Daniel Hubé

How to finish with Armageddon? The history and consequences of industrial-scale, old-ammunition destruction in the interwar period on the western front

Daniel Hubé

To cite this version: Daniel Hubé. How to finish with Armageddon? The history and consequences of industrial-scale, old-ammunition destruction

SMH 2020 Conference April 30-May 3 2020 87th Annual Meeting of the Society for Military History

Hosted by the Army Historical Foundation

CATCHY TITLE: Unexploded Ordnance and Militarized Landscapes

When wars end, states and militaries require comprehensive disarmament and reconstruction strategies that address the colossal political, economic, and social challenges involved in making peace. Scholars such as Michael Neiberg, BLANK, and BLANK, have focused on high-level treaty negotiations, international relations, and veterans rehabilitation, but as the papers in this panel show, the legacies of war also affected the environment and society profoundly.

In the aftermath of war, new national and geographic boundaries are defined, former battlefields are cleaned up, and devastated landscapes are remediated before the mass of displaced people return home. Moreover, the tools of death and destruction do not simply vanish, as captured weapons and surplus ordnance must be collected and catalogued ahead of final disposal.

During the First Word War (WWI), the power, accuracy, firing rates and ranges of artillery reached an unprecedented scale. When hostility ceased 1.7 million tonnes of Excess, Obsolete, Unserviceable Ammunition (EOUA) had been abandoned by all belligerents. Plus around 25% of the one billion fired artillery projectiles had failed to detonate, meaning that the amount of old ammunition (OA) grew when Unexploded Ordnance (UXO) emerged out farm fields when ploughing started again. The destruction of OA quickly became an urgent need during postwar reconstruction, but such a huge task had never been undertaken before. The French and Allied armies started disposal and destruction of OA using common open-detonation methods. Stacked OA was blown in place using an explosive charge. From 1920, civilian companies took over this work on an industrial scale. New methods (including defuzing, open-burning or washing-out) for safely breaking down a large variety of ammunition were developed to recover valuable scrap materials. This paper draws on recent and detailed archive research which indicates that approximately 2.5 to 3 million tonnes of OA were destroyed in France during the interwar period. These operations had serious health and environmental consequences. Numerous workers were wounded or killed during operations that also caused severe soil contamination. To this day, no vegetation grows on the location of some former ammunition destruction factories. Furthermore, there is some evidence indicating that regional scale perchlorate groundwater contamination in northeastern France may be linked to the destruction of OA after WWI. Nevertheless, industrial scale ammunition destruction facilities have disappeared from the collective memory, but the contamination from a hundred years ago still endangers people, threatens land-use planning, and the management of water resources. This original paper discusses the reasons why this history of demilitarization has been forgotten.

TITLE Alex Souchen

During World War II, Allied war factories produced more than 60 billion rounds of ammunition, bombs, and shells. However, when the war ended in 1945, not all of this ordnance was expended or needed for postwar operations, while the bulk of captured enemy weapons added to the surpluses accumulating around the world. Victory triggered a global "disposal problem" that the Allies addressed by dumping hundreds of millions of tons of ordnance into the oceans.

Drawing from a wide range of archival sources and the latest scientific research, my paper will explore the environmental history of underwater munitions by focusing on three related topics. First, it will profile contemporary knowledge of toxicity and the environmental ethics of Allied leaders in order to explain why they chose dumping over other available disposal methods and polluted the oceans for disarmament purposes. Second, the paper will offer an overview of the global scale of dumping operations and highlight particular areas of concern, where large concentrations of chemical and conventional munitions were dumped. Finally, the paper will discuss the evolving ecological and human health risks of underwater munitions. Dumping munitions in the oceans never eliminated their deadly nature; instead it delayed and dispersed the violence across time and space. Corroded bombs can spontaneously detonate or leach toxic chemicals and carcinogens into the food chain. They also threaten the offshore economy and energy industries, while fishermen and tourists are frequently injured by encounters with mustard gas and incendiaries. By shedding light on the history of munitions disposal, my paper offers new insights into the history of warfare and the military's impact on marine environments. In doing so, it discusses the evolution of disposal strategies and identifies the socio-economic costs of military pollution

The Unintended Beauty of War: the Biodiversity of the DMZ Louise Tumchewics After three years of conflict, the Korean War formally came to an end on 27 July 1953. The United Nations Command, Chinese People's Liberation Army, and North Korea People's Army agreed to an armistice. The agreement effectively divided the two Koreas into two countries, separated by a 250-kilometre long (160 mile) 4-kilometre (2.5 miles) wide demilitarised zone, roughly tracing the 38 th parallel. Although most troops and heavy weaponry were removed from the area, it remains one of the mostly heavily mined areas on earth, consequently making a so-called 'demilitarised' zone one of the most militarised landscapes in existence.

Millions of landmines have made the area impassable for humans yet allowed an abundance of animal and plant life to flourishes, undisturbed by human activity. The border region is home to the Siberian flying squirrel, black bears, golden eagles, water deer, and the iconic, and endangered, red crane. War, and its aftermath, have created a rare refuge that preserves that natural history of the Korean peninsula.

For decades, both Koreas view the presence of landmines in the DMZ as integral to their national security. In recent years, South Korea has pursued policies to demine and develop the DMZ and neighbouring regions in an effort to promote détente between the two countries. However, demining could prove a lengthy task, given the sheer volume of UXO, and the movement of the soil in the intervening decades. Development -in the form of roads through the area, and attendant human presence, would disrupt the fragile ecosystem. Areas could be set aside for protection, but North Korea views biosphere reserves in the DMZ as potentially hostile land grabs. Disrupting the mines could disrupt fragile relationships between the two countries. This paper explores the challenges of balancing the preservation of ecological history with the promotion of peace.


Bénédicte Buffin-Meyer

Marcin Tkaczyk

Małgorzata Stańczyk

Benjamin Breuil

Justyna Siwy

Krzysztof Szaflik

Tomasz Talar

Justyna Wojtera

Waldemar Krzeszowski

Stéphane Decramer

Julie Klein

Joost P Schanstra

A single center study to evaluate the efficacy of a fetal urine peptide signature predicting postnatal renal outcome in fetuses with posterior urethral valves

Keywords: Urinary proteome analysis, prenatal, prediction, outcome, chronic kidney disease, rare disease

Background -Posterior urethral valves (PUV) accounts for 17% of pediatric renal failure. The management of pregnancies involving fetuses with PUV is hampered by the fact that current clinical parameters obtained from fetal ultrasound and/or fetal urine biochemistry are insufficient to predict postnatal renal function. We previously have developed a fetal urine peptide signature (12PUV) that predicted with high precision postnatal renal failure at 2 years of age in fetuses with PUV. Here we evaluated the accuracy of this signature to predict postnatal renal outcome in fetuses with PUV in an independent single center study.

Methods -Thirty three woman carrying fetuses with suspected PUV were included. Twenty five fetuses received vesicoamniotic shunts (VAS) during pregnancy. PUV was confirmed postnatally in 23 patients. Of those 23 fetuses, 2 were lost in follow-up. Four and 3 patients died in the pre-and perinatal-period, respectively. Follow-up renal function at 6 month of age was obtained for the remaining 14 patients. The primary outcome was early renal failure, defined by an eGFR<60 mL/min/1.73 before 6 months of age or pre-or perinatal death.

Results -

The peptide signature predicted postnatal renal outcome in postnatally confirmed PUV fetuses with an AUC of 0.94 (95%CI: 0.74-1.0) and an accuracy of 90% (95%CI: 78-100). The signature predicted postnatal renal outcome for the suspected PUV cases with an AUC of 0.89 (95%CI: 0.72-0.97) and an accuracy of 84% (95%CI: 71-97).

Conclusion -

This single center study confirms the predictive power of the previously identified 12PUV fetal urinary peptide signature.

Thirty three woman carrying fetuses with suspected PUV were included. Twenty five fetuses received vesicoamniotic shunts (VAS) during pregnancy. PUV was confirmed postnatally in 23 patients. Of those 23 fetuses, 2 were lost in follow-up. Four and 3 patients died in the pre-and perinatal-period, respectively. Follow-up renal function at 6 month of age was obtained for the remaining 14 patients. The primary outcome was early renal failure, defined by an eGFR<60 mL/min/1.73 before 6 months of age or preor perinatal death.Results -The peptide signature predicted postnatal renal outcome in postnatally confirmed PUV fetuses with an AUC of 0.94 (95%CI: 0.74-1.0) and an accuracy of 90% (95%CI: 78-100). The signature predicted postnatal renal outcome for the suspected PUV cases with an AUC of 0.89 (95%CI: 0.72-0.97) and an accuracy of 84% (95%CI: 71-97).Conclusion -This single center study confirms the predictive power of the previously identified 12PUV fetal urinary peptide signature.

Response to Reviewers:

Reviewers' comments:

Reviewer #1: R1Q1: The authors should supply more clinical data on the patients that died in the perinatal period. Especially cause and time of death should be provided. The authors stated that cause of death was most likely severe renal disease but hypoplasia of the lungs may have accounted for several deaths in the perinatal period. Please clarify. R1A1: Thank you for this comment.

-First of all we would like to correct the numbers of patients that died in the perinatal period (defined by 22 gestational weeks until 7 completed days after birth) which we did not define correctly in the first version of the manuscript. Taken into account this gestational age-range, only 3 patients died in the perinatal period. Hence this was corrected in the text on page 6 and in Table 1.

-Indeed we obtained some information on the non-renal causes potentially involved in the death of those 3 patients in the perinatal period: "Three patients died in the perinatal period (2 patients died within the first 12 hours from delivery and 1 neonate died after 64 hours from delivery). These 3 patients had pulmonary insufficiency, renal insufficiency, secondary cardiac insufficiency with no-response to resuscitation (ventilation) and vasopressors, and neurological complications (intracranial hemorrhage)." This has been added to the results section on page 6 and the comment on the absence of clinical data in the perinatal period was deleted from the discussion section. However no information on the prenatal death other than confirmation of PUV was obtained.

R1Q2: Although the AUC of the ROC analysis carries the highest informative value, the authors should also provide sensitivity and specificity as achieved by the test's cut-off value as used in the results section. R1A2: Sensitivity and specificity has been added to the results section using the cut-off values of the original STM study on page 6 and 7.

Reviewer #2: R2Q1: Introduction: Clarify how better predictors may affect management (prenatal counseling, postnatal follow up) R2A1: We have introduced a paragraph in the introduction describing how improved prediction may affect management on page 3.

R2Q2: Methods: How was simple size determined R2A2: Thank you for this important remark that made us reconsider some of the wording in the manuscript. We did not determine a sample size before starting the study, as the current study was an excellent opportunity to perform a preliminary but small independent "pre-validation" of the signature.

As mentioned in the discussion of the manuscript, a large-scale international multicenter study, called ANTENATAL, is currently ongoing (finishing in 2023) aiming the multicenter validation of the 12PUV fetal urine peptide signature. The power calculation including loss of follow-up and refusal to participate in the ANTENATAL study estimated screening of ~400 patients with PUV for this study. This number is clearly not reachable by a single center for a rare disease such as PUV. Therefore we believe we have not correctly used the word "validation" throughout the current study since this should be reserved for the ANTENATAL study.

As indicated in the present work, the signature displayed similar efficacy in the current single center study to predict outcome compared to the initial STM study. We therefore omitted the word "validation" throughout the revised manuscript and replaced it by "study of efficacy" or similar wording.

R2Q3: Results: What is the value of including infants who didn't have confirmed PUV in the second analysis, this seems to be unnecessary. R2A3: We respectfully disagree with this reviewer that including infants without confirmed PUV is unnecessary. In the clinical situation, where the use of the 12PUV signature is envisaged, management of the disease and parental counselling occur in the prenatal period where PUV is suspected but not yet confirmed (unless laser ablation of the valves is performed in utero). In most cases the presence of valves is only confirmed after birth. We believe that this justification is correctly worded in the discussion section but is only shortly described in the section where we present the results which might have not fully convinced this reviewer. Therefore, we have added an additional justification to the results section as well on page 7.

R2Q4: Discussion: "The 12PUV fetal urine peptide signature-based prediction tends to confirm this by displaying absence of differences in the predictive capacity of the signature between patients that underwent VAS or not." This study was not intended to evaluate that outcome, besides it is a very small number for comparison. R2A5: Although we agree that the study is small scale we believe that it is worth to point out that the majority of the patients (91%) in the current study underwent VAS compared to the initial study (none of the patients was subjected to VAS) on which the 12PUV signature is based. We have therefore slightly reorganized this paragraph and removed the phrase containing "tends to confirm" on page 8.

R2Q5: One of the limitations is the short follow-up period. R2A5: We have added this limitation in the discussion on page 9.

R2Q5: Validating biomarkers in a separate cohort is a strength however the n is small. R2A5: We agree with this reviewer that the n is small. This was already mentioned in the original version of the manuscript as a study limitation in the discussion section. In addition, as discussed above in R2A2, we therefore reconsidered rewording this study as a "validation" study, and to employ the term "study of efficacy" throughout the revised manuscript. Biomarker validation will be performed in the large-scale ANTENATAL study (n=400 Dear Editor, Thank you for sending out our manuscript entitled "Independent single center validation of a fetal urine peptide signature of postnatal renal outcome in fetuses with posterior urethral valves" by Buffin-Meyer and Tkaczyk et al, for review in Pediatric Nephrology.

We found the comments of the reviewers very helpful and we feel they have improved on the reporting of the study. We have replied point-by-point to all comments below and the major modifications have been introduced in the text with a bleu font.

The major changes are:

-Following the comment of reviewer 2 on the sample size, we have omitted the term "validation" from the entire manuscript since the study does not have enough power to use the term "validation.

Validation was replaced by "study of efficacy" or similar wording.

-We wrongly defined the perinatal period and patients are now better divided in the perinatal (22 weeks of gestation to 7 days after birth) and the prenatal period.

-We obtained information on lesions other than in the kidney for the 3 patients that died in the perinatal period as requested.

-A comparison with the previous study has been added to Table 2.

We hope that these modifications make the study suitable for publication in Pediatric Nephrology.

Best regards Joost Schanstra

______________________________________________

Reviewers' comments:

Reviewer #1: R1Q1: The authors should supply more clinical data on the patients that died in the perinatal period.

Especially cause and time of death should be provided. The authors stated that cause of death was most likely severe renal disease but hypoplasia of the lungs may have accounted for several deaths in the perinatal period. Please clarify. R1A1: Thank you for this comment.

-First of all we would like to correct the numbers of patients that died in the perinatal period (defined by 22 gestational weeks until 7 completed days after birth) which we did not define correctly in the first version of the manuscript. Taken into account this gestational age-range, only 3 patients died in the perinatal period. Hence this was corrected in the text on page 6 and in Table 1.

-Indeed we obtained some information on the non-renal causes potentially involved in the death of those 3 patients in the perinatal period: "Three patients died in the perinatal period (2 patients died within the first 12 hours from delivery and 1 neonate died after 64 hours from delivery). These 3 patients had pulmonary insufficiency, renal insufficiency, secondary cardiac insufficiency with noresponse to resuscitation (ventilation) and vasopressors, and neurological complications (intracranial hemorrhage)." This has been added to the results section on page 6 and the comment on the absence of clinical data in the perinatal period was deleted from the discussion section. However no information on the prenatal death other than confirmation of PUV was obtained.

R1Q2: Although the AUC of the ROC analysis carries the highest informative value, the authors should also provide sensitivity and specificity as achieved by the test's cut-off value as used in the results section. R1A2: Sensitivity and specificity has been added to the results section using the cut-off values of the original STM study on page 6 and 7.

Reviewer #2: R2Q1: Introduction: Clarify how better predictors may affect management (prenatal counseling, postnatal follow up) R2A1: We have introduced a paragraph in the introduction describing how improved prediction may affect management on page 3.

R2Q2: Methods: How was simple size determined R2A2: Thank you for this important remark that made us reconsider some of the wording in the manuscript.

We did not determine a sample size before starting the study, as the current study was an excellent opportunity to perform a preliminary but small independent "pre-validation" of the signature.

As mentioned in the discussion of the manuscript, a large-scale international multicenter study, called ANTENATAL, is currently ongoing (finishing in 2023) aiming the multicenter validation of the 12PUV fetal urine peptide signature. The power calculation including loss of follow-up and refusal to participate in the ANTENATAL study estimated screening of ~400 patients with PUV for this study. This number is clearly not reachable by a single center for a rare disease such as PUV. Therefore we believe we have not correctly used the word "validation" throughout the current study since this should be reserved for the ANTENATAL study.

As indicated in the present work, the signature displayed similar efficacy in the current single center study to predict outcome compared to the initial STM study. We therefore omitted the word "validation" throughout the revised manuscript and replaced it by "study of efficacy" or similar wording.

R2Q3: Results: What is the value of including infants who didn't have confirmed PUV in the second analysis, this seems to be unnecessary. R2A3: We respectfully disagree with this reviewer that including infants without confirmed PUV is unnecessary. In the clinical situation, where the use of the 12PUV signature is envisaged, management of the disease and parental counselling occur in the prenatal period where PUV is suspected but not yet confirmed (unless laser ablation of the valves is performed in utero). In most cases the presence of valves is only confirmed after birth. We believe that this justification is correctly worded in the discussion section but is only shortly described in the section where we present the results which might have not fully convinced this reviewer. Therefore, we have added an additional justification to the results section as well on page 7.

R2Q4: Discussion: "The 12PUV fetal urine peptide signature-based prediction tends to confirm this by displaying absence of differences in the predictive capacity of the signature between patients that underwent VAS or not." This study was not intended to evaluate that outcome, besides it is a very small number for comparison. R2A5: Although we agree that the study is small scale we believe that it is worth to point out that the majority of the patients (91%) in the current study underwent VAS compared to the initial study (none of the patients was subjected to VAS) on which the 12PUV signature is based. We have therefore slightly reorganized this paragraph and removed the phrase containing "tends to confirm" on page 8.

R2Q5: One of the limitations is the short follow-up period.

R2A5: We have added this limitation in the discussion on page 9.

R2Q5: Validating biomarkers in a separate cohort is a strength however the n is small. R2A5: We agree with this reviewer that the n is small. This was already mentioned in the original version of the manuscript as a study limitation in the discussion section. In addition, as discussed above in R2A2, we therefore reconsidered rewording this study as a "validation" study, and to employ the term "study of efficacy" throughout the revised manuscript. Biomarker validation will be performed in the large-scale ANTENATAL study (n=400).

Reviewer

Editorial Office:

EO-Q1: Your methods section is largely copied from one of your previous papers. While this paper is OpenAccess, rather than just reusing the same text, please either rewrite, or refer to the methods 'as previously described in ... ' and cite the paper, then possibly adding a brief description. EO-A1: The section "Fetal urinary peptidome analysis" in the methods section was rewritten as suggested in the revised version of the manuscript on page 4.

EO-Q2: Please supply your manuscript in an editable format, eg MS Word, rather than PDF EO-A2: An MS Word version of the revised manuscript was submitted.

EO-Q3: Please revise your abstract to include the following category headings -Background, Methods, Results, Conclusions EO-A3: We inserted these headings in the revised version of the abstract.

EO-Q4: Please include a conflict of interest statement in your manuscript. EO-A4: We have inserted the following COI on page 9 of the revised manuscript for Justyna Siwy (JS) "JS is an employee of Mosaiques diagnostics GmbH (Hannover, Germany) that markets urinary peptide-based diagnostics products."

EO-Q5: In your reference list, please ensure that journal titles are correctly abbreviated in line with Index Medicus, eg as used on PubMed EO-A5: We have double checked and correct abbreviations were used. E.g. ScientificWorldJournal is correct and is used as one word in Pubmed.

EO-Q6: Please define the abbreviations used in each table in table legends.

Introduction

Posterior urethral valves (PUV) is a congenital anomaly associated with a wide spectrum of outcomes ranging from extremely severe phenotypes with prenatal death to live born children with normal renal function. Exclusively found in males, PUV is the major cause of severe obstructive nephropathy in children. Although a rare disease (ORPHA:93110, with a prevalence of 1-9 / 100 000), it accounts for 17% of pediatric end-stage renal disease (ESRD) [START_REF] Agarwal | Urethral valves[END_REF][START_REF] Hodges | Posterior urethral valves[END_REF][START_REF] Krishnan | The anatomy and embryology of posterior urethral valves[END_REF].

The management of PUV is hampered by the absence of tools for adequate antenatal screening of PUV fetuses at high risk for developing renal failure. Current clinical practice is based on prenatal ultrasound and fetal urine biochemistry but this is clearly insufficient to predict postnatal renal outcome with any accuracy in PUV patients as shown in several (meta) studies [START_REF] Morris | Systematic review of accuracy of fetal urine analysis to predict poor postnatal renal function in cases of congenital urinary tract obstruction[END_REF][START_REF] Morris | Antenatal ultrasound to predict postnatal renal function in congenital lower urinary tract obstruction: systematic review of test accuracy[END_REF]. This lack of efficient prognostic methods leads to many parents opting for termination of pregnancy [START_REF] Morris | Systematic review of accuracy of fetal urine analysis to predict poor postnatal renal function in cases of congenital urinary tract obstruction[END_REF][START_REF] Morris | Antenatal ultrasound to predict postnatal renal function in congenital lower urinary tract obstruction: systematic review of test accuracy[END_REF][START_REF] Hogan | Renal outcome in children with antenatal diagnosis of severe CAKUT[END_REF]. Better predictors would allow adequate selection of fetuses that would potentially benefit from prenatal intervention (vesicoamniotic shunting (VAS) or laser ablation of valves). In addition, when predicted in the high-risk stratum, clear-cut knowledge of the outcome of the disease will give time to the future parents to psychologically accept [START_REF] Stevenson | Ethical dilemmas in the delivery room[END_REF] the fact that they will have a child with chronic, potentially severe disease and decide whether they would like their newborn to be offered palliative care or renal replacement therapies (i.e. dialysis or transplantation) [START_REF] Lantos | The evolving ethics of infant dialysis[END_REF]. Hence, new predictive strategies are necessary to allow optimal diagnostic and therapeutic management of the disease.

Urinary peptidome analysis has emerged as a potent tool for the discovery and validation of biomarkers of renal disease [START_REF] Klein | The role of urinary peptidomics in kidney disease research[END_REF], both in adults [START_REF] Schanstra | Diagnosis and Prediction of CKD Progression by Assessment of Urinary Peptides[END_REF][START_REF] Pontillo | A urinary proteome-based classifier for the early detection of decline in glomerular filtration[END_REF] and in children [START_REF] Decramer | Predicting the clinical outcome of congenital unilateral ureteropelvic junction obstruction in newborn by urinary proteome analysis[END_REF][START_REF] Drube | Urinary proteome analysis identifies infants but not older children requiring pyeloplasty[END_REF][START_REF] Schanstra | Proteomic urinary biomarker approach in renal disease: from discovery to implementation[END_REF]. In the setting of PUV, we identified a fetal urinary peptide signature called 12PUV that predicted in utero the postnatal renal function of PUV fetuses with high accuracy [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF], outperforming routine ultrasound and fetal urine biochemistry. Here, as a first step in the validation process, we evaluated the accuracy of the 12PUV signature to predict postnatal renal outcome in an independent single center study including 33 suspected PUV pregnancies.

Material and Methods

Clinical data and sample collection

Patients were included in the study upon suspicion of PUV due to the presence of a megabladder and/or oligohydramnios/anhydramnios. Prenatal ultrasound findings obtained at the time of fetal urine sampling were recorded. The study endpoint was the renal status after 6 months of postnatal follow-up. For live born children, renal function was estimated at 6 months of age using serum creatinine concentrations according the Schwartz method [START_REF] Schwartz | New equations to estimate GFR in children with CKD[END_REF]. Because there are no definite predictive factors of pregnancy survival -just recommendations or suggestions -, our team of gynecologists offered VAS as an option to all patients with lower urinary tract obstruction (LUTO). Patients with confirmed megabladder (with or without bilateral hydronephrosis) and amniotic fluid reduction (AFI 8 or less) during 2 separate ultrasound assessments qualified for intervention. Almost all approached patients opted for intervention.

The study was performed in accordance with the Declaration of Helsinki [START_REF]World Medical Association declaration of Helsinki. Recommendations guiding physicians in biomedical research involving human subjects[END_REF] and with Good Clinical Practice guidelines. The research was approved by the local (Lodz, Poland) ethics committee (N° 1/2016) and informed consent was obtained from each participant.

Fetal urinary peptidome analysis

Fetal urine was collected from the bladder of the fetus under ultrasound guidance and directly frozen.

Samples were shipped on dry-ice to Toulouse (France). Samples were prepared for capillary electrophoresis coupled to mass spectrometry (CE-MS) analysis as described in detail [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF]. Briefly, proteins were eliminated by dilution of the fetal urine sample in ammonium hydroxide buffer containing SDS to decrease the interaction of peptides and proteins followed by centrifugation using a 20-kDa cut-off filter. In the next step salts were removed from the filtrate by gel filtration followed by lyophilization of the sample. The lyophilized samples were stored at 4°C until use.

Shortly before CE-MS analysis, samples were re-suspended in 10 µL HPLC-grade water. CE-MS analyses were performed as previously described [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF]. Briefly, CE-MS analyses were performed using a Beckman

Coulter Proteome Lab PA800 capillary electrophoresis system on-line coupled, using an electro-ionization sprayer (Agilent Technologies), to a micrOTOF II MS (Bruker Daltonic). Spectra were accumulated every 3 s, over a range of m/z 350 to 3000.

Mass spectral ion peaks representing identical molecules at different charge states were deconvoluted into single masses using MosaiquesVisu software as described [START_REF] Neuhoff | Mass spectrometry for the detection of differentially expressed proteins: a comparison of surface-enhanced laser desorption/ionization and capillary electrophoresis/mass spectrometry[END_REF]. CE migration time was calibrated by local regression using over 1700 reference signals. Normalization of the amplitude of the fetal urine peptides is based on sequenced endogenous "housekeeping" peptides as described in [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF]. Raw peptide data for each sample can be found in the Supplementary Table 1.

Statistical analysis

calculated using receiver operating characteristic (ROC) plots (MedCalc version 19.0.3, MedCalc Software). Paired comparison of ROC curves was also carried out in MedCalc using one-sided tests and according to [START_REF] Delong | Comparing the areas under two or more correlated receiver operating characteristic curves: a nonparametric approach[END_REF] in order to evaluate whether the fetal urine peptide signature outperformed clinical parameter-based prediction. A one-sided P-value <0.05 was considered as statistically significant.

Results

Study population

Patients were recruited in the Lodz region (Poland) from January 2016 to September 2017. Thirty-three patients were included in the study on the basis of PUV suspicion (Figure 1). PUV was confirmed after birth in 23 patients (Table 1).

In these confirmed PUV pregnancies, the major cause for the discovery of PUV was the presence of a megabladder (83%). Most kidneys showed increased echogenicity (70%) and 57% of the pregnancies presented oligohydramnios. All but two (91%) patients received a vesicoamniotic shunt (VAS, Table 1).

Evaluation of the predictive performance of the 12PUV fetal urinary signature in patients with postnatally confirmed PUV

Ninety one % (21/23) of the patients with postnatally confirmed PUV were followed up to the age of 6 months postnatally (Figure 1A). This time of follow-up is much shorter than in the initial study where we identified the 12PUV predictive peptide model based on an end point of ESRD at two years of age [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF].

Therefore the end-point in the current study was modified to prenatal and perinatal death or at least stage 3 CKD at postnatal month 6 (eGFR<60 mL/min/1.73, Schwartz formula), considering that having CKD stage 3 renal disease at 6 months is a predisposition to progress to severe renal failure at 2 years of age.

Four patients died in the prenatal period (at 16 (n=2), 19 and 21 gestational weeks). Three patients died in the perinatal period (2 patients died within the first 12 hours from delivery and 1 neonate died after 64 hours from delivery). These 3 patients had pulmonary insufficiency, renal insufficiency, secondary cardiac insufficiency with no-response to resuscitation (ventilation) and vasopressors, and neurological complications (intracranial hemorrhage). Nine live-born had CKD stage ≥3 (mean GFR: 26, range 5-47, mL/min/1.73) and 5 others had a normal or moderately reduced renal function (CKD stage <3, mean GFR: 95, range 68-130, mL/min/1.73) at 6 months postnatally (Table 1).

Application of the previously identified 12PUV fetal urine peptide signature to this new, temporally and geographically, independent cohort classified the patients based on their postnatal renal outcome with an AUC 0.94 (95%CI: 0.74-1.00), similar to the predictive values of the original study (AUC of 0.94 [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF]) (Figure 2). Using the cut-off 12PUV score of 0 as previously defined [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF], the sensitivity was 100% (95%CI: 79-100) and the specificity was 60% (95%CI: 15-95). The low specificity, as exemplified by the large confidence interval, is due to the low number of PUV pregnancies with a favorable outcome. However, the overall resulting accuracy of 90% (95%CI: 78-100) is again close to the calculated accuracy of the previous study of 92% (95%CI: 84-100).

Comparison of the predictive capacity of the fetal urine peptide signature with the clinical parameters showed that the fetal urine peptide signature predicted with a higher AUC postnatal renal outcome (Table 2), except for AF volume where the significance was not reached (p=0.06). Again similar to the initial study [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF].

Use of the signature in the context of suspected PUV

The context in which the 12PUV signature was developed is not entirely representing the clinical situation encountered. Indeed one would need to predict the postnatal renal outcome in the prenatal period in "suspected PUV" instead of "confirmed PUV pregnancies" since the presence of valves is only confirmed after birth in the majority of the cases. We therefore re-analyzed the performance of the 12PUV signature adding the 10 postnatally non-confirmed suspected PUV fetal urine samples (Figure 1B). Interestingly, in this context the predictive value of the 12PUV signature was close to that measured for the confirmed PUV pregnancies with an AUC 0.89 (95%CI: 0.72-0.97) (Figure 3). Again when using the cut-off 12PUV score of 0 as defined in the original study [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF], sensitivity was high (92% (95%CI: 74-99) and specificity was low with a large confidence interval (50% (95%CI: 12-88)), but resulting in an accuracy of 84% (95%CI: 71-97)

Discussion

In the current independent single center study we observed that the previously identified 12PUV fetal urine peptide signature predicted with high precision postnatal renal outcome in fetuses with PUV. An important aspect of the current work is that this is an independent study with respect to: i) time of recruitment (several years separate the studies), ii) independence of the recruiting center (the Lodz center did not participate in the original study), and iii) the geographical origin of the patients (the patients from the original study all came from France while the current patients all come from Poland).

This small scale single center study is therefore an important premise for the recently started prospective large-scale study, named ANTENATAL (clinicaltrials.gov NCT03116217; "multicentre vAlidatioN of a fetal urine pepTidome-based classifiEr to predict postnatal reNAl function in posterior ureThral vALves"), aiming to validate the peptide signature in a much larger multicenter setting. The ANTENATAL trial will enroll 400 PUV pregnancies screened in >30 European centers with involvement of two large European Reference Networks on rare diseases over a period of 4 years and with a patient follow-up until the child's age of 2 years [START_REF] Buffin-Meyer | The ANTENATAL multicenter study to predict postnatal renal outcome in fetuses with posterior urethral valves: objectives and design[END_REF]. The ANTENATAL study will also be an excellent opportunity to evaluate the potential of adding other fetal urinary omics traits to peptide markers for kidney disease stratification, including proteins, metabolites and miRNAs, as we observed in a recent study [START_REF] Buffin-Meyer | Combination of the fetal urinary metabolome and peptidome for the prediction of postnatal renal outcome in fetuses with PUV[END_REF] based on a recently developed urinary metabolome analysis pipeline [START_REF] Boizard | A capillary electrophoresis coupled to mass spectrometry pipeline for long term comparable assessment of the urinary metabolome[END_REF]. In addition, the ANTENATAL trial will allow exploring the possibility of extending the omics analysis to amniotic fluid for a less invasive prediction of postnatal renal function in PUV pregnancies.

The majority of the confirmed PUV patients in the current work underwent VAS (91%), while none of the fetuses were subjected to VAS in the previous study. Although several studies suggest that VAS is beneficial for the perinatal renal survival, it appears that it is not modifying the long-term (>6 months) outcome [START_REF] Nassr | Effectiveness of vesicoamniotic shunt in fetuses with congenital lower urinary tract obstruction: an updated systematic review and meta-analysis[END_REF] since the 12PUV signature displayed similar predictive capacity of postnatal renal outcome 6 months after birth in both current and previous studies.

The 12PUV signature was designed in the context of lower urinary tract obstruction (LUTO) with the focus on PUV, being the most frequently encountered etiology. However, unless laser ablation of the valves is performed in utero allowing the confirmation of PUV prenatally, the presence of valves is only confirmed after birth. Therefore, to be of clinical utility, the signature has also to perform well in any suspected PUV (i.e. in all LUTO). Indeed, we observed that the 12PUV signature displayed excellent predictive capacity in the suspected PUV population. This might be explained by the fact that the 12PUV peptide signature, for the major part composed of collagen fragments, is most likely not representing the molecular signature of the bladder obstruction, but more likely the molecular changes due to tissue remodeling in the kidney as exemplified by the presence of dys/hypoplasia, cysts etc… in LUTO. We have observed similar molecular changes in amniotic fluid of congenital anomalies of the kidney and the urinary tract that were also independent of the underlying etiology (Klein, Buffin-Meyer et al., unpublished observations).

The present study has several limitations. The study is single center and uses a relatively small cohort with short follow-up. However, it should be kept in mind that PUV is a rare disease (ORPHA:93110) with a prevalence of 1-9/100 000. Such limitations should be removed in the future by the ongoing multicenter ANTENATAL study [START_REF] Buffin-Meyer | The ANTENATAL multicenter study to predict postnatal renal outcome in fetuses with posterior urethral valves: objectives and design[END_REF]. In addition, although fetal biochemistry was performed (sodium and chloride), fetal urine β2-microglobulin, known to be also associated to postnatal renal outcome in PUV [START_REF] Dreux | Urine biochemistry to predict long-term outcomes in fetuses with posterior urethral valves[END_REF], was not available for comparison.

In conclusion we have observed in an independent study that the previously identified 12PUV fetal urine peptide signature predicted with high precision the postnatal renal outcome in fetuses with confirmed PUV. The signature also performed very well in suspected PUV pregnancies with an AUC of 0.89.

Abbreviations: PUV, posterior urethral valves; CKD, chronic kidney disease; eGFR, estimated glomerular filtration rate. [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF]; ** Versus 12PUV; Abbreviations: 12PUV, 12 fetal urine peptide signature previously identified in [START_REF] Klein | Fetal Urinary Peptides to Predict Postnatal Outcome of Renal Disease in Fetuses with Posterior Urethral Valves (PUV)[END_REF]; AF volume, amniotic fluid volume.

Figure 1 :

 1 Figure 1: Description of patient flow and fetal urine peptidome analysis of PUV pregnancies with A) postnatally confirmed PUV and B) all suspected PUV pregnancies.

Figure 2 :

 2 Figure 2: ROC curve of the prediction of postnatal renal outcome (severe versus normal to moderately reduced renal function) of confirmed PUV pregnancies (n=21) at 6 months postnatally by the previously established 12PUV fetal urinary peptide signature [15].

Figure 3 :

 3 Figure 3: ROC curve of the prediction of postnatal renal outcome (severe versus normal to moderately reduced renal function) of all suspected PUV pregnancies (n=31) at 6 months postnatally by the previously established 12PUV fetal urinary peptide signature [15].

  ). In your reference list, please ensure that journal titles are correctly abbreviated in line with Index Medicus, eg as used on PubMed EO-A5: We have double checked and correct abbreviations were used. E.g. ScientificWorldJournal is correct and is used as one word in Pubmed.

	rewrite, or refer to the methods 'as previously described in ... ' and cite the paper, then
	possibly adding a brief description.
	EO-A1: The section "Fetal urinary peptidome analysis" in the methods section was
	rewritten as suggested in the revised version of the manuscript on page 4.
	EO-Q2: Please supply your manuscript in an editable format, eg MS Word, rather than
	PDF
	EO-A2: An MS Word version of the revised manuscript was submitted.
	EO-Q3: Please revise your abstract to include the following category headings -
	Background, Methods, Results, Conclusions
	EO-A3: We inserted these headings in the revised version of the abstract.
	EO-Q4: Please include a conflict of interest statement in your manuscript.
	EO-A4: We have inserted the following COI on page 9 of the revised manuscript for
	Justyna Siwy (JS) "JS is an employee of Mosaiques diagnostics GmbH (Hannover,
	Germany) that markets urinary peptide-based diagnostics products."
	EO-Q5:
	Reviewer #3:
	R3Q1: The authors should mention the parameters used to recommend VAS in fetuses
	with LUTO.
	R3A2: Adequate changes were made to the text on page 4 stating "Because there are
	no definite predictive factors of pregnancy survival, just recommendations or
	suggestions, our team of gynecologists offered VAS as an option to all LUTO patients.
	Patients with confirmed classical symptoms during 2 separate ultrasound assessments
	(megabladder with or without bilateral hydronephrosis) and amniotic fluid reduction
	(AFI 8 or less) were qualified. Almost all approached patients opted for intervention."
	R3Q2: I suggest to include a table showing the comparison between the first study
	and the present study in regard to ultrasonography findings, fetal urine biochemistry,
	performance of 12PUV and outcome.
	R3A2: We have added this comparison in Table 2 in the revised version of the
	manuscript.
	Editorial Office:
	EO-Q1: Your methods section is largely copied from one of your previous papers.
	While this paper is OpenAccess, rather than just reusing the same text, please either
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EO-Q6: Please define the abbreviations used in each table in table legends. EO-A6: Abbreviations were added to the table legends. Comment to Editorial office: For clarity, we added in the text and Table 1 the two patients that were lost in follow-up which were only mentioned in fi Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation

  #3: R3Q1: The authors should mention the parameters used to recommend VAS in fetuses with LUTO. R3A2: Adequate changes were made to the text on page 4 stating "Because there are no definite predictive factors of pregnancy survival, just recommendations or suggestions, our team of gynecologists offered VAS as an option to all LUTO patients. Patients with confirmed classical symptoms during 2 separate ultrasound assessments (megabladder with or without bilateral hydronephrosis) and amniotic fluid reduction (AFI 8 or less) were qualified. Almost all approached patients opted for intervention." R3Q2: I suggest to include a table showing the comparison between the first study and the present study in regard to ultrasonography findings, fetal urine biochemistry, performance of 12PUV and outcome. R3A2: We have added this comparison in Table 2 in the revised version of the manuscript.

Table 1 Characteristics of the study population with confirmed PUV.

 1

	Study population

Table 2 Comparison of the 12PUV fetal urine peptide signature predictive performance with clinical parameters in postnatally confirmed PUV pregnancies and comparison with previous study in 2013 [15].

 2

		Current study	Previous 2013 study*
		AUC (95%CI)	One sided P-	AUC (95%CI)	One sided P-
			value**		value**
	12PUV	0.94 (0.74-1.00)		0.94 (0.82-0.99)
	Fetal urine sodium	0.70 (0.46-0.88) 0.031	0.78 (0.62-0.90)	0.025
	Fetal urine chloride	0.70 (0.46-0.88) 0.042	Not available	-
	Fetal urine osmolality	0.72 (0.48-0.89) 0.041	Not available	-
	Reduced AF volume	0.81 (0.58-0.94) 0.061	0.53 (0.36-0.69)	<0.0001
	Increased echogenicity	0.69 (0.45-0.87) 0.0009	0.56 (0.39-0.72)	<0.0001
	Cysts	0.56 (0.33-0.77) <0.0001	0.63 (0.46-0.78)	<0.0001

* Using the 38 patients of the validation set of the 2013 study

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Funding sources

The study was supported by Polish Mothers Memorial Hospital Research Institute (internal grant number 2016/IV/54-GW), by the "Fondation pour la Recherche Médicale" (grant number DEQ20170336759) and by the "Agence de la Biomédecine" (METAPhOR project).

EO-A6: Abbreviations were added to the table legends.

Comment to Editorial office: For clarity, we added in the text and Table 1 the two patients that were lost in follow-up which were only mentioned in figure 1 in the initial version of the manuscript.

Conflict of interest

JS is an employee of Mosaiques diagnostics GmbH (Hannover, Germany) that markets urinary peptidebased diagnostics products.

Supplementary Tables

Supplementary Table : Normalized abundance of each of the ~5616 fetal urinary peptides in each sample.