Increase in short telomeres during the third trimester in human placenta.

An increase in telomere shortening in gestational tissues has been proposed as a mechanism involved in the timing for the initiation of parturition. An increase in very short telomeres with increasing gestational age has been observed in mice; this study sought to explore this phenomenon in human pregnancies. Specifically, this study addressed the hypothesis that prior to labor, the quantity of very short telomeres (<3 kilobase (kb) lengths) increases in human placental tissue as term gestation approaches. The primary outcome was the quantity of very short telomeres present in placental tissue. Quantitative measurements of very short telomeres were performed using real-time polymerase chain reaction (qPCR) adaptation of the telomere restriction fragment technique. Placental tissue from 69 pregnant individuals were included. Mean gestational age was 39.1 weeks (term) and 36.2 weeks (preterm). For term versus preterm placentas, the observed increase in very short telomeres were as follows: 500 bp telomeres increased by 1.67-fold (p < 0.03); 1 kb telomeres increased 1.67-fold (p < 0.08); and 3 kb telomeres increased 5.20-fold (p < 0.001). This study confirms a significant increase in very short telomeres in human placental tissue at term; thereby supporting the hypothesis that telomere shortening at term contributes to the mechanism that determine the length of pregnancy thereby leading to onset of parturition.

Introduction

Despite advancements in the understanding of the molecular, endocrine and inflammatory mechanisms occurring during the initiation of human labor (parturition), the biologic clock mechanism(s) that determine the length of gestation leading to the onset of human parturition are not well understood. Telomere length, specifically telomere shortening over time, has been observed to be a major component of the biologic clock that determines life-span in adult humans [1, 2]. Using this premise, we have proposed a telomere gestational clock hypothesis in which shortening of telomeres in placental trophoblasts and fetal membranes (i.e., gestational tissues) leads to cellular apoptosis, resulting in the release of increasing amounts of cell-free DNA that stimulates a pro-inflammatory response [3, 4], ultimately leading to the spontaneous onset of labor as extensively discussed in the recently published review by Phillippe [5].

The average telomere lengths in blood and skin samples among newborn humans range from about 9 to 12.5 kilobases (kb) [6]. Previous work has demonstrated that placental telomere length decreases with increasing gestational age [7]. Prior studies of telomere lengths in human pregnancies have largely focused on measuring average telomere length [7-10], rather than the proportion of “very short” telomeres (VST) (i.e., telomere segments at or less than 3 kb in length). Some studies have also reported telomere shortening as a percentage of total telomere length in gestational tissue in response to maternal obstetric complications, such as gestational diabetes [11]. However, increases in the quantity of short telomeres, and not reductions in the average telomere lengths, are thought to be the effectors of telomere dysfunction, leading to cellular senescence, apoptosis, aging, and tissue dysfunction [12]. The increase of VST during late gestation has, therefore, been proposed as a biologic clock signal leading to apoptosis in the gestational tissues and the stimulation of proinflammatory signaling ultimately leading to labor.

In mouse studies, very short telomere segments in the placenta and fetal membranes have been observed to increase at the end of gestation compared to mid-gestation [13]. To date, only one recently published report has described a significant increase in short telomeres at term in human placental and chorioamniotic membrane tissues compared to 18 weeks of gestation [13]. The purpose of this study was to further test the telomere gestational clock hypothesis in human pregnancy by assessing the relative quantity of VST during the middle of the third trimester of pregnancy (average 36 weeks), as compared to the end of the third trimester (average 39 weeks) (i.e. a time period preceding the spontaneous onset of labor (parturition)).

Materials and methods

Placental samples were collected immediately following Cesarean delivery at two hospitals within the Mass General Brigham HealthCare System in Boston, MA. Inclusion criteria for samples were as follows: singleton pregnancies between 32 to 41 weeks and delivery by cesarean section prior to the onset of labor. Women in labor were excluded to avoid capturing any potential effect of labor on telomere length. Institutional review board approval was granted by the Partners Human Research Committee, and the need for informed consent was waived and thus not required to be obtained. Clinical and demographic data were collected by medical record review. 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. After collection, the placental tissues were immediately rinsed in phosphate-buffered saline and stored at -80° C until utilized for DNA extraction.

The DNA was extracted from placental tissues using High Pure PCR Template Prep kits (Roche Applied Science) according to the manufacturer’s protocol. The concentration and quality (based on the 260/280 nm ratio) of the isolated DNA was determined using a NanoDrop spectrophotometer (ThermoFisher Scientific). To quantify the increase in very short telomere segments (i.e., 500 base, 1 kb, and 3 kb lengths), a quantitative PCR (qPCR) modification of the telomere restriction fragment (TRF) technique was utilized [14]. Specifically, the DNA underwent restriction endonuclease treatment using Hinf I and Rsa I to digest the genomic DNA (but not the telomere DNA), thereby releasing intact telomere segments. The treated DNA was then size fractionated using the E-Gel Power Snap Electrophoresis system (ThermoFisher Scientific) with collections of approximate 500 base, 1 kb, and 3 kb DNA fractions. Real-time qPCR was performed in triplicate using the telomere PCR primers reported by Gil and Coetzer [15], the SsoAdvanced Universal SYBR Green Supermix (BioRad), and 5 ng DNA aliquots. The qPCR cycles were run using the CFX96 Touch Real-Time PCR Detection System as follows: 40 cycles at 95°C x 10 sec and 55.7°C x 30 sec. Using the same DNA samples, real-time qPCRs were performed using primers and amplification cycles optimal for the human GAPDH gene, which served as the DNA control.

The Pfaffl Method [16] was used to calculate the increase in the relative quantity of very short telomere DNA at term compared to preterm. Based on prior mouse data [14], power calculations suggested that a sample size of 13 would be needed to detect a two-fold difference in very short telomeres between term and preterm groups. The data were analyzed using the Kruskal-Wallis ANOVA on Ranks and multiple comparisons tests (using the Dunn method) with significance at p ≤ 0.05.

Results

Placental tissue was analyzed from 58 term pregnancies (≥37 weeks) and 11 preterm (<37 weeks) pregnancies. The mean gestational ages at delivery were 39 weeks 1 days (term) and 36 weeks 2 days (preterm). Maternal and fetal characteristics between the term and preterm pregnancies did not differ significantly (Table 1). The mean maternal age was 33.2 years for the preterm group, and 34.6 years for the term group. Pre-pregnancy Body Mass Index (BMI) was 25.1 in the preterm group and 27.3 in the term group. Hypertension was present among 2/11 (18%) of the preterm group and only 2/58 (3%) of the term group. Smoking was uncommon between both groups, with no smoking in the preterm group and only 2/58 (3%) in the term group. As expected, the average birthweights were lower in the preterm group: 2977 grams for the preterm versus 3545 grams for the term group. Indications for delivery by Cesarean were similar between the two groups, including fetal malpresentation, prior Cesarean delivery, and prior uterine surgery.

Table 1

Maternal and fetal characteristics of term and preterm placentas.

	Preterm (<37 weeks)	Term (≥37 weeks)
	n = 11	n = 58
Gestational age at delivery	36 weeks 2 days	39 weeks 1 days
Maternal age (years)	33.2	34.6
Pre-pregnancy BMI (kg/m2)	25.1	27.3
Tobacco use	0 (0%)	2 (3%)
Hypertension	2 (18%)	3 (5%)
Race Caucasian	6 (55%)	40 (69%)
Fetal sex male	2 (18%)	29 (50%)
Insurance private	7 (64%)	30 (52%)
		28 (48%)
Indications for delivery	Breech	Prior Cesarean delivery
	Prior Cesarean delivery	Prior myomectomy
		Breech

The relative quantities of very short telomeres (VST) segments were significantly increased in term as compared to preterm placentas as follows: 500 bp telomere segments = 1.67-fold increase (median, interquartile range 0.86–4.16, p < 0.03) and 3 kb telomere segments = 5.20-fold increase (median, interquartile range 3.01–8.41, p < 0.001), as shown in Fig 1 and Table 2. There was a similar, but not statistically significant trend in the relative increase in the quantity of 1 kb telomere segments for the term compared to the preterm placental groups (Table 2).

Fig 1

Relative telomere quantity in preterm versus term human placental tissue by telomere lengths.

Relative quantity of very short telomeres (VST), stratified by telomere length, preterm group serves as reference for Pfaffl calculation. Data in mean ± S.D. (standard deviation), N = 11 for preterm and N = 58 for term samples. (·) indicates p < 0.05 comparing term to preterm.

Table 2

Relative quantity of very short telomeres (VST) in human placental tissue.

Telomere fragment size	Preterm (<37 weeks)	Term (≥37 weeks)	P-value
	N = 11	N = 58
	Median, (IQR)	Median, (IQR)
500 bp size	0.76 (0.27–2.08)	1.67 (0.86–4.16)	0.03
1 kb size	0.93 (0.48–1.47)	1.67 (0.87–3.48)	0.08
3 kb size	1.2 (0.85–1.45)	5.2 (3.01–8.41)	<0.001

Results presented as median (interquartile range) of Pfaffl relative quantity of telomere length.

Discussion

For human placental tissue, we have observed that the relative quantity of 500 bp and 3 kb length telomere segments were significantly higher in term compared to preterm placental tissues. This observation supports the hypothesis that telomere shortening in the placenta occurs with the progression of gestational toward term in our cohorts of placentas obtained from pregnant women delivered pre-labor.

The phenomenon of telomere shortening prior to the onset of labor, with an increase in VST, may help to better understand the pathways that lead to the onset of parturition. Physiologic senescence of the gestational tissues (i.e. the placenta and fetal membranes) occurs as term approaches and is accelerated by the increased oxidative stress of term pregnancy [17]. Progressive aging of the gestational tissues, cellular apoptosis and the release of proinflammatory mediators (including cell-free DNA) have been associated with the onset of parturition [18]. Our studies reported here have shown a parallel increase in short telomeres at term in human placental tissue, thereby supporting the hypothesis that placental senescence in term pregnancy may be physiologic and play a role in determining the timing for the initiation of parturition.

Most prior work on telomeres in human placental tissue examined average telomere lengths and reported an average shortening of telomere length across pregnancy [7, 9, 10]. However, a recent study by Lai et al. [13] has evaluated short telomere segments in human gestational tissues, observing a significant increase of telomere segments less than 3 kb in both term placentas and chorioamniotic membranes when compared to these same tissues obtained at 18 weeks of gestation. Interestingly, these investigators did not observe a corresponding decrease in mean telomere lengths in their term placental tissues; thereby confirming the limited utility of measuring average telomere lengths to assess changes in short telomeres. While our cohort was limited to the third trimester of pregnancy, our observation that even in the last weeks of human pregnancy, there is a significant increase in VST is consistent with the findings from Lai and colleagues [13]. One important difference between our current study and prior studies, including the one reported by Lai et al., is that the placental tissues in the prior studies were collected after the onset of labor [13, 19], making it difficult to determine if the process of undergoing labor had any effect on telomere lengths. By including only placentas from women who underwent Cesarean delivery prior to the onset of labor in our cohort, we were able to avoid this potentially confounding issue in our analysis.

Several obstetric factors have been shown to accelerate shortening of mean telomere length in human placenta and fetal cord blood, some of which include fetal growth restriction [20-22], maternal smoking [23], maternal air quality exposure [24], and fetal male sex [10]. Our study design did not allow us to evaluate the impact of these factors on VST between preterm and term placenta, because we did not include any growth restricted pregnancies and did not have maternal air quality data. Because of the small number of women in the other categories, our study was also not able to assess the effects of maternal smoking or fetal sex on the placental telomere lengths.

Our study has several strengths. First, this is a novel adaptation of telomere restriction fragment technique to assess the relative quantity of short telomeres in human tissue, previously demonstrated only in mouse tissue. Second, this study included a greater number of samples than most other published studies of telomere length in human placental tissue. Our study is one of the only telomere placental studies that exclusively examined pre-labor samples, allowing us to rule out labor itself as a confounder in regard to the observed telomere alterations.

The limitations of this study include a gestational age clustering of samples close to 36 weeks in the preterm group, as opposed to an even distribution across the third trimester. Our sample collection represents the demographics of our region of practice and may not be generalizable to large US or international populations. Our study did not include additional maternal or fetal tissue types leading to the inability to perform non-gestational tissue control analyses. Finally, our sample collection may have been susceptible to confounding by indication for delivery, which may have been incompletely captured by demographic and clinical data alone.

Conclusions

In conclusion, VSTs are increased in term placentas compared to preterm placentas prior to the onset of labor, suggesting that placental telomeres progressively shorten as pregnancies approach term, i.e., 40 weeks of gestation. Further characterization of the biologic role of short telomeres in the placenta and their relationship with the timing for labor have the potential to reveal fundamental insights about the biologic clock that determines the onset of parturition, along with the abnormal timing events leading to spontaneous preterm birth.

3 May 2022

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,

Khursheed Iqbal, Ph.D

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. 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.

If you are reporting a retrospective study of medical records or archived samples, please ensure that you have discussed whether all data were fully anonymized before you accessed them and/or whether the IRB or ethics committee waived the requirement for informed consent. If patients provided informed written consent to have data from their medical records used in research, please include this information.

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 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.

Reviewer #1: No

Reviewer #2: No

[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.

16 Jun 2022

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.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

30 Jun 2022

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:

5 Jul 2022

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