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,
From the * Department of Environmental Health
Sciences, University of Michigan, Ann Arbor, Michigan; the
Department of Bioengineering, University of
Washington, Seattle, Washington; the
Department of Environmental Health, Harvard
School of Public Health, Boston, Massachusetts; and the
Vincent Memorial Obstetrics and Gynecology
Service, Andrology Laboratory and In Vitro Fertilization Unit, Massachusetts
General Hospital, Boston, Massachusetts.
| Correspondence to: Dr John Meeker, Department of Environmental Health Sciences, University of Michigan School of Public Health, 6635 SPH Tower, 109 S Observatory St, Ann Arbor, MI 48109 (e-mail: meekerj{at}umich.edu). |
| Received for publication October 30, 2007; accepted for publication March 3, 2008. |
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Abstract |
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Key words: Chromatin, comet assay, epidemiology, hormone, human
Endogenous hormones are critical to spermatogenesis and maintenance of male reproductive function, although many of the basic mechanisms involved are still not completely understood (Lo and Lamb, 2004). Follicle-stimulating hormone (FSH), luteinizing hormone (LH), inhibin B, and testosterone all serve important and well-known functions in the male hypothalamopituitary-gonadal axis and male reproduction (Lo and Lamb 2004), but in recent years important roles for estradiol (O'Donnell et al, 2001; Hess, 2003; Akingbemi, 2005) and thyroid hormones (Jannini et al, 1995; Krassas and Pontikides, 2004; Trokoudes et al, 2006) in spermatogenesis, germ cell survival, and apoptosis have been described. However, much remains unknown about the relationships and potential roles of hormones in human sperm DNA integrity and damage. Two previous studies assessed the relationship between hormone levels and DNA damage, as measured by the sperm chromatin structure assay (SCSA), with conflicting results. Richthoff et al (2002) reported that sperm DNA damage was inversely correlated with both testosterone and estradiol levels among 267 healthy young Swedish men, whereas Appasamy et al (2007) reported no associations between sperm DNA damage and FSH, inhibin B, or testosterone in 129 men from a United Kingdom infertility clinic.
To our knowledge, no human studies have assessed the relationship between hormone levels and sperm DNA damage measured using assays other than SCSA (eg, terminal deoxyribonucleotide transferase–mediated nick-end labeling or comet assay). In addition, no previous studies have been found that explored the association between thyroid hormone levels and sperm DNA damage. The present study was conducted to assess the relationships between serum hormone levels and sperm DNA damage, as assessed by the neutral comet assay, in men recruited from an infertility clinic. The neutral comet assay has proven useful in epidemiologic studies because it is a simple and sensitive assay that measures DNA double-strand breaks, a critical DNA lesion for normal cell survival (Singh and Stephens, 1998).
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Methods |
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Semen Sample Collection
Semen was collected on site at MGH in sterile plastic specimen cups after a
recommended period of abstinence of 48 hours. After liquefaction at 37°C
for 30 minutes, semen parameters and characteristics were measured.
Measurement of the semen parameters (sperm concentration, motility, and
morphology) has been described previously
(Hauser et al, 2003). Briefly,
we measured sperm count and motility by computer-aided semen analysis using
the Hamilton Thorne IVOS 10 Analyzer (Hamilton-Thorne Research, Beverly,
Massachusetts). To assess sperm morphology, we evaluated 200 sperm using the
Tygerberg strict criteria (Kruger et al,
1988). The remaining unprocessed semen was frozen in 0.25-mL
cryogenic straws (CryoBiosystem, San Diego, California) by immersion of the
straws directly into liquid nitrogen (–196°C). Previous work in our
laboratory showed that this freezing method produced comet assay results that
were highly correlated with results from fresh, unfrozen samples
(Duty et al, 2002). Semen
samples were later analyzed in batches. Straws were thawed by gently shaking
in a 37°C water bath for 10 seconds, and the semen was immediately
processed for the comet assay.
Neutral Comet Assay
The comet assay procedure used in the present study has been previously
described (Singh and Stephens,
1998; Duty et al,
2002). Briefly, 50 µL of a semen/agarose mixture (0.7% 3:1
high-resolution agarose; Amresco, Solon, Ohio) was embedded between 2
additional layers of agarose on microgel electrophoresis glass slides (Erie
Scientific, Portsmouth, New Hampshire). Slides were then immersed in a cold
lysing solution to dissolve the cell membrane and make chromatin accessible
for the enzyme digestion steps. After 1 hour of cold lysis, slides were
transferred to a solution with 10 µg/mL of RNase (Amresco) for enzyme
treatment and incubated at 37°C for 4 hours. Slides were then transferred
to a second enzyme treatment with 1 mg/mL proteinase K (Amresco) and incubated
at 37°C for 18 hours. The slides were placed on a horizontal slab in an
electrophoretic unit, were equilibrated for 20 minutes, and underwent
electrophoresis for 1 hour. DNA in the gel was then precipitated, fixed in
ethanol, and dried. Slides were stained and observed with fluorescence
microscopy. Comet extent, tail-distributed moment (TDM), and percent DNA
located in the tail (Tail%) were measured on 100 sperm in each semen sample
using VisComet software (Impuls Computergestutzte Bildanalyse GmbH, Gilching,
Germany). Comet extent is a measure of total comet length from the beginning
of the head to the last visible pixel in the tail. Tail% is a measurement of
the proportion of total DNA that is present in the tail. TDM is an
integrated value that takes into account both the distance and intensity of
comet fragments:
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I is the sum of all intensity values that belong to the
head, body, or tail and X is the x-position of the intensity value.
Comets with high DNA damage (CHD), which are cells too long to measure with
VisComet (>300 µm), were counted for each subject and used as an
additional measure of DNA damage.
Serum Hormones
One nonfasting blood sample was drawn between the hours of 9:00
AM and 4:00 PM on the same day that the semen sample was
collected. Blood samples were centrifuged, and serum was stored at
–80°C until analysis. Testosterone was measured directly using the
Coat-A-Count radioimmunoassay kit (Diagnostic Products Corp, Los Angeles,
California), which has an interassay and intraassay coefficient of variation
(CV) of 12% and 10%, respectively, with a sensitivity of 4 ng/dL (0.139
nmol/L). The free androgen index (FAI) was calculated as the molar ratio of
total testosterone to sex hormone–binding globulin (SHBG). SHBG was
measured using a fully automated system (Immulite; Diagnostic Products Corp)
that uses a solid-phase 2-site chemiluminescent enzyme immunometric assay and
has an interassay CV of less than 8%. Inhibin B was measured using a
commercially available, double-antibody, enzyme-linked immunosorbent assay
(Oxford Bioinnovation, Oxford, United Kingdom) with interassay and intraassay
CVs of 20% and 8%, respectively; limit of detection of 15.6 pg/mL, and
functional sensitivity (20% CV) of 50 pg/mL. Serum LH, FSH, estradiol, and
prolactin concentrations were determined by microparticle enzyme immunoassay
using an automated Abbott AxSYM System (Abbott Laboratories, Chicago,
Illinois). The Second International Reference Preparation (World Health
Organization 71/223) was used as the reference standard. The assay sensitivity
for LH and FSH were 1.2 international units (IU)/L and 1.1 IU/L, respectively.
The intraassay CVs for LH and FSH were less than 5% and less than 3%,
respectively, with interassay CVs for both hormones of less than 9%. The
testosterone:LH ratio, a measure of Leydig cell function, was calculated by
dividing testosterone (nmol/L) by LH (IU/L). The assay sensitivity for
estradiol and prolactin were 20 pg/mL and 0.6 ng/mL, respectively. For
estradiol, the within-run CV was between 3% and 11%, and the total CV was
between 5% and 15%. For prolactin, the within-run CV was less than or equal to
3%, and the total CV was less than or equal to 6%.
Free T4, total T3, and thyroid-stimulating hormone (TSH) concentrations were also determined in serum by microparticle enzyme immunoassay (AxSYM Automated System; Abbott Diagnostics). The assay sensitivity for free T4 and total T3 were 0.01 ng/dL and 0.15 ng/mL, respectively. The interassay CVs for both hormones were less than 9%. For TSH, the ultrasensitive hTSH II assay (Abbott Diagnostics) was used and has a functional sensitivity of 0.03 µIU/L and interassay CVs of less than 8%.
Statistical Analysis
Data analysis was performed using SAS version 9.1 (SAS Institute Inc, Cary,
North Carolina). Descriptive statistics on subject demographics were
calculated, along with the distributions of hormone levels and comet assay
measures. Hormone levels and comet measures were stratified by demographic
categories, and a student's t test or 1-way analysis of variance was
conducted to investigate differences between categories and the potential for
confounding. Spearman correlation coefficients were used to determine
correlations among hormones, among comet measures, and between comet
parameters and hormone levels.
Multivariate linear regression was used to explore continuous relationships between hormone levels and measures of sperm DNA damage. Serum concentrations of testosterone, estradiol, inhibin B, free T4, and total T3 closely approximated normality and were used in statistical models untransformed, whereas the distributions of FSH, LH, SHBG, FAI, prolactin, and TSH concentrations were skewed left and transformed to the natural log (ln) for statistical analyses. Comet extent, TDM, and Tail% were modeled untransformed. The number of CHD in each subject's semen sample was not normally distributed, and an arcsine transformation was used (Zar, 1984). To improve interpretability, the regression coefficients were back-transformed and expressed as a change in the dependent variable (ie, comet measures) for an interquartile range (IQR) increase in hormone levels. To explore the shape of hormone-DNA damage relationships, comet measures were also regressed on hormone quartiles. Finally, hormones associated with high levels of each of the DNA damage measures were also explored. For this analysis, each comet measure was divided into quartiles, and multivariate logistic regression was used to calculate odds ratios (ORs) for having a comet measure in the highest quartile among increasing hormone quartiles.
Inclusion of covariates was based on statistical and biologic considerations (Hosmer and Lemeshow, 1989). Age and body mass index (BMI) were modeled as a continuous variable, smoking status was dichotomized by current smoker vs never-smoker or former smoker, and race/ethnicity was categorized into 4 groups: white, African American, Hispanic, and other. The period of abstinence prior to semen sample collection was modeled as a 5-category ordinal variable, and timing of blood sample by season (winter vs spring, summer, or fall) and time of day (9:00 AM–12:59 PM vs 1:00 PM–4:00 PM) were considered for inclusion in the models as dichotomous variables. Semen quality parameters (sperm concentration, motility, or morphology) were additionally considered for inclusion as continuous variables because of relationships with comet measures (Trisini, et al. 2004) and hormone levels (Meeker et al, 2007). Sperm motility and morphology were normally distributed, whereas sperm concentration followed a log-normal distribution and was transformed using ln prior to inclusion in the models.
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Results |
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In preliminary bivariate analyses (Spearman correlations) for the continuous variables, age was positively associated (P < .05 for all associations listed in the text) with FSH and LH levels but inversely associated with estradiol, FAI, free T4, and total T3 levels. BMI was associated with decreased levels of LH, inhibin B, testosterone, and SHBG but increased levels of estradiol, FAI, and TSH. Among comet measures, there was a positive association between age and CHD and an inverse association between BMI and Tail%. Among hormones, FSH was positively correlated with LH, and both FSH and LH were positively correlated with prolactin but inversely correlated with inhibin B. Testosterone was positively associated with LH, SHBG, and estradiol. There was an inverse association between estradiol and inhibin B and positive associations between estradiol and SHBG, free T4, and total T3. In addition, inhibin B was positively associated with SHBG but negatively associated with both prolactin and total T3.
Among the categoric variables, men whose clinic visits occurred in the winter had higher comet extent and lower serum inhibin B and free T4 levels compared with those in the spring, summer, or fall. There were positive associations between the ordinal abstinence period categories and LH and FSH levels—men with abstinence time of longer than 6 days had higher LH and FSH levels than men who abstained 3 or less days. Current smokers had higher total T3 concentrations and lower TSH and prolactin concentrations than former and nonsmokers. Men with blood samples collected in the morning (between 9:00 AM and 12:59 PM) had higher testosterone and TSH levels and lower prolactin levels compared with those with blood samples collected in the afternoon.
Multivariate linear regression results for the association of hormone levels with sperm DNA damage are presented in Table 3, both with and without (ln-transformed) sperm concentration included in the models. The associations of sperm DNA damage with FSH, LH, inhibin B, and total T3 levels but not testosterone, estradiol, FAI, and free T4 levels were confounded by sperm concentration. Following adjustment by sperm concentration, there were significant but inconsistent associations between sperm DNA damage measures and the levels of LH, inhibin B, and testosterone. For comet extent, there were statistically significant or suggestive declines of 8.2 µm (95% confidence intervals [CI]: –12.7 to –3.8 µm), 4.7 µm (–9.7 to 0.3), and 3.9 µm (–8.1 to 0.2) associated with IQR increases in the levels of estradiol, free T4, and total T3, respectively. An IQR increase in estradiol was also associated with a 2.21-µm (–4.0 to –0.4) decline in TDM and a 4.6% (–6.4% to –2.6%) decline in Tail%. Based on the population median values of comet extent (131 µm), TDM (57 µm), and Tail% (28%), for an IQR increase in estradiol, these coefficients represent declines of 6.3% (95% CI: –9.7% to –2.9%), 3.9% (–7.1% to –0.7%), and 16.2% (–22.4% to –9.2%), respectively. Both free T4 and total T3 levels were positively associated with CHD. However, there were suggestive inverse associations between free T4 and total T3 and comet extent and significant inverse associations between both thyroid hormones and Tail%. For an IQR increase in free T4, the coefficients represent declines in the study population median (95% CI) of 3.6% (–7.4% to 0.2%) for comet extent and 24.4% (–31.5% to –17.4%) for Tail%. Results in Table 3 were similar when sperm motility was added to the models in place of sperm concentration (results not shown).
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When sperm DNA damage measures were regressed on hormone quartiles, most of the statistically significant relationships presented in Table 3 remained significant. Regression coefficients followed significant monotonic trends indicative of a dose-dependent relationship. For example, the regression coefficients for a change in comet extent among increasing estradiol quartiles (1 through 4) were 0 (reference group), –8.7 (95% CI: –19.4 to 2.06), –14.4 (–24.9 to –3.98), and –22.4 (–33.7 to –11.2), respectively (P < .0001). However, there were a few differences between the regression analysis using hormone quartiles as compared with regression results when hormone levels were modeled as continuous variables (Table 3). In the quartile analysis, there were statistically significant but nonmonotonic trends in the associations for both free T4 and total T3 levels with CHD. There was also a nonmonotonic positive trend in the relationship between total T3 and comet extent, which was inconsistent with the inverse association between total T3 and comet extent in the original regression analysis (Table 3). In addition, the association between total T3 and Tail% was not found in the quartile regression analysis.
For the assessment of hormonal predictors of sperm DNA damage, adjusted ORs were calculated to assess the odds of being in the highest quartile for each of the DNA damage measures among increasing hormone quartiles. Increased estradiol levels were associated with significantly reduced odds of being in the highest comet extent, TDM, and Tail% quartiles (Figure 1), potentially suggesting a protective association. The adjusted ORs for being in the highest quartile for DNA damage comparing the highest estradiol quartile to the lowest estradiol quartile were 0.19 (95% CI: 0.08–0.47), 0.28 (0.12–0.69), and 0.25 (0.11–0.56) for comet extent, TDM, and Tail%, respectively. Free T4 levels were also associated with lower odds of having high comet extent or Tail% measures but was not associated with TDM (Figure 2). The OR for the highest Tail% quartile among men in the highest compared with the lowest free T4 quartile was highly protective (OR = 0.08; 95% CI: 0.03–0.23). Conversely, total T3 levels were suggestively associated with increased odds of being in the highest quartile for comet extent, TDM, and CHD, although the relationships did not follow a monotonic trend (Figure 3). Of the 4 DNA damage measures, testosterone was only associated with a suggestive decrease in odds for being in the highest Tail% quartile. Adjusted ORs for being above the 75th percentile for Tail% among testosterone quartiles 1 through 4 were as follows: 1.0 (reference group), 0.96 (95% CI: 0.48–1.92), 0.71 (0.33–1.53), and 0.52 (0.22–1.21); P = .09.
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Discussion |
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Several other statistically significant or suggestive associations between hormone levels (FSH, LH, inhibin B, testosterone, and total T3) and sperm DNA damage were observed, although they were not as strong or consistent as those for estradiol and free T4. Confounding by sperm concentration of the associations involving FSH, LH, and inhibin B was not unexpected because associations between semen quality parameters and sperm DNA damage (Trisini et al, 2004) and between these hormones and semen quality (Meeker et al, 2007) were both previously reported in cohorts that overlapped with the men in the present study. However, after sperm concentration or motility was considered in the multivariate models, the strong relationships involving estradiol and free T4 remained, suggesting these associations exist independent of semen quality parameters.
To our knowledge, this is the first human study to explore associations between sperm DNA damage and thyroid hormone levels, and our findings for an inverse association between estradiol and sperm DNA damage are consistent with 1 previous study (Richthoff et al, 2002). These researchers used the SCSA, as opposed to the neutral comet assay used in the present study, and reported a statistically significant inverse correlation between estradiol and DNA fragmentation index (DFI; a measure of DNA denaturation following SCSA) among 278 Swedish military conscripts. They also found that DFI was inversely correlated with testosterone levels, although the correlation was not as strong as with estradiol. Another recent study reported no associations between DFI and FSH, inhibin B, or testosterone among 129 men undergoing infertility evaluation (Appasamy et al, 2007). However, only bivariate relationships were tested, and the study did not account for confounding variables in multivariate analysis.
Estradiol is produced by testosterone aromatization, but individual
differences in aromatase activity results in varying correlations between the
2 hormones across a population. In addition, estradiol in the human male may
have important functions independent of those of testosterone. For example,
low estradiol levels serve as a better predictor than testosterone levels of
bone loss and bone density among elderly men
(van den Beld et al, 2000;
Amin et al, 2006) and of
carotid artery intima-media thickness in middle-aged men
(Tivesten et al, 2006). There
have been recent advances in our understanding of the presence and role of
estradiol in the male. Although traditionally considered the female sex
hormone, through a number of experimental models, it has been shown that
estradiol plays a vital role in normal sperm cell development and function
(O'Donnell et al, 2001;
Hess, 2003;
Akingbemi, 2005). Studies of
estrogen receptor 
(ER) knockout and aromatase knockout mice
first suggested an indirect role of estrogens in male fertility
(Eddy et al, 1996;
Hess et al, 1997;
Robertson et al, 1999). Male
ER knockout mice were infertile, with postpubertal degeneration of the
testes and disrupted spermatogenesis (Eddy
et al, 1996; Hess et al,
1997). Estradiol is produced in the testes from aromatized
testosterone, and progressive disruption of spermatogenesis and infertility
was also observed among aromatase knockout mice
(Robertson et al, 1999). A
direct role of estradiol as a germ cell survival factor was then demonstrated
in the human testis in vitro, where estradiol was shown to inhibit testicular
apoptosis much more effectively (100- to 1000-fold) than testosterone
(Pentikainen et al, 2000).
Estradiol has also been shown to induce spermatogenesis in
gonadotropin-deficient (hpg) mice
(Kula, 1988;
Singh et al, 1995;
Ebling et al, 2000). Results
of the present study, in which we found that estradiol levels were inversely
associated with sperm DNA damage, support the hypothesis that estradiol is
associated with sperm development, maintenance, and function in humans.
Thyroid hormones impact most tissues and systems in the human body, and infertility is a common clinical manifestation of thyroid hormone deficiency in adult males (Nussey and Whitehead, 2001; Krassas and Pontkides, 2004). Although much in this area is still unknown, especially for subclinical alterations in thyroid hormone levels, there is evidence that thyroid hormones have important functions on fetal Sertoli cell maturation (Holsberger and Cooke, 2005) and on Leydig cell differentiation and steroidogenesis in the postnatal testis (Mendis-Handagama and Ariyaratne, 2004) and can stimulate testosterone and estradiol production and secretion by varying the pituitary's responsiveness to LH (Velazquez and Bellabarba Arata, 1997; Maran, 2003). Thyroid hormones may be involved with germ cell survival and mitotic germ cell DNA synthesis through a paracrine signal from the Sertoli cells (Jannini et al, 1993, 1995), which may provide a mechanism for the relationship between thyroid hormone levels and sperm DNA damage in the present study. However, because most experimental studies to date have focused on thyroid hormone action in the developing testes, more work is needed on the role of thyroid hormones in spermatogenesis and reproductive function. The influence of thyroid hormones on steroidogenesis may also partially explain our observation of a correlation between thyroid hormones and circulating estradiol and testosterone. However, despite the correlation between estradiol and thyroid hormones in men from the present study, when both estradiol and free T4 were entered as independent variables into multiple linear regression models, they both remained significantly associated with declined sperm DNA damage with no indication of collinearity.
Inconsistent results between the various DNA damage measures obtained by the neutral comet assay regressed on the same independent variable have been observed in previous studies, and it has been hypothesized that the different comet assay parameters may reflect different types of DNA strand breaks (Meeker et al, 2004). Specifically, because of the lack of correlation between TDM and Tail%, it was hypothesized that a high TDM may be more likely to be associated with double-strand breaks, whereas a high Tail% may reflect single-strand breaks (Meeker et al, 2004). Thus, in the present study, TDM was more highly inversely associated with estradiol, which may reflect a protective relationship between estradiol and double-strand breaks. Conversely, Tail% was more highly inversely associated with free T4, which may signify a protective relationship between free T4 and single-strand breaks. However, because both hormones were significantly and independently associated with Tail%, if this hypothesis holds, then both free T4 and estradiol may have a protective association with single-strand breaks.
In summary, we found statistically significant associations between serum levels of several hormones and sperm DNA damage, most notably protective associations involving estradiol and free T4. Estradiol and thyroid hormone play a role in calcium metabolism (Khosla et al, 1998; Lindblom et al, 2001; Kumar and Prasad, 2003) and may prevent elevated intracellular concentrations of calcium (Hilton et al, 2006; Marino et al, 2006) and subsequent DNA damage (Ray et al, 1993; Liu and Huang, 1996; Bentle et al, 2006). However, at this time it is not known whether hormones are in the causal pathway for sperm DNA damage or if both decreased hormone (estradiol, free T4) levels and increased sperm DNA damage are related to another unmeasured factor that is a common cause of both. For example, oxidative stress was associated with decreased steroidogenesis and increased DNA damage in mouse testes (Kaur and Bansal, 2004), although a role for hormones in the DNA damage causal pathway was not ruled out in the study. Additional studies are needed to elucidate the nature of the relationship between hormones and DNA damage in human sperm.
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Acknowledgments |
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Footnotes |
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