Patrizia Andrea Chiesa

Enrica Cavedo

Simone Lista

Paul M Thompson

Harald Hampel

Revolution of Resting-State Functional Neuroimaging Genetics in Alzheimer's Disease

Keywords: Alzheimer's disease, neuroimaging genetics, functional connectivity, genetic risk, precision medicine

The quest to comprehend genetic, biological, and symptomatic heterogeneity underlying Alzheimer's disease (AD) requires a deep understanding of mechanisms affecting complex brain systems. Neuroimaging genetics is an emerging field that provides a powerful way to analyze and characterize intermediate biological phenotypes of AD. Here, we describe recent studies showing the differential effect of genetic risk factors for AD on brain functional connectivity in cognitively normal, preclinical, prodromal and AD dementia individuals. Functional neuroimaging genetics holds particular promise for the characterization of preclinical populations, a target population for disease prevention and modification trials. To this end, we emphasize the need for a paradigm shift towards integrative disease modeling and neuroimaging biomarker-guided precision medicine for AD and other neurodegenerative diseases.

Pathophysiology, Genetics and Functional Brain Processing Underlying Alzheimer's Disease

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease and commonest type of dementia in people over age 65. Despite enormous efforts in global biomedical research and development, the number of affected individuals with AD is dramatically increasing [START_REF] Reitz | Epidemiology of Alzheimer disease[END_REF]. Therefore, effective prevention and disease-modifying therapies are needed to reduce the future global burden of neurodegenerative diseases and dementia [START_REF] Hampel | Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives[END_REF][START_REF] Hampel | Development of biomarkers to chart all Alzheimer's disease stages: The royal road to cutting the therapeutic Gordian Knot[END_REF]. The genetic, biological, and symptomatic heterogeneity underlying the spectrum of AD clinical phenotypes as well as the complex non-linear progression of the pathophysiological mechanisms are key factors for a decade of failure of AD clinical trials. Once late stage clinical symptoms appear, the disease shows extensive, advanced, and potentially irreversible neuropathological alterations -such as inflammatory changes, neuritic plaques (also called senile plaques) and neurofibrillary tangles [START_REF] Braak | Neuropathological stageing of Alzheimerrelated changes[END_REF] (see Glossary). An emerging exploration of the long and largely uncharted preclinical stages of AD has begun [START_REF] Dubois | Preclinical Alzheimer's disease: Definition, natural history, and diagnostic criteria[END_REF].

To date, the amyloid cascade theory is the prevailing hypothesis on the pathogenesis of AD [START_REF] Hardy | Alzheimer's Disease: The Amyloid Cascade Hypothesis[END_REF]. It postulates that brain β-amyloid (Aβ) accumulation is the primary mechanistic event, or key pathophysiological threshold, impairing synaptic function, later inducing neuronal damage, and finally leading to widespread neurodegeneration and clinical dementia [START_REF] Blennow | Amyloid biomarkers in Alzheimer's disease[END_REF]. The detrimental impact of Aβ is assumed to emerge at the system level, as brain functional and structural connections are progressively disrupted (for review see [START_REF] Pievani | Brain connectivity in neurodegenerative diseasesfrom phenotype to proteinopathy[END_REF]). Moreover, clinical decline has been associated with alterations in both structural and functional brain connectivity, causing abnormal brain integration [START_REF] Matthews | Brain Structural and Functional Connectivity and the Progression of Neuropathology in Alzheimer's Disease[END_REF]. Therefore, AD may be considered a complex brain systems disconnection syndrome [START_REF] Brier | Network dysfunction in Alzheimer's disease: refining the disconnection hypothesis[END_REF]. However, it is still unclear which factors induce such disconnection. So far, it is largely accepted that axonal and synaptic contacts can spread dysfunction from a local site through mechanisms of diaschisis and transneuronal degeneration [START_REF] Fornito | The connectomics of brain disorders[END_REF], generating pathophysiological cascades [START_REF] Klupp | In Alzheimer's Disease, Hypometabolism in Low-Amyloid Brain Regions May Be a Functional Consequence of Pathologies in Connected Brain Regions[END_REF] and, consequently, propagating the disease processes [START_REF] Wu | Neuronal activity enhances tau propagation and tau pathology in vivo[END_REF]. In addition, it is possible that brain regions affected by pathophysiological events respond with compensatory mechanisms such as increased activity or functional connectivity, owing to excess neuronal stimulation, and leading to cell damage or death in functionally connected brain sites [START_REF] Wu | Neuronal activity enhances tau propagation and tau pathology in vivo[END_REF]. Finally, according to evidence derived from studies with AD transgenic mouse models [START_REF] Smith | In vivo axonal transport rates decrease in a mouse model of Alzheimer's disease[END_REF], abnormal neural connectivity could arise from the slowing or interruption of the fast axonal transport, which occurs before Aβ plaques formation [START_REF] Smith | In vivo axonal transport rates decrease in a mouse model of Alzheimer's disease[END_REF] and potentially contributes to transneuronal degeneration [START_REF] Bartzokis | Alzheimer's disease as homeostatic responses to agerelated myelin breakdown[END_REF].

Resting state functional MRI (rs-fMRI) studies, which assess functional synchrony in brain networks using fMRI, provide numerous findings highlighting the deep reshaping of a number of functional connectivity networks at each stage of the full clinical ADspectrum [START_REF] Celone | Alterations in memory networks in mild cognitive impairment and Alzheimer's disease: an independent component analysis[END_REF][START_REF] Buckner | Cortical Hubs Revealed by Intrinsic Functional Connectivity: Mapping, Assessment of Stability, and Relation to Alzheimer's Disease[END_REF][START_REF] Damoiseaux | Functional connectivity tracks clinical deterioration in Alzheimer's disease[END_REF][19], from preclinical to prodromal to AD dementia (Box 1). These changes can occur even in the absence of cognitive impairments or structural neurodegeneration [START_REF] Thomas | Functional Connectivity in Autosomal Dominant and Late-Onset Alzheimer Disease[END_REF]. Although other networks have also been implicated, a recent review [START_REF] Pievani | Brain connectivity in neurodegenerative diseasesfrom phenotype to proteinopathy[END_REF] reported consistently decreased functional connectivity in the Default mode network (DMN) in the full clinical AD-spectrum, including the posterior cingulate cortex (PCC), precuneus (Pcu), lateral temporoparietal cortex, and the medial temporal lobes (MTL) [START_REF] Raichle | The Brain's Default Mode Network[END_REF]. The MTL is considered the most prominent candidate brain region for initial histopathological changes in AD [START_REF] Braak | Neuropathological stageing of Alzheimerrelated changes[END_REF], but the PCC is consistently recognized as one of the earliest sites showing hypometabolism and hypoperfusion [START_REF] Jones | Cascading network failure across the Alzheimer's disease spectrum[END_REF][START_REF] Wang | Correspondence between Resting-State Activity and Brain Gene Expression[END_REF]. Disrupted connectivity between the hippocampus/entorhinal cortex and PCC may perhaps constitute the first neural change in AD pathophysiology [START_REF] Greicius | Default-Mode Network Activity Distinguishes Alzheimer's Disease from Healthy Aging: Evidence from Functional MRI[END_REF].

The genetic makeup has the potential to significantly and differentially modulate functional brain connectivity in normal aging and may directly interact with disease effects [START_REF] Richiardi | Correlated gene expression supports synchronous activity in brain networks[END_REF] (Box 2). Mutations in the amyloid precursor protein (APP), presenilin 1 (PSEN1) or 2 (PSEN2) genes cause early-onset AD dementia, at an unusually early age (around 30-50 years). In contrast, the risk of developing late-onset AD seems to be associated with allelic variations in Apolipoprotein E (APOE), phosphatidylinositol binding clathrin assembly protein (PICALM), clusterin (CLU), and bridging integrator 1 (BIN1) genes. Consequently, these have become the most heavily investigated in functional neuroimaging genetics studies of AD [START_REF] Karch | Alzheimer's disease risk genes and mechanisms of disease pathogenesis[END_REF].

Genetic studies of AD have also attempted to integrate multimodal biomarkers to better characterize and stratify populations at risk of developing AD [START_REF] Hampel | Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives[END_REF]. In this regard, neuroimaging genetics might offer an efficient strategy for characterizing intermediate phenotypes of AD, helping bridge the unexplored biological gap between the cell-level molecular changes and systems-level changes in cognition and behavior. Not surprisingly, several research groups have started to explore the neural underpinnings of genotype-dependent differences in AD.

In the present review, we describe the impact of well-known genetic risk factors of AD on brain functional connectivity alterations in the whole AD-spectrum, and critically discuss the key advantages of investigating functional neuroimaging genetics in AD. In particular, we present studies attempting to develop multimodal markers to detect and predict AD [27]. Indeed, to determine when and how brain functional connectivity begins to diverge from expected age-specific norms in individuals with different genetic profiles at risk for AD might be of great value both for the early AD detection and stratification of target populations in clinical trials. These metrics are assumed to be critical for developing and evaluating clinical interventions, to slow or even prevent cognitive decline. This review is focused on addressing new insights in the study of functional brain dysfunction in individuals with genetic susceptibility to AD (Box 3), since extensive literature on AD genetics [START_REF] Karch | Alzheimer's disease risk genes and mechanisms of disease pathogenesis[END_REF][START_REF] Bettens | Genetic insights in Alzheimer's disease[END_REF] and biomarkers [START_REF] Lista | Biomarkers in Sporadic and Familial Alzheimer's Disease[END_REF] is comprehensively reviewed elsewhere. We provide here a critical overview of recent studies that have addressed the role of AD-related genes in the functional connectivity at rest. In particular, we discuss how autosomal dominant genes APP, PSEN1 and PSEN2, and the major genome wide associated gene risk variants for AD, i.e. APOE, PICALM, CLU, and BIN1, impact resting state functional connectivity in: (a) cognitively normal (CN), (b) preclinical AD individuals (including both asymptomatic at risk for AD and presymptomatic diagnostic categories), and (c) AD dementia patients. This review is restricted to addressing recent advances in examining the genetic impact on the functionally interacting and integrative networks at rest, which provide new insights on large-scale neuronal communication in the human brain.

a) Cognitively normal individuals at genetic risk for AD

Elucidating the neural changes in CN at genetic risk for AD is supposed to provide several advantages: i) different functional brain patterns in mutation carriers may be identified independently from the disease, ii) compared to patients, CN individuals can easily perform tasks, making it possible to explore the effective connectivity related to specific cognitive tasks, iii) the effect of genetic risk variants on brain network functioning can be examined in absence of confounding factors, e.g. illness or medications, iv) all genetic variant profiles are included in the sample, and, finally, v) longitudinal follow-up on CN individuals at increased risk for AD would make it possible to test forms of prevention, trace pathophysiological trajectories from health to dementia, and identify an effective therapeutic window for early preclinical stages of AD.

Here, we present data across the lifespan, from childhood to old age, to point out potential temporal trajectories in CN individuals carrying genetic mutations associated with AD (figure 1) [START_REF] Karch | Alzheimer's disease risk genes and mechanisms of disease pathogenesis[END_REF].

Given the central role of the hippocampus in AD neurodegeneration [START_REF] Beason-Held | Dementia and the Default Mode[END_REF], considerable effort has been devoted to study its possible functional connectivity alterations early in life in CN at genetic risk for AD. The influence of the innate genetic patterns on hippocampal connectivity was reported in young individuals [START_REF] Erk | Hippocampal function in healthy carriers of the CLU Alzheimer's disease risk variant[END_REF][START_REF] Zhang | Impacts of PICALM and CLU variants associated with Alzheimer's disease on the functional connectivity of the hippocampus in healthy young adults[END_REF][START_REF] Zhang | Bridging Integrator 1 (BIN1) Genotype Effects on Working Memory, Hippocampal Volume, and Functional Connectivity in Young Healthy Individuals[END_REF], although results partially disagree. On one hand, carriers of the G-homozygote mutation in BIN1 [START_REF] Zhang | Bridging Integrator 1 (BIN1) Genotype Effects on Working Memory, Hippocampal Volume, and Functional Connectivity in Young Healthy Individuals[END_REF],

and the C allele polymorphism in CLU [START_REF] Erk | Hippocampal function in healthy carriers of the CLU Alzheimer's disease risk variant[END_REF] both showed decreased hippocampaldorsolateral prefrontal cortex (dlPFC) connectivity, while individuals carrying the PICALM risk genotype (G-allele) showed reduced strength connectivity between the hippocampus and both the Pcu and the superior frontal gyrus [START_REF] Zhang | Impacts of PICALM and CLU variants associated with Alzheimer's disease on the functional connectivity of the hippocampus in healthy young adults[END_REF]. On the other hand, increased hippocampal connectivity with widespread DMN regions was found in young CLU-C [START_REF] Zhang | Impacts of PICALM and CLU variants associated with Alzheimer's disease on the functional connectivity of the hippocampus in healthy young adults[END_REF] and APOE ε4 carriers [START_REF] Filippini | Distinct patterns of brain activity in young carriers of the APOE-4 allele[END_REF][START_REF] Westlye | Increased hippocampal default mode synchronization during rest in middle-aged and elderly APOE ε4 carriers: relationships with memory performance[END_REF]. Such hippocampal hyperconnectivity was assumed to reflect a compensatory brain response to decreased white matter connections [START_REF] Braskie | Common Alzheimer's Disease Risk Variant Within the CLU Gene Affects White Matter Microstructure in Young Adults[END_REF][START_REF] Kohannim | Predicting White Matter Integrity from Multiple Common Genetic Variants[END_REF] and may predict future cognitive decline [38][START_REF] Sweet | Effect of Alzheimer's Disease Risk Genes on Trajectories of Cognitive Function in the Cardiovascular Health Study[END_REF][40]. As hippocampal subfields exhibit specific functional connections [START_REF] Strange | Functional organization of the hippocampal longitudinal axis[END_REF][START_REF] Fanselow | Are the Dorsal and Ventral Hippocampus Functionally Distinct Structures?[END_REF], considering the entire hippocampus may be a major methodological limitation of the above studies. In this regard, Trachtenberg and colleague [43] reported differences in the anterior (AHN) and posterior (PHN) hippocampal network. Hippocampal subfields exhibit specific functional connections, and in line with this, the APOE ε4 genotype more severely affects the connectivity of the AHN rather than the PHN [43]. In particular, the APOE ε4 genotype may more severely affect the connectivity of the anterior (AHN) rather than posterior (PHN) hippocampal network [43]. In line with this remark, a variety of parietal and frontal regions -and the basal ganglia -displayed increased connectivity with the AHN and decreased connectivity with the PHN in young CN APOE ε4 carriers. This pattern was recently replicated during memory tasks in a fMRI study with a sample of middle-aged individuals (mean age 65 years) [START_REF] Harrison | Altered memory-related functional connectivity of the anterior and posterior hippocampus in older adults at increased genetic risk for Alzheimer's disease[END_REF]. Interestingly, only individuals from older adult communities, care centers, and memory clinic groups were included, to increase the chance of recruiting participants with age-related memory concerns and with an increased likelihood of at least one copy of the APOE ε4 allele. There may also be an APOE ε4 x gender interaction on the DMN [START_REF] Heise | Apolipoprotein E genotype, gender and age modulate connectivity of the hippocampus in healthy adults[END_REF]46]. Compared to males, female APOE ε4 carriers exhibited reduced functional connectivity of the hippocampus with the posterior regions of DMN (Pcu and PCC) [START_REF] Heise | Apolipoprotein E genotype, gender and age modulate connectivity of the hippocampus in healthy adults[END_REF]. Further testing revealed a significant interaction between APOE genotype and sex in the precuneus, a major DMN hub [START_REF] Heise | Apolipoprotein E genotype, gender and age modulate connectivity of the hippocampus in healthy adults[END_REF]46]. The study by Damoiseaux and colleagues revealed lower DMN connectivity in female ε4 carriers compared to either female ε3 homozygotes or male ε4 carriers, whereas males carrying the ε4 phenotype were marginally different from ε3 homozygotes males [46].

After extending the analyses of functional brain connectivity at-rest in CN middle-aged APOE ε4 carriers to different areas of the DMN, a highly consistent pattern emerged.

On one hand, decreased DMN connectivity was detected in the PCC/Pcu and orbital frontal cortex [START_REF] Fleisher | Resting-state BOLD networks versus taskassociated functional MRI for distinguishing Alzheimer's disease risk groups[END_REF][START_REF] Patel | Default mode network activity and white matter integrity in healthy middle-aged ApoE4 carriers[END_REF]; on the other hand, increased DMN connectivity was found in MTL and PFC structures [START_REF] Fleisher | Resting-state BOLD networks versus taskassociated functional MRI for distinguishing Alzheimer's disease risk groups[END_REF][START_REF] Patel | Default mode network activity and white matter integrity in healthy middle-aged ApoE4 carriers[END_REF]. Almost overlapping results were observed in elderly APOE ε4 carriers [49-53], even before the onset of brain amyloid accumulation processes [START_REF] Thomas | Functional Connectivity in Autosomal Dominant and Late-Onset Alzheimer Disease[END_REF][START_REF] Patel | Default mode network activity and white matter integrity in healthy middle-aged ApoE4 carriers[END_REF].

Nevertheless, the inclusion of both middle-aged adults and elderly in the same sample generated conflicting results: both decreased [START_REF] Chen | Disrupted Functional and Structural Networks in Cognitively Normal Elderly Subjects with the APOE ɛ4 Allele[END_REF] and increased [START_REF] Westlye | Increased hippocampal default mode synchronization during rest in middle-aged and elderly APOE ε4 carriers: relationships with memory performance[END_REF][START_REF] Matura | Recognition memory is associated with altered restingstate functional connectivity in people at genetic risk for Alzheimer's disease[END_REF] connectivity were found in a number of DMN nodes, including MTL, PCC, and Pcu.

The fact that both decreased and increased functional connectivity were found at rest might be due to differences in methods and analyses, such as the choice of seed ROI derived from an event-related fMRI task [START_REF] Matura | Recognition memory is associated with altered restingstate functional connectivity in people at genetic risk for Alzheimer's disease[END_REF], ICA [START_REF] Westlye | Increased hippocampal default mode synchronization during rest in middle-aged and elderly APOE ε4 carriers: relationships with memory performance[END_REF], or graph measures [START_REF] Chen | Disrupted Functional and Structural Networks in Cognitively Normal Elderly Subjects with the APOE ɛ4 Allele[END_REF]. Further investigations are needed to clarify these discrepancies.

It should be highlighted that, as age increased, ε2 carriers presented a grown DMN functional connectivity, while this was decreased in ε4 carriers [54]. This finding corroborates the hypothesis of antagonistic pleiotropic properties of the APOE ε4 allele, stating that APOE ε4 carriers may enjoy some cognitive benefits during early life, but exhibit impaired brain function in late adulthood [START_REF] Tuminello | The Apolipoprotein E Antagonistic Pleiotropy Hypothesis: Review and Recommendations[END_REF].

Further analyses revealed that differences in individuals carrying the APOE ε4 allele are not only limited to the DMN. Young adult APOE ε4 carriers showed increased functional connectivity in the sensorimotor network [START_REF] Filippini | Distinct patterns of brain activity in young carriers of the APOE-4 allele[END_REF] and decreased connectivity between the auditory network and several other brain regions in the frontal, temporal, and parietal cortices, as well as in the basal ganglia [43]. Furthermore, elderly APOE ε4 carriers displayed increased connectivity in the salience network, which is comprised of the dorsal anterior cingulate cortex (dACC), the frontoinsular cortices and subcortical and limbic regions [49,[START_REF] Liang | Frequency Specific Effects of ApoE 4 Allele on Resting-State Networks in Nondemented Elders[END_REF]. Again, a number of additional brain regions, not typically involved in AD, such as the dorsal occipital cortex and the fronto-parietal operculum, showed differences in functional connectivity in CN APOE ε4 carriers compared to noncarriers [START_REF] Sheline | APOE4 allele disrupts resting state fMRI connectivity in the absence of amyloid plaques or decreased CSF Aβ42[END_REF]. The dissimilarities previously described may reflect supplementary effects -either genetically mediated during brain neurodevelopment -or caused by an early low degree of amyloid deposition not yet detectable by PET scanning. Indeed, recent studies demonstrated significant associations between Tau PET uptake or tau protein concentrations in CSF and alterations in functional connectivity [START_REF] Schultz | Phases of Hyperconnectivity and Hypoconnectivity in the Default Mode and Salience Networks Track with Amyloid and Tau in Clinically Normal Individuals[END_REF][START_REF] Sepulcre | Tau and amyloid-β proteins distinctively associate to functional network changes in the aging brain[END_REF]. Therefore, investigation of inter-systems dynamics is warrented, such as the interplay of the genetic, molecular, and functional associations is warranted.

In conclusion, existing evidence described early detectable brain functional connectivity patterns in CN individuals carrying BIN1, CLU-C, PICALM, and APOE genetic polymorphisms that highly correlate with the functional imaging markers found in AD.

In particular, neural changes detected in young carriers may trigger late life functional differences. i.e. APP, PSEN1, or PSEN2 mutations, are defined as "presymptomatic AD", as they inevitably develop neurodegenerative signs [START_REF] Dubois | Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria[END_REF].

Functional brain connectivity in subjects with PSEN1 mutations was recently investigated in children (9-17 years old) with altered blood-based and brain imaging biomarkers. Notably, they showed an increased brain activity in parietal regions during a memory tasks and increased rs-fMRI functional connectivity between PCC and MTL regions [START_REF] Quiroz | Brain Imaging and Blood Biomarker Abnormalities in Children With Autosomal Dominant Alzheimer Disease[END_REF]. Accordingly, young (18-30 years old, [START_REF] Su | Lower functional connectivity of default mode network in cognitively normal young adults with mutation of APP, presenilins and APOE ε4[END_REF]) and middle-aged presymptomatic individuals (mean age 45 years [START_REF] Chhatwal | Impaired Default Network Functional Connectivity in Autosomal Dominant Alzheimer's Disease: Findings from the DIAN Study[END_REF][START_REF] Sala-Llonch | Evolving brain functional abnormalities in psen1 mutation carriers: A resting and visual encoding FMRI study[END_REF][START_REF] Li | The Effects of Gene Mutations on Default Mode Network in Familial Alzheimer's Disease[END_REF]) displayed lower intrinsic connectivity in posterior [START_REF] Su | Lower functional connectivity of default mode network in cognitively normal young adults with mutation of APP, presenilins and APOE ε4[END_REF][START_REF] Chhatwal | Impaired Default Network Functional Connectivity in Autosomal Dominant Alzheimer's Disease: Findings from the DIAN Study[END_REF][START_REF] Sala-Llonch | Evolving brain functional abnormalities in psen1 mutation carriers: A resting and visual encoding FMRI study[END_REF][START_REF] Li | The Effects of Gene Mutations on Default Mode Network in Familial Alzheimer's Disease[END_REF], and temporal [START_REF] Sala-Llonch | Evolving brain functional abnormalities in psen1 mutation carriers: A resting and visual encoding FMRI study[END_REF] nodes of the DMN compared with controls. Significant correlations were observed between rs-fMRI measures (Z-scores) and CSF Aß42, P-tau181p, and T-tau protein concentrations [START_REF] Li | The Effects of Gene Mutations on Default Mode Network in Familial Alzheimer's Disease[END_REF]. Alterations in young and middle-age adults were also observed in frontal regions; however, results are still debated because of decreased [START_REF] Su | Lower functional connectivity of default mode network in cognitively normal young adults with mutation of APP, presenilins and APOE ε4[END_REF] as well as increased DMN connectivity [START_REF] Sala-Llonch | Evolving brain functional abnormalities in psen1 mutation carriers: A resting and visual encoding FMRI study[END_REF] results.

The heterogeneity of evidence in presymptomatic adults might indicate that there is no simple interpretation of autosomal dominant-related changes in resting state functional connectivity. Explanations for such findings may include i) compensatory responses related to individual cognitive reserves, ii) aging-related developmental modifications in the brain networks architecture, independent of the genetic pattern, iii) the interaction with other genes, and iv) neurotransmitter failure, v) differential impact on brain function of the different Mendelian AD mutations on brain function.

Overall, these data indicate the presence of a relevant genetic impact on functional connectivity due to APP or PSEN1/2 mutations. Interestingly, reduced DMN functional connectivity, as detected in individuals carrying autosomal dominant mutations, does not differ from the one observed in APOE ε4 carriers [START_REF] Su | Lower functional connectivity of default mode network in cognitively normal young adults with mutation of APP, presenilins and APOE ε4[END_REF].

Overall, findings in presymptomatic AD individuals suggest that abnormalities in resting-state networks potentially represent a valuable biomarker to detect early preclinical stages of AD (Figure 2).

To the extent of the existing knowledge, the influence of genetics on the functional architecture in the "asymptomatic at-risk state for AD" [START_REF] Dubois | Advancing research diagnostic criteria for Alzheimer's disease: the IWG-2 criteria[END_REF], i.e., CN individuals showing positivity to AD pathophysiological markers, has yet not been examined.

This figure is a derivative of the work created by Vivid Apps and AXS Biomedical Animation Studio for Cold Spring Harbor Laboratory DNA Learning Center (https://www.dnalc.org/resources/3dbrain.html) c) Patients with AD dementia

To date, no published studies have identified effects of specific genotypes on functional connectivity patterns in patients with prodromal-AD [START_REF] Matura | Recognition memory is associated with altered restingstate functional connectivity in people at genetic risk for Alzheimer's disease[END_REF] or with mild cognitive impairment (MCI) due to AD [START_REF] Sepulcre | Tau and amyloid-β proteins distinctively associate to functional network changes in the aging brain[END_REF], i.e. MCI individuals with a positive core biomarker signature positive, who have a high likelihood of progressing to AD dementia within a few years.

The substantial effects of the APOE ε4 allele on the intrinsic functional architecture have been reported in patients with AD dementia (Figure 3). Specifically, AD demented APOE ε4 carriers exhibited a selective weakness in both intra-and inter-network integration that predominantly resided in the posterior part of the DMN [START_REF] Jones | Cascading network failure across the Alzheimer's disease spectrum[END_REF][START_REF] Wang | Correspondence between Resting-State Activity and Brain Gene Expression[END_REF] and in the executive control network [START_REF] Wang | Correspondence between Resting-State Activity and Brain Gene Expression[END_REF]. However, significant results of APOE ε4 effect on the DMN were not consistently reported [START_REF] Jones | Cascading network failure across the Alzheimer's disease spectrum[END_REF][START_REF] Koch | Diagnostic power of default mode network resting state fMRI in the detection of Alzheimer's disease[END_REF][START_REF] Zhao | Disrupted Small-World Brain Networks in Moderate Alzheimer's Disease: A Resting-State fMRI Study[END_REF]. This gap may originate from the high degree of sporadic AD complexity and heterogeneity, which potentially may involve different biological and neurophysiological systems at different levels. For instance, familial autosomal dominant AD individuals with PSEN1 mutations have shown strong decreased frontal connectivity; in contrast, results observed in posterior networks were unclear [START_REF] Chhatwal | Impaired Default Network Functional Connectivity in Autosomal Dominant Alzheimer's Disease: Findings from the DIAN Study[END_REF][START_REF] Sala-Llonch | Evolving brain functional abnormalities in psen1 mutation carriers: A resting and visual encoding FMRI study[END_REF].

In conclusion, these findings further support the belief that differences in genetic predispositions could differentially impact on brain function during cellular/molecular pathophysiological stages. Additional research on the interaction among genetics, biology, and environmental factors as well as their influence on brain functional connectivity in AD needs to be addressed.

This figure is a derivative of the work created by Vivid Apps and AXS Biomedical

Animation Studio for Cold Spring Harbor Laboratory DNA Learning Center (https://www.dnalc.org/resources/3dbrain.html)

Genetics of Brain Biomarkers

Some of the issues related to explicate the functional effects of AD risk genotypes in the brain may be addressed by exploiting large-scale consortia linking the areas of neuroimaging and genetics. The use of genome-wide association studies led to identify over 20 genetic susceptibility loci in AD versus CN individuals [66]. In this regard, the Enhancing Neuro Imaging Genetics through Meta-Analysis (ENIGMA) consortium [START_REF] Hibar | Novel genetic loci associated with hippocampal volume[END_REF][START_REF] Hibar | Common genetic variants influence human subcortical brain structures[END_REF][START_REF] Thompson | ENIGMA and the individual: Predicting factors that affect the brain in 35 countries worldwide[END_REF] (http://enigma.ini.usc.edu/) has recently discovered more than 20 genetic loci that are consistently associated with brain structural MRI-based measures, in over 30,000 individuals worldwide. Loci affecting the risk for neurodegenerative diseases overlap substantially with those affecting brain markers. The authors found that the microtubule associated protein tau gene (MAPT), which is related to Parkinson's disease, contains polymorphic loci that appear to boost intracranial volume early in life [START_REF] Adams | Novel genetic loci underlying human intracranial volume identified through genome-wide association[END_REF]. Similarly, the APOE genotype showed a gradually increasing effect on hippocampal volume ranging from minimal effects in young adults to strong effects in old age [START_REF] Adams | Novel genetic loci underlying human intracranial volume identified through genome-wide association[END_REF]. Such evidence supports the antagonistic pleiotropy that some genetic risk factors for neurodegenerative diseases may have a positive influence early in life.

Efforts to harmonize functional connectivity phenotypes worldwide should soon reveal whether functional networks implicated in AD show similar or different genetic effects to those seen for structural markers of AD. In this regard, normative data compiled over the lifespan will be very useful to stratify into groups with different profiles of genetic risk, as the ENIGMA consortium has done for structural MRI measures. A second benefit of large-scale genetic consortia is their ability to determine the reproducibility of effects in cohorts worldwide. This is crucial as claims of genetic effects in one cohort may not always persist when tested more generally (see, e.g., [START_REF] Jahanshad | Do Candidate Genes Affect the Brain's White Matter Microstructure? Large-Scale Evaluation of 6[END_REF] for an analysis, in over 6,000 individuals, of genetic markers claimed to affect white matter integrity assessed with diffusion MRI).

Concluding Remarks and Future Perspectives

Overall, evidence is building that several genes associated with AD risk are able to differentially disrupt brain functional connectivity at rest in CN, presymptomatic, and symptomatic AD individuals [START_REF] Sanchez-Mut | Human DNA methylomes of neurodegenerative diseases show common epigenomic patterns[END_REF]. Such neural differences are detectable in CN mutation carriers of APOE, PICALM, CLU, and BIN1 genes across the lifespan. Relatively consistent at-rest functional neuroimaging data showed decreased connectivity in the middle and posterior DMN regions, including PCC and Pcu, and increased DMN connectivity in the frontal and lateral structures, such as the middle temporal and the prefrontal cortices.

Additional functional connectivity alterations associated with the APOE polymorphism were identified in the salience [49] and auditory systems [43]. Accordingly, presymptomatic AD individuals exhibited abnormalities in the DMN [START_REF] Chhatwal | Impaired Default Network Functional Connectivity in Autosomal Dominant Alzheimer's Disease: Findings from the DIAN Study[END_REF][START_REF] Sala-Llonch | Evolving brain functional abnormalities in psen1 mutation carriers: A resting and visual encoding FMRI study[END_REF], even at a very young age [START_REF] Quiroz | Brain Imaging and Blood Biomarker Abnormalities in Children With Autosomal Dominant Alzheimer Disease[END_REF][START_REF] Su | Lower functional connectivity of default mode network in cognitively normal young adults with mutation of APP, presenilins and APOE ε4[END_REF]. In contrast, significant results were not consistently reported in symptomatic AD dementia patients [START_REF] Chhatwal | Impaired Default Network Functional Connectivity in Autosomal Dominant Alzheimer's Disease: Findings from the DIAN Study[END_REF][START_REF] Sala-Llonch | Evolving brain functional abnormalities in psen1 mutation carriers: A resting and visual encoding FMRI study[END_REF], despite two studies reported a selective alteration of the DMN [START_REF] Jones | Cascading network failure across the Alzheimer's disease spectrum[END_REF][START_REF] Wang | Correspondence between Resting-State Activity and Brain Gene Expression[END_REF] and the executive control network [START_REF] Wang | Correspondence between Resting-State Activity and Brain Gene Expression[END_REF].

As a result, existent findings seem to converge in proposing a substantial, although not conclusive, relationship between genetics and functional brain networks in the AD clinical spectrum. However, caution in interpreting the reliability of the outcomes is warranted since large-scale replication studies need to be conducted. Notably, no direct genetic effect on neural networks was measured in the above reported studies. Indeed, while they investigated genetic predisposition at the level of polymorphic markers in the genome, complementary data should be produced to identify the gene expression in the known AD functionally-related networks (see Outstanding Questions). In this regard, Richiardi and colleagues [START_REF] Richiardi | Correlated gene expression supports synchronous activity in brain networks[END_REF] indicated a set of 136 genes exhibiting well-orchestrated fluctuations in their expression levels across networks, in healthy adolescents. From a molecular viewpoint, these genes are strictly related to ion channel activity, neurotransmitters, and synaptic function, thus suggesting an intrinsic association of brain functional connectivity with complex synaptic mechanisms. Given the evident convergence of such multimodal dimensions in healthy young individuals, a key future perspective is to define gene expression profiles related to non-pathological variations in structural and functional connectivity networks in CN older adults. Secondly, patterns of altered functional connectivity networks need to be identified in clinical and preclinical cohorts, such as presymptomatic and asymptomatic at-risk for AD individuals (amyloid positive) compared with CN age-matched older controls (amyloid negative). Eventually, the trend in neuroimaging genetics will be to embrace novel approaches, such as the concept of genome-wide association coupled with high-throughput functional neuroimaging [START_REF] Thompson | Genetics of the connectome[END_REF], or even genome-wide connectome-wide screening [START_REF] Jahanshad | Genome-wide scan of healthy human connectome discovers SPON1 gene variant influencing dementia severity[END_REF] to disclose complex genetic traits in CN individuals and across the full AD-spectrum.

The final goal in AD translational bench-to-bedside-to-bench (reverse translation) research is to develop multimodal neuroimaging-genetic-driven personalized signatures and screenings to enable the development of customized and biomarkerguided targeted therapies, thus improving patient care [START_REF] Hampel | Development of biomarkers to chart all Alzheimer's disease stages: The royal road to cutting the therapeutic Gordian Knot[END_REF][START_REF] Hampel | Recent developments of functional magnetic resonance imaging research for drug development in Alzheimer's disease[END_REF]. Recent years have witnessed substantial achievements in biomarker-guided therapeutic strategies in more advanced translational research areas of biomedicine, such as oncology and cardiovascular medicine [START_REF] Hampel | PRECISION MEDICINE -The Golden Gate for Detection, Treatment and Prevention of Alzheimer's Disease[END_REF][START_REF] Hampel | A Precision Medicine Initiative for Alzheimer's disease: the road ahead to biomarker-guided integrative disease modeling[END_REF]. This path to the paradigm of precision medicine (PM) for detecting, treating, and preventing complex multifactorial neurodegenerative diseases, including AD, will likely transform and revolutionize neurology, psychiatry, and neuroscience via breakthrough advances in sensitive, specific and integrated genomic/epigenomic, neuroimaging and biofluid biomarker screening, biological staging and patient subset stratification, and earliest biological detection of pathophysiological mechanisms [START_REF] Hampel | Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives[END_REF][START_REF] Hampel | Development of biomarkers to chart all Alzheimer's disease stages: The royal road to cutting the therapeutic Gordian Knot[END_REF][START_REF] Ewers | Staging Alzheimer's disease progression with multimodality neuroimaging[END_REF][START_REF] Teipel | Relevance of Magnetic Resonance Imaging for Early Detection and Diagnosis of Alzheimer Disease[END_REF]. This will allow both early prevention [START_REF] Teipel | Relevance of Magnetic Resonance Imaging for Early Detection and Diagnosis of Alzheimer Disease[END_REF][START_REF] Ewers | Neuroimaging markers for the prediction and early diagnosis of Alzheimer's disease dementia[END_REF] and, ultimately, successful development of combinatorial disease-modifying treatments based on the individuals genetic and pathophysiological profile [START_REF] Hampel | PRECISION MEDICINE -The Golden Gate for Detection, Treatment and Prevention of Alzheimer's Disease[END_REF][START_REF] Hampel | A Precision Medicine Initiative for Alzheimer's disease: the road ahead to biomarker-guided integrative disease modeling[END_REF].

Significant advances in Drug Discovery & Development programs are still substantially limited by the traditional "one-drug-fits-all" approach, which reductionistically categorizes the continuous genetically and biologically heterogeneous spectrum of different neurodegenerative diseases, including polygenic AD, as hypothesized "homogenous" clinicopathological or clinicobiological entities. In contrast, the emerging PM paradigm aims to overcome these historically grown challenges, notably the reductionistic clinically descriptive disease categories [START_REF] Hampel | PRECISION MEDICINE -The Golden Gate for Detection, Treatment and Prevention of Alzheimer's Disease[END_REF][START_REF] Hampel | A Precision Medicine Initiative for Alzheimer's disease: the road ahead to biomarker-guided integrative disease modeling[END_REF]. Notably, the PM strategy will facilitate a paradigm shift in AD and other neurodegenerative diseases away from the outdated "one-size-fits-all" approach in drug discovery, towards (I) biomarker-guided "molecularly" tailored therapies for precise and effective treatment of molecular pathophysiological pathways associated with AD, and (II) prevention options [START_REF] Hampel | PRECISION MEDICINE -The Golden Gate for Detection, Treatment and Prevention of Alzheimer's Disease[END_REF][START_REF] Hampel | A Precision Medicine Initiative for Alzheimer's disease: the road ahead to biomarker-guided integrative disease modeling[END_REF][START_REF] Ewers | Neuroimaging markers for the prediction and early diagnosis of Alzheimer's disease dementia[END_REF]. As a result, next-generation neurologists and psychiatrists (as the oncologist today), supported by interdisciplinary colleagues, e.g. geneticists, neurochemists, neuroradiologists, neuropsychologists, together with data science specialists and biostatisticians, will be able to precisely deliver biomarker-guided, targeted and timed interventions adapted to the genetic and biological profiles of individuals at the preclinical stage of AD and other neurodegenerative diseases.

Currently, this objective has been conceptualized and operationalized by the international pilot Alzheimer Precision Medicine Initiative Cohort Program (APMI-CP) [START_REF] Hampel | PRECISION MEDICINE -The Golden Gate for Detection, Treatment and Prevention of Alzheimer's Disease[END_REF][START_REF] Hampel | A Precision Medicine Initiative for Alzheimer's disease: the road ahead to biomarker-guided integrative disease modeling[END_REF].

According to the interdisciplinary and translational systems theory -allowing the implementation of novel and original models to elucidate all brain systems levels -and the PM paradigm, genetically and biologically distinct AD individuals may develop and display converging and/or overlapping clinical phenotypes with distinct combinations of underlying structural and functional neuroimaging genetics patterns that may be subject to dynamic variations across all different stages of the chronically evolving disease spectrum [START_REF] Hampel | Development of biomarkers to chart all Alzheimer's disease stages: The royal road to cutting the therapeutic Gordian Knot[END_REF][START_REF] Ewers | Staging Alzheimer's disease progression with multimodality neuroimaging[END_REF][START_REF] Teipel | Relevance of Magnetic Resonance Imaging for Early Detection and Diagnosis of Alzheimer Disease[END_REF]. As a result, integrating functional brain indices as dynamic biological markers -through integrative disease modeling [START_REF] Hampel | PRECISION MEDICINE -The Golden Gate for Detection, Treatment and Prevention of Alzheimer's Disease[END_REF][START_REF] Hampel | A Precision Medicine Initiative for Alzheimer's disease: the road ahead to biomarker-guided integrative disease modeling[END_REF] -will complete and further enhance and differentiate the early identification of disease systems endophenotypes [START_REF] Hampel | PRECISION MEDICINE -The Golden Gate for Detection, Treatment and Prevention of Alzheimer's Disease[END_REF][START_REF] Hampel | A Precision Medicine Initiative for Alzheimer's disease: the road ahead to biomarker-guided integrative disease modeling[END_REF].  Precision Medicine is biomarker-guided approach based on systems-levels that include methodological advancements and findings of a wide-ranging pathophysiological profiles of complex multi-factorial neurodegenerative diseases, such as AD. This may allow to identify and characterize the pathophysiological processes at the preclinical stages, before clinical symptoms Broadly expressed in the brain, where it contributes to retrieve synaptic vesicles, apoptosis, inflammation, clathrin-mediated Aβ [START_REF] Karch | Alzheimer's disease risk genes and mechanisms of disease pathogenesis[END_REF][START_REF] Tan | Bridging integrator 1 (BIN1): form, function, and Alzheimer's disease[END_REF].

Potential influence on functional connectivity: 1) impaired neurite outgrowth; 2) impaired synaptic integration. APP gene: codes for the amyloid precursor protein.

Essential for physiological brain development (neurogenesis and synaptogenesis) and plasticity [START_REF] Karch | Alzheimer's disease risk genes and mechanisms of disease pathogenesis[END_REF][START_REF] Bignante | Amyloid β precursor protein as a molecular target for amyloid β--induced neuronal degeneration in Alzheimer's disease[END_REF].

Potential influence on functional connectivity: 1) Aβ peptide clearance and/or deposition; 2) impaired neurite outgrowth; 3) impaired synaptic integration. PSEN1 and PSEN2 genes: encode for presenilin 1 and presenilin 2.

Presenilins are proteolytic subunits of γ-secretase intramembrane protease complex [START_REF] Karch | Alzheimer's disease risk genes and mechanisms of disease pathogenesis[END_REF].

Potential influence on functional connectivity: 1) Aβ peptide clearance and/or deposition; 2) impaired neurite outgrowth; 3) impaired synaptic integration, 4) calcium dyshomeostasis.

Figure 1

 1 Figure 1 Main effects of genetic risk factors for AD on brain functional connectivity in cognitively normal individuals. Schematic illustration of the main networks influenced by genetic variations in cognitively intact individuals. Mutations in the APOE or CLU genes affect the functional connectivity of (i) the anterior DMN (red), including the anterior cingulate and the middle prefrontal cortices, (ii) the poster DMN (blue), including the posterior cingulate cortex, the precuneus, the inferior parietal lobe and the retrosplenial cortex, and (iii) the hippocampus (green). In contrast, BIN1 and PICALM genetic variations seem to affect essentially the hippocampal connectivity. This figure is a derivative of the work created by Vivid Apps and AXS Biomedical Animation Studio for Cold Spring Harbor Laboratory DNA Learning Center (https://www.dnalc.org/resources/3dbrain.html)

Figure 2

 2 Figure 2 Main effects of genetic risk factors for AD on brain functional connectivity in presymptomatic individuals In presymptomatic individuals, genetic effects of APP, PSEN1, PSEN2, and APOE were shown in the resting-state functional connectivity of the posterior DMN (blue). In addition, while APP, PSEN2, and APOE influence the anterior DMN (red), PSEN1 mutations affect the temporal lobe (purple). APOE variants affect functional connectivity as well, in sensorymotor, auditory and salience networks (not shown).

Figure 3

 3 Figure 3 Main effects of genetic risk factors for AD on brain functional connectivity in AD dementia individuals Neuroimaging genetics results in AD dementia patients are still controversial. However, functional alterations at rest resulted in the posterior DMN (blue) in AD diseased individuals with PSEN1 mutations, and in the anterior DMN (red) in APOE ε4 carriers.

Glossary

  Amyloid beta (Aβ) denotes peptides of different length in terms of amino acids.The 42-amino acid-long Aβ peptide (Aβ1-42) is the major component of the neuritic plaques in AD brains and the core biochemical marker for the amyloidogenic process in AD. It derives from the pathological cleavage of the amyloid precursor protein (APP). Diaschisis: a functional interruption of regions remote from the initial insult, caused by the deafferentation of excitatory inputs. Functional connectivity: the statistically synchronized or temporal coherent blood oxygen level-dependent (BOLD) activity of remote brain regions that thus share a common functional specialization. Functional neuroimaging genetics: identifies genes that contribute to functional alterations in brain networks. Integrative disease modeling: is a multidisciplinary approach, which aims to standardize, manage, integrate, and interpret multiple biological quantitative and qualitative data, by applying computational models to support decisionmaking for translation of patient-specific molecular mechanisms into tailored clinical applications  Intermediate phenotype: often referred to as an endophenotype, is a stable phenotype with a clear genetic connection. Neuritic plaques: abnormal extracellular deposits primarily composed of amyloid beta (Aβ) peptides in the grey matter of the brain, also named senile plaques. Neurofibrillary tangles: intracellular aggregates of hyperphosphorylated tau proteins. They are generated by the excessive phosphorylation (hyperphosphorylation) of a microtubule-associated protein known as tau, causing it to aggregate in an insoluble form. Neuroimaging genetics: methodological approach applied to understand brain structure, function and disease, based on brain imaging modalities and genetic data.



  Resting State Functional Magnetic Resonance Imaging (rs-fMRI): neuroimaging procedure for evaluating synchronous fluctuations of signal intensities across brain regions showing a high degree of temporal correlation, while participants lay with their eyes closed or fix on a visual cue, without performing explicit tasks.  Structural connectivity: anatomical connections of physical white matter tracts.  Transneuronal degeneration: process that evolves over time consisting of a progressive structural deterioration of areas remote from the injured site. The damage might first occur in a postsynaptic target, reducing the trophic support to the presynaptic neuron (retrograde), or, alternatively, one neuron may cause the degeneration of its postsynaptic target (anterograde).

Box 1 :Box 2 :

 12 Clinical diagnostic criteriathree meta categories for the global staging of AD"Preclinical AD: indicates the asymptomatic stage between the earliest neuropathological events and the appearance of AD-related cognitive impairments (clinical stage). Although the preclinical stage of AD represents a continuum, two in vivo preclinical states can be discerned: i) the "asymptomatic at-risk state for AD", which indicates the presence of pathophysiological markers, such as tau pathology (CSF or PET tau) or amyloid pathology (CSF Ab42 or PET amyloid), and ii) "presymptomatic AD", which refers to individuals who will certainly develop AD, because they carry rare autosomal dominant mutations that cause AD, such as APP, PSEN1 or PSEN2.Prodromal AD (or "MCI-due-to-AD"): includes the presence of definite impairment in memory function, e.g. measured by Free and Cued Selective Reminding Test [78], along with in vivo positive pathophysiological markers (CSF or PET tau, CSF Ab42 or PET amyloid). Instrumental activities of daily living are preserved. AD dementia: refers to individuals presenting severe cognitive impairments that interfere with social functioning and instrumental activities of daily living. Clinical symptoms must include progressive deficits in memory and in at least one other cognitive domain, i.e., executive functions, language, or visuospatial abilities. In vivo pathophysiological or topographic markers (e.g., hippocampal atrophy, cortical thickness) are supportive evidence for the diagnosis of AD dementia. Genetic risk factors for AD and their potential functional connectivity counterpart APOE gene: codes for apolipoprotein E. Regulates amyloid-β (Aβ) oligomerization, aggregation and receptor-mediated clearance, brain lipid transport, glucose metabolism, neuronal signaling, and neuroinflammation [26,82,83]. Potential influence on functional connectivity: 1) impaired neurite outgrowth; 2) cytoskeletal disruption and hyperphosphorylation of tau; 3) mitochondrial dysfunction in neurons; 4) impaired synaptogenesis; (5) increased apoptosis in neurons; (6) Aβ peptide clearance and/or deposition. PICALM gene: codes for the phosphatidylinositol binding clathrin assembly protein. Protects neurons from Aβ toxicity by reversing Aβ effects on clathrin-mediated endocytosis and/or by directing amyloid precursor protein transport to the terminal degradation pathway by autophagosomes, which reduces Aβ production [26]. Potential influence on functional connectivity: Aβ peptide clearance and/or deposition. CLU gene: codes for clusterin. Involved in several biological and pathophysiological mechanisms, including cell death and tumor progression. Moreover, CLU assists clearance of Aβ, interacts with ApoE, and promotes neuroinflammation by inhibiting complement activation [26]. Potential influence on functional connectivity: 1) impaired neurite outgrowth; 2) impaired synaptic integration; 3) Aβ peptide clearance. BIN1 gene: codes for the Bridging integrator 1.

Acknowledgements

HH is supported by the AXA Research Fund, the Fondation Université Pierre et Marie Curie and the Fondation pour la Recherche sur Alzheimer, Paris, France. Ce travail a bénéficié d'une aide de l'Etat « Investissements d'avenir » ANR-10-IAIHU-06 (HH). The research leading to these results has received funding from the program "Investissements d'avenir" ANR-10-IAIHU-06(HH). PT is supported in part by NIH grant U54 EB040203 to the ENIGMA Center for Worldwide Medicine, Imaging and Genomics. The authors wish to thank Dr. Michael Greicius, Associate Professor of Neurology and Neurological Sciences at Stanford University, for critically reading, editing and discussing our manuscript.


Nathalie Chabbert-Buffet #

Christian Jamin

Iñaki Lete

Paloma Lobo

Rossella E Nappi

Axelle Pintiaux $

Günther Häusler

Christian Fiala

Nathalie Chabbert-Buffet
email: nathalie.chabbert-buffet@aphp.fr

Missed pills: frequency, reasons, consequences and solutions

Keywords: Compliance, Efficacy, Half-life, Hormone-free interval, Missed pill, Nomegestrol acetate, Non-compliance, Progestin, Regimen

Oral hormonal contraception is an effective contraceptive method as long as a regular daily intake is maintained. However, this routine is a constraint for many women and can lead to missed doses, pill discontinuation and/or unintended pregnancy. Risk factors for missed pills include user characteristics, cultural aspects, a lack of information about the choice of contraceptive options and a lack of involvement in the decision. The formulation of the combined oral contraceptive, including the type of dosing regimen (21-day or 28-day packs), can also negatively influence efficacy and compliance. As ovarian activity is not fully suppressed, the risk of failure is greatest when the hormone-free interval (HFI) is prolonged.

Continuous pill intake or long-cycle regimens can reduce the risks of follicular development and thus the likelihood of ovulation and unintended pregnancy. A shortened or eliminated HFI and a progestin with a longer half-life (providing better suppression of ovarian activity) may be an option to reduce the negative consequences of missed pills in oral contraception.

INTRODUCTION

Fertility has been a concern for people throughout history. Pre-20 th Century contraceptive methods included male condoms and coitus interruptus, and with the development of combined oral contraceptives (COCs) in the 1960s women gained more control [START_REF] Parenthood | the Katharine Dexter McCormick Library and the Education Division of Planned Parenthood Federation of America[END_REF] . However, the high contraceptive efficacy of COCs depends on regular daily intake [START_REF] Grimes | Forgettable contraception[END_REF][START_REF] Halpern | Strategies to improve adherence and acceptability of hormonal methods of contraception[END_REF] and compliance problems are common among all age groups [START_REF] Rosenberg | Causes and consequences of oral contraceptive noncompliance[END_REF] . Efforts were made to improve compliance (including easy use, minimal side effects and better tolerability), but with poor results [START_REF] Halpern | Strategies to improve adherence and acceptability of hormonal methods of contraception[END_REF] .

In this narrative review we will summarize available data from the literature on missed pill occurrence and its consequences, as well as discuss available solutions to this important drawback of COCs. We have used the term 'missed pills' to describe inconsistent COC use, defined as missing one or more pills per 28-day cycle [START_REF] Hall | Studying the use of oral contraception: a review of measurement approaches[END_REF] .

THE PILL AND ITS INCONSISTENT USE

More than 100 million women worldwide used COCs in 2009, according to the United Nations, equating to an 8.8% contraceptive prevalence among women of reproductive age 6 : 18.0% in developed countries alone (up to 59% in Portugal) [START_REF]World Contraceptive Patterns[END_REF] . In 2008, a study assessed women's contraceptive practices and sexual behaviour across 14 European countries [START_REF] Cibula | Women's contraceptive practices and sexual behaviour in Europe[END_REF] . COCs were the most popular contraception method, with usage rates highest in France (49%) and the Czech Republic (44%), and lowest in Russia, the Baltic States and Spain (15-18%). The study concluded that differences in usage may reflect socio-historical developments and availability of contraception [START_REF] Cibula | Women's contraceptive practices and sexual behaviour in Europe[END_REF] . In late 2012, a 'pill scare' (mainly based on the higher risk of venous thromboembolism (VTE) associated with 3rd-and 4th-generation COCs compared to 2 nd -generation COCs) changed many women's perception of COCs. In France, a national survey from 2010-2013 showed a 9% reduction in use of 3rd-and 4 th -generation COCs and was not compensated by increased use of 2 nd -generation COCs (23% in 2013 versus 22% in 2010) [START_REF] Bajos | The French pill scare: towards a new contraceptive model?[END_REF] . In 2013, 229,000 abortions were conducted in France, a rise of 4.5% on 2012 10 : similar outcomes were observed after the 1995 pill scare in Great Britain [START_REF] Dillner | Pill scare linked to rise in abortions[END_REF] .

Forgetting one or more oral contraceptive tablets per cycle is common, regardless of age, but adolescents have the highest risk of non-compliance [START_REF] Rosenberg | Causes and consequences of oral contraceptive noncompliance[END_REF] . They reportedly forget an average of three pills per cycle, with discontinuation rates up to 50% during the first three months of use [START_REF] Balassone | Risk of contraceptive discontinuation among adolescents[END_REF] . In a study comparing acceptability of the vaginal contraceptive ring to COCs, 12% (8/65) of adolescents reported they forgot the pill and 1.5% (1/65) reported forgetting the ring [START_REF] Stewart | Adolescent and young women's experience with the vaginal ring and oral contraceptive pills[END_REF] . In women aged 18 or older, 10% to 51% miss at least three pills a month, depending on electronic or self-reported data [START_REF] Potter | Measuring compliance among oral contraceptive users[END_REF] . One study found 47% of women miss >1 pill per cycle and 22% at least two pills per cycle [START_REF] Rosenberg | Causes and consequences of oral contraceptive noncompliance[END_REF] . A European survey showed that 19% of women aged 16 to 30 years missed one or more pills per cycle, increasing the risk of unintended pregnancy by 2.6 times compared to those who took COCs consistently [START_REF] Rosenberg | Unintended pregnancies and use, misuse and discontinuation of oral contraceptives[END_REF] . In the United States, the probability of pregnancy during the first year of perfect use (i.e. following the directions for use) was estimated at 0.3%. Failure rates reached 9% with typical use, including inconsistent and incorrect use [START_REF] Dinger | Effectiveness of oral contraceptive pills in a large U.S. cohort comparing progestogen and regimen[END_REF][START_REF] Trussell | Contraceptive failure in the United States[END_REF] .

REASONS FOR 'MISSED PILL'

COC compliance rates are similar to those for long-term treatments for chronic conditions [START_REF] Rosenberg | Causes and consequences of oral contraceptive noncompliance[END_REF][START_REF] Cramer | Compliance with contraceptives and other treatments[END_REF][START_REF] Ingersoll | The impact of medication regimen factors on adherence to chronic treatment: a review of literature[END_REF] . Both are influenced by patient, environmental and clinician factors [START_REF] Ingersoll | The impact of medication regimen factors on adherence to chronic treatment: a review of literature[END_REF] . COC regimens also have unique characteristics [START_REF] Ingersoll | The impact of medication regimen factors on adherence to chronic treatment: a review of literature[END_REF] : they are generally preventive rather than curative, and COC users seldom receive ongoing reinforcement, whereas treatment management for chronic conditions may provide symptom relief leading to positive feedback [START_REF] Whelan | Compliance with contraceptive regimens[END_REF][START_REF]Global Library of Women's Medicine. The educational platform for FIGO[END_REF] . COC users have several treatment choices whereas patients treated for a specific condition often have limited or no choice [START_REF] Whelan | Compliance with contraceptive regimens[END_REF] . Also, contraceptive decisions may be affected by interactions between sexual partners, whereas usually just one patient is involved when a disease-related drug is prescribed [START_REF] Whelan | Compliance with contraceptive regimens[END_REF] . Therefore, studying contraceptive compliance is more complex compared with compliance with other medical regimens [START_REF] Whelan | Compliance with contraceptive regimens[END_REF][START_REF]Global Library of Women's Medicine. The educational platform for FIGO[END_REF] .

Women who do not establish a routine or do not succeed in associating COC intake with another daily activity, such as applying make-up or brushing teeth, are 4.6 times more likely to miss two or more pills per month and 3.3 times more likely to be inconsistent users compared with women who have a routine [START_REF] Rosenberg | Unintended pregnancies and use, misuse and discontinuation of oral contraceptives[END_REF] . In addition, women with little or no understanding of COCs and their usage are 2.4 times more likely to miss two or more pills per cycle than more informed women [START_REF] Rosenberg | Unintended pregnancies and use, misuse and discontinuation of oral contraceptives[END_REF] .

Occurrence of side effects is the primary predictor for early discontinuations [START_REF] Rosenberg | Unintended pregnancies and use, misuse and discontinuation of oral contraceptives[END_REF] . Increased risk of missing a pill is associated with women's lack of involvement in choosing a contraceptive method [START_REF] Moreau | Social, demographic and situational characteristics associated with inconsistent use of oral contraceptives: evidence from France[END_REF] , lack of a regular sex partner, limited support from a partner or low socioeconomic status [START_REF] Rosenberg | Unintended pregnancies and use, misuse and discontinuation of oral contraceptives[END_REF][START_REF] De Zarate | Evaluation of factors associated with noncompliance in users of combined hormonal contraceptive methods: a cross-sectional study: results from the MIA study[END_REF] . Cultural aspects can also affect compliance: myths and misinformation are often passed down the generations [START_REF] Cramer | Compliance with contraceptives and other treatments[END_REF] .

Age and the type of physician prescribing the contraceptive were not significantly related to consistent pill use [START_REF] Rosenberg | Unintended pregnancies and use, misuse and discontinuation of oral contraceptives[END_REF] . However, experiencing benefits associated with COC, such as reduced cramping and bleeding, may improve compliance. [START_REF] Robinson | Dysmenorrhea and use of oral contraceptives in adolescent women attending a family planning clinic[END_REF] In adolescents, psychosocial characteristics including low evaluation of personal health, previous abortion and multiple sexual partners greatly influence compliance [START_REF] Durant | Influence of psychosocial factors on adolescent compliance with oral contraceptives[END_REF] . Some adolescents might not want their future threatened by an unwanted pregnancy, yet may continue to seek personal self-fulfilment through childbearing [START_REF] Durant | Influence of psychosocial factors on adolescent compliance with oral contraceptives[END_REF] . 'Magical thinking' is also common: it helps maintain a shield of perceived low susceptibility to pregnancy [START_REF] Cramer | Compliance with contraceptives and other treatments[END_REF] .

In a 2005 American study, the three most frequent reasons for missed pills given by the 141 COC users were 'away from home' (12.9%), 'forgot' (12.9%), and 'no new pack' (10.5%) [START_REF] Smith | Why do women miss oral contraceptive pills? An analysis of women's self-described reasons for missed pills[END_REF] .

If 'unavailable' was given as the reason for missing pills, one missed pill day was more likely to be followed by another (44.3%). If the reason was physiological (i.e. health reasons, side effects or sleep disturbances), the likelihood of missed pills on consecutive days was only 10.7%. The likelihood was significantly increased in case of 'work pressures' (19.3%) and 'no new pill pack' (21.9%) [START_REF] Smith | Why do women miss oral contraceptive pills? An analysis of women's self-described reasons for missed pills[END_REF] .

In a 2013 Spanish multicentre study, forgetfulness was the main reason for omission/delay of using COCs (74.9%) among 8,762 women aged 18-28 years, followed by having little experience of their chosen contraceptive method [START_REF] De Zarate | Evaluation of factors associated with noncompliance in users of combined hormonal contraceptive methods: a cross-sectional study: results from the MIA study[END_REF] . Remembering to use COCs was more difficult on vacation (28.2%), weekends (34.8%), after going out the previous night (15.9%), or on short trips (11.2%) than on weekdays (9.4%) [START_REF] De Zarate | Evaluation of factors associated with noncompliance in users of combined hormonal contraceptive methods: a cross-sectional study: results from the MIA study[END_REF] .

MISSED PILLS AND OVULATION

Estrogen and, above all, progestin in COCs prevent ovulation by inhibiting gonadotropin secretion via a negative feedback effect on the hypothalamo-pituitary axis [START_REF] Wan | Pituitary response to LHRH stimulation in women on oral contraceptives: a followup dose response study[END_REF][START_REF] Speroff | Oral contraception[END_REF][START_REF] Baerwald | Ovarian follicular development during the use of oral contraception: a review[END_REF][START_REF] Bitzer | Current issues and available options in combined hormonal contraception[END_REF] . The contraceptive efficacy is essentially provided by the progestin component, which primarily suppresses luteinizing hormone (LH) secretion necessary for ovulation [START_REF] Speroff | Oral contraception[END_REF][START_REF] Bitzer | Current issues and available options in combined hormonal contraception[END_REF] . The estrogenic component suppresses follicle-stimulating hormone (FSH) secretion, preventing the selection of a dominant follicle [START_REF] Speroff | Oral contraception[END_REF] . Another important role of estrogen and progestin is to stabilize the endometrium and prevent unwanted breakthrough bleeding [START_REF] Speroff | Oral contraception[END_REF] .

Although very effective in preventing pregnancy if correctly and consistently used, COCs do not lead to complete ovarian suppression [START_REF] Spona | Shorter pill-free interval in combined oral contraceptives decreases follicular development[END_REF][START_REF] Willis | Greater inhibition of the pituitary-ovarian axis in oral contraceptive regimens with a shortened hormone-free interval[END_REF] . Follicle growth and concomitant estradiol production usually occur during the hormone-free interval (HFI), first week of medication, or when COCs are missed [START_REF] Fauser | Manipulation of human ovarian function: physiological concepts and clinical consequences[END_REF][START_REF] Kuehl | Pituitary-ovarian hormone levels and symptoms in oral contraceptive users: comparison of a 21/7-day and extended regimen[END_REF][START_REF] Vandever | Evaluation of pituitary-ovarian axis suppression with three oral contraceptive regimens[END_REF] . Studies have shown a gradual decline of gonadotropins in the first week of pill intake, which leads to suppression of non-dominant follicle development [START_REF] Van Heusden | Residual ovarian activity during oral steroid contraception[END_REF] .

However, dominant follicles present on the first day the pill is taken can still increase in diameter [START_REF] Van Heusden | Residual ovarian activity during oral steroid contraception[END_REF] , so the potential to ovulate remains [START_REF] Baerwald | Ovarian follicular development during the use of oral contraception: a review[END_REF] .

A study looked at serum pituitary and ovarian hormone levels during the 7-day HFI among six different COC formulations [START_REF] Cho | Pituitary and ovarian hormone activity during the 7-day hormone-free interval of various combined oral contraceptive regimens[END_REF] . A greater dose-response suppression of LH, FSH, and estradiol was found on day 1 of the HFI, with 30-or 35-g EE COCs than 20g EE COCs.

Higher doses of EE were associated with a more rapid increase in gonadotropin levels from day 1 to day 7 of the HFI; hormone levels during the HFI were not changed by the type and dose of 19-norsteroid progestin [START_REF] Cho | Pituitary and ovarian hormone activity during the 7-day hormone-free interval of various combined oral contraceptive regimens[END_REF] .

Increasing the number of active pills per cycle or adding EE-only pills to shorten the HFI to 3 or 4 days diminishes FSH, LH and estradiol levels [START_REF] Willis | Greater inhibition of the pituitary-ovarian axis in oral contraceptive regimens with a shortened hormone-free interval[END_REF][START_REF] Killick | Extending the duration of the pill-free interval during combined oral contraception[END_REF] . This can provide greater pituitaryovarian inhibition, potentially reducing the risk of ovulation and common withdrawal symptoms [START_REF] Baerwald | Ovarian follicular development during the use of oral contraception: a review[END_REF][START_REF] Spona | Shorter pill-free interval in combined oral contraceptives decreases follicular development[END_REF][START_REF] Willis | Greater inhibition of the pituitary-ovarian axis in oral contraceptive regimens with a shortened hormone-free interval[END_REF][START_REF] Van Heusden | Residual ovarian activity during oral steroid contraception[END_REF][START_REF] Sullivan | Effect of 21-day and 24-day oral contraceptives decreases follicular development[END_REF] . More suppression of ovarian follicular activity is possible by eliminating the HFI completely [START_REF] Kuehl | Pituitary-ovarian hormone levels and symptoms in oral contraceptive users: comparison of a 21/7-day and extended regimen[END_REF][START_REF] Schlaff | Manipulation of the pill-free interval in oral contraceptive pill users: the effects on follicular suppression[END_REF] .

Ovarian suppression increases with the number of pills already taken, with maximum suppression often encountered at the end of the COC cycle [START_REF] Smith | The effect of deliberate omission of Trinordiol or Microgynon on the hypothalamo-pituitary-ovarian axis[END_REF] . After achieving maximum suppression (i.e. active pills taken for seven consecutive days), theoretically up to seven pills can be omitted (the 7-day HFI) without affecting contraceptive efficacy [START_REF] Curtis | Effective use of hormonal contraceptives: Part I: Combined oral contraceptive pills[END_REF] . Risk of pregnancy after missing pills depends on when and how many pills are missed [START_REF]Combined oral contraceptives (the Pill): when to start taking the Pill, and missed pill advice[END_REF] . The first week of the COC cycle is critical [START_REF] Aubeny | The Coraliance study: non-compliant behavior. Results after a 6-month follow-up of patients on oral contraceptives[END_REF] . Evidence suggests missing pills on days not adjacent to the HFI is less critical to missing pills on adjacent days [START_REF] Curtis | Effective use of hormonal contraceptives: Part I: Combined oral contraceptive pills[END_REF] . Eliminating the HFI when one or more days of COCs are missed in Week 2 or Week 3 may reduce the risk of unplanned pregnancy [START_REF] Guilbert | Missed hormonal contraceptives: new recommendations[END_REF] . However, missing three or more pills in a row during Week 3 is likely to impair contraceptive effectiveness, because the HFI comes immediately after Week 3 [START_REF] Guilbert | Missed hormonal contraceptives: new recommendations[END_REF] .

GUIDELINES ABOUT MISSED COCS

Guidelines are conflicting, probably leading to their low acceptance rate by physicians and COC users [START_REF] Jamin | Oublis de la contraception hormonale : réflexions sur leur prise en charge en pratique quotidienne[END_REF] . Revised recommendations were developed based mainly on the evidence reviewed in 2008 by the WHO Expert Working Group, a Cochrane review and the Society of Obstetrics and Gynaecology of Canada [START_REF]Combined oral contraceptives (the Pill): when to start taking the Pill, and missed pill advice[END_REF] . The missed pill window was changed from 12 hours to 24 hours [START_REF]Combined oral contraceptives (the Pill): when to start taking the Pill, and missed pill advice[END_REF] . The number of pills missed and the time in the cycle when this occurs are crucial for considering risk of contraceptive failure [START_REF]Combined oral contraceptives (the Pill): when to start taking the Pill, and missed pill advice[END_REF] . Contraceptive cover is provided: the user should take the last pill missed, even if it means taking two pills in one day, and continue taking the rest of the pack as usual [START_REF]Combined oral contraceptives (the Pill): when to start taking the Pill, and missed pill advice[END_REF] . Missing two or more pills (more than 48 hours late) may affect the contraceptive cover, except in the second week of the pack and could require emergency contraception [START_REF]Combined oral contraceptives (the Pill): when to start taking the Pill, and missed pill advice[END_REF] .

HOW CAN THE 'MISSED PILL ISSUE' BE ADDRESSED?

Several strategies have been proposed to improve contraceptive compliance [START_REF] Rosenberg | Causes and consequences of oral contraceptive noncompliance[END_REF] . HCPs must ensure patients understand the correct usage of COCs and help women with their choice, based on background, individual needs and concerns [START_REF] Rosenberg | Causes and consequences of oral contraceptive noncompliance[END_REF] . Talking about establishing a regular pill-taking routine, discussing possible side effects associated with pill use, providing clear instructions (including missed pill guidance), and using follow-up contact to look for signs of non-compliance are all strategies that may prove helpful, particularly with adolescents who may sporadically use COCs [START_REF] Rosenberg | Causes and consequences of oral contraceptive noncompliance[END_REF][START_REF]Global Library of Women's Medicine. The educational platform for FIGO[END_REF] .

COC users should take the pill at a regular time, preferably as part of a daily routine such as applying make-up, carefully read the instructions that come with the pill package, and know what to do if pills are missed [START_REF] Rosenberg | Causes and consequences of oral contraceptive noncompliance[END_REF] .

There are options for women who want reliable contraception, but have poor compliance: long-acting reversible contraceptives (LARCs), such as an intrauterine system (IUS) and implants, do not depend on daily pill-taking [START_REF] Halpern | Strategies to improve adherence and acceptability of hormonal methods of contraception[END_REF][START_REF] Stoddard | Efficacy and safety of long-acting reversible contraception[END_REF] . LNG IUS is often considered close to an 'ideal' contraceptive, although ≥25% of women discontinue this method within the first year [START_REF] Diaz | Acceptability and performance of the levonorgestrel-releasing intrauterine system (Mirena) in Campinas, Brazil[END_REF] and at five years the discontinuation rate reaches 60%, mostly due to bleeding problems [START_REF] Cox | Clinical performance of the levonorgestrel intrauterine system in routine use by the UK Family Planning and Reproductive Health Research Network: 5-year report[END_REF] .

CAN A COC WITH A LONG HALF-LIFE PROGESTIN AND SHORT HFI IMPROVE THE MISSED PILL ISSUE?

Follicular maturation is most likely to occur during an HFI or following a missed pill [START_REF] Baerwald | Ovarian follicular development during the use of oral contraception: a review[END_REF][START_REF] Van Heusden | Residual ovarian activity during oral steroid contraception[END_REF] . In 2004, a cohort study found that women using pills in a continuous 28-day cycle missed fewer pills in the vulnerable first week than those using a traditional 21-day regimen [START_REF] Aubeny | The Coraliance study: non-compliant behavior. Results after a 6-month follow-up of patients on oral contraceptives[END_REF] . The study concluded that continuous (everyday) cycle regimens provide an undeniable benefit, eliminating the risk of forgetting during the week of discontinuation between two blister packs [START_REF] Aubeny | The Coraliance study: non-compliant behavior. Results after a 6-month follow-up of patients on oral contraceptives[END_REF] . In contrast, a 2014 Cochrane study found no statistically significant difference in compliance rates between traditional monthly cyclic dosing and extended cycles [START_REF] Edelman | Continuous or extended cycle vs. cyclic use of combined hormonal contraceptives for contraception[END_REF] . It has been argued that 28-day packs are much simpler and cause less confusion than 21-day packs [START_REF]Global Library of Women's Medicine. The educational platform for FIGO[END_REF][START_REF] Finlay | Patterns of contraceptive pill taking in an inner city practice[END_REF] . However, there is variation in the onset of withdrawal bleeding and some women may start the next cycle as late as 8-10 days after the previous packet [START_REF]Global Library of Women's Medicine. The educational platform for FIGO[END_REF] .

Additional use of a progestin with a long half-life was suggested to reduce the risk of contraceptive failure during 'typical use', which includes occasional missed pills [START_REF] Dinger | Effectiveness of oral contraceptive pills in a large U.S. cohort comparing progestogen and regimen[END_REF] .

Knowledge of a drug's elimination half-life is useful for making recommendations on how to proceed if the patient has missed one or more doses [START_REF] Gilbert | I've missed a dose; what should I do?[END_REF] . In general, a single missed dose is less problematic for a drug with a long half-life as it will lose therapeutic effect less rapidly than a drug with a shorter half-life [START_REF] Wan | Pituitary response to LHRH stimulation in women on oral contraceptives: a followup dose response study[END_REF] .

A variety of progestins, including testosterone-, progesterone-derived progestins, and derivatives of progesterone are currently used in COCs. They differ in their biological effects (in addition to the basic progestogenic effect), and their pharmacokinetic parameters, including half-life (Table 1) [START_REF] Schindler | Classification and pharmacology of progestins[END_REF][54][55][START_REF] Bouchard | Chlormadinone acetate (CMA) in oral contraception--a new opportunity[END_REF] . Drospirenone (DRSP) is a progestin with a half-life of 40 hours 54 . In astudy, which included intentional dosing error days at the beginning of Cycle 3 (equivalent to missed pill days), it was reported that ovarian suppression was more frequent in the 24/4 regimen group than in the 21/7 regimen: 87.8% vs. 56.0% during Cycle 2 and 55.1% vs. 30.0% during Cycle 3, respectively [START_REF] Klipping | Suppression of ovarian activity with a drospirenone-containing oral contraceptive in a 24/4 regimen[END_REF] . The EE 24/4 regimen COC was also associated with less fluctuation of endogenous estradiol [START_REF] Klipping | Suppression of ovarian activity with a drospirenone-containing oral contraceptive in a 24/4 regimen[END_REF] . It was concluded that, under conditions of imperfect use, a 24-day COC containing a progestin with a long half-life may offer better effectiveness than a 21-day COC containing a progestin with a short half-life [START_REF] Dinger | Effectiveness of oral contraceptive pills in a large U.S. cohort comparing progestogen and regimen[END_REF] .

Nomegestrol acetate (NOMAC) is another progestin that exhibits a long elimination half-life (46 hours) and is used in combination with estradiol (E2) to provide a monophasic regimen COC, NOMAC/E2. NOMAC is a progesterone-derived progestin that binds almost exclusively to the progesterone receptor and does not interfere with other steroid receptors 58- 60 . In a 24-day regimen, NOMAC/E2 was associated with greater inhibition of follicular growth and shorter duration of withdrawal bleeding than a 21-day regimen [START_REF] Serfaty | Comparison of a 24-day and a 21day pill regimen for the novel combined oral contraceptive, nomegestrol acetate and 17bestradiol (NOMAC/E2): a double-blind, randomized study[END_REF] . Withdrawal bleeding duration was significantly shorter in the 24-day group [START_REF] Serfaty | Comparison of a 24-day and a 21day pill regimen for the novel combined oral contraceptive, nomegestrol acetate and 17bestradiol (NOMAC/E2): a double-blind, randomized study[END_REF] .

Following findings that a long half-life may offer greater flexibility in the event of a missed dose compared with COCs containing progestins with shorter half-lives [START_REF] Dinger | Effectiveness of oral contraceptive pills in a large U.S. cohort comparing progestogen and regimen[END_REF] , the summary of product characteristics of the 24/4 regimen of DRSP/EE was updated in 2013 to extend the time window for missed pills from 12 to 24 hours [START_REF] Bayer | Public assessment report of the Medicines Evaluation Board in the Netherlands. Yaz 24+4, film[END_REF] . Two years later, the missed pill window was also changed from 12 to 24 hours for the E2 pill NOMAC/E2 [START_REF]Zoely Procedural steps taken and scientific information after the authorization[END_REF] .

CONCLUSION

Women using COCs differ from other patients in that they make the decision to use a contraceptive to modulate a physiological process without medical indications [START_REF] Cramer | Compliance with contraceptives and other treatments[END_REF] . Noncompliance reveals the difficulties they experience with their daily use of the pill. In the context of high prevalence of both pill omission and the subsequent risk of contraceptive failure, improving COC use may be a responsibility shared among COC users, providers and manufacturers. Shortening the HFI to 3 or 4 days, or even eliminating the HFI completely, can reduce ovarian follicular activity and the risk of ovulation. Pharmacokinetics of COCs and the scheme of combination are characteristics that should be considered. A progestin 64.

Tables 1 2 Table 1: Half-life of various progestins [54][55][START_REF] Bouchard | Chlormadinone acetate (CMA) in oral contraception--a new opportunity[END_REF] Levonorgestrel 20 54 Desogestrel 31 54 Chlormadinone acetate 34 -36 [START_REF] Bouchard | Chlormadinone acetate (CMA) in oral contraception--a new opportunity[END_REF] Cyproterone acetate 38 55 Drospirenone 40 54 Nomegestrol acetate 46 54 7

  . Half-lives can be given in a range of values for a 3 few progestins. Dienogest and nomegestrol acetate are used in combination with estradiol, 4

	5	other progestins in combination with ethinylestradiol.
	6
		Progestins	Half-life (hours)
		Norethisterone	5 -12 54
		Dienogest	11 54
		Norgestimate	12 -30 55
		Gestodene	16 -18 55

with a longer half-life, as well as a shorter HFI, could contribute to greater effectiveness of COCs under conditions of typical use.

ACKNOWLEDGEMENTS

This study was supported by Teva. The manuscript was prepared with writing and editorial assistance from Jean-Louis Carsol, Daniel Loniewski and Tristan Blundell, all employed by Publicis Life Brands International.

Declaration of interest

: NC-B is a member of the European board of Teva/Theramex and of the French board of Gedeon Richter. CJ is a consultant for MSD, Teva and HRA Pharma. IL has a financial relationship (lecturer, member of advisory boards and/or consultant) with MSD, Teva, Gedeon Richter, HRA Pharma and Nordic Pharma. P. L. has received lecture fees, is member of advisory boards and/or consultant with Bayer, Effik, MSD and TEVA/Theramex. REN has a financial relationship (lecturer, member of advisory boards and/or consultant) with Bayer HealthCare, Boehringer Ingelheim, Eli Lilly, Gedeon Richter, HRA Pharma, MSD, Novo Nordisk, Pfizer, Procter & Gamble, Shionogi and Teva. AP is a member of the European Board of Teva without personal gain. CF has been a lecturer for Teva and Bayer HealthCare and is a member of the European scientific advisory board of Teva and Exelgyn.