Human Reproduction Update 2000, Vol. 6 No. 2 pp. 122–131
© European Society of Human Reproduction and Embryology
Smoking and reproduction: gene damage to human
gametes and embryos
Maria Teresa Zenzes*
Division of Reproductive Science, Department of Obstetrics and Gynaecology, University of Toronto, Toronto, Ontario, M5G 1L7, Canada
Received on July 21, 1999accepted on January 13, 2000
Assisted conception is a useful methodology for detecting disturbances in clinical outcome, meiotic maturation, and
genetic integrity of human gametes. Germinal cells are vulnerable to genetic damage from smoking, but can repair
damage during meiosis. In ejaculated spermatozoa, repair capacity declines drastically. Smoking alters the meiotic
spindle of oocytes and spermatozoa, leading to chromosome errors which affect reproductive outcomes. Smoking is
associated with reduced numbers of retrieved oocytes, leading to early age of menopause. Oocyte elimination occurs
preferentially during meiosis I, a period sensitive to genetic damage. Smoking inhibits embryo fragmentation;
inhibition may confer survival advantage to embryos genetically altered. Smoking is associated with low sperm
quality, but clinical effects are not recognized. Cadmium (a heavy metal), nicotine (a toxic alkaloid), and its
metabolite cotinine, are detectable in gonadal tissues and fluids in association with smoking. Cotinine incorporates
into ovarian granulosa–lutein cells, compromising the developmental potential of follicles. Benzo[a]pyrene is a
carcinogenic polycyclic aromatic hydrocarbon resulting from cigarette combustion. Its reactive metabolite binds
covalently to DNA, forming adducts. Smoking-related adducts were detectable in ovarian granulosa–lutein cells,
oocytes, spermatozoa and preimplantation embryos. Transmission of altered DNA from smoking by spermatozoa
was demonstrated in preimplantation embryos and in association with increased risk of childhood cancer.
Key words: adducts/embryos/oocytes/smoking/spermatozoa
TABLE OF CONTENTS
Introduction
122
Vulnerability and repair capacity of germinal cells
122
Smoking and clinical reproductive outcomes—females
123
Smoking and clinical reproductive outcomes—males
125
Effects of smoking on meiotic spindle function
126
Components of cigarette smoke in gonadal fluids and cells 126
Gene damage to spermatozoa from tobacco smoke
128
Oxidative damage from smoking
128
Gametic transmission of gene damage
128
Acknowledgements
129
References
129
Introduction
Although tobacco smoking is a widely recognized health hazard and
a major cause of preventable mortality, consumption of tobacco
remains prevalent in human societies. Approximately one-third of the
world’s population aged ≥15 years smokes (World Health
Organization, 1997). Smoking also affects reproductive health.
Epidemiological studies from the general population of reproductive
age have provided evidence of dose-related effects of smoking
resulting in a conception delay of ~2 months (Zenzes, 1995; Hughes
and Brennan, 1996; Bolumar et al., 1996) and an advance in the age
of menopause by ~2 years (Midgette and Baron, 1990).
This suggests that certain components in cigarette smoke interact,
directly or indirectly, with the gamete cells affecting their function
and viability; the mechanisms involved need to be elucidated.
Because the reproductive system is complex, many sites, from the
hypothalamic–pituitary–axis to the germinal cells, can be
vulnerable to disruption of reproduction. Therefore, clinical and
experimental designs for in-vivo and in-vitro testing can be useful
for detecting such disruptions. In this context, assisted conception
provides an ideal setting for using fluids and cells of the
reproductive system, for providing clinical data on reproductive
performance, and makes it possible to analyse disturbances of
meiotic maturation and genetic integrity in gamete cells. These
aspects are reviewed here, focusing primarily on the mechanisms of
damage to the gametes.
Vulnerability and repair capacity of germinal cells
Gametogenesis, the process of meiotic maturation of germinal cells,
involves two cell divisions: meiosis I and meiosis II. Meiosis I is
reductional and results in oocytes and spermatocytes with haploid
chromosome complements (23 instead of 46 chromosomes). Meiosis
II is equational; the two sister chromatids of each chromosome
* To whom correspondence should be addressed at: Division of Reproductive Sciences, The Toronto General Hospital Research Centre, Room CCRW 1–813,
Toronto, Ontario M5G 1L7, Canada. Fax: 001 416 340 4596; e-mail: [email protected]
Smoking and reproduction: gene damage to human gametes and embryos
separate, leading to four haploid cells. The female meiotic process
(oogenesis) involves nuclear and cytoplasmic maturation; the final
product is one haploid oocyte arrested at metaphase II, which
completes meiosis at fertilization, and three polar bodies (the first
polar body divides shortly after extrusion), which are dysfunctional.
The male meiotic process (spermatogenesis) leads to four haploid
spermatocytes which, in turn, transform during spermiogenesis into
four mature haploid spermatozoa.
Gametogenesis is very sensitive to the risk of damage from
cigarette smoking (Mattison, 1983). Female and male germinal
cells have different periods of sensitivity to external factors during
meiosis I and II, and have different capacities to repair damage. This
is explained below.
Oogenesis
During oogenesis there is a long period of meiotic arrest of primary
(meiosis I) oocytes which starts before birth and lasts until sexual
maturity shortly before each preovulatory surge. Oocytes are
vulnerable when they divide during meiosis I and II and become
metabolically active: firstly, during fetal life before the block at
dictyotene stage (e.g. diplotene) in prophase I; secondly, in mature
life during the preovulatory stage of the menstrual cycle when
oocytes resume meiotic maturation into metaphase II (Mattison,
1993). In mice, diplotene oocytes and, to a lesser extent, oocytes in
metaphase I and II, have an efficient DNA repair system (Ku et al.,
1975; Masui and Pedersen, 1975; Brazill and Masui, 1978; Russell,
1980; Generoso, 1984; Guli and Smith, 1989) so that damage is
repaired before fertilization. Also, fertilized eggs of mice and
hamsters repair genetic damage by pre- and post-replication
mechanisms (Brandriff and Pedersen, 1981; Matzuda and Tobari,
1989; Genescá et al., 1992).
Spermatogenesis
In contrast, in spermatogenesis there is continuous production of
functional male germinal cells from puberty until old age; once
germinal cells enter meiosis (I and II) they become vulnerable to
exposure to environmental toxins (Wyrobek, 1986).
Spermiogenesis, the process of transforming spermatids into
spermatozoa (Russell and Saylors, 1963), is a second period of
sensitivity when de-novo point mutations arise (Generoso et al.,
1979, 1984; Chandley, 1991).
Spermatogonia and spermatocytes have the ability to repair DNA
(Dixon and Lee, 1980), as well as having efficient screening
mechanisms to eliminate aberrant cells with reduced viability
(Brusick, 1978). During the final stages of sperm cell
differentiation, however, spermatids have little if any repair
capacity; once the chromatin has condensed they do not repair DNA
damage (Bentley and Working, 1988). Having no repair capacity,
ejaculated spermatozoa are at risk for transmission of gene damage.
Indeed, it has been postulated that the risk of damage to germinal
cells from exposure to external factors is greater in males than in
females (Lessels, 1997). Mutation rates should be higher in males
than in females (Haldane, 1947) because of a greater number of
meiotic and mitotic divisions when mutations can accumulate. In
mice it was found that the number of divisions from spermatogenesis
to zygote formation (~205), is six times greater than the number from
oogenesis to the zygote (~33; Chang et al., 1994).
123
Smoking and clinical reproductive outcomes—
females
The impact of female smoking on clinical outcomes of IVF/embryo
transfer has been a matter of interest in assisted conception (Trapp
et al., 1986; Augood et al., 1998). Some studies, however, have
poor adjustment of confounding variables (e.g. age), small sample
sizes with insufficient statistical power, and lack information on
smoking habits or concentrations of cotinine, a marker for smoking
(Benowitz et al., 1983), precluding analysis of a dose-related
association. After adjusting for some of these deficiencies, the
overall results clearly implicate two mechanisms: (i) production of
fewer oocytes, and (ii) increased rates of abortion. These are
discussed below.
Reduction in number of oocytes
A relationship in number of retrieved oocytes in female patients
undergoing assisted conception in relation to smoking was analysed
(Harrison et al., 1990; Elenbogen et al., 1991; Pattinson et al., 1991;
Hughes et al., 1992, 1994; Van Voorhis et al., 1992, 1996; Sharara
et al., 1994; Zenzes et al., 1995a; and El-Nemr et al., 1998). Data
from these studies are summarized in Table I. The ratio of mean
number of oocytes of smokers divided by the mean for nonsmokers, which should be exactly one (except for random sampling
error) when there is no effect of smoking, is less than one in nine of
the 10 studies. The weighted overall mean ratio for nine studies
(omitting Pattinson et al., 1991; see Table I) is 0.920; this
corresponds to a mean reduction of number of oocytes of ~8.0%.
Using only the heavier smokers of the three studies in which they
are identified (Harrison et al., 1990, 11–30 cigarettes/day; Hughes
et al., 1992; Zenzes et al., 1995a), the weighted mean ratio is 0.828;
this corresponds to a mean 17.2% reduction in number of oocytes in
these heavy smokers.
This overall very significant reduction in number of oocytes in
association with smoking may have consequences for reproductive
outcomes in assisted conception, but this has not been consistently
demonstrated. Clinical recognition is suggestive from epidemiological studies in natural conception which found a consistent
dose-related smoking effect resulting in a conception delay of ~2
months (Weinberg et al., 1989; Zenzes, 1995; Curtis et al., 1997),
with a common odds ratio for conception in smokers of 0.57 (95%
confidence interval 0.42–0.78; Hughes and Brennan, 1996).
With advancing age the decline in number of retrieved oocytes
was found to be faster in smokers than non-smokers (Harrison et al.,
1990; Hughes et al., 1994; Sharara et al., 1994; Van Voorhis et al.,
1996; Zenzes et al., 1997a; Joesbury et al., 1998), indicating that the
synergistic effects of age and smoking may accelerate the rate of
oocyte destruction. Sharara et al. (1994) reports on a clinically
detectable decreased ovarian response with increasing age of 12%
in smokers, compared with 5% in non-smokers.
These studies are supported by the epidemiological evidence for a
dose-related decrease of age of menopause in smokers, (Jick et al.,
1977; Midgette and Baron, 1990), a clinical manifestation of follicle
and oocyte depletion. Studies in animals exposed to various cigarette
smoke components have also shown increased rates of follicular
depletion (Mattison et al., 1983; Shiromizu and Mattison, 1984).
124
M.T.Zenzes
Table I. Trends of decreasing numbers of oocytes with smoking
Reference
Smoking status No. of cigarettes/day No. of women Mean age (years) Mean no. of oocytes S/NS (oocytes)a
Harrison et al. (1990)
S
–
108
1–10
72
5.4
11–20
31
4.4
21–30
5
3.8
NS
Elenbogen et al. (1991)
S
–
NS
Pattinson et al. (1991)
S
–
NS
Van Voorhuis et al. (1992) S
–
NS
Hughes et al. (1992)
S
–
S
<40
5.1
20
33.5
6.2
21
32.6
6.8
?
33.1
5.1
?
32.6
5.4
18
32.6
13.4*
36
32.3
16.4
59
32.8
5.54c
0.912
0.944b
31.8
≥15
23
34.4
4.90c
163
34.1
5.70c
96
33.5
6.57c*
–
34.3
5.83
29
37.8
8.4
73
37.2
8.6
–
33
32.3
7.67
<15
19
32.2
7.95
≥15
14
32.4
7.29
PS
21
32.1
9.52d
NS
102
33.8
10.02
37
31.5
14.0d
111
33.8
b
15.1
351
32.9
15.4
65
33.3
6.2
108
33.2
11.1
S
–
S
XS
–
–
NS
S
NS
–
0.817
0.972
1.127
c
119
Van Voorhuis et al. (1996) S
El-Nemr et al. (1998)
542
36
NS
Zenzes et al. (1995)
0.980
1–14
NS
Sharara et al. (1994)
5.0
5.95c
NS
Hughes et al. (1994)
<40
0.977
0.765
0.909
0.559
S = smokers: NS = non-smokers; PS = husband smokes but not wife; XS = ex-smokers.
aMean no. of oocytes from smokers divided by the mean for non-smokers. Calculated by present authors from means given by study authors.
Weighting the individual study ratios by the numbers of smoker women or cycles, the overall weighted mean ratio for nine studies (omitting that
of Pattinson et al., 1991, which did not give number of women or cycles) is 0.920 ± 0.008, significantly different from one (no smoking effect) at
P ≤ 0.001. Using only heavier smokers (Harrison et al., 1990, 11–30 cigarettes/day; Hughes et al., 1992, Zenzes et al., 1995), the weighted
mean is 0.828 ± 0.006, significantly different from one at P ≤ 0.001.
bNot used in the calaculation of the overall S/NS ratio.
c Using no. of cycles.
dDose-dependent correlation with pack–years of cigarette smoking (packs/day × years of smoking).
*Significant difference between smokers and non-smokers (P < 0.05).
**Significantly different (P ≤ 0.0001).
Selection for better-quality oocytes
A mechanism for the dose-related reduction of oocytes from
smoking is that oocytes are preferentially eliminated shortly after
resumption of meiotic maturation into metaphase II. This is
suggested from quantitative studies on oocyte maturity (Zenzes
et al., 1995a, 1997a). The first study analysed 253 unfertilized
oocytes for chromosome status in relation to smoking habits.
Smoking and reproduction: gene damage to human gametes and embryos
Smokers had proportionally more (P = 0.03) analysable oocytes
(i.e. metaphase II oocytes of good quality for cytogenetic analysis)
than non-smokers. This observation was extended and confirmed in
a larger study using 2020 oocytes retrieved in relation to
concentrations of cotinine in follicular fluids. Oocytes were
assessed for maturity stage (e.g. immature, intermediate, mature or
post-mature) according to conventional morphological parameters
(Veeck, 1986). After correcting for age, a consistent trend
(P = 0.003) for an increasing proportion of mature oocytes with
increasing cotinine concentrations was found concomitantly with a
consistent trend for decreasing proportions of oocytes of
intermediate maturity (Zenzes et al., 1997a). This suggests that
oocytes are sensitive to damage shortly after they resume meiosis;
this would lead proportionally to deficiency in intermediary oocytes
and excess of mature oocytes. This is supported by a study in human
oocytes (Racowsky and Kaufman, 1992), in which degenerative
changes in chromatin were found predominantly at resumption of
meiosis I (diakinesis), a stage vulnerable to external insults.
In addition to the morphological evaluations of maturity status in
oocytes, Zenzes et al. (1997a) used the fertilization rate (number of
embryos/number of oocytes retrieved) as an independent and
objective measure of oocyte maturity, since mature oocytes have
better chances of fertilization than immature oocytes (Van Blerkom
et al., 1994). The results also showed a strong positive correlation
between cotinine and the rate of IVF (Zenzes et al., 1997a). This
higher rate of fertilization from smoking reflects an increased
incidence of mature, fertilizable oocytes. Higher rates of fertilization
in smokers, compared with non-smokers, were also reported in
several studies, also using relatively large numbers of patients
(n = 54–650; Trapp et al., 1986; Harrison et al., 1990; Pattinson et al.,
1991; Hughes et al., 1992; Van Voorhis et al., 1992; Sterzik et al.,
1996; Zenzes and Reed, 1999), but not in studies with smaller sample
size (n = 41–71; Elenbogen et al., 1991; Rosevear et al., 1992;
Rowlands et al., 1992). Possibly, the much smaller sample sizes in the
latter studies do not provide sufficient statistical power.
Selection for better-quality embryos
Apoptosis (programmed cell death) is a physiological process of cell
death that controls normality of cell populations during
embryogenesis and tissue homeostasis. Interestingly, nicotine and
cotinine were found to inhibit apoptosis in different cell lines (Wright
et al., 1993; Marana et al., 1998), suggesting that this inhibition
confers survival advantage to neoplasic cells and contributes to the
pathogenesis of tobacco-related cancer. Although apoptosis has not
been investigated in this context, two different studies investigated
whether fragmentation in human preimplantation embryos is
associated with smoking (Zenzes and Reed, 1996, 1999).
Morphological assessments in 1094 embryos (Zenzes and Reed,
1996), and in 1682 embryos (Zenzes and Reed, 1999) at 2–8-cell
stage, graded for quality from 1 to 4 (grades 1 and 2; with even-size
blastomeres and no fragments; 0–25% fragments, and grades 3 and 4;
25–45% fragments; >40% fragments) were analysed in relation to
follicular fluid cotinine. The proportion of embryos of grades 1 and 2
increased with increasing cotinine concentrations (P = 0.0001),
without age effect. These results suggest a cotinine-mediated
inhibition of apoptosis in developing embryos of smokers.
Acquisition of resistance to fragmentation from smoking may confer
survival advantage to embryos with genetic alterations.
125
Spontaneous abortion
A relationship between smoking and increased risk of spontaneous
abortion in assisted conception has been investigated by several
authors (Harrison et al., 1990; Pattinson et al., 1991; Hughes et al.,
1992, 1994; Maximovich and Beyler, 1995). Data from these
studies are summarized in Table II. The table shows, for each study,
the probabilities from Fisher’s exact 2 × 2 test for the proportion of
pregnancies terminating in abortion for all smokers and for nonsmokers. Two studies, Harrison et al. (1990) (P = 0.028) and
Maximovich and Beyler (1995) (P = 0.0006) have significant
probabilities (smokers have greater risk of abortion), and Pattinson
et al. (1991) has borderline significance (P = 0.074). The two
studies with clear non-significance (Hughes et al., 1992, 1994) are
also those with the smallest number of abortions, both in smokers
and non-smokers. Comparison of the Hughes studies with the others
in Table II is difficult, since the former studies are based on cycles
and the latter on pregnancies.
This evidence from a relatively small number of studies is
supported by epidemiological evaluations from ~100 000 women
using natural reproduction, in which a small, but significant,
increase in abortion rate with a dose–response effect was found (for
review see Hughes and Brennan, 1996).
Smoking and clinical reproductive outcomes—
males
Research is sparse on effects of paternal smoking in assisted
conception because reproductive problems (e.g. pregnancy loss)
have been traditionally associated with women. The available data
are not conclusive that male smoking affects IVF outcomes
(Pattinson et al., 1991; Hughes et al., 1994; Hughes and Brennan,
1996; Joesbury et al., 1998). The latter authors, addressing paternal
smoking effects in IVF outcomes, report reduced pregnancy rates
associated with male smoking and with increasing age of male
smoking partners; age is considered here a surrogate for duration of
smoking exposure (Joesbury et al., 1998). A limitation of this study
is that information on number of cigarettes smoked per day was
incomplete, precluding analysis of other variables.
Studies on the effect of smoking on conventional seminal
parameters (sperm density, motility, and morphology) give some
indication whether smoking impairs sperm function sufficiently to
be clinically apparent. Studies which adjusted smoke exposure to
cigarettes/year (Chia et al., 1994; Vine et al., 1996), or cotinine
concentrations in seminal plasma (Saaranen et al., 1989; Vine et al.,
1996; Pacifici et al., 1993), demonstrated a dose-related association
of smoking with low quality of spermatozoa. A study in young
healthy teenagers, to avoid a confounding age effect, also found a
trend towards poorer semen quality among smokers in association
with urine cotinine (Rubes et al., 1998). A meta-analysis found that,
in smokers, the sperm concentration is on average 13% lower than
that of non-smokers (Vine et al., 1994, 1996).
The standard parameters of semen analysis, however, cannot
predict the outcome of IVF (Toda et al., 1992). Low correlation
coefficients between sperm parameters and fertilization rates in
assisted conception (Sofikitis et al., 1993) may also be related to a
female factor which cannot be excluded in the analysis. On these
grounds, sperm function tests are more informative. Using the
outcomes of the zona-free hamster oocyte penetration test,
126
M.T.Zenzes
Table II. Smoking and abortion
Reference
Smoking status No. of cigarettes/day No. of pregnancies (cycles) No. of abortions A/P (C)
Smoking effecta
Harrison et al. (1990)
S
Yes (P = 0.028)
–
9
5
0.556
119
24
0.202
19
8
0.421
50
10
0.2000b
–
(77)
2
0.0260
1–14
(47)
1
0.0213
≥15
(30)
1
0.0333
(220)
3
0.0136
13
0
0.000
25
3
0.120
15
11
0.733
65
16
0.246
NS
Pattinson et al. (1991)
S
–
NS
Hughes et al. (1992)
S
NS
Hughes et al. (1994)
S
–
NS
Maximovitch and Beyler (1995) S
–
NS
? (P = 0.074)
No (not significant)
No (not significant)
Yes (P = 0.0006)
S = smokers: NS = non-smokers; A/P (C) = no. of abortions divided by the no. of pregnancies or no. of cycles.
aComparison of A/P (C) for smokers and non-smokers using Fisher’s exact 2 × 2 test (2-tailed probabilities) for an effect of smoking.
b
Authors give 0.189.
hypoosmotic swelling test, and sperm single/double-stranded DNA,
Sofikitis et al. (1995) showed a detrimental effect of male smoking in
such indices, the results of which were lower in heavy smokers (>20
cigarettes per day), than in non-smokers. Using a cytogenetic method,
terminal deoxynucleotidyl transferase-mediated dUTP-biotin endlabelling (TUNEL), to detect DNA fragmentation in oocytes from
intracytoplasmic sperm injection (ICSI), Lopes et al. (1998) found
that spermatozoa with DNA fragmentation were negatively
associated with both fertilization rates and seminal parameters.
Effects of smoking on meiotic spindle function
Disturbances in microtubule polymerization and assembly affect
the pulling of chromosomes from the equator to the poles, leading to
errors in chromosome number (aneuploidies) in the resulting
daughter cells. Because aneuploidy can be induced by chemicals
(for reviews see Dellarce et al., 1985; Tarín, 1996), cytochemical
analyses of the spindle and chromosomes provide useful models for
studying the action of environmental chemicals.
The meiotic spindle in human oocytes can be disrupted by
external factors such as advanced maternal age (Battaglia et al.,
1996), oocyte ageing (Eichenlaub-Ritter et al., 1988), subtle
changes in temperature (Pickering et al., 1990), and cigarette
smoking (Zenzes et al., 1995a). The latter study found increased
proportions of oocytes with diploid chromosome complements (46
instead of 23) in association with number of cigarettes smoked per
day (P = 0.0006), and an increased proportion of triploid zygotes.
Diploid (digynic) oocytes arise from lack of extrusion of first polar
body (Tarín et al., 1991), indicating failure of meiotic spindle
function. It is known that alkaloids bind to tubulin, a protein in the
tubules of spindles (Singer and Himes, 1992; Jordan et al., 1998).
By a similar mechanism of disruption of meiotic spindle function in
spermatozoa, male smoking leads to disturbances of normal
chromosome number. A recent cohort study done in 25 teenage
males, to exclude a possible age effect, found increased rates of
disomic spermatozoa, with disomy for chromosomes Y, X, X-Y, and
8, in association with urine cotinine concentration (Rubes et al.,
1998). A study of 45 healthy male volunteers aged 19–45 years found
a suggestive (P = 0.07) but not consistent association between
smoking ≥20 cigarettes per day and XX,18 aneuploidy (Robbins
et al., 1997).
Components of cigarette smoke in gonadal fluids
and cells
Cigarette tobacco contains several thousand compounds (Dube and
Green, 1982). The bulk of the tobacco consists of carbohydrates and
proteins (~50%). Other significant constituents are alkaloids (0.5–
5%) with nicotine the predominant compound (90–95% of total
alkaloids), polyphenols (0.5–4.5%), phytoteroles (0.1–2.5%),
carboxilic acids (0.1–0.7%), alkanes (0.1–0.4%), aromatic
hydrocarbons, aldehydes, ketones, amines, nitriles, N- and Oheterocyclic compounds, pesticides, alkali nitrates (0.01–5%) and at
least 30 metallic compounds (Wynder and Hoffmann, 1967;
International Agency for Research on Cancer, 1986). More than 40
compounds are known to be chemical carcinogens, most of which
are also considered mutagenic to humans (IARC, 1986). Three
components of cigarette tobacco cadmium, cotinine, and
benzo[a]pyrene are discussed here in relation to their interactions
with human gametic cells.
Cadmium
This heavy metal is present in water, soil, air, foods, and in
cigarettes; smoking constitutes a major source of inhaled cadmium
(Norman, 1977; Saaranen et al.,1989; Staessen et al., 1990).
Cadmium occurs in amounts of 1.0–2.0 µg per cigarette (Nandi et
al., 1969). Up to 70% of this amount is passed through burning into
smoke, and up to 10% of cadmium in a cigarette (0.1–0.2 µg) is
Smoking and reproduction: gene damage to human gametes and embryos
inhaled (Schröeder and Balassa, 1961; Nandi et al., 1969;
Szadkowski et al., 1969).
Cadmium accumulates over time in different organs, and in blood
(Lewis et al., 1972; Watanabe et al., 1983), with a substantial
increase in blood cadmium concentrations in heavy smokers of ≥20
cigarettes per day (Nandi et al., 1969; Lewis et al., 1972; Brockhaus
et al., 1983; Watanabe et al., 1983; Ulander and Axelson, 1984;
Staessen et al., 1990; Chia et al., 1994). In ovarian and testicular
tissue, epididymides, and seminal vesicles, removed at necropsy
from individuals who died suddenly, a linear age-dependent
accumulation of cadmium from smoking was found (Oldereid et al.,
1993; Varga et al., 1993). Cadmium was detected in follicular fluids
of women undergoing assisted conception, with a significant dose
effect in relation to smoking (Zenzes et al., 1995b). In seminal
plasma the concentration of cadmium in heavy smokers is ~1.8
times greater than in non-smokers (Saaranen et al., 1989; Oldereid
et al., 1993, 1994; Chia et al., 1994; Telisman et al., 1997).
Nicotine/cotinine
The principal alkaloid in tobacco, nicotine, is present in cigarettes in
amounts varying from 0.8 to 1.8 mg per cigarette, according to the
brand and size of cigarettes (Benowitz et al., 1983; Rosa et al.,
1992). As much as 1 mg of nicotine can be absorbed by smoking a
single cigarette (Barbieri et al., 1986). Nicotine is a very toxic
alkaloid, and is quickly absorbed through the respiratory track,
mouth mucosa and skin (Gandini et al., 1997). About 80–90% of
nicotine is metabolized mainly by the liver, but also by the kidney
and lung (Armitage et al., 1975).
Cotinine, its major metabolite, is a more stable compound than
nicotine, with a longer half-life of ~20 h (nicotine is 2 h), and its
concentration is not influenced by confounding factors, e.g. diet
(Isaac and Rand, 1972; Matsukara et al., 1979; Rosenberg et al.,
1980; Benowitz et al., 1983; Pojer et al., 1984; Darby et al., 1984;
Pirkle et al., 1996; Davis et al., 1991; Rosa et al., 1992). Cotinine is
therefore used as a marker for recent cigarette smoke exposure and
dose (Benowitz et al., 1983).
Cotinine has been detected in follicular fluids of women
undergoing assisted conception (Weiss and Eckert, 1989; Rosevear
et al., 1992), and in dose-related association with cigarette
consumption (Sterzik et al., 1996; Zenzes et al., 1996). On the male
side, nicotine and/or cotinine were also detected in seminal plasma
of male subjects in dose-related association with cigarette
consumption (Pacifici et al., 1993, 1995; Vine et al., 1993; Zenzes
et al., 1999b). This indicates passage of the metabolite through the
blood–testis barrier.
Cellular components of ovarian follicles are readily permeable to
endogenous and exogenous chemicals (Fabro, 1978). Indeed,
cotinine was found incorporated into ovarian granulosa–lutein cells
in dose-relationship with follicular fluid cotinine (Zenzes et al.,
1997b). Using antiserum to cotinine and immunoperoxidase, a
positive reaction was visualized in the nuclei and cytoplasm of
granulosa–lutein cells. Binding of this alkaloid to nuclear and
cytoplasmic proteins could affect the developmental potential of
maturing follicles and could lead to perturbations in meiotic
maturation of oocytes. When female hamsters were injected with
nicotine, Racowsky et al. (1989) found in their oocytes a significant
delay in meiotic maturation and meiotic arrest, but only at doses
higher than those in heavy smokers of cigarette tobacco.
127
In-vitro studies of smoking on the physiology of human
granulosa–lutein cells are controversial. In-vitro exposure to low
molecular weight constituents of aqueous tobacco smoke extracts
inhibited granulosa cell aromatase; nicotine inhibited steroid
aromatization (Barbieri et al., 1986). In contrast, Weiss and Eckert
(1989) found that neither nicotine nor cotinine altered progesterone
or oestradiol secretion by granulosa cells in vitro in the presence of
serum, while in serum-free medium Bódis et al. (1992) found dosedependent increase in the secretion of oestradiol and decrease in the
secretion of progesterone in serum-free medium. This study
speculated that early abortion in pregnant women who smoke may
be due to nicotine-related corpus luteum insufficiency; however,
other factors may also be involved.
Benzo[a]pyrene
Benzo[a]pyrene belongs to a group of environmental pollutants,
polycyclic aromatic hydrocarbons (PAHs), produced mainly by
combustion of fossil fuels (IARC, 1986). PAHs have the ability to
bind covalently to DNA, giving rise to adducts (Koreda et al., 1978).
Benzo[a]pyrene occurs in cigarettes by combustion in amounts of 6–
40 ng per cigarette; a pack-a-day smoker who consumed 20 cigarettes
in 8 h could be expected to inhale from 0.067 to 0.568 µg of
benzo[a]pyrene (Kaiserman and Rickert, 1992).
Benzo[a]pyrene is highly mutagenic and carcinogenic (IARC,
1986). The carcinogenic metabolite (8a-dihydroxy-9a,10a-epoxy7,8,10-tetrahydro-benzo[a]pyrene) is a diol epoxide derivative of
benzo[a]pyrene (BPDE-I) which binds predominantly to the 2amino group of DNA guanosine, forming adducts (Jeffrey et al.,
1977). These adducts are premutational lesions in guanosine
nucleosides, which, if not repaired, constitute a potential source of
carcinogenic damage (Denissenko et al., 1996).
Immunocytochemical techniques using monoclonal or polyclonal
antibodies directed against PAH–DNA adducts are useful for
quantitative analysis of adducts in different target cells of smoking
individuals. In ovarian tissue, BPDE–DNA adducts were detected
in oocytes, luteal cells, and stromal arteries of postmortem ovaries
(Shamsudin and Ghan, 1988), but no quantitative data on adduct
values were given, and no detailed information on the smokers
status was provided. In granulosa–lutein cells, the mean BPDE–
DNA adduct values were detected in dose-related association with
follicular fluid cotinine (Zenzes et al., 1998). Granulosa–lutein cells
treated in vitro with anti-BPDE monoclonal antibody, which
recognizes and is structurally related to BPDE–DNA adducts and
other PAH-derived DNA adducts (Santella et al., 1970, 1984),
showed positive reactivity confined to the nuclei. A 3-fold and a 2fold increase of adduct concentrations was found respectively, in
active and passive smokers, relative to non-smokers (Zenzes et al.,
1998). Mooney et al. (1995) also found that passive exposure to
smoke at home was significantly associated with PAH–DNA
adducts in leukocytes.
The presence of BPDE–DNA adducts in the ovarian cells of
smokers (Shamsudin and Ghan, 1988; Zenzes et al., 1998) indicates
a potential risk for DNA damage. In hamster cell lines
benzo[a]pyrene induced cytogenetic damage including structural
and numerical chromosomal aberrations and spindle disturbances
(Sbrana et al., 1995; Matzuoka et al., 1997). Administration in high
doses into mice resulted in destruction of primordial oocytes
(Mattison and Thorgeison, 1979; Mattison et al., 1983; 1989),
128
M.T.Zenzes
depending on genetic differences in mice of benzo[a]pyrene
metabolism (Felton et al., 1978).
The impact of smoking on ovarian function is not only through
interactions between gonadal cells and constituents in cigarette
smoke, but also from decreasing oestrogen secretion (Schulman
et al., 1990). Women smokers excrete only about one-third as much
oestrogen during the luteal phase of the ovulatory cycle than nonsmokers or ex-smokers (MacMahon et al., 1982). Cigarette
smoking appears to interfere with biosynthesis of oestrogens,
suggested by lower serum oestrogen concentrations in oestrogentreated women smokers after treatment, compared with their nonoestrogen treated smoker counterparts (Jensen et al., 1985). This
oestrogen deficiency in smokers is linked to an earlier onset of
menopause (Jick et al., 1977).
Gene damage to spermatozoa from tobacco smoke
Tobacco smoke can cause DNA damage or chromosomal damage
in human germinal cells, and in oocytes and spermatozoa (Little and
Vainio, 1994; Zenzes et al., 1995a, 1999b; Fraga et al., 1996;
Robbins et al., 1997; Shen et al., 1997; Rubes et al., 1998).
Experimental work has shown that cigarette smoke constituents
and/or their DNA-reactive metabolic intermediates react directly
with spermatozoa (Fraga et al., 1996; Shen et al., 1997; Zenzes
et al., 1999b). A major form of DNA damage found in lung cells of
smokers, 8-hydroxydexyguanosine (8-OHdG), is an oxidative
lesion of guanine (Asami et al., 1997). Concentrations of 8-OHdG
also occur in spermatozoa in association with smoking and seminal
plasma cotinine (Fraga et al., 1996; Shen et al., 1997).
Using [32P]-postlabelling autoradiograms of DNA isolated from
sperm cells of heavy smokers, light smokers and non-smokers,
Gallagher et al. (1993) did not detect smoking-related adducts.
Using immunoperoxidase on sperm cells pre-treated with a
disulphide reducing agent for accessibility to DNA binding sites,
BPDE–DNA adducts were detected in spermatozoa in dosedependent association with seminal plasma cotinine (Zenzes et al.,
1999b). Inter-individual variations in adduct concentrations were
found both in smokers and non-smokers. This reflects individual
variations in biological response to exposure via life style,
occupation and ambient air pollution, and to variations in
metabolism and detoxification of carcinogens (Niebert, 1991;
Mooney et al., 1997).
Oxidative damage from smoking
Occurrence in cigarette smoke of toxic oxygen reactive species, i.e.
superoxide anion (O2–), hydrogen peroxide (H2O2) and free radical
(OH–)] has been widely demonstrated (Stone and Bermudes, 1986;
Church and Pryor, 1990). These cause pro-oxidant/antioxidant
imbalance, leading to oxidative stress (Fraga et al., 1996; Hulea et al.,
1995). Accumulated oxidative stress induces mitochondrial and nuclear
DNA damage (Ames et al., 1994; Ballinger et al., 1996). Also, a rise in
oxidative stress is associated with in-vitro ageing (Tarín, 1995), and is
bound to produce cytoskeletal alterations in oocytes and embryos
leading to chromosomal aneuploidy and cellular fragmentation.
Oxidative stress can also be induced experimentally in mice oocytes
using oxidizing agents; this leads to errors in chromosome segregation
in meiosis I and II (Tarín et al., 1996).
Figure 1. Diol epoxide derivative of benzo[a]pyrene (BPDE)–DNA adducts in
human preimplantation embryos. (A) Eight-cell embryo from a smoker couple.
(B) Two-cell embryo from a non-smoker couple. Bar = 10 µm. (Courtesy of Dr
Hang Yin, Division of Reproductive Sciences, Department of Obstetrics and
Gynaecology, University of Toronto, Canada).
Smoking-related adducts in spermatozoa (Fraga et al., 1996; Shen
et al., 1997; Zenzes et al., 1999b) arise from oxidative damage.
Oxidant radicals in cigarette tar are assumed to bind, perhaps
covalently, to DNA and to produce DNA nicks (Stone and Bermudes,
1986; Pryor and Stone, 1993). Using TUNEL, fragmentation of DNA
was detected in human embryos from IVF (Jurisicova et al., 1996).
The increased incidence of cellular fragmentation and abnormal
morphology in cultured human embryos often result from free
oxygen radical damage (Tarín, 1996). Increased fragmentation in
embryos was correlated with increased concentrations of H2O2
concentration in the culture medium (Yang et al., 1998).
In spermatozoa, Sun et al. (1997) reported higher proportions of
spermatozoa with DNA fragmentation in male smoker patients in
assisted conception, compared with non-smokers. The results of this
study are, however, inconclusive as no dose-relationships with
smoking were obtained and no data on fertile subjects are shown to
rule out infertility factors.
Gametic transmission of gene damage
Because ejaculated spermatozoa have minimal, if any, repair capacity
(Bentley and Working, 1988), formation of smoking-related adducts
in spermatozoa are a potential source of transmissible prezygotic
DNA damage. Parental transmission of BPDE–DNA adducts was
recently investigated in early human preimplantation embryos
(Zenzes et al., 1999a). In this study of twenty-seven preimplantation
embryos at 4–8-cell stage treated with monoclonal antibody against
benzo[a]pyrene, reactivity was assessed individually in 83
blastomeres. The proportion of reactive blastomeres was higher
(P = 0.007) in embryos of smoker couples (either both members
smoked or only the husband smoked), than in embryos of nonsmokers. A 3.7-fold increase in mean adduct concentrations of
embryos from smokers was found to indicate transmission of
modified DNA from parental smoking. Figure 1 depicts two such
treated embryos. The embryo from a smoking couple shows
reactivity in the nuclei in several blastomeres, in contrast to the
embryo from a non-smoking couple where reactivity is negligible.
In this study, similar mean adduct concentrations were found in
embryos from couples where both parents smoked, or where only
the father smoked. This suggests preferential gametic transmission
Smoking and reproduction: gene damage to human gametes and embryos
of modified DNA from smoking fathers (Zenzes et al., 1999a).
Ideally, embryos of couples where only the wife smoked should
have been included to confirm preferential paternal transmission,
but these were not available.
Paternal transmission of altered DNA may compromise
embryonic development in utero, resulting in failed implantation,
early pregnancy loss, or disturbances of postnatal development. In a
case control population study of 642 individuals of a Chinese
community where female smoking is discouraged (Ji et al., 1997),
paternal smoking during the preconception period was associated
with increased risk of childhood cancer within the child’s first 5
years. This evidence of paternal gametic transmission of genetic
damage from smoking has implications for male reproduction.
Acknowledgements
I wish to thank the patients of the IVF/embryo transfer programme
at the Toronto Hospital for kindly donating spare IVF material and
Dr Robert F.Casper, Division of Reproductive Sciences, University
of Toronto, for access to this material. I am also grateful to my
research associate Dr Ryszard Bielecki, Division of Reproductive
Sciences, University of Toronto, for his intellectual and technical
contribution to this research. My kind appreciation goes also to Dr
T.E.Reed, Department of Zoology, University of Toronto, for help
in the statistical analysis and revisions of the manuscript, and to
Claudina Berlanga, Metropolitan Autonomous University of
Mexico, for useful comments. This study was supported by a Grant
(#MT 12871) from the Medical Research Council, Ottawa, Canada.
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Received on July 21, 1999; accepted on January 13, 2000