Screening of Chorioamnionitis Using Volatile Organic Compound Detection in Exhaled Breath: A Pre-clinical Proof of Concept Study

Chorioamnionitis is a major risk factor for preterm birth and an independent risk factor for postnatal morbidity for which currently successful therapies are lacking. Emerging evidence indicates that the timing and duration of intra-amniotic infections are crucial determinants for the stage of developmental injury at birth. Insight into the dynamical changes of organ injury after the onset of chorioamnionitis revealed novel therapeutic windows of opportunity. Importantly, successful development and implementation of therapies in clinical care is currently impeded by a lack of diagnostic tools for early (prenatal) detection and surveillance of intra-amniotic infections. In the current study we questioned whether an intra-amniotic infection could be accurately diagnosed by a specific volatile organic compound (VOC) profile in exhaled breath of pregnant sheep. For this purpose pregnant Texel ewes were inoculated intra-amniotically with Ureaplasma parvum and serial collections of exhaled breath were performed for 6 days. Ureaplasma parvum infection induced a distinct VOC-signature in expired breath of pregnant sheep that was significantly different between day 0 and 1 vs. day 5 and 6. Based on a profile of only 15 discriminatory volatiles, animals could correctly be classified as either infected (day 5 and 6) or not (day 0 and 1) with a sensitivity of 83% and a specificity of 71% and an area under the curve of 0.93. Chemical identification of these distinct VOCs revealed the presence of a lipid peroxidation marker nonanal and various hydrocarbons including n-undecane and n-dodecane. These data indicate that intra-amniotic infections can be detected by VOC analyses of exhaled breath and might provide insight into temporal dynamics of intra-amniotic infection and its underlying pathways. In particular, several of these volatiles are associated with enhanced oxidative stress and undecane and dodecane have been reported as predictive biomarker of spontaneous preterm birth in humans. Applying VOC analysis for the early detection of intra-amniotic infections will lead to appropriate surveillance of these high-risk pregnancies, thereby facilitating appropriate clinical course of action including early treatment of preventative measures for pre-maturity-associated morbidities.

Chorioamnionitis is a major risk factor for preterm birth and an independent risk factor for postnatal morbidity for which currently successful therapies are lacking. Emerging evidence indicates that the timing and duration of intra-amniotic infections are crucial determinants for the stage of developmental injury at birth. Insight into the dynamical changes of organ injury after the onset of chorioamnionitis revealed novel therapeutic windows of opportunity. Importantly, successful development and implementation of therapies in clinical care is currently impeded by a lack of diagnostic tools for early (prenatal) detection and surveillance of intra-amniotic infections. In the current study we questioned whether an intra-amniotic infection could be accurately diagnosed by a specific volatile organic compound (VOC) profile in exhaled breath of pregnant sheep. For this purpose pregnant Texel ewes were inoculated intra-amniotically with Ureaplasma parvum and serial collections of exhaled breath were performed for 6 days. Ureaplasma parvum infection induced a distinct VOC-signature in expired breath of pregnant sheep that was significantly different between day 0 and 1 vs. day 5 and 6. Based on a profile of only 15 discriminatory volatiles, animals could correctly be classified as either infected (day 5 and 6) or not (day 0 and 1) with a sensitivity of 83% and a specificity of 71% and an area under the curve of 0.93. Chemical identification of these distinct VOCs revealed the presence of a lipid peroxidation marker nonanal and various hydrocarbons including n-undecane and n-dodecane. These data indicate that intra-amniotic infections can be detected by VOC analyses of exhaled breath and might provide insight into temporal dynamics of intra-amniotic infection and its underlying pathways. In particular, several of these volatiles are associated with enhanced oxidative stress and undecane and dodecane have been reported as predictive biomarker of spontaneous preterm birth INTRODUCTION Chorioamnionitis, inflammation associated with an intra-uterine infection of the amniotic fluid and fetal membranes is a major risk factor for preterm birth and an independent risk factor for postnatal disorders such as chronic lung disease, necrotizing enterocolitis, and periventricular leukomalacia (1)(2)(3). Incidences of chorioamnionitis are inversely related to gestational age (GA) at birth ranging from an incidence of >70% at 24 weeks GA to 16% at 34 weeks GA (4).
Only a small proportion of pregnant women with preterm birth show clinical signs of chorioamnionitis such as maternal fever, uterine fundal tenderness, maternal tachycardia, fetal tachycardia, and purulent or foul amniotic fluid (5). However, preterm birth is most frequently the result of a clinically unapparent histological chorioamnionitis (6, 7). In these cases, evidence for the presence of chorioamnionitis becomes available only after delivery. More precisely, post-partum histological examination of the placenta with evidence of inflammation and necrosis throughout the chorionic plate and amnion is currently the gold standard to diagnose chorioamnionitis (5).
There is emerging evidence that the timing and duration of intra-amniotic infections are crucial determinants for developmental injury at birth (8, 9). Moreover, we and others have successfully tested in utero therapies to prevent or treat chorioamnionitis-induced lung and gut injury (10-12). These combined findings highlight that identification of chorioamnionitis at the earliest time point, being in utero is essential for optimal treatment and prevention of organ injury during pregnancy or after birth. Collectively, early identification of chorioamnionitis during pregnancy will extend the time window for clinicians to decide and institute the appropriate clinical course of action to (1) prevent of delay preterm birth and (2) improve outcomes for preterm born neonates.
In recent years, there is increasing attention for noninvasive biomarkers. Exhaled breath consists of volatile organic compounds (VOCs), which are formed during various inflammatory and metabolic processes on a cellular and systemic level (13). In adults and older children, the study of metabolomics using exhaled VOCs is known to be such a safe and non-invasive procedure to evaluate ongoing processes of inflammation and oxidative stress (14), which are key requirements for the induction of cystic fibrosis, asthma and chronic obstructive pulmonary disease (15-17). In addition, profiling exhaled VOCs has been applied successfully in the diagnosis of chronic diseases of the intestine (18, 19), liver (20), kidney (21), pancreas (diabetes) (22, 23) and neurodegenerative diseases (24-26).
Using a well-established pre-clinical model of chorioamnionitis, we questioned whether an intra-amniotic infection could be accurately diagnosed by a specific VOC profile in exhaled breath of pregnant sheep.

Animals
The animal study, including sampling of exhaled breath, and experimental protocols were in line with the guidelines for animal experiments and approved by the Central Authority for Scientific Procedures on Animals and the animal welfare body of Maastricht University. For this proof-of-concept study, we did not perform a power calculation. Within the context of good laboratory animal practice and the 3R principle we did not plan a separate animal study but rather made use of sheep that destined for another study published by Hütten et al. (27). Power calculations were performed for this initial study and a power of n = 6 per experimental group was considered adequate.
Six date-mated Texel ewes (n = 5 singleton; n = 1 twin pregnancy) underwent ultrasound-guided intra-amniotic injection with 5.0 * 10 5 color-changing units (CCU) of Ureaplasma parvum (UP) (strain HPA5) as reported previously (12, 27). In case of twin pregnancy, both uterine horns were inoculated with UP. Amniotic fluid was collected prior to UP injection and during Cesarean section and cultured for UP quantification (28). All animals were group-housed in the same stable under standardized conditions with a 12-h dark/light cycle and had ad libitum access to food and water. During sampling procedure, the animals were restrained by experienced care takers to avoid stress. Six days after intra-amniotic injection (129 days GA) fetuses were delivered preterm by Cesarean section under general anesthesia and used for experimental protocols detailed previously (27). At birth, no sex (3 males and 4 females) and bodyweight (3,270 ± 611 g) differences were observed between all lambs.

Exhaled Breath Sampling
Exhaled breath was collected prior to injection, for baseline (control) measurements, and then daily for 6 days ( Figure 1A) after injection. As such, each sheep serves as its own internal reference. During breath collection, animals were monitored for physical signs of eructation. In case of eructation, the sample was discarded as this might negatively influence the VOC profile (29). Breath was collected via a customized veterinary mouthnose mask (Koo Medical Equipment, Arese, Italy) connected to a non-rebreathing valve (Ruben Valve, Intersurgical Ltd., Wokingham, Berkshire UK) and an inert 5L Tedlar (Tedlar . Prior to (t = 0) and daily for 6 days after injection, exhaled breath was collected by inflation of 5L Tedlar back through a mouth-nose mask connected to a two-way non-rebreathing valve. Six days (t = 6) after intra-amniotic UP-exposure, fetus were delivered by Cesarean section and used for experimented reported elsewhere (27). Group 1 (n = 4) was sampled first, followed by group 2 at a later time point. (B) Illustrative picture of exhaled breath collection in UP-exposed sheep. VOC, volatile organic compound; UP, U. Parvum.
Bag, SKC Ltd., Dorset, UK) gas-sampling bag ( Figure 1B). Immediately after breath sampling, the contents of the bag were transferred onto a stainless steel two-bed desorption tube filled with carbograph 1TD/Carbopack X (Markes International, Llantrisant Business Park, UK) using a pump at a constant flow of = 200 ml/min. The tubes were then air-tight capped and stored at room temperature until further analysis. Between exhaled breath collections, the sampling bags and masks were flushed three times with high-grade nitrogen to ensure that all contaminants were eliminated.
The samples collected at each time point from different animals were collected at the same location in a random order to minimize confounding variable error mediating bias. Timing of exhaled breath collections was between 09.00 and 11.00 to minimize diurnal variations between samplings.
The current study involved two groups of animals, a discovery set to define the parameters and a separate confirmation set to test the rigor of the selected discriminators. All animals followed the same protocol for modeling experimental chorioamnionitis and exhaled breath sampling.

Analysis of Exhaled Breath Samples
The volatile metabolites in exhaled breath were measured using gas chromatography time-of-flight mass spectrometry (GC-tof -MS). All samples were measured in random order.
Before the measurements by GC-tof -MS, all sorbent tubes were purged for 5 min to remove water. To remove the volatile metabolites trapped on the sorption tubes automated thermal desorption TD 100 for industry standard (Markes International, Llantrisant, Wales, UK) under a flow of helium was used. The tubes were heated at 270 • C to release the VOCs. The vapor containing the released VOCs was then divided into two parts. The first part, consisting of 25% of the released VOCs, was collected in a cold trap at 5 • C, whereas the other 75% was recollected into the identical stainless steel two-bed desorption tube. Of the part of VOCs sample collected in the cold trap, 75% was injected in the Gas Chromatogram column at a temperature of 300 • C and separated by capillary gas chromatography (column: RTX-5ms, 30 m × 0.25 mm 5% diphenyl, 95% dimethylsiloxane, film thickness 1 m; Trace 1300GC, Thermo Fisher Scientific, Waltham, Massachusetts). The temperature gradient for the gas chromatograph was programmed in the following manner: 40 • C for 5 min, then raised with 10 • C/min to a maximum temperature of 270 • C, which was maintained for 5 min. Time-of-flight mass spectrometry (tof -MS; Bench TOFdx, Almsco International, Llantrisant, Wales, UK) was used to detect and identify compounds available in the samples. Electron ionization mode was set at 70 eV and the mass range 35-350 m/z was measured. Sample frequency of the mass spectrometer was set to 5 scans/s and analysis run time to 33 min. Following this procedure, a chromatogram was generated for each breath sample of each animal.

Data Pre-processing and Statistical Analysis
The raw chromatograms obtained by GC-tof -MS were first preprocessed to diminish the effect of various artifacts including noise and baseline, column bleeding, and chromatographic shifts. The detailed description of data pre-processing steps can be found elsewhere (30). The GC-tof -MS data after noise removal and baseline correction were further transformed by calculating the area under the peak. These calculated areas for each peak were matched from sample to sample based on the similarity in retention time and Pearson correlation between the mass spectra. A high correlation (>85%) was used to consider peaks as the same compounds. The last step was the creation of a data matrix with samples/animals in rows and relative Six days after intra-amniotic UP-exposure, increased numbers MPO positive cells were observed in the lower crypt. In addition, influx of MPO positive cells into the villi was observed following UP-exposure, which was absent in non-infected historical controls. Images were taken at 100 times magnification, scale bar represents 200 µm. Insert images were taken at 400 times magnification. MPO, myeloperoxidase; UP, U. Parvum.

concentrations of measured volatiles metabolite (ion counts) in columns.
Data obtained for group 1 and group 2 consisted of baseline measurements, pre-injection samples, and six consecutive postinjection sampling days (labeled T1, T2, T3, T4, T5, and T6, Figure 1). The animals from group 1 was used as the discovery set, i.e., finding specific VOCs related to chorioamnionitis, while animals in group 2 served as an independent test set i.e., evaluating the predictive power of the selected exhaled VOCs.
The statistical analysis consisted of performing random forest (RF) analysis (31) with 500 trees using exhaled breath samples of group 1. The classification model comprised of finding the exhaled breath VOCs capable to discriminate pre-infection samples (baseline) and post-infection samples corresponding to development of chorioamnionitis. The classification model was first internally validated using so called out-of-bag (oob) samples. For each RF tree one-third of the training samples were left out and not used in the construction of the classification model. The most discriminatory exhaled VOCs were selected looking at the importance index obtained in RF model (32). The final RF model containing the set of the most discriminatory VOCs was validated using independent test set consisting of samples of group 2. The final performance of the model was expressed in sensitivity, specificity and receiver operating characteristic curve (ROC). To visualize the differences between pre-infection and post-infection samples Principal Component Analysis (PCA) score plot was used. PCA is a workhorse technique within various -omics related fields. This technique reduces data dimensionality by means of Principal Components (PCs), while preserving as much variability as possible. The PCs are the linear combination of the original parameters (here VOCs measured in exhaled breath). The PCA score plot enables representing possible trends, groupings and outliers in the data.

Intra-Amniotic Infection
In accordance with several earlier studies (12, [33][34][35], intraamniotic infection following intra-amniotic UP administration was confirmed by the presence of UP in amniotic fluid during Cesarean section. No endogenous UP was detected in the amniotic fluid prior to inoculation (data not shown). Experimental induction of chorioamnionitis following intrauterine inoculation of UP is a well-established model and has been shown to induce systemic organ inflammation, including in the intestine (12, 36). Consistent with these earlier reports, gut inflammation at 6 days post UP infection was detected, confirming the phenotype of the model (Figure 2).

Exhaled Breath
In this study 544 different VOCs were detected in exhaled breath of 4 animals. To increase the group size, the possibility of combining the baseline and T1 samples to define pre-infection class and T5 and T6 time points into post-infection class was investigated. The differences in breath profile between baseline and T1 samples as well as between T5 and T6 samples were investigated by regularized multivariate analysis of variance (rMANOVA). The rMANOVA indicated no statistical differences between baseline and T1 samples (p-value of 0.15) as well as between T5 and T6 samples (p-value of 0.12).
In Figure 3 the averaged VOC profiles for pre-infection (baseline samples) and post-infection (T6) samples are shown. As could be expected the VOC profiles of baseline samples, i.e., exhaled breath taken before intra-amniotic UP injection and samples taken at T6, i.e., 6 days after intra-amniotic UP injection were similar. Note, that here the overall averaged profile is visually assessed. The largest differences can be seen in the chromatograms at retention time two and 20 min.
The classification model let to the selection of 15 exhaled VOCs. The final RF model was built using the most discriminatory set of 15 VOCs. In order to visualize the differences between pre-infection (baseline and T1) and postinfection (T5 and T6) samples, PCA analysis was performed using the set of 15 discriminatory VOCs. The corresponding PCA score plot is shown in Figure 4A. As can be seen the pre-infection and post-infection samples are separated among PC1, which explains almost 35% of the data variance (i.e., information contained in the data). Since PC1 always explains the majority of the variance in the data, this result indicates that it corresponds to the differences in the development of infection. The validity of the selected set of 15 discriminatory compounds was tested using the independent test set, consisting of new group of animals sampled following the same protocol as the discovery group (group 2). The external validation of the findings resulted in the ROC curve with an area of 0.93 and sensitivity and specificity of 83.3 and 71.4%, respectively ( Figure 4B). The previous time points, T2 and T3, did not show a clear distinction of UP infection using the set of 15 VOCs. The following time point, T4, showed good accuracy of predicting UP infection with accuracy of 0.7. The PCA score plot obtained for pre-infection and post-infection data with projected samples from T4 is shown in Figure 5.
The putative chemical identification of the set of the 15 most discriminatory VOCs was performed using spectrum recognition using the National Institute of Standards and Technology library in combination with spectrum interpretation by an experienced mass-spectrometrist and identification based on retention times of compounds. From a set of 15 VOCs it was possible to chemically identify 11 of them while four remain unknown. These 4 compounds could not be identified due to insufficient mass spectrum, overlap in the retention time or absence of mass spectrum in the library. Moreover, six compounds could be only identified as their global formula and the exact chemical structure remains uncharacterized. Table 1 shows the list of 15 identified VOCs and their relative concentrations change in breath samples obtained from pre-infection and post-infection samples. Up or down regulation of relative VOC concentration is indicated as (+) or (-), respectively, with reference to the infected animal. As can be observed the discriminatory compounds belong mostly to different alkanes and aldehydes.
To further investigate whether the relative amounts of the individual VOCs are significantly different between preinfection and post-infection samples, a rank test was used. The corresponding p-values are indicated in Table 1. As can be observed the discriminatory compounds belong mostly to different alkanes, alcohol and aldehydes, with the relative concentration of the majority of the compounds being elevated in exhaled breath of the post-infection samples.

Discussion/Conclusion
Chorioamnionitis is an important risk factor for preterm birth and prematurity-associated disorders such as bronchopulmonary dysplasia, necrotizing enterocolitis, and neurological injury (1,3,37). Currently, chorioamnionitis is diagnosed by postnatal histological assessment of the placenta and membranes. Consequently, data on the presence of chorioamnionitis become available postnatally, and do not provide information on timing of onset and duration of infection. This latter aspect is of key importance since information on timing and duration of infection are critical determinants for initiation of optimal treatment regimes in preterm infants. Moreover, timely diagnosis and treatment of intra-amniotic infections as the main underlying mechanisms of preterm birth using antibiotic treatment and fetal delivery has shown to improve maternal and neonatal outcomes (38).
Several pre-clinical studies showed that interventions at the earliest possible moment, being in utero, have been successfully tested (10-12, [39][40][41][42][43][44]. Successful development and implementation of such treatment strategies in clinical care is currently impeded by a lack of diagnostic tools for early (antenatal) detection and surveillance of intra-amniotic infections. As such, we aimed to develop a diagnostic tool for early (antenatal) recognition of this distinct risk factor in utero in a safe and non-invasive manner.
In the current proof-of-concept study, we demonstrated that intra-amniotic UP infection induces a distinct VOC-signature in exhaled breath of pregnant sheep. Based on the identified profile of 15 discriminatory volatiles, animals could correctly be classified as either infected or not with good sensitivity (83.3%) and specificity (71.4%). Importantly, we were capable of differentiating UP infection with very good accuracy of 0.  swabs can be used to predict group B streptococcus infection during pregnancy (45) and detect bacterial vaginosis which was linked to preterm labor (46).
Among the identified VOCs associated with UP-infection are various alkenes, aldehyde and alcohols which are primary and secondary products of the lipid peroxidation. The elevated level of the lipid peroxidation marker nonanal and various hydrocarbons including the identified n-undecane and ndodecane is in line with the concept that intra-uterine infection induces higher level of oxidative stress. The presence of these volatiles indicates the occurrence of oxidative stress, a process defined as an imbalance between the production of and the protection against reactive oxygen species in favor of the first (47) and associated with inflammation (48). Chorioamnionitis-induced oxidative stress has been shown to play an important role in preterm labor (49) and its occurrence has recently been underlined in a human study associating placental lesions due to chorioamnionitis with higher levels of oxidative stress biomarkers in cord blood of neonates (50). More closely to our setup, the induction of systemic oxidative stress and modest lung oxidative stress, potentially contributing to bronchopulmonary dysplasia (BPD), by intra-amniotic endotoxin has been demonstrated in fetal lambs (51,52). This alleged involvement of oxidative stress in chorioamnionitisinduced preterm labor and potential development of BPD implies prospective antioxidant supplementation might be a good strategy in the future to protect preterm newborns from severe lung damage (48). This approach highlights the role of antenatal identification of women at risk of chorioamnionitis using the non-invasive markers, such as VOC profiles in breath.
Interestingly, due to the direct link between inflammation and oxidative stress, the volatile undecane has been associated with both processes in general. This is especially the case for infections caused by viruses though, as they do not produce their own metabolites but instead hijack the metabolism of the host, thereby enhancing processes such as glycolysis and oxidative stress (53). Although various studies have already been performed to identify volatile markers unique for specific bacterial strains, n-undecane has not yet been reported as possible biomarker specific for any of the pathogens tested until now (54)(55)(56). However, UP has not 1 | A list of 15 discriminatory VOCs, their putative identification, statistical significance (indicated as p-value obtained from rank test after correction for multitesting with Benjamin-Hochberg) and their relative concentration change in post-infection group with respect to pre-infection samples.

Nr.
Compound name Compound change in infected group been included in such investigations thus far, indicating it might be useful to investigate whether undecane is indeed specifically produced by this pathogen or a byproduct of the oxidative stress induced by this bacterium instead. Finally, the volatiles undecane and dodecane that were discriminative between pre-infection and post-infection samples in our study have recently been reported as predictive biomarker of spontaneous preterm birth in humans (57). In that study, the maternal serum metabolome was studied at 15-and 20weeks' gestation in an attempt to identify biomarkers for an increased risk of spontaneous preterm birth, which may lead to enhanced neonatal morbidities including BPD, in asymptomatic nulliparous women in 2 different cohorts. In one of the cohorts, the alkanes undecane, dodecane, and decane were associated with spontaneous preterm birth whereas these elevated levels could not be observed in the other cohort. Consequently, the study did not provide enough evidence yet to use these alkanes as clinical predictor but does underline once more the relationship between oxidative stress and the risk of neonatal morbidity and mortality (57).
Strengths of the current study are (1) the controlled environment of the established model that is free of confounding factors including behavioral and dietary influences; (2) establishment of individual baseline measurements prior to intra-amniotic infection; (3) the use of a precisely regulated amount of UP, which are the most common microorganisms isolated in cases of spontaneous (asymptomatic) preterm birth (58)(59)(60) and significantly associated with histological chorioamnionitis (60); (4) the use of a sampling technique that is used in clinical practice; and (5) the use of a simple, non-invasive, and repeatedly obtained sample medium, being exhaled air, which will appeal to patients. A limitation is that a number of VOCs remained incompletely characterized. Still, the identified VOCs could serve as specific markers for chorioamnionitis, and UP infections specifically, and could thus be applied for future human exhaled breath studies. Future pre-clinical studies should extend on the current findings and look for further discriminative markers by (1) increasing the experimental power; (2) taking the polymicrobial nature of intra-amniotic infections into account; and (3) focus on discerning timing of onset of infection or duration of infection, which are essential elements that determine organ outcomes at birth. Moreover, combining exhaled VOCs with peripartum diagnostic tools, including amniotic fluid parameters, might aid in the diagnosis of (subclinical) chorioamnionitis. Furthermore, extending the time window of VOC profiling post-partum might provide essential insight into maternal recovery from chorioamnionitis and contribute to risk profiling for women at risk of developing post-partum endometritis, which is the most common post-partum complication following chorioamnionitis, thereby allowing selective use of prophylactic antibiotics (61,62).

CONCLUSION
With 15 million cases globally, preterm birth remains a major health care problem (63) with chorioamnionitis as its most important risk factor. Analyses of VOCs in exhaled breath of pregnant sheep shows great potential to identify pregnancies complicated by intra-amniotic infections and clinical implementation would be an immense breakthrough in perinatal diagnostics. Early detection of intra-amniotic infections with point-of-care VOC testing, potentially combined with peri-partum amniotic fluid biomarkers indicative for intraamniotic infection (64), will lead to optimal surveillance of these high-risk pregnancies and will facilitate appropriate clinical management including antibiotic treatment and timely treatment or preventative measures for pre-maturity-associated morbidities. A clinical study in a high risk pregnancy population, defined by (1) chorioamnionitis in previous pregnancies; (2) asymptomatic cervical shortening; and (3) prolonged rupture of membranes has been planned to verify the results from this proof-of-concept study in combination with intra-and postpartum diagnostic techniques that are currently in practice, including clinical parameters, amniotic fluid parameters, and placenta histology (5, 64).

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

ETHICS STATEMENT
The animal study was reviewed and approved by Central Authority for Scientific Procedures on Animals and the animal welfare body of Maastricht University.

AUTHOR CONTRIBUTIONS
DO, AB, and TW conceived original idea. DO and MH performed ultrasound-guided injections with UP, which was provided by OS. DO collected and processed exhaled breath for laboratory analyses. AB, AS, and F-JS supervised laboratory analyses on exhaled breath. DO, AB, MH, SA-N, RJ, OS, F-JS, AS, and TW contributed to the interpretation of the results. DO and AB wrote the manuscript with input from all authors. DO and TW supervised the project. All authors read, and approved the submitted version.