Identification of Novel Syncytiotrophoblast Membrane Extracellular Vesicles Derived Protein Biomarkers in Early-onset Preeclampsia: A Cross-Sectional Study

Reviewer #1 (Public Review):

The authors primary objective in this study was to identify differences between patients with preeclampsia and normal patients with respect to the placental syncytiotrophoblast extracellular vesicle proteome.

A strength of this study is that the authors identified novel STB-EV protein markers that are more abundant in the placenta of patients with preeclampsia compared with normal controls. This contributes a little more to what is already known about STB-EV markers and preeclampsia. If these markers can be shown to be more abundant in maternal plasma of preeclampsia patients, it would be very useful for identifying patients who are at high risk for developing early-onset preeclampsia.

Weaknesses include:
(1) The small sample size. There were only 6 patients in the study group and 6 normal controls. However, this can be considered as a pilot study.
(2) The normal controls were not matched with the study patients and the authors did not state how the controls were selected.
(3) The authors state that the placenta samples were obtained at the time of elective cesarean section. However, it is likely that all the preeclampsia patients were delivered for clinical indications rather than electively. This should be clarified.

Reviewer #2 (Public Review):

Summary:

Preeclampsia is a disorder of pregnancy that affects 4-5% of pregnancies worldwide. Identifying this condition early is clinically relevant as it will help clinicians to make management decisions to prevent adverse outcomes. The placenta holds a key to many pregnancy-related pathologies including preeclampsia and studies have shown many differences in the placenta of women with preeclampsia as compared to controls. However as the placenta cannot be collected directly during pregnancy, the exosomes secreted by it are considered a good alternative to tissue biopsy. In this study, the authors have compared the proteins in different sizes of exosomes from the placenta of women with and without preeclampsia. The idea is to eventually use these as biomarkers for early detection of preeclampsia.

Strengths:

The novelty factor of this study is the use of two different-sized exosomes which has not been achieved earlier.

Weaknesses:

The study measured the proteins at only a single time point after the disease has already occurred. However, the placenta is an ever-changing tissue throughout pregnancy and different proteins can come up at different times in pregnancy. Thus serial measurements are necessary and a single time point measurement. The has not validated the identified biomarkers in plasma or circulating placental exosomes from women with and without preeclampsia. Thus the utility of these findings in real-life situations can not be judged from this work.

Author Response

The following is the authors’ response to the original reviews.

Reviewer #1

(1) Since you only included patients with early-onset preeclampsia in the study, I suggest revising the title to "Identification of novel syncytiotrophoblast membrane extracellular vesicle derived protein biomarkers in early-onset preeclampsia...."

We have changed our title to early-onset preeclampsia.

(2) Under methods, you state that placenta was obtained from women undergoing elective cesarean section. Was this because all the study patients were delivered before the onset of labor? Or were laboring patients specifically excluded from the study?

Indeed, labor influences the extracellular vesicles (EVs) generated. To ensure consistency in our samples and avoid this variable, we chose placentas obtained from elective cesarean sections (CS) for our study.

(3) In Table 1 on page 10, the 8th row (Birth weight grams) needs to be reformatted. The mean birthweights for normal pregnancy and preeclampsia should be the same.

We have reformatted the table and using ranges instead of brackets.

(4) In the legend for Table 1, the sentence beginning on page 10, line 227, and continuing onto page 11, line 228, does not make sense. Part of the sentence was omitted inadvertently.

We have modified this sentence to :

Detergent treatment, which could break down EVs, with NP-40 confirmed that the majority (99%) of our samples were largely vesicular since only 0.1 ± 0.12% of BODIPY FL N-(2-aminoethyl)-maleimide and PLAP double-positive events were detected (a reduction of 99%) (Figure 1E and 1H).'

(5) As you acknowledge, the sample size (12 patients) was small. This is understandable because early-onset preeclampsia occurs in <1% of parturients. You could collaborate with other centers in future studies to increase the sample size.

Thank you very much for your comment. We are willing to cooperate on future research and will try to expand our sample size in subsequent studies.

Reviewer #2 (Recommendations For The Authors):

(1) This is one of the many "catalogue" papers where placental exosome proteins in preeclampsia are profiled. Thus, the manuscript lacks novelty. The only novelty factor is the authors have isolated exosomes by a different method and even separated the small and large exosomes. However, there is no mention of how these exosomes differ from each other in terms of their functionality. Thus it is hard to judge the biological significance of this work.

We appreciate your insights regarding the novelty of our study. While numerous papers have profiled placental exosome proteins in preeclampsia, our methodology for enriching sSTB-EVs (exosomes) offers a distinct perspective. We believe that the separation of sSTB-EVs (exosomes) and medium/large STB-EVs (microvesicles) introduces a differentiation that extends beyond mere profiling, with implications for their functionality. There are previous studies showed that the different sizes of placenta EVs have distinct characteristics (Zabel RR, et al. Enrichment and characterization of extracellular vesicles from ex vivo one-sided human placenta perfusion. Am J Reprod Immunol. 2021 Aug;86(2)). Furthermore, the way cells internalize and respond to EVs may depend on the size of the EV (Zhuang X et al. Treatment of brain inflammatory diseases by delivering exosome encapsulated anti-inflammatory drugs from the nasal region to the brain. Mol Ther. 2011 Oct;19(10).) Therefore, it would be important for future studies to distinguish different sizes of EVs for the research.

(2) The authors must demonstrate that these two types of EVs are also produced in vivo by detecting them in the serum of women.

Thank you for the comment. Many previous studies have shown the two types of placental EVs in women's blood. Nakahara et al.'s (PMCID: PMC7755551) extensive review compiles studies that have specifically isolated various subtypes of placenta-derived EVs from maternal circulation. We have also readdressed it in the introduction.

(3) The authors must compare the proteomes of serum-derived placental exosomes and the proteome of the STBs isolated from the perfusion experiments to judge how overlapping the outcomes are from those produced naturally and those produced under ex vivo conditions.

We appreciate the reviewer's suggestion to compare the proteomes of serum-derived placental sSTB-EVs (exosomes) with those from STBs isolated through perfusion experiments. Indeed, such a comparison would provide valuable insights into the similarities and differences between naturally produced and ex vivo-generated sSTB-EVS (exosomes). However, isolating placental EVs from maternal circulation for comprehensive proteomic profiling presents challenges. It requires a significant amount of serum or plasma sample that will be sufficient to enable the isolation of placenta-specific EVs amongst numerous EVs in the circulation. In addition, it will require multiple intricate steps such as ultracentrifugation followed by immunoprecipitation. Each of these steps can potentially lead to the loss of EVs. Additionally, given the high concentration of lipoproteins in plasma relative to EVs, there's a significant risk of obtaining low-purity isolates from the outset. These challenges might compromise the comparability of results between placenta-specific EVs from maternal circulation and those from ex vivo perfusion. Nevertheless, we acknowledge the value of such an endeavor and will consider incorporating this aspect in future studies as the EV and proteomic methodology and technology improve and become more sensitive.

(4) I have a major issue with the chosen study subjects. While the study title and the manuscript mention preeclampsia, as per the inclusion criteria mentioned in lines 88-90, the patients will be HELLP syndrome. Please clarify what was used and modify the manuscript accordingly.

Thank you very much for finding this error. Our patients had none of the features that would qualify them for HELLP syndrome. We have edited to:

PE was defined as new (after 20 weeks) systolic blood pressure of 140 mmHg or diastolic pressure of 90 mmHg, proteinuria (protein/creatinine ratio of 30 mg/mmol or more). None of our patients had maternal acute kidney injury, liver dysfunction, neurological features, hemolysis, or thrombocytopenia.

(5) It is hard to reconcile how only 15 proteins were identified in the placental extract while 300+ in EVs. There is a methodological issue in the mass spec or extraction. With such widely different denominators in the total proteins identified, it is hard to compare the outcomes in terms of the three sample types.

We acknowledge the reviewer's concerns regarding the disparity in protein counts between the placental extract and the EVs. Ultimately, more is not necessarily better. Several factors might contribute to this discrepancy. Firstly, it is plausible that certain proteins exhibit selective affinity to varying sizes of EVs, leading to a more diverse range of proteins than the placental extract. We were also stringent in our analysis to enable us to select proteins whose biological differences are more likely to be reproducible with a different validatory method like a western blot. Additionally, although the placental extract might contain a higher total protein concentration, it doesn't necessarily translate to a richer diversity of disease-specific proteins. Considering these nuances when comparing protein outcomes across sample types is helpful.

(6) I am unable to understand the terms least differentially expressed and most differentially expressed. Do the authors mean upregulated and downregulated? Please clarify and use the terms appropriately by providing fold change values.

We appreciate the reviewer's request for clarification. We intended to provide a relative measure of expression for the terms 'least differentially expressed' and 'most differentially expressed'. The terms are roughly equitable to down- and upregulated. Regarding EVs, we avoid using the terms 'upregulated' and 'downregulated' as EVs act as transporters and do not possess regulatory functions per se. However, for the placenta, we recognize the relevance of these terms.

(7) The data presented is very superficial and lacks methodological details. The authors should provide the total number of targets achieved after mass spec. The cutoffs used the FDRs and other details.

We apologize for the omission. We have added these details to the method section.

(8) It is not clear how were these differentially abundant proteins identified. What was the cutoff used? Was it identified in all the replicates?

We apologize for the omission. We have added these details to the method section.

(9) How many samples were subjected to the discovery cohort, and how many were in the validation cohort? Were they the same or different? If the samples were different, how many PE samples had differentially abundant proteins by both methods?

The study utilized 12 samples for initial discovery and another 12 for western blot validation. The validation samples specifically targeted proteins of interest, rather than undergoing another comprehensive mass spectrometry analysis.

(10) It is striking that the authors report the expression of prostatic acid phosphatase in the placenta. In my understanding of placental biology, this gene or protein is not known to be expressed by the placenta. Please perform immunofluorescence to demonstrate that this protein is indeed produced in the STBs

Research has revealed that even though it's called prostate-specific antigen, it's created in tissues other than the prostate, such as the placenta. Here are a couple of references to support this claim: PMID: 10634405, PMID: 7533063, PMID: 8939403, and PMID: 8945610. Hence it is likely not beneficial to demonstrate what many researchers have already demonstrated.

(11) Please validate the differential abundance of these proteins in the exosomes isolated from the plasma of women with and without preeclampsia. A serial measurement will be of high value to determine how early as compared to hypertension, these biomarkers can predict preeclampsia.

We are validating each EV-carried marker individually in the circulation (plasma or serum), localizing them in the placenta, and performing downstream functional analysis. This article is already lengthy and would likely be too cumbersome to include the details of all individual proteins in this manuscript. However, we have already published papers on Siglec 6 (PMID: 32998819) and Neprilysin (PMID: 30929513), and others will be published soon. We agree that there will be a lot of value to serial measurement, not just in terms of how early as compared to hypertension, these biomarkers can predict preeclampsia but also as potentially a more sensitive or specific test. This would be the subject of subsequent papers.

(12) The authors are recommended to carry out immunofluorescence to localize the differentially abundant proteins in the placental sections and show that they are specific to STBs.

We have already provided a similar response earlier (see response to point 11). In addition, while it is preferable, the biomarkers don't necessarily need to be specific to STB. Not all biomarkers are mechanistic agents/targets, and not all mechanistic agents are biomarkers. However, mechanistic agents should preferably be placental-specific. For example, the total sFLT1, the most studied biomarker, is not exclusively synthesized in the placenta, even though the placental-specific isoform represents a small fraction of the total sFLT-1. For example, in the non-placental world, alkaline phosphatase (ALP) is not exclusively produced by the liver but is a ‘biomarker’ of cholestatic disease.

(13) Table 1 should give the range and SD could be given as + instead of the bracket.

Thank you for your suggestion. We have edited it accordingly.

(14) It is necessary to provide the gestational age of the onset of hypertension to get a judgment of how long these women were preeclamptic, culminating in HELLP.

We want to emphasize that none of our patients experienced HELLP syndrome. In the results section, we have included the gestational age at the time of diagnosis in the table for preeclampsia. It's crucial to understand that the gestational age at diagnosis is distinct from the gestational age when hypertension initially appeared. Detecting the exact gestational age of hypertension onset would be challenging, and it would likely require a prospective or randomized clinical trial with continuous monitoring, possibly on a daily basis. However, our study is retrospective. Thus we can only comment on the gestational age at diagnosis

(15) For newborns the term Sex is used and not gender

Thank you for your suggestion. We have edited it accordingly.

(16) Figure 2 is stretched and hard to read

Thank you for your suggestion. We have edited it accordingly by creating two separate images to promote readability.

(17) Line 278 change the sentence "there fifteen (15) proteins in the placenta" to "there were fifteen (15) proteins in the placenta"

Thank you for your suggestion. We have edited it accordingly.

(18) Line 288 you mean least and not lease

Thank you for your suggestion. We have edited it accordingly.