Original article 1

Androgen receptor gene CAG repeat length as a modifier ofthe association between persistent organohalogen pollutantexposure markers and semen characteristicsAleksander Giwercmana, Lars Rylanderb, Anna Rignell-Hydbomb, Bo A.G.Jonssonb, Henning S. Pedersenc, Jan K. Ludwickif, Vladimir Lesovoyg, ValentynaZvyezdayh, Marcello Spanoi, Gian-Carlo Manicardij, Davide Bizzarok, Eva C.Bonefeld-Jørgensend, Gunnar Tofte, Jens Peter Bondee, Charlotte Giwercmana,Tarmo Tiidoa, Yvonne Lundberg Giwercmana and INUENDOl

Objectives Exposure to persistent organohalogen pollu-

tants was suggested to impair male reproductive function.

A gene–environment interaction has been proposed. No

genes modifying the effect of persistent organohalogen

pollutants on reproductive organs have yet been identified.

We aimed to investigate whether the CAG and GGN

polymorphisms in the androgen receptor gene modify the

effect of persistent organohalogen pollutant exposure on

human sperm characteristics.

Methods Semen and blood from 680 men [mean (SD) age

34 (10) years] from Greenland, Sweden, Warsaw (Poland)

and Kharkiv (Ukraine) were collected. Persistent organo-

halogen pollutant exposure was assessed by measuring

serum levels of 2,20, 4,40,5,50-hexachlorobiphenyl (CB-153)

and dichlorodiphenyl dichloroethene (p,p0-DDE). Semen

characteristics (volume, sperm concentration, total count,

proportion of progressively motile and morphology) and

DNA fragmentation index (DFI) were determined. CAG and

GGN repeat lengths were determined by direct sequencing

of leukocyte DNA.

Results A statistically significant interaction was found

between the CB-153 group and CAG repeat category in

relation to sperm concentration and total sperm count

(P = 0.03 and 0.01, respectively). For p,p0-DDE, in the

European cohorts a significant interaction was found in

relation to DFI (P = 0.01). For CAG < 20, sperm concentra-

tion and total sperm count were 35 and 42% lower,

respectively, when the group with CB-153 exposure above

median was compared with that below the median. DFI was

40% higher in the high p,p0-DDE exposure group for

CAGr21.

Conclusions This study indicated that the androgen re-

ceptor CAG repeat length might modify the susceptibility of

an individual to the adverse effects of persistent organo-

halogen pollutant exposure on semen quality. Other

studies regarding this matter are warranted. Pharmaco-

genetics and Genomics 00:000–000 �c 2006 Lippincott

Williams & Wilkins.

Pharmacogenetics and Genomics 2006, 00:000–000

Keywords: androgen receptor, CAG repeats, gene–environment interaction,persistent organohalogen pollutants, semen quality

aMolecular Reproductive Medicine Research Unit, Department of ClinicalSciences, Fertility Centre, Malmo University Hospital, Lund University, Malmo,bDivision of Occupational and Environmental Medicine and PsychiatricEpidemiology, Lund University, Lund University Hospital, Lund, Sweden, cCentrefor Arctic Environmental Medicine, Nuuk, Greenland, dInstitute of Public Health,Department of Occupational and Environmental Medicine, University of Aarhus,eDepartment of Occupational Medicine, Aarhus University Hospital, Aarhus,Denmark, fDepartment of Environmental Toxicology, National Institute of Hygiene,Warsaw, Poland, gRegional Clinical Center of Urology and Nephrology,hLaboratory of Human Reproduction, Kharkiv State Medical University, Kharkiv,Ukraine, iSection of Toxicology and Biomedical Sciences, BIOTEC-MED, ENEACasaccia Research Centre, Rome, jUniversity of Modena and Reggio Emilia,Reggio Emilia, kIstituto di Biologia e Genetica Universita Politecnica delle Marche,Ancona, Italy and lwww.inuendo.dk.

Correspondence and requests for reprints to Professor Aleksander Giwercman,Fertility Centre, Malmo University Hospital, SE 205 02 Malmo, SwedenTel: + 46 40 33 79 14; fax: + 46 40 33 82 66;e-mail: aleksander.giwercman@med.lu.se

The INUENDO Project: Biopersistent organochlorines in diet and human fertility.Epidemiological studies of time to pregnancy and semen quality in Inuit andEuropean populations, is supported by The European Commission to the 5thFramework Programme Quality of Life and Management of living resources, Keyaction four on environment and health, (Contract no. QLK4-CT-2001–00202),running 01.01.02–30.06.05. www.inuendo.dk. The Ukrainian part of the studywas supported by a grant from INTAS (Contract no. 2001 2205). The Swedishpart of the study has also been funded by the Swedish Medical Research Council(Grant No: 521-2004-6072) and the Swedish Research Council for Environment,Agricultural sciences and Spatial Planning.

Received 28 April 2006 Accepted 18 July 2006

IntroductionThe alarming reports on a time-related impairment of

male reproductive function, together with an increased

incidence of testicular cancer, cryptorchidism and hypos-

padias and a possible decline in sperm counts, have drawn

attention to the effects of ‘endocrine disrupters’ [1]. The

endocrine disrupters include a long list of chemicals with

agonistic or antagonistic properties with respect to the

action of endogenous hormones. Among the endocrine

disrupters, persistent organohalogen pollutants (POPs)

have been considered as strong candidates for interfering

with the male reproductive system.

CE: ayesha ED: raji Op: vp FPC: lww_fpc_200712

1744-6872 �c 2006 Lippincott Williams & Wilkins

POPs, for example, polychlorinated dibenzofurans

(PCDFs), polychlorinated dibenzo-p-dioxins (PCDD),

polychlorinated biphenyls (PCBs), dichlorodiphenyl tri-

chloroethane (DDT), and dichlorodiphenyl dichlor-

oethene (p,p0-DDE), the most stable metabolite of

DDT, are ubiquitous environmental contaminants. As a

result of the hydrophobic and lipophilic nature of these

chemicals and their long half-times, these compounds are

highly persistent, with a tendency to bioaccumulate and

biomagnify in the food chain. Studies have shown

measurable levels of PCBs and p,p0-DDE in a large

proportion of the general population almost everywhere

[2]. Some of these POPs can disrupt multiple endocrine

pathways and induce a wide range of toxic responses [1].

A number of studies have demonstrated their estrogenic,

anti-estrogenic, dioxin-like and androgen competing

properties [3–6].

A finding of significant ethnical and geographical

differences in semen quality and in the incidence of

testicular cancer and of congenital malformations raises

the question of an interaction between genetic and

environmental/lifestyle-related factors, certain genotypes

making individuals more susceptible to adverse exogen-

ous exposures [7,8]. Studying such interactions has

biological, epidemiological and public health-related

implications. It could help us in understanding the

background for the defects in the male reproductive

organs, facilitate proper design of epidemiological studies

and add to identifying individuals susceptible to certain

environmental and lifestyle-related hazards. Such ‘sus-

ceptibility genes’ need to be identified, those involved in

the action of sex steroids being strong candidates.

Serum concentrations of 2,20,4,40,5,50-hexachlorobiphenyl

(CB-153) and p,p0-DDE are suitable biomarkers for the

assessment of POP exposure. The PCB congener CB-153

is highly correlated with total PCB concentration [9,10],

the estimated 2,3,7,8-tetrachlorodibenzo-p-dioxin equiva-

lent (TEQ) from PCB, and the total POP-derived TEQ

[11], respectively. Likewise, the major DDT metabolite

p,p0-DDE, which is an antiandrogenic compound [4], is

another good indicator of exposure. Previous studies

indicate that the exposure levels for p,p0-DDE still are

considerably high in many countries [11,12]. Recently, as

a part of a study supported by the European Union

(www.inuendo.dk), the impact of POP exposure on

semen characteristics in four populations was investi-

gated: Swedish fishermen and spouses to pregnant

women from Greenland, (Warsaw) Poland and (Kharkiv)

Ukraine [13,14]. The major findings of this study were

negative association between serum CB-153 and sperm

motility and sperm DNA integrity, respectively [13,15–

17].

As androgens play a crucial role in the development and

function of male reproductive organs, polymorphisms in

the androgen receptor gene (AR) might modify the effect

of POP on semen characteristics. The fine-tuning of the

AR function is regulated by two polymorphic sequences

in the transactivating part of the receptor; that is,

polyglutamine encoding CAG repeats and polyglycine

encoding GGN triplets [18,19]. Previous studies have

indicated that the CAG and the GGN number can

influence the functional status of the AR. In Caucasian

men, the CAG number is normally distributed with a

median length of 22 and normal range between 12 and 30

[20]. Men with more than 40 CAGs suffer from

Kennedy’s disease characterized by neuromuscular symp-

toms and hypogonadism [21]. In-vitro [19] and in-vivo

[22,23] studies, however, have indicated that even within

the normal CAG length range, there is an inverse

correlation between CAG number and receptor function,

including lower sperm counts in those with long CAGs.

Less information is available regarding the physiological

role of the GGN triplets. In a Caucasian population, the

lengths of 23 and 24 encounters for 50 and 35% of all

genotypes, respectively. In-vivo data indicate that the

length of 23 is associated with superior receptor activity,

with shorter and longer GGNs giving less well functioning

AR [24–26].

Apart from regulating the sensitivity to endogenous

androgens, however, AR polymorphisms might influence

the effect of endocrine disrupters acting through this

receptor. Our main objective was, therefore, to investi-

gate whether the length of these repeats modified the

effect of POP exposure on semen characteristics. We also

used the data set to assess whether there were any

associations between CAG and GGN repeat lengths and

semen characteristics.

Study participants and methodsThe details of the study populations, methods applied

and the data on association between exposure and semen

characteristics have been published previously [14,17].

Study populations

The present study is part of a European study on fertility

(INUENDO) using a uniform protocol for data collection

in Greenland, Sweden, Kharkiv in Ukraine and Warsaw in

Poland [14]. The participation rates ranged from 7% in

the Swedish fishermen, 29% in Warsaw, 33% in Kharkiv to

79% in Greenland.

The populations were selected to represent different

exposure scenarios of relatively high POP exposure in the

populations from Greenland and Sweden, with high

consumption of POP-contaminated seafood. The Ukrai-

nian population was expected to be exposed to POPs

2 Pharmacogenetics and Genomics 2006, Vol 00 No 00

mainly from previous agricultural use of organochlorine

pesticides, whereas the exposure level in Poland was

expected to be in the lower range and similar to other

European populations without specific exposure sources.

Men were recruited for the study as partners of pregnant

women who were consecutively listed when a pregnancy

was recorded at the participating hospitals, except for the

Swedish men who were recruited from a previously

established population of fishermen, independent of

fertility. Seventy-nine percent of the Swedish fishermen,

however, had fathered a child. In all populations, the men

were consecutively asked to donate a semen sample until

approximately 200 men had agreed to participate. All

included men were 18 years or more at the time of

enrolment.

The men gave their informed consent and were asked to

deliver a semen sample and a blood sample for CB-153

and p,p0-DDE analyses as well as for extraction of

leukocyte DNA.

In total, 798 men delivered a semen sample. Among

those, CB-153 and p,p0-DDE data were lacking in 35. Of

the remaining 763 men, DNA for genotyping of the AR

was available in 680 who, thus, comprised the study

population. Semen characteristics included volume,

concentration, total sperm count, proportion progres-

sively motile and percentage morphologically normal;

sperm chromatin characteristics did not differ between

the 680 men included in the study and the 118 excluded

owing to lack of genetic and/or exposure data (data not

shown). Similarly, there were no differences in CB-153 or

p,p0-DDE levels between the 680 men included and the

83 in whom POP levels, but not genotype, were assessed

(data not shown).

Local ethical committees approved the study.

The background characteristics of the present study

population are given in Table 1.

Table 1 Background characteristics of the 680 men included in the study

Greenland Warsaw, Poland Swedish fishermen Kharkiv, Ukraine All

n 188 167 178 147 680

Exposure measuresCB-153 (ng/g lipid)

Mean (SD) 360 (490) 20 (13) 240 (190) 59 (56) 180 (310)Median (range) 220 (18; 5500) 18 (3.3; 94) 190 (40; 1500) 48 (5.4; 570) 89 (33; 5500)

p,p0-DDE (ng/g lipid)Mean (SD) 900 (170) 570 (300) 320 (290) 1300(1200) 750 (920)Median (range) 610 (5.9; 13000) 500 (140; 2100) 240 (400; 2300) 950 (320; 1200) 530 (5.9; 13000)

Effect modifiersCAG (number)

Mean (SD) 23 (2.4) 22 (3.1) 22 (3.0) 22 (3.2) 22 (2.9)Median (range) 24 (13; 29) 22 (14; 17) 22 (17; 23) 22 (14; 31) 23 (13; 37)

GGN (number)Mean (SD) 23 (0.73) 23 (1.7) 23 (1.9) 23 (1.8) 23 (1.6)Median (range) 23 (20; 26) 23 (16; 30) 23 (10; 29) 23 (10; 26) 23 (10; 30)

ConfoundersAge (years)

Mean (SD) 31 (6.9) 30 (3.9) 47 (9.5) 27 (5.6) 34 (10)Median (range) 30 (19; 51) 30 (24; 46) 47 (24; 68) 25 (8; 45) 31 (18; 68)

Abstinence (days)Mean (SD) 5.5 (19) 6.7 (8.7) 3.7 (2.7) 3.9 (1.9) 4.9 (11)Median (range) 3.0 (0.5; 240) 3.5 (0.1; 60) 3.0 (0.5; 21) 3.0 (1.0; 7.0) 3.0 (0.1; 240)

Outcome variablesSemen volume (ml)

Mean (SD) 3.5 (1.8) 3.8 (1.8) 3.4 (1.7) 3.5 (2.0) 3.6 (1.8)Median (range) 3.2 (0.7; 9.7) 3.6 (0.5; 11) 3.3 (0.3; 8.8) 2.9 (0.7; 11) 3.2 (0.3; 11)

Sperm concentration (106/ml)Mean (SD) 72 (61) 90 (84) 58 (45) 69 (54) 72 (63)Median (range) 53 (0.6; 370) 62 (2.1; 420) 49 (0.0; 220) 57 (0.4; 310) 54 (0.0; 420)

Sperm count (106)Mean (SD) 240 (240) 340 (380) 190 (160) 230 (210) 250 (270)Median (range) 180 (2.4; 1500) 190 (6.0; 2100) 140 (0.0; 900) 170 (2.1; 1200) 170 (0.0; 2100)

Progressively motile (%)Mean (SD) 55 (19) 60 (21) 57 (21) 53 (22) 56 (21)Median (range) 60 (1.0; 87) 63 (0.0; 92) 62 (3.0; 93) 55 (0.0; 90) 61 (0.0; 93)

Normal morphology (%)Mean (SD) 7.0 (3.7) 6.7 (3.8) 8.3 (4.6) 7.4 (4.2) 7.3 (4.1)Median (range) 6.0 (0.0; 20) 6.0 (0.0; 22) 8.0 (0.0; 22) 7.0 (0.0; 18) 7.0 (0.0; 22)

DFI (%)Mean (SD) 9.0 (5.9) 12 (7.8) 19 (13) 13 (8.5) 13 (9.8)Median (range) 7.4 (1.3; 38) 10 (2.9; 41) 15 (2.6; 88) 9.4 (2.4; 44) 9.7 (1.3; 88)

CB-153; 2,2, 4,45,5-hexachlorobiphenyl; p,p0-DDE, dichlorodiphenyl dichloroethene; DFI, DNA fragmentation index.

Androgen receptor polymorphisms and POP exposure Giwercman et al. 3

Semen samples

All semen samples were collected by masturbation at the

residence or in privacy in a room at the hospital. The

participants were asked to abstain from sexual activities

for at least 2 days before collecting the samples, and to

report the actual abstinence time. The samples were

analysed for concentration, motility and morphology

according to a manual for the project based on the latest

version of the World Health Organization manual for basic

semen analysis [27]. Semen samples from Warsaw and

Kharkiv were analysed for concentration and motility at

one central hospital in each region, whereas the samples

from Greenland were analysed at the local hospital or

nursing station in nine municipalities and one settlement

scattered all over the country. The Swedish samples were

collected at the residence of the participants and the

basic semen analyses were performed in a mobile

laboratory. Briefly, the sperm concentration was deter-

mined in duplicate using an improved Neubauer

haemocytometer (Glaswarenfabrik Karl Hecht KG, Sond-

heim, Germany). The sperm motility was determined by

counting the proportion of (a) fast progressive sperms,

(b) progressive sperms, (c) nonprogressive motile sperms

and (d) immotile sperms among 100 spermatozoa within

each of two fresh drops of semen. One person performed

the semen analysis in each of the four regions. These four

persons were previously trained in a quality control

program set up specifically for this study. The median

coefficient of variation among investigators in estimating

sperm concentration and the proportion of progressive

motile sperm was 8 and 11%, respectively [28].

Smears for sperm cell morphology analysis were sent to

the Malmo University Hospital for centralized staining

and evaluation. The morphology was evaluated in at least

200 sperms per sample by two technicians from a

reference laboratory for the NAFA-ESHRE (Nordic

Association for Andrology–European Society of Human

Reproduction and Embryology) external quality control

program.

Sperm DNA integrity was assessed by the Sperm

Chromatin Structure Assay at the Section of Toxicology

and Biomedical Sciences at ENEA Casaccia Research

Centre, Italy. The coded frozen semen samples (200 ml at

– 801C) were shipped on dry ice. Briefly, 1–2� 106

thawed cells were treated with an acid–detergent

solution (0.08 N HCl, 0.15 mol/l NaCl, 0.1% Triton-X

100; pH 1.2) for 30 s and then stained with 6 mg/l of

purified Acridine Orange (Molecular Probes, Eugene,

Oregon, USA) in a phosphate-citrate buffer, pH 6.0. Cells

were analysed by a FACScan (Becton Dickinson, San

Jose, California, USA) equipped with an air-cooled argon

ion laser and standard optical filters to detect green and

red fluorescence. The percentage of abnormal sperm with

detectable DNA fragmentation index (DFI) was calcu-

lated from the DFI frequency histogram obtained from

the ratio between red and total (red + green) fluores-

cence intensity [29].

In an external quality control study that was carried out

within the framework of this project, based on blind

analysis of three aliquots from seven donors not selected

among the participants in current study, the median

intersample variability for DFI, expressed as coefficient

of variation, was 1.5%.

Determination of CB-153 and p,p0-DDE

All analyses of CB-153 and p,p0-DDE in serum were

performed at the Division of Occupational and Environ-

mental Medicine and Psychiatric Epidemiology in Lund,

Sweden, applying solid phase extraction using on-column

degradation of the lipids and analysis by gas chromato-

graphy mass spectrometry as previously described. Levels

of detection, coefficients of variation and participation in

quality control programs have been described in detail

elsewhere [30].

Determination of lipids by enzymatic methods

Serum concentrations of triglycerides and cholesterol

were determined by enzymatic methods as described

elsewhere [30]. Total lipid concentration in serum (g/l)

was calculated by the following equation:

total = 0.96 + 1.28� (triglycerides + cholesterol) [31].

Genetic analyses

Assessment of CAG and GGN repeat number was

performed as previously reported [20].

Statistical analysis

In linear regression models, we evaluated whether CAG

and GGN repeat length modified the impact of CB-153

and p,p0-DDE exposure on semen variables listed in Table

1. CAG repeat numbers were categorized into five groups

of almost equal size: < 20, 20/21, 22/23, 24 and > 24.

GGN lengths were divided into three categories: the

most common length of 23, < 23 and > 23. The

exposure levels were categorized as low and high by

dividing at the medians for the whole INUENDO male

cohort: 83 ng/g lipid for CB-153 and 569 ng/g lipid for p,p0-DDE, respectively.

The distribution of participants in the different genotype

groups by country is given in Table 2.

Model assumptions, assessed by residual analysis, were

better fulfilled for sperm concentration and total sperm

count when these variables were log transformed. In all

analyses, based on former studies in these, population

[13,17] age (continuous) and abstinence time (categor-

ized: r 2, 2 to r 4, 4 to r 6 and > 6 days) were

4 Pharmacogenetics and Genomics 2006, Vol 00 No 00

included as covariates owing to their potential confound-

ing effect.

Primarily, in order to investigate whether the data from

different centres could be pooled, a statistical test for

heterogeneity of risk, including significance test for

interaction terms between centre, AR polymorphism

and exposure level in relation to semen characteristics,

was performed in multiple linear regression models.

The heterogeneity between centres with regard to the

modifying effect of AR polymorphism on the impact of

POP exposure on semen characteristics was only statis-

tically significant for the interaction centre� CAG

category� p,p0DDE low/high (P = 0.03) with DFI as

outcome. The same was true for the interaction centre�GGN category�CB153 low/high (P = 0.048) with semen

volume as outcome. These heterogeneities, however,

were no longer statistically significant when the Inuit

cohort was excluded. Therefore, for the assessment of the

impact of CAG number on the effect of p,p0-DDE

exposure on DFI and of the GGN number on the effect

of CB153 exposure on semen volume, the analysis was

performed separately for the European cohorts and for

the Inuit cohort.

Thereafter, the potential of CAG or GGN number as

modifiers of the effect of exposure was investigated by

including interaction terms in the regression models (i.e.

CAG category� CB-153 low/high, CAG category� p,p0-DDE low/high, GGN category� CB-153 low/high and

GGN category� p,p0-DDE low/high, respectively). For

this analysis, ‘centre’ was included as a potential

confounder.

Subsequently, for each genotype subgroup the level of

particular semen parameter in the low and high exposure

groups were compared, separately for CB-153 and for p,p0-

DDE. The low and high exposure groups were compared

using linear regression and the differences were ex-

pressed both in absolute (Tables 3–6) and in relative

terms (in the Results section). As the Inuit population

differed from the other cohorts with respect to genotype

distribution and exposure level, for those associations in

which significant interaction was found, a separate

analysis was performed for the Caucasian cohorts only.

Similarly, as the inclusion criteria for Swedish fishermen

differed from the other groups, in case of significant

interaction an analysis for the partners of pregnant

women only was also performed. These calculations were

done as supplements to the above-mentioned interaction

analyses in order to exclude the heterogeneity between

the centres as the cause of statistically significant

exposure–genotype interactions.

Finally, in linear regression models, we evaluated the

effect of CAG and GGN repeat length on the semen

outcome variables.

All analyses were carried out by SPSS software (SPSS for

Windows 11.0; SPSS Inc., Chicago, Illinois, USA).

ResultsA statistically significant interaction was found between

CB-153 and CAG repeat category in relation to sperm

concentration and total sperm count (P = 0.03 and 0.01,

respectively; Table 3). When ‘centre’ was included as a

covariate, these interactions were still statistically sig-

nificant (P = 0.049 and 0.03, respectively). For

CAG < 20, the ‘high CB-153’ group presented with a

sperm concentration being 35% [95% confidence interval

(CI): 4.4; 79%] lower and the total sperm count being

42% (95% CI: 8.9; 91%) lower than in the ‘low CB-153’

category (Fig. 1).

Table 2 Distribution of study participants according to the levels of CB-153 and p,p0-DDE, androgen receptor genotype and place of origin

n (%)

Greenland Warsaw, Poland Swedish fishermen Kharkiv, Ukraine All

CB-153Low 20 (11) 165 (99) 18 (10) 121 (82) 324 (48)High 168 (89) 2 (1.0) 160 (90) 26 (18) 356 (52)

p,p0-DDELow 87 (46) 103 (62) 156 (88) 23 (16) 369 (54)High 101 (54) 64 (38) 22 (11) 124 (84) 311 (46)

CAG number< 20 17 (9) 35 (21) 34 (19) 26 (18) 112 (17)20/21 21 (11) 39 (23) 48 (27) 37 (25) 145 (21)22/23 38 (20) 45 (27) 40 (23) 38 (26) 161 (24)24 77 (41) 12 (7.0) 13 (7.0) 8 (5.0) 110 (16)> 24 35 (19) 36 (22) 43 (24) 38 (26) 152 (22)

GGN number< 23 8 (4.0) 13 (8.0) 25 (14) 16 (11) 62 (9.0)23 161 (86) 82 (49) 95 (53) 86 (59) 424 (62)> 23 19 (10) 72 (43) 58 (33) 45 (31) 194 (29)

CB-153; 2,2, 4,45,5-hexachlorobiphenyl; p,p0-DDE, dichlorodiphenyl dichloroethene.

Androgen receptor polymorphisms and POP exposure Giwercman et al. 5

When separate analyses were performed for the Cauca-

sian cohorts only, the ‘low’ and ‘high’ CB-153 exposure

groups still differed with respect to total sperm counts for

CAG < 20 (relative mean difference: 41%; 95% CI: 5.3;

62%) as well as sperm concentration (relative mean

difference: 36%; 95% CI: 1.0; 58%). For the cohorts of

partners of pregnant women (except Sweden), the

corresponding differences were statistically significant

for total sperm number (relative mean difference: 47%;

95% CI: 10; 68%), whereas the difference with regard to

sperm concentration was borderline statistically signifi-

cant (relative mean difference: 36%; 95% CI: – 3.6%;

61%).

For p,p0-DDE, among the Caucasians, a significant

interaction with CAG number was found in relation to

DFI (P = 0.01 without and P = 0.006 with ‘centre’ as a

covariate) (Table 4). The ‘high p,p0-DDE’ exposure

group, as compared with the ‘low p,p0-DDE’ group,

presented with 43% (95% CI: 9.6; 78%) higher DFI for

CAG < 20 and 38% (95% CI: 7.6; 68%) higher DFI for

CAG 20/21, respectively. In the Inuit cohort, the

interaction between CAG category and the effect of

p,p0-DDE level on DFI was not statistically significant

(P = 0.64).

After exclusion of the Swedish fishermen in the CAG

category 20/21, the DFI was significantly higher for ‘high’

p,p0-DDE than for ‘low’ p,p0-DDE (relative mean

difference: 48%; 95% CI: 16; 79%). A similar trend,

without reaching the level of statistical significance

(relative mean difference: 17%; 95% CI: – 13%; 47%),

was seen for CAG < 20.

Furthermore, despite the lack of a statistically significant

interaction between exposure and genotype, a signifi-

cantly lower percentage of progressively motile sperms

was found in ‘high CB-153’ for CAG < 20 (mean

difference 11%; 95% CI: 2.6; 19%) and in ‘high p,p0-

Table 3 Interaction between CB-153 levels (low/high) and categorized CAG repeat length in relation to semen characteristics

CAG repeat length

CB-153 < 20 20/21 22/23 24 > 24 P value for interaction

Semen volume (ml)Low 3.6 3.8 3.4 4.1 3.7 0.67High 3.5 3.9 3.5 3.6 3.5Median (95% CI)a 0.17 ( – 0.55; 0.88) – 0.070 ( – 0.69; 0.55) – 0.094 ( – 0.69; 0.50) 0.48 ( – 0.37; 1.3) 0.24 ( – 0.34; 0.83)

Sperm concentration (106/ml)Low 60b 55 47 47 54 0.03High 38b 60 61 49 62Median (95% CI)a 21 (4.0; 73) – 5.1 ( – 18; 14) – 14 ( – 20; 1.8) – 2.4 ( – 18; 22) – 7.9 ( – 19; 9.6)Sperm number (106)Low 190b 190 140 170 170 0.01High 110b 200 180 160 180Median (95% CI)a 79 (29; 300) – 14 ( – 65; 60) – 42 ( – 64; 10) 14 ( – 56; 130) – 18 ( – 58; 43)Progressively motile (%)Low 60b 59 56 53 57 0.15High 49b 60 55 55 57Median (95% CI)a 11 (2.6; 19) – 0.93 ( – 8.3; 6.4) 0.40 ( – 6.7; 7.4) – 2.2 (12; 7.7) – 0.038 ( – 7.0; 6.9)Normal morphology (%)Low 7.6 7.4 6.8 7.2 7.2 0.05High 6.1 8.9 7.7 6.5 7.9Median (95% CI)a 1.5 ( – 0.15; 3.1) – 1,4 ( – 2.8; 0.036) – 0.82 ( – 2.2; 0.56) 0.67 ( – 1.3; 2.7) – 0.62 ( – 2.0; 0.74)DFI (%)Low 16 14 13 13 15 0.89High 15 14 14 12 14Median (95% CI)a 0.91 ( – 2.9; 4.7) 0.41 ( – 2.9; 3.7) – 0.90 ( – 4.1; 2.3) 0.42 ( – 4.1; 4.9) 1.2 ( – 1.9; 4.4)

CB-153; 2,2, 4,45,5-hexachlorobiphenyl; CI, confidence interval; DFI, DNA fragmentation index.aMean difference (low – high) and 95% CI of this difference. As the values for concentration and total sperm count are back transformed from log10 values, thecalculations of mean difference (95% CI) are based on the ratios between low and high.bP < 0.05 for difference between low and high.The results, adjusted for the effect of age and abstinence time, are expressed as means and the difference between the means (95% CI).

Fig. 1

CAG number

0

50

100

150

200

250

<20 20/21 22/23 24 >24

Tota

l spe

rm c

ount

(×10

6 )

P<0.05

Mean total sperm counts in low (white bars) and high (black bars) 2,2,4,45,5-hexachlorobiphenyl (CB-153) exposure groups in relation toCAG repeat length. Statistically significant differences for low and highexposure are indicated in the figure.

6 Pharmacogenetics and Genomics 2006, Vol 00 No 00

DDE’ for CAG 20/21 (mean difference 7.8%; 95% CI:

0.71; 14%) and 21/22 (mean difference 12%; 95% CI: 5.1;

19%; Table 3).

No significant interaction between POP levels and GGN

category in relation to sperm characteristics was found

(Tables 5 and 6). For GGN = 23, however, significantly

Table 4 Interaction between p,p0-DDE levels (low/high) and categorized CAG repeat length in relation to semen characteristics

CAG repeat length

p,p0-DDE < 20 20/21 22/23 24 > 24 P value for interaction

Semen volume (ml)Low 3.5 4.0 3.4 4.0 3.7 0.60High 3.6 3.7 3.6 3.5 3.5Median (95% CI)a – 0.19 ( – 0.89; 0.51) 0.26 ( – 0.35; 0.86) – 0.16 ( – 0.73; 0.42) 0.46 ( – 1.1; 0.23) 0.16 ( – 0.42; 0.73)

Sperm concentration (106/ml)Low 49 55 56 54 55 0.70High 47 59 51 45 62Median (95% CI)a 2.2 ( – 13; 24) – 4.4 ( – 18; 15) 5.8 ( – 9.6; 28) 8.6 ( – 8.8; 37) – 6.2 ( – 18; 11)

Sperm number (106)Low 150 200 170 200 170 0.63High 140 190 160 140 180Median (95% CI)a 8.7 ( – 42; 85) 8.6 ( – 51; 94) 8.7 ( – 39; 77) 60 ( – 5.1; 220) – 5.7 ( – 52; 58)

Progressively motile (%)Low 54 63b 61b 58 57 0.62High 54 55b 49b 51 56Median (95% CI)a – 0.23 ( – 8.37.9) 7.8 (0.71; 14) 12 (5.1; 19) 6.3 ( – 1.7; 14) 1.2 ( – 5.5; 7.9)

Normal morphology (%)Low 6.8 8.3 7.8 6.7 7.4 0.11High 6.7 8.0 6.6 6.5 7.7Median (95% CI)a 0.075 ( – 1.5; 1.7) 0.32 ( – 1.1; 1.7) 1.1 ( – 0.19; 2.5) 0.26 ( – 1.3; 1.9) – 0.37 ( – 1.7; 0.96)

DFI (%)c

Low 13b 13b 15 16 18 0.01High 19b 18b 14 15 15Median (95% CI)b – 5.8 ( – 10; – 1.2) – 4.8 ( – 8.7; – 0.98) 1.9 ( – 2.1; 6.0) 0.98 ( – 6.9; 8.8) 2.3 ( – 1.6; 6.2)

p,p0-DDE, dichlorodiphenyl dichloroethene; CI, confidence interval; DFI, DNA fragmentation index.aMean difference (low – high) and 95% CI of this difference. As the values for concentration and total sperm count are back transformed from log10 values, thecalculations of mean difference (95% CI) are based on the ratios between low and high.bP < 0.05 for difference between low and high.cBased on the three Caucasian cohorts only.

Table 5 Interaction between CB-153 levels (low/high) and categorized GGN repeat length in relation to semen characteristics

GGN repeat length

CB-153 23 < 23 > 23 P value for interaction

Semen volume (ml)c

Low 3.7 3.9 3.3 0.81High 3.8 3.9 3.6Median (95% CI)a,b – 0.074 ( – 0. 65; 0.50) 0.0095 ( – 0.99; 1.0) – 0.23 ( – 0.90; 0.44)

Sperm concentration (106/ml)Low 52 53 54 0.73High 53 62 54Median (95% CI)a – 1.6 ( – 11; 29) – 8.8 ( – 29; 23) – 0.090 ( – 14; 18)

Sperm number (106)Low 170 190 160 0.69High 160 210 160Median (95% CI)a 3.4 ( – 28; 43) – 24 ( – 100; 103) – 0.083 ( – 45; 63)

Progressively motile (%)Low 55 64 58 0.61High 55 60 55Median (95% CI)a 0.33 ( – 4.1; 4.7) 3.8 ( – 7.0; 15) 3.5 ( – 3.2; 10)

Normal morphology (%)Low 7.1 8.6 7.0 0.31High 7.1 8.1 8.2Median (95% CI)a 0.068 ( – 0.80; 0.93) 0.50 ( – 1.6; 2.6) – 1.2 ( – 2.6; 0.12)

DFI (%)Low 15 15 13 0.66High 14 14 13Median (95% CI)a 1.3 ( – 0.65; 3.3) 1.2 ( – 3.6; 6.1) – 0.26 ( – 3.3; 2.8)

CB-153; 2,2, 4,45,5-hexachlorobiphenyl; CI, confidence interval; DFI, DNA fragmentation index.aMean difference (low – high) and 95% confidence interval (CI) of this difference. As the values for concentration and total sperm count are back transformed from log10

values, the calculations of mean difference (95% CI) are based on the ratios between low and high.bP < 0.05 for difference between low and high.cBased on the three Caucasian cohorts only.The results, adjusted for the effect of age and abstinence time, are expressed as means and the difference between the means (95% CI).

Androgen receptor polymorphisms and POP exposure Giwercman et al. 7

higher (mean difference 6.7%; 95% CI: 2.6; 11%)

percentage progressively motile sperms was found for

‘low’ vs. ‘high’ CB-153 exposure group.

No statistically significant association was found between

the categorized CAG repeat number and any of the

semen parameters tested, except for the proportion of

morphologically normal cells (P = 0.02). This association

was due to a significantly higher percentage of normal

sperms for the CAG length of 20/21 (mean: 8.2%) than

for the CAG length of < 20 (mean difference: 1.4%; 95%

CI: 0.32; 2.7%) and CAG length of 24 (mean difference:

1.7%; 95% CI: 0.62; 2.7%), respectively.

For GGN categories, statistically significant differences

were found for morphology (P = 0.05) and proportion of

progressively motile sperms (P = 0.03). In both cases, the

GGN < 23 group diverged from GGN = 23 by presenting

with more sperms with normal morphology: (mean: 8.4%

vs. 7.0%; 95% CI of difference: 0.23; 2.4%) and

progressive motility (mean: 62 vs. 55%; 95% CI of

difference: 1.7; 12%).

DiscussionThe main objective of this study was to investigate

whether polymorphisms in the AR gene may modify the

effect of POP exposure on semen characteristics. Our

results indicate that the variation in the length CAG

repeats may play a role in the individual sensitivity to the

adverse effects of POP exposure on the male reproduc-

tive system. We found a statistically significant interac-

tion between CAG number and CB-153 levels in relation

to sperm concentration and total sperm number and for

p,p0-DDE levels in relation to sperm DNA integrity. This

is, to our knowledge, the first study showing a gene–

environment interaction in relation to semen character-

istics.

The most consistent finding of the study was that the

difference in semen characteristics between low and high

POP exposure groups was most pronounced in men with

short CAG tracts. In a recently published study, based on

the same populations, no significant associations between

CB-153 or p,p0-DDE levels and sperm concentration and

total sperm count were found [17]. The proportion of

motile sperms, however, was found to decrease with

increasing CB-153 levels. In the current work, we found

that men with CAG < 20 had a 35% reduction in sperm

concentration and 42% reduction in total sperm number

when the group with CB-153 concentration above the

median was compared with those with CB-153 below the

50th percentile. In this CAG group, high CB-153

exposure was also associated with a 11% reduction in

the proportion of progressively motile sperms.

Table 6 Interaction between p,p0-DDE levels (low/high) and categorized GGN repeat length in relation to semen characteristics

GGN repeat length

p,p0-DDE 23 < 23 > 23 P value forinteraction

Semen volume (ml)Low 3.7 4.2 3.5 0.28High 3.6 3.5 3.7Median (95% CI)a 0.10 ( – 0. 25; 0.45) 0.70 ( – 0.20; 1.6) – 0.15 ( – 0.68; 0.38)

Sperm concentration (106/ml)Low 54 48 57 0.12High 51 76 50Median (95% CI)a 2.8 ( – 6.1; 13) – 27 ( – 45; 1.3) 6.5 ( – 7.0; 24)

Sperm number (106)Low 170 190 170 0.49High 160 230 150Median (95% CI)a 18 ( – 1.4; 56) – 45 ( – 120; 78) 20 ( – 25; 81)

Progressively motile (%)Low 58b 63 59 0.62High 51b 60 55Median (95% CI)a 6.7 (2.6; 11) 3.6 ( – 7.0; 14) 4.0 ( – 3.2; 10)

Normal morphology (%)Low 7.4 7.9 7.6 0.46High 6.8 8.7 7.4Median (95% CI)a 0.60 ( – 0.21; 1.4) 0.76 ( – 2.9; 1.3) 0.12 ( – 2.3; 10)

DFI (%)Low 14 14 13 0.83High 14 15 13Median (95% CI)a – 0.14 ( – 2.0; 1.7) – 1.1 ( – 5.9; 3.6) – 0.33 ( – 3.2; 2.5)

CB-153; 2,2, 4,45,5-hexachlorobiphenyl; p,p0-DDE, dichlorodiphenyl dichloroethene; CI, confidence interval; DFI, DNA fragmentation index.aMean difference (low – high) and 95% CI of this difference. As the values for concentration and total sperm count are back transformed from log10 values, thecalculations of mean difference (95% CI) are based on the ratios between low and high.bP < 0.05 for difference between low and high.The results, adjusted for the effect of age and abstinence time, are expressed as means and the difference between the means (95% CI).

8 Pharmacogenetics and Genomics 2006, Vol 00 No 00

No statistically significant interactions were found in

relation to the other AR polymorphism – the GGN

repeats. Although the proportion of progressively motile

spermatozoa was significantly higher for the ‘low’ CB-163

group than for the ‘high’ CB-163 group when GGN was

23, this finding is probably due to the fact that GGN = 23

is the most common genotype for this repetitive

sequence and generally a negative association between

CB-153 levels and sperm motility was found in the

INUENDO population [17]

Spermatogenesis is a highly androgen-dependent process

[32], the concentration of testosterone being more than

100 times higher in the testis than in serum [33]. An anti-

androgenic effect of p,p0-DDE [4] and of CB-153 has

been reported [4,34]. In a reporter in-vitro assay, the

highest anti-androgenic activity was found in the Kharkiv

cohort presenting with high p,p0-DDE levels (T. Kruger,

P.S. Hjelmborg, B.A.G. Jonsson, L. Hagmar, A. Giwerc-

man, G.-C. Manicardi et al., in preparation). This study

does not allow a clarification of the mechanism by which

the CAG repeat length might modify the effect of POP

on semen characteristics. As the three-dimensional

structure of the receptor is affected by the length of

the CAG stretch, however, one could hypothesize that

the strength of the POP binding, or of any necessary co-

factor, is regulated by CAG number. Although we

measured levels of CB-153 and p,p0-DDE, the effect

observed might also be due to the interaction of other

POPs with the AR. CB-153 concentration was reported to

be highly correlated with the total PCB concentration

[9,35], the estimated TEQ from PCB and the total POP-

derived TEQ [11], respectively. As some of these

compounds are known to bind to the arylhydrocarbon

receptor, short CAG lengths might also modify the

interaction between the arylhydrocarbon receptor and

the AR, rather than directly affect the binding of POP to

the latter receptor [36].

Short CAG repeats also seemed to modify the association

between p,p0-DDE and sperm DNA integrity. In the

groups with CAG lengths shorter than the median, DFI

was 40% higher in highly exposed men than in those with

low p,p0-DDE exposure. Although sperm DNA strand

breaks can be caused by post-testicular events, topoi-

somerase II, which is an androgen-dependent DNA

reparation enzyme, also plays an important role for

ensuring sperm DNA integrity [37]. Therefore, it seems

plausible that chemicals interfering with AR function may

also have an impact on sperm number and DNA integrity.

In a recent study on the association between POP

exposure and DFI [13], this association was restricted to

the Caucasian men. The Inuit population was found to

differ significantly from the European men with respect

to sperm DNA integrity, possibly owing to differences in

the genetic background and/or in dietary habits.

Developmental and reproductive disorders are critical

toxic effects caused by POP and most of these effects are

believed to be the result of impaired steroidogenesis and/

or steroid action [1]. The finding of a genetic modifier of

the environmental effects on male reproductive organs

has important implications, not only from a biological

point of view but also from a public health point of view.

In order to prevent any further increases in disorders of

male reproductive organs, it is important to develop tools

for identifying individuals susceptible to adverse effects

of environmental and lifestyle-related factors.

Finally, taking genetically determined susceptibility into

account is also important when planning epidemiological

studies. With respect to this, the INUENDO study failed

to show any negative impact of POP exposure on sperm

numbers [17], whereas inclusion of the AR genotype

allowed the identification of such an effect in a subset of

the same study population.

When evaluating the results of this study, the issue of

potential selection bias needs to be considered. Except

for the Inuit population, the participation rate in the

semen study, in similarity with other similar surveys, was

rather low. With respect to the Swedish fishermen, the

age distributions and the mean number of children were

very similar among the participants and the non-

participants [16]. In the three remaining cohorts, it was

found that time to pregnancy did not differ between

those who delivered semen for analysis and those who did

not [14]. Furthermore, it is difficult to imagine how the

interaction between genotype and environmental expo-

sure should affect the participation rate. Therefore, we

do not consider that selection bias is of major concern.

As multiple comparisons have been performed, in order to

minimize the risk of mass significance, we have focused

on the associations in which a statistically significant

interaction between exposure levels and CAG length was

found. In total, the P value was below 0.05 for three of 12

such tests, or for two of 10, if sperm concentration and

total sperm count were considered as markers of the same

biological mechanism. Although these proportions were

higher than expected if our findings had been due to

chance, mass significance as an explanation of our results

cannot be completely ruled out.

It could be asked whether the results might be

confounded by significant differences in distribution of

AR genotypes and exposure profiles, mainly between the

Inuit cohort and the Caucasians. Significant interaction

between genotype and exposure reported here were not

affected by inclusion of centre as potential confounder.

Additionally, for CAG < 20; the differences between the

groups with ‘high’ and ‘low’ CB-153, with respect to

sperm concentration and total number, persisted even

Androgen receptor polymorphisms and POP exposure Giwercman et al. 9

when the Inuits were excluded from the analysis. The

same was true when the Swedish fishermen, who differed

from the other cohorts as these men were not included as

partners of pregnant women, were not included in the

analysis. Furthermore, no significant interaction between

centre, AR genotype group and exposure level was found

for any of the reproductive endpoints, except for DFI in

relation to p,p0-DDE exposure. As, in the latter case, a

separate analysis was performed for the Caucasians and

the Inuit population, we believe that merging of all

groups was justified in the remaining cases and the

interactions we have observed are due to the modifying

effect of the CAG repeat length on the impact of POP

exposure on semen parameters.

The main focus of this study was to assess the impact of

AR polymorphisms as modifiers of the effect of POP on

semen parameters. It is notable that CAG or GGN

number did not, per se, seem to have any strong

associations with sperm concentration or total number.

Previous studies showed conflicting results regarding the

link between CAG length and sperm number [10,22].

Provided the gene–environment interaction found by us,

one could speculate whether the interpopulation differ-

ences in POP exposure levels might contribute to the

diverging results of studies on CAG repeats and

reproductive characteristics. Thus, if short CAG repeat

renders an individual more susceptible to the effect of at

least some POPs, in highly exposed populations this

effect might counteract the per se beneficial effect of low

CAG number on sperm production.

We found a slightly higher percentage of morphologically

normal spermatozoa for CAG = 20 or 21 and for

GGN < 23. The latter group also presented with a higher

percentage of progressively motile sperms. The biological

significance of these findings, however, is difficult to

interpret as they do not fit with previous data indicating

superior AR activity for the shortest CAG categories and

for GGN = 23 as compared with shorter and longer GGNs

[25,26,38].

In conclusion, our data might indicate that the AR CAG

repeat length influences the impact of POP on sperm

characteristics as sperm number and DNA integrity. More

studies, however, are needed in order to establish

whether the AR or other genetic polymorphisms modify

the effect of endocrine disrupters on male reproductive

function and to enlighten possible mechanisms behind

such gene–environment interaction.

AcknowledgementsProfessor Lars Hagmar, Division of Occupational and

Environmental Medicine and Psychiatric Epidemiology,

Lund University, Lund University Hospital, was one of

the key persons behind the INUENDO project and

contributed significantly to the current manuscript. Lars

Hagmar, however, passed away during the time when the

manuscript was under the review process.

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