Detection of sperm in men with Y chromosome microdeletions of the AZFa,AZFb and AZFc regions

BACKGROUND: Y chromosome microdeletions are associated with severe male factor infertility. In this study, the success rate of testicular sperm retrieval was determined for men with deletions of AZF regions a, b or c. METHODS: AZF deletions were detected by PCR of 30 sequence-tagged sites within Yq emphasizing the AZFa, b and c regions. Semen analysis and diagnostic testis biopsy or testicular sperm extraction (TESE) findings were correlated with the specific AZF region deleted. RESULTS: A total of 78 men with AZF deletions included three with AZFa deletion, 11 with AZFb, 42 with AZFc, 16 with AZFb+c and six with Yq (AZFa+b+c). All men with AZFa, AZFb, AZFb+c and Yq deletions were azoospermic and no sperm were found with TESE or biopsy. Of men with isolated AZFc deletion, sperm were found in 75% (9/12) by TESE and 45% (9/20) on biopsy (56% overall); 62% (26/42) were azoospermic and 38% (16/42) severely oligozoospermic. A total of 7 patients with deletion patterns that included the complete AZFa region and 23 that included the complete AZFb region who underwent TESE or biopsy did not have sperm detected by these surgical measures. CONCLUSIONS: Microdeletion of the entire AZFa or AZFb regions of the Y chromosome portends an exceptionally poor prognosis for sperm retrieval, whereas the majority of men with AZFc deletion have sperm within the semen or testes available for use in IVF/ICSI.     

AZFa deletions in Sertoli cell-only syndrome: a retrospective study

Lack of data on the genotype-phenotype relationship in cases of AZF microdeletions is due to the limited number of histological investigations in human male infertility cases. We investigated the possibility of retrospective detection of Yq11 microdeletions by using DNA extracted from diagnostic testicular biopsies. We used histological criteria to select two series of material: 22 biopsies with Sertoli cell-only syndrome and 14 biopsies with maturation arrest at the spermatocyte I stage. Two markers, DFFRY and DAZ, were tested by nested polymerase chain reaction (PCR) in the two series. In the Sertoli cell-only syndrome series, we found four deletions affecting the DFFRY gene (18.2%). In the second series, no deletions were detected. Two conclusions may be considered, although the number of specimens analysed is limited: (i) the frequency of deletions observed in Sertoli cell-only syndrome allows us to suggest that deletion in the AZFa region may be involved in this pathology; and (ii) retrospective studies may yield some additional elements in our search for eventual genotype-phenotype relationships.     

Cytoplasmic granular change of Sertoli cells in two cases of Sertoli-cell-only syndrome

The testicular biopsy specimens of two cases of Sertoli-cell-only syndrome are described, in which some of the Sertoli cells are filled with coarse cytoplasmic eosinophilic granules. Histochemical stains suggest that the granules contain glycocompounds and phospholipids. On electron microscopy, the granules prove to be densely populated secondary lysosomes, containing membrane fragments, phospholipid-like materials, and other amorphous masses of various densities. Whether the excessive secondary lysosomes arise as a result of some metabolic block in the Sertoli cells, or merely represent autophagy in response to the absence of germ cells, cannot be determined. A search of the literature indicates that this is the first instance, to our knowledge, in which such changes are reported in Sertoli-cell-only syndrome.     

High frequency of XY disomy in spermatozoa of severe oligozoospermic men

Frequencies of disomy and diploidy in spermatozoa for chromosomes X, Y and 18 were compared among severe oligozoospermic men (<5x10(6) spermatozoa/ml), oligozoospermic men (5-20x10(6) spermatozoa/ml), and normospermic men using three-colour fluorescence in-situ hybridization (FISH). Semen samples were collected from 10 severe oligozoospermic men aged 26-49 years, 10 oligozoospermic men aged 27-48 years and seven normospermic men aged 25-31 years. Karyotypes in lymphocytes obtained from peripheral blood were all 46,XY. In severe oligozoospermic men, analysis of 200 interphases per individual using FISH showed XY constitutions for sex chromosomes in all cells. A minimum of 10 000 sperm nuclei per individual for each chromosome was evaluated in severe oligozoospermic men and oligozoospermic men, and a minimum of 6000 sperm nuclei per individual in normospermic men. In total, 245 707 sperm nuclei were evaluated. The hybridization efficiency was 99.8%. The severe oligozoospermic men showed significantly higher frequencies of XY disomy (0.41%) and diploidy (0.49%) compared with oligozoospermic men (0.16%, P < 0.01; 0.22%, P < 0.05) and normospermic men (0.18%, P < 0.05; 0.21%, P < 0.05) (Mann-Whitney U-test). The data suggest that when severe oligozoospermic men undergo intracytoplasmic sperm injection, there can be an increase in the rate of conceptuses with 47,XXY chromosomes.     

High frequency of defective sperm-zona pellucida interaction in oligozoospermic infertile men

BACKGROUND: The ability of sperm to interact with the zona pellucida (ZP) plays a critical role during the process of human fertilization. The aim of this study is to determine frequency of defective sperm-ZP interaction in oligozoospermic infertile men. METHODS: Sperm-ZP binding assays and the ZP-induced acrosome reaction (AR) were performed in 72 infertile men with a sperm concentration <20 x 10(6)/ml. Oocytes that had previously failed to fertilize in a clinical IVF programme were used for the tests. Motile sperm (2 x 10(6)/ml) selected by swim-up from each semen sample were incubated with four oocytes for 2 h. The number of sperm bound per ZP and the ZP-induced AR were assessed. Under these conditions, an average of < or =40 sperm bound/ZP was defined as low sperm-ZP binding and a ZP-induced AR < or =16% was defined as low ZP-induced AR. RESULTS: In the 72 oligozoospermic men, 28% (20/72) had low sperm-ZP binding. Of those with normal sperm-ZP binding, 69% (36/52) had low ZP-induced AR. Overall, 78% (56/72) had either low ZP-binding or normal ZP binding but low ZP-induced AR. This means that only 22% (16/72) had both normal sperm-ZP binding and normal ZP-induced AR. CONCLUSION: Oligozoospermic men have a very high frequency of defective sperm-ZP interaction, consistent with their low natural fertility or low fertilization rate in conventional IVF. Infertile couples with oligozoospermic semen should be treated by ICSI rather than by conventional IVF.     

Sertoli cell types in the Sertoli-cell-only syndrome: relationships between Sertoli cell morphology and aetiology

Histological study of testicular biopsies from infertile men showing Sertoli-cell-only tubules due to hypogonadotropic hypogonadism, cryptorchidism, oestrogen treatment, chemotherapy or Del Castillo's syndrome, revealed four types of Sertoli cells: (1) normal adult mature cells showing an indented nucleus, grossly triangular in shape with a prominent tripartite nucleolus; (2) immature cells with round regularly outlined nuclei and immature cytoplasm; (3) dysgenetic cells showing immature nuclei and a nearly mature cytoplasm with less developed cytoplasmic organelles; and (4) involuting cells with very irregularly outlined nuclei and a mature cytoplasm containing abundant lipid droplets and residual bodies and atypical inter-Sertoli junctional specializations. Testes from men with hypogonadotropic hypogonadism showed only immature Sertoli cells; cryptorchid testes showed dysgenetic cells and occasional normal cells; and after treatment with oestrogens or chemotherapy the testes showed involuting cells and normal cells. The testes of men with Del Castillo's syndrome could be classified into three groups, according to the Sertoli cell type present: mature, dysgenetic and involuting cells. This finding suggests that Del Castillo's syndrome may be due to at least three different aetiologies.     

Incipient germ cell tumor in Sertoli-cell-only syndrome testis, accompanied with retroperitoneal teratocarcinoma and widespread metastases

Incipient germ cell tumor in Sertoli-cell-only syndrome testis was examined in an autopsy case of retroperitoneal teratocarcinoma with widespread metastases. Although both testes of a 28-year-old man had clinically been small and free from tumor mass to palpation, histopathological examinations revealed a malignancy in the right testis with the appearance of Sertoli-cell-only syndrome. The left testis showed solely the histology of Sertoli-cell-only syndrome. The testicular malignancy consisted of undifferentiated, atypical germ cells mainly confined within approximately one-tenth of seminiferous tubules, and only one small cartilage nodule. Some tubules showed intratubular growth pattern suggestive of seminoma. A few syncytiotrophoblast-like giant cells occurred in the tubules. These findings seem to furnish substantial evidence to the concept that atypical germ cells are the origin of testicular germ cell tumors of different types.     

High frequency of tissue-specific mosaicism in Turner syndrome patients

Interphase fluorescent studies of X chromosome aneuploidy in cultured and uncultured blood lymphocytes and oral mucosa epithelial cells using X centromere-specific DNA probe in addition to standard karyotype analysis were performed in 50 females with a clinical suspicion of Turner syndrome. All the patients were previously screened for the presence of 'hidden' Y chromosome mosaicism, using the primers DYZ3 and DYZ. The use of fluorescence in situ hybridization (FISH) analysis of interphase nuclei of tissues from different germ layers (lymphocytes from mesoderm and buccal epithelial cells from ectoderm) improves the accuracy of detection of low-level mosaicism. FISH studies on interphase nuclei revealed that 29% of patients with a pure form of monosomy X detected by metaphase analysis are, in fact, mosaics. The level of cells with the normal chromosomal constitution in lymphocytes of these cases as a rule was low, ranging from 3 to 18%, with an average of 7%. Two false-positive cases and one false-negative case of X monosomy mosaicism determined by standard cytogenetic approach were detected using FISH analysis. The majority of patients (92%) with mosaic form of Turner syndrome have considerable tissue-specific differences in levels of X aneuploidy. Our data indicate that in cases when mosaic aneuploidy with low-level frequency is questionable (approximately 10% and lower), the results of standard metaphase analysis should be supplemented with additional FISH studies of interphase nuclei. Tissue-specific differences in contents of different cell lines in the same patients point to the necessity of studying more than one tissue from each patient.     

应用不依赖序列的单引物扩增技术获得人博卡病毒全基因序列

目的 研究通过不依赖序列的单引物扩增（Sequence independent single primeramplification,SISPA）技术获得腹泻标本中人博卡病毒的全基因序列.方法 筛检仅含有人博卡病毒的标本,经过滤、超速离心富集和核酸酶处理后提取病毒核酸,采用SISPA-PCR技术扩增病毒序列,克隆后测序,经比对分析获得人博卡病毒基因序列,再用DNAstar拼接成全基因序列.结果 获得了人博卡病毒4834bp的序列,仅两端少部分序列未得到.结论 SISPA-PCR技术是一种可直接从样品中获得病毒全基因序列的方法.     

一种导致完全性雄激素不敏感综合征的AR基因移码突变的鉴定

目的 对1个男性假两性畸形完全性雄激素不敏感综合征的家系雄激素受体(androgen receptor,AR)基因进行突变检测,并分析其致病原因.方法 用PCR扩增及DNA测序等技术分析男性假两性畸形先证者候选基因AR的外显子及外显子内含子接头序列,根据检测到的突变位点情况,检测患者及其家系其他成员的相应DNA区段的碱基序列.结果 先证者及其家庭成员共3例患者均为AR基因1910delA的移码突变.其母亲为AR基因突变杂合子,是此疾病的携带者.该突变导致AR基因的N637I(AAU→AUC)、L638*(CTG→TGA)改变,导致AR蛋白283个氨基酸的截短.正常人群未发现该移码突变,该突变尚未见文献报道.结论 基因水平确定了该家系为AR基因突变引起的完全性雄激素不敏感综合征男性假两性畸形家系,同时发现了1种AR基因病理性新突变.     

High frequency of sub-optimal semen quality in an unselected population of young men

Male reproductive function seems to have deteriorated considerably during the past 4-5 decades. However, studies of the reproductive function in unselected populations have not previously been reported. As the large majority of young men in Denmark are subjected to a compulsory medical examination for military service, this provided a unique opportunity to study the reproductive function in an unbiased population. Altogether 891 young men delivered a blood sample in which reproductive hormones were measured. From 708 of these men data were also obtained on semen quality and testis size. The median sperm concentration was 41 x 10(6)/ml (mean 57.4 x 10(6)/ml). Men with ejaculation abstinence above 48 h had slightly higher sperm concentrations (median 45 x10(6)/ml, mean 63.2 x 10(6)/ml), but even in this subgroup, 21 and 43% respectively had sperm counts below 20 x 10(6)/ml and 40 x 10(6)/ml. Among men with no history of reproductive diseases and a period of abstinence above 48 h, as many as 18 and 40% respectively had concentrations below 20 and 40 x 10(6)/ml. Sperm counts were positively correlated with testis size, percentage normal spermatozoa and inhibin B, and negatively correlated with percentage immotile spermatozoa and follicle stimulating hormone. Possible causes for this high frequency of young men with suboptimal semen quality are obscure and need to be explored. Whether these findings apply for young male populations of comparable countries remains to be seen.     

High frequency of BTG1 deletions in acute lymphoblastic leukemia in children with down syndrome

Previous cytogenetic studies of myeloid and acute lymphoblastic leukemias in children with Down syndrome (ML-DS and DS-ALL) have revealed significant differences in abnormality patterns between such cases and acute leukemias in general. Also, certain molecular genetic aberrations characterize DS-related leukemias, such as GATA1 mutations in ML-DS and deregulation of the CRLF2 gene in DS-ALL. Whether microdeletions/microduplications also vary between DS and non-DS cases is presently unclear. To address this issue, we performed single nucleotide polymorphism array analyses of eight pediatric ML-DS and 17 B-cell precursor DS-ALL. In the ML-DS cases, a total of 29 imbalances (20 gains and nine losses) and two partial uniparental isodisomies (pUPDs) were detected. None of the 11 small (defined as <10 Mb) imbalances were recurrent, nor were the pUPDs, whereas of the 18 large aberrations, three were recurrent-dup(1q), +8 and +21. In contrast, several frequent changes were identified in the DS-ALL cases, which harbored 82 imbalances (30 gains and 52 losses) and four pUPDs. Of the 40 large changes, 28 were gains and 12 losses, with +X, dup(Xq), dup(1q), del(7p), dup(8q), del(9p), dup(9p), del(12p), dup(17q), and +21 being recurrent. Of the 40 microdeletions identified, several targeted specific genes, with the following being repeatedly deleted: BTG1 and CDKN2A/B (29% of cases), ETV6, IKZF1, PAX5 and SERP2 (18%), and BTLA, INPP4B, P2RY8, and RB1 (12%). Loss of the SERP2 and INPP4B genes, encoding the stress-associated endoplasmic reticulum protein family member 2 and the inositol polyphosphate 4-phosphatase-II, respectively, has previously never been implicated in leukemia. Although deletions of the other genes have been associated with ALL, the high frequency of BTG1 loss is a novel finding. Such deletions may characterize a clinical subgroup of DS-ALL, comprising mainly boys with a high median age. In conclusion, ML-DS and DS-ALL are genetically distinct, with mainly gains in ML-DS and deletions in DS-ALL. Furthermore, DS-ALL is characterized by several recurrent gene deletions, with BTG1 loss being particularly frequent.     

Interstitial deletion of chromosome 22 in a patient with the DiGeorge malformation sequence

We describe a chromosome 22 deletion in a patient with the DiGeorge malformation sequence as manifested by an interrupted aortic arch, mild thymic hypoplasia, and minor craniofacial anomalies. Although others have reported DiGeorge sequence patients with deletions derived from unbalanced translocations involving the chromosome 22 long arm, the small interstitial deletion described here appears to be unusual for patients with this disorder.     

Myelodysplastic syndrome in a child with 15q24 deletion syndrome

15q24 deletion syndrome is a recently-described chromosomal disorder, characterized by developmental delay, growth deficiency, distinct facial features, digital abnormalities, loose connective tissue, and genital malformations in males. To date, 19 patients have been reported. We report on a 13-year-old boy with this syndrome manifesting childhood myelodysplastic syndrome (MDS). He had characteristic facial features, hypospadias, and mild developmental delay. He showed neutropenia and thrombocytopenia for several years. At age 13 years, bone marrow examination was performed, which showed a sign suggestive of childhood MDS: mild dysplasia in the myeloid, erythroid, and megakaryocytic cell lineages. Array comparative genomic hybridization (array CGH) revealed a de novo 3.4?Mb 15q24.1q24.3 deletion. Although MDS has not been described in patients with the syndrome, a boy was reported to have acute lymphoblastic leukemia (ALL). The development of MDS and hematological malignancy in the syndrome might be caused by the haploinsufficiency of deleted 15q24 segment either alone or in combination with other genetic abnormalities in hematopoietic cells. Further hematological investigation is recommended to be beneficial if physical and hematological examination results are suggestive of hematopoietic disturbance in patients with the syndrome.     

High frequency of well-defined Y-chromosome deletions in idiopathic Sertoli cell-only syndrome

Idiopathic Sertoli cell-only syndrome (SCOS) is characterized by azoospermia, small testes, absence of germ cells in the testes, elevated follicle stimulating hormone and normal testosterone concentrations. The Y-chromosome is involved in the regulation of spermatogenesis and in the pathogenesis of a fraction of idiopathic male infertility. An azoospermia factor (AZF) is present on the Y- chromosome long arm euchromatic region (Yq11) and two gene families (DAZ and RBM) have been identified within this region. The aim of this study was to investigate whether a specific pattern of Yq11 microdeletions may be associated with idiopathic SCOS. Eighteen idiopathic subjects showing a testicular cytological picture of bilateral SCOS were selected and tested by polymerase chain reaction for a set of 29 Y-specific sequence-tagged sites (STS). We found Yq microdeletions in 10 out of 18 patients (55.5%) while the fathers or brothers of six out of 10 patients deleted for Yq were shown to carry an intact Y-chromosome. These deletions may therefore be considered as de-novo deletions and the cause of SCOS. The analysis of the microdeletions allowed us to identify two homogeneous regions that have a high incidence of deletion. The smallest deletion, common to all patients, is located in Yq interval 5. We therefore speculate that there is a relationship between specific, well-characterized Yq11 microdeletions and a testicular picture of SCOS, identifying an Y- related region frequently deleted in this syndrome. In conclusion, the findings of this study demonstrate that a large percentage of idiopathic SCOS may be genetically determined and identify an Y-related region that seems to possess one or more still unknown genes essential for spermatogenesis.      

Behavior in preschool children with the 22q11.2 deletion syndrome

Children with the 22q11.2 deletion syndrome (22q11DS) are at an increased risk of psychiatric problems from pre‐adolescence; little is known, however, about behavioral problems at a preschool age and the relationship between speech and behavior in this group. Parents of 90 children (aged 1.42–5.99 years) with 22q11DS filled out the Child Behavior Checklist, documenting behaviors including speech problems. Their profiles were compared with those of a comparison group consisting of 33 children with nonsyndromic orofacial clefts without 22q11DS, since both children with 22q11DS and children with clefts are expected to have speech problems. In the 22q11DS group, data on intelligence was acquired by means of formal tests. Parents of children with 22q11DS reported significantly higher mean scores on withdrawn behavior, affective problems and pervasive developmental problems compared to children with nonsyndromic clefts. Approximately 30% of children with 22q11DS had a score above the 97th percentile on at least one of the behavior subscales, indicating psychopathology. In children with 22q11DS, the reported behavioral problems were not associated with speech problems. Behavioral problems were found in 30% of young children with 22q11DS and were unlikely to be caused by speech problems. Within the 22q11DS group, behavioral problems were not related to the degree of cognitive impairment. This shows that many children with 22q11DS, known to be at an increased risk of psychiatric problems from pre‐adolescence, already show behavioral problems before the age of 6 years. © 2012 Wiley Periodicals, Inc.