Cancer Research CTRC-AACR San Antonio Breast Cancer Symposium Joint Metastasis Research Society-AACR Conference on Metastasis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Frias, S.
Right arrow Articles by Wyrobek, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Frias, S.
Right arrow Articles by Wyrobek, A. J.
[Cancer Research 63, 44-51, January 2003]
© 2003 American Association for Cancer Research

Epidemiology and Prevention

NOVP Chemotherapy for Hodgkin’s Disease Transiently Induces Sperm Aneuploidies Associated with the Major Clinical Aneuploidy Syndromes Involving Chromosomes X, Y, 18, and 211

Sara Frias, Paul Van Hummelen, Marvin L. Meistrich, Xiu R. Lowe, Fredrick B. Hagemeister, Michael D. Shelby, Jack B. Bishop and Andrew J. Wyrobek2

Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California 94550 [S. F., P. V. H., X. R. L., A. J. W.]; Laboratorio de Citogenetica, Instituto Nacional de Pediatria Secretaria de Salud and Facultad de Ciencias, Universidad Nacional Autónoma de Mexico, Mexico Distrito Federal [S. F.]; Departments of Experimental Radiation Oncology [M. L. M.] and Lymphoma and Myeloma [F. B. H.], The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030; and the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 [M. D. S., J. B. B.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The objective of this research was to determine whether Novantrone, Oncovin, Velban, and Prednisone (NOVP) combination chemotherapy for Hodgkin’s disease increases the frequencies of the specific types of aneuploid sperm that might elevate the risk of fathering a child with one of the major clinical aneuploidy syndromes, i.e., Down (disomy 21 sperm), Edward (disomy 18 sperm), Turner (nullisomy sex sperm), XYY (disomy Y sperm), triple X (disomy X sperm), or Klinefelter (XY sperm). A four-chromosome multicolor sperm fluorescence in-situ hybridization assay that simultaneously evaluates chromosomes 18, 21, X, and Y was applied to semen provided by four healthy men and to repeated samples of eight Hodgkin’s disease patients before treatment, 35–50 days after treatment to examine the effects of treatment on male meiotic cells, and 1–2 years after treatment to measure the persistence of damage. There were chromosome-specific variations in baseline frequencies and significant inductions of all of the detectable types of sperm aneuploidies: XY sperm (14-fold increase), disomy 18 (7-fold), nullisomy sex (3-fold), disomy 21 (3-fold), and disomy X and Y (~2-fold each). Disomy 21 was about twice as frequent as disomy 18, and neither showed a preferential segregation with a sex chromosome. Extrapolating across the genome, ~18% of sperm carried a numerical abnormality after NOVP treatment of meiotic cells. Induced effects did not persist to 1–2 years after treatment, suggesting that persistent spermatogonial stem cells were not sensitive to NOVP. These findings establish the hypothesis that conception shortly after certain chemotherapies can transiently increase the risks of fathering aneuploid pregnancies that terminate during development or result in the birth of children with major human aneuploidy syndromes.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chromosomal abnormalities and gene mutations play major roles in pregnancy loss, birth defects, and genetic disease. At least 15% of clinically recognized pregnancies result in spontaneous abortion; ~30% of these and 0.6% of live births have detectable chromosomal anomalies (1) . Furthermore, ~80% of all chromosome abnormalities and 20% of single gene disorders arise de novo in the germ cells of either parent (2) . It is not well understood whether these defects are spontaneous or mutagen-induced. In mice, parental exposure to irradiation or certain chemical mutagens before mating is known to induce embryo lethality, transmissible chromosomal translocations, and gene mutations, as well as cancer in offspring (3, 4, 5, 6) . Despite considerable indirect evidence linking certain paternal exposures to abnormal pregnancy outcomes and childhood cancer (6, 7, 8, 9) , there is still no conclusive evidence for the existence of any human germ-cell mutagen.

Cancer chemotherapies represent widespread human exposures to high doses of chemicals known to induce chromosomal abnormalities in animal models (5 , 10 , 11) . Epidemiological investigations have provided generally negative data, although potent mutagens are among the drugs involved (12, 13, 14) . However, the conclusions that can be drawn from these studies were very limited in several regards: (a) most had only the statistical power to detect a 2–3-fold or greater increase in abnormal reproductive outcomes; (b) most patients were treated as children; (c) outdated drugs and/or treatment regimens were evaluated; (d) studies mixed patient data from both mutagenic and nonmutagenic treatments; and (e) in general, they only studied conceptions that occurred at long times after treatment. Thus, there remains serious concern over the induction of transmissible mutations in germ cells in patients treated before or during their reproductive years, especially those with newer chemotherapy regimens that have high cure rates. An alternative and, as yet, under-utilized approach to assessing the potential heritable risk would be to first identify specific drugs and treatment regimens that might produce genetic defects in spermatozoa, particularly at the chromosomal level (8 , 15) . Although >100 agents have been evaluated for their toxic effects on human sperm production (~50 diminished semen quality; Ref. 16 ), there are very few data regarding the induction of genetically and chromosomally damaged sperm (6 , 9, 10, 11, 12, 13, 14, 15, 16, 17) .

Unlike most cancers that afflict persons well beyond their reproductive years, HD3 has a bimodal age-incidence curve with a major peak at ~25 years of age; it also has a high cure rate of ~80% (18) . In the past, HD was treated with regimens consisting of high-dose alkylating agents such as nitrogen mustard and procarbazine, but these drugs are now seldom used because of their reproductive toxicities and carcinogenic potential. Newer regimens, such as ABVD, avoid high doses of alkylating agents, have fewer side effects, and show excellent recovery of reproductive function (19) . Other regimens have been developed to additionally minimize reproductive toxicity, such as NOVP (20) . However, both ABVD and NOVP include drugs that produce aneuploidy in model systems and are suspected of having undesirable genetic side effects in human germ cells (21, 22, 23) . Two drugs, vinblastine and vincristine, are known to disrupt the spindle apparatus, prevent tubulin polymerization, cause aneuploidy in somatic cells (24) , and induce chromosome malsegregation at meiosis I and II in female germ cells (7) . In prior studies of NOVP chemotherapy, we reported transient side effects on semen quality (25) , as well as increases in the frequencies of sperm with XY, disomy X, and disomy 8 (15) . These findings raised the clinically relevant question of whether NOVP therapy increased the frequency of the various aneuploid sperm that are associated specifically with the major autosomal and sex-chromosomal aneuploidy syndromes in children, i.e., Down, Edward, Turner, triple X, XYY, and Klinefelter syndromes.

We applied the four-chromosome X-Y-18-21 sperm FISH assay (26) to semen from HD patients who were treated with NOVP to measure the frequencies of 5 types of disomy (for each of the four chromosomes plus XY), 4 types of nullisomy (for each of the four chromosomes), 3 types of diploidy (one from meiosis I and two from meiosis II), plus a large variety of complex genotypes. Our study was designed to address the following questions: (a) do men who receive NOVP chemotherapy for HD produce elevated frequencies of aneuploid sperm that might increase their risk of fathering children with any of the major aneuploidy syndromes; (b) what is the relative variation in baselines and relative induction among these clinically relevant sperm aneuploidies; and (c) is there a lack of persistence of induced aneuploidy across all of the clinically relevant aneuploidies? In addition, we determined whether sperm disomy 21 and 18 were associated with either sex chromosome, as reported previously for Y sperm with disomy 21 (27) . Our findings were then interpreted in light of the risk of fathering pregnancies that terminate spontaneously or result in the birth of a child carrying a major constitutive aneuploidy syndrome.


   SUBJECTS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cancer Patients and Their Specimens.
Twelve specimens were provided by eight patients (26–42 years of age) who were diagnosed with HD stages I–II at the M.D. Anderson Cancer Center. The samples used in this study are a subset of patients who each provided during and post-treatment specimens (15) . This research protocol was approved by Lawrence Livermore National Laboratory and M. D. Anderson Cancer Center Institutional Review Boards. All of the donors gave their informed consent to have their specimens be included in the study. As detailed in Table 1Citation , three groups of specimens were investigated: pretreatment samples (from donors A, B, C, D, and E), during-treatment samples (from donors F, G, and H), and post-treatment samples (from B, F, G, and H). Pretreatment specimens were provided after diagnosis but before initiating therapy. NOVP chemotherapy included three cycles of 21 days, each cycle consisted of: a single day i.v. drip of Novantrone (mitoxantrone) 10 mg/m2 and Oncovin (vincristine) 2 mg/m2 on day 1, single day i.v. drip of Velban (vinblastine) 6 mg/m2 on day 8, and Prednisone 100 mg/day given orally on days 1–5 (25 , 28) . During-treatment specimens contained sperm that had been somewhere during meiosis (i.e., spermatocytes) at the time of drug treatment. Specifically, these samples were provided within a time window of 35 to 50 days after one of the three cycles of therapy. Samples meeting these criteria were included even when the patient had already begun a subsequent cycle of therapy, because it is unlikely that postmeiotic treatment with any of these drugs would affect the frequency of aneuploid sperm. Post-treatment samples were collected between 1 and 2 years after the last cycle of chemotherapy. All of the patients in the post-treatment group received mantle radiotherapy, whereas the gonadal dose received was considered negligible. Only patient F received abdominal radiotherapy 96–124 days after chemotherapy with a cumulative gonadal dose of 26 cGy.


View this table:
[in this window]
[in a new window]
  		Table 1 Disease stage, age, and semen quality in HD patient groups and healthy reference group

This table summarizes the disease stage, age, and conventional semen quality of the patients and healthy donors who participated in this study. Conventional semen quality was determined using WHO criteria.

 
Semen specimens were collected by the patient into sterile plastic containers and permitted to liquefy for up to 2 hours at room temperature before processing. Volume (ml), number of sperm per ml, and motility were determined by conventional analyses using the criteria established by the WHO (29) ; aliquots of specimens were stored at -80°C without cryopreservative and shipped frozen to the Lawrence Livermore National Laboratory for FISH analyses. A concurrently analyzed reference group consisted of four healthy, nonsmoking workers at a biomedical research laboratory, 45–51 years of age, with normal semen quality (Table 1)Citation .

Sperm FISH.
Semen was thawed at room temperature and 5–7 µl was smeared onto ethanol (100%)-cleaned slides. Smears were air-dried overnight and then pretreated for 15 min in methanol (100%), decondensed for 30 min in a 10 mM DTT (Sigma, St. Louis, MO)/0.1 M Tris-HCl on ice, followed by 90 min in 4 mM lithium diiodosalicylate (Sigma)/0.1 M Tris-HCl at room temperature. Smears were denatured in a 70% formamide (IBI, New Haven, CT)/2x SSC [0.3 M NaCl and 0.03 M Na citrate (pH 7.0)] at 76–78°C for 6 min and then dehydrated 2 min in 70%, 85%, and 100% ethanol. The X-Y-18-21 multicolor sperm FISH assay was applied (26) using the following DNA probes obtained from Vysis (Downey Grove, IL): (a) the {alpha} satellite region of chromosome X fluoresced in yellow using an equimixture of the {alpha} centromeric probes CEPX Spectrum Green and CEPX Spectrum Orange; (b) the satellite III of the Y chromosome fluoresced in blue using CEPY Spectrum Aqua; (c) the {alpha} satellite of chromosome 18 fluoresced in green, using CEP18 Spectrum Green; and (d) the chromosome 21 region in 21q22.13-q22.2, (which includes D21S259, D21S341, and D21S342) fluoresced in red using LSI21 Spectrum Orange. These DNA probes were precipitated with 100% ethanol/3 M sodium acetate using a standard protocol, resuspended into 7 µl of hybridization mix (Vysis) and 3 µl of water, denatured in formamide 70%/2x SSC at 76–78°C for 6 min, and immediately put onto smears, which were denatured under the same conditions. Hybridization was performed overnight (moist chamber at 37°C) and washed (10 min in 50% formamide/2x SSC at 45°C, 10 min in 2x SSC at 37°C, and then 10 min at room temperature). Sperm nuclei were counterstained with DAPI 0.01–0.05 µg/ml in Vectashield (Vector Laboratories, Burlingame, CA). Hybridization efficiency (number of cells with at least one fluorescent signal) was >99% for this study.

Microscope Scoring.
Analysis was performed on a Zeiss Axioplan fluorescence microscope equipped with phase contrast optics, HBO 100 W/2 mercury lamp (Osram), and the following filters (Chroma Technology, Brattleboro, VT): DAPI/FITC/Texas red (triple band), FITC-Texas Red, and DAPI-Aqua (double band) filters, and DAPI, FITC, and Texas Red (single band) excitation filters. At least 10,000 cells/specimen were scored using the following protocol: a randomized group of slides were coded by a coder (someone who was not involved in the scoring), 5,000 cells were scored per slide by the scorer on half of the slide, slides were then recoded by the coder, and an additional 5,000 cells were scored on a different area of each slide. The two data sets for each specimen were utilized when the two sets of 5,000 cells were not statistically different using 2 x 2 contingency tables. When there was a statistical difference between the two sets, the recoding and scoring process was repeated.

We applied the strict scoring criteria (26) . For a nucleus to be considered as scorable, it had to be physically separated from other nuclei, be decondensed, and all of its fluorescent domains had to be inside the nuclear perimeter as determined under DAPI filter. To be considered as two separated domains, two domains of the same color had to be separated by a distance of at least half of the average domain, and had to be similar to each other in shape and intensity. Every cell was analyzed under triple band filter to simultaneously visualize the nucleus in blue, as well as the red, green, aqua, and yellow domain signals. Abnormal nuclei were also analyzed under a double band (red and green) and single band filters to discriminate single colors and to determine superposition of signals. Low-intensity, phase-contrast imaging was used to determine the presence of flagella, and to avoid confusion between sperm and somatic cells. A separate 18–21 sperm FISH assay was applied to selected specimens in our study to demonstrate reproducibility of the disomy 18 and 21 data.

Statistics.
Sperm counts were compared using the randomization test (30) . Nonparametric tests, Mann Whitney-U and Kruskal Wallis were used to analyze the frequencies of fluorescent phenotypes among the different patient groups. The paired t test was used for comparisons between samples from the same donor.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Semen Quality in Specimens Analyzed.
Table 1Citation lists the disease stage, age, and semen qualities for the patient and samples used in this study. According to WHO criteria, semen quality was generally within normal ranges for all of the parameters for all of the specimens, including those of the during-treatment group. Although semen volume was higher and sperm concentration was lower for the during-treatment versus the pretreatment group (Table 1)Citation , these differences were not statistically significant (P = 0.1). The essentially normal semen quality in the first 50 days after initiation of chemotherapy and at 1–2 years after chemotherapy emphasizes the importance of understanding the burden of chromosomal abnormalities in sperm, because fertility is possible.

Sperm Aneuploidy Was Induced by NOVP.
As shown in Figure 1Citation , the aggregate frequencies of sperm carrying any of the detectable numerical abnormalities involving chromosomes X, Y, 18, or 21 (sum of all of the detectable disomies, nullisomies, or diploidies) were ~5-fold higher in the during-treatment than in the pretreatment group (fold difference is calculated as the average frequency of the during-treatment group divided by the average frequency of the pretreatment group; P = 0.02). As additional evidence that NOVP induced sperm aneuploidy, there were significant declines in the frequencies of aneuploid sperm over time for each of the three patients who provided specimens in both the during- and post-treatment patient groups (Figure 1Citation ; P = 0.03).



View larger version (14K):
[in this window]
[in a new window]
  		Fig. 1. Effects of NOVP chemotherapy on the overall proportion of sperm carrying any numerical chromosomal abnormalities detectable by the X-Y-18-21 sperm FISH assay (disomy, nullisomy, diploidy, and unusual phenotypes). Y axis represents the frequency of abnormal sperm per 10,000 sperm analyzed per sample. The horizontal continuous and dashed lines represent the mean and mean plus one SD, respectively, of the healthy reference group. Each symbol represents a different patient: patient A; {blacklozenge} patient B; patient C; xpatient D; {square} patient E; {blacksquare} patient F; {blacktriangleup} patient G; and {bullet} patient H. A solid line connects the values for two samples provided by the same donor. Pretreatment specimens (Pre) were provided after diagnosis but before initiating therapy. During-treatment samples (Dur) were provided within a time window of 35–50 days after a cycle of therapy to collect cells that were in meiosis during treatment. Post-treatment samples (Post) were collected between 1 and 2 years after the last cycle of chemotherapy.

 
As shown in Table 2Citation , sperm disomies for chromosomes 18 and 21 were elevated ~7-fold (P = 0.02) and ~3-fold (P = 0.05), respectively, in the during-treatment group (versus pretreatment). The frequencies of nullisomic sperm for both chromosomes were also increased in the during-treatment group, but not significantly (Table 2)Citation . Overall, the frequency of sperm with disomy 21 was 2–5-fold higher than disomy 18 among the various comparison groups (P < 0.05).


View this table:
[in this window]
[in a new window]
  		Table 2 Chromosome 18 and 21 disomy and nullisomy in sperm of HD patients treated with NOVPa

This table summarizes the sperm data for aneuploidies involving chromosome 18 and 21, which are two of the chromosomes used in the four-chromosome sperm FISH assay.

 
Using the X-Y-18-21 assay, the aggregate frequency of sperm with sex-chromosomal aneuploidies (sum of frequencies of sperm with disomy X, disomy Y, XY sperm, and sex-null sperm) were ~4-fold higher in the during- versus pretreatment groups (Table 3Citation ;P < 0.05). All of the categories of sex-chromosomal aneuploidies were significantly induced, but there was substantial variation. Compared with pretreatment values (Table 3)Citation , the frequency of XY sperm was increased ~14-fold in the during-treatment group (P = 0.02), and disomy X plus Y were significantly elevated ~2-fold (P = 0.02). The frequencies of sex-null sperm were also significantly elevated ~3-fold in the during-treatment group (P < 0.05). Thus, our new data with 4-chromosome assay shows that NOVP induced disomy X, XY sperm, disomy Y, and sex-null sperm, as well as sperm with autosomal aneuploidies.


View this table:
[in this window]
[in a new window]
  		Table 3 Sex chromosomal disomy and nullisomy in sperm of HD patients treated with NOVPa

 
Sperm with Diploidies and Complex Phenotypes Were Also Induced by NOVP.
The aggregate frequency of diploid sperm (i.e., sum of XY, XX, and YY diploidies) was at ~7-fold higher frequency in the during-treatment than in the pretreatment group (32.5 ± 7.2 versus 230.4 ± 29.1; P < 0.05). Fig. 2Citation illustrates the variations in induction among the diploidy categories: XY diploidy (~9-fold; 17.5 ± 7.8 versus 157.8 ± 30.7), XX diploidy (~6-fold; 6.7 ± 2.3 versus 39.8 ± 4.5), and YY diploidy (~4-fold; 8.3 ± 2.3 versus 32.7 ± 10.0). Fig. 3Citation suggests that the slightly elevated aneuploidy frequencies among pretreatment patients (P = 0.08 compared with healthy men) may be because of a significant difference in the frequency of diploid sperm (P = 0.05), specifically YY sperm diploidy (P = 0.01).



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2. Meiosis I and II sperm diploidy in HD patients before, during, and after NOVP chemotherapy. Donor symbols as in Fig. 1Citation . A, diploid sperm carrying both X and Y sex chromosomes, which presumably represents a meiosis I error. B, the sum of the frequencies of diploid sperm carrying two X chromosomes plus the diploid sperm carrying two Y chromosomes, both of which represent meiosis II errors.

 


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3. Comparison of the genome-wide frequencies of sperm with disomy and diploidy between a group of healthy donors ({square}) and the group of pretreatment HD patients (). Bars, ±SE. See text for the calculation of genome-wide disomy estimate.

 
Four major categories of sperm with unusual phenotypes were detected using the four-chromosome sperm FISH (Table 4)Citation : (a) diploid cells that lost one chromosome (monosomic for one chromosome); (b) diploid cells that gained one chromosome (trisomic for one chromosome); (c) tetraploid cells; and (d) "others" with unusual combinations of fluorescent domains. The total frequency of unusual sperm (Table 4)Citation was also elevated significantly during treatment (P < 0.02).


View this table:
[in this window]
[in a new window]
  		Table 4 Unusual sperm genotypes detected with the four-chromosome sperm FISH assay in HD patients treated with NOVP

This table summarizes the frequencies of the sperm carrying unusual genotypes as detected by the four-chromosome sperm FISH. These genotypes typically involve at least major types of segregation errors. Every number is the average frequency per 10,000 sperm ± SE.

 
Lack of Persistence of the NOVP Treatment Effects.
Persistence of numerical chromosomal abnormalities was assessed by comparing during- and post-treatment specimens for each patient who gave both samples, and by comparing pre- and post-treatment groups. As shown in Fig. 1Citation , the aggregate frequencies of aneuploid sperm in each of the three men of the during-treatment group diminished significantly and approached pretreatment levels with time for each patient (P < 0.05). Results from patient B, who provided both pre- and post-treatment specimens (but not a during-treatment specimen), also support the finding of a lack of persistence of chromosomal abnormalities by 1 year after NOVP therapy. As shown in Table 2Citation , the frequencies for sperm disomies for chromosomes 18 and 21 were reduced significantly between the during- and post-treatment samples, and were similar in the pre- and post-treatment groups. The same pattern of response was seen for diploid sperm, sperm with sex-chromosomal aneuploidies, and sperm with complex genotypes, i.e., there was a significant reduction between the during- and post-treatment frequencies, whereas the pre- and post-treatment samples were essentially at the same frequencies. However, for the sex-chromosomal aneuploidies and diploidies, there was a consistent trend for most post-treatment samples to be nonsignificantly elevated above their pretreatment controls (Table 3Citation ; Fig. 2Citation ).

Disomy 18 and 21 Was Not Preferentially Associated with Either Sex Chromosome.
No significant difference was found in the frequency of disomic 18 or 21 sperm carrying X versus Y chromosomes among any of the semen specimens analyzed in this study, including those from both HD patients and healthy donors. This is contrary to the finding of Griffin et al. (27) but is consistent with our finding with healthy men (31) . Among all of the HD patients, the mean frequency of sperm with disomy 18 with an X chromosome was 3.7 and with a Y chromosome was 2.8 (per 104 sperm; P = 0.26); the mean frequency of disomy 21 with X was 7.7 and with Y was 7.9 (P = 0.8).


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our study applied a previously validated, four-chromosome sperm FISH assay (26) to demonstrate that: (a) men who received NOVP therapy produced elevated proportions of aneuploid and diploid sperm involving chromosomes 18, 21, X, and Y; and (b) these effects did not persist to 1 year after the end of treatment. The magnitudes of the treatment-induced effects varied among chromosomes tested. The magnitudes of the treatment-induced effects varied among chromosomes tested. The aneugenic effects of NOVP on sex chromosomal aneuploidy appeared to be primarily on male meiosis I rather than II. This confirms earlier findings (15) that NOVP induced disomy X and XY in sperm, and provides new data showing that NOVP also induces sperm with disomy Y, 18, and 21, as well as nullisomy of sex chromosomes. These findings suggest that conception during or shortly after NOVP therapy may raise the risk of fathering a child with any of the major constitutive aneuploidy syndromes.

Special design features were utilized to compensate for the small numbers of patients available for analyses: (a) each of the patients sampled during therapy was also sampled 1 year after therapy to provide within-person comparisons of time-dependent effects (to compare meiotic versus stem cell effects); (b) drug dosimetry was well documented for each patient (data not shown); (c) conventional semen analyses showed that the specimen analyzed had semen quality within normal ranges; (d) the X-Y-18-21 sperm FISH assay provides measurements of multiple categories of aneuploidies and diploidies for making interchromosomal comparisons; and (e) specimens from a healthy reference group were included and analyzed concurrently to determine whether the semen of Hodgkin’s patients might have been atypical even before therapy began.

The effects of NOVP did not persist at 1 year after treatment for any of the aneuploidy or diploidy categories measured by the four-chromosome assay, as determined by group-to-group comparisons and by within-patient changes over time. This is consistent with previous results for disomy X and XY sperm (15) , and now also shows that disomy Y, nullisomy sex chromosomes, disomy 18, and disomy 21 have the same transient pattern. The literature data on persistence of sperm damage after chemotherapy is ambiguous, but the numbers of patients and specimens analyzed per patient were generally very small. Several studies suggest that sperm chromosome damage does not persist after therapy: in one patient studied 9 months after treatment with cisplatin, vinblastine, and bleomycin for seminoma (32) , in one patient studied 3 years after treatment for lymphoma, and in four patients studied 2–13 years after treatment for testicular cancer (33, 34, 35) . Other studies suggest persistence of damage: in one patient treated with cisplatin, vinblastin, bleomycin, ifosphamide, vepesid, dactinomycin, clorambucil, and methotrexate (36) , in six patients 3–20 years after MOPP treatment (nitrogen mustard, oncovin, procarbazine and prednizone; Ref. 22 ), and in one patient treated one year before with bleomycin, etoposide, and cisplatin (37) . A study based on one patient (23) suggest that vinblastine might be aneugenic in spermatogenic stem cells, but this is inconsistent with our current findings and those of Robbins et al. (15) showing that sperm aneusomy returned to baseline values by 100 days after treatment with NOVP, which includes vinblastine. The lack of persistence of chromosomal aneuploidies at 1–2 years after NOVP treatment, suggests that there are no long-term effects of this therapy on sperm aneuploidy in stem cells. This agrees with the findings of Meistrich et al. (25) , who showed that stem cells were not killed by NOVP, with 90% survival after therapy.

Variation in Frequencies of Aneuploid Sperm.
We observed three types of variation among the donors: (a) baseline variations among chromosomes, i.e., pretreatment values; (b) treatment-induced variation among chromosomes; and (c) variations among the men of the during-treatment group. Average baseline frequencies (x10-4) of aneuploid sperm in healthy men varied, in increasing order, from: disomy Y = ~0, disomy 18 = 1.4, disomy X = 3.0, XY aneuploidy = 5.0, and disomy 21 = 6.9. The variation of induction among chromosomes in the during-treatment group ranged from 2- to 14-fold among the five disomy categories: disomies X and Y = ~2-fold each, disomy 21 = ~3-fold, disomy 18 = ~7-fold, and XY = ~14-fold. Robbins et al. (15) showed previously that disomy 8 was induced ~3-fold, which is near the low end of the values for autosomes in the current study. Disomy 21 was consistently two times higher than disomy 18 in the pre-, during-, and post-treatment groups (Table 2)Citation . Baseline frequencies for nullisomy 18 and 21 were generally higher than for the corresponding disomy, but the fold induction was also less, so that the induced values of disomy and nullisomy were similar for these chromosomes (Table 2)Citation .

All of the study groups showed a higher frequency for disomy 21 than for the other chromosomes tested. However, there remains some uncertainty as to whether this represents a true differential susceptibility of chromosome 21 to meiotic nondisjunction. The probe for chromosome 21 is different from the other probes in two relevant ways: its target sequence is relatively short and it is located distally at 21q22.3. Thus, it is possible that tiny splits in the signal or products of chromosome breakage will be incorrectly scored as disomy 21. However, there is corroborating evidence in favor of increased differential susceptibility for chromosome 21 nondisjunction in meiotic cells (37, 38, 39, 40, 41, 42) , whereas the data with the hamster-egg system remain ambiguous: two studies found that disomy 21 sperm were elevated among healthy men (43 , 44) , whereas another (22) found average frequencies among other healthy men. Our sperm FISH data support the hypothesis that chromosome 21 also has an increased susceptibility for nondisjunction in meiosis of male patients treated with NOVP.

There was also variation among the donors within the during-treatment group, especially for disomy 21. This donor-to-donor difference was confirmed using a simpler 18–21 sperm FISH assay performed on separate semen smears from the same men (data not shown). It is unknown whether this reproducible variation among men was because of disease status, treatment details, effects of times of semen collection in relation to treatment, secondary exposures, genetic susceptibility, or other factors. Treatment doses were not sufficiently different to explain these differences. Interestingly, patient H, who had the highest induced aneusomy frequency, was also the only smoker in the group (1 pack/day for 20 years). Cigarette smoking was suggested previously to be associated with higher frequencies of aneuploid sperm (45 , 46) .

Meiotic Stages of Induction of Aneuploid and Diploid Sperm.
Inspection of the data (Table 3Citation ; Fig. 2Citation ) suggests that male meiosis I is more sensitive than meiosis II for the induction of sex-chromosomal aneuploidy and diploidy. No such inference was possible for disomy 18 or 21. NOVP therapy also induced a variety of unusual and complex genotypes in sperm (Table 4)Citation . Trisomies in diploid sperm presumably arise from double errors: a complete failure of meiosis I or II coupled with the nondisjunction of a single chromosome. The same mechanism can lead to monosomies in diploid sperm; however, anaphase delay and lack of hybridization may also be involved in this category of abnormality. Trisomic diploid sperm appear to involve chromosome 21 and the sex chromosomes (but not chromosome 18), and occurred mainly in XY diploid sperm, suggesting that they originated before or during meiosis I. It is not known whether diploid sperm were actually binuclear (47) . Additional studies are needed to determine the etiology of these unusual sperm genotypes.

Pretreatment Patients versus Healthy Donors.
The aggregate frequency of sperm with numerical abnormalities detected by the X-Y-18-21 assay in the pretreatment group appeared to be slightly higher than the frequencies found among healthy men. Fig. 3Citation suggests that this effect is real and because of a significant difference in the subcategory of diploid sperm. Meistrich et al. (25) noticed that pretreatment HD patients had slightly reduced sperm counts as compared with a normal group. Several studies have recently reported associations between poor semen quality and aneuploidy (45 , 48 , 49) , suggesting that this difference may be coupled to poorer semen quality in pretreatment samples. However, the frequencies of sperm with disomy 18, 21, X, Y, and XY did not differ between the pretreatment and healthy groups, and showed frequencies similar to those reported for healthy men (41) . Our data are consistent with those of Robbins et al. (15) , who reported that pretreatment HD patients produced higher frequencies of disomy 8 sperm than did healthy men.

Need for Animal Studies.
Because most chemotherapeutic regimens consist of a mixture of drugs and patients rarely receive single-drug therapies, animal models are needed to evaluate the relative aneugenic mechanisms of individual drugs. We have developed several multicolor sperm FISH methods to detect sperm aneupoidy in mice and rats (17 , 50, 51, 52) . We suggest that these models provide a means to measure the relative aneugenic potency of individual drugs to augment the future design of regimes that minimize chromosomal damage in sperm.

Clinical Implications.
Our data can be used to estimate the potential risk for abnormal pregnancy outcomes after paternal treatments with NOVP (Table 5)Citation . Extrapolating from four chromosomes across the haploid genome (which assumes that the average induction of sperm aneuploidies for the four chromosomes of our study is the same as those for the other 19 chromosomes), we calculate that NOVP produced six times more sperm-carrying disomies than diploidies and that the samples of the during-treatment group contained ~18% of sperm with some defect in chromosome number. Because there is little experimental evidence in support of biological selection against aneuploid sperm during spermiogenesis, fertilization, and the first cell cycle of zygotic development (53) , these sperm may well be fertile. It is estimated that overall, ~95% of conceptions with an abnormal number of chromosomes will abort, whereas only ~5% will be live born (54) . Thus, the men who have received NOVP therapy may have a significantly elevated (but transient) risk of fathering embryos that die prematurely (because of aneuploidy) and an increased risk of fathering children who are born with one of the major constitutive aneuploidy syndromes, namely Down, Edward, Klinefelter, Turner, Triple X, and XYY syndromes. Additional studies are warranted for other chemotherapies where there are only transient declines in semen quality, where men are treated in their reproductive years, and where they may remain fertile after therapy.


View this table:
[in this window]
[in a new window]
  		Table 5 Estimated genome-wide proportions of sperm carrying numerical chromosomal abnormalities in HD patients treated with NOVPa

This table provides conservative estimates of the overall burden of chromosomally abnormal sperm that might be expected in semen specimens of the various patient groups of this study. It assumes that the untested autosomes behaved the same as the average of the chromosomes tested sperm FISH. All sex-chromosomal aneuploidies were directly measured by sperm FISH. It should be noted that the estimated genome-wide anomalies are likely to be an underestimate, and thus a very conservative estimate, because the unusual sperm genotypes of Table 4Citation were not extrapolated to the entire genome.

 
Because of specifics of the sperm FISH assay used in this study, our findings are limited to aneuploidy and cannot be extrapolated to structural chromosomal abnormalities in sperm. Thus, additional studies are needed with separate sperm FISH assays designed to detect chromosomal aberrations, such as partial chromosomal duplications and deficiencies, and chromosome breaks (31 , 41 , 55) to determine whether aberrations are induced and persist after NOVP therapy. In this regard, NOVP includes mitoxantrone, a topoisomerase II inhibitor, which may induce chromosomal breakage, as was demonstrated recently for another topoisomerase inhibitor, etoposide (56) .


   ACKNOWLEDGMENTS
 
We thank Drs. Francesco Marchetti and Eddie Sloter for careful reading and helpful suggestions for this manuscript, and thank Gene Wilson for technical assistance.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was performed by the Lawrence Livermore National Laboratory under auspices of the United States Department of Energy under contract W7405-ENG-48, with support from NIH Superfund Project (to A. J. W. and B. Eskenazi, University of California, Berkeley, coPIs), National Council for Sci. Tech. Mexico Programa de Estancias Postdoctorales, CONTACYT-NIH, Consejo Nacional de Ciencia y Tecnologia, Proyecto 32557-M (to S. F.), National Institute of Environmental Health Sciences Pan American Research Fellowship, United States-Mexico Cooperative Biomedical and Behavioural Sciences Program, Fogarty International Center, CF-ES-15879 (to S. F.), and NIH Grant CA-78973 (to M. L. M.). Back

2 To whom requests for reprints should be addressed, at Biology and Biotechnology Research Program, L-448, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, CA 94550. Phone: (925) 422-6296; Fax: (925) 424-3130; E-mail: wyrobek1{at}llnl.gov Back

3 The abbreviations used are: HD, Hodgkin’s disease; NOVP, Novantrone (Mitoxantrone), (Oncovin) Vincristine, Velban (Vinblastine), and Prednisone; FISH, fluorescence in situ hybridization; ABVD, adriamycin, bleomycin, vinblastine, and dacarbazine; DAPI, 4',6-diamidino-2-phenylindole. Back

Received 2/21/02. Accepted 10/31/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 SUBJECTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hook E. B. Perspectives in mutation epidemiology: 3 contribution of chromosome abnormalities to human morbidity and mortality and some comments upon surveillance of chromosome mutation rates. Mutat. Res., 114: 389-423, 1983.[Medline]
  2. Shelby M. D., Bishop J. B., Mason J. M., Tindall K. R. Fertility, reproduction and genetic disease: studies on the mutagenic effects of environmental agents on mammalian germ cells. Environ. Health Perspect., 100: 283-291, 1993.[Medline]
  3. Bishop J. B., Dellarco V. L., Hassold T., Ferguson L. R., Wyrobek A. J., Friedman J. M. Aneuploidy in germ cells: etiologies and risk factors. Environ. Mol. Mutagen., 28: 159-166, 1996.[Medline]
  4. Marchetti F., Lowe X., Bishop J., Wyrobek A. J. Induction of chromosomal aberrations in mouse zygotes by acrylamide treatment of male germ cells and their correlation with dominant lethality and heritable translocations. Environ. Mol. Mutagen., 30: 410-417, 1997.[Medline]
  5. Witt K. L., Bishop J. B. Mutagenicity of anticancer drugs in mammalian germ cells. Mutat. Res., 355: 209-234, 1996.[Medline]
  6. Wyrobek A. J. Methods and concepts in detecting abnormal reproductive outcomes of paternal origin. Reprod. Toxicol., 7: 3-16, 1993.
  7. Meistrich M. L., Wilson G., Brown B. W., da Cunha M. F., Lipshultz L. I. Impact of cyclophosphamide on long term reduction in sperm count in men treated with combination chemotherapy for ewing and soft tissue sarcomas. Cancer (Phila.), 70: 2703-2712, 1992.[Medline]
  8. Brierley J. D., Rathmell A. J., Gospodarowicz M. K., Sutcliffe S. B., Munro A., Tsang R., Pintilie M. Late effects of treatment for early-stage Hodgkin’s disease. Br. J. Cancer, 77: 1300-1310, 1998.[Medline]
  9. Savitz D. A., Chen J. Parental occupational and childhood cancer: review of epidemiologic studies. Environ. Health Perspect., 88: 325-337, 1990.[Medline]
  10. Parry J. M., Parry E. M., Bourner R., Doherty A., Ellard S., O’Donovan J., Hoebee B., de Stoppelaar J. M., Mohn G. R., Onfelt A., Renglin A., Schultz N., Soderpalm-Berndes C., Jensen K. G., Kirsch-Volders M., Elhajouji A., Van Hummelen P., Degrassi F., Antoccia A., Cimini D., Izzo M., Tanzarella C., Adler I. D., Kliesch U., Hess P. The detection and evaluation of aneugenic chemicals. Mutat. Res., 353: 11-46, 1996.[Medline]
  11. Shelby M. D. Selecting chemical assays for assessing mammalian germ cell mutagenicity. Mutat. Res., 352: 159-167, 1996.[Medline]
  12. Mulvihill J. J., Elizabeth M. A., Rosner F., Zarrabi M. H. Pregnancy outcome in cancer patients. Experience in a large cooperative group. Cancer (Phila.), 60: 1143-1150, 1987.[Medline]
  13. Byrne J., Mulvihill J. J., Myers M. H., Connelly R. R., Naughton M. D., Krauss M. R., Steinhorn S. S. Effects of treatment on fertility in long-term survivors of childhood or adolescent cancer. N. Engl. J. Med., 317: 1315-1321, 1987.[Abstract]
  14. Byrne J., Rasmussen S. A., Steinhorn S. C., Connelly R. R., Myers M. H., Lynch C. F., Flannery J., Austin D. F., Holmes F. F., Holmes G. E., Strong L. C., Mulvihill J. J. Genetic disease in offspring of long-term survivors of childhood and adolescent cancer. Am. J. Hum. Genet., 62: 45-52, 1998.[Medline]
  15. Robbins W. A., Meistrich M. L., Moore D., Hagemeister F. B., Weier H. U., Cassel M. J., Wilson G., Eskenazi B., Wyrobek A. J. Chemotherapy induces transient sex chromosomal and autosomal aneuploidy in human sperm. Nat. Genet., 16: 74-78, 1997.[Medline]
  16. Wyrobek A. J., Gordon L. A., Burkhart J. G., Francis M. W., Kapp R. W., Letz G., Malling H. V., Topham J. C., Whorton M. D. An evaluation of human sperm as indicators of chemically induced alterations of spermatogenic function. Mutat. Res., 115: 73-148, 1983.[Medline]
  17. Wyrobek A. J., Marchetti F., Sloter E., Bishop J. Chromosomally defective sperm and their developmental consequences Anderson D. Karakaya A. E. Sram R. J. eds. . Human Monitoring after Environmental and Occupational Exposure to Chemical and Physical Agents, NATO Science Series, Vol. 313: 134-150, IOS Press Amsterdam, The Netherlands 2000.
  18. Mueller N. E. Hodgkin’s disease Schottenfeld D. Fraumeni J. F. eds. . Cancer Epidemiology and Prevention, 2nd Ed. 893-919, Oxford University Press New York 1996.
  19. Viviani S., Santoro A., Ragni G., Bonfante V., Bestetti O., Bonadonna G. Gonadal toxicity after combination chemotherapy for Hodgkin’s disease. Comparative results of MOPP vs ABVD. Eur. J. Cancer Clin. Oncol., 21: 601-605, 1985.[Medline]
  20. Hagemeister F. B., Purugganan R., Fuller L., McLaughlin P., Swan F., Romaguera J., Rodriguez M., Cabanillas F. Treatment of early stages of Hodgkin’s disease with Novantrone, vincristine, vinblastine, prednisone, and radiotherapy. Semin. Hematol., 31: 36-43, 1994.[Medline]
  21. Genesca A., Caballin M. R., Miro R., Benet J., Bonfill X., Egozcue J. Human sperm chromosomes. Long-term effect of cancer treatment. Cancer Genet. Cytogenet., 46: 51-260, 1990.
  22. Brandriff B. F., Meistrich M. L., Gordon L. A., Carrano A. V., Liang J. I. C. Chromosomal damage in sperm of patients surviving Hodgkin’s disease following MOPP (nitrogen mustard, vincristine, procarbazine and prednisone) therapy with and without radiotherapy. Hum. Genet., 93: 295-299, 1994.[Medline]
  23. Monteil M., Rousseaux S., Chevret E., Pelletier R., Cozzi J., Sele B. Increased aneuploid frequency in spermatozoa from a Hodgkin’s disease patient after chemotherapy and radiotherapy. Cytogenet. Cell Genet., 76: 134-138, 1997.[Medline]
  24. Preston R. J. Aneuploidy in germ cells: disruption of chromosome mover components. Environ. Mol. Mutagen., 28: 176-181, 1996.[Medline]
  25. Meistrich M. L., Wilson G., Mathur K., Fuller L. M., Rodriguez M. A., McLaughlin P., Romaguera J. E., Cabanillas F. F., Ha C. S., et al Rapid recovery of spermatogenesis after mitoxantrone, vincristine, vinblastine and prednisone chemotherapy for Hodgkin’s disease. J. Clin. Oncol., 15: 3488-3495, 1997.[Abstract/Free Full Text]
  26. Van Hummelen P., Manchester D., Lowe X., Wyrobek A. J. Meiotic segregation, recombination and gamete aneuploidy assessed in a t(1:10)(p22.1;q22.3) reciprocal translocation carrier by three- and four-probe multicolor FISH in sperm. Am. J. Hum. Genet., 61: 651-659, 1997.[Medline]
  27. Griffin D. K., Abruzzo M. A., Millie E. A., Feingold E., Hassold T. J. Sex ratio in normal and disomic sperm: evidence that the extra chromosome 21 preferentially segregates with the Y chromosome. Am. J. Hum. Genet., 59: 1108-1113, 1996.[Medline]
  28. Dubey P., Wilson G., Mathur K. K., Hagemeister F. B., Fuller L. M., Ha C. S., Cox J. D., Meistrich M. L. Recovery of sperm production following radiation therapy for Hodgkin’s disease after induction chemotherapy with mitoxantrone, vincristine, vinblastine and prednisone (NOVP). Int. J. Radiat. Oncol. Biol. Phys., 46: 609-617, 2000.[Medline]
  29. Meistrich M. L., Chawla S. P., da Cunha M. F., Johnson S. L., Plager C., Papadopoulos N. E., Lipshultz L. I., Benjamin R. S. Recovery of sperm production after chemotherapy for osteosarcoma. Cancer (Phila.), 63: 2115-2123, 1989.[Medline]
  30. Manly B. F. J. . Randomization, Bootstrap and Monte Carlo Methods in Biology, Chapman & Hall, CRC Press Boca Raton, FL 1997.
  31. Baumgartner A., Van Hummelen P., Lowe X. R., Adler I. D., Wyrobek A. J. Numerical and structural chromosomal abnormalities detected in human sperm with a combination of multicolor FISH assays. Environ. Mol. Mutagen., 33: 49-58, 1999.[Medline]
  32. Jenderny J., Jacobi M. L., Ruger A., Rohrborn G. Chromosome aberrations in 450 sperm complements from eight controls and lack of increase after chemotherapy in two patients. Hum. Genet., 90: 151-154, 1992.[Medline]
  33. Martin R. H., Rademaker A. W., Leonard N. J. Analysis of chromosomal abnormalities in human sperm after chemotherapy by karyotyping and fluorescence in situ hybridization (FISH). Cancer Genet. Cytogenet., 80: 29-32, 1995.[Medline]
  34. Martin R. H., Ernest S., Rademaker A., Barclay L., Ko E., Summers N. Chromosomal abnormalities in sperm from testicular cancer patients before and after chemotherapy. Hum. Genet., 99: 214-218, 1997.[Medline]
  35. Martin R. H., Rademaker E. A., Barclay L., Ko E., Summers N. Analysis of human sperm karyotypes in testicular cancer patients before and after chemotherapy. Cytogenet. Cell Genet., 78: 120-123, 1997.[Medline]
  36. Jenderny J., Rohrborn G. Chromosome analysis of human sperm. Hum. Genet., 76: 385-388, 1987.[Medline]
  37. Martin R. Human sperm chromosome complements in chemotherapy patients and infertile men. Chromosoma (Berl.), 107: 523-527, 1998.[Medline]
  38. Blanco J., Egozcue J., Vidal F. Incidence of chromosome 21 disomy in human spermatozoa as determined by fluorescent in situ hybridization. Hum. Reprod., 11: 722-726, 1996.[Abstract/Free Full Text]
  39. Warburton D., Kinney A. Chromosomal differences in susceptibility to meiotic aneuploidy. Environ. Mol. Mutagen., 28: 237-247, 1996.[Medline]
  40. Guttenbach M., Engel W., Schmid M. Analysis of structural and numerical chromosome abnormalities in sperm of normal men and carriers of constitutional chromosome aberrations. A review. Hum. Genet., 100: 1-21, 1997.[Medline]
  41. Van Hummelen P., Lowe X. R., Wyrobek A. J. Simultaneous detection of structural and numerical chromosome abnormalities in sperm of healthy men by multi-color FISH. Hum. Genet., 98: 608-615, 1996.[Medline]
  42. Shi Q., Martin R. H. Aneuploidy in human sperm: a review of the frequency and distribution of aneuploidy, effects of donor age, and lifestyle factors. Cytogenet. Cell Genet., 90: 219-26, 2000.[Medline]
  43. Martin R. H., Rademaker A. The frequency of aneuploidy for individual chromosomes in 6821 human sperm chromosome complements. Cytogenet. Cell Genet., 53: 103-107, 1990.[Medline]
  44. Templado C., Marquez C., Munne S., Colls P., Martorell M. R., Cieply K., Benet J., Van Kirk V., Navarro J., Estop A. M. An analysis of human sperm chromosome aneuploidy. Cytogenet. Cell Genet., 74: 194-200, 1996.[Medline]
  45. Rubes J., Lowe X., Moore D., Perreault S., Slott V., Evenson D., Selevan S. G., Wyrobek A. J. Smoking cigarettes is associated with increased sperm disomy in teenage men. Fertil. Steril., 70: 715-723, 1998.[Medline]
  46. Shi Q., Ko E., Barday L., Hoang T., Rademaker A., Martin R. Cigarette smoking and aneuploidy in human sperm. Mol. Reprod. Dev., 59: 417-21, 2001.[Medline]
  47. Burkhart J. G., Malling H. V. Differentiation of binuclear spermatozoa in mice. Gamete Res., 22: 399-410, 1989.[Medline]
  48. Moosani N., Pattinson H. A., Carter M. D., Cox D. M., Rademaker A. W., Martin R. H. Chromosomal analysis of sperm from men with idiopathic infertility using sperm karyotyping and fluorescence in situ hybridization. Fertil. Steril., 64: 811-817, 1995.[Medline]
  49. Sloter E., Nath J., Wyrobek A. J. Associations between chromosomal abnormalities and semen quality in healthy men. Environ. Mol. Mutagen., 35 (Suppl. 31): 57 2000.[Medline]
  50. Lowe X., O’Hogan S., Moore D., II, Bishop J., Wyrobek A. J. Aneuploid epididymal sperm detected in chromosomally normal and Robertsonian translocation-bearing mice using a new three-chromosome FISH method. Chromosoma (Berl.), 105: 204-210, 1996.[Medline]
  51. Lowe X. R., de Stoppelaar J. M., Bishop J., Cassel M., Hoebee B., Wyrobek A. J. Epididymal sperm aneuploidies detected in three genetic strains of rats by multi-color FISH. Environ. Mol. Mutagen., 31: 125-132, 1998.[Medline]
  52. Wyrobek A. J., Lowe X. R. Multiprobe fluorescence in situ hybridization methods for detecting chromosomally defective sperm in mice and humans Andreeff M. Pinkel D. eds. . Introduction to Fluorescence In Situ Hybridization: Principles and Clinical Applications, 391-408, Wiley-Liss, Inc. New York 1999.
  53. Marchetti F., Lowe X., Bishop J., Wyrobek A. J. Absence of selection against aneuploid mouse sperm at fertilization. Biol. Reprod., 61: 948-954, 1999.[Abstract/Free Full Text]
  54. Gersen S. L., Keagle M. B. . The Principles of Clinical Cytogenetics, Humana Press Totowa NJ 1999.
  55. Sloter E., Lowe X., Moore D., II, Nath J., Wyrobek A. J. Chromosomal breaks, duplications, deletions, and aneuploidy in the sperm of healthy men. Am. J. Hum. Genet., 67: 862-872, 2000.[Medline]
  56. Marchetti F., Bishop J. B., Lowe X., Generoso W. M., Hozier J., Wyrobek A. J. Etoposide induces heritable chromosomal aberrations and aneuploidy during male meiosis in the mouse. Proc. Natl. Acad. Sci. USA, 98: 3952-3957, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J AndrolHome page
R. E. Brannigan and J. I. Sandlow
Cryopreservation of Sperm After Chemotherapy
J Androl, May 1, 2008; 29(3): e1 - e2.
[Full Text] [PDF]

Home page
 Hum ReprodHome page
H.G. Tempest, E. Ko, P. Chan, B. Robaire, A. Rademaker, and R.H. Martin
Sperm aneuploidy frequencies analysed before and after chemotherapy in testicular cancer and Hodgkin's lymphoma patients
Hum. Reprod., February 1, 2008; 23(2): 251 - 258.
[Abstract] [Full Text] [PDF]

Home page
 Mol Hum ReprodHome page
L. Froenicke, P.-H. Hung, C. A. VandeVoort, and L. A. Lyons
Development of a non-human primate sperm aneuploidy assay tested in the rhesus macaque (Macaca mulatta)
Mol. Hum. Reprod., July 1, 2007; 13(7): 455 - 460.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
O. Stahl, J. Eberhard, K. Jepson, M. Spano, M. Cwikiel, E. Cavallin-Stahl, and A. Giwercman
Sperm DNA integrity in testicular cancer patients
Hum. Reprod., December 1, 2006; 21(12): 3199 - 3205.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
S. J. Lee, L. R. Schover, A. H. Partridge, P. Patrizio, W. H. Wallace, K. Hagerty, L. N. Beck, L. V. Brennan, and K. Oktay
American Society of Clinical Oncology Recommendations on Fertility Preservation in Cancer Patients
J. Clin. Oncol., June 20, 2006; 24(18): 2917 - 2931.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
A. J. Wyrobek, B. Eskenazi, S. Young, N. Arnheim, I. Tiemann-Boege, E. W. Jabs, R. L. Glaser, F. S. Pearson, and D. Evenson
Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm
PNAS, June 20, 2006; 103(25): 9601 - 9606.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
F. Marchetti, F. S. Pearson, J. B. Bishop, and A. J. Wyrobek
Etoposide induces chromosomal abnormalities in mouse spermatocytes and stem cell spermatogonia
Hum. Reprod., April 1, 2006; 21(4): 888 - 895.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
G. Bahadur, O. Ozturk, A. Muneer, R. Wafa, A. Ashraf, N. Jaman, S. Patel, A.W. Oyede, and D.J. Ralph
Semen quality before and after gonadotoxic treatment
Hum. Reprod., March 1, 2005; 20(3): 774 - 781.
[Abstract] [Full Text] [PDF]

Home page
 J Natl Cancer Inst MonogrHome page
A. J. Wyrobek, T. E. Schmid, and F. Marchetti
Relative Susceptibilities of Male Germ Cells to Genetic Defects Induced by Cancer Chemotherapies
J Natl Cancer Inst Monographs, March 1, 2005; 2005(34): 31 - 35.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions