Franck Malige
email: franck.malige@lis-lab.fr

Julie Patris

D Djokic

R Sousa-Lima

H Glotin

Use of recurrence plots for identification and extraction of patterns in humpback whale song recordings

Keywords: Humpback whale, song, recurrence plot, song structure, sound visualization, sound transcription, sound sequences

Humpback whale song is comprised of well-structured distinct levels of organization: combinations of sounds, repetition of combinations, and a sequence of repetitions, which have no clear silent intervals. This continuous sound output can be hard to delimit, rather, it could be interpreted as a long series of states of a system. Recurrence plots are graphical representations of such series of states and have been used to describe animal behavior previously. Here, we aim to apply this tool to visualize and recognize structures traditionally used in inferences about behavior (songs and themes) in the series of units manually extracted from recordings of humpback whales. Data from the Abrolhos bank, Brazil were subjected to these analyses. Our analytical tool has proven efficient in identifying themes and songs from continuous recordings avoiding some of the human perception bias and caveats. Furthermore, our song extraction is robust to errors coming from both manual and automated transcriptions, constructing a level of description largely independent of the first stage of analysis.

HIGHLIGHTS

• The complex hierarchical and cyclical structure of humpback whale song can be visually represented in recurrence plots • Recurrence plots of humpback whale singing provide insights into the intraindividual variation in song structures of a male • Our tool can help extracting structures, from long recordings, removing some human perceptual caveats

Introduction

Songs usually describe sequences of sounds showing some structure. Complex strings or sequences of sounds have evolved in many taxa [START_REF] Kershenbaum | Animal vocal sequences: not the markov chains we thought they were[END_REF]) and animals may sing with varying degrees of complexity. Depending on the research question, one may or may not consider the adjacent acoustic context in which such songs, or sound sequences, are delivered, i.e., some are separated by silent intervals, but some are not. These long and continuous complex sequences of sounds impose the added challenge of limiting when one biological meaningful sequence ends, and another begins. The humpback whale male song is an example of such an animal acoustic output structure which is very hard to characterize due to the issues just described.

1.1. Structure of humpback whales songs [START_REF] Schreiber | Some sounds from marine life in the hawaiian area[END_REF] was the first to describe sounds recorded in the ocean by the U. S. Navy in 1951 which were later attributed to humpback whales, Megaptera novaeangliae [START_REF] Schevill | Whale and porpoises voices: A phonograph record[END_REF]. Nonetheless, the complex structure of the humpback whale acoustic display -the song -was noticed almost a decade later by Katy and Roger Payne in 1969 and formally published by [START_REF] Payne | Songs of humpback whales[END_REF]. Three decades after that, genetic confirmation [START_REF] Darling | Interactions of singing humpback whales with other males[END_REF] supported the behavioral and morphological evidence that only males sing [START_REF] Darling | Migrations, abundance and behavior of Hawaiian humpback whales[END_REF], [START_REF] Glockner | Determining the sex of humpback whales (Megaptera novaeangliae) in their natural environment[END_REF]).

According to Payne and McVay's description, singing males emit sound units that are arranged in phrases that are repeated to form a theme. Themes are sung in a fixed order which is a song, and a song session is the continued rendition of the song. Constant changes in the song throughout the singing season, called song evolution characterize the dynamic of singing activity in humpback whales [START_REF] Payne | Progressive changes in the songs of humpback whales (Megaptera novaeangliae): A detailed analysis of two seasons in Hawaii[END_REF]. This hierarchical song structure that cycles in a fixed order was revised by [START_REF] Cholewiak | Humpback whale song hierarchical structure: Historical context and discussion of current classification issues[END_REF] to incorporate multi-level variation in song structure and to address some caveats with the original structure and order proposition. Fundamental differences between bird song literature that inspired [START_REF] Payne | Songs of humpback whales[END_REF], such as the lack of silences between song renditions, hindered the acknowledgement that boxing humpback whale song into static artificial hierarchical levels was potentially misleading inferences [START_REF] Cholewiak | Humpback whale song hierarchical structure: Historical context and discussion of current classification issues[END_REF]. Specially complicated is to arbitrarily define limits of songs and themes that would vary depending on who was describing it.

Methodology on humpback whale song elements identification (and extraction) is still advancing, as consensus on the best protocols are not yet established. As the song and its elements vary in length and order [START_REF] Cholewiak | Humpback whale song hierarchical structure: Historical context and discussion of current classification issues[END_REF], it is challenging to manually identify them (and their limits) without subjectivity. Automating the process of song structure recognition would significantly reduce the frequent human bias. As a mean of going a step closer to this goal of automation, we propose a method based on the "unit" level of the vocalization. Unit is the best defined element of the humpback whale song, and can be described as "the shortest sound entity recognized by the human ear, separated from other sounds by a short period of silence" [START_REF] Payne | Songs of humpback whales[END_REF]. Even though classifying units can be a tricky process since there is some versatility in the renditions of the same unit, yet as an entity, unit remains the most unambiguous element of the humpback whale song hierarchy.

In order to reliably define patterns of higher hierarchical level -themes and songs, as assemblies of units, we propose a new method to remove some of the human influence in defining the start and end points of songs. This is done by adopting a semi-automated protocol to detect, analyze or extract the aforementioned features from transcribed unit label strings.

Recurrence plots

Recurrence plots are used to visualize and analyze, at a global level, long series of states of a system (see definition by [START_REF] Eckmann | Recurrence plots of dynamical systems[END_REF] in the case of a general dynamical system). This tool and its graphical representation have been used in several scientific topics : first in medicine in [START_REF] Zbilut | Use of recurrence plots in the analysis of heart beat intervals[END_REF] and then in astronomy, neuroscience, mechanics, geology, climate changes (see review by [START_REF] Marwan | Recurrence plots for the analysis of complex systems[END_REF]). This tool has recently been proposed to study structures in animal movements or communication in [START_REF] Ravignani | Measuring rhythmic complexity: A primer to quantify and compare temporal structure in speech, movement, and animal vocalizations[END_REF]. It was used in acoustics -monitoring of air guns [START_REF] Miralles | Characterization of the complexity in short oscillating time series: An application to seismic airgun detonations[END_REF], and bioacoustics -shrimps sound production [START_REF] Hee-Wai | Nonlinear time series analysis of snapping shrimp sounds[END_REF]. A closer application to our problem of analyzing humpback whale songs has been to visualize structures of a music extract [START_REF] Foote | Visualizing music and audio using self-similarity[END_REF] or to cut it automatically as in [START_REF] Foote | Automatic audio segmentation using a measure of audio novelty[END_REF] or [START_REF] Paulus | Audio-based music structure analysis[END_REF]. It has recently been used to study the rhythm of humpback whale sound production in [START_REF] Schneider | Characterizing the rhythm and tempo of sound production by singing whales[END_REF], without focusing on the spectral content of the sounds.

The main topic of this paper is to apply this tool to visualize and recognize the main structures (songs and themes) in unit series of humpback whales. We apply this method to data taken in Abrolhos bank, off the northeastern coast of Brazil (see section 2.1). Theses recordings were manually transcribed into a string of units named as letters (see section 2.2). On this input, a matrix of distances, based on the Levenshtein distance is computed as done in recurrence plots (see section 3). A method for the automatic extraction of songs in the series of units is proposed (see section 4) and tested on our data set in the final section 5.

Data collection and sound units transcription

Data collection

Data was collected in the Abrolhos bank, located off the Northeastern coast of Brazil (17 o S and 38 o W) where humpback whales come during the austral winter and which is considered the main calving grounds for the species in the western south Atlantic Ocean [START_REF] Martins | Aspects of habitat use patterns of humpback whales in the abrolhos bank, brazil, breeding ground[END_REF] and [START_REF] Andriolo | The first aerial survey of humpback whales (Megaptera novaean-gliae) to estimate abundance in the breeding ground off brazil (breeding stock a)[END_REF]). During 2000 and 2001 research cruises, groups of humpback whales were sighted and monitored for acoustic activity using one HTI 90 min hydrophone connected to a portable DAT Sony TCD D-10 (sampling rate 48 kHz). Vocalizing males were then identified and located by monitoring the amplitude decrease of an individual's sounds as it surfaced to breath. Silent approaches to these focal singers were performed using a small zodiac with an improvised sail. Songs were collected from the zodiac at distances that varied from 100-500m to the focal male while its behavioral activity was continuously registered. Several recordings were made but only the best quality ones were used in our analyses. Selected recordings made in September of the year 2000 generated 3 high quality audio files (recordings #1,2,3) lasting respectively 58 minutes, 1 hour 37 minutes and 2 hours and 7 minutes. In September of 2001 the two selected audio files (recordings #4,5) were respectively 26 minutes and 1 hour and 38 minutes long. Another study of song sessions in the same place in 2000 will serve as a comparison for the data we present [START_REF] Arraut | The song of the brazilian population of humpback whale, Megaptera novaeangliae, in the year 2000 : individual song variations and possible implications[END_REF].

Sound unit transcription

Classifying units into specific types is a complex problem, since there is a large versatility in the acoustical properties of a single unit [START_REF] Janik | Pitfalls in the categorization of behaviour: a comparison of dolphin whistle classification methods[END_REF]. First, there is a seemingly infinite number of different unit types used by whales, in an ever-changing song. Second, even the units belonging to the same type may vary to a certain level throughout the same song, in the recordings of different singers, and depending on the quality of the dataset. Thus, a different final product of classification can arise from the same dataset due to differences in the methods used to determine the units. The final number of different unit types in different studies varies greatly, from twelve to more than one-hundred [START_REF] Pines | Mapping the phonetic structure of humpback whale song units: extraction, classification, and shannon-zipf confirmation of sixty sub-units[END_REF]. Challenge to group the units by type (as similar or different), opens the debate on what is the acceptable level of variation within the same unit type.

In order to overcome this difficulty, we used the context i.e. the position of the unit in the song (or more commonly in the phrases within each theme), defined by the arrangement of adjacent units [START_REF] Green | Recurring patterns in the songs of humpback whales (Megaptera novaeangliae)[END_REF]. The context has proven as a good way to help determine the unit type [START_REF] Cholewiak | Humpback whale song hierarchical structure: Historical context and discussion of current classification issues[END_REF]. For this work, all recordings were inspected by hand. Spectrograms were created using 1024 point FFTs, Hann window, 43.1 Hz resolution and 50% overlap (created by software Raven pro 1.5 -Cornell Lab of Ornithology, Ithaca, NY, Program ( 2014)), and subjected to aural and visual inspection by two trained analysts. Each analyst separately inspected and labeled the whole dataset. This procedure is a common protocol adopted by humpback whale song researchers [START_REF] Payne | Songs of humpback whales[END_REF], [START_REF] Darling | Convergence and divergence of songs suggests ongoing, but annually variable, mixing of humpback whale populations throughout the north pacific[END_REF]). Our protocol for unit classification also takes context into consideration, i.e. where the unit is placed, so that other hierarchical levels of organization can help define how much variation is allowed in a single unit type. Note that even when studies carry on unit annotation using computational methods, the results are usually validated using manual classification [START_REF] Pace | Subunit definition and analysis for humpback whale classification[END_REF], [START_REF] Garland | Quantifying humpback whale song sequences to understand the dynamics of song exchange at the ocean basin scale[END_REF], [START_REF] Allen | Using self-organizing maps to classify humpback whale song units and quantify their similarity[END_REF]).

The labeling was done in the following way: every unit type was attributed an alphabet letter based on its distinctiveness from other units [START_REF] Darling | Convergence and divergence of songs suggests ongoing, but annually variable, mixing of humpback whale populations throughout the north pacific[END_REF]. Analysts considered visually and auditory perceptual characteristics for classification such as unit tonal or pulsed quality, its pitch content and frequency modulation pattern, its duration, and its placement within phrases (context). Each time the specific unit would arise in the recording, it was labeled in Raven, according to its type, minding its context (adjacent units). The final product of every separate recording would be a list of N consecutive letters, the way the units appeared in that specific humpback whale vocalization (see figure 1 and supplementary material #1). This list of letters (units) will, in the later steps of the method, serve as an input for computing recurrence plots (section 3). Finally, an inexperienced analyst, using another software (Audacity, using a 8192 points FFT, Hanning window, 99% overlap) independently transcribed part of recording #3 into a series of units as a comparison with the first transcription and a check on the robustness of our method (see section 5.2).

The Levenshtein distance recurrence plots

Definition of the Levenshtein distance recurrence plot of order n

To visualize the structures contained in a recording, we compute a distance matrix, using OCTAVE [START_REF] Eaton | GNU Octave version 3.0.1 manual: a high-level interactive language for numerical computations[END_REF]) in the following way. Each recording is transcribed as a string of N letters representing sound units (see section 2.2). Then we define extract i as an n-letter extract beginning with unit number i. The length of the extracts n is taken much smaller than the total length of the string N . The Levenshtein distances d ij = Levenshtein distance (extract i , extract j ) between all pairs of n-letters extracts are computed. The Levenshtein distance between two strings is the minimum number of insertions, deletions and substitutions necessary to transform one string into the other [START_REF] Levenshtein | Binary codes capable of correcting deletions, insertions, and reversals[END_REF]. Note that the computation of other types of distance matrix could be achieved choosing other distances between two strings of letters : Jaro-Winkler, Damerau-Levenshtein and Hamming distances for example.

For convenience, we defined a correlation index by :

c ij = n -d ij n
Thus the coefficients of the matrix satisfy c ij ∈ [0; 1] . The coefficient 0 means maximum Levenstein distance and thus minimal correlation. The coefficient 1 means zero Levenstein distance and thus maximal correlation.

The resulting matrix (c ij ) is a square matrix of size Nn + 1 ≃ N , symmetrical, and has ones on its diagonal (maximal correlation between one element and itself). As an example, figure 2 shows the graphical representation of this Levenshtein distance matrix of recording #2 (a total of N =1260 units), with 5-letters extracts (n=5).

The result is a recurrence plot of a dynamical system (as defined in [START_REF] Eckmann | Recurrence plots of dynamical systems[END_REF]) in which a state is a vector of n letters. We call this special recurrence plot the Levenshtein distance recurrence plot of order n (LDRP n ). The value of n is chosen by the analyst. In our data, the visual information is basically the same when n varies (see figure 3). For low values of n, there are few levels of Grey. In figure 4, we annotated the two different scales of structures found in all recordings of this study. First, a global pattern can be seen that is reproduced periodically (the "period" can be seen in the horizontal or vertical regularly spaced correlations of parts of the recording). We thus define a song as the largest repeated structure that can be found in a recurrence plot. The song is repeated with a high level of similarity, apparent in the non-zero correlation with other songs. This structure is obvious in our representation, it is also in accordance with the general literature on humpback whale bioacoustics [START_REF] Payne | Songs of humpback whales[END_REF], [START_REF] Mercado-Iii | Stereotypical sound patterns in humpback whale songs : Usage and function[END_REF] and [START_REF] Cholewiak | Humpback whale song hierarchical structure: Historical context and discussion of current classification issues[END_REF]).

A second scale of structure is visible in the LDRP n : each song is itself composed of several auto-similar parts or 'squares' (figure 4). These parts are not repeated within a song (square 1 does not correlate with others during the same song). We define a theme as the string of units corresponding to a "square", or self-similar part of a song. With this definition, we are consistent with general literature on humpback whales [START_REF] Mercado-Iii | Stereotypical sound patterns in humpback whale songs : Usage and function[END_REF] which states that a theme is a repetition of similar phrases composed of similar sound units. Nevertheless, it is difficult to see distinctly visual signature of phrases in the recurrence plot. Anyway, the separation of themes in squares could help a humpback whale song analyst to identify phrases in each series of units corresponding to a square.

Automation of the extraction of songs

Based on the computation of the distance recurrence plots, we wrote a routine that separates songs in a fully automatic way. We defined the song as a structure that is repeated with a good amount of similarity, with the largest possible scale in our recording. The approximate size (in number of units) of a song can be estimated by a 2 dimensional FFT of the Levenshtein distance recurrence plot of order n (see figure 5, left). Then, the following algorithm for song extraction is proposed:

(1) Compute the Levenshtein distance recurrence plot for a given value of n (2) Perform a 2D Fast Fourier transform of the matrix considered as an image. A peak at a frequency inverse to the mean length of the song can be seen (figure 5, left). The abscissa of this peak is then measured to obtain an order of magnitude of the lengths of the songs.

(3) Read the first line of the Levenshtein distance recurrence plot and select the columns with non-zero correlation. Sum all these columns to improve the signal to noise ratio. The resulting column is represented on figure 5, center. (4) Find sudden increases or upward steps in the resulting column using convolution with a step filter (figure 5, right). Keep only the steps compatible with the average song size predicted by the 2D Fourier transform. Here, we defined the margin of acceptability at one third of the average size given by FFT analysis. ( 5) Extract the units transcribed from the recording between two upward steps: this is a song (figure 6).

Results : song extraction

Extraction of songs in series of units from Abrolhos Archipielago in 2000/2001

We applied the algorithm presented in the previous section to the five recordings of our study. Visually, the same features corresponding to our definition of songs and themes appear in each Levenshtein distance recurrence plot. The recording #4 is too short and do not present a complete song and our routine consequently did not extract any song. In all the other recordings, the extraction of songs was achieved successfully.

In recording #5, only one song is present.

We compared the extraction of songs carried out by our routine, based on the Levenshtein distance recurrence plot, and songs delimited by a human expert. Comparison was performed for the recording #3. The analyst followed the instructions described in [START_REF] Cholewiak | Humpback whale song hierarchical structure: Historical context and discussion of current classification issues[END_REF], the choice for the beginning point of the first song was the first complete theme (composed of the same type of letters). The extraction of the songs (presented in supplementary material #2) is equivalent by both methods. The only difference sits in a few transitional units (less than 5 units per song).

It is interesting to note that recurrence plots can show transitional phrases, which are combinations of units from the previous theme with ones from the next theme the male sings [START_REF] Frumhoff | Aberrant songs of humpback whales (Megaptera novaeangliae): Clues to the structure of humpback songs[END_REF], [START_REF] Payne | Progressive changes in the songs of humpback whales (Megaptera novaeangliae): A detailed analysis of two seasons in Hawaii[END_REF], [START_REF] Cholewiak | Humpback whale song hierarchical structure: Historical context and discussion of current classification issues[END_REF]). Transitional phrases appear in our recurrence plots as blurry areas around self-similar "squares" that represent the themes. It is particularly clear in figure 6 (right), where the three squares corresponding to themes number 1,2 and 3 inter-penetrate each other. On the contrary, the transition between themes in figure 6 (left) is quite abrupt : the squares are well separated. Figure 7. Percentage of each unit in the four song occurrences of recording #1 from Abrolhos archipelago, 2000 (colors are red, blue, green and black for song occurrences 1,2,3,4 respectively). The first occurrence of the song in the recording (see figure 4), in red in this figure is quite different from the other occurrences in term of its units, due to boat noise that prevented an accurate identification of several units.

Once the songs are extracted, the automatic measurement of the songs parameters is easily done. As an example, we automatically computed songs' length, alphabet (set of different unitsor letters -used in the song), and importance of units (% of the number of occurrence of a particular unit in a song occurrence compared to the length of this song). These results are presented in table 1 and figure 7.

Figure 7 gives the percentage of each unit in the song occurrence of recording #1. The first occurrence of the song, in red (or circles), is quite different from the other songs. Indeed, during the first song occurrence a series of units are masked by boat noise and were labeled with a special letter (H) in place of a whole theme containing the letters J,K,L,M,O,P. It can be seen in the Levenshtein distance recurrence plot where the first theme of the first rendition of the song does not correlate with any of the themes of other renditions (figure 4). However, the recurrence plot still enabled us to extract the song structure accurately.

The results of table 1, where the mean length of all the songs analyzed is 255, compares well with the results of [START_REF] Arraut | The song of the brazilian population of humpback whale, Megaptera novaeangliae, in the year 2000 : individual song variations and possible implications[END_REF], working with the same data but with manual analysis, where the mean song length is around 250-300 units.

Robustness of the extraction method

In order to test the robustness of our method of songs' extraction, we first checked that the extraction is not dependent on the order n of the Levenshtein distance recurrence plot LDRP n . For each recording (#1 to #3) the number of songs extracted was the same for n going from 4 to 10.

We then checked that the extraction is not heavily dependent on the unit tran- scription. For recording #3, an inexperienced analyst transcribed part of it in a string of units. Even though the confirmed analysts transcribed 1614 units and the inexperienced one 1855, the recurrence plots have the same appearance (figure 8). The automatic extraction of the song was then performed on these two strings of units and gave similar results : four songs are extracted in each case. The ratio r u of the length of the song extracted from the series of units produced by the confirmed analysts and the length of the song extracted from the series of units produced by the inexperienced analyst (measured in unit) is very stable for each song extracted (r u = 0.86 ± 0.006).

We also checked that the determination of the start and end of an automatically extracted song is consistent between the human transcriptions (with a ratio between songs durations (measured in seconds) of r t = 1.01 ± 0.04 between the two human analysts). Thus, our method of song extraction is remarkably robust to differences in unit labeling due to the subjectivity or lack of training of the analyst.

In addition, we checked the robustness of our method to accidental errors in units transcription, not considering whether this was done automatically or manually. We applied the extraction routine to the strings of units of recording #1 and #2, gradually and randomly changing a percentage of these units (replacing it randomly by a unit from the same recording). For each percentage of error, the test was repeated 20 times and the number of extracted songs was noted. The result is visible in figure 9 where, for each percentage of errors in unit identification, the average number of extracted songs is plotted, along with the error bar corresponding to the variation across the 20 draws. We see that for less than 5% of randomly changed units, the results are very coherent with the original recording. Even as the rate of error grows, the number of extracted songs stays close to the nominal one (1 song of difference for up to 20% of errors).

Last, we applied the extraction routine to strings of units of recording #1 where we gradually and randomly removed a percentage of these units. In this case, until 40% of units were removed, the routine extracts the correct number of songs for more than 90% of the tries. Therefore, our extraction method is robust to missing units during the transcription, for example when the signal to noise ratio of the vocalizations of humpback whale is low. However, this was done for randomly chosen units and do not account for systematic errors (such as would happen if one type of unit only would be lost in the noise, which is not an improbable case).

Discussion

Use of recurrence plots

Recurrence plots are a very visual way of representing the sequence of units, allowing immediate global identification of the structures of the highly organized vocalization emitted by male humpback whales, presented in the precedent sections. It helps with getting a qualitative view of their variability as well as the type of transition (abrupt versus gradual change) between these structures. It could be used as a valuable tool for every highly structured type of sound emission by animals and seems particularly well fitted to study the ever changing structures of humpback whales songs.

Recurrence plots can also be used for automatic unsupervised selection of different structure levels in the recording translated as a series of units. The results of the previous section indicate that our method of extraction is quite robust to errors during manual or automatic transcription of the sound units into a string of letters. The quantification of errors detected for song unit classification systems in studies such as [START_REF] Dunlop | The social vocalization repertoire of east australian migrating humpback whales (Megaptera novaeangliae)[END_REF], [START_REF] Ou | Automated extraction and classification of time-frequency contours in humpback vocalizations[END_REF] or [START_REF] Rekdahl | Culturally transmitted song exchange between humpback whales (Megaptera novaeangliae) in the southeast atlantic and southwest indian ocean basins[END_REF] is usually from 10% to 20% which compares well with the percentage of errors that still enables us to automatically extract songs.

The use of recurrence plot to extract songs is still a work in progress but these results are encouraging. Even in the situation of different criteria for unit labeling in different recordings, the song session structure will probably still be visible. This is an additional benefit of the proposed recurrence plot methodology, as it undermines differences in unit labeling among studies. The plot gives a representation of each recording, rather independent on the string it was calculated from. In this way, the recurrence plots provide standardized limits of patterns in the recordings that can be further explored and compared.

This automated song extraction is the first step for many more studies regarding song analysis, and comparison between songs. The automated extraction of other structures (such as themes) could be performed based on this tool but is not as straightforward as songs extraction. Some of the difficulties of theme extraction are : blurred or imprecise transition between themes, unclear definition of a theme, very short or evanescent themes. Thus any method of automated theme extraction would probably be rather ad hoc, which is the reason why we didn't perform it on our limited set of data.

Finally, a matrix comparing two series of units extracted from two different recordings can be computed which shows the similarity between two recordings in term of song rendition. This type of analysis could enable people to compare quickly two song bouts distant in time or space.

Large scale automatic treatment of humpback whales' recordings

This study is a step towards automatic treatment of large scale recordings. Automatic tools to transcribe sounds into a string of letters such as the one developed in [START_REF] Dunlop | The social vocalization repertoire of east australian migrating humpback whales (Megaptera novaeangliae)[END_REF], [START_REF] Glotin | New automatic classification for humpback whale songs[END_REF], [START_REF] Rickwood | Methods for automatically analyzing humpback song units[END_REF], [START_REF] Pace | Hidden markov modeling for humpback whale (Megaptera novaeangliae) call classification[END_REF], [START_REF] Ou | Automated extraction and classification of time-frequency contours in humpback vocalizations[END_REF], [START_REF] Razik | Sparse coding for efficient bioacoustic data mining: Preliminary application to analysis of whale songs[END_REF], [START_REF] Bartcus | Hierarchical dirichlet process hidden markov model for unsupervised bioacoustic analysis[END_REF] or [START_REF] Rekdahl | Culturally transmitted song exchange between humpback whales (Megaptera novaeangliae) in the southeast atlantic and southwest indian ocean basins[END_REF] could be used beforehand. Then our method could be applied to extract, classify and compare songs and their features along with other approaches to visualize the structures of humpback whale's songs.

Finally, analyzing humpback whale song structures with recurrence plots would be difficult in the case of a recording where many singers are vocalizing together. In this case, the separation of these singers is a challenge that has to be addressed. Data collection for this work was possible thanks to the support from Fundação O Boticário de Proteção a Natureza / MacArthur Foundation and the Society for Marine Mammalogy (Small-Grants-in-Aid of Research to RSL). We thank the crew of the boat Tomara and Instituto Baleia Jubarte (PBJ) for logistic support. Projeto Baleia Jubarte is sponsored by Petróleo Brasileiro S. A. (Petrobras). We would like to thank Luana Pini for help in data processing and Leonardo Diniz Soares Eugênio who computed the raven tables used to transcribe recordings into series of units .

Figure 1 .

 1 Figure 1. Time (min:sec) / frequency (kHz) representation of an extract of recording #3 (from Abrolhos Archipelago 2000), 1024 point FFTs, Hann window, 43.1 Hz resolution and 50% overlap. Unit transcription into letters is signaled above the boxes

Figure 2 .

 2 Figure 2. Example of a Levenshtein distance recurrence plot (5 letter extracts) created form recording number #2 from Abrolhos Archipelago (2000). The number of units of this recording is N = 1260. The coefficients of the matrix denote correlation between 5 letter extracts (0 is no correlation, 1 is maximal correlation)

Figure 3 .

 3 Figure 3. Recording #5 (from Abrolhos Archipelago 2001) : Levenshtein distances recurrence plots for different values of the length n of the extract (2,7,12 and 17)

  For high values of n, the resulting figure is more blurred (see figure 3), and the computation of Levenshtein distance is rather time consuming (the time of computation of the distance between two strings of n letters is proportional to n 2 (Wagner and Fischer 1974)). The computation of the LDRP n for n = 17 and N = 800 in figure 3, takes around one hour in a domestic computer. The parameter n can be adapted depending on the use of the LDRP n : visual analysis, extraction of structures, etc...3.2. Visual identification of main structures in the recurrence plot

Figure 4 .

 4 Figure 4. Recording #1 from Abrolhos archipelago, 2000 : Levenshtein distances recurrence plot commented (n=5). The main structures of humpback whale sound production appear : songs in blue, themes in yellow.Figures 2, 3 and 4 show obvious structures in the recording. On the diagonal of the matrix, squares represent auto-similar structures. Rectangles out of the diagonal show whether theses structures correlate with each other. The high contrast of this representation is due to the sparsity of input representation (letters) added to the efficiency of the distance operator.In figure4, we annotated the two different scales of structures found in all recordings of this study. First, a global pattern can be seen that is reproduced periodically (the "period" can be seen in the horizontal or vertical regularly spaced correlations of parts of the recording). We thus define a song as

Figure 5 .

 5 Figure 5. Steps in the extraction of the songs. Left : Step 2 of the algorithm, extract of the 2D fast Fourier transform of the Levenshtein distance matrix (for n = 5, recording #1). A peak is visible at a frequency of about 0.045 units -1 (which means a scale of 200 to 250 units of the recording) Center : Step 3 of the algorithm, the horizontal sum of selected columns of the recording 1, n=5. Right : Step 4 of the algorithm, each peak shows the transition between two songs.

Figure 6 .

 6 Figure 6. Two songs automatically extracted. Left : Recording #1 from Abrolhos archipelago, 2000 : third song automatically extracted from figure 4. In order to see clearly the song, the frame of the figure is chosen a bit larger for representation. Right : Recording #2 from Abrolhos archipelago, 2000 : zoom on the third song occurrence in the Levenshtein distance recurrence plot of figure 2. Different types of transition between themes appear.

Figure 8 .

 8 Figure 8. Recurrence plots of part of the recording #3 from Abrolhos archipelago, 2000, with n = 5. Left : transcription into units by confirmed analysts, Right : transcription into units by an inexperienced analyst

Figure 9 .

 9 Figure 9. Number of song correctly found in function of the percentage of randomly changed units. The test is done on 20 random tries and the error bar quantifies the distribution of the results over the 20 tries. Left : Recording 1 from Abrolhos archipelago, 2000, Right : Recording #2 from Abrolhos archipelago, 2000

Table 1 :

 1 Mean and standard deviation of the songs' length for recordings #1,2,3 and 5

	Recording year nb of songs mean length (in units) standard dev. (in units)
	#1	2000	4	238	22
	#2	2000	6	177	25
	#3	2000	8	301	72
	#5	2001	1	438	none

We thank the research program BRILAM STIC AmSud 17-STIC-01, EADM GDRS CNRS MADICS and ANR-18-CE40-0014 SMILES. The collaboration between the authors has been promoted by the Workshop Listening for Aquatic Mammals in Latin America (LAMLA) sponsored by UFRN, CAPES (AUXPE 541/2016) and the U. S. Office of Naval Research (Award number N62909-16-1-2144).


Mathieu Michaut

Alexandre Steffen

Jean-Marie Contreras

Christophe Morice

Aurélie Paulen

Isabelle J Schalk

Patrick Plésiat

Gaëtan L A Mislin

E. faecium) ≥ 0.5 µg.mL -1

Keywords:

MIC (S. epidermidis)

≥0.06 µg.mL -1 MIC (S. aureus) ≥ 0.125 µg.mL -1 MIC (

a CNRS, UMR7242 Biotechnologie et Signalisation Cellulaire, 300 Boulevard Sébastien Brant, 67400 Illkirch-Graffenstaden, France. b Université de Strasbourg, Institut de Recherche de l'Ecole de Biotechnologie de Strasbourg (IREBS), 67400 Illkirch-Graffenstaden, France. c Laboratoire de Bactériologie, UMR 6249 CNRS Chrono-Environnement, Faculté de Médecine-Pharmacie, Université de Franche-Comté, Besançon, France. d Prestwick Chemical, PC SAS, 220 Boulevard Gonthier d'Andernach, 67400 Illkirch-Graffenstaden, France.

--- Corresponding author. Tel.: +33-368854727; e-mail: mislin@unistra.fr Gold is a precious metal and the symbol of continuity in time and, by extension, of health and eternal youth. For this reason, many civilizations have made use of gold in their traditional medicines. For example, Ayurvedic medicine has used the properties of metals to treat many diseases over thousands of years. Gold has been used to treat cancer, vascular disorders, arthritis and fertility problems. [START_REF] Beaudet | Comparative study on cellular entry of incinerated ancient gold particles (Swarna Bhasma) and chemically synthesized gold particles[END_REF][START_REF] Shah | Antioxidant/restorative effects of calcined gold preparations used in Indian systems of medicine against global and focal models of ischaemia[END_REF][START_REF] Brown | Nanogoldpharmaceutics[END_REF][START_REF] Jain | Seminal gold content in healthy fertile men in India[END_REF] In occidental medicine, chrysotherapy (therapy based on gold compounds) is already used to treat rheumatoid arthritis, 5 asthma 6 and skin diseases [START_REF] Thomas | Gold therapy and its indications in dermatology[END_REF] and promising therapeutic strategies for cancer are also being investigated. [START_REF] Shaw | Gold-Based Therapeutic Agents[END_REF][START_REF] Hickey | Mitochondria-targeted chemotherapeutics: the rational design of gold(I) N-heterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols[END_REF][START_REF] Köster | A spontaneous gold(I)-azide alkyne cycloaddition reaction yields gold-peptide bioconjugates which overcome cisplatin resistance in a p53-mutant cancer cell line[END_REF][START_REF] Chen | A new anti-cancer strategy of damaging mitochondria by pro -apoptotic peptide functionalized gold nanoparticles[END_REF] Gold(I) derivatives have also been reported to have antibiotic activity. [START_REF] Glišić | Gold complexes as antimicrobial agents: an overview of different biological activities in relation to the oxidation state of the gold ion and the ligand structure Dalton Trans[END_REF][START_REF] Mora | Recent advances in gold-NHC complexes with biological properties[END_REF][START_REF] Stenger-Smith | Cationic Au(I) complexes with aryl-benzothiazoles and their antibacterial activity[END_REF][START_REF] Schmidt | Biscarbene gold(i) complexes: structure-activity-relationships regarding antibacterial effects, cytotoxicity, TrxR inhibition and cellular bioavailability[END_REF][START_REF] Fernández | Antibacterial properties of water-soluble gold(I) N-heterocyclic carbene complexes[END_REF][START_REF] Mizdal | The antibacterial and anti-biofilm activity of goldcomplexed sulfonamides against methicillin-resistant Staphylococcus aureus[END_REF][START_REF] Abdelkhalek | Antibacterial and antivirulence activities of auranofin against Clostridium difficile[END_REF][START_REF] Thangamani | Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens[END_REF][START_REF] Patra | Small organometallic compounds as antibacterial agents[END_REF] The conjugation of approved antibiotics with metal complexes, like ferrocenyl, already led to interesting antibacterial compounds. [START_REF] Edward | A new class of semi-synthetic antibiotics: ferrocenyl-penicillins and-cephalosporins[END_REF] However, the conjugation of an antibiotic with gold(I) has never before been reported. We describe here the synthesis and biological evaluation of unprecedented organo-gold(I) derivatives of -lactam antibiotics.

Phosphane gold(I) was added to antibiotics by 1,3-dipolar cycloaddition, with a phosphane gold(I) azide complex and a penam scaffold functionalized with a terminal triple bond. During this reaction, an additional migration of the phosphane gold(I) moiety occurs, resulting in the formation of a 1,2,3triazole ring with the gold center in position 5. [START_REF] Köster | A spontaneous gold(I)-azide alkyne cycloaddition reaction yields gold-peptide bioconjugates which overcome cisplatin resistance in a p53-mutant cancer cell line[END_REF][START_REF] Partyka | Carbon-Gold Bond Formation through [3 + 2] Cycloaddition Reactions of Gold(I) Azides and Terminal Alkynes[END_REF] In the final conjugate, the gold(I) center is connected to the antibiotic through a -gold-carbon bond. The (trimethylphosphine)Au(I) azide 5, the (triethylphosphine)Au(I) azide 6, the (dimethylphenyl)Au(I) azide 7 and the (triphenylphosphine) Au(I) azide 8 were prepared according to a published procedure. [START_REF] Patra | Small organometallic compounds as antibacterial agents[END_REF] Commercially available phosphine gold(I) chlorides 1, 2, 3 and 4 were treated with thallium(I) acetylacetonate, and the resulting phosphine Au(I) acetylacetonates were treated with trimethylsilyl azide to obtain the expected reagents 5, 6, 7 and 8. Three different chemical functional groups were evaluated for connection of the phosphine-gold(I)-triazole moiety to the ampicillin molecule: carbamate (compounds 17 to 19), amide (compound 21) and urea (compound 22). Ampicillin 13 was thus treated with propargyl chloroformate, the pentafluorophenyl ester 10 of pentynoic acid 9, or the p-nitrophenyl chloroformate 12 prepared from propargylamine 11. The resulting alkynes 14, 15 and 16 were then treated with gold(I) azides 5 to 8 to obtain the expected conjugates 17 to 22. (Scheme 1).

A R T I C L E I N F O A B S T R A C T

The fully organic penam 25 was prepared, to made it possible to distinguish the relative importances of the gold ion and the 1,2,3-triazole moiety for antibacterial activity. For this purpose, the 3-(1H-1,2,3-triazol-5-yl)propanoic acid 23 was synthesized according a published procedure. [START_REF] Thomson | Development of autotaxin inhibitors: A series of zinc binding triazoles[END_REF] The carboxyl group of 23 was then activated in the form of a pentafluorophenyl-ester 24 and reacted with ampicillin 13 to generate the expected penam derivative 25 (Scheme 2). The antibacterial properties of the chryso-lactams 17 to 22 were then evaluated with characterized microorganisms and compared with those of ampicillin (Amp) 13, alkyne 14 and fully organic penam 25. Most of the pathogenic bacterial species used in the evaluation assays were Gram-positive (Staphylococcus aureus, Staphylococcus epidermis, Enterococcus faecalis and E. faecium). Minimal inhibitory concentrations (MIC) were also determined for Escherichia coli, a Gram-negative bacterium (Table 1). However, this bacterium turned out to be relatively resistant to the chryso-lactams in the range of concentrations tested. The resistance of Gram-negative bacteria to gold(I) derivatives has been reported to be due mostly to the low permeability of the bacterial outer membrane. Indeed, the presence of a membrane-permeabilizing agent greatly increases the antibacterial activity of gold(I) drugs against Gram-negative pathogens. [START_REF] Thangamani | Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens[END_REF] By contrast, compounds 17 to 22 proved to be more effective than their non-metallic parental compounds against several pathogenic Gram-positive strains. The size of the phosphine ligand of the gold(I) derivative of ampicillin appeared to be crucial for biological activity. Indeed, compound 20, with its triphenylphosphine ligand, generally had lower activity than ampicillin. A decrease in ligand size (dimethylphenylphosphine) increased antibiotic activity, but compound 19 had a level of activity similar to that of ampicillin for only a subset of bacterial strains. The optimal ligand was triethylphosphine, as compound 18 was more active against Staphylococcus species and several Enterococcus strains than ampicillin. Surprisingly, however, the use of a trimethylphosphine ligand (compound 17) did not further increase antibacterial activity against Gram-positive bacteria but resulted in moderate activity against E. coli. This finding is consistent with recently reported data for antibacterial gold(I) derivatives bearing trimethylphosphine as a ligand. [START_REF] Holmes | Gold (I)-phosphine compounds as anti-bacterial agents WO2015181550A[END_REF][START_REF] Wu | Synthesis and Structure-Activity Relationship Study of Antimicrobial Auranofin against ESKAPE Pathogens[END_REF] Conjugates 18, 21 and 22 had similar antibacterial profiles, demonstrating that the nature of the linker attaching the phosphine-Au(I)-triazole moiety to the penam scaffold does not greatly influence biological activity. Finally, purely organic penam derivatives 14 and 25 were less active than ampicillin, highlighting the negligible contribution of the triazole moiety and the major importance of the gold(I) center for antibacterial activity (Table 1). Chryso-antibiotic 18 thus appeared to the most promising of the gold(I) compounds synthesized. Other triethylphosphine gold(I) derivatives, such as auranofin, have already found uses in human medicine. [START_REF] Wu | Synthesis and Structure-Activity Relationship Study of Antimicrobial Auranofin against ESKAPE Pathogens[END_REF][START_REF] Skalska | The Rheumatoid Arthritis Drug Auranofin Has Significant in Vitro Activity in MCL and DLCL and Is Synergistic with a Glutathione Depleting Agent[END_REF] The MICs of compound 18 were low for wild-type susceptible strains of S. aureus, S. epidermis and E. faecium. More importantly, they were not affected by the most prevalent mechanisms of resistance tolactams (e.g., methicillin and oxacillin) in Staphyloccus sp. or known mechanisms of resistance to glycopeptides (e.g., vancomycin) in Enterococcus sp. These data suggest that crossresistance between chryso-lactams and drugs classically used against Gram-positive bacteria should not emerge easily. The activity of compound 18 was similar to that of ampicillin with reference strains of E. faecalis.

The toxicity of gold(I) is a critical issue for the further development of molecules of therapeutic potential, such as chryso-antibiotics. The cytotoxicity of compound 18 was assessed on healthy human hepatocytes and compared with that of ampicillin 13, used as a control. Compound 18 exhibit an EC50 of 16.39 ± 0.67 µM. In detail compound 18 did not affect cell viability at concentrations of up to 10 μM (7.9 µg.mL -1 ), but was toxic at higher concentrations, with only 40% viable cells at 22.5 μM (17.7 µg.mL -1 ) and 10% at 25 μM (19.7 µg.mL -1 ). By contrast, ampicillin remained non-cytotoxic over the entire range of concentrations tested (see ESI). These data clearly characterize an interesting therapeutic window for compound 18 even the strong antibacterial activity of this molecule is counterbalanced by a peripheral toxicity to host cells.

The current antibiotic arsenal mostly consists of purely organic compounds, with carbon, hydrogen, sulfur, oxygen and nitrogen atoms involved in the inhibitory processes. However, the emergence and rise of increasingly resistant bacterial strains, has led to the use of last-resort antibiotics known to have significant adverse effects. [START_REF] Kelesidis | The safety of polymyxin antibiotics[END_REF][START_REF] Zavascki | Nephrotoxicity of Polymyxins: Is There Any Difference between Colistimethate and Polymyxin B?[END_REF] Table 1. Minimal inhibitory concentration (MIC) in µg.mL -1 of ampicillin (Amp), penam 14 and gold(I) conjugates 17 to 22 and 25 for a panel of wild-type and resistant strains. a Bacteria tested Amp( 13 Compounds containing other elements from the periodic table have been developed against cancer, but other applications of these compounds in the treatment of bacterial infections have been limited by peripheral toxicity issues, particularly for molecules administered intravenously. Organometallic compounds should be more and more competitive in terms of the benefit vs risk balance for the patient. Chryso-lactams proved to have a specific activity on Gram-positive microorganisms and the low permeability of Gram-negative bacteria outer envelope seems to impair the antibacterial efficiency of many gold(I) compounds. [START_REF] Thangamani | Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens[END_REF] Therefore, the vectorization of organometallic drugs using nutrient uptake systems, should increase their therapeutic potential (antibacterial activity, cytotoxicity). [START_REF] Laurent | Applying a Trojan Horse Strategy to Ruthenium Complexes in the Pursuit of Novel Antibacterial Agents[END_REF][START_REF] Sauvageot | Antipseudomonal activity enhancement of luminescent iridium(III) dipyridylamine complexes under visible blue light[END_REF] This article describes the synthesis of the first conjugates of lactam antibiotics with phosphine-gold(I) complexes. In these conjugates, ampicillin was connected to the metal complex through a 1,2,3-triazole linker. Triethylphosphine derivative 18 proved to be an effective molecule against Gram-positive bacteria, with an activity 120 times that of ampicillin with certain strains of S. aureus, S. epidermis and E. faecium. This compound escapes the most common mechanisms of resistance to -lactams and glycopeptides. With, for certain bacterial strains, a selectivity index ca 130, compound 18 will serve as lead molecule in the development of the next generation of chryso-lactams. However, the mode of action of compound 18 and its ability to select resistant isolates should now be investigated. This information will be crucial for the further development of chryso-antibiotics with better antibacterial properties, manageable toxicity profile and competitive with strategies based on antibacterial gold nanoparticles. [START_REF] Brown | Nanoparticles functionalized with ampicillin destroy multipleantibiotic-resistant isolates of Pseudomonas aeruginosa and Enterobacter aerogenes and methicillin-resistant Staphylococcus aureus[END_REF][START_REF] Cui | The molecular mechanism of action of bactericidal gold nanoparticles on Escherichia coli[END_REF]

Scheme 1 .

 1 Scheme 1. Synthesis of phosphine gold(I) azides 5, 6, 7 and 8, and synthesis of ampicillin-Au(I) conjugates 17 to 22. i. Tl(acac), toluene, 20°C. ii. TMSN3, toluene/MeOH, 20 °C. iii. C6F5OH, EDCI, CH2Cl2, 20°C. iv. pNO2PhOCOCl, NEt3, THF 0°C to 20°C. v. Propargyl chloroformate, 10 or 12, NEt3, THF/H2O, 0°C to 20°C. vi. 5, 6, 7 or 8, THF, 20°C.

Scheme 2 .

 2 Scheme 2. Synthesis of ampicillin analog 25. i. C6F5OH, EDCI.HCl, CH2Cl2, 20°C. ii. NEt3, THF/H2O, 20°C.

a

  The MIC values displayed are the highest values obtained in two independent experiments. b Not Assessed (NA). c Susceptible strain. d -lactam-resistant strain. e Glycopeptide-resistant strain.

Acknowledgments G.L.A.M. and I.J.S. thank the CNRS for global financial support and TTO Conectus for investment in the valorization and patenting process. A.P thanks the Ministère de l'Education National, de l'Enseignement Supérieur et de la Recherche (MENESR) for a PhD fellowship. M.M. thanks Nathalie Wagner for purifying compounds by semi-preparative HPLC and the entire Prestwick Quality Control team for compound analysis. We thank Mr Loïs Andrey for his technical assistance with drug susceptibility testing. Authors finally thank Pr. Frédéric Laurent (Hospices Civils de Lyon/Université de Lyon I, Lyon, France) and Pr. Vincent Cattoir (CHU Rennes/Université de Rennes 1, Rennes, France) for providing respectively Staphylococcus and Enterococcus strains.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in the present article.

Appendix A. Supplementary Data

Protocols and analytical data concerning compounds 14 to 22 and compound 25, experimental conditions for Minimum Inhibitory Concentration assays, additional MICs on resistant Staphylococcus strains for compound 18 compared to ampicillin, concentrations assessments for the in vitro cytotoxicity of compound 18 and ampicillin can be found online DOI xxxxxxxx


Nicolas Perin

Claire Roger

Grégory Marin

Nicolas Molinari

Alexandre Evrard

Jean-Philippe Lavigne

Saber Barbar

Pierre Géraud Claret

Caroline Boutin

Laurent Muller

Vancomycin Serum Concentration after 48 h of Administration: A 3-Years Survey in an Intensive Care Unit

published or not. The documents may come


Hamouda Abassi

Helena Huguet

Marie-Christine Picot

Marie Vincenti

Sophie Guillaumont

Annie Auer

Oscar Werner

Gregoire De

La Villeon

Kathleen Lavastre

Arthur Gavotto

Pascal Auquier

Pascal Amedro
email: p-amedro@chu-montpellier.fr

Health-related quality of life in children with congenital heart disease aged 5 to 7 years: a multicentre controlled cross-sectional study

Keywords: Health-related quality of life, Patients-reported outcomes, Young children, Congenital heart disease

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Background

In the past two decades, great advances in medical and surgical treatments for patients with congenital heart diseases (CHD) have significantly increased their life expectancy, and currently most children with CHD are expected to reach adulthood without any severe physical impairment [START_REF] Spector | Trends in long-term mortality after congenital heart surgery[END_REF][START_REF] Amedro | Cardiopulmonary fitness in children with congenital heart diseases versus healthy children[END_REF]. Consequently, patients, healthcare professionals and researchers in paediatric cardiology have shown a growing interest in patient related outcomes (PROs), such as health-related quality of life (HRQoL) [START_REF] Boyle | The school age child with congenital heart disease[END_REF].

Previous studies have reported that many children with CHD were concerned with behavioural, emotional, or cognitive issues [START_REF] Marino | Neurodevelopmental outcomes in children with congenital heart disease: evaluation and management: a scientific statement from the American Heart Association[END_REF][START_REF] Mccusker | A randomized controlled trial of interventions to promote adjustment in children with congenital heart disease entering school and their families[END_REF]. Moreover, some of these children may be trapped in the vicious circle of muscular deconditioning and therefore suffer from reduced exercise capacity, resulting in a lower HRQoL, despite the absence of any severe physical impairment [START_REF] Amedro | Cardiopulmonary fitness in children with congenital heart diseases versus healthy children[END_REF][START_REF] Amedro | Correlation between cardio-pulmonary exercise test variables and health-related quality of life among children with congenital heart diseases[END_REF]. In the current era, children with CHD should not be limited in any submaximal exercise performance [START_REF] Müller | Currently, children with congenital heart disease are not limited in their submaximal exercise performance[END_REF] and we recently showed from a large cohort of nearly 800 children that most of them had a normal physical capacity, as measured by their maximal oxygen uptake (VO2 max ) [START_REF] Amedro | Cardiopulmonary fitness in children with congenital heart diseases versus healthy children[END_REF]. Nevertheless, parental overprotection and restriction recommendations by physicians have built some barriers to physical activity for youngsters with CHD [START_REF] Reybrouck | Physical performance and physical activity in grown-up congenital heart disease[END_REF][START_REF] Swan | Exercise prescription in adults with congenital heart disease: a long way to go[END_REF][START_REF] Amedro | Quality of life of children with congenital heart diseases: a multicenter controlled cross-sectional study[END_REF].

In the literature, the existing correlation between HRQoL and the severity of the CHD has mostly been found when considering the physical dimension of HRQoL [START_REF] Amedro | Correlation between cardio-pulmonary exercise test variables and health-related quality of life among children with congenital heart diseases[END_REF][START_REF] Hunter | Quality of life in adults living with congenital heart disease: beyond morbidity and mortality[END_REF][START_REF] Moons | Is the severity of congenital heart disease associated with the quality of life and perceived health of adult patients?[END_REF]. Nevertheless, many patients develop a process of resilience including coping, response shift and sense of coherence, and therefore may have a good HRQoL, especially in the psychosocial dimensions [START_REF] Amedro | Quality of life of children with congenital heart diseases: a multicenter controlled cross-sectional study[END_REF][START_REF] Apers | Sense of coherence and perceived physical health explain the better quality of life in adolescents with congenital heart disease[END_REF]. As a result, depending on which aspect of HRQoL is concerned, some studies have reported a better HRQoL in children with CHD than in the general population [START_REF] Reiner | Quality of life in young people with congenital heart disease is better than expected[END_REF], while other studies have mainly focused on the impact of the CHD severity on children's HRQoL, and therefore reported an impaired quality of life [START_REF] Amedro | Quality of life of children with congenital heart diseases: a multicenter controlled cross-sectional study[END_REF][START_REF] Mellion | Health-related quality of life outcomes in children and adolescents with congenital heart disease[END_REF][START_REF] Raj | Health-related quality of life in infants and toddlers with congenital heart disease: a cross-sectional survey from South India[END_REF][START_REF] Uzark | Quality of life in children with heart disease as perceived by children and parents[END_REF].

Nevertheless, most HRQoL studies with a consistent level of evidence have mainly included teenagers or adults with CHD, and, currently, few HRQoL controlled data is available in young children with CHD, especially in those under the age of 8 years [START_REF] Amedro | Healthrelated quality of life of patients with pulmonary arterial hypertension associated with CHD: the multicentre cross-sectional ACHILLE study[END_REF][START_REF] Werner | Factors influencing the participation of adolescents and young adults with a congenital heart disease in a transition education program: a prospective multicentre controlled study[END_REF]. Yet, many questions emerge during this period of life regarding young CHD children's physical, emotional, and social wellbeing. Moreover, the transition from home to school requires developing new skills, involving physical activity, socialisation, autonomy, and self-confidence. As a result, this period may represent new challenges for young children with CHD, as well as for their families.

In this multicentre prospective controlled study, we aimed to evaluate HRQoL of children with CHD aged 5 to 7, in comparison with contemporary peers recruited in schools. We also intended to evaluate the factors associated with HRQoL in this population.

Methods

Study design

This multicentre controlled prospective cross-sectional study was carried out in two paediatric cardiology tertiary care centres (Montpellier University Hospital, France and St-Pierre Institute, Palavas-Les-Flots, France), and in 6 school classes in the Occitanie region, France, randomly selected from the Department of Education's database.

Patient's population

Children with a CHD, as defined by the ACC-CHD classification [START_REF] Houyel | Population-based evaluation of a suggested anatomic and clinical classification of congenital heart defects based on the International Paediatric and Congenital Cardiac Code[END_REF], and aged 5 to 7 years old, were prospectively recruited in both centres during a paediatric cardiology outpatient visit. Inclusion procedures were beforehand harmonized. We did not include children with any other severe chronic disease (polymalformative genetic syndrome, extra-cardiac organ failure) and children unable to understand the questionnaire (e.g. non-French speakers, severe neurodevelopmental disorder). Children with any recent surgical or interventional cardiac catheterization procedures (6 monthsbeforeinclusionvisit) and hospitalized children were also excluded. Children filled-in their HRQoL self-questionnaire under trained clinical research assistant supervising, and their parents filled in the HRQoL proxy-questionnaire in a separate room, as in our similar previous studies [START_REF] Amedro | Quality of life of children with congenital heart diseases: a multicenter controlled cross-sectional study[END_REF][START_REF] Amedro | Healthrelated quality of life of patients with pulmonary arterial hypertension associated with CHD: the multicentre cross-sectional ACHILLE study[END_REF][START_REF] Amedro | Quality of life in children participating in a non-selective INR self-monitoring VKAeducation programme[END_REF][START_REF] Abassi | Impaired pulmonary function and its association with clinical outcomes, exercise capacity and quality of life in children with congenital heart disease[END_REF].

Control population

In the 6 selected school classes, parents or legal guardians of all children aged 5 to 7 years were offered to participate in the study, with a common recruitment procedure for each class and similar to the one at the hospital. Children completed the HRQoL self-questionnaire under clinical research assistant supervising, at school. Parents filled in separately the HRQoL proxy-questionnaire at home.

HRQoL questionnaires

We used the self and proxy versions of the PedsQL questionnaire dedicated to children aged 5 to 7 years. The PedsQL instrument is a generic paediatric HRQoL instrument, designed for children aged from 2 to 18 years, developed from a large cohort of healthy children as well as children with acute or chronic health conditions [START_REF] Varni | The PedsQL 4.0 as a pediatric population health measure: feasibility, reliability, and validity[END_REF]. The PedsQL questionnaire has four multidimensional scales: physical functioning (8 items), emotional functioning (5 items), social functioning (5 items), and school functioning (5 items). The three summary scores are the total scale score (23 items), the physical health summary score (8 items), and the psychosocial health summary score (15 items). Each item uses a 5-point Likert scale from 0 to 4 (0, never a problem; 1, almost never a problem; 2, sometimes a problem; 3, often a problem; 4, almost always a problem). For the 5-7 years self-report questionnaire, the Likert scale is reworded and simplified into a 3-point scale (0, not a problem; 2, sometimes a problem; 4, a lot of a problem). Items are reversed scored and linearly transformed to a 0-100 scale, higher scores indicating a better HRQoL. This instrument was validated by Varni et al. in healthy and patient populations and its psychometric properties showed reliability, validity and responsiveness to clinical change over time [START_REF] Varni | The PedsQL 4.0 as a pediatric population health measure: feasibility, reliability, and validity[END_REF][START_REF] Varni | The PedsQL: measurement model for the pediatric quality of life inventory[END_REF][START_REF] Varni | PedsQL 4.0: reliability and validity of the Pediatric Quality of Life Inventory version 4.0 generic core scales in healthy and patient populations[END_REF]. The translation and cultural adaptation into French was performed by MAPI Research Institute (www.mapitrust .org), following the international guidelines [START_REF] Guillemin | Cross-cultural adaptation of health-related quality of life measures: literature review and proposed guidelines[END_REF]. The psychometric properties of the French version of the PedsQL appeared to be acceptable [START_REF] Tessier | Propriétés psychométriques du questionnaire générique français[END_REF]. We previously showed the good sensitivity of the PedsQL from several controlled prospective HRQoL studies among healthy controls and children with various chronic diseases [START_REF] Amedro | Quality of life in children participating in a non-selective INR self-monitoring VKAeducation programme[END_REF][START_REF] Amedro | Health-related quality of life among children with turner syndrome: controlled cross-sectional study[END_REF].

CHD severity

The ACC-CHD classification was used to define the type of malformation [START_REF] Houyel | Population-based evaluation of a suggested anatomic and clinical classification of congenital heart defects based on the International Paediatric and Congenital Cardiac Code[END_REF]. The disease severity was assessed by a paediatric cardiologist who was blinded to HRQoL reports. Three classifications were used to define CHD severity: (1) The Ross classification, using four severity groups (from Ross class I to Ross class IV) to grade the severity of congestive heart failure in infants (29); (2) the Bethesda classification, stratifying CHD in three severity categories (mild, moderate, and severe) (30); (3) and the prenatal prognostic scale from Davey et al. grading foetal cardiovascular diseases in 7 levels, from level 1 (cardiovascular finding with minimal, if any, negative impact on well-being), to level 7 (cardiovascular abnormality and complex form of CHD with very poor prognosis) [START_REF] Davey | Development and validation of a fetal cardiovascular disease severity scale[END_REF].

Clinical outcomes

In the CHD group, the following clinical variables were collected: gender, age, age at CHD diagnosis (pre or postnatal diagnosis), number of cardiac surgeries, number of interventional cardiac catheterization procedures, drug medication, and electrocardiographic status (presence of pacemaker or implantable defibrillator), and echocardiography data (left ventricle ejection fraction, right ventricle hypertension, pulmonary arterial hypertension, and mechanical valve).

In the control group, only gender and age were collected.

Statistical analysis

The characteristics of the population were presented using mean and standard deviation for continuous variables, and frequencies and proportions for qualitative variables.

Age-and gender-adjusted means (± standard error of the mean, SEM) of HRQoL scores were measured (total, summary, and for each dimension). To compare HRQoL scores between CHD and controls with adjustment on age and gender, a covariance analysis (ANCOVA) was performed. The same method was used to study the effect of Ross classification (class I, class II, class III, class IV and controls) and Bethesda classification (mild severity, moderate severity, severe severity and controls) on HRQoL scores. For pairwise comparisons, the Holm's correction was taken into account.

To identify the clinical factors associated with HRQoL summary scores (total, physical and psychosocial score) in the CHD group, a multiple linear regression was used. Except for age and gender, which were forced in the models, the other clinically relevant variables with a P value ≤ 0.20 in the univariate analysis were included in the model. The final model was obtained using a backward selection with a removal level of 0.10. Colinearity between factors was tested with variance inflation factors. To test the validity of the model, the normality of residues was tested with the Shapiro-Wilk test. The statistical significance was set at 0.05 and analyses were performed with SAS V.9 software.

Results

Population characteristics

A total of 249 children aged 5 to 7 years were recruited (mean age 6.1 ± 0.8 years, 49% female), including 124 subjects in the CHD group (mean age 6.0 ± 0.8 years, 45% female), and 125 subjects in the control group (mean age 6.2 ± 0.8 years, 54% female). Both groups were similar in terms of age and gender.

In the CHD group, the anomalies of the ventricular outflow tracts represented the most frequent sub-group (n = 44, 36%). In greater detail, the most frequent types of CHD were in the following order: ventricular septal defects (n = 32, 26%), anomalies of the atria and interatrial communications (n = 16, 13%), aortic valve stenosis (n = 14, 11%), and tetralogy of Fallot (n = 12, 10%). The smallest samples were complex anomalies of the atrioventricular connections (n = 2) and heterotaxy (n = 2). Most children in the CHD group had no congestive heart failure (Ross class I, 91%), nearly half of them had a low severity status (Bethesda mild severity, 47%), and nearly half of them had a good prognosis (Davey prognosis classification level 2, 48%). CHD diagnosis was performed prenatally in nearly one out of three cases. A total of 58 subjects (47%) underwent at least one cardiac surgical procedure and 29 subjects (24%) underwent at least one interventional cardiac catheterization procedure. Most patients had no cardiac medication and a normal left ventricular systolic function. Clinical characteristics of the CHD group were detailed in Table 1.

HRQoL in children with CHD compared to control children

No significant differences were founded between CHD and control groups in self-reported HRQoL total score (73.5 ± 1.2 vs. 72.8 ± 1.2, P = 0.7, respectively) as in all dimensions (Fig. 1 and Additional file 1: Table 1). Mother-reported HRQoL was lower in the CHD group than in controls for total score (76.1 ± 1.1 vs. 81.1 ± 1.1, P = 0.002, respectively), and in all dimensions (P < 0.05) except for emotional functioning (Fig. 2 and Additional file 1: Table 1). Father-reported HRQoL was lower in the CHD group than in controls for total score (79.2 ± 1.2 vs. 83.7 ± 1.1, P = 0.006, respectively), physical functioning, school functioning, and psychosocial summary functioning (Fig. 3 and Additional file 1: Table 1).

Relation between HRQoL and CHD severity

In terms of CHD severity, as assessed by the Ross classification, HRQoL analyses were performed between three groups of severity including the control group (controls, Ross class I and Ross class II), as no patient included in the study presented with Ross class III or IV status. Self-reported HRQoL was statistically different between those three severity groups, overall (controls, 72.9 ± 1.2; Ross I, 74.3 ± 1.2; Ross II, 63.4 ± 3.9; P = 0.03), in emotional functioning (P = 0.05), and in psychosocial summary functioning (P = 0.03) (Fig. 4). Motherreported HRQoL scores were different between those three severity groups, overall (controls, 81.1 ± 1.1; Ross class I, 77.4 ± 1.2; Ross class II, 63.0 ± 3.8; P < 0.0001), as well as in all other dimensions (physical functioning, P < 0.0001; emotional functioning, P < 0.01; social functioning, P = 0.01; school functioning, P < 0.01; psychosocial functioning, P < 0.0001) (Fig. 5). Father-reported HRQoL was significantly different between the three severity groups, overall (controls, 83.7 ± 1.1; Ross class I, 79.2 ± 1.3; Ross class II, 79.2 ± 4.5; P = 0.02) and in physical functioning (P = 0.02) (Fig. 6). In the CHD group, the self-and mother-reported HRQoL scores (overall, physical health summary and psychosocial health summary) were affected by Ross classification (in univariate and multivariate analyses). The father reported HRQoL scores were not affected by this classification (Table 2, Additional file 1: Tables 2 and3).

In terms of CHD severity, as assessed by the Bethesda classification, self-reported HRQoL scores comparisons were significant between the four severity classes (controls, mild severity, moderate severity, and severe severity) in social functioning (P = 0.03) and psychosocial functioning (P = 0.05). However, no differences were found in the total scale score (controls, 72.9 ± 1.2; Fig. 1 Self-reported HRQoL in children with CHD compared to control children. HRQoL health-related quality of life, NS non-significant mild severity, 76.4 ± 1.7; moderate severity, 71.8 ± 1.9; severe severity, 68.6 ± 3.2; P = 0.1). Mother-reported HRQoL was different between the four severity groups, overall (controls, 81.1 ± 1.1; mild, 77.9 ± 1.7; moderate, 75.2 ± 1.8; severe, 72.9 ± 3.1; P = 0.01), as well as in physical functioning (P = 0.02), school functioning (P = 0.01), and psychosocial functioning (P = 0.02). Father-reported HRQoL scores comparisons were different between the four classes, overall (controls, 83.7 ± 1.1; mild, 79.5 ± 1.8; moderate, 79.8 ± 2.0; severe, 76.5 ± 3.2; P = 0.04), and in physical functioning (P = 0.05). Among CHD patients, Bethesda classification univariate and multivariate analyses found no association with self and proxy-HRQoL scores (Table 2 and Additional file 1: Tables 2 and3).

Effect assessment of CHD severity on HRQoL according to the prenatal prognostic scale from Davey et al. could not be performed, given the small number of subjects in each one of the 7 scales.

Relation between HRQoL and clinical outcomes in children with CHD

When considering the three PedsQL summary scores (e.g. total scale score, physical health summary score, and psychosocial health summary score), self-reported HRQoL was affected by the number of cardiac surgical procedures in the total scale score and in the psychosocial functioning (Table 2). Mother-reported HRQoL scores were affected by the administration of a cardiac medication on the physical, psychosocial and total scores (Additional file 1:Table 2).

Father-reported HRQoL was affected by the presence and the number of cardiac surgical procedures in psychosocial functioning, and in the total scale score, in the univariate analysis (Additional file 1: Table 3).

HRQoL was not significantly affected by the remaining clinical outcomes, such as the moment when the diagnosis of CHD was performed (prenatal vs. postnatal diagnosis), or the presence of a pulmonary arterial hypertension, or an impaired left ventricular function (Table 2, Additional file 1: Tables 2,3).

Discussion

This multicentre prospective controlled study of a cohort of 249 children aged 5 to 7 years old assessed HRQoL of 124 children with CHD in comparison with 125 controls. This study also evaluated the relation between CHD severity and HRQoL and investigated some clinical determinants of HRQoL in young children with CHD.

This study highlighted that self-reported HRQoL in young children with CHD was good, with a total PedsQL Fig. 2 Mother-reported HRQoL in children with CHD compared to control children. HRQoL health-related quality of life, NS non-significant score of 73.5 ± 1.2 (out of 100), and therefore similar to that of their contemporary peers recruited in schools (e.g. 72.8 ± 1.2). Such HRQoL levels probably reflect all tremendous medical advances in the screening and care of CHD in the last decades, in terms of prenatal diagnosis, non-invasive real-time imaging, neonatal cardiac surgery, paediatric intensive care, and cardiac catheterization. Indeed, in the current era, over 90% of children with a CHD are expected to survive more than 30 years after first cardiac surgery [START_REF] Spector | Trends in long-term mortality after congenital heart surgery[END_REF]. In our study, 91% of children with CHD had no symptoms of heart failure (Ross class I). As a result, more attention has been recently given to patient related outcomes in the field of congenital cardiology, beyond the classical cardiac morbidity and mortality outcomes [START_REF] Berkes | Measuring health-related quality of life in Hungarian children attending a cardiology clinic with the Pediatric Quality of Life Inventory[END_REF][START_REF] Spijkerboer | Long-term behavioural and emotional problems in four cardiac diagnostic groups of children and adolescents after invasive treatment for congenital heart disease[END_REF]. Yet, more consistent HRQoL data from controlled studies are necessary in young children with CHD. Nevertheless, our results are in line with the study from Pilla et al., reporting that psychosocial domain of self-reported HRQoL was similar between children with CHD and healthy children aged from 3 to 7 years old [START_REF] Pilla | Healthrelated quality of life and right ventricular function in the midterm follow-up assessment after tetralogy of fallot repair[END_REF]. Conversely, the single-centre controlled study from Uzark et al. concluded that HRQoL in children aged 2 to 17 y.o. in the USA was significantly impaired, however the 5-7 y.o. age group represented the smallest sample of the study [START_REF] Uzark | Quality of life in children with heart disease as perceived by children and parents[END_REF]. From a general perspective, French healthcare system, as in most European countries, provides full coverage from the social security for patients with CHD, which is not the case in the USA. Therefore, we should be aware that HRQoL in chronic diseases also reflects the quality of a health care system, beyond the medical care itself [START_REF] Cramm | The role of disease management programs in the health behavior of chronically ill patients[END_REF][START_REF] Alonazi | Quality of care and quality of life: convergence or divergence?[END_REF]. Moreover, modern medical care strategies in paediatric cardiology involve educational programs, shorter hospital stays, and home-based healthcare programs, which may contribute to achieve good HRQoL levels [START_REF] Werner | Factors influencing the participation of adolescents and young adults with a congenital heart disease in a transition education program: a prospective multicentre controlled study[END_REF][START_REF] Amedro | Quality of life in children participating in a non-selective INR self-monitoring VKAeducation programme[END_REF][START_REF] Amedro | Impact of a centre and home-based cardiac rehabilitation program on the quality of life of teenagers and young adults with congenital heart disease: the QUALI-REHAB study rationale design and methods[END_REF]. Classically, patients with CHD are prone to cope with their health condition, and develop adaptive skills such as reponse shift [START_REF] Pike | Quality of life, health status, and depression: comparison between adolescents and adults after the Fontan procedure with healthy counterparts[END_REF] or sense of coherence [START_REF] Apers | Sense of coherence and perceived physical health explain the better quality of life in adolescents with congenital heart disease[END_REF][START_REF] Müller | Sense of coherence, rather than exercise capacity, is the stronger predictor to obtain health-related quality of life in adults with congenital heart disease[END_REF], which may result in good HRQoL levels, especially in the psychological domains [START_REF] Amedro | Healthrelated quality of life of patients with pulmonary arterial hypertension associated with CHD: the multicentre cross-sectional ACHILLE study[END_REF]. This adaptation has been particularly shown in non-progressive health conditions, such as cerebral palsy, or in children with a correctable congenital anomaly, such as CHD, but less so for children with a progressive disease [START_REF] Jardine | Selfreported quality of life of young children with conditions from early infancy: a systematic review[END_REF]. To our knolewdge, adaptation processes have not been fully investigated yet in young children with CHD, but our results may also suggest an early process of "resilience".

When focusing on proxy reports, our study found that parents-reported HRQoL was significantly lower in the CHD group than in controls, with an approximate Fig. 3 Father-reported HRQoL in children with CHD compared to control children. HRQoL health-related quality of life, NS non-significant mean difference of 5 points (out of 100) in both mother and father reports. Our results are consistent with previous similar studies, but few HRQoL data involving both mothers and fathers of the same child have been reported in paediatric cardiology [START_REF] Mellion | Health-related quality of life outcomes in children and adolescents with congenital heart disease[END_REF][START_REF] Uzark | Quality of life in children with heart disease as perceived by children and parents[END_REF][START_REF] Amedro | Healthrelated quality of life of patients with pulmonary arterial hypertension associated with CHD: the multicentre cross-sectional ACHILLE study[END_REF][START_REF] Amedro | Healthrelated quality of life of patients with pulmonary arterial hypertension associated with CHD: the multicentre cross-sectional ACHILLE study[END_REF][START_REF] Amedro | Quality of life in children participating in a non-selective INR self-monitoring VKAeducation programme[END_REF][START_REF] Amedro | Health-related quality of life among children with turner syndrome: controlled cross-sectional study[END_REF][START_REF] Raj | Health-related quality of life (HRQOL) in children and adolescents with congenital heart disease: a cross-sectional survey from South India[END_REF]. Nevertheless, parents-reported HRQoL scores in our study ranged from 74 to 84 points (out of 100), which is good and remains higher than scores reported in previous studies [START_REF] Amedro | Quality of life in children participating in a non-selective INR self-monitoring VKAeducation programme[END_REF]. We may hypothesize that leaving home and attending kindergarten or the first years of elementary school may be a challenge for parents of children with a CHD [START_REF] Tong | Health care transitions for adolescents with congenital heart disease: patient and family perspectives[END_REF]. Indeed, our results found lower proxyreported HRQoL levels in school and psychosocial domains. Parents of children with CHD classically overprotect them and may fear that their children will be stigmatized, bullied or put on the sideline at school [START_REF] Amedro | Healthrelated quality of life of patients with pulmonary arterial hypertension associated with CHD: the multicentre cross-sectional ACHILLE study[END_REF][START_REF] Berkes | Measuring health-related quality of life in Hungarian children attending a cardiology clinic with the Pediatric Quality of Life Inventory[END_REF][START_REF] Raj | Health-related quality of life (HRQOL) in children and adolescents with congenital heart disease: a cross-sectional survey from South India[END_REF][START_REF] Macran | The development of a new measure of quality of life for children with congenital cardiac disease[END_REF]. In general, parents of children with chronic health diseases are prone to developp stress and anxiety, undermining the well-being or overall stability of their family, as well as their ability to cope [START_REF] Brosig | Psychosocial outcomes for preschool children and families after surgery for complex congenital heart disease[END_REF][START_REF] Compas | Coping with chronic illness in childhood and adolescence[END_REF][START_REF] Rahimianfar | Anxiety determinants in mothers of children with congenital heart diseases undergoing cardiac surgery[END_REF]. In paediatric cardiology, factors contributing to parental stress may involve a past trauma, as after prenatal diagnosis, an anguish of death during cardiac surgery, and an inability to empower their child [START_REF] Werner | Parental anxiety before invasive cardiac procedure in children with congenital heart disease: contributing factors and consequences[END_REF]. Therefore, it is necessary to integrate psychosocial support dedicated to such parents [START_REF] Uzark | Quality of life in children with heart disease as perceived by children and parents[END_REF][START_REF] Uzark | The Pediatric Quality of Life Inventory TM in children with heart disease[END_REF] and develop multidisciplinary family-centred psychosocial care for patients with CHD, as recommendated by the AEPC psychosocial working group [START_REF] Utens | Multidisciplinary family-centred psychosocial care for patients with CHD: consensus recommendations from the AEPC Psychosocial Working Group[END_REF].

Our results also suggested that young children with the more severe CHD had significantly poorer HRQoL, as reported by themselves and their parents. In previous studies, disease severity has been found to impact the HRQoL of children with CHD [START_REF] Macran | The development of a new measure of quality of life for children with congenital cardiac disease[END_REF]. Indeed, HRQoL is significantly impaired in children with hypoplastic left heart syndrome, which is considered as the most severe CHD [START_REF] Dempster | Children with hypoplastic left heart syndrome have lower quality of life than healthy controls and children with other illnesses[END_REF]. In their study, Dempster et al. related HRQoL to the levels of impaired adaptive behavior, behavioral symptoms, and functional status [START_REF] Dempster | Children with hypoplastic left heart syndrome have lower quality of life than healthy controls and children with other illnesses[END_REF]. Similarly, HRQoL in children aged 2 to 18 y.o. after palliative single ventricle surgical repair, remains lower than in CHD eligible to biventricular surgical repair [START_REF] Brosig | Psychosocial outcomes for preschool children and families after surgery for complex congenital heart disease[END_REF], such as tetralogy of Fallot [START_REF] Pilla | Healthrelated quality of life and right ventricular function in the midterm follow-up assessment after tetralogy of fallot repair[END_REF]. As in our previous studies in older children, this study confirms the negative impact of repeated invasive cardiac procedures (surgery or catheterization) on HRQoL [START_REF] Amedro | Quality of life of children with congenital heart diseases: a multicenter controlled cross-sectional study[END_REF][START_REF] Ladak | Health-related quality of life in congenital heart disease surgery in children and young adults: a systematic review and meta-analysis[END_REF]. Moreover, children with a CHD may suffer from an impaired physical capacity, which directly impacts the physical dimension of their HRQoL [START_REF] Amedro | Correlation between cardio-pulmonary exercise test variables and health-related quality of life among children with congenital heart diseases[END_REF]. Some children with CHD may develop an unpleasant feeling of dyspnoea, related to muscular deconditioning or restrictive lung function, which disrupts their physical well-being [START_REF] Abassi | Impaired pulmonary function and its association with clinical outcomes, exercise capacity and quality of life in children with congenital heart disease[END_REF]. Most of those children remain eligible to all sports, therefore healthcare providers, families and teachers should contribute to physical activity promotion in this population from an early age, to avoid the vicious deconditioning circle [START_REF] Amedro | Impact of a centre and home-based cardiac rehabilitation program on the quality of life of teenagers and young adults with congenital heart disease: the QUALI-REHAB study rationale design and methods[END_REF]. Children with complex CHD may also present with an impaired HRQoL, as they are at higher risk of neurological or behavioural problems, including attention disorders, than children with a simple CHD [START_REF] Brosig | Psychosocial outcomes for preschool children and families after surgery for complex congenital heart disease[END_REF].

In our study, HRQoL was predicted by the disease severity based on the infant heart failure classification from Ross et al. [START_REF] Ross | Grading the severity of congestive heart failure in infants[END_REF]. The Ross classification may therefore be more discriminating than the Bethesda classification [START_REF] Warnes | Task force 1: the changing profile of congenital heart disease in adult life[END_REF] or the foetal prognosis classification from Davey 31). In the adult population, the strong correlation between NYHA functional status and HRQoL has been widely reported, in chronic heart failure [START_REF] Izawa | Healthrelated quality of life in relation to different levels of disease severity in patients with chronic heart failure[END_REF], CHD [START_REF] Bredy | New York Heart Association (NYHA) classification in adults with congenital heart disease: relation to objective measures of exercise and outcome[END_REF] or pulmonary arterial hypertension [START_REF] Amedro | Healthrelated quality of life of patients with pulmonary arterial hypertension associated with CHD: the multicentre cross-sectional ACHILLE study[END_REF]. The Ross classification is mostly used in paediatric drug trials, therefore our results suggest using this scale in routine follow-up, to assess both functional status and HRQoL. We also suggest using self-reported HRQoL outcomes in paediatric cardiology trials.

Study limitation

HRQoL in this cohort of children with CHD was good and similar to controls but patients were recruited from outpatient visits in expert centres. Therefore, severe conditions have been underestimated (hospitalization, other severe comorbidities, Ross class III and IV), as well nonsevere conditions (no follow-up in a tertiary care centre). However, we previously reported HRQoL data on young children with severe conditions [START_REF] Amedro | Quality of life in children participating in a non-selective INR self-monitoring VKAeducation programme[END_REF] and most paediatric cardiologists in our region have been involved in the study.

Many patients with a CHD may also be concerned with a genetic syndrome or a neurological deficit, with a potential impact on their HRQoL. They have purposely not been included in this study for methodological issues (inability to understand and/or fill-in the questionnaires). However, investigating HRQoL in this specific population using appropriate measurement tools remains necessary.

Currently, nearly half CHD are diagnosed prenatally, most future children are expected to benefit from an efficient treatment with a low morbidity and mortality. As a result, many parents ask the following question during prenatal diagnosis: "what will be the quality of life of my future child?". Therefore, future research, using large international registries, should investigate the reliability of prenatal prognosis assessment, such as the scale from Davey et al., in terms of HRQoL prediction.

Conclusion

HRQoL in young children with CHD aged 5 to 7 years old was good and similar to controls. HRQoL in this population correlated with CHD severity, especially when assessed by the infant heart failure Ross classification. This study contributed to the growing body of knowledge on HRQoL in congenital cardiology and emphasized the need for child and family support in the most complex CHD. In the current era, prognosis in CHD is excellent, therefore the beneficial role of HRQoL assessment in routine clinical practice for screening and decision-making is of great importance. However, other domains remain under-investigated in the CHD population, such as the impact of psychomotor and cognitive developmental disorders on HRQoL.

Fig. 4

 4 Fig. 4 Self-reported HRQoL scores in the CHD group based on Ross severity class. HRQoL health-related quality of life. *0: Controls, 1: Ross class I, 2: Ross class 2

Fig. 5

 5 Fig. 5 Mother-reported HRQoL scores in the CHD group based on Ross severity class. HRQoL health-related quality of life. *0: Controls, 1: Ross class I, 2: Ross class 2

Fig. 6

 6 Fig. 6 Father-reported HRQoL scores in the CHD group based on Ross severity class. HRQoL health-related quality of life. *0: Controls, 1: Ross class I, 2: Ross class 2

Table 1 CHD children' medical characteristics

 1

	Variables

ACC-CHD anatomic and clinical classification of congenital heart disease, CHD congenital heart disease

Table 2 Self-report QoL explanatory variables in the CHD group

 2 Values are linear regression coefficients β (Standard Error) and p-values. Significant p-values are marked in italicCHD congenital heart disease, PAH pulmonary arterial hypertension, SD standard error

*

Candidate variables for multivariate analysis $ "-" Not maintained in the model (P > 0.10)

Variables Physical functioning Psychosocial functioning Total scale score Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis Estimation (SE) p-value p-value $ Estimation (SE) p-value p-value $ Estimation (SE) p-value p-value $

	0.93	0.17	-			0.03
	0.50 *	0.46 *	0.08 *			0.01 *
	-1.0 (1.4) -1.8 (2.4) 7.4 (3.6)	3.1 (3.7)		10.5 (4.1)
	0.46	0.34	-			0.04
	0.31 *	0.88 *	0.05 *			0.02 *
	-1.6 (1.6) -0.4 (2.7) 9.2 (4.0)	4.4 (4.1)		11.2 (4.6)
	0.72	0.07	-			0.05
	0.89 *	0.16 *	0.54			0.08 *	0.26
	0.3 (1.8)	-4.3 (3.1) 3.9 (4.7)	0.7 (4.8)		9.2 (5.3)	3.9 (3.5)
		Female versus male	Low versus severe	moderate versus	severe	Class I versus Class II	Postnatal versus	prenatal
	Age	Gender	Bethesda severity	classification		Ross classification	Age at CHD diag-	nosis

Acknowledgements

We thank children and their families who kindly participated in the study and the National Ministry of Education, for the collaboration to perform the study in schools.

Trial registration This study was approved by the institutional review board of Montpellier University Hospital (2019_ IRB-MTP_02-19) on 22 February 2019 and was registered on ClinicalTrials.gov (NCT03931096) on 30 April 2019, https :// clini caltr ials

Funding

This work was supported by University Hospital of Montpellier for logistical support, project management, data management, and statistical analyses.

Availability of data and materials

All data generated or analyzed during this study are included in this published article. The datasets used for those published data are available from the corresponding author on reasonable request.

Supplementary information

Supplementary information accompanies this paper at https ://doi. org/10.1186/s1295 5-020-01615 -6.

Additional file 1: Supplementary

Ethics approval and consent to participate

This study complies with the Declaration of Helsinki. It was approved by the institutional review board of Montpellier University Hospital (2019_IRB-MTP_02-19) on 22 February 2019 and was registered on ClinicalTrials.gov (NCT03931096) on 30 April 2019. Schools' participation was supported by the Department of Education Regional Authority. Informed consent and child assent were obtained from the participating families.

Consent for publication

This manuscript does not contain any individual person's data. Informed consent was obtained from all parents or legal guardians.

Competing interests

Authors declare that they have no competing interests.

• fast, convenient online submission • thorough peer review by experienced researchers in your field • rapid publication on acceptance

• support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year

•

At BMC, research is always in progress.

Learn more biomedcentral.com/submissions

Ready to submit your research Ready to submit your research ? Choose BMC and benefit from:

? Choose BMC and benefit from:

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.