Chemotherapy induces transient sex chromosomal and autosomal aneuploidy in human sperm.

Each year more than 20,000 children and young persons of reproductive age are exposed to known mutagens in the form of chemo- and/or radiotherapy for cancer in the States. As more of these treatments are effective there is growing concern that genetic defects are introduced in the germ cells of these young patients. It is well documented for male rodents that treatment with chemo- and radio-therapeutic agents before mating can cause genetic damage in the germ line, and the magnitude of heritable effects depends on the spermatogenic cell stage treated. Similar germinal effects are suspected to occur in humans but remain unproven. Hodgkin's disease (HD) is an example of a malignancy which is typically diagnosed during a patient's reproductive years. In our study we observed eight male HD patients who were treated with NOVP (Novanthrone, Oncovin, Vinblastine, Prednisone) chemotherapy. We evaluated sperm aneuploidy using multi-colour fluorescence in situ hybridization (FISH), and found approximately 5-fold increases in sperm with disomies, diploidies and complex genotypes involving chromosome X, Y and 8. Increases in sex chromosome aneuploidies arose from segregation errors at meiosis I as well as meiosis II. The aneuploidy effects were transient, however, declining to pretreatment levels within approximately 100 days after the end of the therapy. When compared with normal men, some HD patients showed higher proportions of certain sperm aneuploidy types even before their first therapy.

Effect of lifestyle exposures on sperm aneuploidy.

Lifestyle exposures including cigarette smoke, alcohol, and caffeine have all been studied in relationship to male reproductive health. Over the years the focus has primarily been on semen quality and/or fertility. More recently, literature evaluating direct adverse effects of lifestyle exposures on sperm chromosomes and chromatin has grown due to concern that induced damage could be transmitted to offspring causing transgenerational health effects. In this paper we present a new analysis that summarizes published studies of smoking effects on sperm chromosome number and demonstrates a statistically significant increase in sperm disomy among smokers compared to nonsmokers (P < 0.001). In addition, new data on the effect of alcohol intake on sperm chromosome number are presented showing a rate ratio of 1.38 (95% CI 1.2, 1.6) for XY frequency in sperm of alcohol drinkers compared to nondrinkers.

Organophosphorous pesticide exposure increases the frequency of sperm sex null aneuploidy.

It has been estimated that 4 of 1,000 live births and 35% of spontaneous abortions are aneuploid and that an important proportion of embryo and newborn aneuploidy is of paternal origin. Exposure to organophosphorous pesticides (OP) has been associated with sperm hyperploidy/polyploidy. Therefore, we aimed to assess the frequency of sperm aneuploidy (X, Y, and 18) and its relationship with urinary OP metabolites in agricultural workers. We performed multicolor fluorescence in situ hybridization on samples from nine men obtained before and during the pesticide spraying season to assess sperm aneuploidy. We measured urinary OP metabolite levels by gas-liquid chromatography. Aneuploidies were found in 0.67% of total sperm nuclei. The most frequent aneuploidy was the lack of a sexual chromosome or sex null (0.19%), followed by XY18 (0.15%) and XY18-18 (0.06%). OP metabolites detected at higher concentrations were dimethylthiophosphate, dimethyldithiophosphate, and diethylphosphate (DEP). There were no differences in average aneuploidy frequency or urinary metabolite levels between samples collected before and after exposure. However, Poisson regression analysis adjusted for age, alcohol intake, and sperm concentration showed significant associations between OP metabolite concentrations and increased frequency of sperm aneuploidies. The association was more evident between DEP and sex null, and the risk increased further during the spraying season. Thus, OP exposure could interfere with sperm chromosome segregation and increase the risk for genetic syndromes, such as Turner's. Further studies are required to assess the prevalence of spontaneous abortions, birth defects, and genetic syndromes in agricultural communities.

Detection of sex chromosomal aneuploidies X-X, Y-Y, and X-Y in human sperm using two-chromosome fluorescence in situ hybridization.

Sex chromosome aneuploidy is the most common numerical chromosomal abnormality in humans at birth and a substantial portion of these abnormalities involve paternal chromosomes. An efficient method is presented for using air-dried smears of human semen to detect the number of X and Y chromosomes in sperm chromatin using two-chromosome fluorescence in situ hybridization. Air-dried semen smears were pre-treated with dithiothreitol and 3,4-diiodosalicylate salt to decondense the sperm chromatin and then were hybridized with repetitive sequence DNA probes that had been generated by PCR and differentially labeled. Hybridizations with X and Y specific probes showed the expected ratio of 50%X:50%Y bearing sperm. Sperm carrying extra fluorescence domains representing disomy for the X or Y chromosomes occurred at frequencies of approximately 4 per 10,000 sperm each. Cells carrying both X and Y fluorescence domains occurred at a frequency of approximately 6/10,000. Thus, the overall frequency of sperm that carried an extra sex chromosome was 1.4/1,000. The frequencies of sperm carrying sex chromosome aneuploidies determined by hybridization did not differ statistically from those reported from the same laboratory using the human-sperm/hamster-egg cytogenetic technique. Multi-chromosome fluorescence in situ hybridization to sperm is a promising method for assessing sex-ratio alterations in human semen and for determining the fraction of sperm carrying sex or other chromosome aneuploidies which may be transmissible to offspring.

Use of fluorescence in situ hybridization (FISH) to assess effects of smoking, caffeine, and alcohol on aneuploidy load in sperm of healthy men.

Aneuploidy is a common cause of poor reproductive outcomes in humans and is associated with severe medical problems in liveborn offspring, yet little is known about its underlying cause. A substantial amount of aneuploidy is known to be contributed by the father through cytogenetically abnormal sperm. The purpose of this cross-sectional, observational study was to investigate the potential contribution of common lifestyle exposures (smoking, caffeine, and alcohol) to the aneuploidy load in sperm from 45 healthy male volunteers 19-35 years of age. Sperm FISH (fluorescence in situ hybridization) was used to determine aneuploidy and diploidy frequencies for chromosomes X, Y and 18 across varying exposure levels of smoking, caffeine, and alcohol. Caffeine was significantly associated with increased frequencies of sperm aneuploidy XX18 and XY18, diploidy XY18-18 and the duplication phenotype YY18-18 controlling for alcohol, smoking and donor age. Alcohol was significantly associated with increased frequencies of sperm aneuploidy XX18, diploidy XY18-18 and the duplication phenotype XX18-18 controlling for caffeine, smoking and donor age. There was a suggestive, but unstable, association between smoking and XX18. Even within our truncated age range, we were able to confirm an increased risk for XX18 aneuploidy with increasing donor age. Sperm FISH proved to be a useful biomarker to detect and compare numerical cytogenetic abnormalities in human sperm cells across differing levels of exposure to smoking, caffeine, and alcohol.

Evaluation of a container for collection and shipment of semen with potential uses in population-based, clinical, and occupational settings.

Large, population-based studies of semen quality are encumbered by the logistics and expense of obtaining semen samples from men who live in a variety of locations. A prototype semen collection and transportation kit, the TRANSEM100, can be distributed to study participants and then directly shipped to a central laboratory for analysis. This study was designed to evaluate the ability of male volunteers to correctly use the kit. Thirty volunteers aged 20 to 44 years with no history of diabetes, recent chemotherapy, fertility problems, or vasectomy were recruited through a newspaper advertisement, interviewed to obtain demographic information, and instructed on the use of the kit. Twenty-six of the initial subjects provided at least 1 semen specimen using the kit and returned the specimens by overnight delivery to the laboratory for analysis, 25 completed a follow-up interview on the use of the collection kit, and 20 submitted a second semen sample using the same method. The average volunteer was white, 27.8 years old, and held at least a college degree. Forty percent of the volunteers were married. In general, participants correctly followed the instructions for collecting, packaging, and shipping the semen samples. Volunteers were instructed to collect samples after at least 2, but no more than 7 days of abstinence. For the first and second samples submitted, participants collected semen samples after an average of 3.3 and 3.9 days of abstinence, respectively. Seventeen (65%) of the samples from the first sampling period and 16 (80%) of the samples from the second period were received in the laboratory the day after they had been collected. In summary, the TRANSEM100 may prove to be useful for collecting human semen in field studies. Further testing of this method is warranted to evaluate preservation of sample quality and use of the kit by men among diverse socioeconomic groups.

Cytogenetic damage measured in human sperm following cancer chemotherapy.

Germ-line cytogenetic damage is well documented in laboratory animals exposed to anti-cancer agents, but has been harder to verify in the human. This paper reviews published studies demonstrating cytogenetic damage in human sperm following exposure to anti-cancer chemicals, as measured by the human-sperm/ hamster-egg cytogenetic technique and fluorescence in situ hybridization. These two assays have provided important information on one step in the pathway leading to induced, transmissible germ line damage in the human. By way of introduction, a short review of the traditional human endpoints used to address the question of induced, transmissible genetic damage in human germ cells (mutation epidemiology) related to anti-cancer chemicals is presented.

Three-probe fluorescence in situ hybridization to assess chromosome X, Y, and 8 aneuploidy in sperm of 14 men from two healthy groups: evidence for a paternal age effect on sperm aneuploidy.

The method of simultaneous three-chromosome fluorescence in situ hybridization (FISH) was developed using repetitive DNA sequence probes for chromosomes 8, X and Y and applied to semen of 14 men from two healthy groups who differed in their average ages (46.8 +/- 3.1 years, n = 4 v. 28.9 +/- 5.0 years, n = 10). The frequencies of disomic sperm determined by FISH compared well with frequencies obtained using the hamster-egg technique for human-sperm cytogenetics and with the frequencies of disomic and diploid sperm reported in previous FISH studies in this laboratory. The two groups of men did not differ in their baseline frequencies of sperm disomic for chromosome 8 (approximately 6.5 per 10(4) sperm), sperm with XY8 aneuploidy (approximately 9.5 per 10(4) sperm), or sperm with autodiploidy XX88 or YY88 (approximately 2 per 10(4) sperm). However, the older group had statistically higher frequencies of sperm carrying sex chromosomal disomy than the younger group (5.1 v. 2.2 per 10(4) sperm for XX8; 5.9 v. 2.0 per 10(4) sperm for YY8; P < 0.005). A recent report from this laboratory of sex-chromosomal aneuploidy in sperm of aged mice provides inter-species corroborating evidence for this preliminary finding of a paternal age effect on sperm aneuploidy in human males.

Accounting guidelines for HMOs: issues and practices.

HMOs are fast becoming an important part of the healthcare industry today. Unfortunately, specific accounting guidelines have not yet been established for them. This concern has led the AICPA to describe and offer recommendations concerning preferred accounting techniques for HMOs. This article looks at the issues raised by the AICPA and how current HMO financial officers feel about the practices that are recommended. It is suggested that the organization, taxability, and other attributes of an HMO must be clearly understood before strict accounting guidelines are imposed.

Cost variances in health care: when should managers investigate?

When costs must be controlled, variance analysis can be a useful tool to implement that control. Variance analysis compares a standard of performance against actual results and investigates those differences that are felt to be the result of inefficient performance. The question becomes, which variances should be investigated? Using a decision model based on probability theory, variances can be identified that are statistically significant and require further investigation.

Accounting practice diversity in the healthcare industry.

A recent study examining accounting practices currently being used to prepare annual hospital financial statements indicates relatively little diversity, regardless of organizational type or size. The study's findings should interest those concerned with healthcare accounting and financial reporting issues, especially healthcare administrators and members of standards setting boards who participate in accounting policy deliberations.

Evaluation of aneuploidy and DNA damage in human spermatozoa: applications in field studies.

With the goal of incorporating measures of sperm nuclear integrity in an epidemiology study, semen samples from young Czech men were analysed for sperm aneuploidy and sperm chromatin structure in addition to routine measures of sperm production and quality. The exposure in question was to high seasonal air pollution containing reactive polyaromatic hydrocarbons potentially capable of affecting spermatogenesis and damaging sperm DNA. The sperm aneuploidy assay uses fluorescence in situ hybridization to label selected sperm chromosomes; as applied in this study, the sex chromosomes (X,Y) and chromosome 8 were targeted. The sperm chromatin structure assay detects sperm nuclei with increased susceptibility to denaturation, a feature that is associated with DNA damage. Logistically, these assays were relatively easy to incorporate into the study design. The aneuploidy assay provided information suggesting that exposure to high levels of air pollution may increase the risk of sperm aneuploidy and that it is important to control for exposure to cigarette smoke and/or alcohol in such studies. The sperm chromatin structure assay provided valuable baseline information about Czech semen donors and data suggestive of an adverse effect of smoking and air pollution on spermatozoa that merits further investigation.

Detection of aneuploid human sperm by fluorescence in situ hybridization: evidence for a donor difference in frequency of sperm disomic for chromosomes 1 and Y.

Fluorescence in situ hybridization with repetitive-sequence DNA probes was used to detect human sperm disomic for chromosomes 1 and Y in three healthy men. Data on these same men had been obtained previously, using the human-sperm/hamster-egg cytogenetic technique, providing a cytogenetic reference for validating sperm hybridization measurements. Air-dried smears were prepared from semen samples and treated with DTT and lithium diiodosalicylate to expand sperm chromatin. Hybridization with fluorescently tagged DNA probes for chromosomes 1 (pUC177) or Y (pY3.4) yielded average frequencies of sperm with two fluorescent domains of 14.2 +/- 2.4/10,000 and 5.6 +/- 1.6/10,000 sperm, respectively. These frequencies did not differ statistically from frequencies of hyperploidy observed for these chromosomes with the hamster technique. In addition, frequencies of disomic sperm from one donor were elevated approximately 2.5-fold above those of other donors, for both chromosomes 1 (P = .045) and Y (P = .01), consistent with a trend found with the hamster technique. We conclude that fluorescence in situ hybridization to sperm chromosomes provides a valid and promising measure of the frequency of disomic human sperm.

The role of cochlear implants in deaf children.

Advances in technology regarding cochlear implantation and electroacoustic stimulation of the inner ears of deaf children has created cautious enthusiasm. The safety of cochlear implantation is now reasonably well established and language performance measures made over time are encouraging. When coupled with an effective (re)habilitation program, the cochlear implant promises to favorably influence the potential of deafened children.

Antiretroviral therapy effects on genetic and morphologic end points in lymphocytes and sperm of men with human immunodeficiency virus infection.

Many human immunodeficiency virus (HIV)-infected persons receive prolonged treatment with DNA-reactive antiretroviral drugs. A prospective study was conducted of 26 HIV-infected men who provided samples before treatment and at multiple times after beginning treatment, to investigate effects of antiretrovirals on lymphocyte and sperm chromosomes and semen quality. Several antiretroviral regimens, all including a nucleoside component, were used. Lymphocyte metaphase analysis and sperm fluorescence in situ hybridization were used for cytogenetic studies. Semen analyses included conventional parameters (volume, concentration, viability, motility, and morphology). No significant effects on cytogenetic parameters, semen volume, or sperm concentration were detected. However, there were significant improvements in sperm motility for men with study entry CD4 cell counts >200 cells/mm(3), sperm morphology for men with entry CD4 cell counts < or =200 cells/mm(3), and the percentage of viable sperm in both groups. These findings suggest that nucleoside-containing antiretrovirals administered via recommended protocols do not induce chromosomal changes in lymphocytes or sperm but may produce improvements in semen quality.