Male Infertility: A Clinical Reflection

The introduction of intracytoplasmic sperm injection (ICSI) has led to an inappropriate decrease in interest in male fertility. It is apparent that light microscopy provides limited information and molecular techniques show that DNA abnormalities need to be considered further. Abnormalities include not only Yq11 deletions but also DNA strand breaks. Increases in advanced glycation end-products in sperm from well controlled diabetics may provide a mechanism for this damage in non-diabetics. In addition, much publicity is given to decreased male fertility: this is NOT confirmed as technical variations and differences in study populations make it difficult to draw conclusions. The generation of stem cell derived germ cells provides hope for men without germ cells but this is currently only experimental.

The genetic basis of infertility in men.

Subfertility in men is a heterogeneous syndrome, its pathophysiology remaining unknown in the majority of affected men. A large number of genes and loci are associated with sterility in experimental animals, but the human homologues of most of these genes have not been characterized. A British study suggested that, in a large proportion of men with idiopathic infertility, the disorder is inherited as an autosomal recessive trait; this provocative hypothesis needs confirmation. Because normal germ cell development requires the temporally and spatially co-ordinated expression of a number of gene products at the hypothalamic, pituitary and testicular levels, it is safe to predict that a large number of autosomal, as well as X- and Y-linked, genes will probably be implicated in different subsets of male subfertility.

Genetic disorders and spermatogenesis.

Male infertility affects one man in twenty and a genetic basis seems likely in at least 30% of those men. Genetic regulation of fertility involves the inter-related processes of testicular development, spermatogenesis (involving germ cell mitosis, meiosis and spermatid maturation), and their endocrine and paracrine regulation. In regard to spermatogenesis, particular attention has been given to the Yq11 region, where some spermatogenesis genes ('azoospermia factors') appear to be located. Several candidate genes have been identified but have not been shown to have a defined or essential role in spermatogenesis. Microdeletions of Yq11 are found in approximately 15% of azoospermic or severely oligospermic men. The complexity of the genetic control of male fertility is demonstrated by the evidence for genes involved in spermatogenesis and sexual differentiation on the X chromosome and autosomes. Better understanding of the genetic regulation of normal spermatogenesis will provide new probes for clinical studies; however, at present the majority of spermatogenic failure remains without an identified genetic linkage. The advent of intracytoplasmic sperm injection permits fertility in many previously sterile men and presents the possibility of their transmission of infertility; appropriate counselling is required.

Localization of follistatin in the rat testis.

The cellular localization of the activin-binding protein, follistatin, in the rat testis has been a matter of some controversy with different investigators claiming that Sertoli cells, Leydig cells or germ cells are the primary cell types containing this protein. The localization of mRNA encoding follistatin was re-examined using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization as well as the distribution of follistatin by immunohistochemistry. The results demonstrate that mRNA encoding follistatin is located in many germ cells including type B spermatogonia, primary spermatocytes with the exception of the late leptotene and early zygotene stages, and spermatids at steps 1 to 11. It is also found in Sertoli cells and endothelial cells but not in Leydig cells. Immunohistochemistry, using two different antisera to follistatin, showed that this protein was localized to spermatogonia, primary spermatocytes at all stages except the zygotene stage, spermatids at all stages and to endothelial cells and Leydig cells in the intratubular regions. The failure to detect mRNA for follistatin in Leydig cells using RT-PCR and in situ hybridization suggests that the immunohistochemical localization in these cells reflects binding of follistatin produced elsewhere. The widespread localization of follistatin, taken together with its capacity to neutralize the actions of activin, may indicate that follistatin modulates a range of testicular actions of activin, many of which remain unknown.

The role of Y chromosome deletions in male infertility.

Male infertility affects approximately 2-7% of couples around the world. Over one in ten men who seek help at infertility clinics are diagnosed as severely oligospermic or azoospermic. Recent extensive molecular studies have revealed that deletions in the azoospermia factor region of the long arm of the Y chromosome are associated with severe spermatogenic impairment (absent or severely reduced germ cell development). Genetic research into male infertility, in the last 7 years, has resulted in the isolation of a great number of genes or gene families on the Y chromosome, some of which are believed to influence spermatogenesis.

Intragenic SNP haplotypes associated with 84dup18 mutation in TNFRSF11A in four FEO pedigrees suggest three independent origins for this mutation.

Familial expansile osteolysis (FEO) is a rare disorder causing bone dysplasia. The clinical features of FEO include early-onset hearing loss, tooth destruction, and progressive lytic expansion within limb bones causing pain, fracture, and deformity. An 18-bp duplication in the first exon of the TNFRSF11A gene encoding RANK has been previously identified in four FEO pedigrees. Despite having the identical mutation, phenotypic variations among affected individuals of the same and different pedigrees were noted. Another 18-bp duplication, one base proximal to the duplication previously reported, was subsequently found in two unrelated FEO patients. Finally, mutations overlapping with the mutations found in the FEO pedigrees have been found in ESH and early-onset PDB pedigrees. An Iranian FEO pedigree that contains six affected individuals dispersed in three generations has previously been introduced; here, the clinical features of the proband are reported in greater detail, and the genetic defect of the pedigree is presented. Direct sequencing of the entire coding region and upstream and downstream noncoding regions of TNFRSF11A in her DNA revealed the same 18-bp duplication mutation as previously found in the four FEO pedigrees. Additionally, eight sequence variations as compared to the TNFRSF11A reference sequence were identified, and a haplotype linked to the mutation based on these variations was defined. Although the mutation in the Iranian and four of the previously described FEO pedigrees was the same, haplotypes based on the intragenic SNPs suggest that the mutations do not share a common descent.

Protein expression pattern of CDK11(p58) during testicular development in the mouse.

Protein kinases are important signalling molecules critical for normal cell growth and development. CDK11(p58) is a p34(cdc2) related protein kinase, and plays an important role in normal cell cycle progression. In this study, we mainly characterized the protein expression of CDK11(p58) during postnatal development in mouse testes and examined the cellular localization of CDK11(p58) and cyclinD3, which was associated with CDK11(p58) in mammalian cells. Western blot analysis revealed that CDK11(p58) was present in the early stages of development. It gradually increased and reached a peak in adult testes. The protein expression of CDK11(p58) was further analysed by immunohistochemistry due to its developmentally regulated expression. The variable immunostaining patterns of CDK11(p58) were visualized during different developmental periods and, in adult mouse, different stages of seminiferous tubules. CDK11(p58) expression was detected in proliferating germ cells in the early stages of developing testes. In adult testes, the protein was expressed in pachytene primary spermatocytes from stage VII to XI of spermatogenesis and in postmeiotic spermatids in all stages at different levels. The colocalization of CDK11(p58) and cyclinD3 in the adult testis was revealed by immunofluorescence analysis.

Persistently raised C-reactive protein levels are associated with advanced periodontal disease

Objective: The aim was to investigate whether there was an association between periodontitis or tooth loss in a homogeneous group of 60-70-year-old Western European men and either a sustained high or low level of C-reactive protein (CRP).
Material and Methods: Men enrolled in a cohort study of cardiovascular disease in Northern Ireland were screened in 1990-1994 and rescreened in 2001-2004, when a periodontal examination was completed. High-sensitivity CRP was measured from fasting blood samples. There were 806 men with six or more teeth who had either a high level (>3 mg/l) or a lower level of CRP at both time points. Multivariate analysis was carried out using logistic regression with adjustment for possible confounders. Models were constructed with the CRP level as the outcome variable and various measures of periodontal status (low and high threshold periodontitis) or tooth loss as predictor variables. Confounders included in the analysis were known cardiovascular risk factors of age, smoking, diabetes, BMI and socioeconomic status.
Results: There were 67 men who had a high value of CRP (>3 mg/l) and 739 men who had a CRP value =3 mg/l at both time points. The unadjusted odds ratio (OR) for advanced periodontitis to be associated with high CRP was 3.62, p=0.0003. The association was somewhat attenuated but remained significant (OR=2.49, p=0.02) after adjustment for confounders. A high level of tooth loss was also associated with high CRP with an adjusted OR of 2.17, p=0.008. Low threshold periodontitis was not associated with the level of CRP.
Conclusion: There was an association between advanced periodontitis and elevated CRP levels as measured at two time points at a 10-year interval in the 60-70-year-old European males investigated. This association was adjusted for various cardiovascular risk factors. There was also an association between high levels of tooth loss and high CRP in the men studied.

Long-term outcomes for cross-arch stabilizing bridges in periodontal maintenance patients - a retrospective study

Background: Cross-arch bridges are used to stabilize teeth for patients with reduced periodontal support. Little is known about technical or biological complications, whether teeth and implants can be combined in this type of bridge and the long-term effects on tooth loss.

Quantification of all-ceramic crown margin surface profile from try-in to 1-week post-cementation

To use profilometry to assess the margin surface profile of all-ceramic crowns (ACC’s) at try-in and 1-week after cementation with dual-cured resin (DC, RelyX ARC, 3 M ESPE, St. Paul, MN, USA), self-adhesive dual-cured resin (SADC, RelyX Unicem, 3 M ESPE), light-cured resin (LC, RelyX Veneer, 3 M ESPE) or chemically cured resin-modified glass ionomer (RMGI, RelyX Luting Plus, 3 M ESPE) luting cement. Methods: Forty, sound, extracted, human, premolar teeth underwent a standardised preparation for ACC’s. IPS Empress (Ivoclar-Vivadent, Liechtenstein) crowns of standard dimensions were fabricated and 10 luted with each cement and stored in water for 7 days. Three groups of serial profiles were taken, the first of the tooth preparation, the second of the crown margins at try-in and lastly of the crown margins after cementation and 7 days water storage. Results: There were no significant differences in the crown margin surface profile between the four cement groups at try-in. The change in crown margin position between try-in and post-cementation was significantly greater for DC than for LC and RMGI. SADC was not significantly different to the other cements. There were no significant differences in the crown margin extensions between the four cement groups, however most of the IPS Empress ACC’s in this study were underextended but this was not statistically significant. Conclusions: IPS Empress ACC’s seated more fully with LC and RMGI than with DC cement

Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis

Papillon-Lefevre syndrome, or keratosis palmoplantaris with periodontopathia (PLS, MIM 245000), is an autosomal recessive disorder that is mainly ascertained by dentists because of the severe periodontitis that afflicts patients(1,2). Both the deciduous and permanent dentitions are affected, resulting in premature tooth loss. Palmoplantar keratosis, varying from mild psoriasiform scaly skin to overt hyperkeratosis, typically develops within the first three years of life. Keratosis also affects other sites such as elbows and knees. Most PLS patients display both periodontitis and hyperkeratosis. some patients have only palmoplantar keratosis or periodontitis, and in rare individuals the periodontitis is mild and of late onset(3-6). The PLS locus has been mapped to chromosome 11q14-q21 (refs 7-9). Using homozygosity mapping in eight small consanguineous families, we have narrowed the candidate region to a 1.2-cM interval between D11S4082 and D11S931. The gene (CTSC) encoding the lysosomal protease cathepsin C (or dipeptidyl aminopeptidase I) lies within this interval. We defined the genomic structure of CTSC and found mutations in all eight families. In two of these families we used a functional assay to demonstrate an almost total loss of cathepsin C activity in PLS patients and reduced activity in obligate carriers.

Fasciola hepatica: Comparative effects of host resistance and parasite intra-specific interactions on size and reproductive histology in flukes from rats infected with isolates differing in triclabendazole sensitivity

The efficacies of putative fasciolicides and vaccines against Fasciola hepatica are frequently monitored in clinical and field trials by determination of fluke egg output in host faeces and by worm counts in the host liver at autopsy. Less often used are parameters based on fluke size and histology, yet these can provide important indications of specific effects on the development of particular germ-line or somatic tissues, especially in relation to the timing and profligacy of egg production. In this study. F. hepatica metacercariae of two distinct isolates, the triclabendazole (TCBZ)-sensitive Cullompton isolate and the TCBZ-resistant Oberon isolate, were administered to rats as single-isolate or mixed-isolate infections. At autopsy 16 weeks later individual adult flukes were counted, measured and the reproductive organs were examined histologically. The degree of development of the testis tubules in each fluke was represented by a numerical score, based on the proportion of the histological section profiles occupied by testis tissue. The level of anti-F. hepatica antibody in the serum of each rat was determined by ELISA. It was found that Cullompton flukes were significantly larger than Oberon flukes, and that significantly more Cullompton metacercariae developed to adults than Oberon metacercariae. The Cullompton flukes showed histological evidence of aspermy and spermatogenic arrest, which was reflected in quantitatively reduced testicular development, as compared with the Oberon isolate. In Cullompton flukes, parthenogenetic egg development is implied. The size of Cullompton and Oberon flukes was significantly related to the number of adult flukes recovered, to the number of metacercariae administered, and to the percentage success of infection. The testis development score in both isolates was significantly related to the number of adult flukes recovered but not to the number of metacercariae administered, or to the percentage success of infection. Fluke size was positively related to testis score for both isolates, and a significant negative relationship was found between percentage success of infection and metacercarial dose. The results are interpreted in terms of differing interactions between various numbers of young flukes and host immunity during invasion of and migration in the hepatic parenchyma, and of fluke intra-specific (possibly pheromonal) stimulatory effects in the final stages of development, within the host bile ducts. No significant relationships were found between host antibody levels and fluke size or testis score. False positive serological reactions were found in some rats that had been infected, but found to harbour no flukes at autopsy. Clearly the act of eliminating the flukes involved generation of an immune response. (C) 2011 Elsevier B.V. All rights reserved.

Long Term Use of HU210 Adversely Affects Spermatogenesis in Rats by modulating the Endocannabinoid System

Recent societal acceptance of cannabinoids as recreational and therapeutic drugs has posed a potential hazard to male reproductive health. Mammals have a highly sophisticated endogenous cannabinoid (ECS) system that regulates male (and female) reproduction and exo-cannabinoids may influence it adversely. Therefore it is imperative to determine their effects on male reproduction so that men can make informed choices as to their use. Here, an animal model was used to administer HU210, a synthetic analogue of ?9-tetrahydrocannabinol (THC) and potent cannabinoid receptor (CB) agonist to determine its effects on reproductive organ weights, spermatogenesis, testicular histology and sperm motility. Its effects on the physiological endocannabinoid system were also investigated. Spermatogenesis was markedly impaired with reductions in total sperm count after 2 weeks of exposure. Spermatogenic efficiency was depleted, and Sertoli cell number decreased as exposure time increased with seminiferous tubules showing germ cell depletion developing into atrophy in some cases. Sperm motility was also adversely affected with marked reductions from 2 weeks on. HU210 also acted on the sperm’s endocannabinoid system. Long term use of exo-cannabinoids has adverse effects on both spermatogenesis and sperm function. These findings highlight the urgent need for studies evaluating the fertility potential of male recreational drug users.

Population-based study reveals new risk-stratification biomarker panel for Barrett's esophagus

BACKGROUND & AIMS: The risk of progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) is low and difficult to calculate. Accurate tools to determine risk are needed to optimize surveillance and intervention. We assessed the ability of candidate biomarkers to predict which cases of BE will progress to EAC or high-grade dysplasia and identified those that can be measured in formalin-fixed tissues. METHODS: We analyzed data from a nested case-control study performed using the population-based Northern Ireland BE Register (1993-2005). Cases who progressed to EAC (n = 89) or high-grade dysplasia =6 months after diagnosis with BE were matched to controls (nonprogressors, n = 291), for age, sex, and year of BE diagnosis. Established biomarkers (abnormal DNA content, p53, and cyclin A expression) and new biomarkers (levels of sialyl Lewis(a), Lewis(x), and Aspergillus oryzae lectin [AOL] and binding of wheat germ agglutinin) were assessed in paraffin-embedded tissue samples from patients with a first diagnosis of BE. Conditional logistic regression analysis was applied to assess odds of progression for patients with dysplastic and nondysplastic BE, based on biomarker status. RESULTS: Low-grade dysplasia and all biomarkers tested, other than Lewis(x), were associated with risk of EAC or high-grade dysplasia. In backward selection, a panel comprising low-grade dysplasia, abnormal DNA ploidy, and AOL most accurately identified progressors and nonprogressors. The adjusted odds ratio for progression of patients with BE with low-grade dysplasia was 3.74 (95% confidence interval, 2.43-5.79) for each additional biomarker and the risk increased by 2.99 for each additional factor (95% confidence interval, 1.72-5.20) in patients without dysplasia. CONCLUSIONS: Low-grade dysplasia, abnormal DNA ploidy, and AOL can be used to identify patients with BE most likely to develop EAC or high-grade dysplasia.

Frequency and distribution of group I intron genotypes of Candida albicans colonising critically ill patients

A study is performed to examine the distribution and frequency of 25S rRNA intron genotypes of Candida albicans isolated from different anatomical sites of patients in an intensive care unit (ICU) setting. Germ-tube positive Candida isolates (n=65) from 65 patients are included and isolates are characterised by 25S intron genotyping, whereby all can be subdivided into four genotypes (A-D). Results demonstrated that there were no significant differences between the frequency and genotype distribution of the Candida isolates and the anatomical site of colonisation. Furthermore, analysis of the transposable intron region in the 25S rRNA gene demonstrated equal distribution, regardless of age and anatomical site of isolation (groin, throat, etc.). Therefore, there does not appear to be any selective pressure associated with any anatomical site, resulting in an ecological shift in the frequency of genotypes present. This suggests that C. albicans intron genotypes equally colonise those sites of the body examined in this study. Although such an ecological finding as this is interesting, it perpetuates the continued need to find a genotypic typing scheme that helps to identify the source (nosocomial or endogenous) and mode of entry of C. albicans into patients in the ICU setting, resulting in C. albicans bloodstream infection.