-->PONE-D-26-05691-->-->Reproducibility of fetal global longitudinal strain measured with speckle tracking echocardiography-->-->PLOS One

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Reviewers' comments:

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: I have carefully reviewed the manuscript entitled “Reproducibility of fetal global longitudinal strain measured with speckle tracking echocardiography” . Overall, I find it to be a well-conducted and relevant study addressing an important limitation in fetal cardiology, namely the variability of fSTE measurements that currently limits clinical implementation.

The manuscript is generally well structured, with a clear objective and a solid methodological approach. I particularly appreciate the use of a standardized protocol and the decision to analyze the same heart cycle within a single DICOM clip, which represents a meaningful methodological strength. The relatively large sample size further enhances the reliability of the findings.

The results are clearly presented and show that reproducibility is variable, with better performance in the left ventricle and before 32 weeks of gestation. The finding of higher inter-observer than intra-observer reproducibility is interesting and deserves further emphasis. What I found particularly valuable is the clarity with which the methodological steps are described, especially the detailed explanation of the fSTE analysis workflow. This makes the study easy to follow and gives confidence in the reproducibility of the approach.

That said, a few aspects could be improved. The introduction could better highlight the novelty of the study, and the methods section would benefit from more detail regarding operator experience and selection criteria. In the discussion, some sections are slightly repetitive, and the clinical implications of the findings could be more clearly articulated. Additionally, limitations related to generalizability and software dependence should be expanded.

The manuscript includes a limited number of citations from the authors’ previous work, which appear appropriate given the continuity of the research and the use of an established cohort and methodology.

In conclusion, this is a valuable and methodologically sound study. With minor to moderate revisions, particularly to improve clarity and strengthen the discussion, the manuscript would be suitable for publication.

Reviewer #2: The authors make a pretty reasonable atempt to separate the soruces of variability in fSTE by analyzing the same cardiac cycle in the same DICOM clip, which by itself is valuable and honestly more precise then many earlier studies.

My comments on the manuskript:

The authors state that the cohort included 124 women, while the inter-observer analysis was performed on 136 independent ultrasound clips, and at the same time they write they used “only independent data, with one DICOM clip per patient”. These three pieces of information cannot really be reconciled logicaly.

The authors write that for the sample size calculation they set threshold = 0.75 and expected reliability = 0.60. In the classical aproach to planning an ICC reliability study, the expected value should be higher then the minimally acceptable one, not lower. In the present wording it looks like the study was powered to show that the expected reliabilty is worse than the preset threshold. This suggests either the parameters were described in reverse, or the calculation itself was done incorretly.

The authors admit that the clips for the inter-observer analysis came from the gestational age category with the highest reproducibility in the intra-observer subanalysis, and in the end they mostly covered 25 to 30 weeks, with predominance of week 27. This means the inter-observer analysis was not a representative assesment of the whole 18 to 41 week range, but rather an assesment done in a priviliged, preselected fragment of the data.

In both the intra-observer and inter-observer settings, the authors efectively freeze steps 1 and 2, meaning clip selection and cardiac cycle selection. The second intra-observer analysis starts from step 3, and in the inter-observer setting the second observer uses the clip and cycle previously selected by the first observer. So in practice the study does not assess full reproducibility of GLS measurement, but only reproducibility of manual segmentation/contouring and GLS calculation after the clip and cycle had already been fixed. This does not fully invalidate the work, but it needs a more honest naming. The current title and abstract are too broad. This should already be stated in the title, abstract and conclusions.

In the discussion the authors themself write that observer one became more precise after her second own measurement and then trained observer two, while the inter-observer analysis was calculated using the second measurement of observer one and the measurement of observer two. This is not a neutral circumstnace. It means the observers were not simply two independent readers, but that the second observer entered the analysis after a stage of harmonisation with the first one. Such a setup may artifically increase agreement and should be treated as a serious limitation, not only as a possible explanation of the resutls.

The authors correctly criticize the literature for imprecise ICC reporting, but they themself do not provide the most important information, namely which exact ICC model was used. The mere statement that ICC was estimated by LMM does not solve the interpretative problem. Also, p-values are attached to tables labeled as ICC tables, but it is not clearly stated whether these are p-values for the ICC test or for the paired t-test related to bias.

The whole intra-observer analysis includes 632 clips from 124 patients, so the observations are not independent. The authors use LMM for ICC, which is reasonable, but the Bland-Altman plots and paired t-tests are described as if all clips were treated like independent pairs of measurments. With repeated measurements from the same fetuses, the classical Bland-Altman method may give incorrect limits of agreement if the repeated-measures structure is not taken into account. In this work either a repeated-measures Bland-Altman aproach or a mixed-effects aproach to agreement is needed, or at least a clear discussion of this limitation.

The most striking aspect of the intra-observer results is not the ICC itself, but the absolute width of the LoA. For the LV, the mean difference is 0.16, but the limits of agreement go to about -11.34 and +11.67, and for the RV about -11.71 to +12.34. With mean GLS values around -21% and -19%, this is a very wide spread. In other words, even with a “moderate” ICC, the clinical interchangebility of single measurements may still be weak.

The conclusion about the “highest reproducibility before 32 weeks” is too strong. The authors show descriptively that some ICC values are highest in the 23 to 27 and 28 to 32 week groups. But they did not perform a formal comparison of ICCs between gestational age groups, they did not show an interaction test, and the confidence intervals overlap to a large extent. In additon, the inter-observer analysis was intentionally built on material from exactly this range. In this situation, the statement in the abstract and conclusion that reproducibility is highest before 32 weeks is more narrative then evidence-based. It should be softened to a descriptive observation.

In the limitations section the authors write that both intra- and inter-observer reproducibility analyses demonstrated statistically significant ICCs, which is not consistant with the tables, where p-values for intra-observer are 0.499 and 0.221, and for inter-observer LV 0.298.

In the discussion there is a sentence saying that “two of the eight studies” did not report 95% CI, but three papers are cited there: [14], [22], and [26].

Table 4 does not match the authors’ own results. The row “Our study” contains numbers and CIs that do not correspond to Tables 1 and 3. In particular, RV inter-observer in Table 4 is given as 0.71 [0.61-0.78], while Table 3 shows 0.625 [0.426-0.777].

In the Methods, the authors refer to an earlier description of the cohort, but even in this manuskript the basic information is still needed: exact inclusion and exclusion criteria, number of women assessed for eligibilty, number excluded, number of failed or non-analyzable clips, distribution of number of visits per patient, reasons for missing data, and ideally a flow diagram. Without this, it is imposible to judge whether the analyzed clips were a representative subset of the material or rather a selected set of technically best examinations.

The data statement says: “All relevant data are within the manuscript and its Supporting Information files”, but in the submitted draft there are no actual source data allowing the reader to reproduce the reproducibility analyses for 632 clips and 3 reading series. If the relevant dataset is supposed to be in the supplementary material, it should be actually attached and described. Otherwise the statement is incomplete or misleaading.

The authors did not consider angle of insonation because previous data supposedly suggested angle-independence. However, their own bibliography includes papers dealing with the effect of angle and frame rate on GLS, and in the discussion they themself admit that previous studies also differed in this respect.

Overall, I think the manuskript may become suitable for publication, but only after an apropriate major revision.

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Reviewer #1: Yes:  Cerghit-Paler Andreea

Reviewer #2: No

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Reproducibility of fetal global longitudinal strain measured with speckle tracking echocardiography using a fixed cardiac cycle

PONE-D-26-05691R1

Dear Dr. Meireson

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Academic Editor

PLOS One