifies the fluorescence of the reporter molecule (streptavidin R-phycoerythrin) [4]. Therefore, with this method, seven STS markers, including sY84 for AZFa, sY127 and sY134 for AZFb, sY254 and sY255 for AZFc, sex-determining region Y (SRY or sY14) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene can be detected simultaneously in a single well.

2 Materials and methods

2.1 Patients and sample collection

In total, 312 infertile men with a normal 46,XY karyotype including 178 with azoospermia (non-obstructed) and 134 with severe oligozoospermia (semen count less than 5 × 10⁶/mL, non-obstructed) aged from 16 to 48 years were recruited from the Affiliate Hospital of Sichuan Genitalia Hygiene Research Center (Chengdu, China) between June 2005 and August 2006. All patients underwent semen analysis at least twice. In addition, 40 fertile age-matched men with a 46,XY karyotype and one normal woman with a 46,XX karyotype were selected as controls. Informed consent was required from each subject to participate in the present study.

2.2 DNA isolation

Genomic DNA was isolated from peripheral blood lymphocytes by phenol-chloroform extraction.

2.3 Primers selection and multiplex PCR

Primers covering only hot spot regions were chosen according to previous reports [5_7]. A series of five STS from the AZF region on the long arm of the Y chromosome were used to detect microdeletions. These STS included sY84 for AZFa, sY127 and sY134 for AZFb, sY254 and sY255 for AZFc. In addition, testing for the Yp was performed using sY14, an STS located within the gene SRY (internal positive control) (Table 1). Fertile male and female samples were used as positive and negative controls. The GAPDH gene was used to detect the validity of the templates. Seven pairs of primers were amplified in a single reaction.

Multiplex PCR amplifications were carried out in a total volume of 25 µL buffered solution containing about 200 ng of genomic DNA, 800 µmol/L deoxyribonucleotide triphosphates (dNTP), 1.5 mmol/L Mg²⁺, 10 pmol/L of each primer and 2.5 U Taq polymerase. The cycling conditions were as follows: 94ºC for 10 min followed by 94ºC for 30 s, 55ºC for 30 s and 72ºC for 45 s for 35 cycles, with a final extension at 72ºC for 5 min.

2.4 Coupling of oligonucleotide probes

The 5' amino-modifier C-12-linked oligonucleotide probes (Table 2) corresponding to seven STS, respectively, were coupled to carboxylated beads by a carbodiimide-based coupling procedure. For each combination of probe and bead set, 2.5 million carboxylated beads were suspended in 25 µL of 0.1 mol/L 2-(N-morpholino) ethane-sulfonic acid (MES), pH 4.5. Probe oligonucleotides (400 pmol) and 200 µg of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (EDC) were added and thoroughly mixed with the beads. Incubation was performed in the dark under agitation for 30 min and was interrupted by a thorough mixing step after 15 min. The addition of EDC and incubation steps were repeated twice, and the coupled beads were finally washed once with 0.5 mL of 0.02% Tween-20 and once with 0.5 mL of 0.1% sodium dodecyl sulfate before being stored in 100 µL Tris-EDTA buffer at 4ºC in the dark.

2.5 Hybridization assay and bead analysis

Following PCR amplification, 2.5 µL of each reaction mixture was transferred to a tube containing 33 µL of 1.5 × tetramethylammonium chloride (TMAC) hybridization solution and a mixture of 2000 probe-coupled beads of each set. TE buffer (14.5 µL) was added, followed by gentle mixing with a pipette. The mixture was heated to 95ºC for 10 min. Then hybridization was performed at 55ºC for 15 min. Subsequently, 25 µL of 10 µg/mL streptavidin-R-phycoerythrin diluted with 1 × TMAC hybridization solution was added to each tube and mixed. The mixture was incubated at 55ºC for 5 min. Beads were analyzed for internal bead color and R-phycoerythrin reporter fluorescence on a Luminex 100 analyzer. The median reporter fluorescence intensity (MFI) of at least 100 beads was computed for each bead set in the sample.

3 Results

3.1 Assay optimization

Final assay conditions used for the present study were established after systematic variation of the following parameters: coupling procedure of probes to beads (EDC and probe input); temperature, salt concentration and hybridization conditions; Strep-PE concentration; incubation time for staining; concentrations of different blocking substances in the washing buffer; and number of wash cycles and wash buffer composition (see Materials and methods).

3.1.1 Optimization of hybridization time

To identify the optimum hybridization time for multiplex PCR products with bead-conjugated specific probes, we incubated samples of the mixture for 10, 15, 20, 25 and 30 min in 55ºC. The results (Figure 1) at sequential time points indicated that 15 and 20 min showed similar optimal levels of MFI for the detection of microdeletions. Therefore, we have chosen the earlier time point (15 min) for the detection of microdeletion assay.

3.1.2 Optimization of single and multiplex PCR detection

Single (one set of primer) and multiplex PCR (five mixed sets of primer) were performed using the same STS marker primers (SRY, GAPDH, sY84, sY127, sY134, sY254 and sY255).

In a comparison between single and multiplex detection MFI, the MFI of five STS markers (SRY, GAPDH, sY84, sY127, sY134, sY254 and sY255) in single detection were slightly higher than those in multiplex detection (Figure 2). However, there is no deterrent in multiplex detection that prevents it from detecting distinct deletions or presence of STS markers on the Yq chromosome, and this form of detection provides the means by which to measure multiple analytes simul-taneously, potentially saving time and use of expensive resources. Therefore, we have decided to pursue the multiplex PCR detection system for Yq microdeletion analysis in infertile males.

3.1.3 Verifying the reproducibility of the assay

To verify the reproducibility of the hybridization assay, multiplex PCR products were used in the experiments. These products were hybridized to the probe-coupled bead mix. Every experiment was repeated five times, and the results were recorded and analyzed by the software SPSS version 11.0 (SPSS, Chicago, IL, USA) and Origin 6.0 (Microcal Software, Northampton, MA, USA). The results are shown in Table 3.

3.1.4 Determining the specificity of the assay

To determine the specificity of the assay, seven probes (SRY, GAPDH, sY84, sY117, sY134, sY254 and sY255) were coupled individually to define bead sets (7-plex) and hybridized to 2.5 µL of singleplex and multiplex PCR products, respectively. The results showed that the assay was highly specific (Table 4).

3.1.5 Analysis of data validity

Microdeletion can be easily measured by comparing the MFI score with the MFI scores of positive and negative controls. From multiplex bead array screening, we determined a cut-off value of 100 MFI for the STS.

3.2 Application of the assay in detecting male infertility

Screening for AZF microdeletions by multiplex PCR-MASA was performed in a total of 312 patients, including 178 infertile patients with azoospermia and 134 infertile patients with oligozoospermia who had a normal karyotype as well as 40 fertile man controls. As shown in Table 5, 36 (11.5%) of 312 patients were found to have deletions in the AZF region. The microdeletion frequency was 14% (25/178) in the azoospermic group and 8.2% (11/134) in the oligospermic group. Among 36 patients with microdeletions, 19 had deletions in the AZFc region, seven had deletions in AZFa and six had deletions in AZFb. In addition, four patients had both AZFb and AZFc deletions. No deletion in the AZF region was found in the 40 fertile controls. Some examples are shown in Figure 3.

To determine the validity of the MASA results, all samples were detected by classic PCR and identified by polyacrylamide gel electrophoresis (PAGE). The results were coincident with those detected by MASA technology.

4 Discussion

Although multiplex PCR is the most frequently used method in the detection of Y chromosome microdeletions, many problems remain in its practical application such as the specificity, sensitivity and throughput of the method. In general, the multiplex PCR method has a maximum of 1_2 h of running time for the separation of multiple bands, especially when the sizes of PCR products are close to each other. In addition, analysis of multiplex PCR products on an agarose gel based on molecular size and intensity is sometimes complicated when there are non-specific products if the primers for each STS are not correct. So the result is often dependent on the experience of the investigator. In contrast, there are some benefits of suspension array technology, including rapid data acquisition, excellent sensitivity and specificity and multiplexed analysis capability. The MASA system incorporates 5.6 µm polystyrene microspheres that are internally dyed with two spectrally distinct fluorochromes. Using precise amounts of each of these fluorochromes, an array is created consisting of 100 different microsphere sets with specific spectral addresses. Each microsphere set can possess a different reactant on its surface. Because microsphere sets can be distinguished by their spectral addresses, they can be combined, allowing up to 100 different analytes to be measured simultaneously in a single reaction vessel. Also, the MFI of at least 100 beads is computed for each bead set in the sample, which means that each sample is detected at least 100 times. Therefore, this MASA technology might give more accurate results than gel electrophoresis analysis because of the sequence-specific hybridizations with numerical values. Additional benefits of the assay were a significant decrease in labor and turnaround time, flexibility to allow testing of 1 to 96 reactions without increase in labor time or cost per isolate, and it was less technically demanding. The cost per well (per patient) in reagents and consumables (DNA isolation, PCR, array microspheres, plasticware, etc.) is no more than USD4. And the detection can be completed in less than 30 min. This compares favourably with other commercially available AZF assays by gel analysis.

The basic components of nucleic acid detection methods are assay chemistry and analysis platform. Characteristic genotyping technologies include both solid phase (gels, DNA chips, glass slide arrays) and homogeneous solution assay formats (mass spectrometry, capillary electrophoresis). As compared to planar microarrays, suspension arrays have the benefits of convenience, low cost, statistical superiority, faster hybridization kinetics and more flexibility in array preparation. MASA technology is being used in a variety of applications, such as single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, human leukocyte antigen (HLA) DNA typing and microbial detection [4, 8].

Since 1976, when Tiepolo et al. [9] recognized that deletion in the long arm of the Y chromosome is associated with spermatogenic failure, there have been numerous studies on the association of AZF microdeletions with male infertility. The spermatogenesis locus AZF in Yq11 has been mapped to three non-overlapping regions designated as AZFa, AZFb, and AZFc. Many genes on the Y chromosome have been identified. It is currently accepted that AZFa contains two genes (USP9Y and DBY), AZFb contains eight protein-coding genes (CDY2, EIF1AY, HSFY, PRY, RBMYL1, RPS4YS, SMCY and XKRY) and AZFc contains five such genes (BPY2, CDY1, CSPG4LY, DAZ and GOLGA2LY), which are all transcribed in testicular tissue and, therefore, are all candidate genes for some functions in human spermatogenesis [10]. AZF microdeletions are caused by intrachro-mosomal recombination events between large homologous repetitive sequence blocks [11], and AZFc microdeletions are now recognised as the most frequently known genetic lesion causing male infertility.

Y chromosome microdeletions have been of increasing interest to clinicians and scientists since ICSI was introduced to be the main treatment option for severe male factor infertility. The frequency of deletions was reported to be in the range of 0.7% to 34.5%, with an average frequency of 8.2% [1, 2]. In the present study, the frequency of AZF microdeletions was 11.5% (40/312) in infertile patients with azoospermia or oligozoospermia, which is little higher than the average value. Our data also revealed that there was a high prevalence rate of AZF microdeletion in severe oligozoospermic patients (8.2%) as well as in azoospermic patients (14%), which is not in agreement with some studies considering the low prevalence of AZF microdeletion in severe oligozoospermia [12_15]. These differences might be explained by selection criteria of the patients, methodological aspects, population/ethnic variances, particular Y chromosome haplotypes, genetic background and environmental influences. The microdeletions reported by other investigators in China [6] and other countries such as Turkey, France and Holland vary from our observations, because China is a country with a lot of minority groups. The discrepancy could also be related to the selection of patient groups with varied clinical criteria and the set of markers used.

In the present study, microdeletions in the AZFc region were the most prevalent (52.8%), followed by the AZFa (19.4%), AZFb (16.7%) and AZFb/AZFc combination (11.1%). However, the frequency of AZFa deletions was higher than other reports [16_18] and AZFa deletions were detected both in the patients with azoospermia and oligozoospermia. Although deletions occurring in AZFa are mostly associated with Sertoli cell syndrome [19], oligozoospermia in our patients with AZFa deletions was not surprising. Although it has been reported that only complete AZFa deletion is associated with the absence of spermatozoa, there have been cases of spermatozoa retrieval with partial AZFa deletions [2, 19]. Furthermore, sY84 has been previously considered as a polymorphic locus of the Y chromosome that is not associated with sterility phenotypes in men [20]. More recently, the presence of sY84 false-positive results has been related to an alteration in a PCR primer of this STS marker [21]. In the research of Buch et al. [22], there was a patient, previously diagnosed in another laboratory as AZFa microdeleted (absence of sY84 marker), who was checked using their real time PCR protocal. No microdeletions were observed in this patient using selected markers from the AZFa locus (sY81, sY82 and sY182). Increasing STS density with two additional AZFa STS markers, flanking sY84 (DYS388 and sY745), failed to identify any AZFa locus alteration in the patient. So, in the beginning of our study, we planned to employ sY86 as the second STS marker for AZFa evaluation. Unfortunately, we did not find any probes specific to sY86, considering the conditions of multiplex PCR with another six STS primers. In addition, the results of the MASA could be confirmed by classic PCR analysis and polyacrylamide gel electrophoresis, including sY86. Of the seven samples with sY84 microdeletion detected by MASA, all were sY84 microdeletions and four of them were sY86 detected by PAGE.

To make the MASA more useful, we will optimize the STS marker and employ other STS markers that could help to detect false positives related to the sY84 marker.

In the present study, the deletion of AZFc was the most common AZF microdeletion in patients with azoospermia and oligozoospermia. The reason why the deletion of AZFc is more frequent is still not clear. One possible explanation is a repetitive sequence of the genes. Some candidate genes, like DAZ, are known to be repetitive on the Y chromosome. Infertility may be caused by the loss of the repetitive DAZ gene clusters [23, 24].

There are some familiarities between the research of Yeoma et al. [25] and our paper. Both studies used the same technology to detect AZF microdeletions. But there are still differences between their paper and ours. First, we employed different fluorochrome-labeling methods for the multiplex bead array system. In their paper, they used Cy3-labeled reverse primers to perform the PCR reaction. Instead, we employed the conventional streptavidin-R-phycoerythrin method. Although the Cy3 method can reduce the number of experimental steps, the conventional streptavidin_R-phycoerythrin method is five times higher in fluorescence intensity than Cy3 fluorescence intensity. Streptavidin-R-phycoerythrin is also more stable than Cy3. Also, we aimed to establish a high throughput sensitive method for detecting the deletion of the Y chromosome that is specific for Chinese infertile men. So we selected different STS markers and employed different probes based on the characteristic of the Chinese population. Although our methods are similar, the parameters of reaction are different owing to different primers and probes.

In conclusion, we developed a novel method to scan for AZF region microdeletions on the Y chromosome. Further, our data, based on a Chinese population, add to the evidence that there is a cause and effect relationship between Y chromosome microdeletions and azoospermia or oligozoospermia. It is suggested that the screening of Y chromosome microdeletion should be conducted in infertile men with azoospermia and oligozoospermia before ICSI/IVF.

Acknowledgment

This work was supported by the funds of Population and Family Planning Commission of Chongqing Municipality, China (No. 2001-06).

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