Dioxins in the semen of men with infertility

Abstract

The purpose of the present study was to assess ejaculate contamination by polychlorinated dibenzo-p-dioxins/furans in male infertility. The database of 168 infertile and 49 fertile men was included in the study. Dioxin content was determined using gas chromatography/high-resolution mass spectrometry (GC/HRMS). In the ejaculate of infertile men, the content of dioxins and furans was 2.2-2.3 times higher than in fertile donors. The maximum level of the most toxic dioxin congener was detected in pathospermia. Contamination of semen of infertile men by polychlorinated dibenzo-p-dioxins/furans supports the hypothesis about the relationship between environmental factors and reproductive health.

Full text

12TH IHPA FORUM AND SELECTED STUDIES ON POPS
Dioxins in the semen of men with infertility
E. F. Galimova &Z. K. Amirova &Sh. N. Galimov
Received: 21 March 2014 /Accepted: 26 May 2014 / Published online: 5 June 2014
#Springer-Verlag Berlin Heidelberg 2014
Abstract The purpose of the present study was to assess
ejaculate contamination by polychlorinated dibenzo-p-
dioxins/furans in male infertility. The database of 168 infertile
and 49 fertile men was included in the study. Dioxin content
was determined using gas chromatography/high-resolution
mass spectrometry (GC/HRMS). In the ejaculate of infertile
men, the content of dioxins and furans was 2.2–2.3 times
higher than in fertile donors. The maximum level of the most
toxic dioxin congener was detected in pathospermia. Contam-
ination of semen of infertile men by polychlorinated dibenzo-
p-dioxins/furans supports the hypothesis about the relation-
ship between environmental factors and reproductive health.
Keywords POP .Dioxins .Furans .Male infertility .
Ejaculate
Introduction
At present, the degradation of male reproductive health is
being observed in industrialized countries, and environmental
pollution is supposed as the main reason for it (Sharpe 2010).
Markers of anthropogenic influence are pathology of sper-
matogenesis, delay of puberty period, bias of sex ratio of
newborns, growth of incidence of cancer of testis and prostate
gland, and an increase of frequency of cryptorchidism and
hypospadias (Ryan et al. 2002;Hauseretal.2005;Delbèsetal.
2010;Zandenetal.2012). Dioxins and dioxin-like compounds
(polychlorinated dibenzo-para-dioxins and furans—PCDD/Fs)
refer to reprotoxic pollutants (Mocarelli et al. 2011;Schecter
2012); these substances exert their cellular and metabolic
effects via the interaction with arylhydrocarbon receptor
(AhR) associated with xenobiotic- (XRE) and antioxidant
responsive (ARE) DNA elements (Nguyen et al. 2009). How-
ever, some researchers have doubts about both the “crisis of
spermatozoid”and its relations to environmental health risks
(Fisch 2008; Te Velde and Bonde 2013). The purpose of the
present study was to compare the concentration of dioxin-like
compounds in ejaculate with fertility pathology.
The city of Ufa was selected due to a dioxin pollution “hot
point”situated close to the residential area—this is the terri-
tory of the Khimprom plant (Maystrenko et al. 1998). The
plant produced 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)
during the 60s until the late 80s, chlorine, and some herbi-
cides—up to its shutdown in 2004. For 55 years of plant
operation, several chlororganic products have been
manufactured at large scale—2,4-dichlorophenoxyacetic acid
(2,4-D), 2,4,5-trichlorphenol and trichlorphenolate of copper,
orthochlorphenol. In 1964–1967, the production of butyl ether
of 2,4,5-trichlorphenoxyacetic acid was organized. 2,4,5-
Trichlorphenol and 2,4,5-trichlorphenolate of copper were
produced in 1962–1987 (Amirova et al. 2006). The polluted
territory still remains a source of PCDD/Fs emission spread-
ing over residential areas. The area of the city with the popu-
lation of 1.1 million is 753.7 km
2
.Thelargelandsizeofthe
city and the prevailing winds allow for a high gradient of soil
pollution—from background levels (2–4 ppt) to scores of ppb
(Amirova et al. 2012). In the higher-risk zone (3 km away
Responsible editor: Leif Kronberg
E. F. Galimova (*)
Central Research Laboratory, Bashkir State Medical University,
450000 Ufa, Russia
e-mail: efgalimova@mail.ru
Z. K. Amirova
State-financed Institution of the Republic of Bashkortostan
Environmental Research and Protection Center, 450075 Ufa, Russia
e-mail: ecocnt@ufanet.ru
S. N. Galimov
Department of Biochemistry, Bashkir State Medical University,
450000 Ufa, Russia
e-mail: sngalim@mail.ru
Environ Sci Pollut Res (2015) 22:14566–14569
DOI 10.1007/s11356-014-3109-z

from the plant), about 2,500 people are living in the area
within 7 km—over 300,000 people. Concentration of dioxins
in breast milk, blood, and adipose tissue donors from this area
ranges from 28 to 62 pg/g lipid (WHO-TEQ). Increased
PCDD/Fs level for inhabitants of the industrial zone confirm
the high background level for the city in the whole. PCDD/Fs
concentrations in blood and breast milk exceed the back-
ground level in the region by 30–40 % (Amirova and
Kruglov 2001,2005, Amirova et al. 2007).
Materials and methods
One hundred and sixty eight married men aged 22–41 years—
patients of subsidiary reproductive clinics—were examined.
All of them experienced infertility for 1–10 marriage years.
Exclusion criteria were severe somatic pathology, diseases of
the testes, and their adnexa. The control group comprised 49
fertile males having 1 to 3 healthy children and matched the
infertile group by age, growth, and other parameters. Exami-
nation included spermogram analysis and identification
of PCDD/Fs congeners in the ejaculate. The sperm
assessment was performed according to the WHO re-
quirements (WHO, 2010). Standard ejaculation analysis
includes sperm counts, amounts of progressive motility,
and abnormal forms. The study procedures were ap-
proved by the institutional review board; the written
informed consent was obtained from all subjects. The
data were processed with Statistica software package
(StatSoft, Tulsa, OK, USA). Medians, means (M), and
standard deviations (SD) were calculated; the signifi-
cance of differences was assessed according to Student’s
ttest.
Infertile men were divided into two groups. Group I com-
prised patients without changes in their spermogram
(normospermia)—63 men. Group II consisted of 105 males
with symptoms of pathospermia. Before dioxin determination,
the samples of ejaculate from each group were pooled, 100 ml
of the summary pool were used for analysis.
All samples had been frozen at −18 °C and kept in this state
up to the time of analysis. Lipids from semen were extracted
by the mixture of hexane/diethylether/ethanol. The amount of
lipids was determined gravimetrically. The cleanup procedure
was performed by classical methods, namely multi-layer
SiO
2
,Al
2
O
3
, and Carbopac-C/Celite columns. For the sepa-
ration of high-molecular compounds in the sample prepara-
tion, the method of gel-chromatography (Envirogel TM GPC
Cleanup, Waters) was used.
For measuring PCDD/Fs and DL-PCBs, HRGC/HRMS
(RTX-Dioxin, 60 m, Autospec-Ultima, Waters, UK) and a
series of isotope-labeled standards (CIL Corp.) were used in
compliance with the US EPA 1613B methods. For calculating
TEQ, the scales TEF-2005 were used.
Results and discussion
We present parameters of spermogram of infertile patients in
Tab le 1. Spermogram parameters of 63 infertile men (group I,
38 % of all infertile men) were within WHO norms. Terato-
zoospermia of different degrees combined with oligo- and/or
asthenospermia was diagnosed in 105 patients (group II, 62 %
of all infertile men).
PCDD/Fs analysis revealed a number of specificities
concerning their content and distribution in the ejaculate
(Table 2).
Dioxin/furan level in seminal fluid of fertile controls was
lower than that of infertile men. At the same time, there was no
significant difference in total concentrations of these pollut-
ants in infertile patients with normo- and pathospermia.
It was found that the highest concentration of 2,3,7,8-
TCDD, the most toxic congener, was found in the ejaculate
of men with pathospermia. Paradoxically, 2,3,7,8-TCDD
made no considerable contribution to the total level of dioxin
load; its share was 12 % of the equivalent dose, whereas the
major part of toxicity was determined by the presence of
chlorinated dibenzofurans. This aspect is not easily explained.
There may be unidentified sources of PCDD/Fs emission to
the environment. As a whole, the profile of PCDD/Fs conge-
ners in the ejaculate was close to the range of PCDD/Fs in the
process of incineration and does not contradict the
technogenic nature of reproductive pathology.
Interpretation of our results when comparing dioxin con-
centration with other biological liquids is not easy. The lipid-
adjusted concentration of dioxins in the sperm is higher than
in breast milk and close to occupationally exposed subjects.
However, the wet weight dioxin concentration in semen,
taking into account low lipid levels in this liquid (1.04 g/l in
pathospermia and 1.46 g/l in normospermia), appears to be
considerably lower.
To understand the exposure mechanism of dioxin-like
compounds on sperm, we calculated dioxin content per one
gamete. We found that in patients with normospermia, there
were about 16 molecules per one spermatozoon. In patients
with pathospermia, however, there were 72 molecules that
documents health hazard for future generation.
Tabl e 1 Spermogram parameters of infertile males studied
Parameter Infertile men
Normospermia
Group I (n=63)
Pathospermia
Group II (n=105)
Concentration (10
6
per mL) 47.3±2.4 12.6±2.0*
Abnormal forms (%) 41.5±5.3 90.1±5.8*
Progressive motility (%) 45.8±6.1 21.3±1.9*
*The differences between the table groups are significant according to
Student’s test with p<0.05
Environ Sci Pollut Res (2015) 22:14566–14569 14567

Our data show that PCDD/Fs can pass across the
hematotesticular barrier. This is confirmed by the fact that
their concentrations in the ejaculate are comparable with those
in blood serum (Schecter et al. 1996). Taking into account the
high biological activity of PCDD/Fs and the involvement of
the AhR system responsible for their bioreception in provid-
ing the reproduction function (Brokken and Giwercman
2014), it is possible to suggest that dioxins as “endocrine
disruptors”are directly or indirectly involved in the chain of
events in reproductive organs resulting in the abnormality of
the fecundating ability of spermatozoon. Our previous exper-
iments showed the accumulation of other chlororganic pollut-
ants—polychlorinated biphenyls—in the male reproductive
system (Gromenko et al. 2008).
The toxic action of dioxins is mediated by the AhR/ARNT
receptor complex. In this connection, it is important that AhR
and ARNT are expressed in all seminiferous tubule stages of
the human testes (Karman et al. 2012). AhR are localized in
acrosome and the principal piece of the sperm flagella in
normal sperm which are key regulators of reproductive pro-
cesses and play an important role in normal sperm develop-
ment (Hansen et al. 2014). AhR activation may result in
inflammation, apoptosis, and oxidative stress in sperm leading
to DNA damage (Matsumura 2009). Given that numerous
metals, solvents, drugs, pesticides, and other chemical com-
pounds and metabolites have been found in human seminal
fluid (Figà-Talamanca et al. 2001), AhR may contribute to
xenobiotic metabolism in sperm and may be important for
fertilization. For example, 2,3,7,8-tetrachlorodibenzo-p--
dioxin (TCDD) can increase intracellular calcium concentra-
tion (National Research Council 2013), which could impair
sperm capacitation.
AhR/ARNTcomplex is associated with the redox-sensitive
Keap1/Nrf2/ARE defense pathway (Qiang Ma and Xiaoqing
He 2012). The Keap1/Nrf2/ARE redox-sensitive signal sys-
tem plays the function of a molecular sensor for homeostasis
shifts and is responsible for cellular protection against stress
(Lushchak 2011). Disarrangement of this complicated hierar-
chic system in stress situations may result in its switching off,
deficit of mechanisms for xenobiotics neutralization, activa-
tion of free radical processes, disturbances of homeostasis in
male genital organs, and the development of reproduction
pathology (Bozhedomov et al. 2009; Aitken et al. 2012).
The hypothesis that the male reproductive function deteri-
orated during the past 50 years is the subject of lively scientific
debate. Despite the fact that there have been new studies
documenting the reduction in ejaculate quality in diverse
countries (Mukhopadhyay et al. 2010; Iwamoto et al. 2013;
Rolland et al. 2013), on the whole, this hypothesis is not
considered to be cogent enough. Meanwhile, as compared
with advances in the treatment of female infertility, therapy
for male infertility remains ineffective, empirical with unex-
pected results (Galimov et al. 2012;Koetal.2012) since the
underlying cause of male infertility is often not clear (the so-
called idiopathic infertility). Understanding mechanisms of
the development of spermatogenesis pathology is necessary
for the prevention of male infertility and efficiency of its
treatment. The results of our research as well as data of other
authors (Foster et al. 2010;Manikkametal.2012; Vandenberg
et al. 2012)suggest effect of low concentrations of POPs on
male reproductive status and call for rational preventive
measures.
Conclusions
An increased level of polychlorinated dibenzo-para-dioxins
and furans in the ejaculate of infertile males compared with
fertile donors suggests a relationship between environmental
pollution and reproduction function. The detection of a typical
profile of dioxin/furan congeners corresponding to contempo-
rary industrial manufacture confirms technogenic nature of
sperm contamination.
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Tabl e 2 PCDD/PCDF content in semen of the males studied (TEQ, рg/g lipids)
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