Effects of lead exposure on sperm concentrations and testes weight in male rats: a meta-regression analysis.

Abstract

The correlation between exposure to lead (PB) and sperm concentrations and testes weight are important in risk assessment of metal toxicity to male reproductive functions. The authors carried out two systematic reviews and meta-analyses of rats. In addition, a meta-regression analysis was taken to explore a dose-response relationship between the mean difference of testes weight (MD(testes)) and available confounding factors. Data were obtained from computerized literature searches of several databases from their inception through December 2005. The reference lists of identified articles were examined further for relevant articles. The authors identified 6 and 12 studies, separated into 16 and 28 subgroups, in the two meta-analyses. The authors conducted random- or fixed-effect models toward the effect size. Heterogeneity between study results was explored through chi-square tests and meta-regression. Results showed that a decrease in sperm concentrations was found as low as mean difference of sperm concentrations (MD(sperm)) = 30.9 and 95%CI = (25.43-36.37) in a fixed-effect model or MD(sperm) = 35.47 and 95%CI = (15.27-55.68) in a random-effect model after Pb exposure. Similarly, a significantly lower testes weight was also evident: MD(testes) = 0.033 and 95%CI = (0.021-0.046) in a fixed-effect model or MD(testes) = 0.047 and 95%CI = (0.0044-0.089) in a random-effect model. In the meta-regression analysis, two confounders, age and body weight, explained part of the observed heterogeneity. The body weight after Pb exposure was inversely associated with MD(testes). These findings support the notion that Pb exposure produced decreased sperm concentrations and testes weight in rats.

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Effects of Lead Exposure on Sperm Concentrations and
Testes Weight in Male Rats: A Meta-regression Analysis
Lu Wangab; Pengcheng Xuna; Yang Zhaoa; Xinru Wanga; Ling Qianc; Feng
Chena
aDepartment of Epidemiology and Biostatistics, School of Public Health, Nanjing
Medical University, Nanjing
bWuxi Center for Disease Control and Prevention, Wuxi
cNational Institute for Health Education, Chinese Center for Disease Control and
Prevention, Beijing, China
Online Publication Date: 01 January 2008
To cite this Article: Wang, Lu, Xun, Pengcheng, Zhao, Yang, Wang, Xinru, Qian, Ling and Chen, Feng (2008)
'Effects of Lead Exposure on Sperm Concentrations and Testes Weight in Male Rats: A Meta-regression Analysis',
Journal of Toxicology and Environmental Health, Part A, 71:7, 454 — 463
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454
Journal of Toxicology and Environmental Health, Part A, 71: 454–463, 2008
Copyright © Taylor & Francis Group, LLC
ISSN: 1528-7394 print / 1087-2620 online
DOI: 10.1080/15287390701839331
UTEH
Effects of Lead Exposure on Sperm Concentrations and
Testes Weight in Male Rats: A Meta-regression Analysis
Lead-Induced Effects on Reproductive System
Lu Wang1,2, Pengcheng Xun1, Yang Zhao1, Xinru Wang1,
Ling Qian3, and Feng Chen1
1Department of Epidemiology and Biostatistics, School of Public Health, Nanjing Medical University,
Nanjing, 2Wuxi Center for Disease Control and Prevention, Wuxi, and 3National Institute for Health
Education, Chinese Center for Disease Control and Prevention, Beijing, China
The correlation between exposure to lead (PB) and sperm con-
centrations and testes weight are important in risk assessment of
metal toxicity to male reproductive functions. The authors carried
out two systematic reviews and meta-analyses of rats. In addition,
a meta-regression analysis was taken to explore a dose-response
relationship between the mean difference of testes weight
(MDtestes) and available confounding factors. Data were obtained
from computerized literature searches of several databases from
their inception through December 2005. The reference lists of
identified articles were examined further for relevant articles.
The authors identified 6 and 12 studies, separated into 16 and
28 subgroups, in the two meta-analyses. The authors conducted
random- or fixed-effect models toward the effect size. Heterogene-
ity between study results was explored through chi-square tests
and meta-regression. Results showed that a decrease in sperm
concentrations was found as low as mean difference of sperm con-
centrations (MDsperm) = 30.9 and 95%CI = (25.43-36.37) in a
fixed-effect model or MDsperm = 35.47 and 95%CI = (15.27-55.68)
in a random-effect model after Pb exposure. Similarly, a signifi-
cantly lower testes weight was also evident: MDtestes = 0.033 and
95%CI = (0.021-0.046) in a fixed-effect model or MDtestes = 0.047
and 95%CI = (0.0044-0.089) in a random-effect model. In the
meta-regression analysis, two confounders, age and body weight,
explained part of the observed heterogeneity. The body weight
after Pb exposure was inversely associated with MDtestes. These
findings support the notion that Pb exposure produced decreased
sperm concentrations and testes weight in rats.
The roles that heavy metals play in the etiology of repro-
ductive pathogenesis have been debated for several decades.
Lead (Pb) is a well-known environmental toxicant that affects
multiple organ systems (Marcos et al., 2004). Exposure to Pb
has been associated with many adverse effects on the male
reproductive system in both humans and rodents (Sokol, 1987;
Sokol & Berman, 1991; Apostoli et al., 1998). Although the regu-
latory mechanisms of the male reproductive system are com-
plex and not fully understood, there is no doubt that Pb alters
the functions of human reproductive organs in a dose-related
fashion (Lataillade et al., 1993; Corpas et al., 1995). Monitor-
ing organ weight during treatment provides an index of the
general health status of the animals, and this information may
be important for the interpretation of adverse reproductive
effects (Bagchi and Preuss, 2005). A direct testicular toxicity
may occur via the hypothalamic-pituitary-testicular axis (Klein
et al., 1994; Sokol et al., 1994). Testes weight data may be
presented as absolute weights and/or relative weights. Evalu-
ation of data on absolute organ weight basis is important,
because a decrease in a reproductive organ weight may occur
that was not necessarily related to a reduction in body weight
gain. The organ-weight-to-body-weight ratio may show no
significant difference if both body weight and organ weight
change in the same direction, masking a potential organ weight
effect (U.S. EPA, 1996). Other parameters that are important
for the reproductive risk assessment are sperm evaluations, includ-
ing sperm number, sperm morphology, and sperm motility.
Among these measures, sperm concentrations (count) were
the most frequently reported semen variable in the literature
in humans (Wyrobek, 1983).
The adverse effects of Pb on testes weight and sperm con-
centrations were reported by a number of investigators, but the
results are mixed and conflicting. Sokol et al. (1994) found a
reduction in sperm concentrations after exposing animals to Pb
for 14, 30, and 60 days, whereas Coffigny et al. (1994) and
Manlay et al. (1995) reported that sperm concentrations did not
change significantly after metal exposure. Similar results were
Received 28 August 2007; accepted 19 September 2007.
This study was supported by a National Key Basic Research
Program grant (2002CB512910), a National Natural Science
Foundation grant (30571619), and a Natural Science Foundation of
Jiangsu Province grant (04KJB310081). The authors thank Qingyi
Wei for his critical comments and expert editing of this article.
Address correspondence to Dr. Feng Chen, Department of
Epidemiology and Biostatistics, School of Public Health, Nanjing
Medical University, 140 Hanzhong Road, Nanjing, Jiangsu Province,
People’s Republic of China. E-mail: fengchen@njmu.edu.cn

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LEAD-INDUCED EFFECTS ON REPRODUCTIVE SYSTEM
455
also found in studies of Pb effects on testes weight
(Chowdhury et al., 1984; Manlay et al., 1995; Marchlewicz
et al., 2004; Ronis et al., 1998). In order to evaluate the current
knowledge in the field, a comprehensive literature review was
undertaken and a meta-analysis of the effects of Pb exposure
on sperm concentrations and testes weight in rats was per-
formed.
MATERIALS AND METHODS
Search Strategy
A systematic review and meta-analysis was performed in
accordance with the QUOROM (Quality of Reporting of Meta-
analyses) guidelines for meta-analyses of observational studies
(Moher et al., 1999). A Medline search, which included papers
published between 1980 to 2005, was carried out with a combi-
nation of the following keywords: lead, Pb, reproductive toxic-
ity, testicular, testes, sperm reserves, sperm concentrations,
male, animal, and rats. Potentially relevant references were
evaluated by examining the titles and abstracts of all references
obtained, and these publications were obtained for a closer
examination. Besides the database search, the reference lists of
the selected papers were also screened for other potential arti-
cles. The search and evaluation was conducted between
September and December 2005.
The following inclusion and exclusion criteria were used for
the meta-analysis: (1) The article should be published in
English between January 1980 and December 2005; (2) Pb
should be the only metal used in the experiments, and the com-
binations of Pb with other test materials, such as grain alcohol,
aluminum, and zinc were not included (Saxena et al., 1989;
Giridhar and Isom, 1990; Batra and Nehru, 1998); (3) only
in vivo experiments on rat models were considered; (4) the arti-
cle should report sperm concentrations or absolute testes
weight of both sides of the animals before and after Pb expo-
sure; and (5) those publications that presented data allowing
such outcomes to be derived were also included.
After the search, 40 published papers associated with ani-
mal studies of Pb exposure were selected. These papers were
viewed in accordance with the criteria just listed, and seven
papers were excluded initially as they used mice as experiment
models (Johansson et al., 1986; Zuhair et al., 1998; Johansson
1989; Lataillade et al., 1995; Wadi et al., 1999; Graca et al.,
2004; Pace et al., 2005). Among the others, 27 studies focused
only on the testicular system, while 3 studies reported only
sperm concentrations, and another 3 papers reported both testes
weight and sperm concentrations (Lataillade et al., 1993;
Coffigny et al., 1994; Manlay et al., 1995).
In the study by Hsu et al. (1998), the effects of Pb were
combined with vitamin E and/or C. Because some studies on
sperm concentrations did not provide information on testes
weight, subgroup analysis following Pb exposure only was
conducted. Thus, five studies, including Sokol and Berman
(1991), Sokol et al. (1994), Lataillade et al. (1993), Coffigny
et al. (1994), and Manaly et al. (1995), and one subgroup from
Hsu et al. (1998) were included in our meta-analysis of sperm
concentrations.
Among the 30 papers focused on testes, 13 papers were
excluded initially because they did not provide arithmetic means
or standard deviations of testes weight before and after metal
exposure, and this information could not be further derived from
the published data. The study by Sokol and Berman (1991) only
reported weight of one testis, but not the pair, and the standard
deviation of testes weight could not calculated from the study by
Sokol et al. (1985). In the Chowdhury et al. (1986) study, the
related data were expressed in illustrations from which real data
were difficult to derive. Another two papers (Murthy et al., 1991;
Boscolo et al., 1988) focused only on ultrastruture of the testes and
thus were not selected. Hence, 12 papers were included in our
meta-analysis of testes weight, including a total of 518 rats.
Data Extraction
To minimize the bias and improve reliability, all potentially
relevant studies were checked independently by two reviewers.
In addition to the available data on the means of sperm concen-
trations and paired testes weight with or without Pb exposure
and their corresponding standard deviations, data on the fol-
lowing characteristics were also extracted: first author, dates
on which the study was published, strains of rats, and age and
body weight at time rats were sacrificed.
A standardized procedure was used to estimate standard devia-
tion from the standard error displayed in several of the papers. If
the study provided stratum information, each stratum of datum
was entered separately to make a full use of the data (Zhao et al.,
2007). As a result, 14 and 28 subgroups were identified in the
meta-analysis of sperm concentrations and testes weight, respec-
tively (Tables 1 and 2). All selected studies were controlled under
standard animal experimental conditions. From these reports, data
were abstracted in duplicate, using a standardized form.
Some of the studies reported the exact data on age and body
weight of the rats before and after exposure, but some other
papers only provided a crude description, such as “mature,” for
age (Chowdhury et al., 1984; Nathan et al., 1992; Machlewicz
et al., 2004). According to the sexual cycle of the rats, 21 days
was regarded as the weaned age and 80 days as the mature age.
In this case, the age was recoded as a quantitative scale. In
cases where age at the time when rats were sacrificed was
equal to or older than 80 days, the variable “age” was given a
value of 2; otherwise it was given a value of 1.
Methods for Quantitative Synthesis
Mean differences and their 95% confidence intervals (CI) in
sperm concentrations and paired testes weight in each study
and their standard deviations were calculated directly from
data given in the article; that is, the effect size considered in
our study was the occurrence of a mean difference.

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L. WANG ET AL.
In the analysis of pooled data, three different approaches were
used: (1) a fixed-effect model, (2) a random-effect model, and (3) a
meta-regression model (Derry and Loke, 2000; Houwelingen et al.,
2002). A fixed-effect model was first performed, assuming the
same homogeneity of the effect size across all of studies, followed
by a random-effect model under the assumption of heterogeneity.
Both within-study variability and between-study variability were
considered in the random-effect model, and the effect size was
assumed to be drawn from a distribution with a specific mean and
variance (Bagnardi et al., 2004). Finally, meta-regression tech-
niques were used to assess the observed between-subgroups heter-
ogeneity by the inclusion of known study features (Biggeri et al.,
2005; Harder et al., 2005; Alder et al., 2006). In the meta-analysis
of testes weight, age and body weight were considered as covari-
ates to establish the regression model. The regression coefficient
with its 95% confidence interval was reported for data presentation.
Test for heterogeneity between study results was also per-
formed with a chi-square test. A funnel plot was drawn to
assess the publication bias, and the test suggested by Egger and
Smith (1997) was used to test for the funnel plot symmetry.
The test involves a regression model using the standardized
estimate of the size effect as the dependent variable and the
inverse of the standard error as an independent variable. If
the intercept is significantly different from zero, the estimate of
the effect is considered biased.
All of the statistical analyses were performed with Statisti-
cal Analysis System software (version 9.1.3, SAS Institute,
Inc., Cary, NC) and STATA software (version 7.0, Stata
Corporation, College Station, TX).
RESULTS
Literature Search
The search strategy recommended by the QUOROM is
shown in Figure 1. A set of 6 published papers on sperm
concentrations was reviewed, among which 5 studies with
13 subgroups met the inclusion criteria. In the study by Hsu
et al. (1998), the effects of Pb were combined with vitamin E
and/or C. In order to make full use of the data, one subgroup
was extracted that was affected by Pb only, resulting in a total
of 14 subgroups in the database (see Table 1). It was found that
sperm concentrations in 6 of the 14 subgroups (Sokol &
Berman, 1991; Sokol et al., 1994) were significantly reduced
after metal exposure. Although six subgroups (three from
Sokol & Berman (1991), one from Manlay et al. (1995), one
from Coffigny et al. (1994), and one from Hsu et al. (1998))
showed lower sperm concentrations after exposure to Pb, the
association was not statistically significant. In contrast, in the
study of Lataillade et al. (1993), sperm concentrations after Pb
exposure were higher than prior to exposure.
TABLE 1
Characteristics of Studies Included in the Meta-Analysis of Sperm Concentrations
Unexposed Exposed
Sperm concentrations
(106/sperm/g testes)
Sperm concentrations
(106/sperm/g testes)
AuthorsYear Strain Agea
n
Mean SD
n
MeanSD
Sokol and Berman 1991Wister 729
9
8
8
172.6
172.6
238.4
238.4
162.2
162.2
166
136
71
442
467
410
141.11
530
38.4
38.4
46.1
46.1
27.2
27.2
58.14
40
18.71
15.87
23.81
13.23
15.81
100
10
10
155.2
151.1
166.1*
117*
86*
142.4
178
157
66
360*
418*
383*
137
480
31.63
24.35
16.12
15.27
18.24
38.26
26.83
48
10.39
13.23
21.17
13.23
18.03
150
828
8
10010
10
20
16
14
11
10
20
16
12
Lataillade et al. 1993SD160
Manlay et al.
Sokol et al.
1995
1994
SD
SD
115
114
130
160
90
91
7
7
7
7
7
7
Coffigny et al.
Hsu et al.
1994
1998
SD
SD
1013
66
Note. Asterisk indicates significant difference from unexposed, p < .05.
aAge at the end of experiments, when rats were sacrificed.

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LEAD-INDUCED EFFECTS ON REPRODUCTIVE SYSTEM
457
In the next analysis, 12 studies with 28 subgroups were identi-
fied that met our inclusion criteria in the meta-analysis of paired
testes weight. Table 2 indicates which papers were related to each
subgroup and summarizes the characteristics of these subgroups.
Four of the 28 subgroups exhibited a decreased testes weight after
Pb exposure, and others did not show any significant changes, of
which two subgroups (Marchlewicz et al., 1993; Coffigny et al.,
1994) did not assess these results with a significance test.
In animal experiments, several factors interfere with the
results: (1) variability within species, (2) age at the start of
experiments, (3) duration of exposure, (4) body weight, and (5)
other biological variations. Of the 12 studies reviewed in this
paper, 8 had stated a numeric age at the start of experiments. In
another three studies, age at the start was only described as
“mature” (Chowdhury et al., 1983; Nathan et al., 1992;
Machlewicz et al., 2004), and the remaining one study reported
age as “weaned” (Corpas et al., 2002). After considering the
duration of exposure, the age at the time when rats were sacri-
ficed was calculated and given as “mature” or “immature.”
Another major issue that needs to be taken into account was the
body weight. Since all 10 studies with 20 subgroups reported
the exact data, a weighted meta-regression with body weight as
covariate was performed by a random-effect model.
Quantitative Data Synthesis
Study characteristics of the included reports are displayed in
Tables 1 and 2. Figure 2 shows the forest plot of the pooled
TABLE 2
Characteristics of Studies Included in the Meta-Analysis of Paired Testes Weight
Authors YearStrain Agea
Unexposed Exposed
n
Body
weight (g)
Testes weight (g)
n
Body
weight (g)
Testes weight (g)
Mean SDMeanSD
Manlay et al.
Marchlewicz et al.
Ronis et al.
1995
2004
1996
SD115 d
Mature
74 d
74 d
85 d
42 d
14
10
5
10
11
527
470
341.12
337.14
378.5
1.96
1.94
3.65
3.54
3.52
0.48
0.48
0.48
1.80
1.80
1.80
1.80
1.92
1.92
1.92
1.92
1.94
2.03
3.27
1.54
1.49
1.64
1.81
1.82
1.36
1.36
1.36
0.12
0.14
0.14
0.29
0.16
0.2
0.02
0.02
0.02
0.17
0.17
0.17
0.17
0.2
0.2
0.2
0.2
0.2
0.04
0.64
0.08
0.19
0.21
0.21
0.05
0.58
0.58
0.58
0.04
12
10
5
11
7
379
465
333.33
281.74
299.01
1.84
1.97
3.60
3.24*
3.02*
0.48
0.47
0.48
1.93
1.76
1.77
1.80
1.86
1.84
1.72
1.67
2.02
2.05
3.46b
1.55
1.53
1.55
1.93
1.73b
1.28
0.96
0.68*
0.08*
0.17
0.45
0.22
0.23
0.29
0.04
0.04
0.02
0.17
0.21
0.14
0.26
0.18
0.11
0.24
0.04
0.2
0.22
0.86
0.08
0.11
0.19
0.05
0.03
1.71
0.1
1.02
0.03
Wistar
SD
Adhikari et al.2001 Druckray5
5
5
8
8
8
8
4
4
4
4
5
5
5
8
7
8
7
5
8
7
4
Nathan et al.1992 SDmatured 465
465
465
465
530
530
530
530
518
557
525
503
441
439
405
532
531
506
544
495
538
500
Fowler et al.1980 SD9 mo
Lataillade et al.1993 SD160 d 16
20
13
16
20
13 Marchlewicz et al.
Sokol
1993
1990
Wistar
Wistar
12 mo
59 d
66 d
82 d
112 d
90 d
Mature
7
7
7
7
7
7
7
7
Coffigny et al.
Chowdhury et al.
1994
1984
SD10
10
10
10
24
13
10
10
10
30
Albino 83
83
83
41.63
77.14
76.42
74.28
39.98Corpas et al.2002 Wistarweaned
Note. Asterisk indicates significant difference from unexposed, p < .05.
aAge = age at the end of experiments, when rats were sacrificed.
bNo significant test provided in the study.

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458
L. WANG ET AL.
estimate and 95% confidence interval for the reduction in the
sperm concentrations after Pb exposure, calculated from a ran-
dom-effect model. Sperm concentrations were significantly
decreased after exposure to Pb, and the pooled mean difference
of sperm concentrations (MDsperm) was 35.47 × 106/sperm/g
testes (95%CI: 15.27–55.68). A fixed-effect model revealed a
similar pooled estimate and 95% CI (MDsperm = 30.90,
95%CI: 25.43–36.37). More detailed data are presented in
Figure 3. Since a significant heterogeneity between subgroups
was found, the results of a random-effect model might be more
reliable.
In the meta-analysis of paired testes weight, heterogeneity
between the included 28 subgroups was significant, and thus a
random-effect model was used throughout the analysis. In
order to compare the differences, the results of both fixed- and
random-effect models were reported. Figure 4 shows the forest
plot with mean difference in paired testes weight (MDtestes)
with 95% CI in the subgroups included in the meta-analysis.
FIG. 1.
Process of inclusion of studies in the meta-analysis. In total, 18 studies were included in the two analyses.
Studies about both sperm
concentrations and testes system for
more detailed evaluation (n = 3)
Potentially relevant studies identified
(n = 40)
Studies retrieved for more detailed
evaluation (n = 33)
Studies excluded due to use mice
as experiment models (n = 7)
Studies about testes system for
more detailed evaluation (n = 27)
Studies about sperm concentrations
for more detailed evaluation (n = 3)
Studies included in meta-analysis of
sperm concentrations (n = 6)
One subgroup excluded because
interaction of lead and VE
Potentially appropriate studies to be
included in meta-analysis (n = 17)
Studies excluded because no desired
outcome measurements (n = 13)
Studies withdrawn, and lack of
comprehensive data (n = 5)
Studies included in meta-analysis of
testes weight (n = 12)
FIG. 2.
performed by a random-effect model.
Forest plot of MDsperm with their 95% CIs in the 14 subgroups
Mean difference in sperm concentrations (MDsperm)
MDsperm (95%CI)
17.40 (–14.10, 48.90)
21.50 (–7.09, 50.09)
72.30 (38.46, 106.14)
121.40
155.05)
76.20 (56.57, 95.83)
19.80 (–9.30, 48.90)
5.00 (–6.92, 16.92)
–12.00
16.06)
– 21.00 (–51.62, 9.62)
49.00 (25.40, 72.60)
82.00 (66.70, 97.31)
27.00 (13.14, 40.86)
4.11 (–10.00, 18.22)
50.00
194.25)
(87.75,
(–40.06,
(–94.25,
–150 –100 –50500100150 200250
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Overall

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LEAD-INDUCED EFFECTS ON REPRODUCTIVE SYSTEM
459
Table 3 shows results of the meta-analysis. A fixed-effects
model was used to calculate the estimate of MDtestes and 95%
CI (MDtestes = 0.033, 95%CI: 0.021–0.046). The maximum
likelihood estimate (ML-estimate) of MDtestes was 0.047 with
standard error 0.021, and 95%CI was 0.0044 to 0.089 under the
random-effect model. The ML-estimate of between subgroups
variance was 0.0053.
A regression analysis for MDtestes on age was carried out
by constructing a mixed model. The regression coefficients for
age1 (age < 80 d) and age2 (age ≥ 80 d) in the fixed-effect
model were significant, 0.023 and 0.0073. In the random-effect
model with adjustment for age, that is, in a weighted meta-
regression, the corresponding coefficients for age1 and age2
were 0.025 and 0.06, respectively. The weights were the
inverse squared standard error of MDtestes. The residual
between-subgroup variance in this model was 0.0039, smaller
than the between-subgroup variance (0.0053) in a random-
effect model described earlier without the covariate age in the
model. Thus, data indicated that age may partially explain the
between-subgroup variance.
In only 10 of these studies (20 subgroups) were the exact
data for body weight provided. Considering body weight as an
important factor on the alterations of testes weight, the meta-
regression was performed to identify the relationship between
body weight and mean difference in paired testes weight
(Figure 5). Results indicated that body weight was
significantly negatively correlated to MDtestes (regression
coefficients for the intercept and for bodyweight were, respec-
tively, –0.00038 and 0.34). After considering both covariates,
age and body weight, models for age groups could be written
as follow (more details are presented in Table 4).
FIG. 3.
performed by a fixed-effect model.
Forest plot of MDsperm with their 95% CIs in the 14 subgroups
Mean difference in sperm concentrations (MDsperm)
MDsperm (95%CI)
17.40 (–14.10, 48.90)
21.50 (–7.09, 50.09)
72.30 (38.46, 106.14)
121.40 (87.75, 155.05)
76.20 (56.57, 95.83)
19.80 (–9.30, 48.90)
5.00 (–6.92, 16.92)
–12.00 (–40.06, 16.06)
–21.00 (–51.62, 9.62)
49.00 (25.40, 72.60)
82.00 (66.69, 97.31)
27.00 (13.14, 40.86)
4.11 (–10.00, 18.22)
50.00 (–94.25, 194.25)
30.90 (25.43, 36.37)
–150 –100 –50050100150200250
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Overall
FIG. 4.
effect models without adjustment; n means the total number included in each subgroup.
Forest plot of MDtestes with their 95% CIs including 28 subgroups in the meta-analysis. At the bottom of the figure are the results of fixed- and random-
–202
MDtestes
SE
95%CI
1 (n = 26)
2 (n = 20)
3 (n = 10)
4 (n = 21)
5 (n = 18)
6 (n = 10)
7 (n = 10)
8 (n = 10)
9 (n = 16)
10(n = 15)
11(n = 16)
12(n = 15)
13(n = 9)
14(n = 12)
15(n = 11)
16(n = 8)
17(n = 32)
18(n = 40)
19(n = 26)
20(n = 14)
21(n = 14)
22(n = 14)
23(n = 14)
24(n = 23)
25(n = 20)
26(n = 20)
27(n = 20)
28(n = 54)
(Fixed-effect model)
(Random-effect model)
0.12
–0.03
0.05
0.30
0.50
0.00
0.01
0.00
–0.13
0.04
0.03
0.00
0.06
0.08
0.20
0.25
–0.08
–0.02
–0.19
–0.01
–0.04
0.09
–0.12
0.09
0.08
0.40
0.68
0.04
0.033
0.047
0.06
0.15
0.16
0.09
0.11
0.02
0.02
0.01
0.08
0.10
0.08
0.11
0.13
0.09
0.14
0.10
0.07
0.05
0.30
0.04
0.08
0.11
0.08
0.02
0.57
0.19
0.37
0.01
0.0061
0.021
(0.00, 0.25)
(–0.33, 0.28)
(–0.32, 0.43)
(0.12, 0.48)
(0.26, 0.74)
(–0.04, 0.05)
(–0.03, 0.06)
(–0.02, 0.03)
(–0.30, 0.05)
(–0.16, 0.25)
(–0.13, 0.20)
(–0.23, 0.24)
(–0.23, 0.36)
(–0.11, 0.28)
(–0.11, 0.52)
(0.00, 0.50)
(–0.21, 0.06)
(–0.11, 0.08)
(–0.79, 0.42)
(–0.09, 0.08)
(–0.21, 0.14)
(–0.13, 0.32)
(–0.29, 0.06)
(0.06, 0.12)
(–1.11, 1.28)
(0.01, 0.79)
(–0.09, 1.46)
(0.02, 0.06)
(0.021, 0.046)
(0.0044, 0.089)
Model for age1 (age < 80 d):
MDtestes
=
0 22.body we
−×
0 00058.i ight
Model for age2 (age
MDtestes
=
80 d):
body we
≥
−×
0 34.0 00058.i ight

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460
L. WANG ET AL.
No evidence of publication bias was observed in the meta-
analysis of either MDsperm or MDtestes, as indicated by
symmetric funnel plots (Figures 6 and 7). Furthermore, the
Egger’s test showed an intercept of 17.83 for the meta-analysis
of MDsperm and an intercept of 0.072 for MDtestes, suggest-
ing there was no existence of publication bias in both analyses.
DISCUSSION
Our meta-analysis focused on the alterations of sperm con-
centrations and paired testes weight under the influence of Pb
exposure in animal experiments. Semen analysis is consid-
ered to be an effective method to evaluate the reproductive
abnormality produced by physical or chemical factors,
because semen identifies the transmitted genetic damage with
high specificity (Sokol, 1987). General semen analysis
includes a total sperm count, sperm concentrations, sperm
motility, and alterations of sperm chromatin structure (Williams
et al., 1990; Hsu et al., 1997), and thus alterations in these
parameters indicates reproductive damage, such as infertility or
adverse reproductive outcomes. In our meta-analysis of sperm
concentrations with 14 subgroups from published reports, a
reduction in sperm concentrations was found with both ran-
dom and fixed-effect models after Pb exposure. This suggests
that Pb may exert an effect on sperm concentrations and thus
is likely to damage reproductive functions by interfering with
sperm production. However, ejaculated sperm number from
FIG. 5.
proportional to inverse squared standard error of MDtestes.
Meta-regression for MDtestes against body weight. Size of circle is
Body Weight
0100200 300 400500 600
–0.5
0
0.5
1
MDtestes
TABLE 3
Results of Both Fixed- and Random-Effect Models for MDtestes of 28 Subgroups
Model
Pooled estimate
of MDtestes (g)
Standard
error (g)95% CI
p
Fixed-effect model
Random-effect model
Fixed-effect model with adjustment for agea
0.033
0.047
0.073
−0.050
0.060
−0.035
0.006
0.021
0.013
0.015
0.026
0.037
(0.021, 0.046)
(0.004, 0.089)
(0.046, 0.099)
(−0.081,−0.019)
(0.008, 0.110)
—
<.0001
.032
<.0001
.003
.027
—
Intercept
Age 1 (age < 80 d)
Intercept
Age 1 (age < 80 d)
Random-effect model with adjustment for agea
aAge 2 (age ≥ 80 d) was considered as control group in the model.
TABLE 4
Results of the Meta-Regression on Age and Body Weight of 20 Subgroups
Model
Pooled estimate of
MDtestes (g)
Standard
error (g)95% CI
p
Random-effect model
Random-effect model with adjustment
only for body weight
0.0890.036(0.014, 0.17).023
Intercept
Body weight
0.24
−0.00038
0.097
0.00023
(0.035, 0.44)
—
.024
—
Random-effect model with adjustment
for both body weight and agea
Intercept
Body weight
Age 1 (age < 80 d)
0.34
−0.00058
−0.12
0.15
0.00032
0.13
(0.033, 0.65)
—
—
.032
—
—
aAge 2 (age ≥ 80 d) was considered as control group in the model.

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LEAD-INDUCED EFFECTS ON REPRODUCTIVE SYSTEM
461
any species is influenced by several variables, including the
length of abstinence and the ability to obtain the entire ejacu-
late. Intra- and interindividual variation are often high, but
are reduced somewhat if ejaculates are collected at regular
intervals from the same male (Williams et al., 1990). The
ability to detect a decrease in testicular sperm production may
be enhanced if spermatid counts are available.
Normal testis weight varies within a given test species
(Schwetz et al., 1980). This relatively low interanimal variabil-
ity suggests that absolute testis weight may be a reliable indica-
tor of gonadal injury. A significantly lower MDtestes was
found in the meta-analysis of paired testes weight after Pb
exposure (Chemishanska et al., 1984). Under heterogeneity of
the effect size in each study, the pooled estimate of MDtestes
shown by the random-effect model was significantly lower
after metal exposure. These results indicate that Pb exposure
produced degeneration of the testes and support the hypothesis
that a direct testicular toxicity may occur via the hypothalamic-
pituitary-testicular axis. Significant changes in absolute or rela-
tive male reproductive organ weights may constitute an
adverse reproductive effect. Such changes also may provide a
basis for obtaining additional information on the reproductive
toxicity of this agent. However, damage to testes may be
detected as a weight change only at doses higher than those
required to produce significant effects on other parameters of
gonadal status (Berndtson, 1977; Foote et al., 1986). This con-
tradiction may arise from several factors, including a delay
before cell death is reflected in a weight decrease (due to pre-
ceding edema and inflammation, cellular infiltration, etc.) or
Leydig-cell hyperplasia (Gorbel et al., 2002). Significant
changes in other important endpoints that are related to repro-
ductive function may not be reflected as organ weight data.
Therefore, lack of an organ weight effect may not be used to
negate significant changes in other endpoints that may be more
sensitive (U.S. EPA, 1996).
In case of substantial heterogeneity between the 28 subgroups,
a meta-regression model was used to investigate the influence of
several confounding variables. This model assumed a fixed effect
for the covariates and treated the study effects as random varia-
tions around a population mean (Houwelingen et al., 2002). Two
covariates, age and body weight, were considered to correct the
treatment effect and assess existing heterogeneity between each
pair of subgroups. Using the meta-regression, it was found that
body weight was inversely and linearly associated with the
MDtestes. This indicated that Pb exposure may exert weaker pro-
ductive effects on rats with high body weight than on those with
low body weight. It should be noted that because of the lack of
information, the confounder age was included as a binomial vari-
able. For this type of data, a common solution is to perform the
subgroup analysis suggested by Oxman and Guyatt (1992).
Indeed, combining specific subgroup data across studies may
provide further insight into heterogeneity, but meta-regression
analysis is known to be highly flexible with regard to the shape of
a dose-response relationship. In general, the results of the meta-
regression analysis are compatible with that of subgroup analysis.
In our meta-analysis, the pooled estimated mean difference in tes-
tes weight in the fixed-effect model with adjustment for age was
nearly the same as that of subgroup analysis for age (i.e., a fixed-
effect model). The pooled estimates of MDtestes and their 95%
CI were nearly identical (Figure 8).
Though the heterogeneity was partly explained by the two
covariates included in the meta-regression, there must be other
potential confounders that were not fully presented in the pub-
lished papers. For example, the testes Pb content and doses of
metal used also need to be considered. Still, many of the limita-
tions inherent in the study design exist (Murthy et al., 1991; Ronis
et al., 1996; Gandley et al., 1999; Arrieta et al., 2004). Therefore,
one should be cautious when interpreting the results. If the covari-
ates in the model might not explain heterogeneity between studies,
a nonlinear mixed-effect model based on some biological mecha-
nisms needs to be considered. This sort of model might explain the
heterogeneity in the highest measure. Other available methods,
such as the multilevel models and Bayesian hierarchical models,
might be considered in future investigations.
FIG. 6.
concentrations.
Funnel plot for MDsperm in the meta-analysis of sperm
020
Standard Error of MDsperm
40 6080
–100
0
100
200
MDsperm
FIG. 7.
Funnel plot for MDtestes in the meta-analysis of paired testes weight.
Standard Error of MDtestes
00.20.40.6
–1
0
1
MDtestes

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462
L. WANG ET AL.
Because the papers included in our meta-analysis were
limited to those published in English and in the period between
1980–2005, it is possible that some relevant published studies
were not included, which may have biased the results. Although
the test for publication bias showed no significant results, possi-
ble bias, especially the outcome-reporting bias, still can not be
ruled out because of the low power of such assessment.
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0.02 (0.01, 0.04)
80d)
0.12 (0.00, 0.24)
–0.03 (–0.32, 0.26)
0.50 ( 0.27, 0.73)
0.06 (–0.19, 0.31)
0.08 (–0.09, 0.25)
0.20 (–0.08, 0.48)
0.25 (0.05, 0.45)
–0.08 (–0.22, 0.06)
–0.02 (–0.12, 0.08)
–0.19 (–0.77, 0.39)
0.09 (–0.12, 0.30)
–0.12 (–0.28, 0.04)
0.09 ( 0.06, 0.12)
0.08 (–1.04, 1.20)
0.41 (0.04, 0.77)
0.68 (–0.05, 1.40)
–0.13 (–0.30, 0.04)
0.04 (–0.15, 0.23)
0.03 (–0.12, 0.18)
0.00 (–0.22, 0.22)
0.07 (0.05, 0.10)
Overall
0.03 (0.02, 0.05)

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