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Review
Peer-Review Record

The Placenta as a Target of Epigenetic Alterations in Women with Gestational Diabetes Mellitus and Potential Implications for the Offspring

by Dennise Lizárraga and Alejandra García-Gasca *
Reviewer 1: Anonymous
Reviewer 2: Anonymous
Reviewer 3: Anonymous
Submission received: 26 March 2021 / Revised: 23 April 2021 / Accepted: 26 April 2021 / Published: 10 May 2021
(This article belongs to the Collection Epigenetic Mechanisms in Diabetes Research)

Round 1

Reviewer 1 Report

Overview and general recommendation:

The authors provide a clear and concise review covering current research in Gestational Diabetes Mellitus. While there have been recent reviews on this topic, I believe the author’s limit of scope to two epigenetic marks and attention to human studies offers a focus that would be useful to researchers. This review could be further improved with the addition of more perspective from the authors, as well as the inclusion of research covering the analysis of inherited epigenetic alterations in newborns and a short summary of other epigenetic changes beyond miRNA and methylation.

Major concerns

The review title and introduction suggest a major focus on Potential Implications to the offspring but research covering epigenetic changes in the offspring is scarcely cited (line 352-354). More expansion of this in sections 4.2, 4.3 or in the conclusion is suggested to accurately fulfill the aims of the review.

No reference is made to a recent study on the meta-analysis of Seven Pregnancy Cohorts from the Pregnancy and Childhood Epigenetics (PACE) consortium that investigates Epigenome association between maternal DGM and offspring health (Howe CG et al., Diabetes Care, 2020 DOI: 10.2337/dc19-0524). This study, which analyzed a large sample size and adjusted for multiple covariates, is impactful and should be included.

In order to offer greater clarity to the readers, more background on the biological mechanism of genes discussed would be beneficial. The authors give a nice overview on the involvement of TNF-α in insulin sensitivity (line 85). Similarly, more background is suggested for Mecp2 (line 264), LEP (line 211) and NR3C1 (line 228) genes for example.

The authors make several references to “transgenerational inheritance” throughout the review (lines 50, 154, 190 and 349). There is some dispute on the validity of the theory of transgenerational inheritance in humans. Although it has been demonstrated in animal models and plants, the differentiation of epigenetic inheritance from the impact of environmental factors and its occurrence in humans is still unclear. For this reason, it would be helpful and interesting for the authors discuss this uncertainty, either in the discussion or upon mentioning of transgenerational inheritance throughout the text.  

Likewise, the visualization of the impact of environmental factors in Figure 2 (line 150), “Bad eating habits – sedentary lifestyle – stress”, appears to suggest a causative role of these factors in developing chronic diseases, while research suggests more of an increase susceptibility as a result of environment. It might be more accurate for the researchers to rename this to “Environmental factors, e.g. diet, activity, stress” and to mention in the figure description that environmental factors may increase the likelihood of disease development.

Although the authors state clearly that the review focuses on the most researched epigenetic alterations, methylation and miRNA deregulation, it would be beneficial and improve the review if the authors would mention, albeit briefly, other noteworthy alterations (e.g. histone modifications) or up-and-coming epigenetic research (e.g. lncRNA, single cell RNA-seq) to allow the reader a general overview on other aspects of epigenetic changes. The authors might also speculate on whether research on the wider epigenetic field is likely to improve the understanding of GMD and the mechanism of its impact on the offspring.

Minor Concerns

The introduction and abstract would be more attractive if they included incidence (e.g. 1 in 10 pregnancies) and importance of GMD research. The authors do mention the importance of this in line 119. This line may be better placed in the introduction and abstract sections.

Table 1 has the potential to be a useful reference resource for readers. It could be improved by the inclusion of methylation changes as well. The authors might consider changing the organization of the table to lead first with a “Gene column” and follow on with “Epigenetic Changes” (whether methylation or associated miRNA) and continue with other details already included. Another option may simply be to include another table, similar to the miRNA table, for methylation and other epigenetic changes.

Line 114-115 “some pregnant women do not have access to the medical services for detecting diabetes on time, or the diagnostic criteria are not correct” may need clarification. It is unclear to me whether the women do not meet the diagnostic criteria or whether the diagnostic criteria used is itself is incorrect.

In lines 137-138, the statistic given references risk for offspring exposed to maternal diabetes instead of GMD specifically. If possible, the authors could comment on how this increase is broken down for offspring whose who were exposed to GMD during pregnancy. This would improve consistency for the reader.

The authors could expand on lines 179-180 covering the compensation mechanism of alterations in the placenta. This is an interesting theory and they might pose the question on whether epigenetic alterations are likewise compensatory and what pathways particularly would be likely to work in this way?

Further explanation on how CpG methylation modulates gene expression, including the idea of heterochromatin and transcriptional repression would be useful for the reader in better understanding section 4.2 (lines 202-205).

Lines 210-215: the authors could offer some perspective the discrepancy between the two studies investigating epigenetic alterations at the LEP gene. They might similarly comment on whether studies on epigenetic marks may benefit from validation at the gene level due to multi-level and overlapping epigenetic regulation.

Lines 292-293: Inconsistent referral of the sample size of the study (e.g. n=202). The author does not use sample size in other studies discussed. If this is particularly of interest for this study the author should discuss why.

Author Response

The authors would like to thank the reviewers for their constructive comments to this manuscript. We believe their comments and suggestions substantially improved the document. All modifications are highlighted in yellow to facilitate the review.

 

Reviewer 1

Overview and general recommendation:

The authors provide a clear and concise review covering current research in Gestational Diabetes Mellitus. While there have been recent reviews on this topic, I believe the author’s limit of scope to two epigenetic marks and attention to human studies offers a focus that would be useful to researchers. This review could be further improved with the addition of more perspective from the authors, as well as the inclusion of research covering the analysis of inherited epigenetic alterations in newborns and a short summary of other epigenetic changes beyond miRNA and methylation.

- Thank you for your comments. We have addressed all your suggestions and answered one by one.

Major concerns

The review title and introduction suggest a major focus on Potential Implications to the offspring but research covering epigenetic changes in the offspring is scarcely cited (line 352-354). More expansion of this in sections 4.2, 4.3 or in the conclusion is suggested to accurately fulfill the aims of the review.

- Thank you for the recommendation. We added subsection 4.4 entitled “Epigenetic alterations in the offspring and potential alterations”

 

No reference is made to a recent study on the meta-analysis of Seven Pregnancy Cohorts from the Pregnancy and Childhood Epigenetics (PACE) consortium that investigates Epigenome association between maternal DGM and offspring health (Howe CG et al., Diabetes Care, 2020 DOI: 10.2337/dc19-0524). This study, which analyzed a large sample size and adjusted for multiple covariates, is impactful and should be included.

- Reference to this work was included in subsection 4.4

 

In order to offer greater clarity to the readers, more background on the biological mechanism of genes discussed would be beneficial. The authors give a nice overview on the involvement of TNF-α in insulin sensitivity (line 85). Similarly, more background is suggested for Mecp2 (line 264), LEP (line 211) and NR3C1 (line 228) genes for example.

- Gene information was included for all genes mentioned in the document. Thank you.

 

The authors make several references to “transgenerational inheritance” throughout the review (lines 50, 154, 190 and 349). There is some dispute on the validity of the theory of transgenerational inheritance in humans. Although it has been demonstrated in animal models and plants, the differentiation of epigenetic inheritance from the impact of environmental factors and its occurrence in humans is still unclear. For this reason, it would be helpful and interesting for the authors discuss this uncertainty, either in the discussion or upon mentioning of transgenerational inheritance throughout the text.  

- The reviewer is right. We have modified the text in the Introduction section accordingly: “Some theories have been proposed to explain how an individual could acquire these epigenetic marks, including 1) direct exposure to the environment throughout life, 2) events occurring during gestation affecting the fetus, and 3) transgenerational inheritance from the parental germline [17]. These modifications are not permanent but could persist for generations [18] as observed in plants and animal models. Whether transgenerational inheritance of epigenetic marks occurs in humans, and how it differentiates from the impact of the environment and the lifestyle remains unclear and requires further investigation [19].”

 

Likewise, the visualization of the impact of environmental factors in Figure 2 (line 150), “Bad eating habits – sedentary lifestyle – stress”, appears to suggest a causative role of these factors in developing chronic diseases, while research suggests more of an increase susceptibility as a result of environment. It might be more accurate for the researchers to rename this to “Environmental factors, e.g. diet, activity, stress” and to mention in the figure description that environmental factors may increase the likelihood of disease development.

- We modified the legend of Figure 2 and the figure according to the reviewer’s observations: “GDM and obesity are chronic diseases often present in pregnant women. These may induce an aberrant epigenetic flow, triggering pregnancy complications and postnatal metabolic disorders for the baby, such as macrosomia, hypoglycemia, and hyperinsulinemia. Environmental factors (such as diet, activity, or stress) may increase the incidence of type 2 diabetes mellitus (T2DM), obesity, cardiovascular diseases (CVD), and metabolic syndrome; they may also promote the inheritance of aberrant epigenetic marks.”

 

Although the authors state clearly that the review focuses on the most researched epigenetic alterations, methylation and miRNA deregulation, it would be beneficial and improve the review if the authors would mention, albeit briefly, other noteworthy alterations (e.g. histone modifications) or up-and-coming epigenetic research (e.g. lncRNA, single cell RNA-seq) to allow the reader a general overview on other aspects of epigenetic changes. The authors might also speculate on whether research on the wider epigenetic field is likely to improve the understanding of GMD and the mechanism of its impact on the offspring.

- This information was added to subsection 4.1 “These stable changes include several mechanisms such as DNA and RNA methylation, chromatin remodeling, histone modifications, and the expression of non-coding RNAs (ncRNAs). The placenta presents its own epigenetic programing during gestation “ and subsection 4.3: “Investigations regarding ncRNAs (other than miRNAs) in the placenta under GDM conditions are scarce. The whole transcriptome of the maternal surface of the placenta was recently analyzed by high-throughput sequencing [93]. The work shows that the placentas from women with GDM present 290 differentially expressed ncRNAs compared to the control group. Up-regulated ncRNAs under GDM conditions were composed of 2 miRNAs, 86 long non-coding RNAs (lncRNAs), and 55 circular RNAs (circRNAs), while down-regulated ncRNAs included other 2 miRNAs, 86 lncRNAs, and 59 circRNAs. This reveals a differential transcriptomic profile in the placenta under GDM conditions compared with normoglycemic conditions. circRNA expression profiles of GDM have also been sequenced in the placenta. Yan et al [94] identified a total of 482 circRNAs differentially expressed in the placental villi of GDM women, of which 227 were up-regulated and 255 were down-regulated. With the aid of Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes, the authors determined that these circRNAs were related to glucose and lipid metabolism. The characterization of histone modifications and emerging epigenomic research including RNA methylation [95] and single-cell RNA sequencing [96] in the placenta are needed to further understand GDM and its impacts on the offspring.”

 

Minor Concerns

The introduction and abstract would be more attractive if they included incidence (e.g. 1 in 10 pregnancies) and importance of GMD research. The authors do mention the importance of this in line 119. This line may be better placed in the introduction and abstract sections.

- We placed the required information in the Abstract and Introduction: “Gestational Diabetes Mellitus (GDM) is a worldwide health concern affecting pregnant women and their offspring. In 2019, this pregnancy complication affected 17 million newborns globally [1]. The Diabetes Atlas published in 2019 by the International Diabetes Federation [1] reports that 15.8% of live births in 2019 were affected by hyperglycemia during pregnancy, from which 83.6% were due to gestational diabetes mellitus, 8.5% were due to diabetes first detected in pregnancy, and 7.9% were due to diabetes detected before pregnancy, increasing susceptibility of developing chronic diseases in their adult life [2].”

 

Table 1 has the potential to be a useful reference resource for readers. It could be improved by the inclusion of methylation changes as well. The authors might consider changing the organization of the table to lead first with a “Gene column” and follow on with “Epigenetic Changes” (whether methylation or associated miRNA) and continue with other details already included. Another option may simply be to include another table, similar to the miRNA table, for methylation and other epigenetic changes.

-We added the information regarding epigenetic changes in the supplementary file, in a separated sheet named “Placental DNA methylation”.

 

Line 114-115 “some pregnant women do not have access to the medical services for detecting diabetes on time, or the diagnostic criteria are not correct” may need clarification. It is unclear to me whether the women do not meet the diagnostic criteria or whether the diagnostic criteria used is itself is incorrect.

-The paragraph in Section 3 has been rephrased: “some pregnant women do not have access to medical services for detecting diabetes on time, or the criteria used to diagnose the pathology is not appropriate.”

 

In lines 137-138, the statistic given references risk for offspring exposed to maternal diabetes instead of GMD specifically. If possible, the authors could comment on how this increase is broken down for offspring whose who were exposed to GMD during pregnancy. This would improve consistency for the reader.

- Also in Section 3, we included the information regarding GDM only: “Specifically, in the gestational diabetes group, the incidence of cardiovascular disease in the offspring was 1.60 per 1000 person-years.”

 

The authors could expand on lines 179-180 covering the compensation mechanism of alterations in the placenta. This is an interesting theory and they might pose the question on whether epigenetic alterations are likewise compensatory and what pathways particularly would be likely to work in this way?

- In Section 4, we added some information regarding the role of the placenta in ensuring fetal survival by adapting to intrinsic or extrinsic factors, and how in GDM, alterations in the placenta might be interpreted as adaptations to protect the fetus from an aberrant environment, impacting nutrient and molecule transport that may result in neonate complications and diseases later in life. We also added information regarding the three-hit hypothesis at the end of section 4 which may explain (at least in part) the complexity of epigenetic modifications due to genetic and environmental factors.

 

Further explanation on how CpG methylation modulates gene expression, including the idea of heterochromatin and transcriptional repression would be useful for the reader in better understanding section 4.2 (lines 202-205).

- This information was included in subsection 4.2 as suggested: “DNAm occurring in regulatory sequences is associated with chromatin structure; hypermethylation of regulatory elements causes gene silencing through blocking transcription factor-binding sites, or recruiting proteins with a methyl-CpG binding domain (MBD) and histone modification enzymes, causing a tightly packed chromatin conformation (heterochromatin); however, when the regulatory elements are hypomethylated, the chromatin relaxes (euchromatin) enabling gene expression”

 

Lines 210-215: the authors could offer some perspective the discrepancy between the two studies investigating epigenetic alterations at the LEP gene. They might similarly comment on whether studies on epigenetic marks may benefit from validation at the gene level due to multi-level and overlapping epigenetic regulation.

- This information was also included in subsection 4.2:” Leptin regulates energy balance suppressing food intake, meaning that a decrease in LEP expression would favor obesity [57]; however, the authors [56] did not evaluate the expression of the LEP gene, which is important to properly interpret the results, since gene expression might be regulated by different epigenetic mechanisms. Hence, there is still controversy on placental leptin production, since other authors observed that the expression of leptin and its receptor increased in placentas of women with GDM compared with placentas of women without GDM [58]. Despite the controversy, placental leptin plays an important role in modulating implantation, placentation, and cell proliferation, as well as the mobilization of maternal fat”

 

Lines 292-293: Inconsistent referral of the sample size of the study (e.g. n=202). The author does not use sample size in other studies discussed. If this is particularly of interest for this study the author should discuss why.

- The sample size was eliminated to keep consistency.

Reviewer 2 Report

The review article of Lizárraga and García-Gasca focusses on the role that Gestational Diabetes Mellitus plays increasing both the mother and child’s susceptibility to develop chronic diseases later in life. The authors examine the role of the hyperglycaemic intrauterine environment, fatty acid, pro-inflammatory cytokines, as well as the functional (morphological, structural, and molecular) modifications that GDM induces in the placenta, The authors review molecular (epigenetic) alterations including DNA and RNA methylation, chromatin remodelling and histone modifications. Unfortunately, the authors include the expression of non-coding RNAs (ncRNAs) that are not genuine epigenetic mechanisms, but post-transcriptional mechanisms. Overall, the authors can be proud of a well-written, clear, concise, well-illustrated manuscript. There are many minor points that need addressing that will improve the manuscript.

 

 

  1. « exposition » is not the correct word. It should be exposure.
  2. Please explain what you mean by epigenetic flow.
  3. Text that can be included to make the manuscript better….Maternal IR ensures utilisation of more fats compared to carbohydrates to provide the mother with energy so that the foetus can use the available carbohydrate. This feature (IR) during pregnancy ensures a ready supply of energy to the foetus. (PMID: 25584208, 10102701)
  4. Line 71-72 -> What is meant by “correct” rise of glucose….
  5. Line 112-113 (If pregnant women…first detected in pregnancy) -> this line is not very clear (can be worded better).
  6. Line 110-111 -> …diagnosed in the second or third trimester of pregnancy “in women who were not diabetic prior to pregnancy”… please add highlighted text
  7. Line 126-127 ->women have a higher…preterm birth during GDM (and not pregnancy) [3] – please modify and make clearer
  8. For lines 128-130 -> [36] -> Please mention that the limitation of this study was that they were unable to distinguish between GDM and pre-gestational diabetes.
  9. Line 184 would be a good place to elaborate a little on the 3 hit hypothesis and mention that Foetus exposed to GDM have already been subjected to hit 2.
  10. Line 213 -> Please mention that LEP induces reduction in food intake and thus energy consumption, meaning that a decrease in LEP will tip off the energy balance in favour of obesity (a risk factor).
  11. Line 217 mentions LPL which is responsible for clearance of lipid from the blood…in GDM cases there is an overexpression of LPL …what is the biological significance of this?
  12. Please explain the functions of all the genes mentioned (such as Mecp2, PAK1, ).
  13. Line 232-233 -> Please mention that the status of ALU repetitive region methylation acts a proxy for whole genome methylation and changes in ALU for GDM cases is thus indicative of alterations genome-wide.
  14. Line 313-314 -> please provide citations for these pathways.
  15. It is worth mentioning a small paragraph with histone/chromatin modifications happening during GDM as PTMs are also epigenetic alterations. – at the same time, the authors much acknowledge that ncRNA are post-transcriptional regulators, not epigenetic changes7regulation per se.
  16. In GDM cases, the placenta undergoes morphological changes. Thus some of the epigenetic changes might be a result of the changed placental morphology and may have nothing to do with diabetes and this is difficult to dissect. This needs tio be highlighted, and the authors need to mention that it would be worth investigating cases where diabetic women have had children by surrogacy to see and compare the results (this has a lot of confounding factors)..or to perform embryo transfer experiments in animal models of GDM to see (also transferring a normal embryo to a GDM mother) which genes flag up. Similarly, what does the literature say about epigenetic changes in twin pregnancies? They usually have two independent placentas, but the same environment. This should narrow dawn the epigenetic changes to those that are genuinely induced by GBM. If there is no literature, this needs to be given as a strong potential research direction/perspective.

Author Response

The authors would like to thank the reviewers for their constructive comments to this manuscript. We believe their comments and suggestions substantially improved the document. All modifications are highlighted in yellow to facilitate the review.

Reviewer 2

The review article of Lizárraga and García-Gasca focuses on the role that Gestational Diabetes Mellitus plays increasing both the mother and child’s susceptibility to develop chronic diseases later in life. The authors examine the role of the hyperglycaemic intrauterine environment, fatty acid, pro-inflammatory cytokines, as well as the functional (morphological, structural, and molecular) modifications that GDM induces in the placenta. The authors review molecular (epigenetic) alterations including DNA and RNA methylation, chromatin remodelling and histone modifications. Unfortunately, the authors include the expression of non-coding RNAs (ncRNAs) that are not genuine epigenetic mechanisms, but post-transcriptional mechanisms. Overall, the authors can be proud of a well-written, clear, concise, well-illustrated manuscript. There are many minor points that need addressing that will improve the manuscript.

- Thank you for the constructive comments. We have addressed all the observations and differentiated ncRNAs from epigenetic mechanisms in subsection 4.3 as suggested.

 

 « exposition » is not the correct word. It should be exposure.

- “exposition” was replaced by “exposure”. Thank you.

 

Please explain what you mean by epigenetic flow.

- We defined epigenetic flow in the last paragraph of the Introduction: “all epigenetic alterations induced by the mother’s condition, reaching the fetus through the placenta.”

 

Text that can be included to make the manuscript better….Maternal IR ensures utilisation of more fats compared to carbohydrates to provide the mother with energy so that the foetus can use the available carbohydrate. This feature (IR) during pregnancy ensures a ready supply of energy to the foetus. (PMID: 25584208, 10102701)

- The information was added in Section 2: “Maternal insulin resistance (IR) ensures the utilization of fats rather than carbohydrates to provide the mother with energy and, at the same time, make carbohydrates available to the fetus. This feature during pregnancy ensures a ready supply of energy to the fetus”

 

Line 71-72 -> What is meant by “correct” rise of glucose….

- The paragraph was replaced by the information suggested above.

 

Line 112-113 (If pregnant women…first detected in pregnancy) -> this line is not very clear (can be worded better).

- The phrase was deleted.

 

Line 110-111 -> …diagnosed in the second or third trimester of pregnancy “in women who were not diabetic prior to pregnancy”… please add highlighted text

- The phrase was modified as suggested.

 

Line 126-127 ->women have a higher…preterm birth during GDM (and not pregnancy) [3] – please modify and make clearer

- The phrase was modified as suggested in section 3.

 

For lines 128-130 -> [36] -> Please mention that the limitation of this study was that they were unable to distinguish between GDM and pre-gestational diabetes.

- We mentioned this limitation in Section 3: “…however, one limitation of this study is that it did not distinguish pregestational diabetes from gestational diabetes.”

 

Line 184 would be a good place to elaborate a little on the 3 hit hypothesis and mention that Foetus exposed to GDM have already been subjected to hit 2.

- We included the three-hit hypothesis at the end of Section 4 as suggested: “Some investigations suggest that this programming occurs due to epigenetic changes during gestation and early postnatal life [15]. In this context, the “three-hit” hypothesis explains the predisposition to certain phenotypes due to the [sequential] interaction of genetic and environmental factors [52]. Briefly, this hypothesis mentions that the genetic predisposition (hit-1) interacts with the early-life environment (hit-2, which may include epigenetic modifications during gestation), programming the fetus to a certain phenotype, followed by exposure to environmental factors later in life (hit-3). Thus, under GDM conditions, the fetus has already been subjected to hit-2, and the phenotype will vary depending on the environmental factors to which the individual is exposed throughout life (hit-3).”

 

Line 213 -> Please mention that LEP induces reduction in food intake and thus energy consumption, meaning that a decrease in LEP will tip off the energy balance in favour of obesity (a risk factor).

- This information was included in subsection 4.2: “Leptin regulates energy balance suppressing food intake, meaning that a decrease in LEP expression would favor obesity [57]; however, the authors [56] did not evaluate the expression of the LEP gene, which is important to properly interpret the results, since gene expression might be regulated by different epigenetic mechanisms.”

 

Line 217 mentions LPL which is responsible for clearance of lipid from the blood…in GDM cases there is an overexpression of LPL …what is the biological significance of this?

Please explain the functions of all the genes mentioned (such as Mecp2, PAK1, ).

- We have explained the biological significance of LPL in subsection 4.2, as well as all genes/proteins mentioned in the document: “LPL is an extracellular enzyme whose key function is triglyceride hydrolysis into fatty acids in the bloodstream [61], thus increased LPL expression levels in the placenta may indicate a major supply of fatty acids to the fetus.”

 

Line 232-233 -> Please mention that the status of ALU repetitive region methylation acts a proxy for whole genome methylation and changes in ALU for GDM cases is thus indicative of alterations genome-wide.

- The information was added in subsection 4.2: “Finally, lower DNA methylation in ALU repeats may indicate genome-wide alterations under GDM conditions. ALU repeats are transposable elements and hypomethylation is associated with epigenome instability and disease”

 

Line 313-314 -> please provide citations for these pathways.

- Citations were provided.

 

It is worth mentioning a small paragraph with histone/chromatin modifications happening during GDM as PTMs are also epigenetic alterations. – at the same time, the authors much acknowledge that ncRNA are post-transcriptional regulators, not epigenetic changes7regulation per se.

- We added the information at the beginning of subsection 4.3: “While epigenetic mechanisms involve modifications in DNA and RNA (methylation), and/or histones (methylation, acetylation or other post-translational modifications) to regulate gene expression, ncRNAs are involved in gene regulation at the transcriptional and post-transcriptional levels, and may not be considered epigenetic regulators per se, but they do present altered expression profiles in GDM...”

 

In GDM cases, the placenta undergoes morphological changes. Thus some of the epigenetic changes might be a result of the changed placental morphology and may have nothing to do with diabetes and this is difficult to dissect. This needs to be highlighted, and the authors need to mention that it would be worth investigating cases where diabetic women have had children by surrogacy to see and compare the results (this has a lot of confounding factors)..or to perform embryo transfer experiments in animal models of GDM to see (also transferring a normal embryo to a GDM mother) which genes flag up. Similarly, what does the literature say about epigenetic changes in twin pregnancies? They usually have two independent placentas, but the same environment. This should narrow dawn the epigenetic changes to those that are genuinely induced by GBM. If there is no literature, this needs to be given as a strong potential research direction/perspective.

- Thank you for the observations. We added this information in Section 5 (Concluding remarks): “These approaches also carry some limitations such as epigenetic changes that may be due to alterations in placental or cellular morphology or culture conditions and not to GDM itself. In addition, genetic predisposition may influence the epigenetic signature; thus, it would be interesting to investigate cases where diabetic women are pregnant by surrogacy, as well as the epigenetic alterations in placentas from twins, to investigate individual changes occurring in the same environment. These and other investigations will help identify those epigenetic modifications in the placenta that are genuinely induced by GDM.”

Reviewer 3 Report

In this review paper, the authors summarized and discussed recent findings in epigenetic alterations in human placenta under GDM conditions and its potential implications to the offspring. The review is well organized and the quality of figures are good. The included information is helpful for researchers of the field of epigenetics and diabetes.

 

I have minor comments.

 

-In Fig 1. (3) “Glycogen synthesis”, the direction of arrow seems to be reversed.

-English should be checked carefully. 

-Line222. promotor->promoter?

Author Response

The authors would like to thank the reviewers for their constructive comments to this manuscript. We believe their comments and suggestions substantially improved the document. All modifications are highlighted in yellow to facilitate the review.

Reviewer 3

In this review paper, the authors summarized and discussed recent findings in epigenetic alterations in human placenta under GDM conditions and its potential implications to the offspring. The review is well organized and the quality of figures are good. The included information is helpful for researchers of the field of epigenetics and diabetes.

 I have minor comments.

 -In Fig 1. (3) “Glycogen synthesis”, the direction of arrow seems to be reversed.

- The reviewer is right. The direction of the arrow was corrected. Thank you.

 

-English should be checked carefully. 

- English was carefully revised, thank you.

 

-Line222. promotor->promoter?

- “promotor” was replaced by “promoter”. Thank you.

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