Genetic abnormalities leading to qualitative defects of sperm morphology or function.

Infertility, defined by the inability of conceiving a child after 1 year is estimated to concern approximately 50 million couples worldwide. As the male gamete is readily accessible and can be studied by a simple spermogram it is easier to subcategorize male than female infertility. Subjects with a specific sperm phenotype are more likely to have a common origin thus facilitating the search for causal factors. Male infertility is believed to be often multifactorial and caused by both genetic and extrinsic factors, but severe cases of male infertility are likely to have a predominant genetic etiology. Patients presenting with a monomorphic teratozoospermia such as globozoospermia or macrospermia with more than 85% of the spermatozoa presenting this specific abnormality have been analyzed permitting to identify several key genes for spermatogenesis such as AURKC and DPY19L2. The study of patients with other specific sperm anomalies such as severe alteration of sperm motility, in particular multiple morphological anomalies of the sperm flagella (MMAF) or sperm unability to fertilize the oocyte (oocyte activation failure syndrome) has also enable the identification of new infertility genes. Here we review the recent works describing the identification and characterization of gene defects having a direct qualitative effect on sperm morphology or function.

Single gene defects leading to sperm quantitative anomalies.

Azoospermia, defined by the absence of sperm in the ejaculate, is estimated to affect up to 1% of men in the general population. Assisted reproductive technologies have revolutionized the treatment of infertility, and some azoospermic men, those with a post-meiotic defect, can conceive following the use of viable spermatoza recovered from testicular or epididymal biopsies. Although male infertility is a multifactorial disease, it is believed that genetic factors are predominant in the etiology of azoospermia and severe oligozoospermia. Despite that assumption, substantiated by the high number of infertile knockout (KO) mice and the even higher number of genes expressed essentially in the testis, little is known about the pathophysiology of reduced sperm production, its primary causes or the genetic and epigenetic consequences for the gamete and the future conceptus. The identification of genetic abnormalities is therefore paramount to understand spermatogenesis, to adopt the best course of action for the patient and to provide adequate genetic counseling. We provide here a review of the recent literature on the genetics of azoospermia and oligozoospermia, focusing on defects directly altering sperm production. New sequencing technologies are contributing to the rapid evolution of the recent field of infertility genetics.

Mercury proxies and mercury dynamics in a forested watershed of the US Northeast.

Although many studies focus on mercury (Hg) and methylmercury (MeHg) dynamics in streams, challenges remain in identifying the relative importance of land cover and seasonality at regulating Hg and MeHg dynamics at the watershed scale. Developing robust proxies for Hg and/or MeHg determination also remains a challenge. Our study used Hg, MeHg, and dissolved organic carbon (DOC) concentration measurements and various DOC fluorescence indices to characterize Hg and DOC dynamics in a forested watershed of the US Northeast. Principal component analysis indicated that land cover/landscape position (i.e., headwater vs. wetland-influenced area vs. lake-influenced area) explained 44 % of the variance in Hg, MeHg, DOC concentrations, and DOC quality during the snow-free season, while seasonality (i.e., air temperature and discharge) explained only 21 % of the variance in the results. Furthermore, finding a good proxy for Hg that is valid across a range of landscape positions remains a challenge; however, regression analysis indicated that the fluorescence peak Humic C (excitation = 350 nm; emission = max (420-480)), which corresponds to the presence of melanoidins in water, explained 21 % of the variability in MeHg concentrations across both space and time (p = 0.001), and thus appears to be a possible proxy for MeHg determination in our study watershed. From a management perspective, land cover modifications (lake, reservoir, and wetland) are likely to play more important roles at regulating Hg, MeHg, and DOC exports at the watershed scale than long-term changes in the climate of this region.

A Y-encoded subunit of the translation initiation factor Eif2 is essential for mouse spermatogenesis.

In mouse and man, deletions of specific regions of the Y chromosome have been linked to early failure of spermatogenesis and consequent sterility; the Y chromosomal gene(s) with this essential early role in spermatogenesis have not been identified. The partial deletion of the mouse Y short arm (the Sxrb deletion) that occurred when Tp(Y)1CtSxr-b (hereafter Sxrb) arose from Tp(Y)1CTSxr-b (hereafter Sxra) defines Spy, a Y chromosomal factor essential for normal spermatogonial proliferation. Molecular analysis has identified six genes that lie within the deletion: Ube1y1 (refs. 4,5), Smcy, Uty, Usp9y (also known as Dffry), Eif2s3y (also known as Eif-2gammay) and Dby10; all have closely similar X-encoded homologs. Of the Y-encoded genes, Ube1y1 and Dby have been considered strong candidates for mouse Spy function, whereas Smcy has been effectively ruled out as a candidate. There is no Ube1y1 homolog in man, and DBY, either alone or in conjunction with USP9Y, is the favored candidate for an early spermatogenic role. Here we show that introduction of Ube1y1 and Dby as transgenes into Sxrb-deletion mice fails to overcome the spermatogenic block. However, the introduction of Eif2s3y restores normal spermatogonial proliferation and progression through meiotic prophase. Therefore, Eif2s3y, which encodes a subunit of the eukaryotic translation initiation factor Eif2, is Spy.

Factors contributing to variability of quantitative viral PCR results in proficiency testing samples: a multivariate analysis.

While viral load testing has gained widespread acceptance, a primary limitation remains the variability of results, particularly between different laboratories. While some work has demonstrated the importance of standardized quantitative control material in reducing this variability, little has been done to explore other important factors in the molecular amplification process. Results of 185 laboratories enrolled in the College of American Pathologists (CAP) 2009 viral load proficiency testing (PT) survey (VLS) were examined. This included 165 labs (89.2%) testing for cytomegalovirus (CMV), 99 (53.5%) for Epstein-Barr virus (EBV), and 64 (34.6%) for BK virus (BKV). At the time of PT, laboratories were asked a series of questions to characterize their testing methods. The responses to these questions were correlated to mean viral load (MVL) and result variability (RV). Contribution of individual factors to RV was estimated through analysis of variance (ANOVA) modeling and the use of backward selection of factors to fit those models. Selection of the quantitative calibrator, commercially prepared primers and probes, and amplification target gene were found most prominently associated with changes in MVL or RV for one or more of the viruses studied. Commercially prepared primers and probes and amplification target gene made the largest contribution to overall variability. Factors contributing to MVL and RV differed among viruses, as did relative contribution of each factor to overall variability. The marked variability seen in clinical quantitative viral load results is associated with multiple aspects of molecular testing design and performance. The reduction of such variability will require a multifaceted approach to improve the accuracy, reliability, and clinical utility of these important tests.

Molecular characterization of 11 novel mutations in patients with heterozygous and homozygous FV deficiency.

Coagulation factor V (FV) has an important role in the blood coagulation cascade, in both the pro- and anticoagulant pathways. FV deficiency is a rare bleeding disorder with variable phenotypic expression. We report a cohort of 10 patients with mild-severe FV deficiency in whom a total of 11 novel mutations were identified. Three patients were compound heterozygous for two mutations, whereas each of the remaining patients had a single heterozygous variant. FV levels did not correlate with either the type of mutation identified or the bleeding diathesis exhibited by the patients. Although considered to have an autosomal recessive mode of inheritance, patients with a single missense mutation may present with a significant bleeding history. The addition of a significant number of previously unidentified mutations to the public domain will contribute to the knowledge and understanding of the molecular pathology of this rare disorder.

Analysis of sex chromosome abnormalities using X and Y chromosome DNA tiling path arrays.

BACKGROUND: Array comparative genomic hybridisation is a powerful tool for the detection of copy number changes in the genome. METHODS: A human X and Y chromosome tiling path array was developed for the analysis of sex chromosome aberrations. RESULTS: Normal X and Y chromosome profiles were established by analysis with DNA from normal fertile males and females. Detection of infertile males with known Y deletions confirmed the competence of the array to detect AZFa, AZFb and AZFc deletions and to distinguish between different AZFc lesions. Examples of terminal and interstitial deletions of Xp (previously characterised through cytogenetic and microsatellite analysis) have been assessed using the arrays, thus both confirming and refining the established deletion breakpoints. Breakpoints in iso-Yq, iso-Yp and X-Y translocation chromosomes and X-Y interchanges in XX males are also amenable to analysis. DISCUSSION: The resolution of the tiling path clone set used allows breakpoints to be placed within 100-200 kb, permitting more precise genotype/phenotype correlations. These data indicate that the combined X and Y tiling path arrays provide an effective tool for the investigation and diagnosis of sex chromosome copy number aberrations and rearrangements.

Reading the prognosis of the individual with breast cancer.

AIM: To obtain better survival estimates for the individual than is provided by placement in an NPI group. METHOD: Consecutive primary operable breast cancers treated at Nottingham City Hospital 1990-1999. Ten year % actuarial survivals plotted for 10 ranges of NPI from 2.0 to 6.9. There is an excellent inverse correlation between median NPI value for each range and survival at 10 years. To enable estimation of survival for all individual values of NPI, a curve fitting technique applied to these results (by G.B.) gave the formula to estimate survival from the individual's NPI score: 10 year % survival for the individual=-3.0079 x NPI(2)+12.30 x NPI+83.84. This gave an r(2) of 0.98. RESULTS AND CONCLUSION: Greater accuracy in individual survival prediction is obtained by dividing women into 10 groups by NPI scores than in the originally described six groups; rank order of survival in relation to NPI score is preserved. A curve fitting technique has been applied to these data to give a formula for the prediction of 10 year survival for every 0.1 value of NPI.

Survival of invasive breast cancer according to the Nottingham Prognostic Index in cases diagnosed in 1990-1999.

UNLABELLED: The Nottingham Prognostic Index (NPI) is a well established and widely used method of predicting survival of operable primary breast cancer. AIMS: Primary: To present the updated survival figures for each NPI Group. Secondary: From the observations to suggest reasons for the reported fall in mortality from breast cancer. METHODS: The NPI is compiled from grade, size and lymph node status of the primary tumour. Consecutive cases diagnosed and treated at Nottingham City Hospital in 1980-1986 (n=892) and 1990-1999 (n=2,238) are compared. Changes in protocols towards earlier diagnosis and better case management were made in the late 1980s between the two data sets. RESULTS: Case survival (Breast Cancer Specific) at 10 years has improved overall from 55% to 77%. Within all Prognostic groups there are high relative and absolute risk reductions. The distribution of cases to Prognostic groups shows only a small increase in the numbers in better groups. CONCLUSION: The updated survival figures overall and for each Prognostic group for the NPI are presented.

Relative activity of structural analogues of amsacrine against human leukemia cell lines containing amsacrine-sensitive or -resistant forms of topoisomerase II: use of computer simulations in new drug development.

Anilino analogues of amsacrine showed increased activity against amsacrine (AMSA)-resistant cell lines when compared with the parent compound, but the mechanisms of amsacrine resistance in these lines were unknown (Finlay, G. J., Baguley, B. C., Snow, K., and Judd, W., J. Natl. Cancer Inst., 82: 662-667, 1990). We tested the cytotoxic and DNA-cleaving activities of two amsacrine analogues which were derivatives of 9-anilinoacridine (1'-methylcarbamate and 1'-benzenesulfonamide) against an amsacrine-resistant human leukemia cell line (HL-60/AMSA) whose resistance is due to an amsacrine-resistant topoisomerase II. Neither agent could overcome the amsacrine resistance of HL-60/AMSA. Neither agent could induce HL-60/AMSA topoisomerase II-mediated cleavage of DNA in an isolated biochemical system, although at high concentrations the two analogues could inhibit HL-60/AMSA topoisomerase II-mediated DNA strand passage. Both analogues were at least as active, if not more active, than amsacrine against amsacrine-sensitive HL-60 and its topoisomerase II. Comparison of the cellular and biochemical results with those from computer simulation of the energy-minimized structures of amsacrine, its inactive isomer o-AMSA, and the two new active analogues suggests the following possibilities: (a) the positioning of the potential topoisomerase II-binding site (1'-anilino group) of the two new drugs resembles the positioning of this site in amsacrine; (b) the HL-60 topoisomerase II has a binding site which interacts with amsacrine and the two anilino analogues but not with o-AMSA, an analogue with altered positioning of the methoxy group; (c) the HL-60/AMSA topoisomerase II interacts with reduced affinity with amsacrine and the two anilino analogues, although HL-60/AMSA topoisomerase II still interacts with the structurally distinct topoisomerase II-reactive nonintercalator, etoposide; (d) because of their higher DNA binding affinity or the greater possible positions of their side groups in comparison to amsacrine, the two analogues can, at high concentrations, inhibit the strand-passing activity of HL-60/AMSA topoisomerase II.

High dose angiotensin-converting enzyme inhibition prevents fluid volume expansion in heart transplant recipients.

OBJECTIVES: We sought to test the hypothesis that plasma volume (PV) expansion in heart transplant recipients (HTRs) is caused by failure to reflexively suppress the renin-angiotensin-aldosterone (RAA) axis. BACKGROUND: Extracellular fluid volume expansion occurs in clinically stable HTRs who become hypertensive. We have previously demonstrated that the RAA axis is not reflexively suppressed by a hypervolemic stimulus in HTRs. METHODS: Plasma volume and fluid regulatory hormones were measured in eight HTRs (57+/-6 years old) both before and after treatment with captopril (225 mg/day). Antihypertensive and diuretic agents were discontinued 10 days before. The HTRs were admitted to the Clinical Research Center (CRC), and, after three days of a constant diet containing 87 mEq/day of Na+, PV was measured by using the modified Evans blue dye dilution technique. After approximately four months (16+/-5 weeks), the same HTRs again discontinued all antihypertensive and diuretic agents; they were progressed to a captopril dose of 75 mg three times per day over 14 days, and the CRC protocol was repeated. RESULTS: Captopril pharmacologically suppressed (p<0.05) supine rest levels of angiotensin II (-65%) and aldosterone (-75%). The reductions in vasopressin and atrial natriuretic peptide levels after captopril did not reach statistical significance. The PV, normalized for body weight (ml/kg), was significantly reduced by 12% when the HTRs received captopril. CONCLUSIONS: Extracellular fluid volume is expanded (12%) in clinically stable HTRs who become hypertensive. Pharmacologic suppression of the RAA axis with high-dose captopril (225 mg/day) returned HTRs to a normovolemic state. These findings indicate that fluid retention is partly engendered by a failure to reflexively suppress the RAA axis when HTRs become hypervolemic.

Sequence family variant loss from the AZFc interval of the human Y chromosome, but not gene copy loss, is strongly associated with male infertility.

BACKGROUND: Complete deletion of the complete AZFc interval of the Y chromosome is the most common known genetic cause of human male infertility. Two partial AZFc deletions (gr/gr and b1/b3) that remove some copies of all AZFc genes have recently been identified in infertile and fertile populations, and an association study indicates that the resulting gene dose reduction represents a risk factor for spermatogenic failure. METHODS: To determine the incidence of various partial AZFc deletions and their effect on fertility, we combined quantitative and qualitative analyses of the AZFc interval at the DAZ and CDY1 loci in 300 infertile men and 399 control men. RESULTS: We detected 34 partial AZFc deletions (32 gr/gr deletions), arising from at least 19 independent deletion events, and found gr/gr deletion in 6% of infertile and 3.5% of control men (p>0.05). Our data provide evidence for two large AZFc inversion polymorphisms, and for relative hot and cold spots of unequal crossing over within the blocks of homology that mediate gr/gr deletion. Using SFVs (sequence family variants), we discriminate DAZ1/2, DAZ3/4, CDY1a (proximal), and CDY1b (distal) and define four types of DAZ-CDY1 gr/gr deletion. CONCLUSIONS: The only deletion type to show an association with infertility was DAZ3/4-CDY1a (p = 0.042), suggesting that most gr/gr deletions are neutral variants. We see a stronger association, however, between loss of the CDY1a SFV and infertility (p = 0.002). Thus, loss of this SFV through deletion or gene conversion could be a major risk factor for male infertility.

Influence of ageing of residues on the availability of herbicides for leaching.

Losses by leaching of chlorotoluron, isoproturon and triasulfuron from small intact columns of a structured clay loam and an unstructured sandy loam soil were measured in five separate field experiments. In general, losses of all three herbicides were greater from the clay loam than from the sandy loam soil and the order between herbicides was always triasulfuron >> isoproturon > chlorotoluron. Differences between experiments were also consistent for every soil/herbicide combination. There was no relationship between total loss and either total rainfall or cumulative leachate volume. When weighting factors were applied to the rainfall data to make early rainfall more important than later rainfall, there were significant positive relationships between cumulative weighted rainfall and total losses. Also, there were significant negative correlations between total losses and the delay to accumulation of 25 mm rainfall (equivalent to one pore volume of available water) in the different experiments. In laboratory incubations, there was a more rapid decline in aqueous (0.01 M calcium chloride) extractable residues than in total solvent extractable residues indicating increasing sorption with residence time. However, the rate of change in water extractable residues could not completely explain the decrease in leachability with ageing of residues in the field. Short-term sorption studies with aggregates of the two soils indicated slower sorption by those of the clay loam than by those of the sandy loam suggesting that diffusion into and out of aggregates may affect availability for leaching in the more structured soil. Small scale leaching studies with aggregates of the soils also demonstrated reductions in availability for leaching as residence time in soil was increased, which could not be explained by degradation. These results therefore indicate that time-dependent sorption processes are important in controlling pesticide movement in soils, although the data do not give a mechanistic explanation of the changes in leaching with ageing of residues.

The identification and classification of 41 novel mutations in the factor VIII gene (F8C).

Hemophilia A is a bleeding disorder caused by a quantitative or qualitative deficiency in the coagulation factor VIII. Causative mutations are heterogeneous in nature and are distributed throughout the FVIII gene. With the exception of mutations that result in prematurely truncated protein, it has proved difficult to correlate mutation type/amino acid substitution with severity of disease. We have identified 81 mutations in 96 unrelated patients, all of whom have typed negative for the common IVS-22 inversion mutation. Forty-one of these mutations are not recorded on F8C gene mutation databases. We have analyzed these 41 mutations with regard to location, whether or not each is a cross-species conserved region, and type of substitution and correlated this information with the clinical severity of the disease. Our findings support the view that the phenotypic result of a mutation in the FVIII gene correlates more with the position of the amino acid change within the 3D structure of the protein than with the actual nature of the alteration.

Ouabain binding in the human brain. Effects of Alzheimer's disease and aging.

We studied Na+,K+-adenosine triphosphatase by assaying specific tritiated ouabain binding in the frontal cortex, temporal cortex, hippocampus, putamen, cerebellum, and cerebral microvessels in subjects with Alzheimer's disease and control subjects. Ouabain binds specifically, in a saturable manner, and with a high affinity to a single class of binding sites in all the tissues studied. The density of ouabain binding sites was highest in cerebellum and frontal cortex (approximately 40 pmol/mg of protein); intermediate in temporal cortex, hippocampus, and putamen; and lowest in brain microvessels (approximately 8 pmol/mg of protein). The dissociation constant of binding was about 30 nmol/L in all tissues. In control subjects, there were no age-related alterations in ouabain binding, nor was there any correlation between ouabain binding and postmortem delay. However, there was a marked decrease in brain ouabain binding in subjects with Alzheimer's disease when compared with age-matched controls, especially in the cerebral cortex. Ouabain binding was also significantly decreased in the cerebellum and putamen of subjects with Alzheimer's disease even though these brain regions are not particularly affected in this disease. Ouabain binding to brain microvessels, which constitute the blood-brain barrier, was not significantly decreased in subjects with Alzheimer's disease. The decreased specific ouabain binding in the brain of subjects with Alzheimer's disease probably reflects the loss of neuronal membranes.

Assessment of painful late effects of lumbar spinal fusion with SPECT.

UNLABELLED: The authors reviewed planar, SPECT and other contemporaneous radiologic images of the spine and the medical records of 33 patients with back pain after lumbar fusion surgery in order to determine the value of SPECT in the assessment of painful late effects of spinal fusion surgery. METHODS: Twenty-one patients had lateral fusion, nine patients had posterior fusion only and three patients had anterior and posterior fusions. There were 24 patients who had surgery more than 4 yr ago (late group, mean 11.8 yr) and 9 patients who had surgery less than 4 yr ago (early group, mean 17.8 mo). RESULTS: The most common SPECT abnormality in patients in the late group were lesions in the vertebral bodies and apophyseal joints in the free motion segments adjacent to the fused segments (62.5% of patients). Such lesions occurred in 46% of patients after lateral fusion, in 87.5% of patients after posterior fusion and in 67% of patients after posterior and anterior fusions. No SPECT abnormalities were detected in the fused segments in patients in the late group with solid lateral fusion but were detected in three patients with solid posterior fusion. These results correlate with biomechanical studies that have shown posterior fusion to produce the largest amount and lateral fusion to produce the least amount of stress in the free segments adjacent to the fusion. Lateral fusion was found to have a more stabilizing effect than posterior fusion. CONCLUSION: In addition to the already established value of SPECT in detecting painful pseudoarthrosis, our results indicate that SPECT is of value in the assessment of painful late effects of fusion.