[CANCER RESEARCH 63, 44 –51, January 1, 2003]
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.]
diation 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– 6). Despite
considerable indirect evidence linking certain paternal exposures to
abnormal pregnancy outcomes and childhood cancer (6 –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 (12–14). 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treated as children; (c) outdated drugs and/or treatment regimens were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 –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 (21–23). 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
ABSTRACT
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 fourchromosome 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
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 irraReceived 2/21/02; accepted 10/31/02.
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.).
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:
[email protected].
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.
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NOVP-INDUCED MAJOR SPERM ANEUPLOIDIES
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.
Conventional semen analysisa
Patient codeb
HD stage
Age
Semen volume
Sperm concentration
Sperm motility
IIB
IA
IIB
IA
IIB
40
40
30
26
25
3.5
5.7
5.2
2.1
1.1
45
204
28.8
50.2
107
71
74
76
69
50
IIA
IIA
IIA
27
31
35
5.1
6.9
4.9
13.7
16.8
61.6
64
69
37
IA
IIA
IIA
IIA
42
28
32
36
4.3
2.1
2.4
2.6
83.5
12.3
64.2
9.7
65
70
43
48
n⫽4
47.2 ⫹/⫺ 1.5
2.5 ⫹/⫺ 0.7
162 ⫹/⫺ 28
—
Pretreatment
A
B
C
D
E
During-treatmentc
F
G
H
Post-treatmentd
B
F
G
H
Healthy donors
Average ⫹/⫺ SE
a
Age at sampling in years, volume in ml, concentration in millions of sperm per ml, and motility in percentage (25).
b
Identical capital letters represent samples from the same patient.
c
Semen samples were provided by patients F, G, and H at 45, 41, and 49 days, respectively, after chemotherapy.
d
Post-treatment samples were provided by patients B, F, G, and H at 2.0, 1.0, 1.5, and 1.0 years after end of NOVP chemotherapy, respectively.
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.
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.
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).
Sperm FISH. Semen was thawed at room temperature and 5–7 ␮l 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)/2⫻ 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 ␣ satellite region of
chromosome X fluoresced in yellow using an equimixture of the ␣ 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
␣ 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 ␮l of hybridization mix
SUBJECTS AND METHODS
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 1, three groups of specimens were
investigated: pretreatment samples (from donors A, B, C, D, and E), duringtreatment samples (from donors F, G, and H), and post-treatment samples
(from B, F, G, and H). Pretreatment specimens were provided after diagnosis
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NOVP-INDUCED MAJOR SPERM ANEUPLOIDIES
RESULTS
Semen Quality in Specimens Analyzed. Table 1 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 duringtreatment versus the pretreatment group (Table 1), 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
1, 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 duringtreatment 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 1; P ⫽ 0.03).
As shown in Table 2, 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).
Overall, the frequency of sperm with disomy 21 was 2–5-fold higher
than disomy 18 among the various comparison groups (P ⬍ 0.05).
Using the X-Y-18-21 assay, the aggregate frequency of sperm with
sex-chromosomal aneuploidies (sum of frequencies of sperm with
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: L patient A; } patient B; ✚
patient C; ⴛ patient D; 䊐 patient E; ■ patient F; Œ patient G; and F 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. Duringtreatment 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.
(Vysis) and 3 ␮l of water, denatured in formamide 70%/2⫻ 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/2⫻ SSC at 45°C, 10 min in 2⫻
SSC at 37°C, and then 10 min at room temperature). Sperm nuclei were
counterstained with DAPI 0.01– 0.05 ␮g/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 DAPIAqua (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 ⫻ 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.
Fig. 2. Meiosis I and II sperm diploidy in HD patients before, during, and after NOVP
chemotherapy. Donor symbols as in Fig. 1. 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.
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NOVP-INDUCED MAJOR SPERM ANEUPLOIDIES
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.
Chromosome 18
Subject codes
Pre-treatment patients
A
B
C
D
E
Average ⫾ SE
During-treatment patients
F
G
H
Average ⫾ SE
Post-treatment patients
B
F
G
H
Average ⫾ SE
Reference group
1
2
3
4
Average ⫾ SE
a
b
c
Disomyb,c
Null
Chromosome 21
Totalb,c
Disomyb,c
Totalb,c
Null
2.0
4.0
2.0
2.0
1.0
2.2 ⫾ 0.5
2.9
8.9
1.9
2.9
1.9
3.7 ⫾ 1.3
4.9
12.9
3.9
4.9
2.9
5.9 ⫾ 1.7
16.9
8.0
5.9
11.0
3.9
9.1 ⫾ 2.2
8.9
11.9
0.0
1.9
4.9
5.5 ⫾ 2.1
25.8
19.9
5.9
12.9
8.8
14.6 ⫾ 3.6
14.0
14.6
18.7
15.8 ⫾ 1.4
7.0
6.6
9.3
7.6 ⫾ 0.8
21.0
21.2
28.0
23.4 ⫾ 2.2
26.0
12.6
50.0
29.5 ⫾ 10.9
8.0
9.3
28.0
15.1 ⫾ 6.4
34.0
21.9
78.0
44.6 ⫾ 17.0
2.0
5.0
3.0
10.0
5.0 ⫾ 1.8
3.9
2.9
1.0
1.0
2.2 ⫾ 0.7
5.9
7.9
4.0
11.0
7.2 ⫾ 1.5
4.6
16.0
11.9
20.0
13.1 ⫾ 3.3
2.6
2.0
0.0
10.0
3.6 ⫾ 4.3
7.2
18.0
11.9
30.0
16.7 ⫾ 4.9
2.0
0.0
2.0
1.9
1.4 ⫾ 0.5
4.9
8.9
4.9
2.6
5.3 ⫾ 1.3
6.9
8.9
6.9
4.5
6.8 ⫾ 0.8
3.0
8.9
6.9
9.1
6.9 ⫾ 1.4
10.9
0.0
6.9
4.5
5.7 ⫾ 2.3
13.9
8.9
13.8
13.6
12.6 ⫾ 1.2
Frequency per 10,000 sperm determined by four chromosome FISH.
During-treatment group differs from reference group; P ⬍ 0.05.
During-treatment group differs from pretreatment group; P ⬍ 0.05. No differences were found between pretreatment and post-treatment groups.
disomy X, disomy Y, XY sperm, and sex-null sperm) were ⬃4-fold
higher in the during- versus pretreatment groups (Table 3; 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), 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 ⬃3fold 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.
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. 2 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. 3 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).
Four major categories of sperm with unusual phenotypes were
detected using the four-chromosome sperm FISH (Table 4): (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) was also elevated significantly during treatment
(P ⬍ 0.02).
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. 1, 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 2, 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 posttreatment samples to be nonsignificantly elevated above their pretreatment controls (Table 3; Fig. 2).
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
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
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Table 3 Sex chromosomal disomy and nullisomy in sperm of HD patients treated with NOVPa
Disomy
Subject codes
Pretreatment patients
A
B
C
D
E
Average ⫾ SE
During-treatment patients
F
G
H
Average ⫾ SE
Post-treatment patients
B
F
G
H
Average ⫾ SE
Reference group
1
2
3
4
Average ⫾ SE
XY
b,c
XX
b,c
YYb,c
Total disomyb,c
Nullisomyb,c,d
Total sex aneuploidyb,c
1.0
4.0
0.0
6.0
4.9
3.2 ⫾ 1.1
1.0
3.0
4.9
7.0
3.9
3.9 ⫾ 1.0
1.0
5.0
0.0
4.0
0.0
1.9 ⫾ 1.0
3.0
12.0
4.9
17.0
8.8
9.1 ⫾ 2.4
11.9
8.0
8.9
22.9
11.8
12.7 ⫾ 2.6
14.9
20.0
13.8
39.9
20.6
21.8 ⫾ 4.7
36.0
16.6
70.7
41.0 ⫾ 15.8
13.0
7.3
6.7
8.9 ⫾ 2.0
7.0
2.0
4.7
4.5 ⫾ 1.4
56.0
25.9
82.0
54.6 ⫾ 16.2
38.0
15.3
59.3
37.5 ⫾ 12.7
94.0
41.2
141.4
92.1 ⫾ 28.9
4.6
11.0
8.9
40.0
16.1 ⫾ 8.0
5.9
4.0
4.0
3.0
4.2 ⫾ 0.6
2.6
2.0
1.0
4.0
2.4 ⫾ 0.6
13.1
17.0
13.9
47.0
22.7 ⫾ 8.1
6.6
39.9
14.9
16.0
19.3 ⫾ 7.1
19.7
56.9
28.8
63.0
42.1 ⫾ 10.5
3.0
6.9
3.0
7.1
5.0 ⫾ 1.1
3.0
3.0
3.0
3.2
3.0 ⫾ 0.06
0.0
0.0
0.0
0.0
0.0
6.0
9.9
6.0
10.3
8.0 ⫾ 1.2
15.0
11.9
5.0
9.7
10.3 ⫾ 2.1
21.0
21.8
11.0
20.0
18.3 ⫾ 2.5
a
Frequency per 10,000 sperm determined by four chromosome FISH. Tail presence confirmed by phase contrast imaging.
During-treatment group differs from reference group P ⬍ 0.05.
During-treatment group differs from pretreatment group P ⬍ 0.05. No differences were found between pretreatment and post-treatment groups.
d
Sex chromosomal nullisomy is represented by fluorescent phenotypes 0-18-21.
b
c
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–
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
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
Fig. 3. Comparison of the genome-wide frequencies of sperm with disomy and
diploidy between a group of healthy donors (䊐) and the group of pretreatment HD patients
(u). Bars, ⫾SE. See text for the calculation of genome-wide disomy estimate.
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NOVP-INDUCED MAJOR SPERM ANEUPLOIDIES
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.
Patient groupsa
Ploidy group
Healthy reference
group
Pre
During
Post
Diploid sperm with following additional abnormalities
Monosomy 21
Monosomy 18
Monosomy of sex chromosomes
Trisomy 21
Trisomy of either sex chromosome
Tetraploid cells
Others
Total unusualb
0.2 ⫾ 0.2
1.6 ⫾ 0.8
1.1 ⫾ 0.5
0.5 ⫾ 0.3
0.2 ⫾ 0.3
0.5 ⫾ 0.3
8.9 ⫾ 3.0
13
1.7 ⫾ 0.5
2.4 ⫾ 1.2
3.3 ⫾ 0.9
0
0.8 ⫾ 0.7
0.2 ⫾ 0.2
6.4 ⫾ 1.7
14.8
9.9 ⫾ 3.5
12.8 ⫾ 1.7
17.0 ⫾ 2.8
2.0 ⫾ 1.6
2.0 ⫾ 1.6
10.3 ⫾ 4.3
54.6 ⫾ 17.1
108.6
1.3 ⫾ 0.6
2.3 ⫾ 0.7
5.7 ⫾ 1.5
1.0 ⫾ 0.5
0
1.2 ⫾ 0.6
9.2 ⫾ 2.5
20.7
a
Sum of the frequencies of monosomies and trisomies 21 and monosomic 18 that were found in diploids ⫹ monosomies and trisomies of sex chromosomes in diploids ⫹ tetraploid
cells ⫹ others that corresponds to miscellaneous abnormal fluorescent phenotypes. Trisomies of chromosome 18 were not found among diploid sperm.
b
During-treatment values were significantly higher than those of all other groups, for every abnormality but not for trisomies (Mann Whitney U; P ⬍ 0.02).
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 3; Fig. 2) 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). 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. 3 suggests
that this effect is real and because of a significant difference in the
subcategory of diploid sperm. Meistrich et al. (25) noticed that pre-
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 (⫻10⫺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).
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).
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– 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 duringtreatment 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
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 4 were not extrapolated to the entire genome.
Patient groups
Group
Autosomal aneuploidyb
Sex chromosome aneuploidyc
Diploidyd
Unusuale
Estimated percent abnormal
sperm extrapolated across the genomef
Reference
group
Pre
During
Post
2.2
0.2
0.1
0.1
2.6
3.8
0.2
0.3
0.1
4.4
13.9
0.9
2.3
1.0
18.1
4.6
0.4
0.6
0.2
5.8
a
Data in percent.
Extrapolated from data for chromosomes 8, 18, and 21 (Ref. 15; Table 2) using
⌺freq(8,18,21)/3 ⫻ 2 ⫻ 22/100.
c
Sum of total sex chromosomal disomy plus nullisomy (Table 3).
d
Total sperm diploidy.
e
Total unusual fluorescent phenotypes (Table 4).
f
Sum of above, but does not include an estimate of the unusual anomalies extrapolated
across the genome.
b
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NOVP-INDUCED MAJOR SPERM ANEUPLOIDIES
treatment 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 singledrug 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 –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). 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.
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).
2. Shelby, M. D., Bishop, J. B., Mason, J. M., and 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.
3. Bishop, J. B., Dellarco, V. L., Hassold, T., Ferguson, L. R., Wyrobek, A. J., and
Friedman, J. M. Aneuploidy in germ cells: etiologies and risk factors. Environ. Mol.
Mutagen., 28: 159 –166, 1996.
4. Marchetti, F., Lowe, X., Bishop, J., and 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.
5. Witt, K. L., and Bishop, J. B. Mutagenicity of anticancer drugs in mammalian germ
cells. Mutat. Res., 355: 209 –234, 1996.
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., and 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.
8. Brierley, J. D., Rathmell, A. J., Gospodarowicz, M. K., Sutcliffe, S. B., Munro, A.,
Tsang, R., and Pintilie, M. Late effects of treatment for early-stage Hodgkin’s disease.
Br. J. Cancer, 77: 1300 –1310, 1998.
9. Savitz, D. A., and Chen, J. Parental occupational and childhood cancer: review of
epidemiologic studies. Environ. Health Perspect., 88: 325–337, 1990.
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., and Hess, P. The detection and evaluation of aneugenic chemicals.
Mutat. Res., 353: 11– 46, 1996.
11. Shelby, M. D. Selecting chemical assays for assessing mammalian germ cell mutagenicity. Mutat. Res., 352: 159 –167, 1996.
12. Mulvihill, J. J., Elizabeth, M. A., Rosner, F., and Zarrabi, M. H. Pregnancy outcome
in cancer patients. Experience in a large cooperative group. Cancer (Phila.), 60:
1143–1150, 1987.
13. Byrne, J., Mulvihill, J. J., Myers, M. H., Connelly, R. R., Naughton, M. D., Krauss,
M. R., and 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.
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., and
Mulvihill, J. J. Genetic disease in offspring of long-term survivors of childhood and
adolescent cancer. Am. J. Hum. Genet., 62: 45–52, 1998.
15. Robbins, W. A., Meistrich, M. L., Moore, D., Hagemeister, F. B., Weier, H. U.,
Cassel, M. J., Wilson, G., Eskenazi, B., and Wyrobek, A. J. Chemotherapy induces
transient sex chromosomal and autosomal aneuploidy in human sperm. Nat. Genet.,
16: 74 –78, 1997.
16. Wyrobek, A. J., Gordon, L. A., Burkhart, J. G., Francis, M. W., Kapp, R. W., Letz,
G., Malling, H. V., Topham, J. C., and Whorton, M. D. An evaluation of human
sperm as indicators of chemically induced alterations of spermatogenic function.
Mutat. Res., 115: 73–148, 1983.
17. Wyrobek, A. J., Marchetti, F., Sloter, E., and Bishop, J. Chromosomally defective
sperm and their developmental consequences. In: D. Anderson, A. E. Karakaya, and
R. J. Sram (eds.). Human Monitoring after Environmental and Occupational Exposure
to Chemical and Physical Agents, NATO Science Series, Vol. 313, pp. 134 –150.
Amsterdam, The Netherlands: IOS Press, 2000.
18. Mueller, N. E. Hodgkin’s disease. In: D. Schottenfeld, J. F. Fraumeni (eds.). Cancer
Epidemiology and Prevention, 2nd Ed., pp. 893–919. New York: Oxford University
Press, 1996.
19. Viviani, S., Santoro, A., Ragni, G., Bonfante, V., Bestetti, O., and 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.
20. Hagemeister, F. B., Purugganan, R., Fuller, L., McLaughlin, P., Swan, F.,
Romaguera, J., Rodriguez, M., and Cabanillas, F. Treatment of early stages of
Hodgkin’s disease with Novantrone, vincristine, vinblastine, prednisone, and radiotherapy. Semin. Hematol., 31: 36 – 43, 1994.
21. Genesca, A., Caballin, M. R., Miro, R., Benet, J., Bonfill, X., and 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., and 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.
23. Monteil, M., Rousseaux, S., Chevret, E., Pelletier, R., Cozzi, J., and Sele, B. Increased
aneuploid frequency in spermatozoa from a Hodgkin’s disease patient after chemotherapy and radiotherapy. Cytogenet. Cell Genet., 76: 134 –138, 1997.
24. Preston, R. J. Aneuploidy in germ cells: disruption of chromosome mover components. Environ. Mol. Mutagen., 28: 176 –181, 1996.
25. Meistrich, M. L., Wilson, G., Mathur, K., Fuller, L. M., Rodriguez, M. A., McLaughlin, P., and 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.
26. Van Hummelen, P., Manchester, D., Lowe, X., and 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.
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.
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.
50
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.
NOVP-INDUCED MAJOR SPERM ANEUPLOIDIES
27. Griffin, D. K., Abruzzo, M. A., Millie, E. A., Feingold, E., and 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.
28. Dubey, P., Wilson, G., Mathur, K. K., Hagemeister, F. B., Fuller, L. M., Ha, C. S.,
Cox, J. D., and 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.
29. Meistrich, M. L., Chawla, S. P., da Cunha, M. F., Johnson, S. L., Plager, C.,
Papadopoulos, N. E., Lipshultz, L. I., and Benjamin, R. S., Recovery of sperm
production after chemotherapy for osteosarcoma. Cancer (Phila.), 63: 2115–2123,
1989.
30. Manly, B. F. J. Randomization, Bootstrap and Monte Carlo Methods in Biology. Boca
Raton, FL: Chapman & Hall, CRC Press, 1997.
31. Baumgartner, A., Van Hummelen, P., Lowe, X. R., Adler, I. D., and 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.
32. Jenderny, J., Jacobi, M. L., Ruger, A., and 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.
33. Martin, R. H., Rademaker, A. W., and 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.
34. Martin, R. H., Ernest, S., Rademaker, A., Barclay, L., Ko, E., and Summers, N.
Chromosomal abnormalities in sperm from testicular cancer patients before and after
chemotherapy. Hum. Genet., 99: 214 –218, 1997.
35. Martin, R. H., Rademaker, E. A., Barclay, L., Ko, E., and Summers, N. Analysis of
human sperm karyotypes in testicular cancer patients before and after chemotherapy.
Cytogenet. Cell Genet., 78: 120 –123, 1997.
36. Jenderny, J., and Rohrborn, G. Chromosome analysis of human sperm. Hum. Genet.,
76: 385–388, 1987.
37. Martin, R. Human sperm chromosome complements in chemotherapy patients and
infertile men. Chromosoma (Berl.), 107: 523–527, 1998.
38. Blanco, J., Egozcue, J., and Vidal, F. Incidence of chromosome 21 disomy in human
spermatozoa as determined by fluorescent in situ hybridization. Hum. Reprod., 11:
722–726, 1996.
39. Warburton, D., and Kinney, A. Chromosomal differences in susceptibility to meiotic
aneuploidy. Environ. Mol. Mutagen., 28: 237–247, 1996.
40. Guttenbach, M., Engel, W., and 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.
41. Van Hummelen, P., Lowe, X. R., and 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.
42. Shi, Q., and 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.
43. Martin, R. H., and Rademaker, A. The frequency of aneuploidy for individual
chromosomes in 6821 human sperm chromosome complements. Cytogenet. Cell
Genet., 53: 103–107, 1990.
44. Templado, C., Marquez, C., Munne, S., Colls, P., Martorell, M. R., Cieply, K., Benet,
J., Van Kirk, V., Navarro, J., and Estop, A. M. An analysis of human sperm
chromosome aneuploidy. Cytogenet. Cell Genet., 74: 194 –200, 1996.
45. Rubes, J., Lowe, X., Moore, D., Perreault, S., Slott, V., Evenson, D., Selevan, S. G.,
and Wyrobek, A. J. Smoking cigarettes is associated with increased sperm disomy in
teenage men. Fertil. Steril., 70: 715–723, 1998.
46. Shi, Q., Ko, E., Barday, L., Hoang, T., Rademaker, A., and Martin, R. Cigarette
smoking and aneuploidy in human sperm. Mol. Reprod. Dev., 59: 417–21, 2001.
47. Burkhart, J. G., and Malling, H. V. Differentiation of binuclear spermatozoa in mice.
Gamete Res., 22: 399 – 410, 1989.
48. Moosani, N., Pattinson, H. A., Carter, M. D., Cox, D. M., Rademaker, A. W., and
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.
49. Sloter, E., Nath, J., and Wyrobek, A. J. Associations between chromosomal abnormalities and semen quality in healthy men. Environ. Mol. Mutagen., 35 (Suppl. 31):
57, 2000.
50. Lowe, X., O’Hogan, S., Moore, D., II, Bishop, J., and Wyrobek, A. J. Aneuploid
epididymal sperm detected in chromosomally normal and Robertsonian translocationbearing mice using a new three-chromosome FISH method. Chromosoma (Berl.),
105: 204 –210, 1996.
51. Lowe, X. R., de Stoppelaar, J. M., Bishop, J., Cassel, M., Hoebee, B., and Wyrobek,
A. J. Epididymal sperm aneuploidies detected in three genetic strains of rats by
multi-color FISH. Environ. Mol. Mutagen., 31: 125–132, 1998.
52. Wyrobek, A. J., and Lowe, X. R. Multiprobe fluorescence in situ hybridization
methods for detecting chromosomally defective sperm in mice and humans. In: M.
Andreeff, and D. Pinkel (eds.). Introduction to Fluorescence In Situ Hybridization:
Principles and Clinical Applications, pp. 391– 408. New York: Wiley-Liss, Inc., 1999.
53. Marchetti, F., Lowe, X., Bishop, J., and Wyrobek, A. J. Absence of selection against
aneuploid mouse sperm at fertilization. Biol. Reprod., 61: 948 –954, 1999.
54. Gersen, S. L., and Keagle, M. B. The Principles of Clinical Cytogenetics. Totowa, NJ:
Humana Press, 1999.
55. Sloter, E., Lowe, X., Moore, D., II, Nath, J., and Wyrobek, A. J. Chromosomal
breaks, duplications, deletions, and aneuploidy in the sperm of healthy men. Am. J.
Hum. Genet., 67: 862– 872, 2000.
56. Marchetti, F., Bishop, J. B., Lowe, X., Generoso, W. M., Hozier, J., and 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.
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 2003 American Association for Cancer
Research.
NOVP Chemotherapy for Hodgkin's Disease Transiently
Induces Sperm Aneuploidies Associated with the Major
Clinical Aneuploidy Syndromes Involving Chromosomes X,
Y, 18, and 21
Sara Frias, Paul Van Hummelen, Marvin L. Meistrich, et al.
Cancer Res 2003;63:44-51.
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