特发性无精症和严重少精症患者Y染色体微缺失的分子检测

目的 :研究特发性无精症和严重少精症患者与 Y染色体微缺失的关系 ,建立无精症和严重少精症患者 Y染色体微缺失的分子检测方法。方法 :应用 PCR技术对 1 0 0例无精症和严重少精症患者 (其中无精症 72例 ,严重少精症 2 8例 )进行 Y染色体 AZFa、AZFb、AZFc/DAZ、SRY的微缺失检测。结果 :1 2例患者 (1 2 % )有 AZFc的微缺失 (其中无精症 8例 ,占 1 1 .1 % ;严重少精症 4例 ,占 1 4.3% ) ,且其中 1例无精症患者为 AZFb、AZFc双重缺失 ;所有病例未发现有 AZFa的缺失 ;SRY基因 PCR扩增均为阳性。6 0例已有生育的正常男性均无 AZFa、AZFb、AZFc、SRY微缺失。结论 :Y染色体微缺失 ,特别是 AZFc/DAZ的缺失是引起无精和严重少精、造成男性不育的重要原因之一 ,在进行遗传咨询和行卵细胞质内注入精子术 (ICSI)时 ,有必要对不明原因的不育男性患者进行 Y染色体微缺失的分子检测     

Study of Microdeletions in the Y Chromosome of Infertile Men with Idiopathic Oligo- or Azoospermia

Purpose: To determine the relationships between idiopathic oligo- or azoospermia and microdeletions of the Y chromosome. Methods: Eighteen Y-linked sequence-tagged sites (STSs) in AZF (Azoospermia Factor) region were screened by means of multiplex PCR (Polymerase Chain Reaction) in 50 idiopathic infertile men, including 16 patients with azoospermia, 13 severe oligospermia, and 21 oligospermia. Results: Microdeletions in the genomic DNA were observed in 8 of 50 cases, 3 with azoospermia, 1 severe oligospermia, and 4 oligospermia. Total deletion rate was 16.0% (8/50). The deletion regions were concentrated on AZFd and AZFc. Conclusions: Microdeletions of the Y chromosome are an important cause for idiopathic oligo- or azoospermia. Multiplex PCR is a useful technique for detecting the microdeletions. To avoid transmission to their offspring, patients with idiopathic oligo- or azoospermia should be screened for microdeletions of the Y chromosome before ICSI treatment for infertility.     

Genetic screening for chromosomal abnormalities and Y chromosome microdeletions in Chinese infertile men

Purposes

To investigate the frequency and type of both chromosomal abnormalities and Y chromosome microdeletions and analyze their association with defective spermatogenesis in Chinese infertile men.

Methods

This is a single center study. Karyotyping using G-banding and screening for Y chromosome microdeletion by multiplex polymerase chain reactio(PCR)were performed in 200 controls and 1,333 infertile men, including 945 patients with non-obstructive azoospermia and 388 patients with severe oligozoospermia.

Results

Out of 1,333 infertile patients, 154(11.55%) presented chromosomal abnormalities. Of these, 139 of 945 (14.71%) were from the azoospermic and 15 of 388 (3.87%) from the severe oligozoospermic patient groups. The incidence of sex chromosomal abnormalities in men with azoospermia was 11.53% compared with 1.03% in men with severe oligozoospermia (P < 0.01). Also 144 of 1,333(10.80%) patients presented Y chromosome microdeletions. The incidence of azoospermia factor(AZF) microdeletion was 11.75% and 8.51% in patients with azoospermia and severe oligozoospermia respectively. Deletion of AZFc was the most common and deletions in AZFa or AZFab or AZFabc were found in azoospermic men. In addition, 34 patients had chromosomal abnormalities among the 144 patients with Y chromosome microdeletions. No chromosomal abnormality and microdeletion in AZF region were detected in controls.

Conclusions

There was a high incidence (19.80%) of chromosomal abnormalities and Y chromosomal microdeletions in Chinese infertile males with azoospermia or severe oligozoospermia. These findings strongly suggest that genetic screening should be advised to infertile men before starting assisted reproductive treatments.     

Molecular and cytogenetic studies of 101 infertile men with microdeletions of Y chromosome in 1,306 infertile Korean men

Objectives

To determine the prevalence of Y chromosome microdeletions in infertile Korean men with abnormal sperm counts and to assess the clinical features and frequency of chromosomal abnormalities in Korean patients with microdeletions.

Methods

A total of 1,306 infertile men were screened for Y chromosome microdeletions, and 101 of them had microdeletions. These 101 men were then retrospectively studied for cytogenetic evaluation, testicular biopsy and outcomes of IVF and ICSI.

Results

The overall prevalence of Y chromosome microdeletions in infertile men was 7.7 % (101/1,306). Most microdeletions were in the AZFc region (87.1 %), including deletions of AZFbc (24.7 %) and AZFabc (8.9 %). All patients with AZFa, AZFbc and AZFabc deletions had azoospermia, whereas patients with an AZFc deletion usually had low levels of sperm in the ejaculate or in the testis tissues. Chromosomal studies were performed in 99 men with microdeletions, 36 (36.4 %) of whom had chromosomal abnormalities. Among the infertile men with Y chromosome microdeletions in this study, the incidence of chromosomal abnormality was 48.6 % in the azoospermic group and 3.7 % in the oligozoospermic group. Among the 69 patients with microdeletions and available histological results, 100.0 % of the azoospermic group and 85.7 % of the oligozoospermic group had histological abnormalities. The frequency of both chromosomal abnormalities and histological abnormalities was higher in the azoospermic group compared to the oligozoospermic group. Thirty-four ICSI cycles with either testicular (n = 14) or ejaculated spermatozoa (n = 20) were performed in 23 couples with men with AZFc microdeletion. Thirteen clinical pregnancies (39.4 %) were obtained, leading to the birth of 13 babies.

Conclusions

The study results revealed a close relationship between microdeletions and spermatogenesis, although IVF outcome was not significantly affected by the presence of the AZFc microdeletion. Nevertheless, Y chromosome microdeletions have the potential risk of being transmitted from infertile fathers to their offspring by ICSI. Therefore, before using ICSI in infertile patients with severe spermatogenic defects, careful evaluations of chromosomal abnormalities and Y chromosome microdeletions screening should be performed and genetic counseling should be provided before IVF-ET.     

Azoospermia factor and male infertility

Recently, work has shown that azoospermia factor (AZF) microdeletions result from homologous recombination between almost identical blocks in this gene region. These microdeletions in the Y chromosome are a common molecular genetic cause of spermatogenetic failure leading to male infertility. After completion of the sequencing of the Y chromosome, the classical definition of AZFa, AZFb, and AZFc was modified to five regions, namely AZFa, P5/proximal-P1, P5/distal-P1, P4/distal-P1, and AZFc, as a result of the determination of Y chromosomal structure. Moreover, partial AZFc deletions have also been reported, resulting from recombination in their sub-ampliconic identical pair sequences. These deletions are also implicated in a possible association with Y chromosome haplogroups. In this review, we address Y chromosomal complexity and the modified categories of the AZF deletions. Recognition of the association of Y deletions with male infertility has implications for the diagnosis, treatment, and genetic counseling of infertile men, in particular candidates for intracytoplasmic sperm injection.     

Role of the AZFd locus in spermatogenesis

To determine the prevalence of Y-chromosome microdeletions among infertile men and to correlate the clinical presentation of the men with specific deletions, microdeletion analysis in 53 infertile men (30 nonobstructive azoospermic, 23 severely oligozoospermic patients), and 100 age-matched, fathered normospermic men who had fathered children was performed by the multiplex PCR with 18 different Y-chromosome-specific STS primer sets, spanning the AZFa, AZFb, AZFd, and AZFc regions. Detection of the same locus deletion of the AZFd region in three cases indicated the possible importance of the genes located in this region in spermatogenesis.     

Y染色体基因微缺失与男性不育的关系

目的 :探讨男性不育患者尤其是特发性无精子症、严重少精子症及双侧输精管缺如与 Y染色体基因 (无精子因子 ,AZF)微缺失的关系。方法 :对 97例男性不育患者及 2 0例正常男性采用多重聚合酶链反应法进行基因微缺失检测。结果 :36例特发性无精症患者中存在 3例缺失 ,占 8.33% ;1 4例双侧输精管缺如患者存在 2例缺失 ,占 1 4.2 9% ;2 7例严重少精症患者中存在 2例缺失 ,占 7.41 %。2 0例精子数正常的男性不育患者及 2 0例正常男性对照无 AZF缺失。缺失以 AZFa,AZFc区为主 ,AZFb区无缺失。结论 :Y染色体 AZF微缺失可能是导致男性特发性无精症、少精症的原因之一 ,双侧输精管缺如患者也存在 Y染色体的基因微缺失     

The Y chromosome

The Y chromosome has evolved to provide sex determination in mammals. In association with its evolution, genes important for spermatogenesis have been sequestered on this chromosome. Further, X chromosome inactivation has developed as a mechanism to prevent over-expression of genetic factors important for somatic function in females, with maintenance of their activity in males. The multi-repeat organization of the Y chromosome and limited regions of crossover with other chromosomes predisposes it to internal recombination and loss of genes that may be important for spermatogenesis. Y chromosome microdeletion testing of infertile men with non-obstructive azoospermia provides prognostic information useful for management of these patients. In the presence of a complete deletion of the azoospermic factor a (AZFa) or AZFb regions, sperm retrieval is highly unlikely. Recent advances in our understanding of the organization and function of the Y chromosome are likely to enhance further the role of the Y chromosome in normal spermatogenesis and fertility.     

Y染色体微缺失与辅助生殖技术关系的研究进展

Y染色体是男性特有的染色体,其长臂上的无精子因子(AZF)区域具与男性不育密切相关的基因,目前将该区域分为AZFa、AZFb、AZFc和AZFd4个区域。AZF缺失是导致男性不育的重要因素之一,可以通过辅助生殖技术(ART)遗传给下一代引起不育。研究Y染色体微缺失分类与表型关系,可以为临床治疗各种男性不育症提供分子或细胞水平的依据。Y染色体微缺失发生频率存在种族差异性;目前Y染色体微缺失的检测方法仍然以多重PCR为主;对于ICSI助孕的男性后代是否会出现新发Y染色体微缺失仍然存在争论。     

Microarray detection of Y chromosome deletions associated with male infertility

Array-comparative genomic hybridization (CGH) has emerged as a powerful new molecular tool for the high-resolution analysis of copy-number variation and breakpoint analysis. In this study, array-CGH was used to analyse known Yq deletions associated with male infertility. A microarray platform encompassing probes for chromosomes 13, 14, 21, X and Y was developed in-house and was used to detect different Yq deletion types. The successful application of this array for the detection of Yq deletions involving either the AZFb or AZFc region was demonstrated. Partial and complete AZF deletions were correctly detected in 13 patients with Yq deletions previously identified by multiplex polymerase chain reaction (PCR). This study demonstrates that array-CGH may be an alternative approach to multiplex PCR for the diagnosis of known Yq deletions and potentially a useful tool for the discovery of other Y chromosome deletions/polymorphisms associated with defective spermatogenesis.     

Useful marker for the estimation of a recombination pair in the partial azoospermia factor c (gr/gr) deletion using quantitative real-time polymerase chain reaction

Background and aims:  Azoospermia factor c (AZFc) microdeletions are associated with male infertility and are caused by intrachromosal recombination between homologous repetitive sequence segments. Partial AZFc deletion (gr/gr) has been reported in male factor infertility. In the present study, we established detecting the copy number using quantitative real-time polymerase chain reaction (qRT-PCR) with the genome DNA, and assessed the association of the recombination pair set of gr/gr deletion and deleted in azoospermia copies. Furthermore, we determined the clinical significance of differential recombination patterns of gr/gr deletion, and compared them with azoospermia and proven fertile volunteers, with both groups having gr/gr deleted Japanese subjects.
Materials and methods:  A total of 16 Japanese subjects with idiopathic azoospermia, and 13 proven fertile men with gr/gr deletion, were studied. qRT-PCR was used for the estimation of an identical site number.
Results:  The g1/g2 deletion was found in 69.2% (9/13) in proven fertile men and in 75% (12/16) of idiopathic infertile men. The gr/gr deletion could result in the recombination of g1/g2 segments. Furthermore, there was no difference in the position of deletion between azoospermic patients and controls ( P  = 0.59).
Conclusion:  There was no association between the loss of DAZ cluster and azoospermia in gr/gr deletion. This suggests that most of the partial deletions are neutral variants.     

Non-chimerism and chimerism pseudo dicentric Y chromosome: two case reports about azoospermia and cytogenetic/molecular genetic analysis in the Chinese population

Objective

To report the cytogenetic and molecular genetic analysis of the first two cases of non-chimerism and chimerism karyotype in Chinese male patients who suffer from azoospermia, which may be caused by pseudo dicentric Y chromosomes.

Design

Case study.

Setting

Academic reproductive medicine center.

Patients

Two male patients with azoospermia, carrying pseudo dicentric Y chromosome.

Interventions

Review the records of inquiry, testicular biopsy, pathological examination, semen routine examination, endocrine evaluation, cytogenetic chromosomal analysis, and FISH detection of peripheral blood to evaluate Y chromosome deletion.

Main outcome measures

To investigate the possible association among pseudo dicentric Y, chimeric status and azoospermia.

Results

Two patients were both diagnosed with azoospermia by a variety of andrology inspections. Further chromosomal analysis and FISH indicated their pseudo dicentric Y chromosome and different chimerism status. PCR confirmed simultaneous deletions of AZFb and AZFc regions in the Y chromosome of both patients.

Conclusions

Pseudodicentric Y chromosome affecting the long arm may lead to a male phenotype by duplicating the sex-determining region of Y chromosome (SRY) fragment and chimeric status may further impact patient's hormone levels, which obstruct spermatogenesis. However, the deletion of the azoospermia factor (AZF) is likely the key factor that causes azoospermia.     

Male infertility and microdeletions of the Y chromosome

It is estimated that about 10% of men suffer from male infertility. Male infertility is associated with a reduction in the quantity, reduced mobility or abnormal morphology of sperm. In about 50-60% of cases the etiology can be identified. When the cause is unknown, it is referred to as idiopathic infertility. A genetic cause is suspected in some of the latter cases since chromosome anomalies and familial forms of male infertility have been reported. Three different regions of the Y chromosome, termed AZFa, AZFb and AZFc are recurrently deleted in about 15% of cases of idiopathic azoospermia or severe oilgozoospermia. AZFc deletions form the majority of these deletions. The presence of a Y microdeletion does not seem to alter the fertilisation of the oocyte or the development of the embryo. However, if the child is a boy, he will inherit the deletion from his father and will most likely be infertile when he is an adult. In the absence of any other information concerning an association between Y chromosome microdeletions and other development anomalies of the child, in genetic counselling the principal risk for male offspring appears to be infertility.     

A novel, rapid, and accurate method for detecting microdeletion involving the DAZ gene in infertile men

OBJECTIVE: To report on a novel, accurate method for detecting microdeletion involving the DAZ gene in infertile men. DESIGN: Retrospective clinical study. SETTING: University Infertility Center of Cochin Hospital, Paris, France. PATIENT(S): Infertile patients (n = 25) consulting our infertility department during 1998. The patient cohort included subjects with nonobstructive azoospermia and oligoasthenospermia. INTERVENTION(S): Blood samples were collected from each subject. MAIN OUTCOME MEASURE(S): DNA analysis using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). RESULT(S): We used a new molecular genetic strategy to rapidly identify deletions of the Y chromosome that include the DAZ locus. The experiment consists of amplifying simultaneously exon 4 of the DAZ and DAZLA genes with the use of specific primers that are complementary to intronic sequences of these genes. DGGE was used to separate the two PCR products, with good resolution. In infertile men with a microdeletion of the DAZ gene, this method allows amplification of an internal control when a deletion of that portion of the Yq chromosome is observed on a single amplification. CONCLUSION(S): This PCR-DGGE method for detection of DAZ gene deletion is simple and fast and does not require the use of radioactive elements. Compared with the classic PCR approach, this new method allows the amplification of the DAZLA copy to be used as an effective internal control in infertile men with microdeletion of the DAZ locus. This procedure could be particularly useful in screening for the DAZ locus in the diagnostic workup of nonobstructive azoospermia and severe oligoasthenoteratozoospermia.     

Molecular and cytogenetic investigation of Y chromosome deletions over three generations facilitated by intracytoplasmic sperm injection

BACKGROUND: The azoospermic factor (AZF) region is critical for normal spermatogenesis since microdeletions and partial deletions have been associated with infertility. We investigate the diagnostic ability of karyotyping in detecting clinically relevant Y chromosome deletions. The clinical significance of heterochromatin deletions, microdeletions and partial AZFc deletions is also evaluated. METHODS: A patient with a Yq deletion, affected by severe oligoasthenoteratozoospermia, underwent intracytoplasmic sperm injection (ICSI) which resulted in the birth of a healthy baby boy. The patient, his father and his son underwent Y chromosome microdeletion and partial AZFc deletion screening. We also studied the aneuploidy rate in the sperm of the patient by fluorescent in situ hybridization. RESULTS: AZF microdeletions were absent in the family. However, microdeletion analysis confirmed that the Yq deletion was limited to the heterochromatin. We found a partial AZFc gr/gr deletion in all three family members. We observed an increased rate of sex chromosome aneuploidy in the infertile patient. CONCLUSIONS: Cytogenetic analysis was misleading in identifying the Yq breakpoint. Infertility observed in the patient was associated with the gr/gr partial deletion. However, because of the incomplete penetrance of gr/gr deletions, the consequence of the vertical transmission of the deletion through ICSI remains unknown.     

Prenatal diagnosis of two rare de novo structural aberrations of the Y chromosome: cytogenetic and molecular analysis.

Two rare de novo structural aberrations of the Y chromosome were detected during routine prenatal diagnosis: a satellited non-fluorescent Y chromosome (Yqs), the first de novo Yqs to be reported in a fetus, and a terminal deletion of the Y chromosome long arm del(Y)(q11). In both cases detailed cytogenetic and molecular analyses were undertaken. In the case of the Yqs it was demonstrated by fluorescence in situ hybridization (FISH) that the satellites were derived from chromosome 15. In the case of the del(Yq), it was shown with molecular analysis by polymerase chain reaction (PCR) amplification of sequence-tagged sites (STS-PCR) that the deleted portion of the long arm of chromosome Y included the azoospermia factor loci, AZFb and AZFc. The clinical significance of these findings is discussed.     

男性不育中Y染色体微缺失的研究进展

近年来对男性不育的遗传学因素的广泛研究显示Y染色体微缺失是导致不同程度生精障碍从而引起男性不育的第二大遗传学病因。无精子症因子区(AZF区)由近至远包含3个不同的亚区:AZFa、AZFb和AZFc,不同缺失类型的表型不同。目前常采用PCR法进行Y染色体微缺失的检测,其缺点是准确度低、特异性差、耗时。而基因芯片技术虽能克服上述缺点,但目前成本过高。通过检测能预测患者男性后代的遗传风险,有助于患者选择辅助治疗的方式。虽然Y染色体微缺失的严重不育患者能通过辅助生殖技术成功获得后代,但有可能将遗传缺陷传给男性后代,使之获得相同的Yq微缺失和不育。     

Microdeletions within the azoospermia factor subregions of the Y chromosome in patients with idiopathic azoospermia.

OBJECTIVE: To examine the patterns of submicroscopic DNA deletions in the AZF (AZoospermia Factor) subregions of the Y chromosome in patients with idiopathic azoospermia. DESIGN: Controlled clinical study. SETTING: University-based infertility clinic. PATIENT(S): Infertile men (n = 40) with nonobstructive, idiopathic azoospermia. The control group consisted of proven fathers (n = 14) and healthy women (n = 4). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Semen analysis; polymerase chain reaction amplification of the 37 loci spanning the AZFa, AZFb, and AZFc subregions of the Y chromosome; serum FSH, LH, and testosterone levels; and testicular histologic analysis. RESULT(S): Testicular histologic analysis of the subjects revealed Sertoli cell-only syndrome (n = 36) and spermatogenic arrest (n = 4). Microdeletions of the Y chromosome were found in eight (20%) of the patients with azoospermia. All eight affected patients had interstitial microdeletions within the AZFc subregion. Patients with Sertoli cell-only syndrome had additional microdeletions in regions distal to DAZ (Deleted in Azoospermia), although DAZ deletion was observed in seven of the eight affected patients. In five patients, microdeletions were found in the AZFb region containing RBM (RNA Binding Motif). CONCLUSION(S): Our results add to the evidence supporting the current suggestion that there is a cause-and-effect relation between Yq11 microdeletions in the AZF region and azoospermia.     

Cytogenetic and Y chromosome microdeletion screening studies in infertile males with Oligozoospermia and Azoospermia in Southeast Turkey

Purpose

In view of the genetic risks for the next generation, the importance of careful evaluation of karyotypes and AZF microdeletions in male infertility prior to assisted reproduction is evident. In the present study, it is aimed to investigate the frequency and types of both major chromosomal abnormalities by using standard cytogenetic methods and Y chromosome microdeletions of infertile males with azoospermia and oligozoospermia to give appropriate genetic counseling before assisted reproduction techniques in southeast Turkey.

Methods

A total of 80 infertile males (52 were azoospermic, 25 oligospermic and 3 asthenospermic) were studied for the cytogenetic evaluation and molecular AZF screening program prior to use of assisted reproduction techniques. A detailed history was taken for each man. Karyotyping was performed on peripheral blood lymphocytes according to standard methods. Polymerase chain reaction (PCR) amplification by using 15 Y-specific sequence-tagged sites of AZF region was performed to screen the microdeletions in the AZF region of Y chromosome.

Results

Of 80 cases, 71 had normal karyotype (46,XY). The total prevalence of chromosomal abnormalities was found to be 11.2% (9/80), including seven patients with Klinefelter syndromes and two patients with balanced autosomal rearrangements. All of the patients with Klinefelter Syndrome had azoospermia, but carriers with translocation had oligospermia. The deletions of Y chromosome were seen in one patient (1.3%) with features of normal karyotype and azoospermia. Microdeletions were seen in the AZFc and AZFd regions. Neither AZFa nor AZFb microdeletions were detected.

Conclusions

The occurrence of chromosomal anomalies and Y chromosome microdeletions among infertile males strongly suggests the need for routine genetic testing and counseling prior to employment of assisted reproduction techniques.