Expression of the O-Glycosylation Enzyme GalNAc-T3 in the Equatorial Segment Correlates with the Quality of Spermatozoa.

We question whether the expression of GalNAc-T3, the only known O-GalNAc-transferase present in germ cells, is correlated with qualitative and functional parameters of spermatozoa. We investigated the expression of GalNAc-T3 in ejaculated spermatozoa with immunocytochemistry in swim-up purified and acrosome-reacted spermatozoa from quality-control semen donors and in semen samples from 206 randomly selected men representing a broad spectrum of semen quality. Using donor ejaculates and immunofluorescence detection we found that expression of GalNAc-T3 and the presence of the immature O-glycans Tn and T localized to the equatorial segment of spermatozoa. The proportion of GalNAc-T3-positive spermatozoa in the ejaculate increased after swim-up and appeared unaffected by induction of acrosomal exocytosis. The fraction of spermatozoa with equatorial expression of GalNAc-T3 correlated with classical semen parameters (concentration p = 9 × 10-6, morphology p = 7 × 10-8, and motility p = 1.8 × 10-5) and was significantly lower in men with oligoteratoasthenozoospermia (p = 0.0048). In conclusion, GalNAc-T3 was highly expressed by motile spermatozoa and the expression correlated positively with the classical semen parameters. Therefore, GalNAc-T3 expression seems related to the quality of the spermatozoa, and we propose that reduced expression of GalNAc-T3 may lead to impaired O-glycosylation of proteins and thereby abnormal maturation and reduced functionality of the spermatozoa.

Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

Fragile X-related element 2 methylation analysis may provide a suitable option for inclusion of fragile X syndrome and/or sex chromosome aneuploidy into newborn screening: a technical validation study.

PURPOSE: We show that a novel fragile X-related epigenetic element 2 FMR1 methylation test can be used along with a test for sex-determining region Y (SRY) to provide the option of combined fragile X syndrome and sex chromosome aneuploidy newborn screening. METHODS: Fragile X-related epigenetic element 2, SRY, and FMR1 CGG repeat analyses were performed on blood and saliva DNA, and in adult and newborn blood spots. The cohort consisted of 159 controls (CGG <40), 187 premutation (CGG 56-170), and 242 full-mutation (CGG ~200-2,000) males and females, 106 sex chromosome aneuploidy individuals, and 151 cytogenetically normal controls. RESULTS: At the 0.435 threshold, fragile X-related epigenetic element 2 analysis in males was robust on both blood DNA and newborn blood spots, with specificity and sensitivity of ~100% for full-mutation genotype. In females, the specificity was 99%, whereas half of full-mutation females were above the 0.435 threshold in both blood DNA and newborn blood spots. Furthermore, at this threshold, the test could not differentiate individuals with Klinefelter syndrome from female controls without using the SRY marker. When combined with SRY analysis, the test was consistent with most results for sex chromosome aneuploidies from karyotyping. CONCLUSION: Setting specific thresholds for fragile X-related epigenetic element 2 analysis and including the SRY marker provides the option to either include or exclude detection of sex chromosome aneuploidies as part of fragile X syndrome newborn screening.

The psychosocial impact of Klinefelter syndrome and factors influencing quality of life.

PURPOSE: There is considerable information regarding the medical and cognitive aspects of Klinefelter syndrome yet little research regarding its psychosocial impact. This study investigates the personal impact of Klinefelter syndrome and the influence of age at diagnosis, clinical, social, and demographic factors on adult quality of life outcomes. METHODS: Men from across Australia, diagnosed with KS at different ages, were recruited through multiple sources. Participants completed a questionnaire assessing subjective well-being, body image, self-esteem, mental health, social support, and general health. RESULTS: Eighty-seven individuals self-completed the questionnaire. All outcomes were much poorer for the study population than for the general male population. Individuals diagnosed later in life reported many of the same symptoms as those diagnosed at younger ages. Employment status, social support, and phenotypic features were the strongest predictors of psychosocial outcomes. Age at diagnosis was not as influential because it did not correlate with phenotypic severity score. CONCLUSION: This is the first quantitative study to show Klinefelter syndrome has a significant personal impact. Men diagnosed with Klinefelter syndrome later in life reported similar difficulties as those at younger ages, suggesting that they would benefit from early detection and intervention. Understanding factors influencing this can assist in providing adequate services to individuals with Klinefelter syndrome, their partners, families, and the health professionals caring for them.

The prevalence and diagnosis rates of Klinefelter syndrome: an Australian comparison.

OBJECTIVE: To determine the prevalence and diagnosis rates of Klinefelter syndrome (KS) in Victoria, Australia, and compare these to previous international findings. DESIGN, SETTING AND PARTICIPANTS: A Victorian population-based descriptive study of all cytogenetic examinations resulting in a diagnosis of KS, including prenatal diagnoses from 1986 to 2006 and postnatal diagnoses from 1991 to 2006. MAIN OUTCOME MEASURES: Birth prevalence and diagnosis rates of KS. RESULTS: The birth prevalence of KS in Victoria is estimated to be 223 per 100,000 males (95% CI, 195-254), with about 50% of cases remaining undiagnosed. CONCLUSIONS: KS may be occurring more frequently than has been reported previously, yet many cases remain undiagnosed. Our results highlight the need for increased awareness leading to timely detection.

Postnatal screening for Klinefelter syndrome: is there a rationale?

UNLABELLED: Diagnosis of Klinefelter syndrome (KS) allows for timely beneficial interventions across the lifespan. Most cases currently remain undiagnosed because of low awareness of KS amongst health professionals, the hesitancy of men to seek medical attention and its variable clinical presentation. Given these barriers, population-based genetic screening provides an approach to comprehensive and early detection. We examine current evidence regarding risks and benefits of diagnosing KS at different ages. CONCLUSION: There is a lack of evidence regarding the influence of age at diagnosis on adult outcomes that can only be obtained through a pilot screening programme.

CDH1 (E-cadherin) in testicular germ cell neoplasia: suppressed translation of mRNA in pre-invasive carcinoma in situ but increased protein levels in advanced tumours.

E-cadherin (CDH1) is a transmembrane glycoprotein involved in cellular adhesion. In our recent microarray studies of testicular germ cell tumours (TGCTs) and the common precursor carcinoma in situ (CIS), CDH1 mRNA was highly expressed in CIS and embryonal carcinoma. It has previously been reported that the CDH1 protein is not expressed in CIS. To resolve the discrepancy, we performed a detailed analysis of the expression of CDH1 mRNA and protein in a series of normal and neoplastic testes. High expression of CDH1 mRNA in CIS was confirmed by real-time PCR and in situ hybridisation. At the protein level, however, CDH1 was only detected with one of three tested antibodies, but Western blotting analysis with this antibody showed additional bands, suggesting unspecific staining. The levels of a CDH1 protein fragment in serum samples from 58 patients with TGCTs were analysed by ELISA; we found significantly higher levels in patients with advanced disease (stage II/III) when compared to healthy individuals and patients with stage I TGCT. In conclusion, despite high mRNA levels, the CDH1 protein is not expressed in CIS, suggesting translational suppression of CDH1 protein expression. CDH1 serum levels may be a serological marker for staging of TGCT patients.

Identity of M2A (D2-40) antigen and gp36 (Aggrus, T1A-2, podoplanin) in human developing testis, testicular carcinoma in situ and germ-cell tumours.

Testicular germ-cell tumours of young adults are derived from a pre-invasive intratubular lesion, carcinoma in situ (CIS). In a recent genome-wide gene expression screening using cDNA microarrays, we found PDPN over-expressed in CIS compared to normal adult testis. PDPN encodes podoplanin (Aggrus, human gp36, T1A-2), a transmembrane glycoprotein expressed in lymphatic endothelium and various solid tumours. To examine a potential role for PDPN in testicular neoplasms and during testicular development, we investigated its expression pattern during the development of human testis and in a series of testicular CIS, gonadoblastoma and overt germ-cell tumours. We established by RT-PCR and by immunohistochemistry with a gp36 antibody that PDPN mRNA and the protein product were expressed in testes with germ-cell neoplasms but not in the normal adult testis. We also found gp36 expression in early foetal gonocytes and immature Sertoli cells, similar to the expression pattern of M2A antigen, a previously identified marker for CIS and seminoma. This reinforced our previous proposal that M2A (D2-40) antigen was identical to gp36 (podoplanin, Aggrus, T1A-2). Our findings also suggest that podoplanin has a function in developing testis, most likely at the level of cell-cell interactions among pre-meiotic germ cells and immature Sertoli cells.

Screening for Klinefelter syndrome.

PURPOSE OF REVIEW: To examine recently published evidence that may inform the need for population screening of Klinefelter syndrome; by far the most common chromosomal disorder in males, which most often escapes diagnosis throughout the lifespan. RECENT FINDINGS: Research regarding the prevalence and characteristics of developmental and learning difficulties in Klinefelter syndrome emphasize the importance of early intervention with likely subsequent psychosocial and other health benefits. Testosterone treatment will always need to be individualized, but there is growing evidence for the benefits of intervention from the time of puberty, and possibly in early childhood. Discussion of fertility options is now essential given the advent of surgical sperm retrieval and intracytoplasmic sperm injection. SUMMARY: Despite increasing knowledge of the natural history of the Klinefelter syndrome spectrum, beneficial interventions and when they should occur, most opportunities are missed due to nondiagnosis. Population screening is arguably the only way of ensuring timely detection of individuals with Klinefelter syndrome. The technologies and structures for such a program already exist. This field now requires a pilot program to further define the risks, benefits and psychosocial and ethical dimensions of screening.