Detection of microplastics in the feline placenta and fetus

Ilaria Ferraboschi; Francesca Canzolino; Elena Ferrari; Cristina Sissa; Matteo Masino; Matteo Rizzi; Simona Bussolati; Giuseppina Basini; Simone Bertini; Stefano Grolli; Roberto Ramoni; Francesco Di Ianni; Alessandro Vetere; Enrico Bigliardi

doi:10.1371/journal.pone.0320694

Peer Review History

Original SubmissionDecember 4, 2024
4 Dec 2024 Author Response https://doi.org/10.1371/journal.pone.0320694.r001
2 Jan 2025 Decision Letter - Yousuf Dar Jaffer, Editor PONE-D-24-55176Detection of microplastics in the feline placenta and fetusPLOS ONE Dear Dr. Vetere, 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. Please submit your revised manuscript by Feb 16 2025 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'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Yousuf Dar Jaffer Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that your Data Availability Statement is currently as follows: All relevant data are within the manuscript and its Supporting Information files. Please confirm at this time whether or not your submission contains all raw data required to replicate the results of your study. Authors must share the “minimal data set” for their submission. PLOS defines the minimal data set to consist of the data required to replicate all study findings reported in the article, as well as related metadata and methods (https://journals.plos.org/plosone/s/data-availability#loc-minimal-data-set-definition). For example, authors should submit the following data: - The values behind the means, standard deviations and other measures reported; - The values used to build graphs; - The points extracted from images for analysis. Authors do not need to submit their entire data set if only a portion of the data was used in the reported study. If your submission does not contain these data, please either upload them as Supporting Information files or deposit them to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of recommended repositories, please see https://journals.plos.org/plosone/s/recommended-repositories. If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. If data are owned by a third party, please indicate how others may request data access. [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: N/A 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: The work by Ferraboschi et al. describes a very worrisome finding, i.e. the presence of microplastics in foetuses and placentae of stray cats. The object of the investigation is very innovative and interesting. Unwanted feline pregnancies, surgically interrupted through ovariohysterectomy respecting ethical issues, were used to detect microplastic contamination. The work is rather clearly written, with some repetitions that should be deleted (Page 7, lines 115-117 and again lines 130-131) (Page 7, lines 128-129 and again on Page 8, lines 145-146). My doubts: The authors should clarify why a single foetus with his placenta was included from each pregnancy and what criterion was used to select it (always the same position within the uterus?). Foetal age should be reported for the 8 pregnancies, notwithstanding the fact that no gestation age-related condition can be tested when only 8 pregnancies are included. I wonder how an intracardiac injection can be done in 15-day embryos/foetuses (see Knospe (2002) Periods and Stages of the Prenatal Development of the Domestic Cat). Is it true that a coloured particle for which the polymer matrix cannot be identified should be more correctly identified as a ‘suspect’ MNP. If so, this should be reported, modifying the results from line 261 to 271, Page 15. I would add some comments on the possibility of contamination (see your Reference n 32 on human placentae collected at C-sections). On page 15 (line 261) I do not understand where number 5 comes from: four pregnancies were contaminated by coloured MNPs (cats 4,5,6 and 7). In two cases (cat 4 foetus and cat 5 placenta) PE was detected. I cannot see 5. On page 16 (lines 290-291), there is a wrong reference number and a wrong citation: if the correct number is 13 and not 31 (please, add brackets), tap water resulted contaminated with higher levels of MNPs than bottled water. I would stress in the conclusions the alarming findings of this work, that should be a stimulus to drastically limit/eliminate the use of plastic and, for scientists, to find valid alternatives to it. Specific comments Page 4, line 58: ‘(MPs’ delete ‘(‘ Page 7 line 121-122: Propofol is not part of premedication, but it is used for anaesthesia induction. Page 12, line 212: ‘is’ instead of ‘in’ Line 294: do the two ranges refer to the heart? It is not clear. Table 1: The columns do not correspond to what is indicated in the legend Last: Why two corresponding authors?! It is rather unusual Reviewer #2: The manuscript was well written and this can be published in esteemed journal of PLOS ONE.------------------------------------------------------------------------------------------------------------------ ****** 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: Yes: Akbar Abbasi ******** [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.] 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. https://doi.org/10.1371/journal.pone.0320694.r002
Revision 1
10 Feb 2025 Author Response Reviewer Questions and Responses Question Response The authors should clarify why a single foetus with his placenta was included from each pregnancy and what criterion was used to select it (always the same position within the uterus?). The separation of the placenta from the endometrium during an early stage of pregnancy is particularly challenging due to the fragility of the tissues, as well as the uneven number and distribution of placentas. To ensure uniformity in the samples, it was decided to use only one fetus for which complete separation of the placenta was successfully achieved. Line 150-156 Foetal age should be reported for the 8 pregnancies, notwithstanding the fact that no gestation age-related condition can be tested when only 8 pregnancies are included. Foetal ages for the 8 pregnancies have been reported as follows: 15, 20, 27, 27, 28, 29, 30, and 30 days, with a mean age of 26.0 days. (Line 110-111) I wonder how an intracardiac injection can be done in 15-day embryos/foetuses (see Knospe (2002) Periods and Stages of the Prenatal Development of the Domestic Cat). Thank you for your important observation. The injections were performed in the thoracic region in the bigger fetuses; however, given the extremely small size of the fetuses and the inability to confirm the actual distribution of the drug within the circulatory system, it is more accurate to refer to these as intracoelomic injections. We have updated the phrasing accordingly. Is it true that a coloured particle for which the polymer matrix cannot be identified should be more correctly identified as a ‘suspect’ MNP. If so, this should be reported, modifying the results from line 261 to 271, Page 15. We thank the referee for this important comment. We agree that the colored particles for which we were unable to identify the polymer matrix should be classified as suspect MNPs, and we have revised the discussion accordingly. Furthermore, we have moved the discussion about the Raman spectra of dyes/polymer matrix to this paragraph. I would add some comments on the possibility of contamination (see your Reference n 32 on human placentae collected at C-sections). Thank you for your comment. Throughout all stages of processing, plastic instruments were never used. The laboratory environments were controlled. Digestion procedures were performed under a fume hood. It has been reported that microplastics are present in the air within various indoor environments (Zhang et al., 2020). To address this concern, uteri were carefully washed with sterile saline solution prior to dissection to minimize the risk of external contamination. We added few lines in the discussion section. Lines 315-320 On page 15 (line 261) I do not understand where number 5 comes from: four pregnancies were contaminated by coloured MNPs (cats 4,5,6 and 7). In two cases (cat 4 foetus and cat 5 placenta) PE was detected. I cannot see 5. The number of analysed sample is 16 (8 placentas and 8 fetuses). Out of these, we found 5 contaminated samples, belonging to 4 animals (in one case we found that both placenta and fetus were contaminated). In the text, we specified that the 5 samples belong to 4 animals. On page 16 (lines 290-291), there is a wrong reference number and a wrong citation: if the correct number is 13 and not 31 (please, add brackets), tap water resulted contaminated with higher levels of MNPs than bottled water. Thank you for your comment. The correct reference was added. I would stress in the conclusions the alarming findings of this work, that should be a stimulus to drastically limit/eliminate the use of plastic and, for scientists, to find valid alternatives to it. Thank you for your suggestion. Added at line 311-315. Page 4, line 58: ‘(MPs’ delete ‘(‘ Done Page 7 line 121-122: Propofol is not part of premedication, but it is used for anaesthesia induction. Thank you for your comment. Corrected. Line 142-143 Page 12, line 212: ‘is’ instead of ‘in’. Corrected Line 294: do the two ranges refer to the heart? It is not clear. The typo was corrected Table 1: The columns do not correspond to what is indicated in the legend. Corrected. In the legend, columns 2 and 3 were mistakenly swapped. Why two corresponding authors?! It is rather unusual. Given that the work was carried out by researchers from different areas of expertise (clinical and spectroscopic laboratory), it was decided to designate two corresponding authors, each representing their respective field of expertise. https://doi.org/10.1371/journal.pone.0320694.r003
18 Feb 2025 Decision Letter - Yousuf Dar Jaffer, Editor PONE-D-24-55176R1Detection of microplastics in the feline placenta and fetusPLOS ONE Dear Dr. Vetere, 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. Please submit your revised manuscript by Apr 04 2025 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'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols . We look forward to receiving your revised manuscript. Kind regards, Yousuf Dar Jaffer Academic Editor PLOS ONE Additional Editor Comments: Dear Alessandro, At this stage, I still see the manuscript has a scope of improvement. Please address the comments from reviewer 1 except the last comment on having two corrosponding authors. This decision relies on publisher. Best regards, Jaffer [Note: HTML markup is below. Please do not edit.] [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.] 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. https://doi.org/10.1371/journal.pone.0320694.r004
Revision 2
18 Feb 2025 Author Response Question: The authors should clarify why a single fetus with his placenta was included from each pregnancy and what criterion was used to select it (always the same position within the uterus?). Answer: The separation of the placenta from the endometrium during an early stage of pregnancy is particularly challenging due to the fragility of the tissues, as well as the uneven number and distribution of placentas. To ensure uniformity in the samples, it was decided to use only one fetus for which complete separation of the placenta was successfully achieved (Lines 150-156). Question: Foetal age should be reported for the 8 pregnancies, notwithstanding the fact that no gestation age-related condition can be tested when only 8 pregnancies are included. Answer: Foetal ages for the 8 pregnancies have been reported as follows: 15, 20, 27, 27, 28, 29, 30, and 30 days, with a mean age of 26.0 days (Lines 110-111). Question: I wonder how an intracardiac injection can be done in 15-day embryos/foetuses (see Knospe (2002) Periods and Stages of the Prenatal Development of the Domestic Cat). Answer: Thank you for your important observation. The injections were performed in the thoracic region in the bigger fetuses; however, given the extremely small size of the fetuses and the inability to confirm the actual distribution of the drug within the circulatory system, it is more accurate to refer to these as intracoelomic injections. We have updated the phrasing accordingly. Question: Is it true that a coloured particle for which the polymer matrix cannot be identified should be more correctly identified as a ‘suspect’ MNP. If so, this should be reported, modifying the results from line 261 to 271, Page 15. Answer: We thank the referee for this important comment. We agree that the colored particles for which we were unable to identify the polymer matrix should be classified as suspect MNPs, and we have revised the discussion accordingly. Furthermore, we have moved the discussion about the Raman spectra of dyes/polymer matrix to this paragraph. Question: I would add some comments on the possibility of contamination (see your Reference n 32 on human placentae collected at C-sections). Answer: Thank you for your comment. Throughout all stages of processing, plastic instruments were never used. The laboratory environments were controlled. Digestion procedures were performed under a fume hood. It has been reported that microplastics are present in the air within various indoor environments (Zhang et al., 2020). To address this concern, uteri were carefully washed with sterile saline solution prior to dissection to minimize the risk of external contamination. We added a few lines in the discussion section (Lines 315-320). Question: On page 15 (line 261) I do not understand where number 5 comes from: four pregnancies were contaminated by coloured MNPs (cats 4,5,6 and 7). In two cases (cat 4 foetus and cat 5 placenta) PE was detected. I cannot see 5. Answer: The number of analyzed samples is 16 (8 placentas and 8 fetuses). Out of these, we found 5 contaminated samples, belonging to 4 animals (in one case we found that both placenta and fetus were contaminated). In the text, we specified that the 5 samples belong to 4 animals. Question: On page 16 (lines 290-291), there is a wrong reference number and a wrong citation: if the correct number is 13 and not 31 (please, add brackets), tap water resulted contaminated with higher levels of MNPs than bottled water. Answer: Thank you, corrected. Question: I would stress in the conclusions the alarming findings of this work, that should be a stimulus to drastically limit/eliminate the use of plastic and, for scientists, to find valid alternatives to it. Answer: Thank you for your suggestion. Added at line 311-315. Question: Page 4, line 58: ‘(MPs’ delete ‘(‘ Answer: Done Question: Page 7 line 121-122: Propofol is not part of premedication, but it is used for anaesthesia induction. Answer: Thank you for your comment. Corrected. Line 142-143 Question: Page 12, line 212: ‘is’ instead of ‘in’. Answer: Corrected Question: Line 294: do the two ranges refer to the heart? It is not clear. Answer: The typo was corrected Question: Table 1: The columns do not correspond to what is indicated in the legend. Answer: Corrected. In the legend, columns 2 and 3 were mistakenly swapped. Question: Why two corresponding authors?! It is rather unusual. Answer: Given that the work was carried out by researchers from different areas of expertise (clinical and spectroscopic laboratory), it was decided to designate two corresponding authors, each representing their respective field of expertise. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0320694.r005
23 Feb 2025 Decision Letter - Yousuf Dar Jaffer, Editor Detection of microplastics in the feline placenta and fetus PONE-D-24-55176R2 Dear Dr. Alessandro Vetere 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Yousuf Dar Jaffer Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0320694.r006
Formally Accepted
Acceptance Letter - Yousuf Dar Jaffer, Editor PONE-D-24-55176R2 PLOS ONE Dear Dr. Vetere, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Yousuf Dar Jaffer Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0320694.r007

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .