Maternal stress and placental function; ex vivo placental perfusion studying cortisol, cortisone, tryptophan and serotonin

Line Mathiesen; Cecilie Bay-Richter; Gregers Wegener; Nico Liebenberg; Lisbeth E. Knudsen

doi:10.1371/journal.pone.0233979

Peer Review History

Original SubmissionJanuary 20, 2020
8 Apr 2020 Decision Letter - Frank T. Spradley, Editor PONE-D-20-01763 Maternal stress and placental function; ex vivo placental perfusion studying cortisol, cortisone, tryptophan and serotonin. PLOS ONE Dear Mathiesen, 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 ALL the points raised during the review process. We would appreciate receiving your revised manuscript by May 23 2020 11:59PM. When you are 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. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. 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We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere. "Yes, the individual AFCE ratios and the Stress levels of the participating women are published as a part of a another paper as the participants in this paper are a subset of a previously studied population. The current studied subset is unidentifiable from the previous publication with is referenced in the text, and the study presented here is completely different. " Please clarify whether this [conference proceeding or publication] was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. 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: Yes Reviewer #2: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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: No ****** 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: An innovative and valuable way to test the validity of the results in placenta-fetal metabolism in laboratory animals has been devised and used to study long term consequences of the gestational milieu. In this case it tests the impact of human maternal stress on the key modulators of the HPA axis in the fetus. Reviewer #2: REVIEW PONE Investigators collected term placentas from 22 women, and performed ex vivo perfusion concurrently (simultaneously) with cortisol, 5HT and tryptophan with and without inhibitors for treatments for 6 h successfully on 16 placentas. Cortisol was converted to cortisone by placenta, and was somewhat inhibited by carb, resulting in increased cortisol on fetal side. PCBA had no effect, whereas fluoxotine increased fetal side 5HT. There is some merit in the manuscript, but the quality of writing is OK at best, and sloppy with an overuse abbreviations making it impossible to read fluidly and understand. I am not sure any of the maternal ‘stress’ information adds to the paper, as the sample size was too small to do analysis, and so mentioning it detracts from findings, etc., you may be better off indicating that placentas were collected as part of a larger study that was focused on understanding role of maternal stress on offspring development. There are no indicators of statistical significance of changes in hormone levels in response to treatments, that that because there are none? Please state within files. line 17 which can cause changed health conditions, what is meant by this? I think you mean ‘has potential long-term effects on offspring’s development and health’ line 31 maternal 'stress' the n was really too small to analyze this line 36, edit seen to 'evident' needs to be define, 11BHSD2, line 59 line 67, as written suggests that 5HT is transported across placenta by SERT, whereas uptake of 5HT by SERT targets it for degradation via the pathway referred. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084180/ fetal 5HT source is not of maternal origin. Using additional genetic strategies, a new technology for studying placental biology ex vivo, and direct manipulation of placental neosynthesis, we investigated the nature of this exogenous source and uncovered a placental 5-HT synthetic pathway from a maternal tryptophan precursor, in both mice and humans. although this paper suggests SERT SNP, may affect fetal exposure levels. https://www.ncbi.nlm.nih.gov/pubmed/27550733 ine 103, twenty-two line 103-105, sentence needs editing for sense line 127, define HAS line 142, cortisole=cortisol line 146, space between test line 208 what was the number? Line 226, define tendency, what was p-value, what was difference? Line 232, the graphs show line 241 at the end of line 264 subtract ‘from’ the line 340 write out TDo, over use of abbreviations line 349, what is normal? line 354, write out LOQ As written the conclusion is confusing and not easy to follow line 366, an hour after beginning perfusion of fluoxetine. ****** 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: Yes: Elise P. Gomez-Sanchez Reviewer #2: Yes: Theresa M Casey [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0233979.r001
Revision 1
29 Apr 2020 Author Response Reviewers' comments: 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: No Corrections are made as suggested by the reviewer Reviewer #1: An innovative and valuable way to test the validity of the results in placenta-fetal metabolism in laboratory animals has been devised and used to study long term consequences of the gestational milieu. In this case it tests the impact of human maternal stress on the key modulators of the HPA axis in the fetus. We thank the reviewer for this comment, we also believe the data presented are valuable in its’ field. Reviewer #2: REVIEW PONE Investigators collected term placentas from 22 women, and performed ex vivo perfusion concurrently (simultaneously) with cortisol, 5HT and tryptophan with and without inhibitors for treatments for 6 h successfully on 16 placentas. Cortisol was converted to cortisone by placenta, and was somewhat inhibited by carb, resulting in increased cortisol on fetal side. PCBA had no effect, whereas fluoxotine increased fetal side 5HT. There is some merit in the manuscript, but the quality of writing is OK at best, and sloppy with an overuse abbreviations making it impossible to read fluidly and understand. We thank the reviewer for this comment, and have tried to improve the quality of writing and avoid the overuse of abbreviations. I am not sure any of the maternal ‘stress’ information adds to the paper, as the sample size was too small to do analysis, and so mentioning it detracts from findings, etc., you may be better off indicating that placentas were collected as part of a larger study that was focused on understanding role of maternal stress on offspring development. We thank the reviewer for this comment. We believe the presentation of the maternal stress –though not showing significant result, could demonstrate the methods used and inspire future researchers in this area, if only to show that future research needs to include a larger population sample or more variation in population stress levels. There are no indicators of statistical significance of changes in hormone levels in response to treatments, that that because there are none? Please state within files. The statistical significance of treatments is not tested due the few repetitions, only observations are made. This is now stated in the manuscript. line 17 which can cause changed health conditions, what is meant by this? I think you mean ‘has potential long-term effects on offspring’s development and health’ The change is made as suggested. line 31 maternal 'stress' the n was really too small to analyze this The statement “with no effect of adding PCPA” is deleted. line 36, edit seen to 'evident' needs to be define, The change is made as suggested. 11BHSD2, line 59 The name of the enzyme is written in full at the first mention as suggested. line 67, as written suggests that 5HT is transported across placenta by SERT, whereas uptake of 5HT by SERT targets it for degradation via the pathway referred. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3084180/ fetal 5HT source is not of maternal origin. Using additional genetic strategies, a new technology for studying placental biology ex vivo, and direct manipulation of placental neosynthesis, we investigated the nature of this exogenous source and uncovered a placental 5-HT synthetic pathway from a maternal tryptophan precursor, in both mice and humans. although this paper suggests SERT SNP, may affect fetal exposure levels. https://www.ncbi.nlm.nih.gov/pubmed/27550733 Thank you for this comment. We realize that the literature differs in the origin of the fetal serotonin. This manuscript presents data that shows the possible effect of increased levels of maternal serotonin causing a transient increased level of serotonin on the fetal side of placenta, thus supporting some correlation between maternal and fetal serotonin levels. If this increase is caused by transport via the SERT remains to be established. ine 103, twenty-two The correction is made as suggested. line 103-105, sentence needs editing for sense The sentence is edited as follows: Of these, 2 perfusions had a leak of medium from fetal circulation and were unsuccessful, 5 perfusions showed a leak of medium from the fetal circulation after 4 hours perfusion and were thus unsuccessful after this time-point, and 15 were successful 6-hour perfusions. line 127, define HAS HSA is written out as human serum albumin. line 142, cortisole=cortisol The change is made as suggested. line 146, space between test The change is made as suggested. line 208 what was the number? The number is added (n=22). Line 226, define tendency, what was p-value, what was difference? The statement is an observation and does not include statistical measures, the term “tendency” is deleted. Line 232, the graphs show The change is made as suggested. line 241 at the end of The change is made as suggested. line 264 subtract ‘from’ the The change is made as suggested. line 340 write out TDo, over use of abbreviations The change is made as suggested. line 349, what is normal? The term normal means relatively unselected in this case, and is therefore deleted. line 354, write out LOQ The change is made as suggested. As written the conclusion is confusing and not easy to follow Thank you for this observation, the conclusion is rewritten in part to aid understanding: Perfusions were performed on twenty-two placentas studying the effect of maternal stress levels and relevant enzymes on the placental transfer and metabolism on four chosen hormones: cortisol, cortisone, tryptophan and serotonin. The addition of carbenoxolone inhibitis the metabolism of cortisol in the placenta, and this increased the fetal levels of cortisol in our experiments as expected. Unexpectedly we saw a peak in fetal levels of serotonin an hour after beginning perfusion, in placental perfusions of serotonin with added fluoxetine that inhibits the serotonin transporter. This was observed together with a delayed decrease in serotonin concentration on maternal side which suggests that the increased fetal levels of serotonin is caused by increased serotonin concentration on the maternal side of the placenta, perhaps enabling other transplacental serotonin transport routes than through the SERT. The effect of maternal stress on placental transporters and enzymes, represented in this study by adding inhibitors, can thus affect the fetal exposure to maternal cortisol and serotonin levels. The more direct interpretation is increased transient exposure to both cortisol and serotonin when treating pregnant women with enzyme inhibitors. The increased fetal exposure to serotonin when inhibiting the SERT is an interesting finding, especially as regards to pregnant women needing treatment with SSRIs due to major depressive disorder and PTSD. No effect on placental kinetics was seen on maternal stress levels during the pregnancy, which is probably due to small group size in this study and the low to normal stress levels in the majority of most participating mothers. line 366, an hour after beginning perfusion of fluoxetine. The sentence is changed as follows: an hour after beginning perfusion, in placental perfusions of serotonin with added fluoxetine... Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0233979.r002
18 May 2020 Decision Letter - Frank T. Spradley, Editor Maternal stress and placental function; ex vivo placental perfusion studying cortisol, cortisone, tryptophan and serotonin. PONE-D-20-01763R1 Dear Dr. Mathiesen, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Frank T. Spradley Academic Editor PLOS ONE 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: All comments have been addressed ******** 2. 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: N/A ****** 4. 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 #2: Yes ****** 5. 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 #2: Yes ****** 6. 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 #2: The authors addressed my comments--but I suggest a very thorough read theough and editing by a native English speaker, there are still problems here and PLoSOne does not copy edit. For example line 202 'are' shuld be were--as this was done in past/....authors use 'are' are' are, and then switch tense. Again the writing is quite sloppy and not a good reflection of qulaity. ****** 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 https://doi.org/10.1371/journal.pone.0233979.r003
Formally Accepted
20 May 2020 Acceptance Letter - Frank T. Spradley, Editor PONE-D-20-01763R1 Maternal stress and placental function; ex vivo placental perfusion studying cortisol, cortisone, tryptophan and serotonin. Dear Dr. Mathiesen: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Frank T. Spradley Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0233979.r004

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