Development of the fetal myocardium and changes in myocardial fibers orientation

Biagio Castaldi; Marny Fedrigo; Daniela P Boso; Irene Cattapan; Elena De Filippi; Francesca M Susin; Paolo Peruzzo; Giulia Comunale; Paola Veronese; Ornella Milanesi; Annalisa Angelini; Giovanni Di Salvo

doi:10.1371/journal.pone.0315691

. 2025 Apr 11;20(4):e0315691. doi: 10.1371/journal.pone.0315691

Development of the fetal myocardium and changes in myocardial fibers orientation

Biagio Castaldi ^1,^*, Marny Fedrigo ², Daniela P Boso ³, Irene Cattapan ¹, Elena De Filippi ³, Francesca M Susin ³, Paolo Peruzzo ³, Giulia Comunale ³, Paola Veronese ¹, Ornella Milanesi ¹, Annalisa Angelini ², Giovanni Di Salvo ¹

Editor: Dhruba Shrestha⁴

PMCID: PMC11990767 PMID: 40215235

Abstract

Background and aims

The mature left ventricular myocardium is arranged in a complex three-dimensional network of fibers that form a counterclockwise helix in the endocardial layer and a clockwise helix in the epicardial layer. There are no data in the literature on the development of left ventricular myocardium during the fetal life. The aims of this paper were to study the physiological maturation steps of the LV myocardium in fetuses from 17 to 40 gestational weeks, by means of speckle tracking applied to the endocardial and epicardial aspect of the left ventricle, and, to confirm our finds, through the histologic study of the myocardium of demised fetuses.

Methods and results

We studied longitudinal endocardial and epicardial strain by echocardiography in 105 fetuses. Twenty non-diseased fetal hearts from autopsies were selected to assess the layer thickness and cardiac fiber orientation in relation to gestational age. Echocardiography showed a progressive increasing of epicardial/endocardial longitudinal strain ratio with gestational age (r=0.51; p<0.0001). The strain rate E/A ratio increased over time (r=0.27; p=0.018). Histological data revealed that during the same gestational period, the proportion of the epicardial layer increased fourfold, the mesocardiac layer decreased and the endocardial layer remained stable. We found an excellent correlation between the epicardial to endocardial strain ratio and epicardial to endocardial wall thickness (r=0.950, p<0.001).

Conclusions

Left ventricular myocardium maturation begins early during fetal life. As the fetus develops, both the relative tissue volume and peak systolic strain rates shift together from the endocardium towards the epicardium. It is a slow process, completed late in fetal life.

Introduction

The left ventricular myocardium is arranged in a complex three-dimensional network of fibers that form a counterclockwise helix in the endocardial layer and a clockwise helix in the epicardial layer [1]. This peculiar organization is fundamental to myocardial functionality because the left ventricular contraction is both longitudinally and radially oriented, and characterized by ventricular twisting [2]. This architecture may change in congenital and acquired heart diseases [3,4].

Whereas the embryological development of the human heart is well documented, much less is known about fetal myocardial development and the mechanism underlying this process. Using fetal magnetic imaging, Mekkaoui et al. [5] demonstrated that the myocardial fiber orientation of the left ventricle changes during gestation, however these data were not validated by histological investigations or confirmed by a similar study [5]. On the other hand, it has been demonstrated that myocardial function changes in the first days after birth thereby promoting changes of the left ventricular twist and longitudinal strain [6,7].

With the advent of speckle-tracking echocardiography (STE), endocardial and epicardial function can now be assessed separately in vivo [8,9]. Although strain Doppler and STE are widely used to study the left ventricular function in fetal hearts [10–12], no study has yet evaluated the left ventricular longitudinal strain in the two layers separately.

The aims of this study were to assess in vivo the endocardial and epicardial longitudinal strain in fetuses of different gestational ages by STE as surrogate of layer maturation, and to confirm, on histological sections, the relative wall thickness and fiber orientation of the endocardial and epicardial layers at different gestational ages.

Methods

Informed consent was obtained from each patient or their parents. The study protocol conforms to the ethical guidelines (Declaration of Helsinki 1975) and approved by the Institution’s human research committee. The data that support the findings of this study are available from the corresponding author upon reasonable request.

This study consists of two parts: in vivo study, and histological evaluation of normal fetal hearts. One hundred and five fetuses between 17 and 40 gestational weeks were enrolled in the in vivo study. They were selected among healthy singleton pregnancies undergoing general obstetrical ultrasound screening, and they agreed to undergo fetal echocardiography voluntarily. All fetuses examined were free from heart disease and extracardiac pathology, demonstrated normal intrauterine growth and had a normal newborn examination. The pregnant women were otherwise healthy and on no medications other than vitamins. In particular, women with known hypertension, auto-immune diseases, obesity (BMI>30 kg/m² before pregnancy), hypertension during pregnancy or previous pregnancies complicated by gestational diabetes, hypertension, pre-eclampsia or eclampsia were excluded from the study. Maternal diabetes was excluded by oral glucose tolerance test performed between the 26^th and 28^th gestational weeks in all the patients. The normal heart anatomy and function were confirmed after the birth. Neonatological evaluation was unremarkable in all the fetuses selected. The acoustic window and fetal position were ideal for STE assessment: the image quality was good, and the left ventricle was acquired in vertical position. The long axis of the ventricle formed an angle with the probe vertical line of -30/+30°. The sector width was adjusted to obtain the highest frame rate possible (between 90 and 150 fps)

For the histological sections, we obtained 20 hearts without any cardiac diseases from fetal autopsies (gestation age: 13–40 week) available in the Veneto Region Registry of Cardiovascular Pathology Unit of our Hospital. Fetuses were referred to our Cardiovascular Pathology Department for post-mortem investigation in 2015 and 2016. Indications for autopsy were: miscarriage (4 cases), voluntary abortion for social reasons (4 cases), intrauterine death (11 cases) and perinatal death (1 case). We excluded cases with intrauterine growth restriction and cases with placental malperfusion pathologies. According to fetal anthropometric indexes these hearts were within a normal range for the gestational weeks.

Echocardiography

Speckle-tracking was performed on apical 4-chamber views. We used a GE Vivid E9 imaging ultrasound system (General Electric, Waukesha, WI, USA), the 5-MSc and 6S probes. Images were processed offline with a commercially available software (EchoPac v. 11.2, General Electric, Waukesha, WI, USA). To evaluate fetal hearts, we gated the cardiac cycle on the mitral valve opening and closure, while we used ECG in neonates. The endocardial and epicardial borders of the left ventricular wall were traced in an end-systolic frame. The software automatically selects six equidistant tissue-tracking regions of interest in the myocardium (manually adjustable if needed). To measure strain, the myocardium was automatically divided into endocardial and epicardial layers (not further modifiable). To measure strain rate, we recorded the mean S, E and A peak values and the E/A ratio. Visual control of tracking quality was performed and optimized if required, by adjusting the region of interest or manually correcting the contour to ensure adequate automatic tracking. All data were acquired at a frame rate from 90 to 150 frames per second (mean 110 fps, between 45 and 55 frames for cardiac cycle). Strain and strain rate values were obtained from three consecutive cardiac cycles.

Left ventricular volume and mass were calculated applying the Cylinder hemi-ellipsoid Bullet method using the apical 4 chambers view [13]. We also recorded the mean left ventricle end diastolic wall thickness, the left ventricular end diastolic diameter, the left ventricular longitudinal end diastolic diameter and the left ventricular longitudinal to transverse length ratio. Interobserver variability, expressed as coefficient of variation, was assessed by two independent investigators who analyzed 20 randomly chosen cine-loops. For intra-observer variability, 10 cine loops were analyzed by the same investigator twice within an interval of 4 weeks. The second round of intra-observer measures was blinded to results from initial measures.

Histology

A normal heart anatomy was confirmed in all the specimens selected. For each heart we cut two sequential blocks at middle-basal left ventricle free wall one longitudinal and one transverse. The samples were included in paraffin, cut into 5 µm slices, and then stained with Azan-Mallory for measurement of layer thickness and myofibers orientation. We acquired images of each histological section using a Zeiss Video camera connected to a light microscopy (Zeiss Camera, Axio Com color 412–312) and a computer supporting Image Pro Plus morphometric software v. 6.0 (Media Cybernetics, Silver Spring, MA). Longitudinal sections were evaluated to quantify each layer’s thickness, while transversal sections were analyzed to determine the orientation of myofibers.

Longitudinal sections were captured at a magnification of 2.5x and the program was calibrated. Each layer thickness was assessed according to fiber orientation: starting from the non-compacted concave part we defined the innermost longitudinal fibers as endocardium, the medial perpendicularly-cut fibers as mesocardium and the outermost longitudinal fibers as epicardium (Fig 1). The layers were identified according to different orientation of the fibers: vertically cut fibers of middle myocardium appeared lighter in color, because of the major amount of Orange G staining cytoplasm, making it possible to recognize them from the other layers. In the section it was therefore possible to clearly distinguish the longitudinal fibers of the endocardium and epicardium, colored purple red, and the fibers of the mesocardium, which presented a lighter shade as well as a different pattern. Using the same software, we were able to trace line segments for which the program gave us numerical values in micrometer (μm). We measured total wall thickness, non-compacted thickness, endocardial thickness, middle myocardium thickness and epicardial thickness. We carefully avoided artifacts. The measurements were repeated 5 times for each level and in different parts of the specimen. We recorded the mean value for each layer. In addition, the relative layer thickness was calculated.

To study the fibers’ angulation, we applied Streeter’s methodology to transversal sections (Fig 2) [14,15]. Transversal sections were acquired through consecutive sampling moving from the inner side trough the outer one. Magnification used was 20x and morphometric software was properly calibrated. Every sampling area was divided into seven equal regions of interest [16]. Using the function “measurements” we reported the mean orientation of myocardial fibers considering the angle formed between fibers and plan of section for each layer. Then, we averaged the data collected for each layer.

After histopathological assessment, the slices were examined by a pathologist who was blinded to previous findings.

Statistical analysis

The distribution of echocardiographic data was normal as assessed by the Kolmogorov–Smirnov test. Quantitative values are reported as mean ±1 Standard Deviation. The correlation between echo data and gestational age was evaluated with the Pearson test and regression plots, including the research of the more appropriate regression model (linear, quadratic, cubic, logarithmic, logistic, and exponential). The correlation between epicardial to endocardial ratio assessed by speckle tracking and histology was performed by using Spearman’s Rank Test. Data were clusterized for gestational week. Thus, we calculated the mean echocardiographic epi/endo ratio and compared echo data with histological data at any gestational week. The null hypothesis was rejected for a P-value <0.05. All analyses were performed using a commercially available package (SPSS, Rel 18.0 2009. Chicago: SPSS Inc.).

Results

Echocardiography

Table 1 shows the mean values of longitudinal epicardial and endocardial strain at different gestational ages. Endocardial strain, from the 24^th week of gestation, progressively decreased with gestational age (r=0.35; p=0.001), whereas epicardial strain remained substantially unchanged (r=0.04; p=0.698). Therefore, the epicardial to endocardial longitudinal strain ratio progressively increased (r=0.51; p<0.0001; y=0.007x+0.48) (Fig 3. As shown in Fig 4, the S peak by strain rate decreased with gestational age (r=0.385; p=0.001), whereas the E remained quite stable (r=-0.19; p=0.10) and the A wave progressively decreased (r=- 0.56; p<0.001) with gestational age. The E/A ratio increased over time (r=0.27; p=0.018). As expected, wall thickness and diameters grew following a linear regression model, while the left ventricular volume increased based on a cubic regression model (Fig 5).

Table 1. Endocardial and epicardial longitudinal strain and epicardial to endocardial longitudinal strain ratio at different gestational ages [in the square bracket the sample size]. Endocardial strain: r=0.35; p=0.001. Epicardial strain: r=0.04; p=0.698. Epicardial to Endocardial strain ratio (Epi/Endo): r=0.51; p<0.0001.

Gestational weeks	Endocardial S	Epicardial S	Epi/Endo Ratio
17.0-23.0 [n=34]	-27.48±5.98	-17.11±4.01	0.623±0.074
23.1-28.0 [n=22]	-27.05±5.17	-17.67 ±4.55	0.652±0.104
28.1-33.0 [n=29]	-26.53±3.90	-18.15±3.33	0.681±0.067
33.1-40.0 [n=20]	-24.82±3.68	-18.60±2.54	0.748±0.036

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Fig 3 — On the top, left ventricular strain curves. Bottom, plots of endocardial longitudinal strain values (a), epicardial longitudinal strain values (b), and epicardial to endocardial longitudinal strain ratio (c) at different gestational ages.

Fig 4 — Bottom, strain rate plots at different gestational ages: systolic peak (a), early diastolic peak (b), active atrial contraction peak (c) and early diastolic to atrial contraction peak ratio (d).

Fig 5 — (a), left ventricular wall thickness (as mean of basal septum and basal lateral wall end diastolic thickness) (b), and left ventricular end diastolic volume (c) [in logarithmic scale] at different gestational ages.

Histology

The thickness of the left ventricular wall increased with gestational age, from 1305 μm at 13 weeks of gestation to 5782 μm at 40 weeks of gestation. The relative proportion of the endocardial layer remained stable, representing around the 50% of the total myocardial thickness (r=0.058; p=0.8). In contrast, the proportion of epicardial layer increased four-fold, representing 7% of the fetal myocardium at 13 weeks and increasing to 24% by 37 weeks (20 weeks 17%, 30 weeks 22%, 40 weeks 30%), with a significant increase epicardial to endocardial wall thickness with gestational age (r=0.76; p<0.0001). Finally, the relative thickness of the mesocardium decreased with gestation (r=0.51; p=0.02). Like speckle-tracking echocardiography data on epicardial to endocardial strain ratio, the histological relative epicardial to endocardial wall thickness ratio progressively increased with gestational age (Figs 6 and 7) (r=0.76; p<0.0001; y=0.011x+0.05). We found an excellent correlation between the epicardial to endocardial strain ratio and epicardial to endocardial wall thickness (r=0.950, p<0.001).

Fig 6 — (a), epicardial (b) and mesocardium (c) by histological evaluation. The proportion of trabeculated myocardium (d) remained unchanged during gestational age.

The angle of the fibers progressively decreased from 90 to 0 from the endocardium to the mesocardium and from 0 to -90° from the mesocardium to the epicardium. We divided myocardium in seven layers, in Fig 8 was shown the mean angle for the single layer. The angle gradient was stable in the endocardial layer, while was steeper in the epicardium at 13 weeks of gestation, reducing the angle gradient proportionally with the thickening of the epicardial layer.

Fig 8 — The myocardium was divided in seven layers. Layers from 1 to 3 (dashed lines) represent endocardial layers, layer 4 (continue line) is the mesocardial layer, the layers 5 to 7 (dotted lines) become epicardial layer.

Discussion

Normal fetal myocardial development & function

The heart develops from a tubular structure, which by a process of looping and torsion, evolves in a four-chamber structure [17]. The macroscopic anatomy of the heart is almost complete at eight weeks of gestation, whereas the ultrastructure continues to evolve up to birth and beyond. In fact, all the fibers of the embryological heart are arranged radially, whereas the adult heart is highly anisotropic [14,18]. Besides being arranged in two layers, the fibers undergo a toroidal rotation within the layers [19]. Thus, the inner fibers are more longitudinal, becoming transverse in the middle part to return progressively more longitudinal toward the epicardium. Using 3 Tesla MRI, Mekkaoui et al. [5] studied the myocardial tissue of 4 hearts from 10 weeks of gestation to birth. They found that myocardial fibers gradually develop from a single transverse layer to a double longitudinal layer at birth. These results agreed with our histological data.

In our paper, for the first time, the layer specific longitudinal strain and the correlation between fiber orientation, histological changes and in vivo functional changes were studied in fetal hearts. The data found confirm that the endocardial longitudinal layer develops first and is well represented since the first evaluation at 17 GW, while the epicardial longitudinal layer maturation is slower and completed just at the end of the gestation. These histological changes, associated to extracellular matrix development and to the cardiomyocyte maturation [20,21], might suggest a progressive improvement on systolic left ventricular function, as demonstrated in studies on preterm infants [6–7]. The transition from the fetal circulation to the postnatal circulation generates deep changes in the left ventricular function. James et al. [6] demonstrated a progressive improvement of left ventricular twist in the first week of life in infants less than 29 weeks of gestation, confirming the fast changes in left ventricular mechanics driven by the dramatic changes in the newborn heart function. Our previous study demonstrated that the mechanism driving the changes in the left ventricular rotation and twist in preterm newborns was the improvement of the epicardial longitudinal strain [7], according to the delayed epicardial development during fetal life. These findings may be at least in part responsible of the lower tolerance to pre-load and after-load variation in preterm newborns [6,22].

Despite several studies were addressed to estimate normal longitudinal strain values during pregnancy, there are still conflicting data on the physiologic myocardial longitudinal function development and normal range values [23,24]. These differences may be explained with different study design, different machine used and different software type and version available [24,25]. In particular, a high frame rate (>60–80 fps) should be obtained to have an adequate number of frames for heart beat. Thus, a dedicated protocol of image acquisition is mandatory for this kind of studies. In addition, in the earliest versions, the software for speckle tracking analysis were designed and validated for adult left ventricular function assessment [11]. This means that their ideal range of myocardial wall thickness was between 4 and 12 mm. A too large region of interest can be the reason of too low longitudinal strain values at 20–24 weeks found in earliest studies addressed on the normal strain values during fetal growth. Third, the lack of ECG gating requires a manual adjustment of the heart cycle and exclude the possibility to analyze simultaneously more than one heart cycle. The extensive “off-label” use of speckle tracking echocardiography for the assessment of right ventricle and atria function promoted variations and improvement on the software so that the wall thickness can be now manually adjusted and tailored on the single segment and not just pre-fixed by the software. In conclusion, in expert hands, the software available are now ready for fetal heart speckle tracking analysis. In addition, our study for the first time used a layer-specific approach to study the fetal heart and validated the capability of speckle tracking software to distinguish between longitudinal endocardial and epicardial deformation in fetal hearts with a histological study focused on the fiber orientation and the layer thickness of the left ventricular wall. Our data showed that the sub-endocardial strain decreases from the 17^th to the 40^th week of gestation (from -27.5% to -24.8%). On the other hand, the sub epicardial longitudinal strain increases from -17.1% to -18.6%.

Relevance of findings to clinical pathology

Speckle tracking echocardiography was recently used to provide new insights into the understanding of fetal heart function in specific disease states. Miranda et al [26] used speckle tracking to study fetuses of mother with diabetes, demonstrating a biventricular diastolic dysfunction and right ventricular systolic dysfunction by deformation analysis in the third trimester of pregnancy. DeVore et al. [27] proposed a multi-parametric approach, including longitudinal strain, to study the left and right contractility in fetuses with intrauterine growth restriction. They concluded that high rates of abnormal ventricular contractility were present in fetuses with an estimated fetal weight <10th centile, irrespective of the Doppler findings of the pulsatility index of the umbilical artery, and/or cerebroplacental ratio [27].

Speckle tracking echocardiography was used to predict the post-natal outcome of congenital heart diseases. Cohen et al. [28] found that fetuses with pulmonary atresia and intact ventricular septum (PAIVS) had a reduced left ventricle global longitudinal strain value compared to normal, and the patient with post-natal evidence of right ventricle dependent coronary circulation had the worst values compared to PAIVS patient without sinusoids (-15.8±1.2% vs -17.9±1.7%, p=0.009; normal -23.7±2.0%).

Wohlmuth et al [29] studied the outcome of fetal balloon aortic valvuloplasty with Tissue Doppler (TDI). They found that the fetuses that improved TDI data (S’, E’, E’/A’, E/E’ and TDI-based myocardial performance index) had a post-natal biventricular physiology outcome. On the other hand, no changes in TDI data predicted a single ventricle physiology correction.

In congenital heart disease like PAIVS and aortic stenosis, the sub-endocardial ischemia and fibroelastosis play a central role in the loss in function of the left ventricle [30]. Fibrous tissue has isotropic elastic properties while myocardial fibers are longitudinally orthotropic and transversally isotropic [31]. Myocardial fibroelastosis replaces the endocardial longitudinal layer and, by increasing the stiffness of the endocardial layer. This process results in a restrictive pattern with high end-diastolic pressures. Based on these data, the endocardial longitudinal strain ratio could predict the amount of endocardial fibroelastosis and/or the response to fetal balloon valvuloplasty, improving the patient stratification for this complex and risky procedure [32].

Limitations

One limitation of our study is that the anatomical and functional data were not obtained from the same patients. We collected in vivo echocardiographic data from cardiovascular healthy fetuses between 17 and 36 weeks of gestation and we compared with post-mortem data of fetuses (without cardiovascular abnormalities and with normal growth before the death) between 13–40 weeks of gestation. Although only well-preserved fetal hearts were selected for histological study, we were unable to evaluate the angle of myofibers due to tissue maceration in 4 cases. Furthermore, the sample size was too small to develop myocardial fiber angulation plots for gestational age. Fig 8 simply represents the value obtained from the single sample at a given gestational age.

Recently, Semmler et al demonstrated angle dependence of speckle tracking in a fetal population by using a Vitrea Software (Canon® Medical System) [33]. However, in our study we selected only vertically oriented apical 4 chamber view with a good or excellent acoustic window. We specifically avoided the analysis of transverse apical 4 chamber view. Finally, this study for the first time used layer specific longitudinal strain on fetal hearts. Thus, the standard deviation was higher than values calculated in full-thickness strain values. However, the image acquisition and post-processing protocol was already validated on small animals and rodent [34] and data obtained were validated by histologic data.

Conclusions

Acknowledgments

We thank Jean Ann Gilder (Scientific Communication srl., Naples, Italy) for text editing.

Abbreviations

BMI: body mass index
ECG: electrocardiogram
GW: gestational weeks
LV: left ventricle
MRI: magnetic resonance imaging
PAIVS: pulmonary atresia with intact ventricular septum
STE: Speckle tracking echocardiography
TDI: tissue Doppler imaging

Data Availability

All relevant data were added on a supporting information file

Funding Statement

The authors received no specific funding for this work.

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PLoS One. 2025 Apr 11;20(4):e0315691. doi: 10.1371/journal.pone.0315691.r001

Author response to Decision Letter 0

7 Jan 2022

PLoS One. doi: 10.1371/journal.pone.0315691.r002

Decision Letter 0

Peter H Backx

12 May 2022

PONE-D-22-00611Development of the Fetal Myocardium: The Impact of Fiber Orientation on Left Ventricular MechanicsPLOS ONE

Dear Dr. Castaldi,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Your paper has been reviewed by two experts in the field of heart development. Though the paper was considered interesting, concerns were expressed regarding several technical aspects of your study

that call into question the conclusions of the paper. In particular, concerns were raised regarding the reliability of the STE measures, the lack of other (additional) measures of maturation and the usefulness of the simulations

in relationship to the experimental measures as well as the histological measurements. It was suggested that the author attempt to validate their findings in an animal model and consider additional factors in the faetal demise.

The Editors concur with these comments and invite the authors to address comments made

Please submit your revised manuscript by Jun 26 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Peter H. Backx

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The authors formulated the following 3 Aims:

1. "to assess in vivo the endocardial and epicardial longitudinal strain in fetuses of different gestational ages by STE as surrogate of layer maturation"

2. to confirm, on histological sections, the relative wall thickness of the endocardial and epicardial layers at different gestational ages.

3. to evaluate in silico the effect of different myocardial fiber arrangements on diastolic performance.

The endocardial and epicardial strain measurements using STE by their nature are very noisy. During early phases of gestation, when the heart is small the level of noise should be even higher. It is not clear what was the point of using strain as a measure of maturation given there are more common and more reliable measures of maturation such as ventricle volume, diameter or wall thickness. Figure 2 and Table 1 contain overlapping information. Table 1 is not necessary and should be omitted.

It is not clear what histological studies were intended to confirm. The criteria used to identified the boundaries between different layers are not described. Neither is clear how the fiber angle was measured and quantified in particular in situations illustrated in Figure 1,b3.

Simulations utilize a 7-layer mechanical model, which is loosely connected to the rest of the study. It simulates pressure-volume curves which were not measured in this study. Reference 37 cited in the legend to figure 7 is not present in the list of references. It is not clear how the experimental values of fiber angle were determined. According to the methods sections histological analysis involved only 4 layers.

Overall the study is poorly designed and conclusions are not supported by the experimental information.

Reviewer #2: This is an interesting paper that seeks to characterize the fiber orientation in the fetal myocardium and understand its impact on LV mechanics.

1. There are small grammatical issues throughout paper and abstract that should be addressed.

2. Use of histological sections from fetal hearts. A portion of these were from fetal demise- do you know cause? . While structurally normal heart development, issues with placenta and vascularization can affect myocardial development prior to fetal demise. This may affect some of your models.

3. limitations related to angle dependence acknowledge. Are you able to use data from well characterized animal models with histology to validate the in silico model

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

PLoS One. 2025 Apr 11;20(4):e0315691. doi: 10.1371/journal.pone.0315691.r003

Author response to Decision Letter 1

8 Jul 2022

Reviewer #1:

The authors formulated the following 3 Aims:

1. "to assess in vivo the endocardial and epicardial longitudinal strain in fetuses of different gestational ages by STE as surrogate of layer maturation"

2. to confirm, on histological sections, the relative wall thickness of the endocardial and epicardial layers at different gestational ages.

3. to evaluate in silico the effect of different myocardial fiber arrangements on diastolic performance.

Thank you for your comment. STE suffers of some inter- and intra- observer variability. However, it is comparable/lower to other echocardiographic parameters. There are several papers demonstrating the feasibility of STE in fetal hearts, since 16 WoG. However, we stated some issues in the limitations (page 15, lines 430-436). On the other hand, strain imaging still remains an effective tool to study in vivo and not-invasively myocardial function. We reported common measures of heart maturation (page 9 lines 242-244 and figure 4), however, these parameters do not allow to study the fibers distribution. On the other hand, STE is a validated method to estimate fiber arrangement in the heart.

Figure 2 and Table 1 contain overlapping information.

Table 1 is not necessary and should be omitted.

Sorry, I didn’t understand. Figure 2 indicates longitudinal strain values by echo; table 1 is the fibers’ angles by histological measures.

It is not clear what histological studies were intended to confirm. The criteria used to identify the boundaries between different layers are not described. Neither is clear how the fiber angle was measured and quantified in particular in situations illustrated in Figure 1,b3.

We would like to thank the reviewer for raising the point related to the methodological aspects of histology which were not reported in the first version of the paper. We have specified how we proceeded for quantification and identification of the fiber orientations and angle evaluation in the material and methods session on page 7 and 8 on line 175-209. We added two reference on this topic, too.

We believe that the histological study was important for supporting the in silico modelling of different layers orientation to characterize the fiber orientation in the fetal myocardium and understand its impact on LV mechanics.

Sorry for the mistake, we added ref. 37 (now 39) to the bibliography. The 7 layers model was used to create different models by changing fibers angulation. According to our and previous studies, the fiber angulation changes progressively from the endocardium to the epicardium. A 7 layers model was considered the most appropriate compromise between simplification of the model to allow data elaboration and adherence to in-vivo model. The angle of the single layer was attributed according to those found by histological study at the given gestational age. The models were tested by comparing the physiologic pressure-volume plots to that generated by the computational model in order to verify the goodness to fit. Figure 7 demonstrate the adequate adherence of the model to the physiological pressure-volume plot found in animal models and in neonates from previous studies.

In figure 8, we started from the 40GW arrangement and then we changed the fiber orientation to verify alternative setting and the role of alternative fibers arrangements on the pressure-volume plot.

Overall the study is poorly designed and conclusions are not supported by the experimental information.

Thank you for your comment. This is the first study facing the fetal development of myocardium in humans. To build an animal model in this setting is very difficult, and to obtain invasive data on healthy fetal heart is impossible, so we did the best (based on our knowledge) to confirm in vivo data with histological and computational models. Computational models are a validated alternative to animal model, at least to build an adequate hypothesis before to plan further and more invasive or ethical challenging studies.

We removed the last sentence of the conclusion, that might sound a little beat speculative.

Reviewer #2:

This is an interesting paper that seeks to characterize the fiber orientation in the fetal myocardium and understand its impact on LV mechanics.

1. There are small grammatical issues throughout paper and abstract that should be addressed.

Thank you for this comment. We reviewed the English language.

2. Use of histological sections from fetal hearts. A portion of these were from fetal demise- do you know cause? While structurally normal heart development, issues with placenta and vascularization can affect myocardial development prior to fetal demise. This may affect some of your models.

We would like to thank the reviewer for raising these important aspects, which we had considered in the selection of our cases but we did not specify in the first version. We excluded cases with intrauterine growth restriction and cases with maternal malperfusion vascularization. Moreover, according to fetal anthropometric indexes the selected hearts were within a normal range for the gestational weeks. We have specified it in the material and method session on page 6 lines 144-146.

As for the cause of intrauterine death of our cases: 4 were unknown, 1 for volvulus, 1 for uterine rupture, 3 for cordonal issues, 2 for infection; among the miscarriage 2 for cervical incontinence, 1 for early placenta abruption, 1 for superficial placental implantation.

3. limitations related to angle dependence acknowledge. Are you able to use data from well characterized animal models with histology to validate the in silico model.

Thank you for this suggestion. We agree that an animal model might improve the validation of in-silico model. Unfortunately, to build a fetal animal model is outside the purpose of this study, also because a very special ethical authorization should be needed to study fetuses of superior mammalians. In addition, before to build an animal model, a clear hypothesis should be presented to the ethical committee, so a pilot study is mandatory, and this study works in this direction.

Finally, the development of a similar animal study is too long to add those data to this paper.

Attachment

Submitted filename: Plos One rebuttal letter rev 2.docx

pone.0315691.s002.docx^{(17.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0315691.r004

Decision Letter 1

Peter H Backx

5 Feb 2023

PONE-D-22-00611R1Development of the Fetal Myocardium: The Impact of Fiber Orientation on Left Ventricular MechanicsPLOS ONE

Dear Dr. Castaldi,

Your paper has been reviewed again. Only one reviewer previously reviewed you manuscript. I apologize for the long period taken to get your paper reviewed.

As you can see from the reviews, both reviewers are asking for major changers to the paper. If you wish to revise the manuscript, the following changes must be made before the paper will be accepted:

1) "Reframing the scientific question and using the in silico model as the central focus" is recommended in order to address the many limitations and assumptions made in analyzing and interpreting the histological results.

2. Providing additional clear details on the histological analyses and results (addressing questions such as: a) how the various layers identified? and b) what were the thicknesses of the various layers?

3) Clarification on how the histological data was incorporated into the in silico calculations and a discussion of the relationship between the histological measures and in silico results.

Please ensure that your decision is justified on PLOS ONE’s publication criteria and not, for example, on novelty or perceived impact.

For Lab, Study and Registered Report Protocols: These article types are not expected to include results but may include pilot data.

==============================

Please submit your revised manuscript by Mar 22 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Peter H. Backx

Academic Editor

PLOS ONE

Additional Editor Comments:

Your paper has been reviewed again. Only one reviewer previously reviewed you manuscript. I apologize for the long period taken to get your paper reviewed.

As you can see from the reviews, both reviewers are asking for major changers to the paper. If you wish to revise the manuscript, the following changes must be made before the

paper will be accepted:

histological results.

3) Clarification on how the histological data was incorporated into the in silico calculations and a discussion of teh relationship between the histological measures and in silico results.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: N/A

Reviewer #3: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

Reviewer #3: No

**********

6. Review Comments to the Author

Reviewer #2: The authors have resubmitted their manuscript with changes in grammar and clarification of some of the reviewers comments.

The use of STE , histology and in silico models have been used to try to approximate fiber orientation over fetal development

Limitations of the study exist and many assumptions and approximations are made given the issues obtaining fetal tissue and testing hypothesis.

As the study stands, it is not fully supported by its scientific design. Reframing the scientific question and using the in silico model as the central focus and using either animal and/ or the human data as potential supporting evidence may help strengthening the paper

Reviewer #3: The authors attempt to assess the histological and physiological maturation steps of the LV myocardium during the fetal development. In-silico calculations are also performed.

Measurements/Calculations:

1) longitudinal endocardial and epicardial strain by echocardiography in 105 fetuses.

2) layer thickness and cardiac fiber orientation in 20 “non-diseased” fetal hearts.

3) in-silico models of the left ventricular myocardium corresponding

Results: Progressive increases in epicardial/endocardial longitudinal strain ratio were seen with gestational age. Histological data revealed proportional increases in the epicardial layer during development. In silico calculations showed compliance reduction as the longitudinal fibers were more vertical.

Conclusion: “Left ventricular myocardium maturation begins early during fetal life and starts from the differentiation of a subendocardial layer. The development of the epicardial layer is slower and it is completed late in fetal life.”

Assessment

This is revised manuscript. I did not review the paper in the first round. I believe that the major concerns raised in the previously reviews were only slightly addressed. The remaining concerns include the following:

1) Details of the histological analyses and results remain missing, as pointed out by the previous reviewer 1. Were the various layers identified? How were they identified? What were the thicknesses of the various layers?

2) How (and what part of) the histological data was incorporated into the in silico calculations is not described or discussed. It seems the in silico calculation are performed without any specific regards for histological results (i.e. fiber orientations). Would it not be critical to incorporate these histological measures and see how this impacts on calculated function? I would expect that these calculations would require some assumptions about tissue compliance in the fiber direction but there is no description of this.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #2: No

Reviewer #3: No

**********

PLoS One. 2025 Apr 11;20(4):e0315691. doi: 10.1371/journal.pone.0315691.r005

Author response to Decision Letter 2

1 Jul 2023

Reviewer #3 comments:

• This is revised manuscript. I did not review the paper in the first round. I believe that the major concerns raised in the previously reviews were only slightly addressed. The remaining concerns include the following:

o 1) Details of the histological analyses and results remain missing, as pointed out by the previous reviewer 1. Were the various layers identified? How were they identified? What were the thicknesses of the various layers?

Thank you for your comment. We described more in detail in the Methods (lines from 152 to 186 – tracked changes file). In particular, we described how we computed the thickness of the layer and how we calculated the angle. The layers were distinguished according to the different orientation of the fibers, causing a different grade of orange (lighter in longitudinal fibers). In addition, we measured the angle of the fibers in the different layers. We specified that the myocardium was divided in seven layers to calculate the different angle of the fibers, similarly to Streeter et al. We divided the myocardium in 7 layers to have an adequate sample size for repeated measures of the fibers’ angle and to match the results found from histology with the in-silico model. In the results, we reported the values of wall thickness (from 1305 �m at 13 weeks of gestation to 5782 �m at 40 weeks of gestation) and the changes in fibers’ angulation in the seven layers (figure 6). The relative layer thickness was reported in Figure 5.

o 2) How (and what part of) the histological data was incorporated into the in silico calculations is not described or discussed. It seems the in silico calculation are performed without any specific regards for histological results (i.e. fiber orientations). Would it not be critical to incorporate these histological measures and see how this impacts on calculated function? I would expect that these calculations would require some assumptions about tissue compliance in the fiber direction but there is no description of this.

Thank you for this question. We specified that in silico model was based on the histological data, in particular on the fibers’ orientation found in the different layers. Thus, the mesocardial thickness changed over the time, as well as the epicarial layer. On the other hand, endocardial layer remained unchanged because the relative wall thickness of that layer remained stable since the 13th GW. We built a 7 layers model to merge at the best of our possibilities histological data and computational methods. In addition, data on LV volumes at different gestational ages were acquired from fetal echo. Data on elastic properties of myocardial fibers, and shear stress and pressure-volume plots data were available from previous studies (ref. 17-19 and 38-39) on humans or from animal fetuses. For ethical reasons, the acquisition of these data was not feasible in human fetuses. We already stated this point in the limitations (lines 379-384 tracked changed version). However, we believe that these data were adequate for our model, and the pressure-volume plots obtained from our model was in agreement with normal physiology of the LV.

Attachment

Submitted filename: Plos One Rebuttal Letter.docx

pone.0315691.s003.docx^{(15.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0315691.r006

Decision Letter 2

Peter H Backx

12 Jul 2023

PONE-D-22-00611R2Development of the Fetal Myocardium: The Impact of Fiber Orientation on Left Ventricular MechanicsPLOS ONE

Dear Dr. Castaldi,

Please submit your revised manuscript by Aug 26 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Peter H. Backx

Academic Editor

PLOS ONE

Additional Editor Comments:

Your paper has been reviewed by one of the previous Reviewers. The paper continues to have major deficiencies. In the 2 previous review cycles, the Reviewers were highly critical of the in-silico results.

Please address completely the comments made by the attached Review. Without fully addressing all the Reviewers comments, your paper will be be deemed inadequate for publication.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: I Don't Know

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: No

**********

6. Review Comments to the Author

Reviewer #3: The manuscript has been improved marginally in revision.

I have taken the time to provide extensive and specific guidance for revising the paper.

As stated by previous reviewers, the value and details of the in silico resuts need to be better presented. If the authors are unable to do this, then I recommend removing all these results.

The following changes need to be made carefully and completely to the manuscript.

1. In the Abstract, there are numerous punctuation errors and run-on sentences. Please correct. Also you have included descriptions of the methods in the section dedicated to Aims of the studies. Please rearrange. The Abstract uses abbreviation that are not appropriately defined (what is STE and GW, for example).

2. What is meant by “Using the function “measurements” ”? Why are you uses quotes. Do you mean the data from the echo measurements? If so, just state this.

3. It would be useful to accurately define the “mean S, E and A peak values and the E/A ratio. Show a typical epicardial and endocardial Doppler signal for a typical heart that was used to assess the S waves. Place this in Figure 2.

4. The authors need to state how they were able to align the histology measurements with the echo measurements. Some details on this was achieved using the “software” is needed. What assumptions were made? What is the resolution of the echo in comparison with the histology. I am not convinced this possible.

5. Table 1 has an incorrect title. You state “Muscle fiber orientation in the left ventricle layers of a fetus at three gestational ages based on hystological data.” Hystological is incorrectly spelled. It is histological. The angle are not based on specific data; it is based on assumptions. You are better to something like “The angles were assumed and are overall consistent with the histological measurements”.

6. Put statistics into Table 2 and also include the correlation data.

7. What is being shown in Figure 6? Is this based on Echo data? How can such changes “in fibers’ angulation during gestational age” be measured with any level of certainty using echo? If this data comes from echo measurements, I have significant doubts that these results are meaningful. Please explain, justify and defend.

8. Figure 7 is of limited value and needs to be removed. It lacks dimension etc.

9. The in silico model is difficult to understand without further explanation. Are the pressures plotted in Figure 8, systolic or diastolic? Since the histology measurements are all with hearts in diastole, it would only make sense to explore the results of an in silico model for diastolic pressures and volumes. Yet, the description of the results does make this clear.

10. The authors state “As shown in Figure 8d and 8e, the shape of the left ventricle was deeply unrealistic, and compliance of radially oriented fibers was much lower.” Which result? Both the 0O and 90O cases? It is not at all obvious that the volumes shown in Figure 8 a-c are realistic. For a 40GW heart, the volumes for diastolic volumes seem to be unexpectedly small. Do experimental data exist in order to compare to the results of Figure 8?

11. I see little point in illustrating the data in Figure 9. What is the purpose of these results? In particular, the authors state “As shown in Figure 9 (dashed lines), the early development of the endocardial layer resulted in a pressure volume plot similar to the

definitive array”. What is the definitive array? The meaning of this statement is unclear. The authors then state “the inverse plot resulted in poor compliance.” Again, what is the basis for such a statement? Finally, the authors state “The comparison between the 90°/0° and 0°/90° cases showed that the inner layer plays the major role in ventricle stiffness.” Once again, the basis for such a conclusion is a mystery.

In summary, unless the authors are willing to present their data in a careful, coherent and thoughtful manner, and unless the authors are better able to justify their insilico model with additional details in describing the result, I cannot be supportive of publication. I note that this paper has been reviewed twice previously and my comments align well with previous critiques.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #3: No

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PLoS One. 2025 Apr 11;20(4):e0315691. doi: 10.1371/journal.pone.0315691.r007

Author response to Decision Letter 3

17 Aug 2023

Reviewer #3: The manuscript has been improved marginally in revision.

I have taken the time to provide extensive and specific guidance for revising the paper.

As stated by previous reviewers, the value and details of the in silico resuts need to be better presented. If the authors are unable to do this, then I recommend removing all these results.

The following changes need to be made carefully and completely to the manuscript.

Thank you. We deeply revised the abstract. In particular, we rebuilt the background and aims section, we removed the abbreviations (GW and LV) and we checked orthography.

2. What is meant by “Using the function “measurements” ”? Why are you uses quotes. Do you mean the data from the echo measurements? If so, just state this.

Thank you for this comment. It refers (line 171, page 7) to histology section. So the measure was referred to specimens. We changed the sentence in: “Using the same software, we…” (Image Pro Plus, cited in line 160…).

Thank you for this suggestion. The figure was available in Supplementary Data, Figure A. Accordingly, we added the strain curves to figure 2 and strain rate curves to figure 3. To be more clear, we changed strain rate S, E and A value in strain rate S’, E’ and A’ values, most frequently used to identify myocardial velocities.

Speckle tracking echocardiography is a functional in vivo assessment. It is routinely used to study left ventricular mechanics. In pathological settings, decrease in strain values corresponds to loss in function of that myocardial fibers. In physiological fetal maturation of myocardium, the change in strain values can be due to a change in fiber arrangement. By histology, we confirmed that in vivo changes in strain values were associated to anatomical changes in fibers’orientation and relative thickness of endocardial and epicarial longitudinal layers. Thus, we can just correlate (rather than to align) in-vivo and in-silico data.

This is the reason of the sentence “Like speckle-tracking echocardiography data on epicardial to endocardial strain ratio, the histological relative epicardial to endocardial wall thickness ratio progressively increased with gestational age (Figure 5) (r=0.76; p<0.0001; y=0.011x+0.05).” (lines 236-238 tracked changes file)

Post-natal myocardial structure was already documented, while the fetal myocardial development was studied on animal models or (in humans) on few patients by 3T MRI. For the first time, we studied myocardial structure at different gestational age on humans. Our data were in agreement with models hypothesized models [Mekkaoui C, Porayette P, Jackowski MP, Kostis WJ, Dai G, Sanders S, Sosnovik DE. Diffusion MRI Tractography of the Developing Human Fetal Heart. PLoS One 2013;8.]. In our study, changes in layer specific speckle tracking data correspond to changes in relative layer thickness by histology. Of course, we agree that is impossible to compare in vivo function and histology from the same patient for ethical reasons.

Thank you for your suggestions. We modified the title as follows: “Muscle fiber orientation in the left ventricle layers of a fetus at three gestational ages (20, 30 and 40 gestational weeks) used in the in-silico model. The angles were assumed and were overall consistent with the histological measurements. Positive angle: counterclockwise rotation; negative angle: clockwise rotation, zero degree: circumferential direction.”

6. Put statistics into Table 2 and also include the correlation data.

Thank you for your suggestion. Data were available in the text (lines 216-219). However, we added to the table, too.

Sorry for the misunderstanding. Figure 6 was cited in the histology section (line 240). It was a specific request from the last review. Of course, these data cannot be obtained by echo. To be more clear, we modified the title as follows “Changes in fibers’ angulation during gestational age based on histology.”

8. Figure 7 is of limited value and needs to be removed. It lacks dimension etc.

We removed it, accordingly.

We agree with your comment. Pressures plotted in figure 8 (now figure 7) were diastolic. As specified in the aims and in the text, by in silico model we studied the diastolic function. However, we changed the first sentence of the in-silico section in “In-silico models were able to compare diastolic function at different gestational ages and with different myocardial fibers’ arrangements.”, and we modified the figure 7 (former figure 8) title in “(a), (b) and (c) diastolic pressure-volume plots at different gestational ages”

Due to the changes described above, Figure 8 is now mentioned as Figure 7.

We modified the sentence in lines 250-251: “As shown in Figure 7d and 7e, the shape of the left ventricle was far from the normal shape in both the cases”.

Volumes used in figures 7a-c were based on echocardiographic data, as stated in the methods (lines 197-202). The values were absolute volumes and referred to fetal volumes and not to neonates. This might be the reason why the volumes seems to be unexpectedly small. Our data were in agreement with previous studies on fetal volumes nomograms [Messing B, Cohen SM, Valsky DV, Rosenak D, Hochner-Celnikier D, Savchev S, Yagel S. Fetal cardiac ventricle volumetry in the second half of gestation assessed by 4D ultrasound using STIC combined with inversion mode. Ultrasound Obstet Gynecol. 2007 Aug;30(2):142-51. doi: 10.1002/uog.4036. PMID: 17566143][ Simioni C, Nardozza LM, Araujo Júnior E, Rolo LC, Zamith M, Caetano AC, Moron AF. Heart stroke volume, cardiac output, and ejection fraction in 265 normal fetus in the second half of gestation assessed by 4D ultrasound using spatio-temporal image correlation. J Matern Fetal Neonatal Med. 2011 Sep;24(9):1159-67. doi: 10.3109/14767058.2010.545921. Epub 2011 Jan 21. PMID: 21250911.]

Thank you for your comment. We changed the text as follows: “As shown in Figure 8 (dashed lines), the early development of the endocardial layer resulted in a pressure volume plot similar to the adult-type array, while the inverse plot model (epicardial layer develops first) showed a lower compliance. (line 260-263).

The results found in figure 8 (former figure 9) were based on in-silico model. These data were generated by the computer, based on the variables used to build the model, as stated in the methods. Of course, it is impossible to manipulate myocardial fibers orientation in humans or in animal models to verify different myocardial fiber arrangements.

We hope that we have answered the reviewer's questions in a comprehensive manner. In the last revision, many comments were addressed to histology section and how histological data were used in in-silico model. We hope that, following the reviewer’s suggestion, the paper is more clear, now.

PLoS One. doi: 10.1371/journal.pone.0315691.r008

Decision Letter 3

Peter H Backx

13 Nov 2023

PONE-D-22-00611R3Development of the Fetal Myocardium: The Impact of Fiber Orientation on Left Ventricular MechanicsPLOS ONE

Dear Dr. Castaldi,

The paper has again been reviewed by 2 Reviewers. Reviewer #2 reviewed the last version of the paper and Reviewer #1 is a new Reviewer giving a fresh look at the paper.

Both Reviewers are critical of the in silico data. If you are unable to better justify the model calculation, I strongly suggest yu remove this data. If you choose to continue presenting these findings, you need to thoroughly address the Reviewers comments.

Reviewer #1 also had requests for changes to the sections related to the histology and echo. Please address those completely.

It is also noted that the last revised manuscript did not identify the changes made to the manuscript. Please do this in the following submssion.

Please submit your revised manuscript by Dec 28 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Peter H. Backx

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

Reviewer #4: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Partly

Reviewer #4: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: I Don't Know

Reviewer #4: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: No

Reviewer #4: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: Yes

Reviewer #4: Yes

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6. Review Comments to the Author

Reviewer #3: In my previous review I suggested a number of changes. A number of the suggested changes are inadequately addressed.

Strengths

The Abstract is much improved.

Remaining issues

1. The authors should better (more fully explain) how they aligned the histology measurements with the echo measurements.

2. I still see little point in illustrating the data in Figure 8. Without a better description of what goes into the model and how you incorporated the histological data into the model, along with a careful discussion/explanation of the assumptions, I still think this data/analysis adds little of value to the paper. As a results and as I stated in my last review the basis for conclusions derived from these simulations remains a mystery.

Reviewer #4: The paper studied echo and histology of fetal hearts, and peformed diastolic pressurization on finite element model. There seems to several rounds of review by now. I try here to suggest mostly writing amendments.

1. There is some value in the data in terms of histology measurements for later stages of fetuses, which is lacking in the literature.

- However, the authors should detail the sample sizes of that investigation. How many samples were investigated for each age for both the thickness measurements and fiber orientation measurements?

- The authors provided r and p values for trend over age for thicknesses, but we typically expect a plot of meaurements over time and a best fit line across it. This is important to gain credibility and confidence in the conclusions.

- authors should also state clearly what was their crieteria for differentiating different layers from histology, it doesnt seem quite obvious from the histology images in supplementary figure. How did they choose which tissue is in which layer?

- There is no statistics of trend over age for fiber orientation. This should be attempted if possible. If samples are too low for this, authors should state this. even low sample size data is valuable when there is no other data around.

- The fiber orientation description in lines 231-232 is not turue. the fibers dont go to + 90 or -90 according to figure 6. For a paper that focussed on fiber orientation, not being able to characterize fiber orientation properly will be very negative to a reader/reviewer.

2. The in silico work

- The model is not explained well. what software was used? FeBio? FEniCS? Abaqus? Ideally the simulation settings files should be shared for others to verify. The geometry assumed for the heart in the simulation is very consequential. it could complete change outcomes one way or another. How thick were the walls assumed in the simulations? are they realistic? from figure 7 they look very thin, might no be realistic. Otherwise, from experience, it should be very difficult to get those results in figure 7d, if we have a normal stiffness model and physiologic geometry.

- there should be limitations on how adult canine stiffness model is used here for fetal hearts. Should the fetal heart be less stiff?

- there should be limitation text on how active tension and systole was not simulated. For a proper comparison with echo, you need this, as including systolic behaviour can change comparisons with echo.

- I think there are several sweeping claims that should be removed:

a. line 273, claim that there is correlation between fiber orientation, histology and in vivo functional changes. The simulations did not account for active contraction, and contractility can change things. No formal correlation analysis is done.

b. line 277, histology shows improvement on LV function. how is this demonstrated? strains and most strain rates seems to decrease. How is improved function shown? histology cannot show function, the simualtions lacked active tension, and cannot prove that the histology should lead to increased function.

c. lines 350 - 355, claims that pressure volume plots with different fibers can explain histological changes in heart failure. Authors have not shown fiber orientation change in heart failure, and there can be a confusion between fetal heart investigation here, and heart failure in adult settings as it is. There is no investigation of stiffness, and so authors should not use stiffness to support claims here.

3. the echo measurements

- the measurements seem large from past reviews. but the variability is large (van Oostrum NH, European Journal of Obstetrics & Gynecology and Reproductive Biology. 2020 Jul 1;250:178-87.). The current measurements at the endo layer are beyond the higher end of that review. Given the high variability of measurements out there, this might be ok, but should be explicitly addressed in the text.

- discussion section lines 303-310 seems overly optimistic on speckel tracking for echo strain meaurements. As the cited paper above showed, variability in hands of many different experts lead to highly varying strain results. I think the authors should acknowledge that there is a lot of variability out there, and use that to justify that their strains is higher than others.

Finally: The title of the paper is about myofiber and LV mechanics, but the value of the paper seems to be more on the histology and echo measurements, less on the biomechanics and simulations, given that there's no active tension or systolic simulations. authors could conder address this.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #3: No

Reviewer #4: No

**********

PLoS One. 2025 Apr 11;20(4):e0315691. doi: 10.1371/journal.pone.0315691.r009

Author response to Decision Letter 4

9 Apr 2024

Review Comments to the Author

Reviewer #3: In my previous review I suggested a number of changes. A number of the suggested changes are inadequately addressed.

Strengths

The Abstract is much improved.

Remaining issues

1. The authors should better (more fully explain) how they aligned the histology measurements with the echo measurements.

Thank you for this comment. We added Figure 7 to show the increase of epi/endo thickness over gestational age, and we added a statistical test to compare echo by histology.

Statistical analysis: “The correlation between epicardial to endocardial ratio assessed by speckle tracking and histology was performed by using Spearman’s Rank Test. Data were clusterized for gestational week. Thus, we calculated the mean echocardiographic epi/endo ratio and compared echo data with histological data at any gestational week.”

Results: “We found an excellent correlation between the epicardial to endocardial strain ratio and epicardial to endocardial wall thickness (r=0.950, p<0.001).”

According with the Editor, we removed in-silico model section from our paper.

1. There is some value in the data in terms of histology measurements for later stages of fetuses, which is lacking in the literature.

- However, the authors should detail the sample sizes of that investigation. How many samples were investigated for each age for both the thickness measurements and fiber orientation measurements?

Thank you for this comment. We were able to collect about one patient for gestational week. Unfortunately, is difficult to have normal specimens eligible for this kind of study. This is the reason why figure 6 (now Figure 8) doesn’t show SD lines plot.

Thank you for this comment. We further specified in the methods how we measured relative wall thickness. Of course, the contour might be irregular. Thus, multiple measurement were performed.

“The layers were identified according to different orientation of the fibers: vertically cut fibers of middle myocardium appeared lighter in color, because of the major amount of Orange G staining cytoplasm, making it possible to recognize them from the other layers. In the section it was therefore possible to clearly distinguish the longitudinal fibers of the endocardium and epicardium, colored purple red, and the fibers of the mesocardium, which presented a lighter shade as well as a different pattern. Using the same software, we were able to trace line segments for which the program gave us numerical values in micrometer (μm). We measured total wall thickness, non-compacted thickness, endocardial thickness, middle myocardium thickness and epicardial thickness. We carefully avoided artifacts. The measurements were repeated 5 times for each level and in different parts of the specimen. We recorded the mean value for each layer. In addition, the relative layer thickness was calculated.”

Thank you for this comment. We were able to build the plot available in figure 8, where the reader can see how the angle in the seven layers changes over the gestation. In particular, the epicardial layer is 90° oriented at 13 GW, the second layer (over 7) starts to be longitudinal at the 20th GW, the third layer begins at 30 GW. Thus, we added this point in the limitations.

“Furthermore, the sample size was too small to develop myocardial fiber angulation plots for gestational age. Figure 7 simply represents the value obtained from the single sample at a given gestational age.”

- The fiber orientation description in lines 231-232 is not true. the fibers dont go to + 90 or -90 according to figure 6. For a paper that focused on fiber orientation, not being able to characterize fiber orientation properly will be very negative to a reader/reviewer.

Thank you for this comment. Figure 6 (now figure 8) represents a mean value. If we analyze the most endocardial myocytes, they are 90° oriented. This can be seen also in the ref (Streeter DD, Spotnitz HM, Patel DP, Ross J, Sonnenblick EH. Fiber orientation in the canine left ventricle during diastole and systole. Circ Res 1969;24(3):339–47.)

We better specified this point in the text.

“The angle of the fibers progressively decreased from 90 to 0 from the endocardium to the mesocardium and from 0 to -90° from the mesocardium to the epicardium. We divided myocardium in seven layers, in Figure 7 was shown the mean angle of the single layer. The angle gradient was stable in the endocardial layer, while was steeper in the epicardium at 13 weeks of gestation, reducing the angle gradient proportionally with the thickening of the epicardial layer.”

2. The in silico work

- there should be limitations on how adult canine stiffness model is used here for fetal hearts. Should the fetal heart be less stiff?

- I think there are several sweeping claims that should be removed:

According with the Editor, we removed this part of the paper.

3. the echo measurements

We used a layer specific software; thus, the region of interest was smaller, this is the reason why the endocardial layer showed a slightly higher SD. We already stated this in limitations, however, we better specified this point.

“Thus, the standard deviation was higher than values calculated in full-thickness strain values.”

We partially agree on this. It’s true that layer specific strain is more complex. However, “global wall” strain is more robust, when acoustic window is adequate. As mentioned above, we added in the limitations the higher variability of layer specific strain.

We agree that the title should change now: “Development of the Fetal Myocardium and Changes in Myocardial Fibers Orientation”

Attachment

Submitted filename: Rebuttal Letter Fetal heart Rev 5.docx

pone.0315691.s005.docx^{(21.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0315691.r010

Decision Letter 4

Dhruba Shrestha

6 Aug 2024

PONE-D-22-00611R4Development of the Fetal Myocardium and Changes in Myocardial Fibers OrientationPLOS ONE

Dear Dr. Castaldi,

==============================

Here are the Comments from the reviewer: :

Reviewer 3 has asked for minor changes in the manuscript.

The authors examine the developmental changes in LV myocardial structure (histology) and function (speckle tracking) during fetal development. The expected progressive changes in both structure and function were observed. The authors conclude that “Left ventricular myocardium maturation begins early during fetal life and starts from the differentiation of a subendocardial layer. The development of the epicardial layer is slower and it is completed late in fetal life.”

The paper has been improved and simplified by removal of the simulation data. I support publication provided the final adjustment are made.

1. I believe that the term “differentiation” that is used in the conclusion is not very informative. The conclusion would better read (and by more accurate) by stating: “As the fetus develops, both the relative tissue volume and peak systolic strain rates shift together from the endocardium towards the epicardium”.

2. To measure strain, the myocardium was automatically divided into endocardial and epicardial layers.

What does the term “automatically” mean? Please provide details.

3. The authors state “All samples were included in paraffin, cut into 5 µm slices, and then stained with Azan-Mallory for measurement of layer thickness and myofibers orientation.” At this thickness, the term “all” implies thousands of slices. Do the authors mean that several transverse slices were examined from “all” the fetal hearts examined?

4. The sentence lacks a verb. “As expected, wall thickness and diameters while the left ventricular volume increased based on a cubic regression model (Figure 5). “

5. The word “determinate” is an adjective not a verb. I think the word “suggests” might be considered.

6. The statement “These findings may be at least in part responsible of the lower tolerance to pre-load and after-load variation in preterm newborns.” I do not understand this conclusion.

Reviewer 4 has accepted the manuscript for publication but has dropped few suggestions that needs to be addressed.

I thank the authors for responding well. Removing the simulations was a very good move. The focus on the echo and histology measurements is better now, and it covers a gap in the literature. I recommend publication of the article.

I just want to point out that the last paragraph of introduction stated the objective differently from the title of the paper. it stated quantifying thicknesses only, and did not mention fiber orientations.

May I further suggest to the authors that one reason for the differential strains between epi and endo is likely due to the the curved surface of the heart and the radial expansion during systole. here's a reference to check out:

Ren et al., Journal of the American Society of Echocardiography. 2023 May 1;36(5):543-52.

==============================

Please submit your revised manuscript by Sep 20 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Dhruba Shrestha, MD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

There are minor changes to be made before deciding for publication. After going through the article, I have one query about the speckle tracking of fetal myocardium. Since this is a very new concept, is there any standard values for the fetus? How is it possible to do a statistical analysis if there are no standard values.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #3: Yes

Reviewer #4: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #3: I Don't Know

Reviewer #4: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #3: No

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #3: The authors examine the developmental changes in LV myocardial structure (histology) and function (speckle tracking) during fetal development. The expected progressive changes in both structure and function were observed. The authors conclude that “Left ventricular myocardium maturation begins early during fetal life and starts from the differentiation of a subendocardial layer. The development of the epicardial layer is slower and it is completed late in fetal life.”

The paper has been improved and simplified by removal of the simulation data. I support publication provided the final adjustment are made.

2. To measure strain, the myocardium was automatically divided into endocardial and epicardial layers.

What does the term “automatically” mean? Please provide details.

4. The sentence lacks a verb. “As expected, wall thickness and diameters while the left ventricular volume increased based on a cubic regression model (Figure 5). “

5. The word “determinate” is an adjective not a verb. I think the word “suggests” might be considered.

6. The statement “These findings may be at least in part responsible of the lower tolerance to pre-load and after-load variation in preterm newborns.” I do not understand this conclusion.

Reviewer #4: I thank the authors for responding well. Removing the simulations was a very good move. The focus on the echo and histology measurements is better now, and it covers a gap in the literature. I recommend publication of the article.

Ren et al., Journal of the American Society of Echocardiography. 2023 May 1;36(5):543-52.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #3: No

Reviewer #4: No

**********

PLoS One. 2025 Apr 11;20(4):e0315691. doi: 10.1371/journal.pone.0315691.r011

Author response to Decision Letter 5

18 Sep 2024

Reviewer 3 has asked for minor changes in the manuscript.

The paper has been improved and simplified by removal of the simulation data. I support publication provided the final adjustment are made.

Thank you for the suggestion, we modified either in the abstract and in the conclusion.

2. To measure strain, the myocardium was automatically divided into endocardial and epicardial layers.

What does the term “automatically” mean? Please provide details.

Automatically means that it was not manually adjustable/modifiable. Echopac does not allow to modify the relative thickness of the layers, it just divides in inner and outer. Other software (i.e. Tomtec) permit to modify manually this parameter. Anyway, we specified what is manually modifiable and what not in methods.

Thank you for the comment. All might be misleading, we modified “The samples were included in paraffin…”. All means that all the samples were managed in the same way.

4. The sentence lacks a verb. “As expected, wall thickness and diameters while the left ventricular volume increased based on a cubic regression model (Figure 5). “

Sorry, probably, the sentence was accidentally truncated. The complete sentence was:

“As expected, wall thickness and diameters grew following a linear regression model, while the left ventricular volume increased based on a cubic regression model”

5. The word “determinate” is an adjective not a verb. I think the word “suggests” might be considered.

Thank you, we changed accordingly.

6. The statement “These findings may be at least in part responsible of the lower tolerance to pre-load and after-load variation in preterm newborns.” I do not understand this conclusion.

Several studies demonstrated that LV deformation properties are impaired in premature newborns compared to normal (at term) neonates. On the other hand, extreme premature newborns show a reduced tolerance to pre-load and after-load variations (most commonly seen in PDA – low systemic output for steal before, post ligation syndrome after closure, evidenced only in prematures and not in older infants), as well as a reduced response to inotropes. Based on our study, and on similar studies in premature newborns, these clinical evidence might be, at least in part, justified by an incomplete myocardial development in these subjects.

Reviewer 4 has accepted the manuscript for publication but has dropped few suggestions that needs to be addressed. I thank the authors for responding well. Removing the simulations was a very good move. The focus on the echo and histology measurements is better now, and it covers a gap in the literature. I recommend publication of the article.

Thank you for the suggestion, we better specified in the aim this point.

We completely agree. Based on previous reviewers suggestions, we removed the in-silico model analysis, where, at least in part, we described this point. It is well known that strain depends not just on the relative angulations but also on the shape of the ventricle and on the curvature of the surface. Thus, we did not specified further in this paper. This is the reason why endocardial strain paradoxically decreases over the time (L/T ratio decreases over the time, in particular in the second trimester of gestation) and epi/endo strain ratio increases, accordingly with the epi/endo histologic relative wall thickness.

Attachment

Submitted filename: Rebuttal letter PONE v. 6.docx

pone.0315691.s006.docx^{(15.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0315691.r012

Decision Letter 5

Dhruba Shrestha

29 Nov 2024

Development of the Fetal Myocardium and Changes in Myocardial Fibers Orientation

PONE-D-22-00611R5

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PLoS One. doi: 10.1371/journal.pone.0315691.r013

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Dhruba Shrestha

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PERMALINK

Development of the fetal myocardium and changes in myocardial fibers orientation

Biagio Castaldi

Marny Fedrigo

Daniela P Boso

Irene Cattapan

Elena De Filippi

Francesca M Susin

Paolo Peruzzo

Giulia Comunale

Paola Veronese

Ornella Milanesi

Annalisa Angelini

Giovanni Di Salvo

Roles

Abstract

Background and aims

Methods and results

Conclusions

Introduction

Methods

Echocardiography

Histology

Fig 2. The ventricular myocardium was divided into seven layers to model the transmural variation of fiber orientation (A).

Statistical analysis

Results

Echocardiography

Fig 3. Speckle tracking echocardiography.

Fig 4. On the top, strain rate curves with S, E and A peaks.

Fig 5. Left ventricular end diastolic diameter.

Histology

Fig 6. Relative wall thickness of endocardial.

Fig 7. Endocardial to epicardial wall thickness ratio by histology. There was a progressive increase over gestational weeks (GW).

Fig 8. Changes in fibers’ angulation during gestational age based on histology.

Discussion

Normal fetal myocardial development & function

Relevance of findings to clinical pathology

Limitations

Conclusions

Acknowledgments

Abbreviations

Data Availability

Funding Statement

References

Author response to Decision Letter 0

Decision Letter 0

Peter H Backx

Roles

Author response to Decision Letter 1

Decision Letter 1

Peter H Backx

Roles

Author response to Decision Letter 2

Decision Letter 2

Peter H Backx

Roles

Author response to Decision Letter 3

Decision Letter 3

Peter H Backx

Roles

Author response to Decision Letter 4

Decision Letter 4

Dhruba Shrestha

Roles

Author response to Decision Letter 5

Decision Letter 5

Dhruba Shrestha

Roles

Acceptance letter

Dhruba Shrestha

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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