Alexandre Bruckert

Lucie Lévêque

Matthieu Perreira

D A Silva

Patrick Le Callet

A Dataset of Gaze and Mouse Patterns in the Context of Facial Expression Recognition

Keywords: CCS Concepts:, Human-centered computing → Human computer interaction (HCI);, Applied computing → Computers in other domains emotions, facial expression recognition (FER), eye-tracking, BubbleView, dataset

Facial expression recognition is an important and challenging task for both the computer vision and affective computing communities, and even more specifically in the context of multimedia applications, where audience understanding is of particular interest. Recent data-oriented approaches have created the need for large-scale annotated datasets. However, most existing datasets present some weaknesses, because of the collecting methods used. In order to further highlight these issues, we investigate in this work how human visual attention is deployed when performing a facial expression recognition task. To do so, we carried out several complementary experiments, using the eye-tracking technology, as well as the BubbleView metaphor, both under laboratory and crowdsourcing settings. We show significant variations in gaze patterns depending on the emotion represented, but also on the difficulty of the task, i.e., whether the emotion is correctly recognised or not. Moreover, we use these results to propose recommendations on the ways to collect label data for facial expression recognition datasets.

INTRODUCTION

As emotions play a crucial role in human communication, decisions, and perception, they have been under investigation in many different fields, including psychology, sociology, and neuroscience. Thanks to technological advances, new domains have arisen, such as affective computing [START_REF] Picard | Affective computing[END_REF]. The latter corresponds to systems able to recognise, analyse, model, and express human emotions.

In the framework of interactive media experiences, emotions can be of particular interest in a broad range of ways, and at many levels. Emotions and users engagement are indeed strongly linked, as audience engagement can be defined as the "cognitive, emotional, or affective experiences users have with media content" [START_REF] Broersma | Audience engagement. The international encyclopedia of journalism studies[END_REF]. Several research works have been conducted with a view to study such relationship, e.g., to increase audience engagement in live interactive performance [START_REF] Damiano | Studying and designing emotions in live interactions with the audience[END_REF], or even to investigate the role of emotions in live streaming [START_REF] Lin | Happiness begets money: Emotion and engagement in live streaming[END_REF].

Automatic emotion recognition refers to the way a machine is able to identify the emotional states of human beings on the basis of various types of physiological or non-physiological signals. Amidst non-physiological measures, facial expressions are probably the most studied modality of non-verbal expression of emotions [START_REF] Mehrabian | Some referents and measures of nonverbal behavior[END_REF]. In 1969, Ekman identified six so-called "basic emotions", including happiness, anger, disgust, fear, sadness, and surprise -on top of these can be Permission to make digital or hard copies of all or part of this work for personal or classroom use is granted without fee provided that copies are not made or distributed for profit or commercial advantage and that copies bear this notice and the full citation on the first page. Copyrights for components of this work owned by others than ACM must be honored. Abstracting with credit is permitted. To copy otherwise, or republish, to post on servers or to redistribute to lists, requires prior specific permission and/or a fee. Request permissions from permissions@acm.org. © 2023 Association for Computing Machinery. Manuscript submitted to ACM added the neutral emotion [START_REF] Ekman | Pan-cultural elements in facial displays of emotion[END_REF]. Automatic facial expression recognition (FER) therefore corresponds to classification algorithms used to put a name on each emotion by associating known characteristics on human faces. Such algorithms, made possible with advances in computer vision technologies, are trained on large amounts of data.

Facial expression recognition appears amongst the most demanding tasks in social communication [START_REF] Michael | A survey on human face expression recognition techniques[END_REF]. As with many other tasks in machine learning, one of the main challenges remains the quality of datasets. Zeng et al. found annotations in public datasets (e.g., AffectNet [START_REF] Mollahosseini | Affectnet: A database for facial expression, valence, and arousal computing in the wild[END_REF]) to be inconsistent [START_REF] Zeng | Facial expression recognition with inconsistently annotated datasets[END_REF]. Similarly, Lévêque et al. showed in a previous study that the labels of widely used FER datasets (e.g., FER-2013 [START_REF] Goodfellow | Challenges in representation learning: A report on three machine learning contests[END_REF]) can be put into question, as they are mostly defined based on image search or by a single annotator [START_REF] Lévêque | Comparing the robustness of humans and deep neural networks on facial expression recognition[END_REF]. Emotions can be perceived differently from one individual to another; annotating facial expressions can therefore be extremely tedious.

The study of human visual attention when performing a FER task can bring some insights on the analysis of such inconsistency. It has indeed been widely shown in the literature that both eye movements and positions can provide useful information on human perception and cognition [START_REF] Wolfe | Visual attention[END_REF]- [START_REF] Frischen | Gaze cueing of attention: visual attention, social cognition, and individual differences[END_REF]. The eye-tracking technology, i.e., the process of measuring where people look in a visual field, is commonly used to scrutinise how humans process visual information.

Due to the COVID-19 pandemic (amongst other factors), several metaphors have been proposed with a view to collect visual attention data without using eye-tracking devices. For instance, the BubbleView metaphor consists in blurring the image, except for the area around the mouse cursor. This metaphor has shown very good correlation with eye-tracking data in the literature [START_REF] Kim | Bubbleview: an interface for crowdsourcing image importance maps and tracking visual attention[END_REF]- [START_REF] Ellahi | Evaluation of the bubble view metaphor for the crowdsourcing study of visual attention deployment in tone-mapped images[END_REF]. Consequently, it allows for the collection of large visual saliency datasets, like the Saliency in Context (SALICON) dataset [START_REF] Jiang | Salicon: Saliency in context[END_REF].

Previous studies have shown that human visual attention tends to prioritise emotional content (e.g., cute kittens) over non-emotional (i.e., neutral) stimuli [START_REF] Brosch | The perception and categorisation of emotional stimuli: A review[END_REF]- [START_REF] Gall | Emotion regulation, procrastination, and watching cat videos online: Who watches internet cats, why, and to what effect?[END_REF]. Yet, to the best of our knowledge, little research has analysed the relationships between facial expression recognition (FER) tasks and visual attention.

In this article, we present three experiments which were conducted under different settings, i.e., eye-tracking in lab, BubbleView in lab, and BubbleView in crowdsourcing, in order to better understand where humans look when asked to perform a FER task. More specifically, our contributions are the following:

• We propose a new dataset dedicated to study visual attention in the context of facial expression recognition tasks. More specifically, this dataset includes both eye-tracking and mouse-tracking patterns. This dataset is made publicly available at [link removed for anonymization].

• We highlight links between the way we visually explore human faces, and the emotions displayed.

• We discuss ways of leveraging visual attention data in order to improve the collection of large-scale facial expression recognition datasets, and more specifically their reliability.

MATERIAL AND METHODS

In this work, we conducted an eye-tracking experiment, as well as two BubbleView experiments, whose characteristics are summarised in Table 1 and further developed in the following subsections.

Stimuli

The source images used in our experiments consist of a total of 200 images of faces randomly taken in the AffectNet dataset [START_REF] Mollahosseini | Affectnet: A database for facial expression, valence, and arousal computing in the wild[END_REF]. AffectNet contains about one million of in-the-wild facial images collected using search engines. Half of the images were manually annotated (each image by a single annotator) using the seven discrete facial expressions model, while the other half were automatically annotated. More specifically, we selected fifty images labelled as "happy", fifty as "sad", fifty as "surprised", and fifty as "angry".

These four emotions were selected out of the seven basic ones as it was shown in the literature that they were less subject to recognition errors [START_REF] Lévêque | Comparing the robustness of humans and deep neural networks on facial expression recognition[END_REF]. Amongst the fifty images of each emotion, half of them were tagged as "manually annotated" in the AffectNet dataset, whereas the other half were automatically annotated, using a ResNeXt neural network. We resized all images to 720×720 pixels, as their original resolution was different from each other.

Eye-tracking experiment

For the eye-tracking experiment, participants were presented 120 different stimuli (from the 200 previously selected) in a random order. he eye-tracking subset was still balanced in terms of emotions, i.e., it was composed of thirty images per emotion. Each stimulus was displayed for four seconds on a full HD 1920×1080 monitor screen with a grey background (as they were resized to 720×720), and followed by a one-second grey screen. After viewing a stimulus, the participants had to answer the following question: "Which facial expression was represented on the image?", by choosing one of the following four options on the screen: happiness, sadness, surprise, and anger. Once their choice was made and validated, they had to fix a target at the centre of the screen to move to the next image.

The experiment was conducted in a room with a low surface reflectance and constant ambient light. The screen luminance was set at 200 cd/m2, and the luminance for the room's walls was measured at 30 cd/m2. The viewing distance was maintained around 70 cm. The eye movements of the participants were recorded using a Tobii Pro Fusion eye-tracker, at a sampling rate of 120 Hz. At the beginning of a session, i.e., for each participant, the eye-tracking system was calibrated using a 9-point calibration protocol, ensuring a precision and accuracy under 0.5 degrees of visual angle.

Before the start of the experiment, participants were given written instructions about the procedure, and a training session was provided, with a view to allow participants to familiarise themselves with the stimuli and the question asked. The four stimuli used in the training session were different from those used during the real experiment.

A total of fifty participants were involved in the eye-tracking experiment. Data from one participant were removed due to external issues during the procedure. Among the remaining participants were 31 females and 18 males, aged between 20 and 65 (M: 34.9, STD: 14.6). All participants had normal (or corrected-to-normal) vision. Note this experiment was approved by our local ethics committee.

BubbleView experiments

As introduced previously, the BubbleView metaphor is a "mouse-contingent, moving-window interface in which participants are presented with a series of blurred images and click to reveal bubbles" [START_REF] Kim | Bubbleview: an interface for crowdsourcing image importance maps and tracking visual attention[END_REF]. The Bubbleview methodology "replaces" eye-tracking with mouse clicks. We conducted two BubbleView experiments, one in laboratory conditions, and the other one on a crowdsourcing platform. Such platforms allow conducting large-scale experiments with reduced costs and efforts [START_REF] Estellés | Towards an integrated crowdsourcing definition[END_REF]. A total of 160 images, selected from the original 200 stimuli, were used for the BubbleView experiments. Amongst these, eighty are common to the images used for the eye-tracking experiment. These 160 images were carefully divided into four playlists of forty images, balanced in terms of emotions. Two playlists (i.e., eighty images) were randomly presented to in-lab participants, while only one playlist (i.e., forty images) was shown to the online participants, as crowdsourcing sessions need to last less than fifteen minutes. Participants were asked to answer the same question, i.e., to recognise the facial expression represented.

In order to set up the BubbleView metaphor, several parameters need to be defined, i.e., the size of the bubble, the blur sigma, and the display time of the stimulus. The size of the bubble shall approximate the size of the fovea [START_REF] Kim | Bubbleview: an interface for crowdsourcing image importance maps and tracking visual attention[END_REF].

Therefore, it was set to 1.5 degrees of visual angle, i.e., 94 pixels. The blur sigma needs to be derived to approximate the visual acuity, i.e., 12 pixels in our case [START_REF] Gerald Westheimer | Visual acuity and hyperacuity: resolution, localization, form[END_REF]. As for the viewing time, it was set at seven seconds, as some research works showed that a longer presentation time was need for BubbleView compared to eye-tracking experiments [START_REF] Ellahi | Evaluation of the bubble view metaphor for the crowdsourcing study of visual attention deployment in tone-mapped images[END_REF].

Sixty subjects participated in the laboratory experiment, including 34 females and 26 males. They all had normal, or corrected-to-normal vision. As for the online experiment, a total of 240 participants (note one was further discarded)

were recruited using the Prolific platform [START_REF] Palan | Prolific. ac-A subject pool for online experiments[END_REF]. Contrary to other platforms, Prolific takes into account researchers' needs by maintaining a recruitment process which is close to that of a laboratory experiment [START_REF] Peer | Beyond the turk: Alternative platforms for crowdsourcing behavioral research[END_REF]- [START_REF] Ak | Rv-tmo: Large-scale dataset for subjective quality assessment of tone mapped images[END_REF]. Like for the eye-tracking experiment, both BubbleView experiments were approved by the local ethics committee.

EYE-TRACKING EXPERIMENT: RESULTS

With this first experiment, using the eye-tracking technology, we aimed to study the way people look at human faces when trying to understand their emotional state.

Emotion recognition labels

After viewing each image, participants were asked to label it depending on the emotion they recognised (i.e., happiness, sadness, anger, or surprise).

As defining which emotion is displayed on a face can be a difficult task [START_REF] Lévêque | Comparing the robustness of humans and deep neural networks on facial expression recognition[END_REF], we first evaluated the amount of agreement amongst observers watching the same image. The amount of agreement 𝑃, based on Fleiss' kappa, was computed as such:

𝑃 = 1 𝑁 (𝑁 -1) 4 ∑ 𝑖=1 𝑛 𝑖 (𝑛 𝑖 -1)
where 𝑁 is the total number of observers (i.e., 𝑁 = 49), and 𝑛 𝑖 the number of observers who assigned the emotion 𝑖 among the four possible choices to a given image. We also compared the annotations we gathered with the original labels of the AffectNet dataset. medium agreement between participants. It is also interesting to note that, amongst these 18 images, 7 were originally annotated manually by a single observer, while 11 were annotated by an automatic model [START_REF] Mollahosseini | Affectnet: A database for facial expression, valence, and arousal computing in the wild[END_REF].

Eye-tracking data processing

During the experiment, gaze samples were recorded at 120 Hz. First, we discarded all gaze samples where the eye was not detected, due to tracking errors or blinks. We applied a first filter to eliminate data from an observer on an image if more than 10% of gaze samples had been discarded. This way, we eliminated 14 scanpaths, over 12 images.

Eye fixations were then extracted using a thresholding algorithm, relying on motion, velocity, and acceleration [START_REF] Salvucci | Identifying fixations and saccades in eye-tracking protocols[END_REF].

We also removed fixations lasting less than 80 ms, i.e., roughly the minimal time required to process foveal information [START_REF] Manor | Defining the temporal threshold for ocular fixation in free-viewing visuocognitive tasks[END_REF]. Gaze points detected as part as the same fixation were aggregated in a single fixation point, located at the barycentre of all said gaze points.

We defined a saccade as the motion occurring between two successive fixations, provided that they last less than 150 ms, and that no tracking error or blink happened in between.

Visual saliency maps were obtained by aggregating the fixation points of all observers on a binary map, and applying a 2D gaussian kernel which standard deviation was set to represent one degree of visual angle, i.e. 94px, to account for both the size of the fovea and the precision of the eye-tracking device.

Fixation and saccade patterns

Over the 120 images and 49 observers, we gathered a total of 58 273 valid fixations, and 44 321 valid saccades. On average, a fixation lasted 315.5 ms (STD: 251.0 ms), and 9.55 fixations per stimulus per observer were recorded (STD: 0.58). We found an inverse correlation (𝜌 = -0.48) between the number of fixations recorded and the agreement score 𝑃, indicating that participants tended to explore a stimulus more when the emotion was not easy to recognise. Figure 2 shows the distributions of fixation duration (a), saccade amplitudes (b), saccade orientations (c), and angle between two successive saccades (d). The average saccade amplitude is 1.38 degree of visual angle, which is quite low compared to what can be usually observed in natural scenes exploration [START_REF] Roman Von Wartburg | Size matters: Saccades during scene perception[END_REF], but can be explained by the nature of the stimuli. Indeed, the distance between the features of the presented faces (eyes, mouth, nose, and eyebrows) is roughly in the order of two degrees of visual angle. We can also observe a relatively high amount of vertical saccades (Figure 2 (c)), indicating a high number of saccades going from mouth to eyes, and vice-versa).

Facial features and areas of interest

In order to distinguish specific gaze patterns relatively to the displayed emotion, we first segmented each image into areas of interest based on six facial features, i.e., left and right eyes, left and right eyebrows, nose, and mouth. To do so, we used the BiSeNet semantic segmentation model [START_REF] Yu | Bisenet: Bilateral segmentation network for real-time semantic segmentation[END_REF], fine-tuned on the CelebAMask-HQ dataset [START_REF] Cheng-Han Lee | Maskgan: Towards diverse and interactive facial image manipulation[END_REF], using the implementation and weights in [START_REF] Zllrunning | face-parsing.pytorch[END_REF]. Figure 3 shows visual examples of such segmented faces.

We then counted the amount of gaze fixations falling into each region of interest. As shown in Table 2, we can observe significant differences in the distribution of fixations amongst the different facial features areas. For instance, the amount of fixations happening on the eyebrows is significantly higher in images displaying anger or surprise (11.73% on average for both left and right eyebrows) compared with faces displaying happiness (5.33%). Similarly, the amount of fixations located on the mouth are higher on surprised faces (26.32%) than on any other faces. These results are coherent with the general idea we have on facial expressions (e.g., surprise is usually linked to an open mouth).

BUBBLEVIEW EXPERIMENT: RESULTS

With this experiment, we aimed to evaluate whether or not the BubbleView metaphor could be reliably used to collect data that would be analogous to eye-tracking scanpaths in the context of facial expression recognition. To this aim, 160 images were used, in two different settings, i.e., in lab and through crowdsourcing.

Emotion recognition labels

Similarly to the eye-tracking experiment, participants were asked to label the image they were shown, with the same four emotion categories.

In laboratory settings, the average 𝑃 agreement was 0.710 (STD: 0.235). Over the 160 images, 49 (30.62%) had an agreement score over 0.9, and 45 (28.12%) had an agreement under 0.5, which remains consistent with the scores obtained during the eye-tracking experiment.

We observed very similar results in crowdsourcing settings, where the average 𝑃 agreement was 0.671 (STD: 0.222), 33 images (20.6%) had an agreement score over 0.9, and 46 (28.75%) had an agreement under 0.5. We believe that the lower amount of very high agreement images is just due to the higher number of annotators, and the fact that they might have different cultural backgrounds, meaning that they could interpret facial expression slightly differently.

Only 4 images (2.5%) were labeled differently by the majority of participants between laboratory and crowdsourcing settings. All of those images had an agreement score under 0.3 (both in-lab and in crowdsourcing), indicating that these cases were particularly difficult to classify, most likely because they did not fit within one of the four proposed categories (i.e., happiness, sadness, surprise, and anger). We also observe the exact same labeling by the majority of in-lab and crowdsourcing participants compared with eye-tracking experiment participants on the 80 common stimuli,

showing that the collected labels are independent of the conditions in which they were obtained.

BubbleView data processing

For this study, we chose to rely on the continuous BubbleView metaphor, i.e., using solely continuous mouse tracking and not relying on clicks, in order to account for both bottom-up and top-down visual processes. In this way, the whole image was blurred, except for a circular area located around the position of the mouse cursor on screen. For each image and each observer, we removed the mouse tracks from the study when less than 50 points were recorded, indicating that the mouse was not moved during extended periods of time.

To infer visual saliency maps from mouse tracking data, we relied on the work presented in [START_REF] Ellahi | Evaluation of the bubble view metaphor for the crowdsourcing study of visual attention deployment in tone-mapped images[END_REF]. The value of each pixel of the map was set to be the total amount of time spent with the mouse cursor at this location, aggregated over all observers. This map was then convolved with the same 2D gaussian kernel used in the eye-tracking experiment, representing one degree of visual angle.

With a view to validate the soundness of the BubbleView metaphor in this context, we compared the saliency maps obtained with this method to ground-truth eye-tracking data, using three metrics commonly used to evaluate visual saliency maps, i.e., Pearson's correlation coefficient (CC), Normalized Scanpath Saliency (NSS), and Borji's Area Under Curve (AUC-B) [START_REF] Le | Methods for comparing scanpaths and saliency maps: strengths and weaknesses[END_REF]. Over the 80 images that are common to the BubbleView and eye-tracking experiments, we can observe a CC value of 0.84, a NSS value of 2.43, and an AUC-B value of 0.87, indicating a very high similarity between both ways of collecting data. We also observe no significant difference between the maps collected in laboratory and in crowdsourcing settings (CC = 0.98). Consequently, we merge these two sources of data in the following.

Figure 4 however highlights one of the main differences between eye-tracking and BubbleView saliency maps:

eye-tracking maps seem to focus much more on the areas between the eyes, as well as on the nose, which might be indicative of intermediate fixations happening during the visual path from one eye to the other, or from the eyes to the mouth.

Mouse track patterns

During the BubbleView experiments, the position of the mouse was recorded every time the mouse was moving, with a maximum sampling rate of 250 Hz (i.e., corresponding to one point every 4ms). On average, one point was recorded every 20.64ms, and we gathered a total of 3 834 077 points over the 160 images.

Figure 5 shows the distribution of mouse velocities and accelerations. We observe that, overall, mouse velocity did not vary much, indicating that participants explored the stimuli in a relatively smooth and continuous way, which is also supported by low values of mouse acceleration.

Similarly to the eye-tracking patterns, we fond an inverse correlation (𝜌 = -0.45) between the total distance of the mouse path on a stimulus and the agreement score, meaning that an ambiguous stimulus caused observers to explore a face more thoroughly in order to discriminate between the four possible labels.

Facial features and areas of interest

As for the eye-tracking experiment, we used the semantic segmentation of the stimuli to evaluate the importance of facial features in the classification task. In this case, instead of counting the number of recorded points falling in the different regions, we rather counted the amount of time during which the mouse was located on each facial feature area.

Similarly to the saliency maps created from mouse-tracking, this was done to avoid an over-representation of transition points relatively to "fixations" where the mouse could stay still at a given location, and thus creating only a single point.

Table 3 shows the distribution of time spent on each of the considered facial feature. It should be noted that, as illustrated in Figure 4, most of the time was spent on these given features rather than on the rest of the face: 95.4% on average, compared to 77.6% of eye fixation points falling in these areas. We observe a distribution of attention between the feature areas that is quite similar to the one obtained in the eye-tracking experiments: "Happiness" stimuli show a high proportion of fixations on the eyes, "Anger" stimuli draw more attention on the eyebrows, and "Surprise" stimuli focus more on mouths and eyes. This supports the hypothesis that different emotions are linked to different distinguishable attention patterns, highlighting the interest of such a multi-modal dataset.

DISCUSSION

With this new dataset, we have proposed to consider visual attention and facial expression recognition (FER) as a whole, showing links between attention patterns obtained through eye-tracking or the BubbleView metaphor, and the annotation task of assigning the right emotion label to pictures of faces. In the following, we discuss several ways to make use of the existence of such relationship, and propose perspectives for future works.

Reliability of FER annotations

As discussed in several previous works, e.g., [START_REF] Zeng | Facial expression recognition with inconsistently annotated datasets[END_REF][START_REF] Lévêque | Comparing the robustness of humans and deep neural networks on facial expression recognition[END_REF], existing FER datasets often present inconsistent, or unreliable labels.

For instance, over the 200 images that we used for this work, 32 (16%) presented discrepancies between the original label of the AffectNet set and the most-voted label in our data collection.

This can be explained by various factors, related to the way of collecting such data, but also to the intrinsic nature of the very definition of ground-truth in the context of facial expressions. It can indeed be noted that the AffectNet dataset was annotated with a single human annotator per image on half of the set, and a combination of predictions of deep neural networks for the remaining half. This way of collecting data, while enabling the large scale of the dataset, allows for numerous cases of misclassification.

As highlighted previously, we showed the existence of a relationship between human visual attention, through gaze and mouse patterns, and the emotion recognised, as well as with the difficulty of the FER task -that is to say on whether an emotion was easily recognised or not. Consequently, such patterns could be used as a mean to evaluate the confidence level of human annotators when associating an emotion to a given portrait.

In practice, the annotation of images of faces would include, on top of the FER task, recording the gaze patterns of annotators using the eye-tracking technology, or their mouse patterns using the BubbleView metaphor. This way, FER datasets could be more reliable and would include objective levels of confidence for each image.

Human and machine attention in FER tasks

Recently, the computer vision and machine learning fields have drawn inspiration on the human mechanism of visual attention, i.e., the selection of the most task-relevant areas of an image before further cognitive process. By implementing or learning such mechanisms, some models offer an additional layer of explicability, by providing saliency maps indicating the areas of interest the most responsible for the final decision [START_REF] Lai | Understanding more about human and machine attention in deep neural networks[END_REF][START_REF] Hassanin | Visual attention methods in deep learning: An in-depth survey[END_REF].

Such saliency maps can then be compared to human visual attention ground-truth when performing similar tasks.

Previous works showed that, for high-level tasks, the closeness between human and machine attention leads to better performances of the model [START_REF] Lai | Understanding more about human and machine attention in deep neural networks[END_REF]. We would argue that facial expression recognition is an excellent example of such high-level vision tasks. Therefore, our dataset could be used to improve FER models relying on attention mechanisms, e.g., [START_REF] Li | Attention mechanism-based cnn for facial expression recognition[END_REF][START_REF] Zhu | A cascade attention based facial expression recognition network by fusing multi-scale spatio-temporal features[END_REF], for instance by adding a regularisation term to the loss function to penalise the generated attention map should it be too far away from human ground truth.

Moreover, considering the relationship between attention patterns and facial expressions, we believe that deep learning models could rely on a common set of extracted features for both attention prediction and FER tasks. Consequently, a multi-task learning framework, where a model would learn to predict the displayed emotion as well as visual saliency or scanpaths using the same feature extractor, seems to be an interesting approach, for which our dataset -or extensions of it -would prove particularly useful.

Figure 1 (

 1 Figure 1 (a) represents examples of faces ordered by their agreement scores. Over the 120 images, only 29 of them (24.2%) had an agreement value over 0.9, indicating that observers strongly agreed on the label, while 31 images (25.8%) presented an agreement value under 0.5, indicating that the emotion displayed on these images was not easily recognised by the participants.

Figure 1 (Fig. 1 .

 11 Fig. 1. Illustration of a sample of images based on the degree of agreement between participants in terms of labels. More particularly: (a) examples ordered by agreement score, (b) examples of disagreement between the majority vote and original labels.

Fig. 2 .

 2 Fig. 2. Illustration of distributions of (a) fixation durations, (b) saccade amplitudes, (c) saccade orientations, and (d) angle between successive saccades over the 120 images of the eye-tracking dataset.

Fig. 3 .

 3 Fig. 3. Illustration of examples of face semantic segmentation.

Fig. 4 .

 4 Fig. 4. Illustration of examples of eye-tracking (middle line) and BubbleView (bottom line) saliency maps.

Fig. 5 .

 5 Fig. 5. Illustration of the distributions of (a) mouse velocities and (b) mouse accelerations over the 160 images in the BubbleView dataset.

Table 1 .

 1 Characteristics of the eye-tracking and BubbleView experiments.

	Features	Eye-tracking	BubbleView in lab	BubbleView online
	Number of images	120	80 (2 playlists of 40)	160 (4 playlists of 40)
	Screen resolution	1920×1080	1920×1080	Depending on participants
	Display time (sec)	4	7	7
	Number of observers	50	60	240
	Eye-tracker	Tobii Pro Fusion	/	/
	Bubble size (px)	/	94	94
	Blur sigma (px)	/	12	12

Table 2 .

 2

	Happiness	21.43	21.08	2.71	2.62	7.55	19.78
	Anger	18.26	18.17	6.80	6.76	5.23	19.07
	Sadness	20.59	20.35	3.26	3.38	6.17	20.70
	Surprise	23.87	24.01	4.97	4.93	4.28	26.32

Amount (in percent) of gaze fixations located on the area of each facial feature, for each emotion.

Left eye Right eye Left eyebrow Right eyebrow Nose Mouth

Table 3 .

 3

	Happiness	28.21	27.03	3.64	3.16	9.83	22.31
	Anger	20.98	21.09	9.71	9.90	7.74	24.50
	Sadness	25.27	24.88	4.13	5.82	6.26	25.87
	Surprise	27.07	27.18	5.24	5.10	5.27	31.38

Amount (in percent) of time spend by the mouse cursor on the area of each facial feature, for each emotion.

Left eye Right eye Left eyebrow Right eyebrow Nose Mouth


Bérangère Coutanceau

Esther Dos Santos

Nelly Swierkowski Blanchard

Anne Sanchez Louboutin

Florence Boitrelle

François Margueritte

François Vialard
email: francois.vialard@uvsq.fr

Valérie Serazin

Khadija Fathallah

Esther Dos Santo

Nelly Swierkowski Blanchar

Function of the Endometrial Cytokine Profile? A Single-Center Experience

Keywords: embryo implantation, in vitro fertilization, endometrium, cytokine profile

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Despite recent progress in assisted reproductive technology (ART), today's implantation rates per embryo transferred (20-30%), clinical pregnancy rates per transfer, and live birth rates are unsatisfactory [START_REF] Mascarenhas | regional, and global trends in infertility prevalence since 1990: A systematic analysis of 277 health surveys[END_REF]. Repeated implantation failure (RIF) is defined as the absence of an ongoing clinical pregnancy after the transfer of at least four good-quality embryos [START_REF] Coughlan | Recurrent implantation failure: Definition and management[END_REF]. The management of RIF remains a challenge for ART professionals.

A poor-quality embryo is the most important cause of RIF. Many studies have confirmed the negative impact of embryonic aneuploidies on the likelihood of pregnancy [START_REF] Montag | New approaches to embryo selection[END_REF].

of 11

Although a recent study showed that the routine pre-implantation diagnosis of aneuploidy (PGD-A) enabled pregnancy to be achieved after at least three attempts in more than 90% of cases [START_REF] Pirtea | Endometrial causes of recurrent pregnancy losses: Endometriosis, adenomyosis, and chronic endometritis[END_REF], RIF can sometimes occur even when good-quality embryos are transferred. In fact, the literature data indicate clearly that in humans fewer than 60% of euploid embryos result in pregnancy [START_REF] Scott | Comprehensive chromosome screening is highly predictive of the reproductive potential of human embryos: A prospective, blinded, nonselection study[END_REF].

Along with well-known causes of embryo implantation failure (such as anatomical anomalies [START_REF] Tulandi | Redefining reproductive surgery[END_REF], infectious diseases [START_REF] Giakoumelou | The role of infection in miscarriage[END_REF], hormone disorders [START_REF] Hanna | Decidual NK cells regulate key developmental processes at the human fetal-maternal interface[END_REF], hematological disorders [START_REF] Simone | Antiphospholipid antibodies affect human endometrial angiogenesis: Protective effect of a synthetic peptide (TIFI) mimicking the phospholipid binding site of β(2) glycoprotein I[END_REF], and immune diseases [START_REF] Kwak-Kim | Immunological modes of pregnancy loss: Inflammation, immune effectors, and stress[END_REF]), it appears that endometrial defects constitute a non-negligible risk factor for RIF [START_REF] Revel | Defective endometrial receptivity[END_REF]. For example, it is well known that immune defects can cause obstetric disorders in early pregnancy and during placental development [START_REF] Marron | Detailed endometrial immune assessment of both normal and adverse reproductive outcome populations[END_REF]. Defects in maternal-fetal immune tolerance are thought to be strongly associated with morbidities such as pregnancy-induced hypertension, pre-eclampsia, growth retardation, and preterm delivery [START_REF] Savasi | Maternal and fetal outcomes in oocyte donation pregnancies[END_REF].

Embryo implantation is a complex process during which the embryo adheres to and then penetrates the maternal endometrium. Various adhesion molecules, growth factors, chemokines, and cytokines create a molecular dialogue between the embryo and the endometrium. Two groups of cytokines have different roles in the polarization of T cell secretion: Th1 type cytokines (interleukin 2 (IL2), tumor necrosis factor beta (TNFß), and interferon gamma (IFNγ)) are involved in cell-mediated immune reactions that are harmful for implantation, whereas Th2 type cytokines (IL4, IL5, and CD40) are involved in humoralmediated immune reactions that favor implantation. Furthermore, uterine-specific natural killer (uNK) cells have a different phenotype from other circulating NK cells with a low level of spontaneous cytotoxicity that can be modulated by the cytokine environment [START_REF] Verma | Human decidual natural killer cells express the receptor for and respond to the cytokine interleukin 15[END_REF] and by class 1b major histocompatibility complex molecules (including HLA-G). Overall, the establishment of an equilibrium enables implantation and favors an ongoing pregnancy. Any imbalance will result in implantation failure and rejection of the fetal allograft [START_REF] Perrier D'hauterive | L'implantation: Premier dialogue entre la mère et l'embryon[END_REF]. For example, an abnormally high basal level of uNK cell activity is reportedly a predictor of recurrent miscarriages [START_REF] Aoki | Preconceptional natural-killer-cell activity as a predictor of miscarriage[END_REF].

Following on from these observations, several studies have shown that the expression of IL15 and IL-18 is dysregulated in the endometrium of women with RIF, relative to that of fertile women [START_REF] Lédée-Bataille | Role of the endometrial tripod interleukin-18, -15, and -12 in inadequate uterine receptivity in patients with a history of repeated in vitro fertilization-embryo transfer failure[END_REF]. IL-15 activity is modulated by its receptor fibroblast growth factor-inducible molecule 14 (Fn-14) [START_REF] Petitbarat | Tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor inducible-14 might regulate the effects of interleukin 18 and 15 in the human endometrium[END_REF] and is directly involved in the recruitment and maturation of uNK cells [START_REF] Kitaya | Central role of interleukin-15 in postovulatory recruitment of peripheral blood CD16(-) natural killer cells into human endometrium[END_REF]. Similarly, the Th2-type cytokine IL-18 is immunomodulated by the TNF weak inducer of apoptosis (TWEAK) [START_REF] Croy | Update on pathways regulating the activation of uterine Natural Killer cells, their interactions with decidual spiral arteries and homing of their precursors to the uterus[END_REF]. The expression of IL-18 rises during the implantation window and the cytokine has a primarily angiogenic role [START_REF] Huang | Interleukin-18 system messenger RNA and protein expression in human endometrium during the menstrual cycle[END_REF]. By monitoring the IL-15/Fn-14 mRNA ratio (a biomarker of uNK activation/maturation) and the IL-18/TWEAK mRNA ratio (a biomarker of angiogenesis and Th1/Th2 balance), the human endometrium's immune profile can thus be classified as normal, hyper-activated, or hypo-activated [START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF].

In the context of immune deregulation and RIF, it was first shown that the prednisone treatment of women with a hyper-activated profile led to the normalization of immune biomarker levels [START_REF] Lédée | Impact of prednisone in patients with repeated embryo implantation failures: Beneficial or deleterious?[END_REF]. This normalization was associated with a live birth rate of 39.8% after the first embryo transfer [START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF]. We therefore hypothesized that the correction of an abnormal endometrial immune profile would result in a higher pregnancy rate.

The primary objective of the present study was to describe the management of RIF and the clinical pregnancy rates in our center as a function of the endometrial immune profiles. The secondary objective was to evaluate the impact of the time interval between the diagnosis of endometrial immune dysfunction (following an endometrial biopsy) and the implementation of the ART cycle.

Materials and Methods

We performed an interventional, longitudinal, prospective cohort study at the Poissy-Saint-Germain-en-Laye Hospital over a four-year period (from January 2016 to December 2019). All women with RIF were invited to undergo an endometrial biopsy for an analysis of their cytokine profile. The treatment prior to embryo transfer was then adjusted as a function of the profile (normal, hyper-activated, or hypo-activated). In each group, the ongoing pregnancy rate per transfer cycle was calculated for the 12 months following the biopsy.

We also analyzed the miscarriage rate; the ongoing pregnancy rate per treated patient; the mean time interval between the endometrial biopsy and a pregnancy; the number of patients who received treatment after a biopsy; the number of cycles with a transfer (with one or two embryos); the number of embryos transferred in total and on average per patient after a biopsy; and the mean time interval between the biopsy and the embryo transfer.

Population

In the absence of a consensus of the definition in the literature, we defined RIF as the absence of an ongoing pregnancy at more than 10 weeks of amenorrhea despite embryo transfer at the 2 or 3-day culture stage or four embryos at the blastocyst stage during an in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) procedure. All included patients had to be between 18 and 43 years of age. Patients outside this age range; those having undergone a gamete donation procedure; those who had not undergone an endometrial biopsy during hormone replacement therapy and during the implantation phase (according to the histologic features); and those wishing to stop their treatment were excluded. All included patients gave their written consent to participate in this study.

Endometrial Biopsy

An endometrial biopsy (using a Cornier endometrial suction catheter) was performed in all patients with a spontaneous menstrual cycle during the implant phase (between days 20 and 24 of the cycle, for normally ovulating patients) without anesthesia but with ultrasound guidance, if necessary. The aspirated fragments of uterine mucosa were aliquoted into two different vials: (i) a 4% formalin solution for standard histological analysis, in order to confirm that the specimen was in the secretory phase, and (ii) RNA stabilization solution (RNAlater; Qiagen, Venlo, The Netherlands), to study the expression of the genes of interest.

RNA Quantification

Total RNA from the cells was extracted with the Nucleospin RNA II ® kit (Macherey-Nagel, Düren, Germany) and stored at -80 • C. The RNA was reverse-transcribed using the SuperScript III Reverse Transcriptase kit (Invitrogen, Waltham, MA, USA) and the expression levels of the target gene (coding for IL15, IL18, Fn14, and TWEAK) were quantified with regard to the reference genes (coding for beta 2 microglobulin, ribosomal protein L13A, and TATA box-binding protein). The primer sequences have been described previously [START_REF] Huang | Interleukin-18 system messenger RNA and protein expression in human endometrium during the menstrual cycle[END_REF].

For amplification, the reaction mixture consisted of 4 µL of cDNA diluted 1:20, 0.2 µL of forward primer (25 µm), 0.2 µL of reverse primer (25 µm), 0.6 µL of sterile water, and 5 µL of buffer (2×) containing Sybr ® Green, Taq polymerase, and its buffer (Light Cycler 480 SYBR Green I Master mix, Roche Diagnostics, Basel, Switzerland). Quantitative RT-PCR was performed on a Light Cycler 480 (Roche Diagnostics). The program used included a 7 min denaturation initiation at 95 • C (to activate the Taq polymerase), 44 PCR cycles (10 s of denaturation at 95 • C, the hybridization of the coupled primers at 60 • C for 10 s, and then elongation by Taq polymerase for 15 s), and a melting step (65-95 • C, with five acquisitions/s) and a cooling step at 4 • C. Each run included a cDNA-free control and an inter-run calibrator. The results were interpreted as relative amounts using Light Cycler 480 software 1.5.0 (Roche Diagnostics), as described previously [START_REF] Huang | Interleukin-18 system messenger RNA and protein expression in human endometrium during the menstrual cycle[END_REF][START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF].

We calculated the mean ± standard deviation (SD) IL15/Fn14 and IL18/TWEAK mRNA ratios in endometrial biopsies in a control group of fertile women. For each patient, the mRNA ratio was considered to be low when it was more than one SD below the mean.

Conversely, the ratio was considered to be high when it was more than one SD above the mean.

Immunohistochemistry of uNK Cells

Biopsies were fixed in formalin for 24 h and embedded in kerosene. Histologic sections were stored at 4 • C before use. Labeling was automated with the BENCHMARK XT Ventana system, using the Ultra View Universal DAB Kit and the CD56/NCAM clone 123 C3 Ventana antibody (Abbott, Chicago, IL, USA). The method used to interpret the results has been described previously [START_REF] Lédée | Impact of prednisone in patients with repeated embryo implantation failures: Beneficial or deleterious?[END_REF]. The uNK cell count was calculated as the mean (per field) number of CD56+ cells in four representative fields at a magnification of 400×, on a microscope with a field diameter of 0.62. A uNK cell count of 30 to 60 was considered to be normal; hence, an abnormally low count was <30, and an abnormally high count was >60.

Interpretation of the Cytokine Profile

The cytokine profile was classified as normal, hyper-activation, or hypo-activation according to a previously described decision tree [START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF] (Figure 1) [START_REF] Ledée | Method for Increasing Implantation Success in Assisted Fertilization[END_REF]. To classify the patients, we sequentially used the IL18/TW ratio, the uNK cell count (when the IL18/TW ratio was normal), and the IL15/Fn14 ratio (when the uNK cell count was normal or high).

an inter-run calibrator. The results were interpreted as relative amounts using Light Cycler 480 software 1.5.0 (Roche Diagnostics), as described previously [START_REF] Huang | Interleukin-18 system messenger RNA and protein expression in human endometrium during the menstrual cycle[END_REF][START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF].

We calculated the mean ± standard deviation (SD) IL15/Fn14 and IL18/TWEAK mRNA ratios in endometrial biopsies in a control group of fertile women. For each patient, the mRNA ratio was considered to be low when it was more than one SD below the mean. Conversely, the ratio was considered to be high when it was more than one SD above the mean.

Immunohistochemistry of uNK Cells

Biopsies were fixed in formalin for 24 h and embedded in kerosene. Histologic sections were stored at 4 °C before use. Labeling was automated with the BENCHMARK XT Ventana system, using the Ultra View Universal DAB Kit and the CD56/NCAM clone 123 C3 Ventana antibody (Abbott, Chicago, IL, USA). The method used to interpret the results has been described previously [START_REF] Lédée | Impact of prednisone in patients with repeated embryo implantation failures: Beneficial or deleterious?[END_REF]. The uNK cell count was calculated as the mean (per field) number of CD56+ cells in four representative fields at a magnification of 400×, on a microscope with a field diameter of 0.62. A uNK cell count of 30 to 60 was considered to be normal; hence, an abnormally low count was <30, and an abnormally high count was >60.

Interpretation of the Cytokine Profile

The cytokine profile was classified as normal, hyper-activation, or hypo-activation according to a previously described decision tree [START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF] (Figure 1) [START_REF] Ledée | Method for Increasing Implantation Success in Assisted Fertilization[END_REF]. To classify the patients, we sequentially used the IL18/TW ratio, the uNK cell count (when the IL18/TW ratio was normal), and the IL15/Fn14 ratio (when the uNK cell count was normal or high). The treatment goal was to control the activation of immune cells, promote the invasion by the embryo, and avoid the rejection and apoptosis of the embryo. Our treatment strategy was based on the fact that (i) glucocorticoids have long been used to influence the expression and activity of pro-inflammatory mediators and are key components of many immunomodulatory regimens [START_REF] Krigstein | Prednisolone for repeated implantation failure associated with high natural killer cell levels[END_REF][START_REF] Harrity | ART implantation failure and miscarriage in patients with elevated intracellular cytokine ratios: Response to immune support therapy[END_REF] and (ii) progesterone and estrogen decrease the expression of pro-inflammatory cytokines [START_REF] Lissauer | Progesterone promotes maternal-fetal tolerance by reducing human maternal T-cell polyfunctionality and inducing a specific cytokine profile[END_REF].

We therefore applied a previously described treatment protocol [START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF] with the administration of prednisolone (20 mg per day) and vitamin E (Toco ® 500 mg, twice a day) from day 3 of the cycle onwards. The corticosteroid treatment was continued until 8 weeks after the embryo transfer and was then tapered over a 3-week period if the pregnancy test was positive. Treatment with vitamin E was continued until 10 weeks after transfer. High-dose progesterone supplementation was also initiated (Progestan ® 200 mg, two tablets for intravaginal administration three times a day) on the evening of the oocyte retrieval (for a fresh embryo transfer) or when the uterine mucosa during estrogen treatment was thicker than 7 mm (for a frozen embryo transfer during hormone replacement therapy). Oral estrogen treatment (Provames ® 2 mg, two tablets per day) was initiated on the evening of the oocyte retrieval (for a fresh embryo transfer) or on the first day of the cycle (for a frozen embryo transfer during hormone replacement therapy). These treatments were continued for 10 weeks if the pregnancy test was positive. The patient was instructed to avoid activities that might damage the endometrium during the cycle preceding the transfer, and sexual intercourse was prohibited after the transfer. In fact, seminal plasma has been shown to induce the mobilization and activation of local immune cells [START_REF] Johansson | Semen activates the female immune response during early pregnancy in mice[END_REF]. Lastly, it has been reported that endometrial scratching or other endometrial injury in the middle of the luteal phase of the cycle preceding the embryo transfer activates and stimulates the expression of adhesion molecules and IL-15 [START_REF] Gnainsky | Local injury of the endometrium induces an inflammatory response that promotes successful implantation[END_REF]. Human chorionic gonadotropin (HCG) triggers the proliferation and maturation of uNK cells and induces immune tolerance at the maternal-fetal interface [START_REF] Schumacher | Human Chorionic Gonadotropin-Mediated Immune Responses That Facilitate Embryo Implantation and Placentation[END_REF].

Treatment in the Hypo-Activated Group

The treatment goal was to increase local responsiveness and promote the adhesion of the embryo. Endometrial scratching was performed in the middle of the luteal phase of the cycle preceding the embryo transfer. Luteal phase support with HCG 1500 IU was provided on days 4, 6 and 8 after oocyte retrieval. Sexual intercourse after the transfer was recommended.

Treatment in the Normal-Profile Group

The patients in the normal-profile group did not receive any additional treatments.

Statistical Analysis

First, we analyzed the following characteristics in the three endometrial profile groups (normal, hyper-and hypo-activation group): age, body mass index, tobacco use, the serum anti-Mullerian hormone level, the presence or absence of endometriosis, the type of infertility, parity, gravida, history of miscarriage, rank of the attempt, and the mean number of embryos transferred before the biopsy.

Second, we assessed the number of cycles with a transfer after the biopsy and also the number of embryos transferred after the biopsy for treatment into each group. Moreover, a treated patient could have more than one cycle after the biopsy (depends of the number of attempts) and, within each cycle, more than one embryo transferred. Within each group of the endometrial profile, we assessed the mean number of embryos transferred, the mean time interval between the biopsy and the transfer, and the number of pregnancies and ongoing pregnancies between each cycle with a transfer.

Quantitative variables are expressed as the mean standard deviation, minimum, and maximum, and were compared by the status of the endometrial profile using nonparametric Kruskal-Wallis test. Because of the distribution between groups (such as the hypo group), assessment of normality cannot be performed and non-parametric tests were used. Qualitative variables were compared by endometrial profiles using Fisher exact tests.

The significance threshold retained for all analyses (p) was an alpha risk of 0.05. All analyses were also performed using the STATA© 15.1 IC (StataCorp, College Station, TX, USA).

Results

Between January 2016 and December 2019, 120 patients underwent an endometrial biopsy and a cytokine profile analysis of an indication of RIF. This study's patient flow is summarized in Figure 2. Twenty-one of the 120 patients (17.5%) were subsequently excluded because the endometrium was not in the implantation phase (judging by the histologic features) or the patient had transferred to a gamete donation program. Of the remaining 99 biopsies, 22 (22.2%) did not yield a result (due to a lack of material (in 90.5% of these cases)) or the presence of degraded RNA (in 9.5%C)., whereas 77 (77.8%) yielded a result.

Results

Between January 2016 and December 2019, 120 patients underwent an endometrial biopsy and a cytokine profile analysis of an indication of RIF. This study's patient flow is summarized in Figure 2. Twenty-one of the 120 patients (17.5%) were subsequently excluded because the endometrium was not in the implantation phase (judging by the histologic features) or the patient had transferred to a gamete donation program. Of the remaining 99 biopsies, 22 (22.2%) did not yield a result (due to a lack of material (in 90.5% of these cases)) or the presence of degraded RNA (in 9.5%C)., whereas 77 (77.8%) yielded a result.

Hence, a total of 77 patients were included in the final analysis (Table 1). The mean (range) age of the patients was 34.2 years [START_REF] Ledée | Method for Increasing Implantation Success in Assisted Fertilization[END_REF][START_REF] Krigstein | Prednisolone for repeated implantation failure associated with high natural killer cell levels[END_REF][START_REF] Harrity | ART implantation failure and miscarriage in patients with elevated intracellular cytokine ratios: Response to immune support therapy[END_REF][START_REF] Lissauer | Progesterone promotes maternal-fetal tolerance by reducing human maternal T-cell polyfunctionality and inducing a specific cytokine profile[END_REF][START_REF] Johansson | Semen activates the female immune response during early pregnancy in mice[END_REF][START_REF] Gnainsky | Local injury of the endometrium induces an inflammatory response that promotes successful implantation[END_REF][START_REF] Schumacher | Human Chorionic Gonadotropin-Mediated Immune Responses That Facilitate Embryo Implantation and Placentation[END_REF][START_REF] Bellver | Implantation Failure of Endometrial Origin: What is New?[END_REF][START_REF] Kolanska | Proportion of Cytotoxic Peripheral Blood Natural Killer Cells and T-Cell Large Granular Lymphocytes in Recurrent Miscarriage and Repeated Implantation Failure: Case-Control Study and Meta-analysis[END_REF][START_REF] Dieamant | New therapeutic protocol for improvement of endometrial receptivity (PRIMER) for patients with recurrent implantation failure (RIF)-A pilot study[END_REF][START_REF] Li | Local injury to the endometrium: Its effect on implantation[END_REF][START_REF] Almog | Promoting implantation by local injury to the endometrium[END_REF](36)[START_REF] Gnainsky | Biopsy-induced inflammatory conditions improve endometrial receptivity: The mechanism of action[END_REF][START_REF] Potdar | Endometrial injury to overcome recurrent embryo implantation failure: A systematic review and meta-analysis[END_REF][START_REF] Chomarat | TNF skews monocyte differentiation from macrophages to dendritic cells[END_REF][START_REF] Luster | Immune cell migration in inflammation: Present and future therapeutic targets[END_REF][START_REF] Somigliana | Repeated implantation failure at the crossroad between statistics, clinics and over-diagnosis[END_REF][START_REF] Sar-Shalom Nahshon | The impact of intentional endometrial injury on reproductive outcomes: A systematic review and meta-analysis[END_REF]. The mean number of embryos transferred before the endometrial biopsy was 7.5. Twenty-two of the patients had experienced at least one early miscarriage during their ART program prior to the biopsy (29%). We did not observe any significant differences between the three groups for any of the variables (p > 0.05), except for the AMH serum level, which is the highest in the normal group compared to the others Hence, a total of 77 patients were included in the final analysis (Table 1). The mean (range) age of the patients was 34.2 years [START_REF] Ledée | Method for Increasing Implantation Success in Assisted Fertilization[END_REF][START_REF] Krigstein | Prednisolone for repeated implantation failure associated with high natural killer cell levels[END_REF][START_REF] Harrity | ART implantation failure and miscarriage in patients with elevated intracellular cytokine ratios: Response to immune support therapy[END_REF][START_REF] Lissauer | Progesterone promotes maternal-fetal tolerance by reducing human maternal T-cell polyfunctionality and inducing a specific cytokine profile[END_REF][START_REF] Johansson | Semen activates the female immune response during early pregnancy in mice[END_REF][START_REF] Gnainsky | Local injury of the endometrium induces an inflammatory response that promotes successful implantation[END_REF][START_REF] Schumacher | Human Chorionic Gonadotropin-Mediated Immune Responses That Facilitate Embryo Implantation and Placentation[END_REF][START_REF] Bellver | Implantation Failure of Endometrial Origin: What is New?[END_REF][START_REF] Kolanska | Proportion of Cytotoxic Peripheral Blood Natural Killer Cells and T-Cell Large Granular Lymphocytes in Recurrent Miscarriage and Repeated Implantation Failure: Case-Control Study and Meta-analysis[END_REF][START_REF] Dieamant | New therapeutic protocol for improvement of endometrial receptivity (PRIMER) for patients with recurrent implantation failure (RIF)-A pilot study[END_REF][START_REF] Li | Local injury to the endometrium: Its effect on implantation[END_REF][START_REF] Almog | Promoting implantation by local injury to the endometrium[END_REF](36)[START_REF] Gnainsky | Biopsy-induced inflammatory conditions improve endometrial receptivity: The mechanism of action[END_REF][START_REF] Potdar | Endometrial injury to overcome recurrent embryo implantation failure: A systematic review and meta-analysis[END_REF][START_REF] Chomarat | TNF skews monocyte differentiation from macrophages to dendritic cells[END_REF][START_REF] Luster | Immune cell migration in inflammation: Present and future therapeutic targets[END_REF][START_REF] Somigliana | Repeated implantation failure at the crossroad between statistics, clinics and over-diagnosis[END_REF][START_REF] Sar-Shalom Nahshon | The impact of intentional endometrial injury on reproductive outcomes: A systematic review and meta-analysis[END_REF]. The mean number of embryos transferred before the endometrial biopsy was 7.5. Twenty-two of the patients had experienced at least one early miscarriage during their ART program prior to the biopsy (29%). We did not observe any significant differences between the three groups for any of the variables (p > 0.05), except for the AMH serum level, which is the highest in the normal group compared to the others Fifteen of the 21 patients (71.4%) in the "normal profile" group underwent at least one embryo transfer (fresh or frozen) within 12 months of the endometrial biopsy, with an average of 3.5 embryos transferred per patient treated and a mean time interval between the biopsy and the transfer of 5.1 months (Table 2). Seven ongoing pregnancies were obtained in 33 cycles with a transfer (i.e., a rate of 21.2%). The mean time interval between the biopsy and the pregnancy was 6.3 months. After the implementation of appropriate treatment, 30 of the 53 patients in the "hyperactivation" group (56.6%) underwent an embryo transfer within 12 months of the biopsy. On average, 2.8 embryos were transferred per patient treated, and the mean time interval between the biopsy and the transfer was 6.1 months (Table 2). Fifteen ongoing pregnancies were obtained in 52 cycles with a transfer (i.e., a rate of 28.8). The mean time interval between the biopsy and the pregnancy was 6.8 months.

In the "hypo-activation" group, two of the three patients (66.7%) underwent an embryo transfer after treatment. In five cycles, five embryos were transferred, and one live birth was obtained. The mean time interval between the biopsy and the transfer was 3.8 months for the latter pregnancy (Table 2).

We did not observe any significant differences between the three groups for treated patients within any of the variables (mean number of embryos transferred, mean interval time between a biopsy and a transfer, number of pregnancies).

Discussion

Repeated implantation failure is one of the factors that limits the success of ART. Many lines of research are underway in this field, with a view of increasing the pregnancy rate. In fact, RIF is probably a multifactorial phenomenon. Historically, RIF was considered to have an anatomical cause, such as a congenital or acquired anomaly of the cavity. Today's biological and immunologic approach appears to be opening up interesting new perspectives [START_REF] Bellver | Implantation Failure of Endometrial Origin: What is New?[END_REF], particularly with regard to the endometrium's immune profile.

In the present study, we sought to duplicate research performed in 2016 [START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF] in order to determine whether adapting the treatment of patients with RIF as a function of the endometrial cytokine profile could increase pregnancy rates. We adopted the same definition of RIF as used previously, namely the absence of pregnancy for more than 10 weeks of amenorrhea after the transfer of at least six embryos during a course of IVF/ICSI. Likewise, we used the same algorithm and the same thresholds for the interpretation of the results. The mean number of embryos transferred in our study population of RIF patients was 7.5; this value is similar to those reported in the literature [START_REF] Kolanska | Proportion of Cytotoxic Peripheral Blood Natural Killer Cells and T-Cell Large Granular Lymphocytes in Recurrent Miscarriage and Repeated Implantation Failure: Case-Control Study and Meta-analysis[END_REF][START_REF] Dieamant | New therapeutic protocol for improvement of endometrial receptivity (PRIMER) for patients with recurrent implantation failure (RIF)-A pilot study[END_REF]. However, our interventional, longitudinal, prospective cohort study with the same methodology as the previously reported study also had the same limitations. To validate the treatment strategy, a randomized controlled trial would be required.

As mentioned above, a high proportion (22.2%) of the histologically valid biopsies did not yield a result for the cytokine profile-due mainly to a lack of endometrial material or the presence of degraded RNA. These data emphasize the operator-dependent nature of the endometrial biopsy.

The proportion of abnormal endometrial profiles in our cohort (72.7%) was similar to that reported by Lédée et al. (80%) [START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF]. Conversely, we found a higher proportion of hyper-activation profiles (68.8%, versus 56% for Lédée et al.) and thus a lower proportion of hypo-activation profiles (3.9% versus 25%, respectively). These differences might be due to the small number of patients in our study (120, versus 394 in Lédée et al.'s study).

In our study, the ongoing pregnancy per patient treated rate was 46.7% in the "normal profile" group; despite the presence of RIF, this value is higher than that found in the general population of infertile patients. This relative improvement might be due to an effect of the biopsy or "scratching" on implantation, as often observed in the literature [START_REF] Li | Local injury to the endometrium: Its effect on implantation[END_REF]. This effect might be mediated by the local inflammation created by the invasive procedure [START_REF] Almog | Promoting implantation by local injury to the endometrium[END_REF].

The rate for an ongoing pregnancy per cycle with a transfer (cumulative transfers in the 12 months after the biopsy) was 28% in the hyper-activation and hypo-activation groups together (with an average of 2.8 embryos transferred per patient treated after the biopsy). This value is similar to the 24.2% observed in the general population of infertile women after IVF/ICSI with frozen embryo transfers [36]. Hence, personalized treatment as a function of the endometrial profile might therefore "normalize" the likelihood of im-plantation in a population with RIF.

Nevertheless, this ongoing pregnancy rate is lower than the 40.9% reported in our original study [START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF]. This difference might be due to the severity of RIF in the present study population, as again evidenced by the average of 7.5 fruitless embryo transfers before the biopsy. However, our results are similar to those in two other studies, notably for the "hyper-activation" group treated with prednisone [START_REF] Lédée | Impact of prednisone in patients with repeated embryo implantation failures: Beneficial or deleterious?[END_REF][START_REF] Krigstein | Prednisolone for repeated implantation failure associated with high natural killer cell levels[END_REF].

In the hyper-activation and hypo-activation groups, the pregnancy rate did not differ significantly from that observed in the control group. This similitude indicates that normalization of the immune profile resulted in a "normal" pregnancy rate, although we did not check for normalization in a subsequent endometrial biopsy. In order to confirm this result, one would have to conduct a randomized study in which a group of patients with hyper-activation or hypo-activation is treated for deregulation and another group is not. Nevertheless, given our encouraging results and the great distress of couples faced with RIF, we consider that this option was not ethically feasible. In order to generate biological proof of our treatment's putative ability to normalize the cytokine profile, we could have performed post-treatment biopsies. However, previous studies have demonstrated the beneficial impact of corticosteroids on the normalization of immune biomarkers for immune hyper-activation profiles [START_REF] Lédée | Impact of prednisone in patients with repeated embryo implantation failures: Beneficial or deleterious?[END_REF] and that an endometrial biopsy activates endometrial immune cells [START_REF] Gnainsky | Biopsy-induced inflammatory conditions improve endometrial receptivity: The mechanism of action[END_REF].

With regard to the time interval between biopsy and embryo transfer, the vast majority of published studies focused on the cycle preceding the biopsy [START_REF] Potdar | Endometrial injury to overcome recurrent embryo implantation failure: A systematic review and meta-analysis[END_REF]. However, it has been shown [START_REF] Gnainsky | Local injury of the endometrium induces an inflammatory response that promotes successful implantation[END_REF] that monocytes recruited at the injured sites (which have a role in improving endometrial receptivity) are long lived and are found in the tissues for several months [START_REF] Chomarat | TNF skews monocyte differentiation from macrophages to dendritic cells[END_REF][START_REF] Luster | Immune cell migration in inflammation: Present and future therapeutic targets[END_REF]; this might mediate the biopsy's long-term effect. In our study, the mean time interval between the biopsy to pregnancy was 6.3 and 6.8 months in both the "normal" and "hyper-activation" groups, respectively, and 3.8 months in the "hypo-activation" group. This implies that a short time interval between the biopsy and transfer is not essential for a successful attempt. One could therefore consider not repeating the biopsy in the following 12 months, while continuing to transfer embryos and while applying the recommended treatment scheme as a function of the cytokine profile. However, this approach would also need to be validated in randomized trials with follow-up biopsies at different time intervals.

The main limitation of our study was the small number of participants, given the low incidence of RIF in ART (estimated to be around 10%) [START_REF] Somigliana | Repeated implantation failure at the crossroad between statistics, clinics and over-diagnosis[END_REF]. However, the lack of a consensus of the definition of RIF makes this proportion difficult to estimate. Secondly, a high proportion of the endometrial biopsies did not give an interpretable result due to a lack of material or the absence of an implantation window. Thirdly, we did not calculate the number of participants required prospectively. Fourthly, we did not use diagnose aneuploidies prior to implantation as this technique is still controversial but has been shown to be very effective in increasing pregnancy rates [START_REF] Pirtea | Endometrial causes of recurrent pregnancy losses: Endometriosis, adenomyosis, and chronic endometritis[END_REF]. Lastly, and as mentioned above, this was only an interventional, longitudinal, prospective cohort study that lacked a control group. However, it is very difficult not to suggest a new strategy for couples with RIF.

In terms of study strengths, our population was very homogeneous (despite its small size) and produced results that were consistent with the literature data [START_REF] Lédée | The Uterine Immune Profile May Help Women With Repeated Unexplained Embryo Implantation Failure After In Vitro Fertilization[END_REF]. Our study was also the first to have assessed ongoing pregnancy rates over a long period (12 months) after the endometrial biopsy; most of the previous studies focused solely on the cycle immediately after the biopsy [START_REF] Potdar | Endometrial injury to overcome recurrent embryo implantation failure: A systematic review and meta-analysis[END_REF][START_REF] Sar-Shalom Nahshon | The impact of intentional endometrial injury on reproductive outcomes: A systematic review and meta-analysis[END_REF]. However, this is a pilot study to assess the performance of this strategy. In order to confirm these results, a prospective study based on these findings and with a number of subjects of "needed to treat" is necessary. Then, a prospective randomized study should be performed to confirm the efficacy of this clinical approach, aiming to improve implantation rates per embryo transferred.

Conclusions

Our results indicated a potential positive effect of appropriate treatment on the ongoing pregnancy rate in women with RIF. The results must now be validated in randomized studies with a larger number of well-characterized patients. By applying a previously published decision tree, this treatment approach could be implemented in clinics worldwide.

Figure 1 .

 1 Figure 1. Decision tree for the interpretation of immune deregulation, according to Lédée et al. [24].Figure 1. Decision tree for the interpretation of immune deregulation, according to Lédée et al. [24].

Figure 1 .

 1 Figure 1. Decision tree for the interpretation of immune deregulation, according to Lédée et al. [24].Figure 1. Decision tree for the interpretation of immune deregulation, according to Lédée et al. [24].

2. 6 .

 6 Treatment Regimens in Each Group 2.6.1. Treatment in the Hyper-Activation Group

Figure 2 .

 2 Figure 2. Study flow diagram.

Table 1 .Figure 2 .

 12 Figure 2. Study flow diagram.

Table 1 .

 1 The characteristics of the study participants, according to their endometrial profile.

		Normal Group	Hyper-Activation Group	Hypo-Activation Group
	Endometrial Profile	21/77 (27.3%)	53/77 (68.8%)	3/77 (3.9%)	p Value
		n (%) or µ ± SD	n (%) or µ ± SD	n (%) or µ ± SD
	Age (years) (n = 77)	34 (25-42)	34 (24-41)	32 (30-33)	0.27
	Body mass index (kg/m 2 ) (n = 76)	23 (18-31)	24 (17-34)	22 (19-25)	0.58
	Tobacco use (n = 74)	4 (19.1%)	6 (12.0%)	0 (0.0%)	0.66
	Serum AMH level (ng/dL) (n = 69)	4.18 (0.7-10.6)	2.94 (0.1-14.7)	3.37 (2.0-4.1)	0.04
	Endometriosis (n = 77)	7 (33.3%)	14 (26.4%)	1 (33.3%)	0.81
	Primary infertility (n = 77)	14 (66.7%)	33 (62.3%)	3 (100.0%)	0.62
	Gravida (n = 77)
	G0 G1	7 (33.3) 8 (38.1)	23 (43.4) 17 (32.1)	2 (66.7) 1 (33.3)	0.86
	G2+	6 (28.6)	13 (24.5)	0(0.0)

Table 1 .

 1 Cont.

		Normal Group	Hyper-Activation Group	Hypo-Activation Group
	Endometrial Profile	21/77 (27.3%)	53/77 (68.8%)	3/77 (3.9%)	p Value
		n (%) or µ ± SD	n (%) or µ ± SD	n (%) or µ ± SD
	Parity (n = 77)
	P0 P1	15 (71.4) 6 (28.6)	41 (77.4) 8 (15.1)	3 (100.0) 0 (0.0)	0.45
	P2+	0 (0.0)	4 (7.6)	0 (0.0)
	At least one miscarriage before the ART program (n = 77)	11 (52.4%)	21 (39.6%)	1 (33.3%)	0.63
	At least one miscarriage during the ART program (n = 77)	8 (38.1%)	13 (24.5%)	1 (33.3%)	0.46
	Mean Attempted rank (n = 77)	2.4 ± 0.8	2.5 ± 0.8	2.3 ± 0.6	0.88
	Mean Embryos transferred before the biopsy (n = 77)	7.8 ± 2.3	7.5 ± 2.1	6.3 ± 0.6	0.48

AMH: anti-Mullerian hormone; ART: assisted reproductive technology.

Table 2 .

 2 Cumulative results for transfer within 12 months for treated patients.

		Normal Group	Hyper Group	Hypo Group
		n = 15	n = 30	n = 2	p Value
		n (%) or µ ± SD	n (%) or µ ± SD	n (%) or µ ± SD
	Cycles with a transfer after the biopsy for treated patient (n)	33	52	5
	Embryos transferred after the biopsy for treated patient (n)	52	84	5
	Mean number of embryos transferred per treated patient, after the biopsy *	3.5 ± 2.3	2.8 ± 1.6	2.5 ± 2.1	0.61

Mean time interval between the biopsy and the transfer (months) **

		5.1 ± 3.4	6.1 ± 3.4	4.5 ± 0.7	0.48
	Pregnancies per cycle patients with a transfer **	10/33 (30.3%)	19/52 (36.5%)	1/5 (20.0%)	0.70
	Ongoing pregnancies per cycle with a transfer **	7/33 (21.2%)	15/52 (28.8%)	1/5 (20.0%)	0.84
	Miscarriages per cycle with a transfer **	3/33 (9.1%)	3/52 (5.8%)	0/5 (0.0%)	0.77
	Ongoing pregnancies per treated patient *	7/15 (46.7%)	13/30 (43.3%)	1/2 (50.0%)	1.00
	Mean ± SD time interval between the biopsy and the pregnancy (months) ***	6.3 ± 3.2	6.8 ± 3.5	3.8 ± NA	0.59
	*: per treated patients: normal group n = 15, hyper group n = 30, hypo group n = 2; **: per cycle per treated
	patients: normal group n = 33, hyper group n = 52, hypo group n = 5; ***: per pregnancy: normal group n = 10,
	hyper group n = 19, hypo group n = 1; NA: Not Available, only one patient.

Funding: This research received no external funding.

Institutional Review Board Statement: CPP-IdF11-number 13028.

Informed Consent Statement:

All included patients gave their written consent to participate in this study.

Data Availability Statement:

The data presented in this study are available from the corresponding author upon reasonable request.

Conflicts of Interest:

The authors declare no conflict of interest.


B Najib

T Quibel

A Tessier

J Mortreux

P Bouvagnet

C Cohen

F Vialard

R Dard

Prenatal diagnosis of recurrent hypoplastic left heart syndrome associated with MYH6 variants: a case report

Keywords: Hypoplastic left heart syndrome, MYH6, MYH7, Congenital heart disease, Case report

Background Hypoplastic left heart syndrome (HLHS) is a rare but genetically complex and clinically and anatomically severe form of congenital heart disease (CHD).

Case presentation Here, we report on the use of rapid prenatal whole-exome sequencing for the prenatal diagnosis of a severe case of neonatal recurrent HLHS caused by heterozygous compound variants in the MYH6 gene inherited from the (healthy) parents. MYH6 is known to be highly polymorphic; a large number of rare and common variants have variable effects on protein levels. We postulated that two hypomorphic variants led to severe CHD when associated in trans; this was consistent with the autosomal recessive pattern of inheritance. In the literature, dominant transmission of MYH6-related CHD is more frequent and is probably linked to synergistic heterozygosity or the specific combination of a single, pathogenic variant with common MYH6 variants.

Conclusions

The present report illustrates the major contribution of whole-exome sequencing (WES) in the characterization of an unusually recurrent fetal disorder and considered the role of WES in the prenatal diagnosis of disorders that do not usually have a genetic etiology.

Background

The first-trimester prenatal diagnosis of congenital heart disease (CHD) using transvaginal ultrasound was first reported in 1990s [START_REF] Mcbrien | Early fetal echocardiography[END_REF]. Since then, various groups with a high risk of CHD have been defined: these include increased nuchal translucency in the fetus, certain maternal illnesses (e.g., diabetes mellitus), conception via assisted reproductive technology, exposure to teratogenic drugs, an unambiguous family history and/or previous pregnancies with CHD [START_REF] Donofrio | Diagnosis and treatment of fetal cardiac disease: a scientific statement from the American Heart Association[END_REF]. In the fetal echocardiogram, the ventricles should be symmetric and equivalent in volume; any differences in size are suggestive of disease [START_REF] Smrcek | Early fetal echocardiography: heart biometry and visualization of cardiac structures between 10 and 15 weeks' gestation[END_REF]. However, first-trimester fetal cardiac ultrasound has a limited ability to diagnosing subtle, minor, late-onset or progressive fetal pathologies such as ventricular septal defects, cardiac tumors, valvular stenosis or regurgitation, and coarctation of the aorta [START_REF] Zidere | Comparison of echocardiographic findings in fetuses at less than 15 weeks' gestation with later cardiac evaluation[END_REF].

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of CHD [START_REF] Tomita-Mitchell | Impact of MYH6 variants in hypoplastic left heart syndrome[END_REF]. It accounts for around 4% of cases of CHD but is responsible for 15-25% of the associated deaths [START_REF] Boneva | Mortality associated with congenital heart defects in the United States: trends and racial disparities, 1979-1997[END_REF]. HLHS combines atresia or stenosis of the aortic or mitral valves with hypoplasia of the left ventricle and the ascending aorta [START_REF] Hinton | Hypoplastic left heart syndrome is heritable[END_REF].

Although the cause of HLHS is unknown, severe cardiomyopathy is thought to be a complex genetic disorder [START_REF] Hinton | Hypoplastic left heart syndrome is heritable[END_REF]; the genetic analysis of families with inherited left heart abnormalities (including HLHS, a bicuspid aortic valve, and coarctation of the aorta) have identified a few genes in which mutations are likely to be causative (notably NKX2.5 [START_REF] Elliott | Cardiac homeobox gene NKX2-5 mutations and congenital heart disease: associations with atrial septal defect and hypoplastic left heart syndrome[END_REF], NOTCH1 [START_REF]Mutations in NOTCH1 cause aortic valve disease[END_REF][START_REF] Mcbride | NOTCH1 mutations in individuals with left ventricular outflow tract malformations reduce ligand-induced signaling[END_REF][START_REF] Hrstka | NOTCH1-Dependent Nitric Oxide Signaling Deficiency in Hypoplastic Left Heart Syndrome Revealed Through Patient-Specific Phenotypes Detected in Bioengineered Cardiogenesis[END_REF] and MYH6 [START_REF] Tomita-Mitchell | Impact of MYH6 variants in hypoplastic left heart syndrome[END_REF][START_REF] Theis | Recessive MYH6 Mutations in Hypoplastic Left Heart With Reduced Ejection Fraction[END_REF][START_REF] Theis | Genetic Association Between Hypoplastic Left Heart Syndrome and Cardiomyopathies[END_REF]).

The present report highlights the use of rapid prenatal whole-exome sequencing (WES) to diagnose a severe case of recurrent, neonatal HLHS (cause by compound heterozygous MYH6 variants) early in pregnancy. Written informed consent was obtained from the patient for publication of this case report and any accompanying images.

Case presentation

We report on the case of a 40-year-old, nonsmoking, nulliparous, Caucasian woman (BMI: 31 kg/m 2 ) with an unremarkable medical history other than idiopathic hypertension (treated with labetalol 200 mg once daily). After spontaneous conception, the woman was referred to our center in April 2020 for an undefined fetal cardiopathy. The results of the first-trimester ultrasound scan were normal (crown rump length: 63.68 mm; nuchal translucency: 1.7 mm), with a low risk of trisomy 21 (1 in 1548; PAPP-A: 0.65 multiples of the median, β-HCG: 0.59 multiples of the median) and a negative, non-invasive prenatal test requested by the woman. The diagnosis of severe HLHS with mitral and aortic stenosis was made at 23 weeks of gestation (w.g.) (Fig. 1). The results of a chromosomal microarray (CMA) analysis of an amniotic fluid sample were normal. The couple requested termination of pregnancy at 25 w.g.; given the severe prognosis, the request was approved by the multidisciplinary antenatal board. The couple did not allow a fetal autopsy to be performed.

The parents had normal cardiac ultrasound findings and did not report a family history of CHD or adult cardiomyopathy. The parents were reassured by the initial genetic counselling because HLHS is usually sporadic; in the absence of a family history, the risk of recurrence is 2-4% [START_REF]Hypoplastic left heart syndrome | Genetic and Rare Diseases Information Center (GARD) -an NCATS Program[END_REF].

A year later (in April 2021, after 13 w.g.), the women was referred for an early morphological ultrasound check-up on her next pregnancy, which revealed cardiac asymmetry and aortic stenosis (Figs. 2 and3). The diagnosis of HLHS was confirmed at 15 w.g. Again, the results of a CMA analysis of an amniotic fluid sample were normal.

In view of the recurrence of HLHS, we suggested WES. The couple agreed, and a quartet analysis was performed on fetal DNA stored from the previous pregnancy, DNA from the ongoing pregnancy, and the parents' DNA. WES revealed that both fetuses were compound heterozygous for probably pathogenic MYH6 variants (class 4, according to the American College of Medical Genetics and Genomics (ACMG) classification [START_REF] Richards | Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology[END_REF]) inherited from the mother and the father. The mother carried MYH6 (NM_002471.4) chr14: g.23853649A > T c.5565 + 2 T > A, predicted to be a loss-of-function (splice donor) variant that was not present in population databases [16]. The father carried MYH6 (NM_002471.4) chr14:g.23857395G > T c.4328C > A p.(Ala1443Asp) -a very rare missense variant that is predicted to be pathogenic, according to the ACMG classification and the Varsome database (https:// varso me. com/). Furthermore, the wild-type and mutant amino acids' respective physicochemical properties are very different (Grantham score:

Discussion & conclusions

Cardiac muscle myosin consist of two heavy chain subunits, two light chain subunits, and two regulatory subunits. MYH6 codes for myosin heavy chain α (MHC-α), the major myosin isoform in the fetal heart [START_REF] Hao | Establishment of a human MYH6 compound heterozygous knockout hESC line to model cardiomyopathy and congenital heart defects by CRISPR/Cas9 system[END_REF]. After birth, MHC expression switches to MYH7, located 4 kb downstream of MYH6 and which codes for MHC-β.

Relative to MHC-β, MHC-α has 1.5 to 3 times more contractile velocity and a 0.6-fold shorter actin attachment duration. In the adult heart, MHC-β expression is predominates in the ventricles and MHC-α expression predominates in the atria.

Pathogenic MYH6 variants have been described in several cardiac diseases. For example, it has been shown that heterozygous variants are responsible for moderately severe types of CHD, such as atrial septal defects and AVSD [START_REF] Posch | Cardiac Alpha-Myosin (MYH6) Is the Predominant Sarcomeric Disease Gene for Familial Atrial Septal Defects[END_REF] whereas homozygous or compound heterozygous variants are responsible for severe CHD such as HLHS [START_REF] Liu | Next-generation sequencing in a series of 80 fetuses with complex cardiac malformations and/or heterotaxy[END_REF]. MYH6 is also thought to be involved in adult familial hypertrophic cardiomyopathy. The association between MYH6 variants and HLHS has already been reported; in a study of 190 unrelated HLHS subjects, rare MYH6 variants were observed in around 10% of cases [START_REF] Tomita-Mitchell | Impact of MYH6 variants in hypoplastic left heart syndrome[END_REF]. More recently, whole-genome sequencing revealed a highly significant (p = 0.000068), abnormally high proportion of pathogenic MYH6 variants observed in 197 patients with HLHS (vs. 813 controls) [START_REF] Theis | Genetic Association Between Hypoplastic Left Heart Syndrome and Cardiomyopathies[END_REF]. The frequency of MYH6 variants was also 10% in this population of patients with HLHS. Despite the presence of autosomal dominant transmission of left heart disease/cardiomyopathy in some families, a single pathogenic MYH6 variant alone might not be enough to cause disease: a second hit might be necessary. Interestingly, the second hit might be either a second rare MYH6 pathogenic variant (i.e. compound heterozygosity), a "common" MYH6 variant (minor allele frequency > 0.01) or a variant in a modifier gene like FLNC (i.e. synergistic heterozygosity). These various possibilities might explain the broad phenotypic spectrum associated with MYH6 variants (Fig. 4), which ranges from a normal heart to a severe form of CHD like HLHS. As reported for many genes with a high variant density, mutations in MYH6 and CFTR are probably associated with a broad spectrum of effects and marked phenotypic variability.

Discordant inheritance patterns have been reported in the literature. The versatile nature of MYH6-related inheritance prompts us to think that MYH6 is at the interface between Mendelian autosomal-dominant inheritance and autosomal-recessive inheritance and thus has an unconventional pattern of inheritance.

The "common variant hypothesis" has been further underpinned by several recent reports of an association between rare variants and common variants in MYH6related conditions [START_REF] Anfinson | Significance of α-Myosin Heavy Chain (MYH6) Variants in Hypoplastic Left Heart Syndrome and Related Cardiovascular Diseases[END_REF] and for other genes responsible for congenital left heart defects [START_REF] Mcbride | Association of common variants in ERBB4 with congenital left ventricular outflow tract obstruction defects[END_REF]. This hypothesis is also supported by the results of several genome-wide association studies [START_REF] Eijgelsheim | Genome-wide association analysis identifies multiple loci related to resting heart rate[END_REF][START_REF] Hindorff | Potential etiologic and functional implications of genome-wide association loci for human diseases and traits[END_REF][START_REF] Macarthur | The new NHGRI-EBI Catalog of published genome-wide association studies (GWAS Catalog)[END_REF], in which MYH6 polymorphisms were linked to cardiac disease in the general population; hence, a common variant might have harmful consequences. In the overall genetic landscape and Fig. 4 The genotype-phenotype relationship for MYH6 in the "second variant" hypothesis. mutations in MYH6 is responsible for degenerative heart diseases or cardiac malformations of varying severity. Three main genotypes are thought to be responsible for these diseases: (i) classical autosomal-recessive inheritance of two rare, pathogenic MYH6 variants, responsible for the most severe phenotype; (ii) synergistic heterozygosity, i.e. the combination of a single pathogenic MYH6 variant with a modifier variant in an interacting gene (FLNC in this case), and (iii) variable compound heterozygosity, i.e. the combination of a single MYH6 pathogenic variant with one or more common MYH6 variants, with intrafamily variability. ASD: atrial septal defect, AV: aortic valve, Coarc: coarctation, FLNC: filamin C, HCM: hypertrophic cardiomyopathy, HLHS: hypoplastic left heart syndrome along with environmental factors and modifier genes, the "common variant hypothesis" tends to explain incomplete penetrance and the variable expressivity of Mendelian autosomal-dominant diseases [START_REF] Kingdom | Incomplete Penetrance and Variable Expressivity: From Clinical Studies to Population Cohorts[END_REF].

Cases of compound heterozygosity or homozygosity for MYH6 mutations are rare; to the best of our knowledge, the only literature report concerned cases in four families [START_REF] Posch | Cardiac Alpha-Myosin (MYH6) Is the Predominant Sarcomeric Disease Gene for Familial Atrial Septal Defects[END_REF]. Most of these cases could have been due to synergistic heterozygosity or an association with a common MYH6 variant. It should be noted that MYH6 is one of the most variable genes, and so there are probably many common alleles. In the case described here, neither the fetus nor the parents carried a "common" MYH6 or FLNC variant; this is consistent with the absence of a family history of CHD and hypertrophic cardiomyopathy and the classical autosomal recessive transmission of HLHS.

Moreover, the presence of MYH6 variants is associated with greater expression of MYH7, which results in hypocontractility [START_REF] Tomita-Mitchell | Impact of MYH6 variants in hypoplastic left heart syndrome[END_REF]. According to the literature, CHD is associated with low levels of MYH6 mRNA and higher levels of MYH7 mRNA [26].

There are two possible explanations for a diseasecausing effect of MYH6 variants. Firstly, severe valvular stenosis might led to ventricular dysmorphology and reduced preload [START_REF] Hinton | Hypoplastic left heart syndrome is heritable[END_REF]. Secondly, the defective expansion and/or differentiation of cardiomyocytes might result in ventricular dysmorphology and dysfunction [START_REF] Dealmeida | Increased Ventricular Preload Is Compensated by Myocyte Proliferation in Normal and Hypoplastic Fetal Chick Left Ventricle[END_REF]. In the present case, we observed left hypoplastic heart failure with mitral and aortic stenosis; these observations are compatible with the first hypothesis. The presence of MYH6 variants is strongly associated with MYH7 overexpression in both atrial and ventricular tissues [START_REF] Tomita-Mitchell | Impact of MYH6 variants in hypoplastic left heart syndrome[END_REF], leading to hypocontractility. Delayed fetal blood flow from the right atrium to the left atrium and through the mitral valve would then result in limited filling of the left ventricle and thus HLHS.

The present report is the first to have described the detection of MYH6 variants related to fetal HLHS during an ongoing pregnancy. Our case also emphasizes the value of WES for understanding the unusual recurrence of fetal anomalies during pregnancy. Fetal anomalies are detected in 3% of all pregnancies and are responsible for 20% of prenatal deaths [START_REF] Kurdi | Prenatal exome sequencing and chromosomal microarray analysis in fetal structural anomalies in a highly consanguineous population reveals a propensity of ciliopathy genes causing multisystem phenotypes[END_REF]. Thus, some cases with an abnormal obstetric history and/or ultrasound anomalies but normal karyotyping and CMA results have been underdiagnosed [START_REF] Jelin | Whole Exome Sequencing: Applications in Prenatal Genetics[END_REF]. WES improves the reliability of prenatal diagnosis and facilitates genetic counseling and medical decisions during early pregnancy [START_REF] Jin | Prenatal diagnosis by whole exome sequencing in a family with a novel TBR1 mutation causing intellectual disability[END_REF]. Furthermore, WES improves the diagnostic yield for recurrent fetal structural anomalies and can reveal new genes that are potentially relevant in human development [START_REF] Pauta | Prenatal Exome Sequencing in Recurrent Fetal Structural Anomalies: Systematic Review and Meta-Analysis[END_REF]. The present report illustrates the major contribution of WES in the characterization of an unusually recurrent fetal disorder and considered the role of WES in the prenatal diagnosis of disorders that do not usually have a genetic etiology. A better understanding of genetic mechanisms and WES results is crucial for future medical practice.

Fig. 1

 1 Fig. 1 Severe left hypoplastic heart syndrome

Fig. 2 Fig. 3

 23 Fig. 2 Cardiac asymmetry

Acknowledgements

Not applicable.

Funding

No funding source to declare.

Availability of data and materials

Upon request by contact to the corresponding author Rodolphe.dard@gmail. com.

Abbreviations

HLHS

Declarations

Ethics approval and consent to participate Not applicable.

Consent for publication

Written informed consent for publication of their clinical details and/or clinical images was obtained from the patient.

Competing interests

The authors declare that they have no competing interests.

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