Pelvic organ prolapse (POP) is the descent of the vaginal wall, cervix, uterus, bladder, and rectum downward into the vaginal canal. It occurs owing to the weakness of the structures supporting and keeping pelvic organs in anatomic position. Prolapse occurs due to exposure to risk factors; women in developing countries are highly predisposed to the risk factors of the prolapse. No study assesses POP in Southern Ethiopia.A case-control study design was employed in 369 participants (123 cases and 246 controls) of seven randomly selected Public Hospitals of Southern Ethiopia from February-June, 2020, using a structured questionnaire. All patients diagnosed with prolapse (stage I- IV) were included as cases; patients free of prolapse (stage 0) were taken as controls after physicians had performed a diagnosis and vaginal examination. Bivariate and multivariable logistic regression analyses were performed using SPSS.In this study, after adjusting for covariates, age of the women ≥ 45 years , underweight , low income , parity ≥5 , assisted vaginal delivery , instrumental delivery , sphincter damage and vaginal tear , carrying heavy loads , and prolonged labor ≥24 hours  were significant associated factors of prolapse. The odds of developing prolapse is lower among women attended school. Most(84.55%) of the women with prolapse were delayed for the treatments and only surgical interventions were done as treatments. Most of them claimed lack of social support, lack of money, and social stigma as the main reasons for the delay in seeking treatments.Older age, low educational status, underweight, low income, higher parity, assisted vaginal delivery, prolonged labor, sphincter damage, and carrying heavy loads were significant associated factors of POP. It is better to screen older age women by doing campaigns against the prolapse. Also, responsible bodies should work on raising awareness of women as well as awareness of the community about the prolapse through expanding health education. Moreover, informing women to practice pelvic muscle training daily, raising women’s income to empower them, and help of family members to reduce carrying an overload of mothers are recommended. Pelvic organ prolapse (POP) is defined as the herniation or descent of the vaginal wall, cervix, uterus, bladder, and rectum downward along the vaginal lumen . UniversThe risks of POP are categorized into three. The leading risk factors for POP are obstetric factors , 19. It In Ethiopia, since it is a very low-income country, the women are highly exposed to the above-mentioned risk factors , 17, 21 A multi-center unmatched case-control study design was conducted from February 28 to June 05, 2020, at Public Health Hospitals of Southern Ethiopia. Out of the total fourteen general and referral public health hospitals in southern Ethiopia, seven hospitals  were selected randomly by the simple random sampling method. All available gynecologic patients diagnosed with POP (stage I-IV) at each hospital were taken as cases. To select controls, we used a systematic random sampling method, depending on the gynecologic patient flow of each hospital. Hence, the K value for each hospital varies .1q1 + p2q2) ) / ((p1 - p2)2, n= number of sample size in each group, α = type I error, β = type II error]. To calculate the sample size, we used proportions from a similar study conducted in Bahr Dar City, Ethiopia  more likely to develop POP than women with normal BMI values. Women who earn daily ≤ $1.25 were 2.5 times [AOR = 2.5] more likely to acquire POP than their counterparts. The women who had parity ≥5 were five times [AOR = 5.2 ] more likely to develop POP than their counterparts. Women who delivered through assisted vaginal delivery at their last delivery were four times [AOR = 4 ] more likely to develop POP as compared to women delivered through spontaneous vaginal delivery. The women who had prolonged labor (≥24 hours) at the last delivery were three times [AOR = 3.3 ] more likely to develop POP than those women in labor ≤12 hours. Women who had a history of sphincter damage and vaginal tear were about three times [AOR = 3.2(95% CI: 1.44-7.1)] more likely to have POP than their counterparts. The women who practiced carrying/ heavy loads daily were 2.5 times more likely to develop POP than non-carriers [AOR = 2.5] ] more likely to develop POP as compared to women who were aged <35 years. Women who attended secondary school 68% [AOR = 0.32], and diploma (and above) 83% [AOR = 0.17] were less likely to develop POP as compared with those women who had no schooling at all. The women who had a BMI value ≤ 18.5kg/m, 5.35)] .This study primarily assessed associated factors among women with POP at Public Health Hospitals of Southern Ethiopia. Accordingly, age, educational status, BMI, monthly income, higher parity, mode of delivery, duration of labor, history of sphincter damage, and carrying heavy loads were associated factors of POP.In this study, age ≥45 years increased the risk of developing POP. Accordingly, women who were aged ≥45 years were about 5 times more likely to develop POP. This finding is supported by the study conducted in Bahr Dar , Gondar This study revealed all levels of formal education were significant protective factor of POP. The odds of developing POP were 68%, and 83% lower among women of the secondary, and diploma (and above) education, respectively. This is in line with study findings reported from Bahr Dar  and Kers2) were riskier to develop POP Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. 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: YesReviewer #2: Partly**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?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.  The Reviewer #1: NoReviewer #2: No**********  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: YesReviewer #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. Reviewer #1: Reviewer’s report: Predictors of pelvic organ prolapse among patients at Public Hospitals of Southern Ethiopia: A case-control study designthe aim of this case control study was to examine predictors of pelvic organ prolapse patients at southern Ethiopian public hospitalsAbstract:the conclusion is largely valid but the last sentence is perhaps somewhat speculative. Perhaps the authors might like to modify this.IntroductionThe authors note the highly variable prevalence of prolapse in studies which existed to date perhaps they might confine this detailed discussion to the discussion section and perhaps suggest some explanation as to why this is the case.The authors note established risk factors in other studies, could they therefore suggest why a study in Ethiopia would be of value or might produce different information?Would a study attempting to intervene on the known modifiable factors be more significant and have greater utility than reiterating what is known?the authors make a good point about using the data with “responsible bodies” in playing a role of modification of such factors which would be a useful motivation upon which to buildmethods and materialsIt is good to see an accurate sample size calculation. what was the degree of imprecision in the prevalence estimate?please give the authors describe their random sampling technique For participants and hospitals?Was the Constipation question validated?Who examined the women?The authors are to be congratulated in the conduct of this study in a challenging environment, in particular the governance processes they have put in place to ensure data qualityThe statistical analysis section is entirely appropriate.I note that written informed consent was obtained from study participants, What proportion of women was illiterate and how was consent gained in this case?ResultsFor what reason have the authors included the religion of the women? Was this used as a controlling variable in parity? Did religion and parity interact?Is the proportion of women earning greater than 1200 birrs representative of the Ethiopian general population of women? Likewise, is the proportion having a home delivery typical?Is the history of Sphincter damage and vagina tear when gained from the women reliable?Regarding predictors, were any variables discarded on bivariate analysis? Were there any interactions? For example was high parity associated with relative poverty?Likewise was carrying heavy loads more common in women who were of lower educational attainment or low income?The results section is otherwise extremely well written But I should like to see some comparative analysis of the factors in table one in terms of prevalence of prolapse and say religion occupational status and ethnicity for completions sakeCan the authors tell us what the average delay in seeking treatment was for the women both as a whole and according to income status education status or employment status?This might help to support their argument that women with high income get better health services seek timely treatments and can hire someone to help themDiscussionthe authors present their findings in a logical manner and attempts to explain their findings.Might the authors also consider the amount of physical exertion women perform separate from carrying heavy loans as a potential mitigating factor - as the authors note this might also account for the higher prevalence of prolapse in underweight womenThe authors might consider comparing their sample with the general Ethiopian population to give the reader some frame of reference for example, were the women more educated, or likely to be employed or were they wealthier than average women in Ethiopiathe high parity doesn't necessarily imply low utilization of family planning - the high parity may well be purposeful. do the authors have any evidence to suggest that women want fewer children?The authors have considered their limitations and biases, notably the external validity of this hospital population seeking help versus the general populationto what extent were husbands likely to give reliable information on their wives?In conclusion the authors have identified many potentially remedial factors in the development of prolapse. how likely are women to choose active treatment of their prolapse in Ethiopia. Is operative intervention financially out of reach for the majority of women? What conservative therapies are available?This information would certainly help add context to the paperReviewer #2: The authors investgated in the present study the effect estmate of several known factors that predispose to pop in an ethiopian population. Despite their rigorous methodology i believe that the lack of an appropriate sample size  is the main reason for rejection of this article.**********  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: NoReviewer #2: Nohttps://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.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  26 May 2022Responses to Reviewers: Corrections/revisions for Reviewer 1Abstract:1. The conclusion is largely valid but the last sentence is perhaps somewhat speculative. Perhaps the authors might like to modify this.    Dear reviewer, we found this comment is relevant and we have corrected/revised as your request. Thank you very much for your suggestions and recommendations.�Introduction:2. The authors note the highly variable prevalence of prolapse in studies which existed to date perhaps they might confine this detailed discussion to the discussion section and perhaps suggest some explanation as to why this is the case.    Dear reviewer, the objective of the study is to determine predictors of the pelvic organ prolapse. This why we preferred to discuss in detail about the prevalence of the pelvic organ prolapse in introduction part.�3. The authors note established risk factors in other studies, could they therefore suggest why a study in Ethiopia would be of value or might produce different information? Would a study attempting to intervene on the known modifiable factors be more significant and have greater utility than reiterating what is known? the authors make a good point about using the data with “responsible bodies” in playing a role of modification of such factors which would be a useful motivation upon which to build   Dear reviewer, the most risk factors of the prolapse varies greater from the community to community because of different socioeconomic status, health system development level, and culture or beliefs. According to the literatures, so many factors (over 60) have association with the prolapse. Policy makers and health managers would not intervene all risk factors of the prolapse. This study was conducted with a case control study design to identify which factors are significantly affecting the prolapse in the study areas. Hence, managers and policy makers of the study areas possibly can work on identified factors to modify.�Methods and materials4. It is good to see an accurate sample size calculation. what was the degree of imprecision in the prevalence estimate?    Dear reviewer, previously, we had written the sample size calculation in short and summarized way. Now, on the revised manuscript, we have explicitly explained how the sample size was calculated as following:�   In this study, sample size was calculated by using Epi info version 7 software. For unmatched case-control study design, this software calculates by using double proportion formula . To calculate sample size, we used proportions from the similar study conducted in Bahr Dar City, Ethiopia, as following: (see Table 1)�Risk actors % of case with exposure % of control with exposure Power CI (%) AOR Sample sizeAge >44 71.8 27.1 80 95 6.8 55Formal education 85 58 80 95 4.3 111Parity/delivery 64.5 26.67 80 95 4.5 69Sphincter damage 15.2 2 80 95 8 173Carry heavy object 67.4 40 80 95 3.1 131Family history of POP 22.6 6.2 80 95 4.9 176Underweight 34.5 14 80 95 3.2 164Delivery assisted by non-health personnel 63.4 39.1 80 95 2.6 167   We got maximum sample size with variable of ‘family history of POP’ which is 173, after considering the design effect of two, and a 5% non-response rate, the total sample size became 369 patients (123 cases and 246 controls: in 1 to 2 cases to controls ratio).�5. Please give the authors describe their random sampling technique for participants and hospitals?    Dear reviewer, among 14 governmental general and referral hospitals in SNNPR, seven hospitals were selected by simple random method (lottery method). All patients with a pelvic organ prolapse visiting gynecologic OPD during data collection period were included as cases. To select controls, we used systematic random sampling method, depending on gynecologic patient flow of each hospital. Hence, the K value for each hospital varies  �  �  �  �  �  �  �  �  �  �   N/E/M/M/= Negest Elleni Mohammed Memoral, HU = Hawassa University, Ca=cases, Co=controls�Figure 1: Sampling procedure5. Was the Constipation question validated?    Dear reviewer, we considered constipation lasting longer than three months as a significant enough to contribute for the development of the prolapse. Many other studies also agree with this statement.�6. Who examined the women?    Gynecologists performed physical examination, history taking, and diagnosis as already explained in the manuscript.�7. The authors are to be congratulated in the conduct of this study in a challenging environment, in particular the governance processes they have put in place to ensure data quality. The statistical analysis section is entirely appropriate.    Dear reviewer, we thank you very much for understanding our efforts and challenging environment that we had faced.�8. I note that written informed consent was obtained from study participants, What proportion of women was illiterate and how was consent gained in this case?    Dear reviewer, thank you for your important comments, a written informed consent was obtained directly from study participants who can read and write. For those unable to read and write, first the consent was clearly read to her and then her immediate/ significant family member  will sign it. Even she has not family member, the consent was clearly read to her and then her fingerprints were taken as signature.�Results9. For what reason have the authors included the religion of the women? Was this used as a controlling variable in parity? Did religion and parity interact?    Dear reviewer, religion of the respondents first included as sociodemographic characteristic for the descriptive purpose. Later, its association with the prolapse checked on multivariable logistic regression and it did not show association. We have checked that no interaction of the religion and parity.�10. Is the proportion of women earning greater than 1200 birrs representative of the Ethiopian general population of women? Likewise, is the proportion having a home delivery typical?    Dear reviewer, this study categorizes the income by using World Bank cut off point of poverty line, as operationally defined in the main manuscript. According to World Bank definition of the poverty line, earning below $1.25/day(1200 birr/month) is considered below poverty line. In our study, about 69% of the women with prolapse earn less than $1.25/day; about 17.47% of women from the control earn less than $1.25/day. In our study, 35.58% of the women with prolapse had at least one history of home delivery and about 30% of women from controls had at least one history of home delivery in their life. We don’t think shortage of the income affects home delivery in Ethiopian because medical services related with obstetrics is for free. Rather, home delivery is related to cultures, awareness, and attitude�11. Is the history of Sphincter damage and vagina tear when gained from the women reliable?   Dear reviewer, yes, it is reliable because they never forget history that they had episiotomy or vagina tear. Moreover, their husbands had helped during the interview for some. �12. Regarding predictors, were any variables discarded on bivariate analysis? Were there any interactions? For example was high parity associated with relative poverty?   Variables such as ethnicity, marital status, religion, residency, home delivery, duration of time to return to work after delivery, delivery interval, age at marriage, age at first delivery, age at last delivery, chronic cough and chronic constipation were discarded on bivariate logistic analysis.�13. Likewise was carrying heavy loads more common in women who were of lower educational attainment or low income?    Dear reviewer, yes, carrying heavy loads more common in women who were of lower educational attainment or low income.�14. The results section is otherwise extremely well written But I should like to see some comparative analysis of the factors in table one in terms of prevalence of prolapse and say religion occupational status and ethnicity for completions sake.    Dear reviewer, Table 2 shows these very clearly, and one can easily calculate from the table. We done in such way to reduce space and explain in summarized way..�15. Can the authors tell us what the average delay in seeking treatment was for the women both as a whole and according to income status education status or employment status? This might help to support their argument that women with high income get better health services seek timely treatments and can hire someone to help them   Dear reviewer, we found your concerns as very important and we have included it also in the main manuscript.�   The average delay of the women for the treatments since onset of the symptoms was 36.41 ± 3.94 months. Out of 123 women with the prolapse, 84.55% delayed for the treatments and only surgical interventions done as treatments. Among 104 women delayed for the treatment, 76% of them claimed lack of money as main reason for the delay. Out of 104 women delayed for the treatment, 67.48% (83) did not attend formal school and 69.1% (85) of them earn less than $1.25/ day.�Discussion16. The authors present their findings in a logical manner and attempts to explain their findings.Might the authors also consider the amount of physical exertion women perform separate from carrying heavy loans as a potential mitigating factor - as the authors note this might also account for the higher prevalence of prolapse in underweight women.    Of course, the reviewer is right, but for us it was difficult to measure amount of exertion force other than carrying heavy loads. We considered all type of heavy physical forces as carrying heavy loads.�17. The authors might consider comparing their sample with the general Ethiopian population to give the reader some frame of reference for example, were the women more educated, or likely to be employed or were they wealthier than average women in Ethiopia.    Dear reviewer, we found this a very important comment and we incorporated this comment into the manuscript. �18. The high parity doesn't necessarily imply low utilization of family planning - the high parity may well be purposeful. do the authors have any evidence to suggest that women want fewer children?    Dear reviewer, we appreciate you for rising important idea, and we have made modifications accordingly. �   In Ethiopia, there is low utilization of family planning especially among rural residents. This because women in rural Ethiopia are characterized by low education, low access to health informations, beliefs and cultures that encourages the women to have many children. In the study areas, having many children is considered as proud, dignity, and ultimate blessings from the Creator. Moreover, in the study areas, deciding the number of the children to have exclusively the right of the husbands, women have no right to decide.�19. The authors have considered their limitations and biases, notably the external validity of this hospital population seeking help versus the general populationto what extent were husbands likely to give reliable information on their wives?   During the data collection, we found that husbands’ helps were very important especially when women had forgotten some events in their life history. They knew almost everything about their wives.�20. In conclusion the authors have identified many potentially remedial factors in the development of prolapse. how likely are women to choose active treatment of their prolapse in Ethiopia.    Dear reviewer, thank you for your comment and we included it into main manuscript accordingly.�   Most of the women arrived at hospital at advanced stage of the prolapse. They completely had hidden the problem and they had showed to their close relative when the condition got unbearable. As a result no options than surgical treatments. During the data collection, out of 123 women with prolapse 104 (84.55%) were surgically repaired .�21. Is operative intervention financially out of reach for the majority of women?    Dear reviewer, thank you for your comment and we included it into main manuscript accordingly�   Among 104 women delayed for the treatment, 76% of them claimed lack of money as main reason for the delay. Most of the women with prolapse come to hospital after they had got some financial support from some people who understood their conditions. �22. What conservative therapies are available? This information would certainly help add context to the paper   Dear reviewer, rest of the women with less advanced stages ( stage II) were treated with conservative means of treatments like lifestyle and behavior modification, physical therapy for the pelvic floor, and using vaginal device (Pessary)�Corrections/revisions for Reviewer 21. The authors investgated in the present study the effect estimate of several known factors that predispose to pop in an Ethiopian population. Despite their rigorous methodology i believe that the lack of an appropriate sample size  is the main reason for rejection of this article.    Dear reviewer, we thank you very much for your concern. The most risk factors of the prolapse vary greater from the community to community because of different socioeconomic status, health system development level, and culture or beliefs. According to the literatures, so many factors (over 60) have association with the prolapse. Policy makers and health managers would not intervene all risk factors of the prolapse. This study was conducted with a case control study design to measure which factors are significantly affecting the prolapse in the study areas. Hence, managers and policy makers of the study areas possibly can work on identified factors to modify.�   Regarding sample size calculation,�   Dear reviewer, our sample size very high for case-control study and we calculated and got the value according scientific rule. Everything in this study is scientifically randomized.�   Dear reviewer, previously, we had written the sample size calculation in short and summarized way. Now, on the revised manuscript, we have explicitly explained how the sample size was calculated as following:�   In this study, sample size was calculated by using Epi info version 7 software. For unmatched case-control study design, this software calculates by using double proportion formula or power approach . To calculate sample size, we used proportions from the similar study conducted in Bahr Dar City, Ethiopia, as following: (see Table 1)�   We got maximum sample size with variable of ‘family history of POP’ which is 173, after considering the design effect of two, and a 5% non-response rate, the total sample size became 369 patients (123 cases and 246 controls: in 1 to 2 cases to controls ratio).�Table 1: sample size calculationRisk actors % of case with exposure % of control with exposure Power CI (%) AOR Sample sizeAge >44 71.8 27.1 80 95 6.8 55Formal education 85 58 80 95 4.3 111Parity/delivery 64.5 26.67 80 95 4.5 69Sphincter damage 15.2 2 80 95 8 173Carry heavy object 67.4 40 80 95 3.1 131Family history of POP 22.6 6.2 80 95 4.9 176Underweight 34.5 14 80 95 3.2 164Delivery assisted by non-health personnel 63.4 39.1 80 95 2.6 167AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 16 Aug 2022
 PONE-D-21-33943R1

 Predictors of pelvic organ prolapse among patients at Public Hospitals of Southern Ethiopia: A case-control study design

 PLOS ONEDear Dr. Asfaw,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that there is a little more work to do to fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.==============================

 Reviewer 2 makes useful points about prediction - a term you should probably avoid unless you would like to commit to further analytical models - it may be simpler to avoid the termACADEMIC EDITOR: Please insert comments here and delete this placeholder text when finished. Be sure to:==============================plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by Sep 30 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at
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For instructions see: We look forward to receiving your revised manuscript.Kind regards,Adrian Stuart Wagg, MDAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********  2. 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: YesReviewer #2: No**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?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.  The Reviewer #1: YesReviewer #2: Yes**********  5. 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: YesReviewer #2: Yes**********  6. 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. Reviewer #1: The authors have prepared an excellent response to the reviewers comments and have added in information which makes this an informative paper. The abstract might benefit from adding into the results the delay results – this would then justify their conclusion about awareness raising etcId like to see the consent response added into the paper – this is useful for those conducting research in areas with high levels of illiteracyReviewer #2: The authors investigated in the present study the impact of several factors on the actual incidence of POP. The following comments are necessary to be addressed prior to its acceptance.1) If the predictive value of these factors is to be investigated a ROC and AUC analysis along with sensitivity and specificity of individual factors or the whole logistic regression should be provided. Otherwise the study should be targeted only around the risk factors and not their predictive role. If the predictive role is to be investigated the Hosmer Lemeshow goodness of fit of the logistic regression should be reported.2) Despite having performed a sample size calculation, the confidence intervals that the authors report are rather wide; hence, an accompanying power calculation of their findings could help evaluate the actual robustness of their results and comment on them in the discussion.**********  7. 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: NoYes: Vasilios PergialiotisReviewer #2: **********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.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  27 Sep 2022Response Letter Authors: 1. Mr. Asfaw Borsamo 2. Mr. Mohammed Oumer 3. Mr. Ayanaw Worku 4. Mr. Yared Asmare Editorial-in-Chief of the PLOS ONE JOURNAL October 03, 2022Dear Dr. (Professor) Adrian Stuart Wagg, Please find enclosed a revised manuscript entitled “Associated factors of pelvic organ prolapse among patients at Public Hospitals of Southern Ethiopia: A case-control study design’’ with the kind request to consider it for publication in the Plos One Journal.In the first place, we would like to thank the Esteemed Dr. Adrian Stuart Wagg and Reviewers for your kind and lesson giving comments, create an opportunity to improve our work. We have accepted the comments, and we made modifications accordingly. For the concerns that were raised by the esteemed reviewers, we have given explanations. We have checked that our references list is complete and correct.Journal requirement: We have checked that our references list is complete and correct.Responses to Reviewers: Corrections/revisions for Reviewer 11. The authors have prepared an excellent response to the reviewers’ comments and have added in information which makes this an informative paper. The abstract might benefit from adding into the results the delay results – this would then justify their conclusion about awareness raising etc.   Dear reviewer, we would like to thank for appreciating a previous response and we found the recent comment is also relevant. Hence, we have corrected/revised as your request. Thank you very much for your suggestions and recommendations. Your comments are making our work complete. �2. Id like to see the consent response added into the paper – this is useful for those conducting research in areas with high levels of illiteracy    Dear reviewer, we used the consent form as following:� Consent form English versionI have read or it was read to me (for those who cannot read and write) and understood well the condition stated above and I can withdraw from the study at any time and I understand that there is no risk of participating and no incentive to be given when I participate in the study. Therefore, I am willing to participate in the study. Signature___________________ Date____________  Thank you so much!  Amharic versionየስምምነት ፍቃድ ከላይ የተጻፈውን መረጃ በደንብ አንብቤ ተረድቸው አለሁ፡፡ በጥናቱ ላይ መሳተፍ በኔም ሆነ በልጀ ላይ ምንም አይነት ችግር እንደማያደርስ ፤ የተለየ ጥቅማጥቅምም እንደለለው እንዲሁም በማነኛውም ሰአት ካልተመቸኝ የማቋረጥ መብት እንዳለኝ ተነግሮኛል፡፡ በመሆኑም በጥናቱ ላይ ለመሳተፍ ዝግጁ/ፍቃደኛ ነኝ ፡፡  ፊርማ --------------------- ቀን -------------------------------- ስለተሳትፎዎ እናመሰግን አለን !!! Corrections/revisions for Reviewer 2Thank you very much for your suggestions and recommendations.1. If the predictive value of these factors is to be investigated a ROC and AUC analysis along with sensitivity and specificity of individual factors or the whole logistic regression should be provided. Otherwise the study should be targeted only around the risk factors and not their predictive role. If the predictive role is to be investigated the Hosmer Lemeshow goodness of fit of the logistic regression should be reported.   Dear reviewer, thank you so much for your comment. We have modified our manuscript; we have replaced the term “predictors” with term “associated factors” throughout the manuscript. Mostly in public health research, researchers prefer the term “determinant” for cohort studies, “predictors” for case-control studies and retrospective cohort studies, and “associated factors” for cross-sectional studies. Similarly, we used the term “predictor” simply to show the level of term choice. So, thank you the Esteemed Academic Editor and Reviewer, now use the term “associated factors” Thank you very much for your suggestions and recommendations.�2. Despite having performed a sample size calculation, the confidence intervals that the authors report are ather wide; hence, an accompanying power calculation of their findings could help evaluate the actual robustness of their results and comment on them in the discussion.    Dear reviewer, we have incorporated per your request in the discussion. Thank you very much.�AttachmentResponse to Reviwers.docxSubmitted filename: Click here for additional data file. 17 Nov 2022Associated factors of pelvic organ prolapse among patients at Public Hospitals of Southern Ethiopia: A case-control study designPONE-D-21-33943R2Dear Dr. Asfaw,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.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. 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The interaction between strong correlation and Berry curvature is an open territory of in the field of quantum materials. Here we report large anomalous Hall conductivity in a Kondo lattice ferromagnet USbTe which is dominated by intrinsic Berry curvature at low temperatures. However, the Berry curvature induced anomalous Hall effect does not follow the scaling relation derived from Fermi liquid theory. The onset of the Berry curvature contribution coincides with the Kondo coherent temperature. Combined with ARPES measurement and DMFT calculations, this strongly indicates that Berry curvature is hosted by the flat bands induced by Kondo hybridization at the Fermi level. Our results demonstrate that the Kondo coherence of the flat bands has a dramatic influence on the low temperature physical properties associated with the Berry curvature, calling for new theories of scaling relations of anomalous Hall effect to account for the interaction between strong correlation and Berry curvature. Kondo systems offer a rich platform to study the interplay between strong correlations and topology. Here the authors observe a large anomalous Hall conductivity in a Kondo ferromagnet USbTe, which they attribute to the Berry curvature originating from flat bands induced by the Kondo hybridization. Skew scattering depends on the scattering rate, leading to a quadratic dependence of anomalous Hall conductivity on the longitudinal conductivity, σxx and temperature6.The anomalous Hall effect (AHE) can arise from two different mechanisms: extrinsic processes due to scattering effects, and an intrinsic mechanism connected to the Berry curvature associated with the Bloch waves of electrons. While large Berry curvature arises when the inversion or time-reversal symmetry of the material is broken, in the clean limit skew scattering can dominate and cause large AHE. To distinguish different mechanisms, scaling analysis has been developed and widely used in various systemsThe established scaling relation is based on the Fermi liquid theory and is expected to apply to weakly interacting systems. The intersection of strong electron correlation and the Berry curvature is still an open territory. On the theory side, due to the interplay between Coulomb repulsion and kinetic degrees of freedom for the electrons, the prediction of band structure properties in strongly correlated materials represents a theoretical challenge. On the experiment side, not many strongly correlated systems have been identified to host large Berry curvature. It is highly demanding to investigate the effect of electron correlation on the Berry curvature-induced physical properties, particularly the scaling relation of the AHE in strongly correlated systems.16. A Weyl–Kondo semimetal phase was predicted and observed in non centrysymmetric Kondo semimetal Ce3Bi4Pd321, which exhibits large AHE that is symmetric with respect to applied magnetic fields. Further theoretical studies show that other topological phases, including non-Fermi liquid topological phases, can be driven by the Kondo effect combined with crystalline symmetries23. Note that in the magnetically ordered systems, Kondo hybridization gives rise to non-symmetry-breaking low-temperature coherence phenomena, which are not precluded by the high transition temperature25.Recently Kondo systems have emerged as a promising platform to explore the interaction between strong correlation and Berry curvature associated with Band structure topology. A large anomalous Nernst effect has been observed in a Kondo lattice noncentrosymmetric ferromagnetHere we report the large AHE and breakdown of the scaling relation of AHE in a Kondo lattice ferromagnet, USbTe. The sign change of the AHE upon cooling indicates competing mechanisms from skewing scattering and Berry curvature. The scaling relation between anomalous Hall conductivity and longitudinal conductivity is not valid for a large temperature range. At low temperatures, the scaling relation is recovered which reveals that Berry curvature dominates the AHE. However, Berry curvature contribution has strong temperature dependence and vanishes above the Kondo coherent temperature. This strongly suggests that the Berry curvature in USbTe is hosted by the flat bands at the Fermi level due to Kondo hybridization, consistent with our ARPES measurement and DMFT calculations. Our results demonstrate that the coherence of the flat bands dramatically modifies the scaling relation of AHE, calling for further theoretical investigation of the interaction between strong correlation and Berry curvature-induced physical properties.Tc = 125 K28, evidenced in resistivity, magnetization, and specific heat measurements . U and Te atoms each form planes with mirror and screw nonsymmorphic symmetries. USbTe exhibits typical behaviors of the ferromagnetic Kondo lattice system. The ferromagnetic ground state develops in USbTe below Curie temperature of γ0 = 40 mJ/mol-K2  shows a pronounced convex shape, not typical for metallic ferromagnets. The previous studies excluded structural change below Tc and attributed the convexity to the formation of Kondo coherent scattering28. At even lower temperatures, below 15 K, resistivity shows an upturn, which was also observed in the previous study30. Likely there remains some incoherent single-ion Kondo scattering together with coherent Kondo scattering at low temperatures.The Sommerfeld coefficient of the electronic contribution to the specific heat is ρxy of a USbTe single crystal at 2 K, measured with the magnetic field applied along the c axis and the electric current along the a axis. ρxy exhibits a negative slope indicating a normal Hall effect due to the electron carriers. In addition to the normal Hall effect, a large remnant Hall resistivity at zero-field is observed. The anomalous Hall resistivity Tc of 125 K. Figure 2. However, in our case, magnetization reaches a constant value below 50 K  as a function of ρxx, where M is the magnetization, as shown in Fig. ρxx should follow 36. Previous studies indicate that a(M) is proportional to M linearly37. In general, b(M) could have nonlinear M dependence2. However, M dependence of b(M) will not play a role in two temperature ranges, in which magnetization reaches a saturation value or intrinsic contribution vanishes. In these cases, the slope of Mρxx) vs ρxx plot gives intrinsic Berry curvature contribution, while the intercept gives the skew scattering contribution. Figure Mρxx) is relatively flat between 70 and 100 K, with an unchanged intercept representing skew scattering contribution and no Berry curvature contribution. Below 70 K, the slope starts to deviate from zero, indicating the onset of intrinsic Berry curvature contribution. Below 50 K, the magnetization reaches a constant value. The scaling relation Mρxx) vs ρxx regardless of the M dependence of b(M). However, this scaling relation is not followed for a large temperature ranges below 50 K as seen in Fig. To further extrapolate the Berry curvature contribution and evaluate the scaling relation, we plot entclass1pt{minimaentclass1pt{minimaentclass1pt{minima−1 cm−1 at 2 K, ~0.65 × e2/hd where d is half the lattice constant along the c-axis (there are two uranium layers within each unit cell). The large value of anomalous Hall conductivity is comparable to those obtained in weakly correlated Weyl semimetals14. We want to emphasize that the separation of intrinsic and skew scattering contributions to the AHE in the two temperature ranges of interest, 2–6 K and 70–100 K, is based on the established scaling relation with no additional assumptions. Particularly, M dependence of the intrinsic contribution is irrelevant in these two temperature ranges as discussed above. Between 6 and 50 K, M dependence of the intrinsic contribution is still irrelevant since M is constant. However, the scaling relation is violated. There is no established theory predicting how each part of AHE changes with temperature quantitatively in this scenario. We simply assume that anomalous Hall resistivity due to skew scattering changes linearly with temperature from 6 to 70 K. This is only for simplicity. The exact temperature dependence between 6 and 70 K does not change the main finding of the current study and begs for future theoretical investigation.Based on these scaling analyses, we can estimate the Berry curvature and scattering contributions as shown in Fig. γs = ne2Δy/ℏkF38, where γs is the coefficient for the anomalous Hall resistivity from the side jump y is the amplitude of the side jump in the Hall direction after an impurity scattering event, and is estimated to be 10−11 m38. Using the carrier concentration calculated from normal Hall resistivity  and intersect with heavy bands within EB ~1.0 eV , the light band features appear to have unbroken dispersions that intersect the Fermi level, as traced in black in Fig. EB ~20 meV (traced in white). Dispersion of the flat band becomes apparent upon cooling beneath T < ~50 K and significantly modifies the electronic structure at the Fermi level . Positive and negative magnetic fields were applied in order to antisymmetries the Hall signal. Hall conductivity is calculated using the equation σxy = −ρxy/ and dynamical mean field theory (DMFT) method56. For the self-consistent process of electron charge, a proper k-mesh of 11 × 11 × 6 is adopted and the energy cutoff of plane wave basis is 350 eV. The single-particle mean-field Hamiltonian is extracted by the Wannier90 package from the DFT calculations57, based on which the anomalous Hall conductivity is calculated through the WannierTools package58 with a dense k-mesh of 101 × 101 × 101.The spin polarization calculations including the spin–orbital coupling (SOC) correction based on density functional theory (DFT) have been performed through the Vienna ab initio simulation package (VASP) with the Perdew–Burke–Ernzerhof (PBE) generalized gradient approximation (GGA) exchange-correlation potential. The Coulomb interaction of U atoms is considered through the GGA + U method introduced by ref. Supplementary InformationPeer Review File


The accurate characterizationof prototypical bricks of life canstrongly benefit from the integration of high resolution spectroscopyand quantum mechanical computations. We have selected a number ofrepresentative amino acids  to validate a new computational setuprooted in quantum-chemical computations of increasing accuracy guidedby machine learning tools. Together with low-lying energy minima,the barriers ruling their interconversion are evaluated in order tounravel possible fast relaxation paths. Vibrational and thermal effectsare also included in order to estimate relative free energies at thetemperature of interest in the experiment. The spectroscopic parametersof all the most stable conformers predicted by this computationalstrategy, which do not have low-energy relaxation paths available,closely match those of the species detected in microwave experiments.Together with their intrinsic interest, these accurate results representideal benchmarks for more approximate methods. From theone side, the size of the systems prevents a brute force approachemploying very accurate but very expensive state-of-the-art QC methodologies.12 From the other side, the very powerful local optimization techniquesdeveloped for semirigid systems are not effective for the explorationof rugged potential energy surfaces (PES) characterized by a hugenumber of energy minima possibly separated by low-energy barriers.14Accurate quantum chemical (QC) computations can help to solve thiskind of problem,17 The effective strategy of this kind we have beendeveloping in the past few years starts from a knowledge-based selectionand constrained geometry optimizations of a limited number of conformersemploying a fast semiempirical method.18 Next, an effectiveexploration of the whole conformational PES is performed by the samesemiempirical method guided by a purposely tailored evolutionary algorithmwith the aim of finding other low-lying minima.13 The results of this step are refined by hybrid and thendouble-hybrid density functionals,20 and possiblerelaxation paths between pairs of adjacent energy minima are identified.16 Once a panel of low-energy minima has been defined,accurate relative energies are computed by reduced-scaling compositemethods.26 These results are integrated by zero point energies (ZPE) and thermalcontributions to enthalpies and entropies employing anharmonic approachesrooted in the second order vibrational perturbation theory (VPT2)34 and proper treatment of hindered rotations.36 Finally, accurate spectroscopic parameters of the energy minimawith nonnegligible populations under the experimental conditions ofinterest are computed.37 In the specificcase of rotational spectroscopy, improved equilibrium rotational constantsare obtained by refining the optimized geometries by a linear regressionapproach.38This situation calls for an integrated computational approachemployingQC models of increasing accuracy in the different steps of an exploration/exploitationstrategy guided by machine learning (ML) tools.50 which represent very demanding benchmarks for the a priori prediction of structural and spectroscopic parameters. We have thereforeselected the glycine and alanine prototypes together with a panelof α-amino acids with polar side chains  with the aim of providingbenchmark results allowing unbiased comparisons with experimentalresults. In fact, the current standards for the computation of MWparameters of biomolecule building blocks in the gas phase –1 for both relative energiesand vibrational frequencies . Together with their intrinsicinterest of the studied molecules, these results will provide alsoa reference set for more approximate methods and/or search techniques.Based on thesepremises, the goal of the present study is to improveand validate a general strategy able to find all the conformers detectablein supersonic jet expansions taking also into account fast relaxationprocesses possibly leading to the disappearance of some low-lyingspecies. Unbiased comparison with spectroscopic results is made possibleby the accuracy of the computational results, which will be shownto provide mean unsigned errors (MUEs) within 20 MHz for rotationalconstants and 10 cm252 which is ableto detect characteristic structural motifs and to separate soft  and hard degrees of freedom.Then, a knowledge-based systematic search of soft degrees of freedom14 can be optionally performed, which producesa panel of guess structures . The geometriesof these candidates are next optimized using the fast GFN2-XTB semiempiricalmethod,18 which has been selected becauseit tends to underestimate energy differences , which allows a safer use of energy thresholdsfor further processing. Next, a custom implementation of the islandmodel evolutionary algorithm (IM-EA)53 isemployed to produce other candidates starting from an initial population(P0) generated by the so-called LatinHypercube stratified sampling54 in orderto maximize the diversity of soft degrees of freedom. The chemicaldescriptor (fitness) of each structure is the relative electronicenergy obtained by GFN2-XTB geometry optimizations of the stiff degreesof freedom. Improved populations are then built iteratively for agiven number of cycles by applying, with predetermined probability,different genetic operators, namely, crossover (interpolation of thefeatures of different related structures for creating new ones), mutation(change of one or more soft degrees of freedom with some stochasticrule), and selection (high chance for high fitness structures of propagatingtheir features in the next cycles). In the IM-EA, the different operatorsact separately on disjoint regions of the conformational landscape(islands), which are mixed only at predefined intervals by a dedicatedoperator (migration). Furthermore, some of the best structures foundin each cycle are directly transferred to the next cycle .55 All those choices aredictated by the high cost of evaluating the fitness of a new structureby constrained geometry optimizations. As a consequence, high fitnessstructures are worth being preserved in the population until somesignificantly improved structure is found. Typical values of the initialpopulation, maximum number of cycles, and number of islands are 100,50, and 4, which result in about 1000 constrained geometry optimizationsfor each run of the algorithm. In order to further increase the coverageof the conformational space, 4 runs with different initial populationsare performed for each molecular system. The full set of parametersemployed in the IM-EA algorithm is given in Table S1 of the Supporting Information (SI), while further details are given in refs .At the end of the whole exploration, low-energyconformers withina predefined energy range are selected from the panel of structuresissued from IM-EA and, possibly, knowledge-based steps by eliminatingtoo similar structures  and then performing single pointenergy evaluations at the B3LYP/jun-cc-pVDZ level,61 (hereafter rDSD) for both geometry optimization and evaluation ofharmonic force fields.62 The rDSD functionalhas been selected because several studies have shown that it providesexcellent geometrical structures,38 dipolemoments,63 spectroscopic parameters,37 noncovalent intermolecular interactions,64 and conformational landscapes.66In the next step, structureslying within a smaller energy rangeare optimized at the same level, and the surviving ones define thepanel of candidates for the final structural refinement, which isperformed employing the revDSD-PBEP86-D3BJ/jun-cc-pv(T+d)Z model–1 .16 As a consequence, the typicalthresholds for the acceptance of semiempirical structures, B3 geometryoptimizations, and final rDSD refinement are 2500, 1500, and 1000cm–1, respectively. These choices lead to about100 B3 computations (including both single point and geometry optimizations)and no more than 20 rDSD geometry optimizations for each molecularsystem.This composite strategy allows for stronglyreducing the numberof expensive geometry optimizations by hybrid and, especially, double-hybridfunctionals without any loss of accuracy in the final results. Thedifferent energy thresholds depend on the system and the spectroscopictechnique of interest. For the specific case of rotational spectroscopy,a conservative limit for the relative stability of detectable conformersis around 900 cm–1 being usuallyemployed for discriminating in rotational spectroscopy of amino acidsand related compounds.67 Withthe aim of unraveling fast conformational relaxations, we always performrelaxed torsional scans at the rDSD level in order to obtain preliminaryinformation on low-energy interconversion paths. Next, after preciselocation of transition states (TSs) by full geometry optimizations,their nature is checked by computing Hessian matrices.As mentioned in the 338 are largely sufficient to obtainaccurate relative electronic energies of different conformers by single-pointenergy evaluations using composite methods rooted in the coupled cluster(CC) ansatz.68 In particular, the CC modelincluding single, double, and perturbative estimate of triple excitations(CCSD(T))69 is considered the gold standardfor this kind of computations provided that complete basis set (CBS)extrapolation and core valence (CV) correlation are taken into theproper account. The key idea of the reduced cost Cheap scheme (ChS)is that, starting from frozen core (fc) CCSD(T) computations in conjunctionwith the cc-pVTZ basis set,57 CBS and CVterms can be computed with good accuracy and negligible additionalcost employing second order Møller–Plesset perturbationtheory (MP2).70 Several benchmarks23 have shown that improved noncovalent interactions can be obtainedemploying partially augmented (jun-cc-pV(n+d)Z) basis sets,71 and the corresponding model is labeled junChS. Replacement of conventionalmethods with the explicitly correlated (F12) variants leads to thejunChSF12 model, which is even more accurate without any excessiveadditional cost. In detail, the starting point is the frozen-core(fc) CCSD(T)-F12b(3C/FIX) method74 again in conjunctionwith the jun-cc-pV(T+d)Z basis set.75 The correspondingauxiliary basis sets are also employed for resolution of the identityand density fitting, and the geminal exponent (γ) was fixedto 1.0 a0–1.76 CBS extrapolation is carried out with thestandard n–3 two-point formula77 employing MP2F12/jun-cc-pV(X+d)Z energies withX = T and Q. The CV contribution is then incorporated as the differencebetween all-electron (ae) and fc MP2F12 calculations, both with thecc-pCVW(T+d)Z basis set.78 A systematicstudy of noncovalent intermolecular interactions23 showed that the junChSF12 approach is affected by smallbasis set superposition errors (BSSE), which would be difficult totake into account for intramolecular interactions. Furthermore, comparisonwith the most accurate results available for a panel of representativenoncovalent complexes provided an average absolute error smaller than10 cm–1.23The typicalMUEs of rDSD bond lengths (0.003 Å) and valenceangles  observed in the large SE100databaseΔH0°) or free energies (ΔG°) at a temperature depending on the experimentalconditions. The vibrational contributions to thermodynamic functionsare usually computed by the harmonic oscillator (HO) model, whichshows the largest errors in the high frequency (overestimated contributionsto zero point energies) and low frequency (overestimated contributionsto entropies) regions.To determinethe relative stability of different low-energy minima,one has to move from electronic energy differences to the correspondingrelative enthalpies at 0K (79 Harmonic (rDSD) and anharmonic(B3) contributions are employed in this connection, since a recentbenchmark study has shown that for semirigid molecules the averageabsolute error of zero point energies with respect to accurate experimentalresults is reduced from 53 to 17 cm–1 when goingfrom the HO to the VPT2 anharmonic model.80The first issue is solved in the presentwork by estimating anharmoniccontributions in the framework of second order vibrational perturbationtheory (VPT2), which provides analytical and resonance free expressionsfor the ZPEs.–1) is more involved because different modes need be identified, characterized,and treated by proper variational anharmonic computations.81 In the same benchmark study mentioned above in connection with anharmonicZPEs,80 it has been shown that the simpleone-dimensional hindered rotor model proposed by Ayala82 in conjunction with the VPT2 model for the othervibrational modes leads to remarkably accurate vibrational entropiesfor both semirigid and flexible molecules for which accurate experimentalresults are available. In particular, an average absolute error of3 cm–1 is obtained for the TΔS contribution to free energies at room temperature. Inthe present context, test computations showed that the unbiased detectionof hindered rotations becomes ambiguous for some conformers, so thatwe prefer to resort to the much simpler and black-box quasi-harmonic(QH) approximation.83 In the QH approach, below a givencutoff value, entropic terms are obtained from the free-rotor model,and a damping function is used to interpolate between free-rotor andharmonic oscillator expressions close to the cutoff frequency.The treatment of low frequency contributions , which include vibrational corrections(ΔBvibi) in addition to equilibriumrotational constants (Bei).84 In the framework of the VPT2 approximation,85 the ground-state rotational constants can beexpressed asr’s are the vibration–rotationinteraction constantsand the sum runs over all r vibrational modes. Notedis that the evaluation of the αr’s implies anharmonic force field calculations and that thesum appearing in Bvibi being a small fraction of the corresponding Bei ,88 it can be determinedat an affordable level of theory (B3 in the present context) withoutsignificantly affecting the accuracy of the resulting vibrationalground-state rotational constant.89 At the sametime, inclusion of vibrational corrections is not warranted if theerrors on the computed rotational constants are not much lower than1% (50 MHz for a constant of 5000 MHz). Therefore, equilibrium rotationalconstants require very accurate geometrical parameters, which canbe obtained only with state-of-the-art composite methods incorporatinghigh excitation orders in the correlation treatment. These methodsare able to deliver errors on equilibrium rotational constants aslow as 0.1% .90 The reduced cost junChSF12 composite methoddelivers typical relative errors of 0.2%,91 which are still sufficient for the unequivocalprediction and assignment of different conformers in the MW spectraof flexible molecules. Higher relative errors are obtained at the rDSD level. However, the systematic nature ofthe errors permits geometrical parameters to be obtained and, thus,equilibrium rotational constants, rivaling the accuracy of the jun-ChSF12counterparts by the linear regression approach (LRA). In this model,the computed geometrical parameters (rcomp) are corrected for systematic errors by meansof scaling factors (a) and offset values (b) depending on the nature of the involved atoms and determinedonce for ever from a large database of accurate semiexperimental (SE)equilibrium geometries:92a and b values for differentbonds and valence angles are taken from ref .93The leading terms of MW spectra are the rotational constants ofthe vibrational ground-state .94 Nuclear quadrupole coupling is the interactionbetween the quadrupole moment of a nucleus with nuclear spin I ≥ 1) and the electric gradient at the nucleus itself.86 Since at least one 14N quadrupolarnucleus is present in all amino acids, nuclear quadrupole couplingconstants are important for accurate predictions of rotational spectrabecause they determine a splitting of the rotational transitions,which generates the so-called hypefine structure. Since a systematicstudy of rDSD quadrupole coupling constants has not yet been performed,the comparison with the experimental values for several conformersof different amino acids represents per se an interesting benchmark.We anticipate that vibrational effects on nuclear quadrupole couplingconstants are usually smaller than the uncertainty affecting the computedequilibrium values, and thus they have not been considered in thiswork.Additional parameters of particularrelevance for MW spectroscopyare the nuclear quadrupole coupling constants ,and ω = Cα–C′–O–H)and side chain torsional angles, as shown in the central panel of 2 moiety suggests replacing the customary ϕdihedral angle (H–N–Cα–C′)with ϕ′ = LP–N–Cα–C′= ϕ + 120°, where LP is the nitrogen lone-pair perpendicularto the plane defined by the two aminic hydrogens and the Cα atom.The conformation of isolated amino acids is determinedby bothbackbone , II , or III.4 Higher energy minima can be classified as typeI′ or type III′ . Furthermore, conformers of type I, I′,and III have higher energy counterparts for ω ≈ 0°,labeled in the following as Ic, I′c, and IIIc, respectively.The customary c, g, s, and t labels are used to indicate the cis,gauche, skew, and trans conformations for each dihedral angle in theorder ϕ′,ψ, ω/χ1, ..., χn.The most stable backbone structures involve theformation of hydrogenbonds see 2, which 96For purposes of consistency withthe original experimental studies,capital letters L, M, N, ... are used in some cases to label conformersof amino acids with polar side chains in order of decreasing relativepopulations estimated from MW spectra.55.1101 All the eight conformers mentioned above  have been characterized withfour of them  having a planar backbone (Cs point group) and the otherfour  lacking any symmetry99 (see Table S2 of the SI). Concerning relative stabilities, the junChSF12 modelperforms remarkably well with an average absolute error of 6 cm–1 from the most accurate available results101 (see –1) is observed for the II conformer, which is slightly stabilizedby triple and quadruple excitations. Also the accuracy of the rDSDmodel (maximum error (MAX) and MUE of 29 and 15 cm–1 with respect to the most accurate available results) is largelysufficient for most purposes and gives further support to the useof this computational level for geometry optimizations and harmonicfrequency evaluations.Glycine has been extensively characterizedfrom both experimentaland computational points of view  arrangement of thecarboxylic group in the I form. The role of the arrangement of thecarboxylic group is confirmed by the nearly constant destabilizationof the Ic and I′c forms with respect to their I and I′counterparts (1690 cm–1 for Ic vs I and 1687 cm–1 for I′c vs I′). At the same time, thereduced stability of the III form with respect to I (about 600 cm–1) is related to the lower strength of the bifurcatedNH2···O(H) hydrogen bond with respect toits NH2···O(=C) counterpart for identicalarrangements of the carboxylic moiety. Finally conformers I′and III′ are less stable than their I and III counterparts because a bifurcatedhydrogen bond is replaced by a more conventional single hydrogen bond.This trend could change in the presence of polar side chains becauseit allows the formation of additional backbone (side chain) hydrogenbonds (vide infra).A shorterN···O distance in the II form with respectto I parallels the greater strength of the OH···N hydrogenbond with respect to its NH···O counterpart. Despitethese relative hydrogen-bond strengths, the I conformer is more stablethan II by about 230 cm–1, respectively), whereas structure I′crelaxes to structure Ic (with an energy barrier of about 25 cm–1). Furthermore, the relative stability of structuresIII′ (927 cm–1), Ic (1679 cm–1), and IIIc (2043 cm–1) are too low to permit theirunequivocal characterization by MW spectroscopy. We are thus leftwith only two conformers (I and II), which could be  detected in MW experiments.40Computation of energy barriers ruling theinterconversion betweenpairs of adjacent conformers shows that structures III and I′relax easily to structure I , reachingthe accuracy of state-of-the-art composite methods.102 More generally, all the computed spectroscopic parameters of theI and II conformers are in remarkable agreement with their experimentalcounterparts40  being expected at leastfor structures of II, I′, III′, and I′c type.The number of conformers thus increases to 12, but unconstrained geometryoptimizations lead also to a splitting of structure III into III andIII–, although the energy difference is so tinythat an effective planar structure is expected. In all the energyminima the methyl group is found in a staggered position with respectto the substituents at Cα with rotational barriersof about 1200 cm–1, close to the value of 1140 cm–1 obtained for ethane at a comparable computationallevel.109Moving to alanine,–1) are more than fivetimes smaller than the corresponding B3 values (222.2 and 91.6 cm–1) and less than half the corresponding MP2 values(96.5 and 28.8 cm–1). What is even more important,junChSF12 and rDSD provide the same stability order, whereas B3 andMP2 computations overestimate the stability of type II conformers(see Table S3 of the SI).The MAX and MUE of rDSDcomputations with respect to the junChSF12reference  are too unstable to permit their unequivocal detectionin MW experiments. Furthermore, relaxation of I′ and III′conformers to their more stable I and III counterparts is ruled bylow energy barriers, which are easily overcome in the typical conditionsof supersonic-jet expansion. Low energy barriers govern also the relaxationof III to I and II to II– conformers. As a consequence,only the I and II– conformers could be detectedin MW studies, with the former collecting the populations of I, III–, III, I′, I′–, III′,and III′– conformers and the latter thoseof the II and II– conformers. It is remarkable thatthe relative population of conformer I computed at room temperaturefrom the free energies collected in 41 whereas a significantlylower relative population (54%) would have been predicted neglectingzero point and thermal effects.As already mentioned, the comparison with experimentrequires thecomputation of the relative free energies for the different conformersat the temperature of the carrier gas  and of transition states ruling their interconversion.The results collected in – conformers. A remarkable agreement is notedwith the MAX and MUE of rDSD-LRA/B3 rotational constants (36.1 and10.5 MHz) being even better than those (50.5 and 12.2 MHz) obtainedat the much more expensive CCSD(T)/cc-pVTZ level.106 It is noteworthy that for both conformers of alanine theerror on the B0 rotational constant ismuch higher than those affecting the other two rotational constants,whereas in both the observed conformers of glycine the largest errorwas found for A0.αC′ valence angle, which decreasesby about 3.5° when going from the I to the II– conformer, consistent with the trans-angle rule of hyperconjugativeand steric effects.110 At the same time,the C=O bond length shows the expected lengthening by about0.002–0.003 Å when going from free (structure II–) to hydrogen-bonded (structure I) forms.The geometrical parameter most sensitiveto conformational changesis the NCα–C′bond length (shorter in glycine by about 0.007 Å for both conformers)and the NCαC′ valence angle (narrower in glycineby about 2° for both conformers). Therefore, the main structuraldifferences between glycine and alanine are highly localized at theCα. As already mentioned, the ψ torsional anglecharacterizes the backbone deviation from planarity (see Tables S2 and S3 of the SI). For I conformers, it is exactly equal to 180° in glycine,whereas the lack of any symmetry induces a change of more than 15°in alanine. On the other hand, comparable ψ values are observedfor the II forms of glycine and alanine .The only significantdifferences between the geometrical parametersof glycine and those of alanine concerns the C5.242 isoleucine,43 andleucine44) present two dominant conformersof types I and II, respectively. On the other hand, the conformationallandscape of natural amino acids with polar side chains is much richerdue to the synergy or competition between intrabackbone and backbone(side chain) hydrogen bonds.Systematicinvestigations have revealed that, in analogy with alanine,the natural amino acids containing simple nonpolar side chains , withthe OH moiety able to act either as donor or acceptor in quite strongintramolecular hydrogen bonds.111 The increasednumber of soft degrees of freedom (from 3 to 5) makes this systemsuitable for applying the PES exploration strategy introduced in theprevious sections, which produces 12 low-energy conformers (see Let us start our discussion fromserine (Ser), which has two soft degrees of freedom in its CHmers see 5.–g– conformerrelaxes to the more stable IIg–t form through rotationaround χ2; IIIg–g relaxes to Ig–g through rotation around ψ; the less stableIIg–t conformer relaxes to its more stable counterpartthrough a planar structure ;Igt relaxes to I′gg– through rotation aroundχ2, and Igg relaxes to III′gg through rotationaround ψ. We are thus left with seven conformers possibly detectablein MW experiments: three of type II, two of type III′, andone each for types I and I′  hydrogen bond between the backboneand the side chain in addition to the intrabackbone HNH···OHhydrogen bond.All the most stable conformersare stabilized by both intrabackboneand backbone (side chain) hydrogen bonds see 3. Furthe45 According to both theory and experiments, thefirst four conformers  are significantly more stable than the two conformersof type III′ and a further conformer of type II, which have,in turn, comparable stability  are obtained at the rDSD-LRA level, whose strongly reducedcost has allowed us to compute the spectroscopic parameters of allthe other low-energy conformers. The remarkable agreement betweencomputed and experimental results for all the detected conformersof serine confirms the accuracy of our computational strategy.The rotational constants of the two most stable conformershavebeen recently computed by geometry optimizations at the ChS level,reaching MAX and MUE of 28.7 and 10.6 MHz, respectively.113 in which a methyl group replaces one of the hydrogen atomsbonded to Cβ, leading to the CHCH3OH sidechain which has again two soft degrees of freedom (χ1 = N–Cα–Cβ–Oand χ2 = Cα–Cβ–O–H) since the terminal methyl group is frozen ina staggered conformation with an estimated rotation barrier of 1400cm–1. There is now a second chiral center in additionto the Cα atom, with the natural amino acid being2S,3R-threonine. The conformationallandscape of threonine has been investigated in two different studies,114 which obtained 71 and 56 conformers, respectively, in a range ofabout 4000 cm–1, but the final set of conformerswas the same up to a relative energy of 1600 cm–1. The knowledge-based step of our conformational exploration startedfrom the 12 low-energy conformers of serine collected in –1) collected in The next studied system is threonine (Thr),–g– is too lowto allow its detection in MW experiments,and conformers IIgt and Igt relax easily to conformers IIgg and Ig–g, respectively. We are thus left with the same number(seven) and backbone conformation  of thestructures discussed above for serine, which should be  detected in MW experiments. However,the presence of the β methyl group increases the energy barriergoverning relaxation of the III′g–g conformerto its Ig–g counterpart from about 200 to about800 cm–1 when going from serine to threonine. Asa consequence, the III′g–g conformer is observedin threonine in place of the less stable III′tg– conformer observed in serine (see The predicted populationof conformer IIgrine see 4. At therine see 5.–g) and the threeleast stable  conformers are the same in terms of electronicenergies, enthalpies, or free energies. The relative ordering of thetwo intermediate conformers is, instead, altered by both ZPE and thermalcontributions.The two most stable .One might think that the same conformers should be detected for cysteineand serine. However, the strengths of the H-bonds possibly formedby the thiol group are weaker than those of its alcohol counterpart.Therefore, it is expected that the barriers separating low-lying conformersdecrease and in some instances may even disappear. In ref .Replacement of the oxygen atom in the side chain ofserine by asulfur produces cysteine (Cys), whose CH. In ref , a syste– relaxes easily toits Ig–g counterpart, which has a similar shape.Therefore, the number of detectable conformers reduces to 6: two eachfor types I, II, and III′ , whereas errors as large as40 MHz are obtained for the A0 rotationalconstant. For most conformers, the C–S bond is nearly perpendicularto the average backbone direction .The spectroscopicparameters computed at the rDSD level are inremarkable agreement with their experimental counterpartsectively 10. The etion see 6 and is,2COOH side chain has threedihedral angles  and χ3 = Cβ–Cγ–O–H). However, χ3 is frozen in trans (favored and not explicitly labeled inthe following) or cis (labeled by c in the following) conformations.A recent systematic analysis of the conformational landscape116 identified 19 energy minima in a range of 3500cm–1, and we were able to locate all those minimaby our general exploration strategy with enlarged energy thresholds.Within this panel of candidates, only 9 conformers have electronicenergies lying within 1000 cm–1 above the absoluteenergy minimum  hydrogen bond. The increased stability explainsalso the absence of low-barrier relaxation paths from these conformersto I structures.Both the I′g96 computations forecasted that one or two differentconformers should be experimentally detected and that the spectroscopicconstants obtained at that level show MAX and MUE with respect toexperiment (29.2 and 10.6 MHz) more than three times larger than theirrDSD-LRA counterparts (8.2 and 3.1 MHz). The rDSD MUE  approaches again the accuracy of state-of-the-art compositemethods for small semirigid molecules117 and permits the unbiased assignment of MW spectra.118 The stability order of the six most populated conformersis, however, quite different between theory and experimental estimateswith the strongest discrepancy concerning the inversion of the relativestability of I and II species. Although the experimental populationstake into account also possible relaxation of higher-energy structuresto the most stable conformers, according to the computed free energiesthe initial populations of all the species outside the six most stableones are too low to alter the computed relative populations. Fromanother point of view, the experimental estimates are based on a numberof assumptions, which might not be fulfilled in the present case.Also taking these considerations in mind, the agreement between theoryand experiment concerning the nature and spectroscopic parametersof all the observable species remains remarkable.The spectroscopic parameters collected in 119 containing an amide group. The soft degreesof freedom of the asparagine side chain (CH2CONH2) include two dihedral angles  because the coupled rotation/inversiondisplacements of the NH2 amide moiety from the planar referencestructure can be safely added to the panel of stiff degrees of freedom.The amide moiety can act either as a proton donor or as a proton acceptor,with this increasing the number of possible backbone (side chain)intramolecular hydrogen bonds. Asparagine in the gas-phase has beenwidely studied by both computational120 and experimental121 points of view, but a comprehensive characterization of its structureand conformational landscape has not yet been performed by state-of-the-artquantum chemical methods.The last system considered in this study is asparagine,which isthe only proteinogenic α-amino acid, together with glutamine,–1 above the absolute energy minimum (see 2 amidic moietyin the side chain permits the compensation of the weak hydrogen bondin the carboxylic moiety (lacking in II structures of aspartic acidwith respect to their I counterparts) by a backbone/side chain OH···NH2 hydrogen bond without reducing the local stability of theamide moiety. In fact, an analogous situation would involve a 180°rotation of the OH moiety in the carboxylic group of the side chainin aspartic acid away from its most stable arrangement.The usual exploration/refinement strategyprovides 5 conformerswith rDSD electronic energies within a little more than 1000 cmimum see 13. At thimum see 8 should –t form , which involvesthe best donor and the best acceptor, followed by H2–N···HNH(with an estimated strength of 2400 cm–1). Thosevalues, together with the energy differences among conformers I, II,and III of glycine, permit strengths of about of 1700 and 1100 cm–1 to be estimated for the bifurcated NH2···O=C and NH2···O–Hhydrogen bonds. Furthermore, the difference between the pairs I, I′and III, III′ leads to hydrogen bond strengths of about 1300and 800 cm–1 for the more conventional single H–N–H···O=Cand HNH···O–H hydrogen bonds. Finally, a comparablestrength of about 800 cm–1 is estimated for theH2N···H–S, H–S···H–N–H,and S–H···O=C hydrogen bonds. It is thenquite straightforward to understand why conformer I is more stablethan its II counterpart in the absence of backbone (side chain) hydrogenbonds : in fact, the sum of NH2···O=Cand favorable carboxyl conformation exceeds by about 200 cm–1 the stronger H2N···H–O hydrogenbond but with an unfavorable conformation of the carboxylic moiety.On the other hand, in serine and threonine, conformer II becomes morestable due to the extra stabilization related to an O–H···O=Chydrogen bond involving the backbone and the side chain. The sameoccurs in cysteine, where the 800 cm–1 gained fromthe S–H···O=C hydrogen bond makes theIIgg conformer more stable than the Igg counterpart by about 600 cm–1. An analogous situation is found in aspartic acid,where the amine moiety is involved at the same time in an OH···Nhydrogen bond within the backbone and a HNH···O=Chydrogen bond with the side chain. Finally, IIgg is by far the moststable conformer in asparagine because the presence of an amide groupallows the formation of two additional backbone (side chain) hydrogenbonds. Type III conformers are intrinsically less stable than theirI counterparts (due to the lower strength of NH2···O–Hwith respect to NH2···O=C hydrogenbond), and moreover, they can easily relax to I forms through rotationaround ψ when not locked by additional interactions. However,III′ conformers featuring a single H–N–H···O–Hhydrogen bond can be stabilized and locked into sufficiently deepenergy wells upon involvement of the released N–H bond intoadditional hydrogen bonds with the side chain. This is the case, forinstance, of the III′gg conformer in serine, threonine (H–O···H–N–H···O–H),and cysteine (H–O···H–N–H···S–H).The accurate results obtained for several aminoacids permit thestrengths of the main interactions governing the conformational landscapesof these flexible systems to be estimated. In particular, approximatevalues for the strengths of different hydrogen bonds can be computedfrom prototypical systems and used to rationalize energy differencesamong the conformers of different amino acids in terms of sums ofstabilizations from near-atom interactions. Based on the energy differencebetween Ic and I or I′c and I′ conformers of glycine,for each carboxyl group ω = 180° is more stable than ω= 0° by about 1700 cmHydrogen bonding is surely the driving force ruling the generaltrends of structures and relative stabilities, but the detailed geometryand energy changes between conformers depend strongly on other stereoelectroniceffects like, e.g., hyperconjugation or steric repulsion. For instance,any additive picture based on individual hydrogen bond strengths istuned by the preference of bulky vicinal substituents for trans orgauche conformations, which, in turn, depends on the balance betweenelectrostatic, steric, and hyperconjugative effects. Furthermore,vibrational effects (affecting both ZPEs and entropic contributions)alter the stability order provided by relative electronic energiesand must be taken into the proper account.115 we point out that only the availability of accurateresults including all the stereoelectronic and vibrational effects(like those reported in the present paper) can provide an unbiasedreference for building more realistic models  for the study of flexible biomolecules.While the readeris referred to studies of specific systems formore detailed analyses along these lines,6In this paper, ageneral strategyaimed at the unbiased disentanglementof the conformational bath of flexible biomolecule building blocksin the gas phase has been further improved and validated for the specificcase of representative natural α-amino acids. The use of curvilinearinternal coordinates permits the separation between stiff and softdegrees of freedom. Then, effective exploration of the soft variablescan be performed by purposely tailored evolutionary algorithms, whosefitness scores are obtained by constrained geometry optimizationsof the stiff degrees of freedom employing a fast semiempirical method.Refinement of the energies and structures by a hybrid and then a last-generationdouble-hybrid functional allows very reliable results to be obtainedminimizing the number of expensive computations. Application of theprocedure to supersonic jet experiments requires also the locationof transition states ruling the interconversion between pairs of adjacentenergy minima and the identification of fast relaxation processes.Improved structures and relative energies are obtained by the rDSD-LRAapproach and the junChSF12 composite method, respectively. Finally,the spectroscopic parameters of sufficiently populated conformerscan be safely computed at the rDSD level.The results obtainedfor glycine, alanine, and, especially, differentnatural α-amino acids with polar side chains are in full agreementwith the available spectroscopic data and permit their unbiased interpretationin terms of the cooperation or competition between intrabackbone andbackbone (side chain) hydrogen bonds.Together with the intrinsicinterest of the studied molecules,the results of the present investigation show that highly reliableanalysis of the conformational landscape is today possible for flexiblebuilding blocks of biomolecules in the gas phase. Furthermore, weprovide benchmark results for the validation of cheaper quantum chemicalmethods, which become unavoidable for large biomolecules.