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closeReferee Comments: Referee 2
Posted by PLOS_ONE_Group on 28 Apr 2008 at 19:30 GMT
Referee 2's Review:
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N.B. These are the comments made by the referee when reviewing an earlier version of this paper. Prior to publication the manuscript has been revised in light of these comments and to address other editorial requirements.
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This manuscript is a logical extension on previous work from a well-respected group of researchers with extensive expertise in proteomic analysis of amniotic fluid. In previous studies they have identified proteomic patterns in the amniotic fluid representative of intra-amniotic inflammation that may be predictive of preterm labor. In the present study, the authors attempt to identify biomarkers of preterm labor in the absence of intra-amniotic inflammation.
Overall, the manuscript was well written. The objectives were clear and the methods were appropriate for the design of the study. While the introduction proved informative and composed the necessary background information to link prior studies to that of the present investigation, the authors would benefit from describing expected results in this study and how they could be similar or differ from their previous studies. The proteomic pattern (Q-profile) of amniotic fluid relating to preterm birth in the absence of inflammation or bleeding is intriguing and the 2D-DIGE pathway analysis further supports the theory that multiple pathways are responsible for the initiation of preterm labor.
Major Questions and Comments:
1. The proteomic pattern (Q-profile) of the amniotic fluid without inflammation or bleeding occurred in 17% of the subjects. These subjects possessed symptoms of preterm labor and were assumed to be treated with tocolytic agents. Could it be that any of the given tocolytic agents may be responsible for one or several of the observed peaks in the Q-profile or even in the 2D-DIGE protein analysis? May be informative to know the tocolytic agents given to the subjects in this group.
2. In the author's previous investigation(s) they used similar SELDI / proteomic techniques to determine the biomarker patterns characteristic for intra-amniotic inflammation and decidual bleeding and were able to successfully identify the proteins associated with each peak in the pattern. Why could a similar determination of the peaks in the Q-profile not be performed in this investigation? This information would significantly strengthen the current manuscript.
3. Without the identification of the peaks in the Q-profile, the correlation between the proteins detected from the 2D-DIGE analysis and the Q-profile is highly speculative.
4. The 2D-DIGE analysis appears to be preliminary as the authors have only used a sample size of 3 for the preterm and term group and a 1.5 fold change to detect differences. Most studies would use a 2-fold change for proteins or genes to be considered significantly different.
5. According to the PANTHER analysis, 50% of the total transcripts were classified into the category of protein metabolism and modification. This biological process as well as the others identified in the Figure 5 represent very broad and general biological processes. The authors may also benefit by subjecting these transcripts to other bioinformatic / pathway analysis which offer more specific biological categorizations.
Conclusion
New biomarkers in preterm labor are discovered by proteomic techniques. The proteomic methods used in this study prove important in the identification and diagnosis of idiopathic preterm labor.
Authors' responses to Referee 2
ibuhi001 replied to PLOS_ONE_Group on 03 May 2008 at 15:50 GMT
We thank this reviewer for her/his comments. This reviewer expressed several important points.
1. Tocolysis. The reviewer raises an important point related to administration of tocolytics to patients presenting with preterm labor. In Table 1 have tocolysis was added as a variable for all analyzed groups. There were no differences in the proportion of women who received tocolytics prior to amniocentesis among groups. This can be explained by the fact that per clinical protocols, tocolytics are not administered for women presenting at less than 24 weeks gestational age. Moreover the amniocentesis procedure is generally performed at admission, before tocolytics are initiated. Exceptions are women transferred to our tertiary care facility from outside hospitals where they already have received tocolytic treatment. Most used tocolytics were magnesium sulfate and nifedipine, but this also did not differ among groups.
2. Determination of the peaks in the Q profile. As we mentioned in our original submission at this time we have not been able to identify the specific proteins responsible for the five SELDI peaks of the Q-profile due to the limitation of the current technology. However, the Q profile was able to delineate a subgroup of patients whose amniotic fluid samples were subjected to 2D-DIGE which has the ability to provide protein identity information.
3. Correlation between proteins in the Q profile and 2D-DIGE. In our original submission we specifically made the point that the SELDI peaks comprising the Q profile may not be the ones identified as differentially expressed by 2D-DIGE. There are many reasons why the two techniques differ and sensitivity is only one of them. In the discussion section of the revised manuscript we expanded on this issue.
4. Cut-off. The reviewer was pointing out that in our manuscript we used a cut-off of 1.5-fold to assess for changes in expressed proteins by 2D-DIGE. Indeed most studies on microarray data use an arbitrary 2-fold cutoff. However, in proteomics, the 1.5-fold cut-off is generally used in 2D-DIGE analysis. Other authors used a similar cut-off: (see Michaels JE et al. Comprehensive proteomic analysis of the human amniotic fluid proteome: gestational age-dependent changes. J Proteome Res. 2007:1277-85). (reference 13).
5. Pathway analysis. The reviewer suggested that the transcripts identified in our studies be subjected to other bioinformatic pathway analysis tools under the assumption that these may offer more specific biological categorization. AS suggested by the reviewer we subjected our 2D-DIGE data to Ingenuity Pathway Analysis (Ingenuity Systems, www.ingenuity.com, Yale institutional license). The results were identical albeit only 8 transcripts were matched to either biological processes or pathways. We believe that the above information would not further add to the results obtained through Panther.