Tim Schmoll
email: tim.schmoll@uni-bielefeld.de

A review and perspective on context-dependent genetic effects of extra-pair mating in birds

Keywords: compatible genes, context-dependence, differential investment, extra-pair paternity, genotypeby-environment interaction, good genes, fitness consequences, half-sibling comparison, multiple mating

The evolutionary origin and the maintenance of extra-pair mating in birds has been a major field of study in the last decades, but no consensus has been reached on the adaptive significance of this behaviour for female birds. The genetic benefit hypothesis proposes that extra-pair sires provide alleles of superior quality and/or better compatibility compared to the social mate, resulting in offspring of higher reproductive value. One frequently adopted approach to test this idea compares the performance of maternal half-siblings in broods with multiple paternity. However, results from such comparisons are inconsistent. Here I discuss the idea that the magnitude of genetic fitness benefits from extra-pair mating depends on the environmental context. Context-dependent genetic effects in maternal half-sibling comparisons have been demonstrated for only five passerine bird species up to date. In none of the studies were the crucial environmental conditions experimentally manipulated and the potentially confounding effects of differential maternal investment in relation to paternity were mostly not accounted for either. A number of high-quality data sets on fitness consequences of extra-pair mating behaviour are available that could be (re-) analysed for context-dependence given relevant gradients of the environment have been recorded and their use is well justified a priori.

Such relevant variation may include, for example, the time of breeding in temperate regions, hatching order, but also offspring sex. Primarily, however, experimental approaches are required that systematically and gradually vary fitness-relevant environmental gradients like food availability or parasite abundance and analyse the resulting differential fitness effects while controlling for differential investment. Context-dependence of genetic effects of extrapair mating behaviour may offer an opportunity for reconciling conflicting results from different extra-pair paternity studies within and across species. More generally, it could allow better understanding under which environmental conditions selection may act to maintain a female mating bias towards extra-pair males with potentially far-reaching implications for the ecology and evolution of mating preferences and the maintenance of genetic variation in (sexually) selected traits.

Introduction

Extra-pair paternity (EPP) arises when female birds engage in extra-pair copulations (EPC) with males other than their social mates, frequently resulting in broods with multiple paternity which contain offspring sired by the social male (within-pair offspring, WPO) and offspring sired by extra-pair males (extra-pair offspring, EPO). A large and solid body of evidence has impressively demonstrated that EPP is the rule rather than the exception in birds (especially in passerines), and a number of synthetic contributions have reviewed this field with its various ramifications over the last two decades (e.g. [START_REF] Westneat | The ecology and evolution of extra-pair copulations in birds[END_REF][START_REF] Birkhead | Sperm competition in birds: evolutionary causes and consequences[END_REF][START_REF] Petrie | Extra-pair paternity in birds: explaining variation between species and populations[END_REF][START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF][START_REF] Westneat | Extra-Pair Paternity in Birds: Causes, Correlates, and Conflict[END_REF][START_REF] Akçay | Extra-pair paternity in birds: review of the genetic benefits[END_REF].

As EPP is so widespread and so common and because it must be considered a powerful source of pre-as well as postcopulatory sexual selection, no comprehensive understanding of avian mating systems and sexual selection in birds seems possible without understanding the evolutionary causes and consequences of this phenomenon. However, despite the very substantial research effort in the field, there is still no consensus on a number of fundamental questions related to this topic. For example, there is sustained debate about which sex is actually in control of EPC behaviour and, particularly, about the adaptive significance of extrapair mating behaviour for female birds (e.g. [START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF][START_REF] Westneat | Extra-Pair Paternity in Birds: Causes, Correlates, and Conflict[END_REF][START_REF] Arnqvist | The evolution of infidelity in socially monogamous passerines: The strength of direct and indirect selection on extrapair copulation behavior in females[END_REF][START_REF] Griffith | The evolution of infidelity in socially monogamous passerines: Neglected components of direct and indirect selection[END_REF]; [START_REF] Akçay | Extra-pair paternity in birds: review of the genetic benefits[END_REF][START_REF] Griffith | Female infidelity and genetic compatibility in birds: the role of the genetically loaded raffle in understanding the function of extrapair paternity[END_REF]. A number of different hypotheses have been put forward to explain why female birds mate extra-pair, which are discussed in more detail in the reviews mentioned above. In this contribution I will focus on the idea that has gained most attention and stimulated the most intense and most controversial debate in the past, i.e. that genetic benefits from a mating bias towards extra-pair sires select for extra-pair mating behaviour in females (for a more general review and discussion of genetic benefit models of mate choice see e.g. [START_REF] Jennions | Why do females mate multiply? A review of the genetic benefits[END_REF][START_REF] Mays | Choosing mates: good genes versus genes that are a good fit[END_REF][START_REF] Neff | Genetic quality and sexual selection: an integrated framework for good genes and compatible genes[END_REF].

After briefly introducing the genetic benefit hypothesis of extra-pair mating and the advantages and potential problems of using maternal half-sibling comparisons to test it, I will outline the idea that the magnitude of genetic benefits of extra-pair matings may depend on environmental context and illustrate potential consequences for the evolution of female mating preferences and the maintenance of genetic variation for traits under selection. Then I will briefly summarise and review the main results of the few empirical studies that have demonstrated context-dependent genetic effects of extra-pair matings and suggest potential directions for future research in this field. In line with other recent conceptual contributions (e.g. [START_REF] Greenfield | Genotype-environment interaction and the reliability of mating signals[END_REF][START_REF] Bussière | Mate choice for genetic quality when environments vary: suggestions for empirical progress[END_REF][START_REF] Ingleby | The role of genotype-by-environment interactions in sexual selection[END_REF], this paper aims at promoting an approach that puts work on sexual selection more explicitly into the ecological context within which it operates.

Genetic benefit hypothesis of extra-pair matinggood and/or compatible alleles

The principal idea behind the genetic benefits hypothesis is that, by engaging in extra-pair matings, females express a mating preference that is otherwise constrained, for example because social monogamy precludes many females from mating with the few top genetic quality males in a population or because the best social and the best genetic mate for any individual female are not identical. This hypothesis therefore assumes that female birds have a substantial degree of control over extra-pair fertilizations through either pre-copulatory extrapair mate choice and/or cryptic postcopulatory mechanisms that bias paternity in favour of (particular) extra-pair sires. In the following, I will subsume both these possibilities under the term "mating preference" (for extra-pair males), which does therefore not necessarily imply classic precopulatory mate choice. A further assumption of the genetic benefit hypothesis is that extra-pair mating behaviour is costly for females and needs to be balanced by some sort of fitness benefit. This benefit may lie in the higher viability or fecundity and/or sexual attractiveness of offspring sired through EPCs with males that are of higher genetic quality or of better genetic compatibility compared to the respective social mate. The proposed genetic benefits may therefore be assumed to be additive genetic (acquisition of "good alleles") or represent non-additive effects presupposing an interaction of maternal and paternal genotype (acquisition of "compatible alleles" sensu Kempenaers 2007, i.e. broadly defined as alleles that increase fitness contingent on the genetic make-up of the choosing individual). Thus, a mating preference for mates with compatible alleles may, for example, include biasing paternity to maximise offspring heterozygosity, minimise the risk of close inbreeding, or optimise immunogenetic complementarity. Both types of benefits are not mutually exclusive but could also operate in concert.

Revealing paternal genetic effects on offspring traits by maternal half-sibling comparisons

The most straightforward test of the genetic benefit hypothesis of extra-pair mating is a comparison of the performance of maternal half-siblings from multiply sired broods [START_REF] Sheldon | Paternal genetic contribution to offspring condition predicted by size of male secondary sexual character[END_REF]. Here, potentially confounding, non-genetic effects of a common environment of sire and offspring as well as maternal effects on offspring fitness-related traits are controlled for (but see paragraph on differential maternal investment below). Maternal half-siblings share, by definition, on average half of their maternally inherited nuclear genes and experience the same early environment, including, for example, the same parenting skills of the respective social parents or exposure to the same parasite load in the shared nest. Therefore, any systematic differences in phenotypic traits between the two maternal half-sibships can only be attributed to the differential paternal genetic contribution (differential paternity hereafter) of the respective genetic sires. A consistently superior performance of EPO compared to their WPO maternal half-siblings is thus considered strong evidence in favour of the genetic benefit hypothesis [START_REF] Sheldon | Paternal genetic contribution to offspring condition predicted by size of male secondary sexual character[END_REF][START_REF] Griffith | Extra pair paternity in birds: a review of interspecific variation and adaptive function[END_REF]. In principal, this statement is true regardless of whether the proposed genetic benefits are assumed to represent additive or non-additive effects (but see [START_REF] Griffith | Female infidelity and genetic compatibility in birds: the role of the genetically loaded raffle in understanding the function of extrapair paternity[END_REF].

Extra-pair paternity and genetic benefitsinconsistent results

Evidence for a genetic type of benefit based on maternal half-sibling comparisons is rather mixed across studies. Some highly cited landmark studies spectacularly support the genetic benefit hypothesis. For example, in Collared Flycatchers Ficedula albicollis, EPO were in better body condition near fledging compared to their maternal half-siblings [START_REF] Sheldon | Paternal genetic contribution to offspring condition predicted by size of male secondary sexual character[END_REF], and nestling condition has been shown to positively affect recruitment in this species [START_REF] Lindén | Selection on fledging mass in collared flycatchers and great tits[END_REF]. Furthermore, the difference in mean body condition between half-sibships was predicted by the difference in size of an at least partly intersexually selected male ornament of the respective sires (the white forehead patch, [START_REF] Sheldon | Paternal genetic contribution to offspring condition predicted by size of male secondary sexual character[END_REF]. In Bluethroats Luscinia svecica, EPO nestlings showed better cellular immunocompetence than their maternal and, notably, also their paternal half-siblings, suggesting not only a genetic benefit from an extrapair mating preference, but also an interaction effect of maternal and paternal genotype on offspring immunocompetence [START_REF] Johnsen | Female bluethroats enhance offspring immunocompetence through extra-pair copulations[END_REF]; see also [START_REF] Fossøy | Multiple genetic benefits of female promiscuity in a socially monogamous passerine[END_REF]. Strong support in favour of the genetic benefit hypothesis of extra-pair mating also comes from a study in the Blue Tit Cyanistes caeruleus. Here, EPO sired by non-local extra-pair males showed higher average heterozygosity compared to their maternal half-siblings and individual heterozygosity was positively correlated with a number of fitness-related traits in both sexes [START_REF] Foerster | Females increase offspring heterozygosity and fitness through extra-pair matings[END_REF]. In contrast to these findings, other studies failed to reveal any systematic differences in maternal half-sibling performance, sometimes in the same or in closely related species (e.g. [START_REF] Strohbach | Extrapair paternity in the great tit (Parus major): a test of the "good genes" hypothesis[END_REF][START_REF] Lubjuhn | Extra-pair paternity in great tits (Parus major) -A long term study[END_REF][START_REF] Whittingham | Survival of extrapair and within-pair young in tree swallows[END_REF][START_REF] Schmoll | Long-term fitness consequences of female extra-pair matings in a socially monogamous passerine[END_REF][START_REF] Edly-Wright | Genetic similarity of mates, offspring health and extrapair fertilization in house sparrows[END_REF][START_REF] Rosivall | Effects of extrapair paternity and sex on nestling growth and condition in the collared flycatcher, Ficedula albicollis[END_REF][START_REF] Dunn | Multiple paternity and offspring quality in tree swallows[END_REF]. Furthermore, application of identical experimental protocols (e.g. the phytohemagglutinin test of cellular immunocompetence) in different species with similar extra-pair mating systems produced mixed results [START_REF] Johnsen | Female bluethroats enhance offspring immunocompetence through extra-pair copulations[END_REF][START_REF] Kleven | Extra-pair paternity and offspring immunocompetence in the reed bunting (Emberiza schoeniclus)[END_REF][START_REF] Kleven | No evidence of paternal genetic contribution to nestling cell-mediated immunity in the North American barn swallow[END_REF][START_REF] Fossøy | Multiple genetic benefits of female promiscuity in a socially monogamous passerine[END_REF][START_REF] Wilk | Lack of evidence for improved immune response of extrapair nestlings in collared flycatcher Ficedula albicollis[END_REF][START_REF] Forsman | Female house wrens (Troglodytes aedon) increase the size, but not immunocompetence, of their offspring through extra-pair mating[END_REF][START_REF] Butler | Ambient temperature, but not paternity, is associated with immune response in Savannah Sparrows (Passerculus sandwichensis)[END_REF]). The inconsistencies of results across EPP studies may be caused by methodological challenges and/or reflect true and meaningful differences in the respective study systems. In either case, it is important to address this heterogeneity in order to evaluate how reproducible and how generalisable these results may or may not be.

Effect sizes of genetic benefits likely to be small

The inconsistency of results across EPP studies may be related to the fact that additive genetic fitness benefits from mate choice are likely to be small in general (e.g. [START_REF] Kirkpatrick | The strength of indirect selection on female mating preferences[END_REF][START_REF] Møller | Good-genes effects in sexual selection[END_REF][START_REF] Arnqvist | The evolution of infidelity in socially monogamous passerines: The strength of direct and indirect selection on extrapair copulation behavior in females[END_REF]. Thus, even studies with comparatively large sample sizes may fail to detect any effects of differential paternity on offspring fitness due to a type II statistical error (accepting the null hypothesis when it is wrong), which may contribute to the observed heterogeneity in results across EPP studies.

Given small expected effect sizes, the other side of the same coin is that studies actually demonstrating differences in maternal half-sibling performance based on comparatively small samples sizes may be prone to a type I statistical error (rejecting the null hypothesis when it is true), potentially seconded by differential maternal investment in relation to paternity (see below).

Recent conceptual contributions emphasize the potential of non-additive genetic benefits from extra-pair mating (e.g. [START_REF] Kempenaers | Mate choice and genetic quality: A review of the heterozygosity theory[END_REF][START_REF] Griffith | Female infidelity and genetic compatibility in birds: the role of the genetically loaded raffle in understanding the function of extrapair paternity[END_REF]. For example, a number of studies have reported that EPO were more heterozygous than their WPO maternal half-siblings (e.g. [START_REF] Foerster | Females increase offspring heterozygosity and fitness through extra-pair matings[END_REF][START_REF] Stapleton | Female tree swallows (Tachycineta bicolor) increase offspring heterozygosity through extrapair mating[END_REF][START_REF] Suter | Reed bunting females increase fitness through extra-pair mating with genetically dissimilar males[END_REF][START_REF] Fossøy | Multiple genetic benefits of female promiscuity in a socially monogamous passerine[END_REF]; but see [START_REF] Wetzel | Heterozygosity and extra-pair paternity: biased tests result from the use of shared markers[END_REF] or that EPO not only outperformed their maternal, but also their paternal half-siblings [START_REF] Johnsen | Female bluethroats enhance offspring immunocompetence through extra-pair copulations[END_REF][START_REF] Garvin | Immune response of nestling warblers varies with extra-pair paternity and temperature[END_REF], suggesting non-additive rather than additive genetic benefits of extra-pair matings. However, a recent meta-analytical approach revealed that effect sizes of heterozygosity-fitness correlations are generally positive in sign, but not large either [START_REF] Chapman | A quantitative review of heterozygosity-fitness correlations in animal populations[END_REF]. Accordingly, the magnitude of non-additive fitness benefits to be potentially obtained through extra-pair mating may be expected to be rather small, too. This may not be the case for all types of non-additive genetic effects, however, for example if extra-pair mating helps preventing fertilisations which would lead to a substantial fitness loss due to pronounced inbreeding (e.g. [START_REF] Keller | Inbreeding and its fitness effects in an insular population of song sparrows (Melospiza melodia)[END_REF][START_REF] Kruuk | Severe inbreeding depression in collared flycatchers (Ficedula albicollis)[END_REF] or outbreeding [START_REF] Veen | Hybridization and adaptive mate choice in flycatchers[END_REF] depression.

Differential maternal investment may confound paternal genetic effects

Selection may favour females with the ability to bias resource allocation in favour of offspring descending from preferred sires [START_REF] Burley | Sexual selection for aesthetic traits in species with biparental care[END_REF][START_REF] Møller | Male parental care, differential parental investment by females and sexual selection[END_REF]. If females selectively favour their extra-pair offspring because their sires were of superior genetic quality, superior EPO performance may at least partly be due to maternal rather than paternal genetic effects. Thus, differential maternal investment with respect to paternity may have the potential to confound paternal genetic effects on offspring fitness [START_REF] Mousseau | The adaptive significance of maternal effects[END_REF][START_REF] Møller | Male parental care, differential parental investment by females and sexual selection[END_REF]. On the other hand, it can be argued that a biased investment in favour of EPO may make a case for rather than against the genetic benefit hypothesis, since differential investment solely depends on differential paternity [START_REF] Sheldon | Differential allocation: tests, mechanisms and implications[END_REF]. This argumentation seems less straightforward in light of recent studies suggesting that EPO occur earlier in the laying order and, accordingly, in the hatching sequence than their WPO maternal half-siblings, giving them a head start in sibling competition (e.g. [START_REF] Magrath | Maternal Effects Contribute to the Superior Performance of Extra-Pair Offspring[END_REF][START_REF] Ferree | Hatching order explains an extrapair chick advantage in western bluebirds[END_REF]. For example, [START_REF] Magrath | Maternal Effects Contribute to the Superior Performance of Extra-Pair Offspring[END_REF] not only demonstrated that EPO hatch slightly earlier than WPO, but also that statistically significant differences between maternal half-siblings in morphology and survival until fledging tend to diminish once hatching sequence is statistically controlled for. At present, it is unclear whether females time EPCs to occur early in their laying phase, whether these sequence effects reflect mating effort patterns of social or extra-pair sires, or whether they simply represent a by-product of social or ecological constraints on EPC behaviour (discussed in [START_REF] Magrath | Maternal Effects Contribute to the Superior Performance of Extra-Pair Offspring[END_REF]). In any case, such effects have the potential to confound paternal genetic effects on offspring fitness for both, additive as well as non-additive genetic benefits, possibly leading to an overestimation of the effects of differential paternity.

Context-dependent genetic benefits of mating preferences and their implications

Another explanation for inconsistent results from maternal half-sibling comparisons may be that the genetic benefits of extra-pair matings vary in magnitude in relation to the prevailing environmental conditions (please note that, while in the compatible alleles model the genetic background of a female may well be seen as an "environment" or a "context" within which paternal alleles are expressed and with which they may interact, I reserve the use of these terms for extra-genomic components of the environment). Benefits may be large enough to be detectable in some ecological circumstances, but too small or non-existent in others. Such context-dependent genetic effects presuppose temporal and/or spatial variation in the environment -a ubiquitous feature in most natural bird populations. They also presuppose genotypes to differ in how their fitness is affected by variation in the environment, i.e. genotype-by-environment interactions for fitness must be present. If this is not the case, the relative genetic benefit a female may obtain from choosing among different sires is identical across environments (Fig. 1 a,b). However, whenever such genotype-by-environment interactions exist, there is a potential for context-dependent genetic effects to occur (Fig. 1 c,d). This refers to sire as well as offspring genotypes in the case of additive genetic effects, but only to offspring genotypes in the case of non-additive genetic effects for which offspring fitness is not normally a function of sire genotype but determined by the interaction of maternal and sire genotype.

Two principal cases of genotype-by-environment interactions can be distinguished.

First, if fitness reaction norms do not cross and the fitness rank order of genotypes is preserved across the environments that are experienced, context-dependent genetic effects will affect the magnitude of the genetic benefit a female can potentially obtain, although not its sign (Fig. 1 c).

Single best male genotypes on the level of the population (in case of additive genetic effects) or most compatible male genotypes on the level of the individual female (in case of non-additive genetic effects) may exist and they are transient across these environments. Second, if fitness reaction norms cross and the fitness rank order of genotypes is not preserved across the environments that are experienced, context-dependent genetic effects will not only affect the magnitude of the genetic benefit a female may obtain, but also its sign, rendering preferences for particular sire genotypes adaptive in some of these environments, but not in others (Fig. 1 d, also termed ecological cross-over). Here, no single best male genotypes on either the level of the population or the level of the individual female do exist. Indeed, the terms "good alleles" or "compatible alleles" in reference to any particular male genotype do not make sense under this scenario. In both cases, however, context-dependent genetic effects suggest selection on female mating preferences to vary across environments, which has interesting implications for the evolution of mating preferences and the maintenance of genetic variation in traits that are under sexual selection.

If relevant gradients of the environment that offspring will experience are fairly well predictable, we may expect females to show phenotypically plastic instead of static mating preferences (discussed in [START_REF] Qvarnström | Context-dependent genetic benefits from mate choice[END_REF]). In the case of non-crossing fitness reaction norms, they may be selected to balancing direct costs of choice against expected genetic rewards and hence express any given preference more or less strongly depending on environmental variation. In the case of additive genetic effects, the strength of sexual selection on preferred male traits (secondary sexual characters and genetically correlated traits) will then vary accordingly and episodes of relaxed selection could slow down the depletion of genetic variance in these traits or even contribute to its maintenance by allowing more time for deleterious mutations to occur. In the case of ecological cross-over, plastic preferences would need to be reversed to be adaptive and would hence directly support maintenance of genetic variation in male traits. If the environmental conditions that offspring will experience are largely unpredictable, however, females may not benefit at all from expressing mating preferences for any particular sire genotype. For example, in the case of ecological cross-over in fitness, females have no means to evaluate the relative genetic quality of potential mates and they may rather opt for genetic bet-hedging, mate multiply and thereby sample a number of different sires in order to increase the within-cohort genetic diversity of their offspring (see [START_REF] Yasui | The 'genetic benefits' of female multiple mating reconsidered[END_REF] for more background on this argument and [START_REF] Schmoll | Polyandry in coal tits Parus ater: fitness consequences of putting eggs into multiple genetic baskets[END_REF] for an application to extra-pair mating behaviour in birds).

Context-dependent genetic benefits from mate choice in general have been shown in a range of taxa including insects [START_REF] Jia | When are good genes good? Variable outcomes of female choice in wax moths[END_REF], anurans [START_REF] Welch | Genetic benefits of a female mating preference in gray tree frogs are context-dependent[END_REF][START_REF] Sheldon | Sire coloration influences offspring survival under predation risk in the moorfrog[END_REF], and mammals [START_REF] Mills | Signal reliability compromised by genotype-by-environment interaction and potential mechanisms for its preservation[END_REF]). Furthermore, context-dependent genetic benefits from mate choice may be inferred from studies demonstrating genotype-by-environment interactions on the expression of male sexual signal traits under directional intersexual selection (e.g. [START_REF] Qvarnström | Genotype-by-environment interactions in the determination of the size of a secondary sexual character in the collared flycatcher (Ficedula albicollis)[END_REF][START_REF] Jia | Genetic variance of sexually selected traits in waxmoths: Maintenance by genotype x environment interaction[END_REF][START_REF] David | Condition-dependent signalling of genetic variation in stalk-eyed flies[END_REF][START_REF] Danielson-François | Genotype x environment interaction for male attractiveness in an acoustic moth: evidence for plasticity and canalization[END_REF]; see also [START_REF] Greenfield | Genotype-environment interaction and the reliability of mating signals[END_REF]). In the following paragraph, I will apply the idea of contextdependent genetic effects to extra-pair mating behaviour in birds and briefly review the few studies that have suggested such effects up to date.

Context-dependent genetic effects of extra-pair mating

In order to demonstrate context-dependent additive genetic effects of extra-pair matings, it is necessary to test for a two-way interaction effect of differential paternity and the focal environmental variable on offspring traits, i.e. EPO and WPO "genotypes" are predicted to react in a different manner to different environmental conditions. In a purely operational view, the same is true for non-additive genetic effects, although conceptually, a three-way interaction effect of maternal genotype, paternal genotype and the environment is predicted. Here, the magnitude of the fitness effects of the interaction between maternal and paternal genotypes depends on the environmental context.

As far as I am aware, only five studiesrelating to five different specieshave demonstrated context-dependent genetic effects when testing for maternal half-sibling performance. In the socially monogamous Coal Tit Periparus ater EPO had a higher probability of local recruitment into the breeding population compared to their WPO maternal half-siblings only when they originated from late (i.e. second) broods, but not when they originated from early (i.e. first) broods [START_REF] Schmoll | Paternal genetic effects on offspring fitness are context dependent within the extrapair mating system of a socially monogamous passerine[END_REF]. Recruitment probability in general was significantly reduced in late broods, such that the superior performance of EPO coincides with environmental conditions that are stressful compared to those in early broods [START_REF] Schmoll | Paternal genetic effects on offspring fitness are context dependent within the extrapair mating system of a socially monogamous passerine[END_REF]. First-year reproductive performance was affected in a similar way, reflecting the differential recruitment patterns [START_REF] Schmoll | Paternal genetic effects on offspring fitness are context dependent within the extrapair mating system of a socially monogamous passerine[END_REF]. No context-dependent effects were apparent in the same study population in an analysis of fitness-related traits that are expressed beyond recruitment [START_REF] Schmoll | Lifespan, lifetime reproductive performance and paternity loss of within-pair and extra-pair offspring in the coal tit Periparus ater[END_REF]).

In the socially monogamous Common Yellowthroat Geothlypis trichas, EPO mounted a stronger cellular immune response than their WPO maternal half-siblings only in the colder of two study years, but not in the warmer one [START_REF] Garvin | Immune response of nestling warblers varies with extra-pair paternity and temperature[END_REF]. Low ambient temperatures negatively affect offspring immunocompetence (directly and/or indirectly e.g. via food abundance) such that superior performance of EPO coincides with environmental conditions that are relatively stressful [START_REF] Garvin | Immune response of nestling warblers varies with extra-pair paternity and temperature[END_REF]).

In the socially polygynous Red Bishop Euplectes orix, WPO were found to be more immunocompetent than their EPO maternal half-siblings only in the hotter of two study years, but not in the cooler one [START_REF] Edler | Within-pair young are more immunocompetent than extrapair young in mixed-paternity broods of the red bishop[END_REF]. Compared to the yellowthroat study [START_REF] Garvin | Immune response of nestling warblers varies with extra-pair paternity and temperature[END_REF], the pattern thus appears reversed with respect to both, the relative genetic quality of maternal half-siblings and the effect of ambient temperature as a key environmental variable.

Hotter seasons, however, are likely to provide less favourable breeding conditions in this African species due to a lack of nestling food and increased heat stress [START_REF] Edler | Within-pair young are more immunocompetent than extrapair young in mixed-paternity broods of the red bishop[END_REF].

The fact that WPO instead of EPO perform better under such stressful conditions may be explained by the polygynous mating system of this species. Females paired to highly attractive and therefore more strongly polygynous males face an increased risk that their social mate runs out of sperm and seem to use EPC as a means to insure themselves against their high-quality social mates being temporarily infertile. Consequently, mainly less attractive males of presumably lower genetic quality may be successful in siring EPO [START_REF] Friedl | Extrapair fertilizations in red bishops (Euplectes orix): Do females follow conditional extrapair strategies?[END_REF][START_REF] Edler | Within-pair young are more immunocompetent than extrapair young in mixed-paternity broods of the red bishop[END_REF]. In this mating system, it thus seem to be WPO, not EPO, which are of higher genetic quality, so that the superior performance of offspring of relatively better genetic quality coincides with relatively stressful environmental conditions [START_REF] Edler | Within-pair young are more immunocompetent than extrapair young in mixed-paternity broods of the red bishop[END_REF]).

In the socially monogamous Tree Swallow Tachycineta bicolor, EPO had longer ninth primary feathers at nestling day 16 than their maternal half-siblings only when natural parasite abundance was low and when they had hatched early relative to their WPO maternal halfsiblings [START_REF] O'brien | Context-dependent genetic benefits of extra-pair mate choice in a socially monogamous passerine[END_REF]. The difference in ninth primary length at a particular developmental stage (i.e. near fledging) is seen as reflecting a genetic benefit of extra-pair mating, because nestling wing length predicts time of fledging and wing length at fledging is positively related to recruitment probability in the study species (discussed in O' Brien and Dawson 2007). This study differs from the previous ones not only in that it considers, conceptually, a three-way interaction (the effects of differential paternity were dependent on the interaction of two environmental dimensions, parasite abundance and relative hatch order). In contrast to the former studies, it was also in relatively favourable, not stressful, conditions that EPO have differed from their WPO half-siblings (O'Brien and Dawson 2007).

Finally, in the predominantly socially monogamous House Wren Troglodytes aedon WPO were more immunocompetent than their EPO maternal-half-siblings in two out of three study years, but the publication did not give reference as to whether these differences were related to relevant variation in (the quality of) the environment [START_REF] Forsman | Female house wrens (Troglodytes aedon) increase the size, but not immunocompetence, of their offspring through extra-pair mating[END_REF].

Poor environmental qualityrevealing or concealing paternal genetic effects?

The studies summarised above differ in how they see the role of environmental quality in determining the extent of genetic variation that is expressed in offspring phenotypes. Three out of the five studies that have demonstrated context-dependent genetic effects show a pattern where only comparatively poor environments seem to reveal genetic variation. An explanation for this could be that under beneficial environmental conditions all offspring may perform relatively well irrespective, to some degree, of their genetic quality (i.e. of being sired withinpair or extra-pair). Beneficial environments may thus have the potential to mask genetic variation in fitness-related traits, and, as a consequence, it would be difficult to demonstrate the differences in maternal half-sibling performance predicted by the genetic benefit hypothesis. This is particularly relevant when taking into account that the magnitude of genetic benefits of mate choice must be expected to be small in general (see above). In contrast, comparatively stressful conditions where essential resources are limited may reveal this genetic variation and it is only then that the small genetic differences between maternal half-siblings are reflected in their phenotypic performance and become detectable with limited sample sizes. Along similar lines, inbreeding depression is generally (although not universally) more pronounced under stressful conditions across a wide range of taxa according to a recent meta-analysis [START_REF] Fox | Inbreeding depression increases with environmental stress: an experimental study and meta-analysis[END_REF]. As an example in birds, [START_REF] Keller | Environmental conditions affect the magnitude of inbreeding depression in survival of Darwin's finches[END_REF] report a case of inbreeding-byenvironment interaction in the Cactus Finch Geospiza scandens. For example, inbreeding depression for adult survival was most severe under unfavourable conditions (i.e. dry climate and high population densities), but substantially less pronounced under a range of other conditions [START_REF] Keller | Environmental conditions affect the magnitude of inbreeding depression in survival of Darwin's finches[END_REF]. Similarly, nestling survival in great tits Parus major was negatively affected by parental genetic similarity only late in the season when food resources are likely to be more restricted ( [START_REF] Van De Casteele | Seasonal and lifetime reproductive consequences of inbreeding in the great tit Parus major[END_REF]. Thus in both examples inbred versus outbred genotypes differed in their response to varying conditions with only stressful environments reflecting the existing genetic variation in the phenotype (see also [START_REF] Marr | Interactive effects of environmental stress and inbreeding on reproductive traits in a wild bird population[END_REF][START_REF] Szulkin | The Environmental Dependence of Inbreeding Depression in a Wild Bird Population[END_REF].

In contrast to the former work, the Tree Swallow study by [START_REF] O'brien | Context-dependent genetic benefits of extra-pair mate choice in a socially monogamous passerine[END_REF] found that it is in relatively favourable conditions when half-siblings differ in performance. The difference between this study and the previous ones may be the type of focal trait considered or the type of genetic effect supposed to create the observed differential half-sibling performance.

In the case of a morphological character such as feather length, the underlying type of effect is likely to be additive genetic and the full range of existing genetic variation may only be expressed in the phenotype under favourable conditions, when individual genotypes can max out their genetic potential [START_REF] Hoffmann | Heritable variation and evolution under favourable and unfavourable conditions[END_REF]; see also Qvarnström 1999 reporting higher heritability of a sexually selected feather ornament under beneficial conditions). The other studies did not address differences in morphological characters but traits that seem more closely related to fitness. In contrast to morphometric traits, the phenotypic expression of genetic variation for such traits does not seem to be promoted by favourable conditions [START_REF] Charmantier | Environmental quality and evolutionary potential: lessons from wild populations[END_REF]. Neither do differences in these traits necessarily reflect additive genetic benefits of extra-pair matings. For example, a significantly better performance of EPO not only in maternal, but also in paternal half-sibling comparisons reported in [START_REF] Garvin | Immune response of nestling warblers varies with extra-pair paternity and temperature[END_REF] indicates a non-additive type of benefit in this case.

Conclusions

In conclusion, only five empirical studies have suggested context-dependent genetic effects of extra-pair mating in birds up to date. None of these studies, however, has experimentally manipulated the focal environmental dimensions and hence results are open for alternative explanations. Furthermore, four of the studies have not controlled for maternal effects in general and hatching sequence in particular. This is problematic because differential maternal investment in favour of EPO may not only confound effects of differential paternity in general, but lead to effects similar to those predicted from the context-dependence hypothesis. Finally, it is also mostly not clear, how variation in offspring quality traits relates to variation in fitness and how variation in the environmental variables highlighted in these studies affects offspring quality traits (with year effects being a case in point). Thus evidence for context-dependent genetic benefits from extra-pair matings in birds is suggestive rather than conclusive at present and it is unclear how important and how widespread context-dependent genetic effects of extrapair mating may actually be and how consistently specific (e. g. high-stress versus low-stress) environments may affect the context-dependent expression of genetic variation.

Perspectives

Studies addressing the genetic benefits of extra-pair matings by comparing maternal halfsiblings have generally used a range of different offspring quality traits and the few studies that have demonstrated context-dependent effects have likewise picked a number of different environmental variables to test for interactions with differential paternity (see above). The availability of many such potential response variables and, in particular, the availability of many potentially important predictor variables (i.e. environmental dimensions) harbours the risk of revealing spurious associations when testing for two-way (or even higher-order) interactions with differential paternity. Obviously, focal offspring quality traits must exhibit a verifiably close relationship with fitness, preferably as demonstrated in the very study population, to allow meaningful inference regarding the fitness consequences of extra-pair mating behaviour (frequently this relationship is assumed rather than established). Furthermore, to qualify as a "relevant" dimension of environmental quality, any candidate variable should predictably affect the focal offspring quality trait, preferably in a manner that is also understood mechanistically in some detail. Which offspring quality traits may be regarded and which particular dimension of the environment may be considered "relevant" will ultimately depend on the particular study system, but response and predictor variable(s) should be carefully chosen and well defined a priori for both, the observational as well as the experimental approaches outlined below.

A number of high-quality data sets on maternal half-sibling performance is meanwhile available (or being collected) and may frequently have been sampled across different environmental contexts. These data could be (re-) analysed while explicitly considering relevant environmental variation. Studies that have failed to establish the predicted differences in maternal half-sibling performance may then find that under some circumstances these effects are detectable (note that there was no support for an "overall" (i.e. main) effect of differential paternity in some of the studies that demonstrated context-dependence, cf. Fig. 1 in [START_REF] Schmoll | Paternal genetic effects on offspring fitness are context dependent within the extrapair mating system of a socially monogamous passerine[END_REF]. However, also studies that have shown differences in maternal half-sibling performance may benefit by allowing a deeper understanding when taking ecology into account, for example if an overall effect of differential paternity largely relates to particular sub-samples in the data sets (but see discussion on the risk of spurious findings above). At this stage, reporting a lack of evidence for context-dependence is also of considerable interest (given sample sizes are reasonably large), to avoid publication bias and obtain a balanced view on how common such effects may or may not be in natural populations.

As an example, in many bird populations in temperate regions, environmental conditions deteriorate predictably and substantially over the breeding season while laying and/or hatching date are recorded routinely in many studies. They could thus serve retrospectively as a proxy for ecological contexts of varying quality to compare the relative performance of maternal half-siblings against, even if more meaningful data like quantitative estimates of food abundance are not available. Similarly, available weather data combined with longterm data sets could identify key meteorological variables affecting fitness-related traits and these gradients of environmental variation could be used to test for interations with differential paternity.

Instead of considering laying or hatching order primarily a potentially confounding factor when analysing maternal half-sibling performance (see above), it could also be seen as reflecting yet another relevant dimension of the natural environment. There is evidence that hatching order directly affects early survival prospects in many altricial bird species through asymmetrical sibling competition, particularly when hatching asynchrony is pronounced [START_REF] Magrath | Hatching asynchrony in altricial birds[END_REF]; see also [START_REF] Magrath | Maternal Effects Contribute to the Superior Performance of Extra-Pair Offspring[END_REF]). Thus, once hatching sequence has been recorded in order to control its effects, testing for differential paternity-by-hatching sequence interactions may also represent a valuable option for testing the context-dependence hypothesis.

Different from the truly ecological variables discussed above, offspring sex may also be regarded a relevant dimension of environmental context. Fitness effects of particular alleles may be sex-dependent, for example due to sexually antagonistic selection resulting from intralocus sexual conflict [START_REF] Bonduriansky | Intralocus sexual conflict[END_REF]. Such sexually antagonistic fitness variation could render effects of paternal genetic contribution sex-dependent, too. Thus it may be worthwhile considering offspring sex as an (intragenomic) dimension of the environment within which paternal alleles are expressed, and test for differential paternity-by-offspring sex effects on fitness traits in both, observational and experimental approaches (see below). Of course, the magnitude of such effects may again depend on external dimensions of the environment, which may require testing for three-way interactions of differential paternity, offspring sex and quality of the environment. If the effects of differential paternity were indeed sex-dependent or even sexually antagonistic, it would be adaptive for females to adjust sex allocation in relation to paternity, because only then would they be able to obtain a (maximum) net genetic benefit from extra-pair mating. For example, male extra-pair offspring may inherit alleles from their attractive (i.e. extra-pair) fathers that will make themselves more attractive as extra-pair sires while the same alleles may negatively affect the fitness prospects and thus the reproductive value of female offspring. In general, there is good evidence for the ability of female birds to facultatively bias offspring sex ratio, for example in relation to the sexual attractiveness (e.g. [START_REF] Ellegren | Sex ratio adjustment in relation to paternal attractiveness in a wild bird population[END_REF] or the genetic compatibility (e.g. [START_REF] Pryke | Genetic Incompatibility Drives Sex Allocation and Maternal Investment in a Polymorphic Finch[END_REF] of their social mates. However, in the context of extra-pair mating behaviour, evidence for facultative sex allocation in relation to paternity is rather weak. Only a single study has unequivocally demonstrated the predicted sex ratio bias towards males in extra-pair offspring [START_REF] Johnson | Extra-pair young in house wren broods are more likely to be male than female[END_REF], while many others tried, but failed (e.g. [START_REF] Sheldon | Offspring sex and paternity in the collared flycatcher[END_REF][START_REF] Leech | No effect of parental quality or extrapair paternity on brood sex ratio in the blue tit (Parus caeruleus)[END_REF][START_REF] Dietrich-Bischoff | Extra-pair paternity, offspring mortality and offspring sex ratio in the socially monogamous coal tit (Parus ater)[END_REF]). Thus, if differential paternity was indeed sexually antagonistic in its effects, fitness gains realised via the advantaged sex would possibly have to exceed the fitness costs incurred by the disadvantaged sex to render extra-pair matings adaptive.

Most importantly, however, experimental approaches are needed that systematically manipulate relevant environmental contexts and subsequently test for differential paternity-bytreatment effects. If genetic benefits are assumed to be apparent more clearly under relatively poor conditions, the goal is provoking genetic effects by deteriorating environmental contexts to a degree that provides stressful conditions that could be encountered by the study population (at least from time to time), but that is still acceptable from an ethical point of view (and does also not lead to an overwhelmingly detrimental environmental effect which would also blur differences between genotypes). We may then predict a significant interaction between paternity and experimental treatment with differences between half-siblings being more pronounced under relatively poor conditions. Ideally, any treatment would create more than just two experimental environments to better understand the predicted differential reaction norm trajectories of offspring quality traits (which may not necessarily be linear as assumed in Fig. 1 for the sake of clarity). As expected effect sizes are small, such experiments need to be carried out on a larger scale that maximises the number replicates (i.e. broods with multiple paternity).

Given that differential maternal investment with respect to paternity may lead to the same type of context-dependent effects as predicted by the genetic benefit hypothesis, future studies need to control such effects in general and hatching order in particular.

In the field, brood size manipulation experiments are one straightforward option to manipulate the degree of sibling competition and thus resource availability. Cross-fostering designs may then easily be tailored to create a range of experimental environments of different quality. Furthermore, by separating offspring from their natural parents at an early stage through cross-fostering of entire (half-) sibships, potential post-hatching effects of maternal or paternal differential allocation in relation to paternity could be controlled. Manipulation of parasite prevalence and/or abundance or nest temperature regimes or enforcing replacement clutches while taking advantage of naturally deteriorating conditions in seasonal environments offer further opportunities depending on the biology of the focal species.

In order to unequivocally demonstrate genetic benefits of female mating preferences, it is necessary to allocate paternity randomly to a focal female's offspring, raise her progeny under identical environmental conditions and then test for differences in fitness between maternal half-sibships descending from preferred versus less preferred sires. This is comparatively easily achieved in laboratory studies of externally fertilising species by means of split-clutch in-vitro fertilisations (e.g. [START_REF] Barber | Indirect fitness consequences of mate choice in sticklebacks: offspring of brighter males grow slowly but resist parasitic infections[END_REF][START_REF] Welch | Genetic benefits of a female mating preference in gray tree frogs are context-dependent[END_REF]) and feasible to some degree within captive (semi-natural) populations of certain internally fertilising species (e.g. [START_REF] Petrie | Improved Growth and Survival of Offspring of Peacocks with More Elaborate Trains[END_REF]). However, the use of artificial insemination techniques in laboratory populations would offer interesting perspectives here, too. Such systems would not only exclude differential maternal investment, but also allow for a much better control and resolution of the experimental environments. One drawback here is that in the few established model species such as the zebra finch, EPP may play only a minor role in natural populations [START_REF] Griffith | Low level of extrapair parentage in wild zebra finches[END_REF].

As the magnitude of the genetic benefits a female may obtain from extra-pair mating is expected to be small (see above), comparatively large sample sizes will normally be required to demonstrate them. When analysing the fitness consequences of extra-pair mating, a replicate typically consists of a brood with multiple paternity and it may thus be challenging to achieve substantial samples, especially in species with relatively low frequencies of EPP. Properly controlling potentially confounding effects of differential maternal investment (see above) makes this an even more difficult enterprise. However, in species where EPO have been unequivocally shown to be more heterozygous than their WPO maternal half-siblings (see above), a shortcut may be testing for the context-dependence of the predicted heterozygosityfitness correlations in the study population/study species instead of testing the context-dependence of the fitness effects of differential paternity directly. Similar to context-dependent effects of (close) inbreeding discussed above, heterozygosity-fitness correlations may vary in their strength across environments of different quality, giving rise to heterozygosity-byenvironment interactions (for an interesting example in anurans see [START_REF] Lesbarrères | Environmental and population dependency of genetic variability-fitness correlations in Rana temporaria[END_REF]. This approach would not only allow making use of (much) larger samples by also including offspring from broods without multiple paternity (in observational as well as experimental studies). As we can expect substantial random variation in the degree of individual heterozygosity also within each of the two groups of maternal half-siblings (i.e. WPO and EPO), it might also provide a means to efficiently control differential maternal investment, because significant heterozygosity-fitness correlations as well as heterozygosity-byenvironment interactions within these groups cannot be confounded by differential maternal investment in relation to paternity.

Context-dependent genetic benefits imply that it would be adaptive to express costly mating preferences in a phenotypically plastic manner, depending on the expected net benefit within a given environment [START_REF] Qvarnström | Context-dependent genetic benefits from mate choice[END_REF][START_REF] Welch | Genetic benefits of a female mating preference in gray tree frogs are context-dependent[END_REF] and there are remarkable examples of adaptive phenotypic plasticity in female mate choice in birds (e.g. [START_REF] Qvarnström | Adaptive plasticity in mate preference linked to differences in reproductive effort[END_REF][START_REF] Chaine | Adaptive plasticity in female mate choice dampens sexual selection on male ornaments in the lark bunting[END_REF]. This flexibility presupposes the ability of females to evaluate the specific quality of the environment that their offspring are likely to experience, which should not pose a major problem in seasonal environments that may vary predictably in quality (see above). Assuming extra-pair matings to be costly, we may thus predict adaptive phenotypic plasticity in female propensity to engage in extra-pair matings. For example, in the Coal Tit population referred to above, the frequency of EPP was substantially higher in second broods (relatively stressful environment) compared to first broods (relatively beneficial environment, [START_REF] Dietrich | Pair identity -an important factor concerning variation in extra-pair paternity in the coal tit (Parus ater)[END_REF]). This pattern is compatible with a plastic female response in relation to differential environmental conditions, although other explanations may seem more likely (for example a better availability of extra-pair mating opportunities with preferred sires, [START_REF] Dietrich | Pair identity -an important factor concerning variation in extra-pair paternity in the coal tit (Parus ater)[END_REF]). Nevertheless, comparing the frequency of EPP against the magnitude of genetic benefits (i.e. the difference in half-sibling performance) across different environments (within as well as across populations) may represent a first step to elucidate this idea further.

If context-dependent genetic effects could be demonstrated in a robust and repeatable manner, it may be of interest to determine how frequently environmental contexts arise that allow females to realise a net genetic fitness benefit by mating extra-pair and thus maintain selection for this behaviour in the long run. Analysis of long-term data setswhere available -may help to evaluate this and could possibly be used to parameterise models that address the maintenance of mating preferences for extra-pair sires in heterogeneous environments. This article has focused on how variation in environmental quality may affect the phenotypic expression of the predicted, genetically based differences between EPO and WPO maternal half-siblings, assuming that genotype fitness ranks are maintained across environments (i.e. non-crossing reaction norms as illustrated in Fig. 1c). However, different environments as opposed to environments of different quality (for example in terms of the prevalence of different types of parasites) may also reflect relevant environmental heterogeneity and ecological cross-over of reaction norms in fitness-related traits has been demonstrated in other taxa (e.g. [START_REF] Jia | When are good genes good? Variable outcomes of female choice in wax moths[END_REF][START_REF] Welch | Genetic benefits of a female mating preference in gray tree frogs are context-dependent[END_REF][START_REF] Mills | Signal reliability compromised by genotype-by-environment interaction and potential mechanisms for its preservation[END_REF]) and may also be considered.

The idea of a context-dependence of genetic benefits of extra-pair matings may offer one possible approach for reconciling mixed results from different EPP studies and may thus contribute to resolving the longstanding puzzle why female birds mate extra-pair so frequently.

More generally, it may allow understanding under which environmental conditions selection will act to maintain a female mating bias towards extra-pair males. This has potentially farreaching implications for the ecology and evolution of female mating preferences, the maintenance of genetic variation in (sexually) selected traits and possibly the co-evolution of male sexual signals and female mating preferences. Taking into consideration that evidence from other taxa also suggests that context-dependence of genetic benefits of mate choice may be of considerable importance, I conclude that context-dependent genetic effects of mate choice in birds deserve more attention in general and in the context of extra-pair mating in particular.

Figure captions

Fig. 1 Fitness

 1 Fig. 1 Schematic representation of linear fitness reaction norms of two offspring genotypes (represented by lines) to illustrate potential effects of genotype-by-environment interactions on the genetic benefits of mate choice. a Genotypes show no phenotypically plastic response, the slopes of their reaction norms are identical and equal zero (parallel reaction norms, no genotype-by-environment interaction). b Genotypes show a phenotypically plastic response, the slopes of their reaction norms are identical, but different from zero (parallel reaction norms, no genotype-by-environment interaction). c Genotypes show a phenotypically plastic response, the slopes of their reaction norms are different, but reaction norms do not cross within the range of environmental conditions experienced (genotype-by-environment interaction with non-crossing reaction norms). d Genotypes show a phenotypically plastic response, the slopes of their reaction norms are different, and reaction norms cross within the range of environmental conditions experienced (genotype-by-environment interaction with crossing reaction norms, ecological cross-over). Relative genetic fitness benefits from preferring a male siring either one or the other offspring genotype are identical (a), (b), differ in magnitude (c), or may even be reversed (d) across environments.

Acknowledgments

Thanks to Klaus Reinhold, Peter Korsten and Verena Dietrich-Bischoff who commented on earlier drafts of this manuscript. Thomas Friedl and an anonymous reviewer also provided helpful comments.

Kontextabhängige genetische Effekte von Fremdkopulationen bei


MD Rafael Dal-Ré
email: rafael.dalre@urjc.es

Early -phase drugs and biologicals clinical trials on worldwide leading causes of death: a descriptive analysis

Keywords: Clinicaltrials.gov, drugs and biologicals, healthy volunteers, phase 1 and 2 clinical trials, worldwide mortality

To describe the global effort on currently "open" early-phase clinical trials with medicines on the major causes of mortality.

Methods

16 of the 20 leading causes of death were chosen; 9 of these were also amongst the top ten causes of death in low-income countries. Studies were identified from the Clinicaltrials.gov database to include phase 1 and/or 2 "Interventional""open" trials-i.e. recruiting or about to start recruitment-by Sponsorship -Industry, Universities and other organisations (UNO),and US Federal Agencies (NIH included)-,by gender and age groups, conducted with drugs and/or biologicals. The search was performed on March 2010 Results 2298 (824 phase 1; 1474 phase 2) trials were retrieved. 67% of all were on trachea, bronchus and lung cancers (25%),diabetes mellitus(15%),colon and rectum cancers (14%) and HIV/AIDS (12%). On the contrary, 4% were trials on diarrhoeal disease, nephrosis and nephritis, liver cirrhosis, and prematurity and low birth weight. UNO were the first source of funding. 52% of phase 1 non-cancer trials were on healthy volunteers. 29% of all trials were co-funded. Trials with drugs accounted for 4.6 times as much as those with biologicals. Only 7% were conducted with drugs and biologicals, the majority (78%) on cancers. No discrimination neither by gender nor by age group was observed.

Conclusions

More than 2/3 of early-phase trials were in 4 of the 16 diseases considered. Cancers were a top priority for all sponsors. Increasing attention should be given to conditions with current and projected global high mortality rates that had few "open" early phase trials.

In September 2004 the International Committee of Medical Journal Editors (ICMJE) published a new policy, to become effective in 2005, requiring investigators to register information about their trials in an accepted clinical trials registry before starting patient enrolment [START_REF] De Angelis | International Committee of Medical Journal Editors. Clinical trial registration: a statement from the International Committee of Medical Journal Editors[END_REF]. Soon after, under the World Health Organization (WHO) leadership, a multidisciplinary team agreed the minimum data set about design, conduct and administration of a study to be disclosed. Contrary to the initial ICMJE"s proposal, it was decided that all trials -including phase 1-must be registered before study start [START_REF] Gulmezoglu | WHO facilitates international collaboration in setting standards for clinical trial registration[END_REF][START_REF] Sim | Clinical trial registration: transparency is the watchword[END_REF][START_REF]International Clinical Trials Registry Platform[END_REF]. Data showed that the ICMJE policy dramatically impacted the number of trials registered on the largest registry, Clinicaltrials.gov (CTG) [5]. Thus, one month after the policy went into effect, the number of registered trials jumped from >13000 to close to 23000 [START_REF] Zarin | Trial registration at ClinTrials.gov between May and October 2005[END_REF]. In March 2010, close to 88000 studies were registered in CTG. The increasing registration of trials before starting recruiting subjects in CTG [5] and the publication of the WHO comprehensive report about the leading causes of death [START_REF]Global Burden of Disease[END_REF], made it possible to analyse what was the global effort on early-phase trials devoted to investigate new medicines or new approaches with drugs or biologicals to combat the major causes of mortality on a worldwide basis.

Methods

This analysis was conducted using Clinicaltrials.gov registered studies as of March 24 th , 2010. A careful assessment on the impact of the search fields in order to provide the optimal inclusion of the data for review was carried out. To fulfil the study objective, it was preferred to use the "open" (i.e. "recruiting", "not yet recruiting") rather than the "closed" (i.e. "active, but not recruiting") descriptor, and although studies that were "active, but not recruiting" anymore may be lost, it would avoid the inclusion of studies that have been "terminated" or "completed" some years back. So, the fields chosen were "study type: interventional", "recruitment: open"; "study results: all"; "conditions"; "interventions: drugs and/or biologicals"; "Gender"; "Age group"; "phase:1 and/or 2"; "funded: University/Organization (UNO), Industry (INY) and National Institutes of Health and Other US Federal Agency" (FED) merged together (Appendix A). In addition, "healthy volunteers" was used as a specific "search term". Although CTG has a validation system in place [START_REF] Zarin | Issues in the registration of clinical trials[END_REF], it relies ultimately on the accuracy of the data introduced by thousands of scientists, this being a source of inconsistencies and errors when filling-in the CTG fields. A review of inconsistencies was conducted when reviewing the data retrieved from CTG.

Sixteen of the 20 2004 leading causes of death, accounting for 58% of all worldwide mortality, were included in the analysis [START_REF]Global Burden of Disease[END_REF]. Since WHO and CTG may name diseases or conditions differently, among CTG"s "conditions" the one that could best match each of the WHO"s causes of death was chosen, using ICD10 as guidance [8] (table 1).

However, with ischaemic heart disease, hypertensive heart disease and cerebrovascular disease, more than one "condition" was chosen in order to capture the majority of studies (table 1). On the other hand, WHO"s diarrhoeal diseases is more restrictive that CTG"s diarrhoea; hence, the search was done excluding traveller diarrhoea, antibioticassociated, chemotherapy-induced, radiation, irritable bowel disease, IBS and cancer.

Among the 20 conditions, four were not included in this analysis: three are not suitable for experimental clinical research ("road traffic conditions", "self-inflicted injuries" and "birth asphyxia and birth trauma"), whereas the fourth one ("neonatal infections") had a complex definition -a category that following the WHO description also includes "other non-infectious causes arising in the perinatal period, that are responsible for 20% of deaths in this category" [START_REF]Global Burden of Disease[END_REF], that makes it impossible to match it with CTG conditions.

Results

In 2004 71 "open", "interventional", phase 1, studies with drugs and/or biologicals were registered on CTG, 14 on them on the 16 conditions considered; these figures were 150 and 17 for phase 2 trials, respectively. In 2010, the total "open", "interventional" phase 1 trials and those related to the 16 conditions have climbed to 4145 and 824, respectively; these figures were 6552 and 1474 for phase 2 trials, respectively (table 2). US centres were involved on the 56% of all phase 1 and/or 2 trials on the diseases considered.

Specific data on the number of both phase 1 or 2 trials, with regards to the 16 conditions, sponsorship, gender and age-groups accepted for inclusion are shown in tables 2-4. Sixty seven percent of all phases 1 and/or 2 trials were on trachea, bronchus and lung cancers (25%, 574/2298), diabetes mellitus (15%, 346/2298), colon and rectum cancers (14%, 333/2298) and HIV/AIDS (12%, 283/2298) (table 2). On the contrary, 4% were trials on diarrhoeal disease, nephrosis and nephritis, liver cirrhosis, and prematurity and low birth weight. Malaria was the only condition with more phase 1 than phase 2 trials. Trials with drugs accounted for 4.6 times as much as those with biologicals. Seven percent (161/2298) were conducted with drugs and biologicals, the majority (78%, 126/161) on cancers (table 2). Fifty two percent of phase 1 (non-cancer) trials were conducted on healthy volunteers; 36% of phase 1 trials, were labelled as phase 1 and 2 trials (table 2). UNO were the first source of funding (60%, 1378/2298), co-funding 46% (627/1378) of their sponsored trials. Twenty nine percent (674/2298) of all were co-funded trials (table 3).

Twelve percent (270/2298) of phase 1 and/or 2 trials accepted ≤17-year old subjects.

Respiratory tract infections, diabetes and HIV/AIDS accounted for 54% in both phase 1 (52/97) and 2 (93/173) trials accepting minors (table 4). Seniors were included in almost all diseases except on prematurity and low birth weight trials. Females and males were accepted for inclusion in both phase 1 and 2 trials in a balanced manner in all conditions (table 4).

Discussion

This study provides a snapshot of "open" early-phase clinical research conducted with drugs and/or biologicals in 16 of the 20 2004 worldwide major causes of death as of March 2010.

The most striking finding observed was not the fact that cancers (mainly trachea, bronchus and lung and colon and rectum neoplasms) were leading the interest of all sponsors, but the high percentage of effort devoted to them. Except HIV/AIDS and diabetes mellitus, which were highly interested single conditions for FED and INY, respectively, 45% trials were on cancers. Hence, the global effort on early cancer development trials is really remarkable. There were many medicines (and new combination regimens) in early-phase trials that hopefully will help future patients in high-and middle-income countries in which both trachea, bronchus and lung and stomach cancers are predicted to be among the 10 leading causes of mortality in 2030 [START_REF] Mathers | Projections of global mortality and burden of disease from 2002 to 2030[END_REF]. Of note is also the number of biologicals on early clinical development, expanding the approaches to the treatment of cancer patients. To this end, modern technology (e.g. cancer genomics and proteomics, circulating tumour cells) and the use of validated biomarkers are key to increase the success of cancer treatment [START_REF] Tan | Biomarkerdriven early clinical trials in oncology: a paradigm shift in drug development[END_REF].

On the other hand, there were a number of diseases with almost no interest to public and private sponsors. These included conditions not only prevalent in developing countries (e.g. diarrhoea, prematurity and low birth weight) but also in developed societies (e.g. COPD). It"s obvious that prevention and treatment of these conditions should mainly rely on changes on individual behavioural (e.g. smoking, diet, alcohol intake, physical activity) and community (e.g. water supply, sanitation) changes and this could explain, to some extent, the scarce number of trials initiated with drugs and biologicals.

Data suggests that overall participation of women and men in trials supporting the marketing authorization of new medicines in Europe [START_REF] Müllner | Are women appropriately represented and assessed in clinical trials submitted for marketing authorization? A review of the database of the European Medicines Agency[END_REF] and the US [START_REF] Yang | Participation of women in clinical trials for new drugs approved by the Food and Drug Administration in 2000-2002[END_REF] is comparable. However, significant under-representation of women in early-phase trials has been also observed -Fleish et al showed an overall proportion of only 37% of female participants in phase 1 and 2 trials results published in 2001 in three major Clinical Pharmacology journals [START_REF] Fleish | Women in early-phase clinical drug trials: Have things changed over the past 20 years?[END_REF]. Furthermore, more recent data showed that among 352 phase 1 trials of 30 new molecular entities submitted to the US Food and Drug Administration (FDA) in 2006-2007 for non-sex specific indications, only 31% of participants were women and that 34% of trials had only men volunteers [START_REF] Pinnow | Increasing participation of women in early phase clinical trials approved by the FDA[END_REF]. This analysis shows that there was not a gender bias with regards to early-phase clinical trials selection criteria. This being required, is not enough to ensure women are appropriately represented: female participants should be actively invited to participate in these early phase trials. With this respect, Pinnow et al [START_REF] Pinnow | Increasing participation of women in early phase clinical trials approved by the FDA[END_REF] data are encouraging: the number of trials that enrolled women and the number of women participating in phase 1 has increased over time since 2001, albeit women are still under-represented. Finally, it should be highlighted that this analysis shows no gender discrimination in both healthy volunteers and patient trials, the first step to prevent what was previously observed [START_REF] Fleish | Women in early-phase clinical drug trials: Have things changed over the past 20 years?[END_REF],

i.e. to have higher proportion of women participating in patient trials than in healthy volunteers ones .

This analysis shows that elderly people were not excluded from most of early development trials. In a 2005 editorial, McMurdo et al [START_REF] Mcmurdo | Including older people in clinical research. Benefits shown in trials in younger people may not apply to older people[END_REF] shared their concerns regarding the evidences of having older people excluded from clinical trials. This is more relevant for cancer trials than for the other conditions considered in this study, since volunteers in oncology trials have to be patients from the very beginning of clinical research. Trials submitted to the FDA supporting new cancer therapies [START_REF] Talarico | Enrollment of elderly patients in clinical trials for cancer drug registration: a 7-year experience by the US Food and Drug Administration[END_REF] showed that elderly was under-represented for all cancer treatment except for breast cancer hormonal therapies, despite the fact that seniors are willing to consider participation in cancer trials [START_REF] Townsley | Understanding the attitudes of the elderly towards enrolment into cancer clinical trials[END_REF]. This analysis shows that elderly people could participate in 99% of phase 1 and/or 2 cancer trials. However, what really matters is to actually have enough seniors participating in these trials, to better design phase 3 studies for this patient population.

Minors were accepted in only 12% of all phase 1 and/or 2 trials on drugs and biologicals. This figure could seemed low considering the recent legislative changes in both the US [START_REF]Best Pharmaceuticals for Children Act[END_REF] and the European Union [START_REF]the European Parliament and of the Council of 12 December 206 on medicinal products for paediatric use and amending Regulation (EEC) No 1768/92[END_REF] aiming to increase the development of medicines for use in the paediatric population. However, a number of factors could help to explain this, including that a number of conditions are not prevalent in this population (e.g ischaemic heart disease, cerebrovascular disease), and that it is prudent to gather enough data in adults before trying new medicines in children -although this should not postpone the conduct of appropriate studies. Trials should pursue the acquisition of data, but at the same time should not pose youngsters to unnecessary risks. The fact that 40% of phase 1 and/or 2 trials on malaria were on or accepted minors is encouraging.

In any case, many reports are being published pointing out specific challenges and needs (and approaches to address them) regarding the diseases considered in this analysis in the paediatric population [START_REF] Nader | Type 2 diabetes mellitus in children and adolescents: where do we stand with drug treatment and behavioral management?[END_REF][START_REF] Zwaan | Innovative therapies for children with cancer' European consortium. The role of the 'innovative therapies for children with cancer' (ITCC) European consortium[END_REF][START_REF] Swaminathan | Pediatric tuberculosis: global overview and challenges[END_REF][START_REF] Sohn | Sequencing of antiretroviral therapy in children in low-and middle-income countries[END_REF][START_REF] Theodoratou | The effect of case management on childhood pneumonia mortality in developing countries[END_REF].

How to label a given trial, as phase 1 or as phase 2, could be tricky. When a scientist is filling-in the CTG fields of a trial, he/she could use the rather simple (and short) definitions provided in CTG as guidance (Appendix A). However, scientists could follow more detailed definitions such as the ones provided by the ICH Tripartite Harmonised Guideline [START_REF]International Conference on Harmonisation of technical requirements for registration of pharmaceuticals for human use. ICH Harmonised Tripartite Guideline. General Considerations for clinical trials[END_REF], in which phase 1 are mostly human pharmacology studies whereas phase 2 are typically therapeutic exploratory trials. Excluding cancer trials, this analysis shows that among phase 1 studies there were very similar number of healthy volunteer and patient trials -furthermore, some studies had both healthy and patients subjects-, and that a remarkable number were labelled as phase 1 and 2 trials. Seems that, in practice, the borders between both phases are getting blurred to some extent.

Strengths and limitations

This analysis has the limitation of being based on the trials registered in only one data base (CTG), although this is the largest (the number of registered studies in CTG being more than 10 times as much as the second one, ISRCTN [START_REF]Current controlled trials[END_REF]) and the most international one available. Currently 38% of all "open" "interventional" phase 1 and/or 2 trials with drugs and/or biologicals have no US participation, this figure being 44% when considering only the 16 leading causes of mortality. A second limitation is the difficulty to assess how the "conditions" in CTG correspond exactly to the WHO"s "leading causes of death". In most instances, there was no problem in making this match. However, in diseases like ischemic heart disease and cerebrovascular disease, some trials could have been lost in the search performed in CTG. This, however, should not have impacted the overall results obtained, since to fulfil the aim of the analysis it was not necessary to include all the studies, but the majority.

Finally, another limitation could come from the inconsistencies and errors that scientists could have made when filling-in CTG fields. Thus, inconsistencies could be found in some cases such as, for instance, a phase 1 (NCT00492102) and a phase 2 (NCT00238056) trials on prematurity and low birth weight that accepted seniors. Few trials labelled as phase 1 could be more appropriate to be labelled as phase 2. Thus, for example, study NCT00783107, a randomised, double-blind, placebo-controlled trial aimed to "evaluate the safety and effectiveness of aerosol cyclosporine" in treating COPD patients, or study NCT00476918, a randomised, double-blind, high vs low triamcinolone acetonide dose, to assess the "visual acuity, number of treatments and duration of efficacy" for the treatment of diabetic macular edema, seemed more phase 2 studies than phase 1 ones. Although 136 trials on healthy volunteers were also labelled as phase 1, 122 other healthy volunteer trials were not labelled as such. Among these there were errors like three nephrosis or nephritis trials (NCT00298506, NCT00302536 and NCT00302523) that were labelled as admitting healthy volunteers as participants but were in fact opened only for patients; these three studies were conducted at the same University, two of them by the same study director. Also, occasionally there were errors derived from key words that could prevent a correct search. Thus, for instance, NCT00955344 is a study aiming to assess if a behavioural intervention increases physician rates of colorectal cancer recommendation -but appeared in this search that was focussed only on trials with drugs and/or biologicals. Again, acknowledging this has happened (and will happen in the future) the impact should be limited since the analysis included both phase 1 and 2 trials -and those healthy volunteer ones with no labelled phase. A different scenario would have been if only phase 1 trials were included in the study, due to the confusion that could arise between phase 1 and or 2 in labelling some studies.

It is worth mentioning that the analysis was based in 2004 leading causes of death, which will continue to be an important source of mortality and morbidity in the two decades to come. Thus, 11 of the diseases considered are among the 15 leading causes of mortality projected in 2030 [START_REF] Mathers | Projections of global mortality and burden of disease from 2002 to 2030[END_REF], whereas 5 of them are among the top 10 of the projected major causes of disability adjusted life years [START_REF] Mathers | Projections of global mortality and burden of disease from 2002 to 2030[END_REF]. It should be bore in mind the high burden of disability caused by mental diseases, albeit none of them were among the 20 leading causes of death [START_REF]Global Burden of Disease[END_REF]. With this respect, unipolar depressive disorders are projected to be the second worldwide cause of disability adjusted life years in 2030, and to be among the three top leading causes of disability in high-, middle-and low-income countries [START_REF] Mathers | Projections of global mortality and burden of disease from 2002 to 2030[END_REF].

The number of registered healthy volunteer studies seems rather low and was clustered in three conditions (diabetes, HIV/AIDS and respiratory tract infections) accounting for 71% of all trials. It"s difficult to assess the extent of "open" healthy volunteer studies that were not registered on CTG. Bioequivalence trials (aimed to gather data for generic products marketing authorization) are usually not registered since, most likely, sponsors are not interested in publishing the results in peer-reviewed journals. Even taken this into consideration, 256 healthy volunteers studies seems surprisingly low, with conditions like hypertensive heart disease and cerebrovascular disease with only 1 and4 "open" trials, respectively. Maybe the WHO [START_REF]International Clinical Trials Registry Platform[END_REF] request to register phase 1 trials has not yet been widely followed by researchers when dealing with healthy volunteer trials.

Conclusions

It is of concern to observe that more than 2/3 of currently "open" early-phase trials were in only 4 of the 16 leading causes of death considered in this analysis. Furthermore, 45% of all phase 1 and/or 2 were on cancers, being a top priority for all sponsors.

Although acknowledging that neoplasms will continue to be a leading cause of mortality in the years to come, and that behavioural individual (e.g. smoking, diet, physical activity) and community (e.g. sanitation) interventions have to play a major role in the prevention and management of the vast majority of these 16 diseases, seems rather disappointing to observe that early clinical research with new medicines, new combination regimens and new approaches with drugs and/or biologicals was fairly scarce in conditions afflicting millions of people globally. With this regard, it was surprising the relatively few "open" trials conducted on the three of the five major projected causes of death in 2030 in high-, middle-and low-income countries [START_REF] Mathers | Projections of global mortality and burden of disease from 2002 to 2030[END_REF], i. e. ischaemic heart disease, cerebrovascular disease and COPD. On the contrary, it was encouraging to observe the increasing effort against malaria, although there are yet many issues to be addressed [START_REF] Anon | Malaria 2010: more ambition and accountability please[END_REF].

"Other U.S. Federal Agency" -Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDC), Department of Veterans Affairs (VA), etc. "Industry" -the pharmaceutical companies "University/Organization" -all others, including community-based organizations (…) Table 1.-Sixteen of the 20 World Health Organisation"s "leading causes of death" [START_REF]Global Burden of Disease[END_REF] and corresponding "conditions" as per Clinicaltrials.gov terminologies, respectively Leading causes of death / (% of the total) [START_REF]Global Burden of Disease[END_REF]

  These are included in Total A; (b) Some trials admitted healthy volunteers and patients; (c) These are included in Total B; (d) Study accepting healthy volunteers and patients MIO: Ischaemic heart disease; CBD: Cerebrovascular disease; RTI: Lower respiratory infections; COP: Chronic obstructive pulmonary disease; DIA: Diarrhoeal diseases; HIV: HIV/AIDS; TBC: Tuberculosis; RTN: Trachea, bronchus, lung cancers; PLW: Prematurity and low birth weight; DBM: Diabetes mellitus; HFA: Heart failure; MAL: Malaria; LVC: Cirrhosis of the liver; N&N: nephritis and nephrosis; CRC: Colon and rectum cancers

Conditions (Clinicaltrials.gov)

	Ischaemic heart disease / (12.2) *	Myocardial ischemia, myocardial infarction (MIO)
	Cerebrovascular disease / (9.7) *	Cerebral infarction, stroke (CBD)
	Lower respiratory infections / (7.1) *	Respiratory tract infections (RTI)
	COPD / (5.1) *	COPD, Chronic obstructive pulmonary disease
		(COP)
	Diarrhoeal diseases / (3.7) *	Diarrhea (DIA)
	HIV/AIDS / (3.5) *	HIV/AIDS, Acquired immunodeficiency syndrome
		(HIV)
	Tuberculosis / (2.5) *	Tuberculosis (TBC)
	Trachea, bronchus, lung cancers / (2.3)	Respiratory tract neoplasms (RTN)
	Prematurity and low birth weight / (2.0) *	Prematurity, low birth weight (PLW)
	Diabetes mellitus / (1.9)	Diabetes mellitus (DBM)
	Hypertensive heart disease / (1.7)	Heart failure, Hypertensive heart disease (HFA)
	Malaria / (1.5) *	Malaria (MAL)
	Stomach cancer / (1.4)	Stomach neoplasms (STN)
	Cirrhosis of the liver / (1.3)	Liver cirrhosis (LVC)
	Nephritis and nephrosis / (1.3)	Nephritis, nephrosis (N&N)
	Colon and rectum cancers / (1,1)	Colorectal cancer (CRC)

* Among the 10 top causes of death in low-income countries COPD: Chronic obstructive pulmonary disease HIV: Human immunodeficiency virus AIDS: Acquired immunodeficiency syndrome

Table 2 .

 2 -Number (%) of phase 1 and 2 trials distributed by Clinicaltrials.gov"s "condition" corresponding to World Health Organisation"s "leading causes of death"[START_REF]Global Burden of Disease[END_REF]

	Trials	Trials	MIO	CBD	RTI	COP	DIA HIV	TBC RTN	PLW DBM	HFA MAL	STN	LVC N&N	CRC	Total
	Phase 1 Total A	25

Table 3 .

 3 -Number (%) of phase 1 and 2 trials distributed by Clinicaltrials.gov"s "condition" corresponding to World Health Organisation"s

	"leading causes of death" [7] and by funding source
			MIO CBD RTI	COP DIA HIV	TBC RTN	PLW DBM HFA MAL STN	LVC N&N CRC Total*
	Phase 1	No of trials 25	19	62	12	5	115	17	184	12	153	26	27	31	14	6	116	824
			(3.0)	(2.3)	(7.5)	(1.5)	(0.6)	(14.0)	(2.0)	(22.3)	(1.5)	(18.6)	(3.2)	(3.3)	(3.8)	(1.7)	(0.7)	(14.1)
	Funding FED	3	3	20	2	3	56	6	46	3	27	8	10	8	3	0	25	223
			(1.4)	(1.4)	(9.0)	(0.9)	(1.4)	(25.1)	(2.7)	(20.6)	(1.4)	(12.1)	(3.6)	(4.5)	(3.6)	(1.4)		(11.2)
		INY	6	5	19	7	0	28
			(1.8)	(1.5)	(5.8)	(2.2)		(

Table 4 .

 4 -Number (%) of phase 1 and 2 trials distributed by Clinicaltrials.gov"s "condition" corresponding to World Health Organisation"s "leading causes of death"[START_REF]Global Burden of Disease[END_REF] by age groups and gender Total No of different studies by "condition". Since many studies included different age groups and genders, the sum of the studies by each age group and gender is greater than the total number of studies.

		38	225
	(0.9)	(16.9)

* Total No of different studies by "condition". Since some studies were funded by more than one "funding source", the sum of the studies funded by each source is greater than the total number of studies. MIO: Ischaemic heart disease; CBD: Cerebrovascular disease; RTI: Lower respiratory infections; COP: Chronic obstructive pulmonary disease; DIA: Diarrhoeal diseases; HIV: HIV/AIDS; TBC: Tuberculosis; RTN: Trachea, bronchus, lung cancers; PLW: Prematurity and low birth weight; DBM: Diabetes mellitus; HFA: Heart failure; MAL: Malaria; LVC: Cirrhosis of the liver; N&N: nephritis and nephrosis; CRC: Colon and rectum cancers FED: National Institutes of Health and Other US Federal Agency; INY: Industry; UNO: University/Organization *MIO: Ischaemic heart disease; CBD: Cerebrovascular disease; RTI: Lower respiratory infections; COP: Chronic obstructive pulmonary disease; DIA: Diarrhoeal diseases; HIV: HIV/AIDS; TBC: Tuberculosis; RTN: Trachea, bronchus, lung cancers; PLW: Prematurity and low birth weight; DBM: Diabetes mellitus; HFA: Heart failure; MAL: Malaria; LVC: Cirrhosis of the liver; N&N: nephritis and nephrosis; CRC: Colon and rectum cancers

Competing interests

The author declares having no conflict of interests

Observational: Studies without an intervention. (…)

Expanded Access: Records describing the procedure for obtaining an experimental drug or device for patients who are failing on currently available treatments for their condition and also are unable to participate in ongoing clinical trials. (…)

Conditions.

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Studies are supported by one or more organizations called sponsors. Support includes contributions of facilities, expertise, and/or financial resources. All sponsors in the database have been assigned one of the following organization types: "National Institutes of Health" (NIH)