Endocrine Disrupting Chemical Exposure and Testicular Cancer
Endocrine Disrupting Chemical Exposure and Testicular Cancer
DISRUPT is a Danish nested case-control study that is currently being conducted to explore
 the impact of prenatal exposure to endocrine disrupting chemicals on testicular cancer risk
 (including histological sub-groups) with emphasis on the analysis of exposure mixtures.
 Pregnant mothers provided serum and amniotic fluid at recruitment up to 50 years ago. By
 registry linkage within highly reliable national population and disease registries cancer
 cases and matched controls will be identified. Levels of EDCs including DDT, DDE and other
 organochlorine pesticides, PCBs, PBDEs, PFAS, phthalates and triclosan will be quantified in
 cases whose sons develop testicular cancer during 40 year follow up and compared to controls.
Objectives and background Hypothesis: The central hypothesis is that testicular cancer has a
 fetal origin and that exposure to Endocrine Disrupting Chemicals (EDCs) plays a pivotal role
 in the programming of this disease via disruption of the natural in utero estrogen/androgen
 balance and dysregulation of the epigenetic control of gene expression within a critical
 masculinization time window of testicular differentiation in the developing male fetus from
 gestational weeks 8-14. There will be no (new) medical intervention, no new collection of
 biological material and no other strain on the mothers or sons who have already submitted the
 biological material.

 Objectives: The overall objective of this proposal is to provide unique evidence relating
 pre-natal EDC exposure to the risk of testicular cancer in male offspring. The project will
 focus on exposures taking place in fetal life directly quantified using serum and amniotic
 fluid samples collected from mothers while they were pregnant with the young men now aged 21
 to 40. Additionally, the investigators will use dried heel blood spots collected from the
 newborn sons within 5 days after birth. Only ten studies have investigated environmental risk
 factors and testicular cancer. These were small and addressed ongoing adult EDC exposure
 (often after diagnosis); not reflecting exposure levels at the critical time window in early
 life. This project will focus on exposures taking place in fetal life and hereby for the
 first time address the endocrine disruption hypothesis with respect to testicular cancer in a
 large epidemiological study. The specific aims are to investigate the risk of testicular
 cancer following in-utero exposure to EDCs including DDE
 (1,1-bis-(4-chlorophenyl)-2,2-dichloroethene) (major metabolite of DDT), perfluorinated alkyl
 substances, phthalates, triclosan and cotinine (nicotine metabolite). These compounds are
 selected due to ubiquitous environmental occurrence, detection in humans with evidence of
 transfer over placenta and their endocrine disruptive properties. Thus, the successful
 completion of this study will have an important impact on the epidemiological evidence in
 reproductive male health.

 Background: Although a rare cancer in the general population, testicular cancer is the most
 common malignancy in men aged 20-40 years worldwide, representing around 60% of all cancers
 diagnosed for men in this age group. The incidence of this cancer has increased several folds
 in all industrialized during the 20th century. The reasons for this trend remain largely
 unknown and cannot be explained by established risk factors including cryptorchidism,
 Caucasian ethnicity or personal/family history of the cancer. Environmental exposures to
 chemicals disrupting natural in-utero hormone balance within a critical time of testicular
 differentiation in the developing male fetus may play a role and this is supported by the
 evolution of the incident rates between first and second-generation migrant populations. In
 addition, the large geographical variations in incidence in different European countries and
 the possible increase of environmental exposure to endocrine active xenobiotics within the
 same period in industrialized countries also support this. Given the peak incidence of this
 cancer in young men and the fact that testicular cancer (both seminomas and non-seminomas) is
 shown to develop through a common precursor -the carcinoma-in situ cells of fetal origin; the
 role of early exposures, during critical time windows of testicular differentiation has also
 been hypothesized. This is part of a hypothesis called the testicular dysgenesis syndrome,
 proposing a common origin of male reproductive disorders within fetal life resulting from
 disturbed programming of the development of male reproductive organs as a result of endocrine
 disruption from exogenous compounds.

 Challenges and obstacles The rarity of testicular cancer in the population, the long lag time
 between the presumed sensitive period during fetal development and clinical appearance and
 the to date lack of appropriate animal models for testicular cancer have hindered the
 understanding of the factors involved in the development of this cancer. Therefore, there is
 a need for research with reliable data based on exposure assessment quantified in biological
 pre-natal samples, which are considered the golden standard for exposure assessment. There is
 an evident gap in knowledge regarding evidence to support or refute the role of EDCs in the
 fetal origin of testicular cancer. Based on quantification of EDC exposures in biological
 samples collected during pregnancy and ascertainment of outcomes in high quality, complete
 nationwide Danish registries, this study will provide unprecedented data on risk of
 testicular cancer following pre-natal exposure to EDCs.

 Unique biobank (already established). All Danish women are offered a free screen for
 congenital diseases and chromosomal defects in offspring in the first trimester of their
 pregnancy, consisting of an ultrasound scan and amniotic fluid/blood sample. Samples from
 128,702 of the screened pregnant women were stored for research purposes in the Danish
 National Biobank, in the period from 1976-2004. Within 7 days of birth all live born children
 born to these women had heel-blood sampled and stored as dried blood spots at the same Danish
 National Biobank. This heel-blood collection is part of the national Danish screening for
 congenital diseases.

 The unique infrastructure of Danish registries. In Denmark, information on births, deaths,
 immigration and emigration, disease incidence, education and social conditions has been
 collected in nationwide registries for several decades and high-quality data with complete
 coverage of the whole population is available, offering great possibilities for nationwide
 epidemiological studies of health effects related to environmental and lifestyle exposures
 that are not be feasible in most other countries. In each registry persons are each
 identified by a unique number, called a CPR-number. This number has been given to all
 residents of Denmark since the establishment of the Danish Civil Registration System (CRS)in
 1968 and allows accurate linkage between registries. Using this infrastructure, the
 investigators can identify: (i)Study participants by linking mothers that provided blood and
 amniotic fluid to their live-born sons that provided heel-blood at birth. Using the mother's
 CPR-number that is linked to her samples in the Danish National Biobank at Staten Serum
 Institute the investigators can trace the CPR-number of her son in the Danish Medical Birth
 Registry (MBR). The MBR contains information on all live-and stillbirths in Denmark since
 1973, as well as information on congenital defects including cryptorchidism of the live-born
 sons. Further, the MBR contains information on weight, height, age and ethnicity of mother as
 well as the age, ethnicity and CPR-number of the father. (ii) All sons that have developed
 cancer in later life with accuracy and complete coverage. Using the Danish Cancer Registry
 (DCR), which is a population-based registry containing data on the incidence of all cancers
 since 1943, known for accuracy and complete coverage, the investigators will identify all
 cases of testicular cancer amongst the live-born sons (22 to 45 years old). (iii) Family
 history of testicular cancer with accuracy and complete coverage. Using the DCR and the
 father's CPR-number the investigators will trace family history of testicular disease, a
 known risk factor for testicular cancer in offspring [72]. All other cancers will be
 ascertained for the case-controls. Included controls are free of all cancers and cases with
 another cancer prior to testicular cancer onset will be excluded.

 Expected scientific and public health impact of this proposal. The overall scientific
 contribution of the proposed project is to provide human data to scientists, enabling them to
 corroborate or refute whether fetal exposure to EDCs are associated with increased risk of
 testicular cancer in offspring. Currently there is little that pregnant women (and others)
 can do to avoid EDC exposures, which most often are "invisible" pollutants in food,
 cosmetics, indoor climate, air and water. If the study refutes associations this could ease
 concern and if an association is corroborated the evidence could assist governments and
 environmental protection agencies in introducing new policies that can protect vulnerable
 populations in home settings, schools and workplaces or implement education programs for
 pregnant women such that they can take sensible measures to reduce their exposure.

 Methods The project will make use of maternal (serum, amniotic fluid) and newborn (dried
 heel-blood) samples stored at the Danish National Biobank (cf. biobank). Design (Nested
 case-control).

 Three controls are matched to each case. Controls are free of any cancer and matched by
 maternal-and paternal-age, calendar month of blood sample, ethnicity and age and address at
 time of cancer diagnosis. This information along with family history of testicular cancer,
 history of any cancer in case-control population, and other a-priori determined potential
 confounding variables will be obtained from nationwide registries Exposure assessment (Fetal
 exposure to EDCs). Two sub-contractors will perform all chemical analyses of maternal levels
 of selected EDCs in bio-samples.

 Metabolomics profiling. The Metabolomics Research Group at the Danish Center for Neonatal
 Screening at Statens Serum Institut will perform the analysis of the dried blood spot
 metabolome. A punch of 3.2 mm diameter of the neonatal dried blood spot of each child will be
 used for extraction. Using analytical and computational workflows previously introduced and
 applied by the group, a wide range of several thousands of endogenous as well as exogenous
 metabolites will be measured. Data will be preprocessed, and mass spectral features will be
 annotated to chemical structures using computational metabolomic workflows provided through
 the community-driven Global Natural Products Social Molecular networking (GNPS) platform. The
 resulting metabolite profiles will be used for statistical analysis to identify metabolites
 that are differentially expressed across cases and controls using diverse in-house algorithms
 in R and python. Outcome ascertainment (testicular cancer). Using the CPR-number the
 investigators will link the sons to the DCR to extract data on the first occurrence of
 testicular cancer. Controls are cancer and cryptorchidism free. Cases diagnosed with any
 cancer prior to testicular cancer will be excluded. The project duration is 60 months.

 Statistical analysis Population. The investigators expect 175 testicular cancer cases (both
 seminoma and non-seminomas as these have a common fetal precursor) within this population of
 sons. Each case will be matched with three controls giving a total population of 700 men.

 Statistical analyses. Logistic regression models will be used to estimate risk and 95%
 confidence intervals (CIs) relating compound concentrations with risk of testicular cancer.
 Analyses will be done for individual compounds, compound groups where relevant (total PFASs
 and total phthalates) and compound mixtures. Data will be analyzed with and without
 adjustment for a-priori determined potentially confounding variables including mothers BMI at
 time of the son's birth, history of testicular cancer and cotinine serum levels (marker of
 smoking) adjusted for gestational sampling week.

 Metabolomicsprofiling. Overall variation in the metabolome related to testicular cancer will
 be assessed using multivariate statistical methods such as principal coordinates analyses and
 permutational multivariate analyses of variance (PERMANOVA).
Inclusion Criteria:

 -  Male

 -  Available stored maternal serum and/or maternal amniotic fluid

 Exclusion Criteria:

 -  other cancer