Building an adverse outcome pathway network for estrogen-, androgen- and steroidogenesis-mediated reproductive toxicity.

Introduction: Adverse Outcome Pathways (AOPs) can support both testing and assessment of endocrine disruptors (EDs). There is, however, a need for further development of the AOP framework to improve its applicability in a regulatory context. Here we have inventoried the AOP-wiki to identify all existing AOPs related to mammalian reproductive toxicity arising from disruption to the estrogen, androgen, and steroidogenesis modalities. Core key events (KEs) shared between relevant AOPs were also identified to aid in further AOP network (AOPN) development. Methods: A systematic approach using two different methods was applied to screen and search the entire AOP-wiki library. An AOPN was visualized using Cytoscape. Manual refinement was performed to remove AOPS devoid of any KEs and/or KERs. Results: Fifty-eight AOPs relevant for mammalian reproductive toxicity were originally identified, with 42 AOPs included in the final AOPN. Several of the KEs and KE relationships (KERs) described similar events and were thus merged to optimize AOPN construction. Sixteen sub-networks related to effects on hormone levels or hormone activity, cancer outcomes, male and female reproductive systems, and overall effects on fertility and reproduction were identified within the AOPN. Twenty-six KEs and 11 KERs were identified as core blocks of knowledge in the AOPN, of which 19 core KEs are already included as parameters in current OECD and US EPA test guidelines. Discussion: The AOPN highlights knowledge gaps that can be targeted for further development of a more complete AOPN that can support the identification and assessment of EDs.

Perinatal exposure to the fungicide ketoconazole alters hypothalamic control of puberty in female rats.

Introduction: Estrogenic endocrine disrupting chemicals (EDCs) such as diethylstilbestrol (DES) are known to alter the timing of puberty onset and reproductive function in females. Accumulating evidence suggests that steroid synthesis inhibitors such as ketoconazole (KTZ) or phthalates may also affect female reproductive health, however their mode of action is poorly understood. Because hypothalamic activity is very sensitive to sex steroids, we aimed at determining whether and how EDCs with different mode of action can alter the hypothalamic transcriptome and GnRH release in female rats. Design: Female rats were exposed to KTZ or DES during perinatal (DES 3-6-12µg/kg.d; KTZ 3-6-12mg/kg.d), pubertal or adult periods (DES 3-12-48µg/kg.d; KTZ 3-12-48mg/kg.d). Results: Ex vivo study of GnRH pulsatility revealed that perinatal exposure to the highest doses of KTZ and DES delayed maturation of GnRH secretion before puberty, whereas pubertal or adult exposure had no effect on GnRH pulsatility. Hypothalamic transcriptome, studied by RNAsequencing in the preoptic area and in the mediobasal hypothalamus, was found to be very sensitive to perinatal exposure to all doses of KTZ before puberty with effects persisting until adulthood. Bioinformatic analysis with Ingenuity Pathway Analysis predicted "Creb signaling in Neurons" and "IGF-1 signaling" among the most downregulated pathways by all doses of KTZ and DES before puberty, and "PPARg" as a common upstream regulator driving gene expression changes. Deeper screening ofRNAseq datasets indicated that a high number of genes regulating the activity of the extrinsic GnRH pulse generator were consistently affected by all the doses of DES and KTZ before puberty. Several, including MKRN3, DNMT3 or Cbx7, showed similar alterations in expression at adulthood. Conclusion: nRH secretion and the hypothalamic transcriptome are highly sensitive to perinatal exposure to both DES and KTZ. The identified pathways should be exploredfurther to identify biomarkers for future testing strategies for EDC identification and when enhancing the current standard information requirements in regulation.

Exposure to the pesticides linuron, dimethomorph and imazalil alters steroid hormone profiles and gene expression in developing rat ovaries.

Inhibition of androgen signaling during critical stages of ovary development can disrupt folliculogenesis with potential consequences for reproductive function later in life. Many environmental chemicals can inhibit the androgen signaling pathway, which raises the question if developmental exposure to anti-androgenic chemicals can negatively impact female fertility. Here, we report on altered reproductive hormone profiles in prepubertal female rats following developmental exposure to three pesticides with anti-androgenic potential: linuron (25 and 50 mg/kg bw/d), dimethomorph (60 and 180 mg/kg bw/d) and imazalil (8 and 24 mg/kg bw/d). Dams were orally exposed from gestational day 7 (dimethomorph and imazalil) or 13 (linuron) until birth, then until end of dosing at early postnatal life. Linuron and dimethomorph induced dose-related reductions to plasma corticosterone levels, whereas imazalil mainly suppressed gonadotropin levels. In the ovaries, expression levels of target genes were affected by linuron and dimethomorph, suggesting impaired follicle growth. Based on our results, we propose that anti-androgenic chemicals can negatively impact female reproductive development. This highlights a need to integrate data from all levels of the hypothalamic-pituitary-gonadal axis, as well as the hypothalamic-pituitary-adrenal axis, when investigating the potential impact of endocrine disruptors on female reproductive development and function.

Effects of the Hedgehog Signaling Inhibitor Itraconazole on Developing Rat Ovaries.

Early ovary development is considered to be largely hormone independent; yet, there are associations between fetal exposure to endocrine disrupting chemicals and reproductive disorders in women. This can potentially be explained by perturbations to establishment of ovarian endocrine function rather than interference with an already established hormone system. In this study we explore if Hedgehog (HH) signaling, a central pathway for correct ovary development, can be disrupted by exposure to HH-disrupting chemicals, using the antifungal itraconazole as model compound. In the mouse Leydig cell line TM3, used as a proxy for ovarian theca cells, itraconazole exposure had a suppressing effect on genes downstream of HH signaling, such as Gli1. Exposing explanted rat ovaries (gestational day 22 or postnatal day 3) to 30 µM itraconazole for 72 h induced significant suppression of genes in the HH signaling pathway with altered Ihh, Gli1, Ptch1, and Smo expression similar to those previously observed in Ihh/Dhh knock-out mice. Exposing rat dams to 50 mg/kg bw/day in the perinatal period did not induce observable changes in the offspring's ovaries. Overall, our results suggest that HH signal disruptors may affect ovary development with potential long-term consequences for female reproductive health. However, potent HH inhibitors would likely cause severe teratogenic effects at doses lower than those causing ovarian dysgenesis, so the concern with respect to reproductive disorder is for the presence of HH disruptors at low concentration in combination with other ovary or endocrine disrupting compounds.

Organophosphate ester flame retardants have antiandrogenic potential and affect other endocrine related endpoints in vitro and in silico.

Organophosphate ester flame retardants (OPFRs) are used to prevent ignition and spreading of fire. They are present in various human matrices suggesting adult, fetal, and neonate exposure. Endocrine related effects have been observed in vivo, but information at the molecular level is lacking for some OPFRs. Also, a better understanding of potential contribution from chemical substructures is needed. The aim of this study was to screen OPFRs for endocrine disruptive potential in vitro and in silico. We selected eleven substances to represent some OPFRs with 1) little information on endocrine activity and others to represent 2) varied chemical substructures. We used in vitro assays for androgen receptor (AR), aryl hydrocarbon receptor (AhR), and Nrf2 activity, effects on steroidogenesis, and transthyretin (TTR) binding, as well as in silico models covering estrogen, thyroid, and CYP3A4 induction related endpoints. Ten OPFRs affected AR and AhR activity, seven affected TTR binding, and five affected 17ß-estradiol levels. Several substances had IC50-values below 10 µM and exhibited efficacious effects. These included TPHP, CDP, TMPP, TIPPP, and EHDPP for AR antagonism, suggesting that the degree of arylation and the size of the substance can play a role for the activity. Chlorinated OPFRs had low/no effect on TTR binding. No clear trend was observed for AhR and steroidogenesis, but all arylated OPFRs were predicted to have alert for estrogen receptor binding in an in silico model with metabolism simulator included. Collectively, our data suggest that OPFRs have endocrine disruptive potential warranting further studies to enable human risk assessment.

Using assessment criteria for pesticides to evaluate the endocrine disrupting potential of non-pesticide chemicals: Case butylparaben.

Regulation of chemicals with endocrine disrupting properties depend on the use of the chemical rather than its intrinsic properties. Within the EU, the only criteria currently in place for identifying an endocrine disrupting chemical (EDC) are those developed for biocidal and plant protection products. We argue that ECHA/EFSA guidance for assessing endocrine disrupting properties of biocidal and plant protection products can be applied to all chemicals independent of their intended use. We have assessed the REACH-registered compound butylparaben (CAS 94-36-8), a preservative used primarily in cosmetics. Based on scientific evidence of adverse reproductive effects and endocrine activity, the open literature suggest that butylparaben is an EDC. By applying the ECHA/EFSA guidance for pesticides and biocides, we identify butylparaben as a compound with endocrine disrupting properties. Even though available data is markedly different from that for biocides and pesticides, it was possible to reach this conclusion. More generally, we propose that the ECHA/EFSA guidance can and should be used for identification of EDC regardless of their intended application.

Does human papillomavirus-negative condylomata exist?

Condylomata acuminata is caused by human papillomavirus (HPV). PCR with consensus primers will typically detect HPV in >96% of condylomata. Metagenomic sequencing has found that some "HPV-negative" condylomata do indeed contain HPV. We wished to perform a renewed evaluation of the "HPV-negative" condylomata using deeper metagenomics sequencing. Sequencing of whole genome amplified DNA from 40 apparently "HPV-negative" condylomata detected HPV in 37/40 specimens. We found 75 different HPV types, out of which 43 represented novel putative HPV types. Three types were cloned and established as HPV types 200, 201 and 202. Molluscum contagiosum virus was detected in 24 of the 40 samples. In summary, deep sequencing enables detection of HPV in almost all condylomata. "HPV-negative" condylomata might largely be explained by clinical misdiagnosis or the presence of viral variants, distantly related HPV types and/or low viral loads.

Presence of High-Risk HPV mRNA in Relation to Future High-Grade Lesions among High-Risk HPV DNA Positive Women with Minor Cytological Abnormalities.

OBJECTIVE: Continuous expression of E6- and E7-oncogenes of high-risk human papillomavirus (HPV) types is necessary for the development and maintenance of the dysplastic phenotype. The aim of the study was to determine the sensitivity and specificity of the APTIMA HPV mRNA assay (Hologic) in predicting future development of high-grade cervical intraepithelial neoplasia (CIN) among high-risk HPV-DNA-positive women with atypical squamous cells of undetermined significance (ASCUS) or low-grade squamous epithelial lesion (LSIL) cytology. METHODS: Archived SurePath cervical samples of women ≥ 35 years of age with high-risk HPV DNA-positive ASCUS (n = 211) or LSIL, (n = 131) were tested for the presence of high-risk HPV E6/E7 mRNA using the APTIMA HPV assay, and the women were monitored for development of histopathologically verified CIN2+. RESULTS: Twenty-nine percent (61/211) of the women in the ASCUS group, and 34.3% (45/131) in the LSIL group developed CIN2+ within 4.5 years of follow-up. The prevalence of HPV mRNA was 90.0% (95% CI 85.9-94.0) among women with ASCUS and 95.4% (95% CI 91.8-99.0) among women with LSIL. The presence of HPV E6/E7 mRNA was associated with future development of CIN2+ among women with ASCUS and LSIL (p=0.02). The mRNA assay demonstrated high sensitivity in predicting future CIN2+ and CIN3 for index ASCUS (96.7%; 95% CI 87.6-99.4 and 100%; 95% CI 82.2-100, respectively) and LSIL (97.8%, 95% CI 86.8-99.9 and 100%, 95% CI 79.9-100, respectively). The corresponding specificity was low, 12.7% (95% CI 7.9-19.3) and 5.8% (95% CI 2.2-13.6), for future CIN2+, respectively. The negative predictive value of the HPV mRNA assay for detecting future CIN3 was 100%, since no mRNA-negative woman developed CIN3 (0/27) as compared to 13.6% (43/315) of the mRNA-positive women (p = 0.03). CONCLUSION: The APTIMA mRNA assay demonstrated high sensitivity but low specificity in predicting future CIN2+ among women with minor cytological abnormalities. The assay had high negative predictive value for future CIN3, indicating that HPV-mRNA-negative women are at low risk of progression to high grade CIN.

Perinatal exposure to mixtures of anti-androgenic chemicals causes proliferative lesions in rat prostate.

BACKGROUND: Elevated levels of endogenous or exogenous estrogens during fetal life can induce permanent disturbances in prostate growth and predispose to precancerous lesions. Recent studies have indicated that also early anti-androgen exposure may affect prostate cancer risk. METHODS: We examined the influence of perinatal exposure to mixtures of anti-androgenic and estrogenic chemicals on prostate development. Wistar rats were exposed from gestation day 7 to postnatal day 22 to a mixture of 8 anti-androgenic compounds (AAMix), a mixture of four estrogenic compounds (EMix), or paracetamol or a mixture of all 13 compounds (TotalMix) in mixture ratios reflecting human exposure levels. RESULTS: Ventral prostate weights were reduced by the TotalMix and AAMix in pre-pubertal rats. Histological changes in prostate appeared with increasing age and indicated a shift from the normal age-dependent epithelial atrophy towards hyperplasia. These lesions showed similarities to pre-cancerous lesions in humans. Increased proliferation was observed already in pre-puberty and it was hypothesized that this could be associated with reduced ERß signaling, but no clear conclusions could be made from gene expression studies on ERß-related pathways. The influences of the estrogenic chemicals and paracetamol on prostate morphology were minor, but in young adulthood the estrogen mixture reduced ventral prostate mRNA levels of Igf1 and paracetamol reduced the mRNA level ofPbpc3. CONCLUSIONS: Mixtures of endocrine disrupters relevant for human exposure was found to elicit persistent effects on the rat prostate following perinatal exposure, suggesting that human perinatal exposure to environmental chemicals may increase the risk of prostate cancer later in life.

Human papillomavirus type 197 is commonly present in skin tumors.

Non-melanoma skin cancers commonly contain Human Papillomavirus (HPV), but the types found have varied depending on the polymerase chain reaction (PCR) primer systems used. Whole genome amplified DNA (not amplified by any specific PCR primers) from 91 skin lesions [41 squamous cell skin carcinomas (SCCs), 8 keratoacanthomas, 22 actinic keratoses, 3 basal cell carcinomas and 17 SCCs in situ] were sequenced. All samples were sequenced both at 160 Mb and 1.8 Gb sequencing depth per sample. The sequences from 10 different HPVs in 47/91 specimens were found. Sequences represented four established HPV types (HPV types 16, 22, 120, 124), two previously known putative types (present in GenBank) and four previously unknown HPV sequences (new putative types). The most commonly detected virus was cloned, sequenced and designated as HPV197. Type-specific real-time PCR detected HPV197 in 34/91 specimens. For comparison, a pool of the same samples after general primer PCR amplification was also sequenced. This revealed 40 different HPVs, but only two HPV types were detected both with sequencing without prior PCR and with sequencing PCR amplicons, suggesting that sequencing without prior PCR gives a more unbiased representation of the HPVs present. In summary, it was found that HPV can be sequenced from most skin disease specimens and HPV197 appeared to be the most commonly present virus.

Mixtures of environmentally relevant endocrine disrupting chemicals affect mammary gland development in female and male rats.

Estrogenic chemicals are able to alter mammary gland development in female rodents, but little is known on the effects of anti-androgens and mixtures of endocrine disrupting chemicals (EDCs) with dissimilar modes of action. Pregnant rat dams were exposed during gestation and lactation to mixtures of environmentally relevant EDCs with estrogenic, anti-androgenic or dissimilar modes of action (TotalMix) of 100-, 200- or 450-fold high end human intake estimates. Mammary glands of prepubertal and adult female and male offspring were examined. Oestrogens increased mammary outgrowth in prepubertal females and the mRNA level of matrix metalloproteinase-3, which may be a potential biomarker for increased outgrowth. Mixtures of EDCs gave rise to ductal hyperplasia in adult males. Adult female mammary glands of the TotalMix group showed morphological changes possibly reflecting increased prolactin levels. In conclusion both estrogenic and anti-androgenic chemicals given during foetal life and lactation affected mammary glands in the offspring.

Characterization of human papillomavirus subtype 72b.

We report the characterization of human papillomavirus (HPV) subtype 72b of the genus Alphapapillomavirus isolated from an oral rinse sample of a healthy woman. The HPV72b L1 open reading frame (ORF) was 90.2% identical to that of HPV72, indicating a subtype close to the border of a novel HPV type.

Complete genome sequences of three novel human papillomavirus types, 175, 178, and 180.

We report the characterization of three novel human papillomavirus (HPV) types of the genus Gammapapillomavirus. HPV175 and HPV180 were isolated from a condyloma. HPV178 was isolated from healthy skin adjacent to an actinic keratosis.

The nasal mucosa contains a large spectrum of human papillomavirus types from the Betapapillomavirus and Gammapapillomavirus genera.

BACKGROUND: Human papillomavirus (HPV) types from the Betapapillomavirus and Gammapapillomavirus genera are common at cutaneous sites. The aim of this study was to analyze the prevalence of these HPV types in oral and nasal samples. METHODS: Nasal samples and oral samples were obtained from 312 volunteer Danish healthcare staff (240 women and 72 men), among whom the mean age was 42 years. A total of 311 oral samples and 304 nasal samples were eligible for HPV DNA analysis. HPV types were detected by use of polymerase chain reactions with modified general primers (MGP) and Forslund-Antonsson primers (FAP) and identified by Luminex (for types detected by MGP PCR) or direct sequencing or cloning before sequencing (for types detected by FAP PCR). RESULTS: HPV DNA was detected in 6% of the oral samples and 50% of the nasal samples. Seventy-five diverse HPV types or putative HPV types were identified. HPV types within the Alphapapillomavirus, Betapapillomavirus, and Gammapapillomavirus genera were detected in 3%, 31%, and 23% of the nasal samples, respectively. A putative subtype of HPV76, originally isolated from a feline oral squamous cell carcinoma, was detected in 7 nasal samples. CONCLUSION: A large spectrum of HPV types from Betapapillomavirus and Gammapapillomavirus have tropism for the nasal mucosa. The implication of the relatively high prevalence of these viruses in the nasal mucosa is unknown.

Unbiased approach for virus detection in skin lesions.

To assess presence of virus DNA in skin lesions, swab samples from 82 squamous cell carcinomas of the skin (SCCs), 60 actinic keratoses (AKs), paraffin-embedded biopsies from 28 SCCs and 72 kerathoacanthomas (KAs) and fresh-frozen biopsies from 92 KAs, 85 SCCs and 92 AKs were analyzed by high throughput sequencing (HTS) using 454 or Ion Torrent technology. We found total of 4,284 viral reads, out of which 4,168 were Human Papillomavirus (HPV)-related, belonging to 15 known (HPV8, HPV12, HPV20, HPV36, HPV38, HPV45, HPV57, HPV59, HPV104, HPV105, HPV107, HPV109, HPV124, HPV138, HPV147), four previously described putative (HPV 915 F 06 007 FD1, FA73, FA101, SE42) and two putatively new HPV types (SE46, SE47). SE42 was cloned, sequenced, designated as HPV155 and found to have 76% similarity to the most closely related known HPV type. In conclusion, an unbiased approach for viral DNA detection in skin tumors has found that, although some new putative HPVs were found, known HPV types constituted most of the viral DNA.

Low temperatures enhance the toxicity of copper and cadmium to Enchytraeus crypticus through different mechanisms.

Knowledge about how toxicity changes with temperature is important for determining the extent of safety factors required when extrapolating from standard laboratory conditions to variable field scenarios. In the present study, the authors evaluated the toxicity of Cu and Cd to the potworm Enchytraeus crypticus at 6 temperatures in the range of 11 °C to 25 °C. For both metals, reproductive toxicity decreased approximately 2.5-fold with increasing temperature. This is contrary to what most other studies have found. Measurements of the bioavailable fraction of the metals in the soils and the internal metal concentrations in the worms over time showed that the major cause of change in toxicity with temperature for Cu was the worms' ability to regulate internal concentration at high temperatures. Uptake of Cd increased with time at all temperatures and with higher rates at high temperatures. Hence, the lower toxicity of Cd at high temperatures is proposed to be due to the E. crypticus being more efficient at immobilizing Cd and/or repairing damages at high compared to low temperatures. The present study concludes that no consistent relationship between metal toxicity and temperature across species can be made. The metabolic dependence of the species in terms of regulating metal uptake, excretion, immobilization, damage, and repair processes, will be crucial factors in determining species susceptibility to metals at varying temperatures.

Metagenomic sequencing of "HPV-negative" condylomas detects novel putative HPV types.

Condylomas are caused by human papillomavirus (HPV), but may in rare cases be "negative for HPV" by PCR. Metagenomic sequencing can be used for an unbiased assessment of the presence of virus. Ten swab sample pools, each containing four cases of "HPV-negative" condylomas, were subjected to metagenomic sequencing. One pool contained Molluscum contagiosum. Five pools contained HPV, of which three pools contained novel putative HPV-types. The 12 samples in these three pools were sequenced individually. Six of these contained HPV and two contained Molluscum contagiosum. Altogether, 1337 HPV-related reads were detected, representing 23 novel putative Gammapapillomaviruses, 10 established HPV types (genital HPV types 6, 57, 58 and 66, Betapapillomavirus types 5, 105, 124, and Gammapapillomavirus types 50, 130, 150) and two described HPV sequences (KC7 and FA69). Complete genomes of Gammapillomavirus FA69 and SE87 were compiled. Metagenomic sequencing reveals that seemingly "HPV-negative" condylomas contain known and previously unknown HPV types.

Human papillomavirus typing in reporting of condyloma.

BACKGROUND: Monitoring of condylomas is an early evidence of population effectiveness of human papillomavirus (HPV) vaccination programs. If reporting could include HPV typing, the contribution by vaccine HPV types to condyloma burden could be monitored. METHODS: A sentinel site for reporting of condyloma including HPV typing was established at the Centre for Sexual Health in Malmö, Sweden. In 2006 to 2009, when there were few HPV vaccines, 621 subjects with condyloma were reported and HPV genotyped. RESULTS: Ninety-four percent of the condylomas contained genital HPV types. Thirty-five different genital HPV types were identified, with HPV6 (62%), HPV16 (13%), and HPV11 (10%) being the most common. At least 1 of the 4 HPV types in the HPV6/11/16/18 vaccine was detected in 77%. High-risk HPV types were more common in females (45%) than among males (27%) (odds ratio, 1.9; confidence interval, 1.3-2.8). Extended testing among subjects initially negative for HPV found 21 patients with cutaneous types of HPV, including a novel type (HPV153). CONCLUSIONS: This report provides a baseline distribution of HPV types in condylomas before the introduction of an HPV vaccination program in this population. Human papillomavirus typing is feasible in routine condyloma reporting.

EBV latent membrane protein 1 is a negative regulator of TLR9.

EBV infects most of the human population and is associated with a number of human diseases including cancers. Moreover, evasion of the immune system and chronic infection is an essential step for EBV-associated diseases. In this paper, we show that EBV can alter the regulation and expression of TLRs, the key effector molecules of the innate immune response. EBV infection of human primary B cells resulted in the inhibition of TLR9 functionality. Stimulation of TLR9 on primary B cells led to the production of IL-6, TNF-α, and IgG, which was inhibited in cells infected with EBV. The virus exerts its inhibitory function by decreasing TLR9 mRNA and protein levels. This event was observed at early time points after EBV infection of primary cells, as well as in an immortalized lymphoblastoid cell line. We determined that the EBV oncoprotein latent membrane protein 1 (LMP1) is a strong inhibitor of TLR9 transcription. Overexpression of LMP1 in B cells reduced TLR9 promoter activity, mRNA, and protein levels. LMP1 mutants altered in activating the NF-κB pathway prevented TLR9 promoter deregulation. Blocking the NF-κB pathway recovered TLR9 promoter activity. Mutating the NF-κB cis element on the TLR9 promoter restored luciferase transcription in the presence of LMP1. Finally, deletion of the LMP1 gene in the EBV genome abolished the ability of the virus to induce TLR9 downregulation. Our study describes a mechanism used by EBV to suppress the host immune response by deregulating the TLR9 transcript through LMP1-mediated NF-κB activation.

Common definition for categories of clinical research: a prerequisite for a survey on regulatory requirements by the European Clinical Research Infrastructures Network (ECRIN).

BACKGROUND: Thorough knowledge of the regulatory requirements is a challenging prerequisite for conducting multinational clinical studies in Europe given their complexity and heterogeneity in regulation and perception across the EU member states. METHODS: In order to summarise the current situation in relation to the wide spectrum of clinical research, the European Clinical Research Infrastructures Network (ECRIN) developed a multinational survey in ten European countries. However a lack of common classification framework for major categories of clinical research was identified, and therefore reaching an agreement on a common classification was the initial step in the development of the survey. RESULTS: The ECRIN transnational working group on regulation, composed of experts in the field of clinical research from ten European countries, defined seven major categories of clinical research that seem relevant from both the regulatory and the scientific points of view, and correspond to congruent definitions in all countries: clinical trials on medicinal products; clinical trials on medical devices; other therapeutic trials (including surgery trials, transplantation trials, transfusion trials, trials with cell therapy, etc.); diagnostic studies; clinical research on nutrition; other interventional clinical research (including trials in complementary and alternative medicine, trials with collection of blood or tissue samples, physiology studies, etc.); and epidemiology studies. Our classification was essential to develop a survey focused on protocol submission to ethics committees and competent authorities, procedures for amendments, requirements for sponsor and insurance, and adverse event reporting following five main phases: drafting, consensus, data collection, validation, and finalising. CONCLUSION: The list of clinical research categories as used for the survey could serve as a contribution to the, much needed, task of harmonisation and simplification of the regulatory requirements for clinical research in Europe.