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Date: Dec 18, 2020 To: "Katrien Janssens"
From: "The Green Journal" [email protected] Subject: Your Submission ONG-20-3012
RE: Manuscript Number ONG-20-3012
Performance and diagnostic value of genome wide non-invasive prenatal testing in twin pregnancies Dear Dr. Janssens:
Your manuscript has been reviewed by the Editorial Board and by special expert referees. Although it is judged not acceptable for publication in Obstetrics & Gynecology in its present form, we would be willing to give further consideration to a revised version.
If you wish to consider revising your manuscript, you will first need to study carefully the enclosed reports submitted by the referees and editors. Each point raised requires a response, by either revising your manuscript or making a clear and convincing argument as to why no revision is needed. To facilitate our review, we prefer that the cover letter include the comments made by the reviewers and the editor followed by your response. The revised manuscript should indicate the position of all changes made. We suggest that you use the "track changes" feature in your word processing software to do so (rather than strikethrough or underline formatting).
Your paper will be maintained in active status for 21 days from the date of this letter. If we have not heard from you by Jan 08, 2021, we will assume you wish to withdraw the manuscript from further consideration.
REVIEWER COMMENTS:
Reviewer #1:
1. The abstract is clear and concise. It should also include the results of the vanishing twin data. NIPS (S=screening) preferred, rather than NIPT.
2. The introduction gives a good explanation of the issues associated with analyzing NIPS in twins. Would not say performance in twins poorly documented (lines 67-68). Instead could state that detailed information is limited for twin gestations.
3. The materials and methods section provides a good description of the study procedures. The gestational age range of initial and repeat testing would be of interest, if this data is available.
4. The results are provided in detail. The variation in failure rate between centers (lines 133-46) is attributed to low fetal fraction and/or poor quality. Was maternal weight or gestational age a factor? Were the placentas non mosaic in cultures cells in the two unconfirmed amniocentesis cases (line 151)?
5. The discussion notes that published studies have had limited twin data sets(lines 207-10), but recent large studies have been published, although not always in English (e.g. Yin et al Zhejiang Da Xue Xue Bao Yi Xue Ban 2019 48:403, 2057 twin pregnancies). Thus better to state "one of largest twin datasets". The discussion is appropriately detailed.
6. The references are correct.
7. Table 1's contents are all included in the paper, and so this table can be omitted. Table 2 provides good summary of data. Figure 1 is well discussed within the paper. Organizing the data into a bar chart does not add additional clarity, and thus this can be omitted.
8. The supplementary table is appropriate.
Reviewer #2:
The authors intent to provide more information on the performance of non-invasive prenatal testing is commendable. Non- invasive techniques that can be used early are always welcome in the management of pregnancies. A strength of this study is the large size of the samples available for analysis that covered a relatively long time period. As expected, large
numbers of each of the types of pregnancies of interest were identified.
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In the cases of vanishing twins, was there a correlation between hCG levels and the results of the non-invasive prenatal testing?
As a means of demonstrating the utility of non-invasive prenatal testing in multiple pregnancies, this study achieved its objective. However, the authors do not dwell in depth about the clinical relevance of their findings. They did not confirm any trisomies in the evolutive twins besides Down' syndrome, Edward's syndrome and Patau syndrome. All other trisomies were supposedly in the lost twins. It would have been valuable to describe what management protocol was used in these pregnancies. Were any particular measures taken to ensure that any future health risks for the evolutive twin were addressed. Was there genetic counselling for the parents?
In today's world, it is no longer enough to report methodological quality data, it is important to put the results in the context of every day clinical practice. Busy obstetricians want the full picture regarding the use and usefulness of any diagnostic technique, not just its intrinsic methodological quality.
Methodological questions
What was the rational for not collecting samples from pregnancies under 10 weeks?
Why did the authors choose to exclude gonosomal aneuploidies from the reporting?
Other comments:
Line 133 - should read "…detected among centers,…"
Reviewer #3:
The presented manuscript is analysis of NIPT data focusing on multiple gestations from eight different Belgian genetic centers from 2013-2020. As NIPT testing becomes even more common, it's application in different pregnancy types needs to be further evaluated, so this dataset is valuable. I'd like to see some better clarity throughout the manuscript regarding the outcomes for the different types of multiple gestation.
1. Title: Consider changing to "…in Multiple Gestations" as data on more than just twin pregnancies is included.
2. Abstract: Line 56: Data on vanishing twins was still analyzed, though this sentence makes it sound like they were excluded. Therefore, recommend changing to "…and 756 vanishing twin samples".
3. Introduction: Consider adding in some information about the different types of NIPT, specifically focusing on whole genome analysis.
4. Methods: Lines 98-101: At what gestational age were these ultrasounds performed? Lines 116-117: please give more information on types of pregnancy outcomes that would lend concern for false negative results and how accurate the clinical databases are for documenting these.
5. Results: I'd like to see the results for the different types of multiple gestation presented a bit more clearly. Were vanishing twins included or excluded in the analysis (line 126 vs. line 132)? Were the 525 lacking information on
chorionicity or amionicity included or excluded (line 129)? For the reported failure rates, it would be nice to see this broken down more specifically between the singleton gestations and the different types of multiple gestation. Was it higher in the triplet or quadruplet gestations? For those centers with the higher failure rates, were they overall testing at an earlier gestational age (lines 133-134)? Were the testing platforms similar between the two centers with 0% failure rate?
6. Discussion: Line 210: consider adding ranges of incidences reported in singleton pregnancies for more direct reader comparison. I would like to see more of a discussion of the limitations of the current study and data set.
7. References: The ACOG Committee Opinion #640 has been withdrawn. Consider utilizing the Practice Bulletin #226 from October 2020 "Screening for Fetal Chromosomal Abnormalities".
8. Figure: Please add a title for this figure and label the X- and Y-axes.
STATISTICS EDITOR COMMENTS:
Table 2: As seen by the CIs and due to the small counts of T 21, T18 or T13, The CIs for sensitivity and PPV are very wide.
Since the PPV and NPV are dependent on the prevalence of the conditions, it would be preferable to cite the LR(+) and LR(-), rather than PPV or NPV, since the former are independent of prevalence.
General: Although it is true that the diagnostic test performances shown here are similar to that for singletons, the actual trisomy counts are small and there is insufficient statistical power to generalize that the test performance is the same for
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singletons and twins. It would be prudent to simply state the test performances with CIs.
At multiple places in text, the sensitivity, specificity etc are cited without CIs. This is incomplete and requires CIs for context, since some of the estimates have quite wide CIs.
EDITOR COMMENTS: Thank you for submitting your work to Obstetrics and Gynecology. If you choose to submit a revision, the editors request that you remove all of the information about singletons. The value of this manuscript is in the examination of twin gestations specifically, and as noted by the statistical editor, there is an insufficient number of
participants with a diagnosis of aneuploidy to make comparisons between singletons and twins.
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We thank the Editor and Referees for their careful evaluation of our work and the comments provided. We hereby present a revised version of our manuscript ‘Performance and diagnostic value of genome wide non-invasive prenatal testing in twin pregnancies’. We sincerely hope that in its current form, our manuscript is acceptable for publication in Obstetrics &
Gynecology.
Please note that during the revision of the paper, a few small data inaccuracies were encountered; these were corrected in the revision, but do not affect in any way the overall results or conclusions of the study.
We have read the ‘Instructions for authors’, have filled out the STARD checklist and have made sure that the manuscript meets all requirements of Obstetrics & Gynecology.
REVIEWER COMMENTS:
Reviewer #1:
1. The abstract is clear and concise. It should also include the results of the vanishing twin data. NIPS (S=screening) preferred, rather than NIPT.
The data on vanishing twins was added to the abstract (line 63-64; please note that the line numbers referred to throughout this letter correspond to the lines in the manuscript with the removed text hidden). We opted to use NIPT instead of NIPS as it is a standard and accepted abbreviation listed by the Journal.
2. The introduction gives a good explanation of the issues associated with analyzing NIPS in twins. Would not say performance in twins poorly documented (lines 67-68). Instead could state that detailed information is limited for twin gestations.
This was adapted accordingly (line 73-74).
3. The materials and methods section provides a good description of the study
procedures. The gestational age range of initial and repeat testing would be of interest, if this data is available.
Data on gestational age of initial and repeat testing was not collected upon initial submission.
Before July 2017, a gestational age of ten weeks was the earliest time for patients to receive NIPT, as to minimize the failures due to low fetal fraction. From July 2017 onwards, NIPT is reimbursed in Belgium from 12 weeks of gestation. In response to the reviewer’s request, we asked the participating genetic centers to provide us with the data on gestational age upon initial and repeat testing. We obtained data for 3.057 cases (73.7% of our cohort of all multiple gestations, excluding vanishing twins); the weighted mean of the gestational age upon initial testing was 13.7 weeks. Gestational age data for repeat testing was available for 91 samples, with a weighted mean of 15.9 weeks.
4. The results are provided in detail. The variation in failure rate between centers (lines 133-46) is attributed to low fetal fraction and/or poor quality. Was maternal weight or gestational age a factor?
High maternal weight is not a contraindication for NIPT in Belgium. Some cases are indeed classified under ‘low fetal fraction’, but no systematic correlation between fetal fraction and maternal weight has been detected.
To investigate the effect of gestational age on test failure, we collected these data when available. The weighted mean gestational age of 2.948 successful initial NIPT analyses was
13.7 weeks. For 109 failed tests, the weighted mean gestational age was 13.3 weeks. This difference could indicate a small effect of gestational age on the success rate. However, the low number of failed analyses prevents drawing solid conclusions.
Were the placentas non mosaic in cultured cells in the two unconfirmed amniocentesis cases (line 151)?
Upon reviewing the clinical files of these two cases, we noticed two inconsistencies in our initial data file.
For the trisomy 21 case, no amniocentesis had been performed, but the combination of trisomy 21 in the chorionic villi and an increased nuchal translucency in one of the fetuses provided sufficient evidence to perform selective feticide. Consequently, this case was reconsidered to be a true positive instead of a false positive.
The trisomy 13 case that was unconfirmed by amniocentesis did not have placental follow-up available. Therefore, it is unknown whether the trisomy was present in the placenta.
We apologize for these errors and have revised the manuscript accordingly (line 153-156).
5. The discussion notes that published studies have had limited twin data sets (lines 207- 10), but recent large studies have been published, although not always in English (e.g. Yin et al Zhejiang Da Xue Xue Bao Yi Xue Ban 2019 48:403, 2057 twin pregnancies). Thus better to state "one of largest twin datasets".
This was adapted accordingly (line 212 and line 225).
The discussion is appropriately detailed.
6. The references are correct.
7. Table 1's contents are all included in the paper, and so this table can be omitted. Table 2 provides good summary of data. Figure 1 is well discussed within the paper. Organizing the data into a bar chart does not add additional clarity, and thus this can be omitted.
We moved Table 1 to the Appendix (Appendix 2), as it provides a clear overview of the study population. We respectfully disagree with the reviewer’s point of view on Figure 1, as we feel it allows the reader to go straight to the essence of our results. However, for readability in print, we left out the chromosomes with zero cases.
8. The supplementary table is appropriate.
Reviewer #2:
The authors intent to provide more information on the performance of non-invasive prenatal testing is commendable. Non-invasive techniques that can be used early are always welcome in the management of pregnancies. A strength of this study is the large size of the samples available for analysis that covered a relatively long time period. As expected, large numbers of each of the types of pregnancies of interest were identified.
In the cases of vanishing twins, was there a correlation between hCG levels and the results of the non-invasive prenatal testing?
In Belgium, hCG levels are not routinely determined in pregnant women. Therefore, data on hCG levels are not available.
As a means of demonstrating the utility of non-invasive prenatal testing in multiple pregnancies, this study achieved its objective. However, the authors do not dwell in depth about the clinical relevance of their findings. They did not confirm any trisomies in the evolutive twins besides Down' syndrome, Edward's syndrome and Patau syndrome. All other trisomies were supposedly in the lost twins. It would have been valuable to describe what management protocol was used in these pregnancies. Were any particular measures taken to ensure that any future health risks for the evolutive twin were addressed. Was there genetic counselling for the parents?
We demonstrate the added value of performing NIPT in twin gestations for the detection of trisomy 13, 18 and 21, as screening based on ultrasound and maternal age has an inferior sensitivity and specificity.
Despite the inability to confirm any of the rare autosomal trisomies, the suspected association with an adverse pregnancy outcome warrants extensive follow-up by ultrasound.
In Belgium, pregnancies with a vanishing twin receive the same follow-up as singleton pregnancies in case of a normal NIPT result; no additional genetic counseling apart from the pretest counseling is provided. In case of an abnormal NIPT result, parents do receive posttest genetic counseling explaining that the aberrant NIPT result is presumably derived from the vanishing twin or might be attributable to confined placental mosaicism, but that it cannot be excluded that it is present in the evolutive twin, and invasive testing is offered. In case NIPT detects an autosomal trisomy and amniocentesis is negative, fetal growth and development are monitored closely throughout the remainder of the pregnancy (line 232-238).
In today's world, it is no longer enough to report methodological quality data, it is important to put the results in the context of every day clinical practice. Busy obstetricians want the full picture regarding the use and usefulness of any diagnostic technique, not just its intrinsic methodological quality.
We emphasized the added value of NIPT in multiple gestations for pregnancy management, given the limitations of other screening methods that are currently performed (line 81-84 and 202-208).
Methodological questions
What was the rational for not collecting samples from pregnancies under 10 weeks?
With the implementation of NIPT in Belgium by the genetic centers, 10 weeks was set as the minimal gestational age to perform NIPT to ensure sufficient cell-free fetal DNA would be present in the maternal bloodstream and hence limiting NIPT failures due to low fetal fraction.
As of July 2017, the start date of NIPT reimbursement in Belgium for the entire pregnant population, the minimal gestational age for NIPT has been set at 12 weeks by the Belgian government.
Why did the authors choose to exclude gonosomal aneuploidies from the reporting?
Currently there is no national consensus on the reporting of sex chromosome aneuploidies in Belgium.
Other comments:
Line 133 - should read "…detected among centers,…"
This was adapted accordingly (line 141).
Reviewer #3:
The presented manuscript is analysis of NIPT data focusing on multiple gestations from eight different Belgian genetic centers from 2013-2020. As NIPT testing becomes even more common, its application in different pregnancy types needs to be further evaluated, so this dataset is valuable. I'd like to see some better clarity throughout the manuscript regarding the outcomes for the different types of multiple gestation.
1. Title: Consider changing to "…in Multiple Gestations" as data on more than just twin pregnancies is included.
We respectfully disagree as the number of triplets in our dataset is limited (n=25) and no positive cases were included (line 253-257), which makes it impossible to draw any conclusions on the performance and diagnostic value of NIPT in triplets and higher-order multiples.
2. Abstract: Line 56: Data on vanishing twins was still analyzed, though this sentence makes it sound like they were excluded. Therefore, recommend changing to "…and 756 vanishing twin samples".
This was adapted accordingly (line 56-57).
3. Introduction: Consider adding in some information about the different types of NIPT, specifically focusing on whole genome analysis.
As the introduction cannot exceed 250 words, we chose not to elaborate on the different types of NIPT.
4. Methods: Lines 98-101: At what gestational age were these ultrasounds performed?
The ultrasound to determine chorionicity and amnionicity was determined between six and fourteen weeks of gestation, depending on the hospital. The monitoring for a vanishing twin does not take place at a fixed time point. At first ultrasound examination, the number of embryos is noted; in case a vanishing twin is present, this will be noted as well.
Lines 116-117: please give more information on types of pregnancy outcomes that would lend concern for false negative results and how accurate the clinical databases are for documenting these.
Cases are classified as false negative when invasive prenatal testing or postnatal testing reveals the presence of a common autosomal aneuploidy that was not detected by NIPT. In our study, this was the case for one DCDA twin: NIPT was normal, but developmental anomalies detected through ultrasound warranted an amniocentesis that demonstrated the presence of a trisomy 18 in one fetus. Since all prenatal and neonatal cytogenetic testing in Belgium is performed in one of the genetic centers involved in this study, it is very unlikely that a false negative case is missed. This information was added to the manuscript (line 124- 126).
5. Results: I'd like to see the results for the different types of multiple gestation presented a bit more clearly.
We are somewhat puzzled by the reviewer’s remark that the outcomes of the different type of multiple gestations are not presented in a clear manner. The manuscript focuses on the population of DCDA twins for 2 main reasons: 1) this is the most challenging subgroup of twins, due to the two placentas that contribute to the cell-free DNA fraction; 2) this is our largest
‘subcohort’, allowing for reliable calculation of specificity and sensitivity. We also discuss the results of our vanishing twin subcohort, which is also one of the largest reported to date, but as most of the demised twins could not be tested, test performance cannot be calculated reliably and therefore was not described in detail. The other subcohorts of MCDA twins, MCMA twins and triplets are too small to reliably determine NIPT performance. We tried different formats to depict the results in Figure 1, but the current format seemed the best option to depict twin category, number of cases, affected chromosome and outcome all together.
Were vanishing twins included or excluded in the analysis (line 126 vs. line 132)?
Vanishing twins were included in the analysis but considered as a separate category from the other multiple gestations throughout the whole paper. This section was revised in the paper for clarification (line 133-134).
Were the 525 lacking information on chorionicity or amionicity included or excluded (line 129)?
These 525 cases were included in the analysis.
For the reported failure rates, it would be nice to see this broken down more specifically between the singleton gestations and the different types of multiple gestation. Was it higher in the triplet or quadruplet gestations? For those centers with the higher failure rates, were they overall testing at an earlier gestational age (lines 133-134)?
Unfortunately, data on the failure rate for the different subtypes of multiple gestations were not available. Gestational age (GA) data on all multiple gestations, excluding vanishing twins, was available in 4 genetic centers, as presented in the table below.
CENTER Failure rate Average GA successful NIPT in
weeks
Average GA failed NIPT in weeks
1 2.84% 13.5 12.8
2 0% 12.9 Not applicable
3 5.88% 14.2 13.6
4 4.67% 13.7 14.2
In summary, the weighted mean gestational age of 2.948 successful initial NIPT analyses was 13.7 weeks, while for 109 initial failed tests, it was 13.3 weeks. This difference could indicate a small effect of gestational age on the success rate. However, the low number of failed analyses prevents drawing solid conclusions.
Were the testing platforms similar between the two centers with 0% failure rate?
No, different testing platforms were used in these two centers. One of the centers had a limited number of multiple gestations included (26), which might explain the 0% failure rate.
In the other center without failures (center 2 in the table above), the data were reevaluated
to make sure no mistake had been made; this was indeed not the case. In the centers with failures, reasons for failure were low fetal fraction, low quality, or a combination of both. As mentioned earlier, we did notice a higher failure rate in genetic centers that set a threshold for fetal fraction compared to genetic centers that do not. We have no indication of superior performance of a particular testing platform.
6. Discussion: Line 210: consider adding ranges of incidences reported in singleton pregnancies for more direct reader comparison.
The journal editors requested to remove all the information about singletons as the number of aneuploidy cases in multiple gestations is considered too small to ensure sufficient statistical power to compare singleton and multiple gestations.
I would like to see more of a discussion of the limitations of the current study and data set.
This was added to the discussion of the paper (line 261-263).
7. References: The ACOG Committee Opinion #640 has been withdrawn. Consider utilizing the Practice Bulletin #226 from October 2020 "Screening for Fetal Chromosomal Abnormalities".
The reference has been replaced by Practice Bulletin #226.
8. Figure: Please add a title for this figure and label the X- and Y-axes.
Thank you for pointing this out; a title and axis labels were added.
STATISTICS EDITOR COMMENTS:
Table 2: As seen by the CIs and due to the small counts of T 21, T18 or T13, the CIs for sensitivity and PPV are very wide. Since the PPV and NPV are dependent on the prevalence of the conditions, it would be preferable to cite the LR(+) and LR(-), rather than PPV or NPV, since the former are independent of prevalence.
This was adapted accordingly (Table 1).
General: Although it is true that the diagnostic test performances shown here are similar to that for singletons, the actual trisomy counts are small and there is insufficient statistical power to generalize that the test performance is the same for singletons and twins. It would be prudent to simply state the test performances with CIs.
All information on singletons was removed from the manuscript.
At multiple places in text, the sensitivity, specificity etc are cited without CIs. This is incomplete and requires CIs for context, since some of the estimates have quite wide CIs.
CIs were added wherever necessary (line 57-62 and line 203-208).
EDITOR COMMENTS: Thank you for submitting your work to Obstetrics and Gynecology. If you choose to submit a revision, the editors request that you remove all of the information about singletons. The value of this manuscript is in the examination of twin gestations specifically, and as noted by the statistical editor, there is an insufficient number of participants with a diagnosis of aneuploidy to make comparisons between singletons and twins
All information on singletons was removed from the manuscript.
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Performance of NIPT in twin pregnancies
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The word count is 233.
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OK
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This was adapted accordingly (line 137).
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This was adapted accordingly (line 250).
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This was adapted accordingly (line 212 and line 225).
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The Table Checklist was reviewed. Please note that Appendix 1 does contain the brand names of the cfDNA isolation and library preparation kits and the sequencing platforms used, as they are crucial information in our opinion. Alternatively, we could include this information in the Materials & Methods section of the manuscript, but this would extend this section considerably and decrease its legibility.
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OK
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OK
15. Figure 1: This may be hard to read in print due to its size. Please consider moving to supplemental digital content or abbreviate the amount of information shown (maybe removing items that have a value of 0 and noting that in the legend).
The figure was revised by removing the chromosomes with no trisomy cases; this was noted in the legend.
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