Increase in short telomeres during the third trimester in human placenta

Paula K. Edelson; Michala R. Sawyer; Kathryn J. Gray; David E. Cantonwine; Thomas F. McElrath; Mark Phillippe

doi:10.1371/journal.pone.0271415

Peer Review History

Original SubmissionMarch 1, 2022
3 May 2022 Decision Letter - Khursheed Iqbal, Editor PONE-D-22-06161Increase in short telomeres during the third trimester in human placentaPLOS ONE Dear Dr. Edelson, 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. As you can see the referees find the analysis preliminary and require more data to warrant publication. They raise different concerns that I anticipate that you should be able to address. I would therefore like to invite you to submit a revised version. For such a revision to be successful, it will however be important to fully resolve all of referee #1's concerns. Please submit your revised manuscript by Jun 17 2022 11:59PM. 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Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. MP is supported by Burroughs-Wellcome Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. [Note: HTML markup is below. Please do not edit.] 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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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: Yes Reviewer #2: Yes ****** 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: Here, the authors assessed the ratio of short telomeres in placental tissue of patients who underwent Cesarean section in the third trimester. To quantify the short telomeres, the genomic DNA (gDNA) was isolated from placenta samples and digested by restriction enzymes (HinfI and RsaI) with a 4 and 5 base pair recognition sequence to release the telomeres. The digested gDNA was then size fractionated using a specific electrophoresis system and the fractions of 500 bp, 1 kb and 3 kb were PCR amplified with telomere-specific primers. The authors concluded `that placental telomeres progressively shorten as pregnancies approach 40 weeks of gestation`(ll225-226). A weak point of this study is the strong experimental bias to detected short telomeres, without doing relevant controls. For example, only placental samples were analyzed, why didn´t the authors included chorioamniotic tissue, cord, cord blood, and blood from the mothers? The determination of mean telomere size would also improve the validity of the data. In the applied method the authors cannot discriminate between cell-free DNA and cellular DNA. Thus the first and last sentence in the Abstract - `An increase in telomere shortening in gestational tissues has been proposed as a mechanism for the initiation of parturition` (ll46-47); and `…thereby supporting the hypothesis that increasing short telomeres at term contributes to the mechanism leading to parturition`- are at least not rigorously tested. Sectioning and immunochemical staining of placental tissues was not done. From the Materials and Methods it is unclear how the authors standardized the tissue sampling. The authors do not mention whether there were abnormal placentas, and if so, how many. With regard to the DNA fractionation, what do 500 bp, 1 kb and 3 kb mean?, e.g. is the 1 kb fraction exactly 1 kb, from > 500 bp to 1 kb, or 0 to 1 kb? Would sampling of a 0 to 3 kb fraction also yield an increased short telomere ratio, is there an experimental bias that produces significant increases in the 500 bp and 3 kb fractions? How did the authors confirm the completeness of DNA restriction, and the absence of star activity? Apoptosis is a well described process in placenta maturation, and contribute to DNA fragmentation (also of telomeres). The here presented correlation between increased ratio of short telomeres with increased gestational age cannot elucidate whether this is a specific mechanism of telomer shortening or just an unspecific byproduct of apoptosis. In conclusion, the presented data represent a preliminary draft and need supportive analyses. Reviewer #2: In this manuscript, Edelson et al showed a significant increase in very short telomeres in human placental tissue at term. Authors should describe about the cell types of placenta in material and methods. The experimental content is relatively small, just a description of the phenomenon Its relevance and importance in parturition are too preliminary and speculative. However, I think is not ready for publication. ****** 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. 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Revision 1
16 Jun 2022 Author Response Journal Requirement Comments: 1. Manuscript style requirements: When submitting your revision, we need you to address these additional requirements. Response: We have updated our formatting to reflect the style requirements as laid out in the manuscript formatting guidelines. 2. Please provide additional details regarding participant consent. In the ethics statement in the Methods and online submission information, please ensure that you have specified (1) whether consent was informed and (2) what type you obtained (for instance, written or verbal, and if verbal, how it was documented and witnessed). If your study included minors, state whether you obtained consent from parents or guardians. If the need for consent was waived by the ethics committee, please include this information. Response: In our study, the need for consent was waived by the ethics committee (Institutional Review Board) because placental tissues are routinely discarded. The data used in subsequent analysis which involved abstraction of medical record were anonymized. We have updated the manuscript to clarify this point (Line 108-110). 3. Thank you for stating in your Funding Statement: (KJG is supported by NIH/NHLBI K08 HL146963. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.) Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. MP was supported by Burroughs-Wellcome Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Response: Thank you for bringing this to our attention. We have updated the funding statement as you have recommended above. This is included in the cover letter. 4. Please include your full ethics statement in the ‘Methods’ section of your manuscript file. In your statement, please include the full name of the IRB or ethics committee who approved or waived your study, as well as whether or not you obtained informed written or verbal consent. If consent was waived for your study, please include this information in your statement as well. Response: In our study, the need for consent was waived by the ethics committee (Institutional Review Board) because placental tissues are routinely discarded. The data used in subsequent analysis which involved abstraction of medical record were anonymized. We have updated the manuscript to clarify this point (Line 108-110). Reviewer Comments to the Author: Reviewer #1: A: “Here, the authors assessed the ratio of short telomeres in placental tissue of patients who underwent Cesarean section in the third trimester. To quantify the short telomeres, the genomic DNA (gDNA) was isolated from placenta samples and digested by restriction enzymes (HinfI and RsaI) with a 4 and 5 base pair recognition sequence to release the telomeres. The digested gDNA was then size fractionated using a specific electrophoresis system and the fractions of 500 bp, 1 kb and 3 kb were PCR amplified with telomere-specific primers. The authors concluded `that placental telomeres progressively shorten as pregnancies approach 40 weeks of gestation`(ll225-226). A weak point of this study is the strong experimental bias to detected short telomeres, without doing relevant controls. For example, only placental samples were analyzed, why didn´t the authors included chorioamniotic tissue, cord, cord blood, and blood from the mothers?” Response: The placentas were the tissue of interest collected for this study based on the previously published literature describing telomere shortening in the gestational tissues, especially the placenta. We have updated our limitations section to include the lack of control tissues (Line 225-226). B: “The determination of mean telomere size would also improve the validity of the data.” Response: The focus and novelty of this study was on short telomeres based on previously published reports demonstrating that the effects of telomere shortening are based on the increase in short telomeres, rather than any shift in the average telomere lengths (Hemann et al. Cell 2001;107:67-77). Also, there can occur a significant increase in short telomeres without any change in mean telomere lengths, so we disagree with the reviewer regarding value of having measured mean telomere lengths. C: “In the applied method the authors cannot discriminate between cell-free DNA and cellular DNA.” Response: This statement is not correct. The telomeres were assayed after having extracted DNA from tissue specimens which contain genomic DNA, not cell free DNA. This method for DNA extraction and analysis has been previously published by our group (Phillippe et al. American Journal of Obstetrics and Gynecology 2019:220:496.e1-496.e8). D: “Thus the first and last sentence in the Abstract - `An increase in telomere shortening in gestational tissues has been proposed as a mechanism for the initiation of parturition` (ll46-47); and `…thereby supporting the hypothesis that increasing short telomeres at term contributes to the mechanism leading to parturition`- are at least not rigorously tested.” Response: The reviewer needs to be aware of the large body of scientific evidence already supporting the premise that gestational length (and thus the timing for the onset of parturition) is based on a telomere gestational clock mechanism (ie Lai et al Sci Rep 2021:11:5115,Wilson et al Placenta 2016:48:26-33, Phillippe et al Reprod Sci 2015:22:1186-201, and many others). We acknowledge that this study does not definitively test the hypothesis that short telomeres control parturition, but it does provide novel and important data in support of this association. E: “Sectioning and immunochemical staining of placental tissues was not done.” Response: This is accurate; however, this comment appears irrelevant. These laboratory procedures were not part of the research engaged in our study. F: “From the Materials and Methods it is unclear how the authors standardized the tissue sampling. The authors do not mention whether there were abnormal placentas, and if so, how many.” Response: As described in the Materials and Methods section, placentas were screened for eligibility criteria. Those placentas meeting inclusion criteria were collected and samples were taken from four representative 1-1.5 cm full thickness sections of the placental disk with a scalpel, and then rinsed in phosphate-buffered saline prior to being stored at -80� C. The manuscript has been updated to clarify this point (Line 111-112). The determination of “normal” vs “abnormal” placenta is more challenging, and is outside of the scope of our study. We have included extensive clinical data to correlate pregnancy co-morbid conditions such as hypertension, tobacco use, diabetes, etc., but our specimens did not undergo formal pathology examination by a pathologist to give a formal pathologic diagnosis of the placental tissue. G: “With regard to the DNA fractionation, what do 500 bp, 1 kb and 3 kb mean?, e.g. is the 1 kb fraction exactly 1 kb, from > 500 bp to 1 kb, or 0 to 1 kb? Would sampling of a 0 to 3 kb fraction also yield an increased short telomere ratio, is there an experimental bias that produces significant increases in the 500 bp and 3 kb fractions?” Response: Size fractions were based on standard DNA gel electrophoresis techniques and size markers, so the 500 bp, 1 kb and 3 kb sizes are not exact, but narrow ranges DNA fractions at these sizes. Fraction sizes chosen for this study were arbitrary, but provided representative short telomere fractions compared to mean young adult telomeres in the 10 - 15 kb ranges. Obviously, if we had made our size collection ranges bigger (ie 0 - 500 bp as suggested by the reviewer), we would have measured more short telomeres using our qPCR technique. H: “How did the authors confirm the completeness of DNA restriction, and the absence of star activity?” Response: The restriction digests were performed for the time periods and under the conditions recommended by the manufacturer, and also consistent with previous published reports using the classic telomere TRF (telomere restriction fragment); therefore, we validly assumed that digestions would be complete and absent abnormal enzyme STAR activities. I: “Apoptosis is a well described process in placenta maturation, and contribute to DNA fragmentation (also of telomeres). The here presented correlation between increased ratio of short telomeres with increased gestational age cannot elucidate whether this is a specific mechanism of telomere shortening or just an unspecific byproduct of apoptosis.” Response: This reviewer is correct that there is a close relationship between short telomeres and apoptosis. Our premise is that the short telomeres lead to apoptosis, rather than the reverse. Also, during apoptosis, the DNA is fragmented into much smaller sizes consistent with cell free DNA found in the plasma (i.e. about 200-400 bp sizes). This might spuriously affect our 500 bp measurements, but not the larger 1 and 3 kb measurements. J: “In conclusion, the presented data represent a preliminary draft and need supportive analyses.” Response: We respectfully disagree. Our studies are not definitive, but they are novel and provide the basis for ongoing similar research studies. As we state in our conclusion, the novel observations made in our study support the hypothesis that telomere shortening is associated with placental aging and maturation, but that certainly further characterization of the role of telomere length in influencing the onset of parturition is needed. Reviewer #2: A: “In this manuscript, Edelson et al showed a significant increase in very short telomeres in human placental tissue at term. Authors should describe about the cell types of placenta in material and methods.” Response: As is the case with all tissue biopsy studies for telomere lengths, the tissues are composed of two or more cell types, and the telomere measurements consist of a composite of the mix. The same is true for the placenta which is composed of trophoblast cells, stromal fibroblasts, fetal endothelial cells, immune cells, etc. B: “The experimental content is relatively small, just a description of the phenomenon. Its relevance and importance in parturition are too preliminary and speculative. However, I think is not ready for publication.” Response: As noted above, we respectfully disagree. Our studies are not definitive, but they are novel and provide the basis for ongoing study into this important topic. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0271415.r002
30 Jun 2022 Decision Letter - Khursheed Iqbal, Editor Increase in short telomeres during the third trimester in human placenta PONE-D-22-06161R1 Dear Dr. Edelson, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Khursheed Iqbal, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0271415.r003
Formally Accepted
5 Jul 2022 Acceptance Letter - Khursheed Iqbal, Editor PONE-D-22-06161R1 Increase in short telomeres during the third trimester in human placenta Dear Dr. Edelson: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Khursheed Iqbal Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0271415.r004

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