PhD Roshan A Karunamuni
email: rakarunamuni@health.ucsd.edu

Minh-Phuong Huynh-Le

Chun C Fan

Wesley Thompson

Rosalind A Eeles

Zsofia Kote-Jarai

Kenneth Muir

Artitaya Lophatananon

Catherine M Tangen

Phyllis J Goodman

Ian M Thompson

William J Blot

Wei Zheng

Adam S Kibel

Bettina F Drake

Olivier Cussenot

Géraldine Cancel-Tassin

Florence Menegaux

Thérèse Truong

Jong Y Park

Hui-Yi Lin

Jeannette T Bensen

Elizabeth T H Fontham

James L Mohler

Jack A Taylor

Luc Multigner

Pascal Blanchet

Laurent Brureau

Marc Romana

Robin J Leach

Esther M John

Jay Fowke

William S Bush

Melinda Aldrich

Dana C Crawford

Shiv Srivastava

Jennifer C Cullen

Gyorgy Petrovics

Marie-Élise Parent

Jennifer J Hu

Maureen Sanderson

Ian G Mills

Ole A Andreassen

Anders M Dale

MD Tyler M Seibert
email: tseibert@health.ucsd.edu

Chris O'

Brien Lifehouse

African-specific improvement of a polygenic hazard score for age at diagnosis of prostate cancer

Keywords: Prostate cancer, health disparities, genome wide association study, polygenic risk, genomics, genotypic ancestry, African

Novelty and Impact

We identified three SNPs (rs76229939, rs74421890, and rs5013678), using crossvalidation, that significantly improved performance of a previously published polygenic hazard model (PHS46) in men of African genetic ancestry. The performance of the novel PHS46+African score improved by as much as 79% after incorporation of rs76229939, rs74421890, and rs5013678. PHS46+African significantly improved association between polygenic risk with age at diagnosis of prostate cancer in Africans, a population generally under-served in genome-wide studies, to levels comparable with Europeans.

Introduction

Polygenic models can provide personalized estimates of the risk of developing prostate cancer. In the context of survival analysis, these models can provide insight into age at diagnosis of prostate cancer, and thus could be used to guide decisions on whether and when to offer screening [START_REF] Seibert | Polygenic hazard score to guide screening for aggressive prostate cancer: Development and validation in large scale cohorts[END_REF] . Studies of polygenic models have often included only individuals of European genetic ancestry, owing to greater availability of data from that population [START_REF] Duncan | Analysis of polygenic risk score usage and performance in diverse human populations[END_REF][START_REF] Popejoy | Genomics is falling on diversity[END_REF] . As a consequence, these models have been tailored to identify and estimate coefficients of genetic common variants for that particular population, while potentially missing variants that may hold value in other populations [START_REF] Duncan | Analysis of polygenic risk score usage and performance in diverse human populations[END_REF] . There is concern that using these European-focused models could actually exacerbate health disparities 2- 4 .

As an example, our group recently published on the performance of a polygenic hazard score (PHS) originally developed using a European dataset, in a multi-ethnic dataset consisting of individuals of European, African, and Asian genetic ancestry [START_REF] Huynh-Le | Polygenic hazard score is associated with prostate cancer in multi-ethnic populations[END_REF] . The model (called here PHS46, referred to in the referenced manuscript as PHS2), includes 46 single nucleotide polymorphisms (SNPs) in its calculation and was strongly associated with age at diagnosis in all three genetic populations (p<10 -16 ). However, the hazard ratio for prostate cancer between individuals in the upper 20 th percentile to those in the lower 20 th percentile of PHS46 was approximately half as large for those with African genetic ancestry (2.6) as it was for those with European (5.6) or Asian (4.6) ancestry. A similar pattern was observed for clinically significant prostate cancer and for death from prostate cancer.

In the current study, we attempt to bridge the apparent gap in model performance of PHS46 for individuals with African genetic ancestry. To this end, we used a machine learning approach to systematically search for SNPs that add statistical value to a base model of PHS46 among African men (PHS46+African). By including PHS46 as a covariate in our SNP search, we sought to identify those SNPs that may hold particular value for individuals with African genetic ancestry.

Material and Methods

Study dataset

We obtained genotype and phenotype data from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) [START_REF] Amos | The oncoarray consortium: A network for understanding the genetic architecture of common cancers[END_REF] consortium for this study. Genotyping was performed using the OncoArray platform [START_REF] Amos | The oncoarray consortium: A network for understanding the genetic architecture of common cancers[END_REF][START_REF]Infinium OncoArray-500K BeadChip | Research cancer predisposition and risk[END_REF] and had undergone quality assurance steps, as described previously [START_REF] Leitzmann | Risk factors for the onset of prostatic cancer: Age, location, and behavioral correlates[END_REF] . The study dataset contains no overlap with that used to estimate model coefficients of PHS46. It is a subset of another dataset wherein the performance discrepancy of PHS46 between different genotypic ancestries was first observed [START_REF] Huynh-Le | Polygenic hazard score is associated with prostate cancer in multi-ethnic populations[END_REF] . All 46 SNPs of PHS46 were directly genotyped on the OncoArray platform.

The genotypic ancestry of each individual was also determined previously [START_REF] Amos | The oncoarray consortium: A network for understanding the genetic architecture of common cancers[END_REF][START_REF] Li | FastPop: A rapid principal component derived method to infer intercontinental ancestry using genetic data[END_REF] . In total, the African dataset consisted of data from 6,253 men with African genotypic ancestry. Missing SNP calls were replaced with the mean of the genotyped data for that SNP in the African dataset. The percentage of individuals with missing SNP calls ranged from 0 to 5.9% across the SNPs, while the percentage of SNPs with missing calls ranged from 0.5% to 5.1% across the individuals. Individuals without prostate cancer were censored at age at last follow-up in the Cox proportional hazards models. A description of the PRACTICAL study groups that contributed data towards this analysis are described in Supplementary Table 1. PHS46 risk score for each individual in the African dataset was estimated as the sum of SNP allele counts (X) multiplied by their respective coefficients (β) 5 : SNP-scan A multi-step approach was used to select SNPs, from those directly genotyped on the OncoArray platform, that would improve the performance of PHS46 in the African dataset. Training and testing sets were generated using 10 iterations of a 10-fold crossvalidation structure resulting in 100 total permutations. For each permutation, a multivariable logistic regression model using case/control status as the dependent variable was estimated using each genotyped SNP in turn, adjusting for PHS46 and four principal components based on genetic ancestry, determined previously [START_REF] Li | FastPop: A rapid principal component derived method to infer intercontinental ancestry using genetic data[END_REF] . SNPs with pvalues less than 1x10 -6 were considered for further analysis. In order of increasing pvalue, each SNP was tested in a multiple Cox proportional hazards model, after adjusting for PHS46, four ancestral principal components, and previously selected SNPs. The Cox model in the SNP-scan used age at diagnosis of prostate cancer as the dependent variable. If the p-value of the coefficient of the tested SNP was less than 1x10 -6 , it was considered for the final model in that permutation. SNPs that reached this p-value threshold in more than 50% of the permutations were selected to construct the PHS46+African model, consisting of PHS46 and the newly identified SNPs.

Comparing performance between PHS46 and PHS46+African -Hazard Ratio

For each permutation of the previously described cross-validation structure, an PHS46+African Cox proportional hazards model was estimated in the training set using PHS46 and the selected SNPs as independent predictors. The PHS46+African risk score for each individual is then estimated using the corresponding PHS46 score, selected SNP allele counts (Y) and their respective coefficients (α):

The performance of the PHS46+African and PHS46 models was then determined in the cross-validation testing set, and the resulting hazard ratios (HR) were obtained, as previously described [START_REF] Seibert | Polygenic hazard score to guide screening for aggressive prostate cancer: Development and validation in large scale cohorts[END_REF] . For each model, the PHS risk scores within the cross-validation testing set are assigned to quantile groups identified using the corresponding training set control values. The hazard ratio between two quantile groups, such as those in the top 20% to those in the bottom 20%, is estimated as the exponential of the difference in mean PHS values for each group. In this calculation, the PHS values are linearly scaled by a sample-weight correction factor, to account for case-control sampling [START_REF] Seibert | Polygenic hazard score to guide screening for aggressive prostate cancer: Development and validation in large scale cohorts[END_REF][START_REF] Huynh-Le | Polygenic hazard score is associated with prostate cancer in multi-ethnic populations[END_REF][START_REF] Therneau | Computing the Cox Model for Case Cohort Designs[END_REF] . Three HR were calculated: HR80/20 (top 20% to bottom 20%), HR98/50 (top 2% to middle 40%) and HR20/50 (bottom 20% to middle 40%). The average HR across permutations for both PHS46+African and PHS46 are reported.

To allow for comparisons with previously published results, the performance metrics for PHS46 and PHS46+African were also estimated for age at diagnosis of clinically significant prostate cancer. When estimating performance for clinically significant prostate cancer, controls and non-clinically significant cancers were censored at age of last follow-up and age of diagnosis, respectively. The previously used criteria for clinically significant cancer were any of: Gleason score >=7, stage T3-T4, PSA concentration >= 10ng/mL, pelvic lymph nodal metastasis, or distant metastasis 1 . Paired t-tests were used to test for statistically significant differences (α = 0.05) in HR between PHS46+African and PHS46.

Additionally, in each permutation, the performance of a Cox model consisting of PHS46 and SNPs that were considered in that permutation was also estimated. These results are provided within the Supplementary Table 2 and provide performance estimates that are not prone to information leakage from training to testing set.

Comparing performance between PHS46 and PHS46+African -C-index

In addition to the hazard ratio, the performances of PHS46 and PHS46+African were compared using Harrell's c-index [START_REF] Harrell | Evaluating the Yield of Medical Tests[END_REF] . For each permutation of the aforementioned cross-validation structure, the c-index of PHS46 and PHS46+African scores were estimated in the testing fold using the "coxph" function in the R "survival" package.

Paired t-tests were used to test for statistically significant differences (α = 0.05) between the two models.

Characterization of PHS46+African

Coefficients of the PHS46+African model, consisting of PHS46 and the SNPs selected in the SNP-scan, were estimated using 1000 bootstrapped samples of the African dataset.

Clinical utility of PHS46+African

As an example of the clinical utility of the PHS46+African risk score, the riskequivalent age was estimated for those individuals in the upper 2 percentile of the distribution of PHS46+African risk scores. The risk-equivalent age, as defined previously [START_REF] Huynh-Le | A genetic risk score to personalize prostate cancer screening, applied to population data[END_REF] , is when an individual from a given PHS percentile has prostate cancer risk equivalent to the average 60-year-old man. The age-specific general cumulative incidence curve was generated using data from SEER*Explorer incidence rates by age at diagnosis, 2003-2017 for Black Americans [START_REF]SEER*Explorer: An interactive website for SEER cancer statistics Surveillance Research Program[END_REF] . The corresponding risk-adjusted incidence curve was estimated by multiplying the general cumulative incidence curve by the mean value of HR98/50 for PHS46+African obtained from the analysis of the age-ofdiagnosis of prostate cancer. The risk-equivalent age was then calculated as the age at which the risk-adjusted cumulative incidence curve had the same value as the general cumulative incidence curve at age 60.

Results

Individual and OncoArray characteristics

In total, there were 3,013 men with (cases) and 3,240 men without (controls)

Single Nucleotide Polymorphism (SNP)-scan

Across the 100 permutations of the cross-validation iterations, a total of twelve SNPs were considered for final selection (Supplementary Table 3). Three SNPs were selected in more than 50% of the permutations and included in the final PHS46+African model. By cross-referencing the chromosomal positions against dbSNP [START_REF] Sherry | dbSNP -database for single nucleotide polymorphisms and other classes of minor genetic variation[END_REF] , these variants were identified as rs76229939 15 , rs74421890 16 , and rs5013678 17 . All three SNPs (Table 1) are located on chromosome 8q24, a region of the chromosome previously identified as containing common variants associated with prostate cancer [START_REF] Yeager | Genome-wide association study of prostate cancer identifies a second risk locus at 8q24[END_REF][START_REF] Haiman | Multiple regions within 8q24 independently affect risk for prostate cancer[END_REF] . An examination of the R 2 (Supplementary Table 4) showed little association, ranging from 0.0027 to 0.0057, among genotype data from the three SNPs in the African dataset.

Reference threshold (Supplementary Table 5) and mean (Supplementary Table 6) values for PHS46+African in the African dataset are presented in the Supplemental Data.

HR performance of PHS46+African

Figure 1 demonstrates the difference in HRs between PHS46+African and PHS46 within the African dataset using age at diagnosis of any prostate cancer (Supplementary Table 7). Overall, we observed an improvement in all the metrics calculated: a 75% increase in HR98/50 from 2.10 to 3.67; a 79% increase in HR80/20 from 2.47 to 4.42; and a 23% decrease in HR20/50 from 0.65 to 0.51. We also observed improvements in all performance metrics when using age at diagnosis of clinically significant prostate cancer: 103% increase in HR98/50 from 1.91 to 3.88, 113% improvement in HR80/20 from 2.21 to 4.71, and 29% improvement in HR20/50 from 0.70 to 0.50. All observed changes in HR were statistically significant (p < 1x10 -16 ).

C-index of PHS46+African

The mean c-indices of PHS46 and PHS46+African across the cross-validation folds were estimated as 0.55 and 0.58 (p < 1x10 -16 ), respectively.

Risk-equivalent age for PHS46+African

The risk-equivalent age for those individuals in the top 2 percentiles of the distribution of PHS46+African scores was estimated as 50 years old, suggesting that a man with a PHS46+African score in the top 2 percentile reached a prostate cancer detection risk equivalent to that of a standard 60-year-old roughly 10 years earlier, at an age of 50 years. The corresponding risk-equivalent age when using PHS46 scores was

Discussion

Using a cross-validated search of a dataset made up entirely of men with African genetic ancestry, we were able to identify three SNPs that substantially improved the performance of PHS46 in this population to levels that are comparable to those observed in Europeans and Asians. Performance improvements were observed in hazard ratios tracking risk between PHS groups, concordance-indices tracking the overall utility of PHS as a continuous variable, and risk-equivalent age tracking the potential clinical utility of PHS. The three SNPs, rs76229939, rs74421890, and rs5013678, are all located on chromosome 8q24 -a region of the genome where variants have been associated with prostate cancer in both the general population and specifically in men with African genetic ancestry [START_REF] Haiman | Multiple regions within 8q24 independently affect risk for prostate cancer[END_REF][START_REF] Han | Prostate cancer susceptibility in men of African ancestry at 8q24[END_REF] . Despite the relative proximity of the three SNPs on chromosome 8, their genetic data was not strongly associated in our dataset, suggesting that each SNP provides non-redundant information for an individual's genetic score.

Each of the three SNPs have been previously identified in the literature to be associated with prostate cancer: rs76229939 is an intron variant of the prostate-cancerassociated transcript 2 (PCAT2) gene, while rs74421890 and rs5013678 are both noncoding transcript variants of the prostate-cancer-associated non-coding RNA 1 (PRNCR1) gene. The minor allele frequencies of rs76229939 and rs74421890 in Europeans, as reported by dbSNP [START_REF] Sherry | dbSNP -database for single nucleotide polymorphisms and other classes of minor genetic variation[END_REF] , are approximately zero to three decimal places, which may explain why they were not selected in the original formulation of PHS46.

This study is not meant to be an exhaustive search for all possible SNPs that are associated with the age of diagnosis of prostate cancer in individuals with African genetic ancestry. Our study is also limited by the small number of available observations relative to those often found in many genome-wide association studies, which can have tens or hundreds of thousands of individuals. However, we were able to extract information that is likely robust by employing a cross-validated search for those SNPs that specifically add value to the performance of PHS46, and not simply independently associated with prostate cancer. Future analysis will include a more detailed analysis of the 8q24 region, including SNPs that are imputed using TOPMed reference panels. We also note that no SNP score, including PHS46 and PHS46+African, has been shown to discriminate men at risk of aggressive prostate cancer from those at risk of indolent prostate cancer. Finally, the performance metrics reported in this study may be biased by the leakage of information across cross-validated folds of the data when identifying those SNPs to include in the final African-PHS model. This bias is expected to be similar for all SNPs and should not have influenced selection of the three SNPs included in the final model over those not selected.

In conclusion, we identified three SNPs (rs76229939, rs74421890, and rs5013678) on 8q24 that substantially improved the performance of PHS46 in a dataset of men with African genetic ancestry. The addition of these SNPs to the polygenic risk score substantially improved its association with age at diagnosis of prostate cancer in Africans, to levels comparable with those found in Europeans.

  prostate cancer in the African dataset. The mean [95% CI] ages of cases and controls were 62.4 [62.1, 62.7] and 61.8 [61.4, 62.1] years respectively. The OncoArray genotypic data, after the quality assurance process, included 444,323 SNPs.

Table 1 .

 1 Characteristics of PHS46+African SNPs. RS-ID, chromosome and base-pair position (based on version 37), effect and reference alleles, bootstrap-estimated beta, and effect allele frequencies in aggregated Africans from 1000 Genomes (referenced from dbSNP) of the three SNPs selected for addition to PHS46

Figure 1

 1 Figure 1 Comparison between PHS46 and PHS46+African. Mean hazard ratio

uk/our-research/research-divisions/division-of-genetics-andepidemiology/oncogenetics/research-projects/ukgpcs/ukgpcs-collaborators

CRUK and PRACTICAL consortium This work was supported by the Canadian Institutes of Health Research, European Commission's Seventh Framework Programme grant agreement n° 223175 (HEALTH-F2-2009-223175), Cancer Research UK Grants C5047/A7357, C1287/A10118, C1287/A16563, C5047/A3354, C5047/A10692, C16913/A6135, and The National Institute of Health (NIH) Cancer Post-Cancer GWAS initiative grant: No. 1 U19 CA 148537-01 (the GAME-ON initiative). We would also like to thank the following for funding support: The Institute of Cancer Research and The Everyman Campaign, The Prostate Cancer Research Foundation, Prostate Research Campaign UK (now Prostate Action), The Orchid Cancer Appeal, The National Cancer Research Network UK, The National Cancer Research Institute (NCRI) UK. We are grateful for support of NIHR funding to the NIHR Biomedical Research Centre at The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust. The Prostate Cancer Program of Cancer Council Victoria also acknowledge grant support from The National Health and Medical Research Council, Australia (126402, 209057, 251533, , 396414, 450104, 504700, 504702, 504715, 623204, 940394, 614296,), VicHealth, Cancer Council Victoria, The Prostate Cancer Foundation of Australia, The Whitten Foundation, PricewaterhouseCoopers, and Tattersall's. EAO, DMK, and EMK acknowledge the Intramural Program of the National Human Genome Research Institute for their support. Genotyping of the OncoArray was funded by the US National Institutes of Health (NIH) [U19 CA 148537 for ELucidating Loci Involved in Prostate cancer SuscEptibility (ELLIPSE) project and X01HG007492 to the Center for Inherited Disease Research (CIDR) under contract number HHSN268201200008I]. This study would not have been possible without the contributions of the following: Coordination team, bioinformatician and genotyping centers: Genotyping at CCGE, Cambridge: Caroline Baines and Don Conroy Funding for the iCOGS infrastructure came from: the European Community's Seventh Framework Programme under grant agreement n° 223175 (HEALTH-F2-2009-223175) (COGS), Cancer Research UK (C1287/A10118, C1287/A 10710, C12292/A11174, C1281/A12014, C5047/A8384, C5047/A15007, C5047/A10692, C8197/A16565), the National Institutes of Health (CA128978) and Post-Cancer GWAS initiative (1U19 CA148537, 1U19 CA148065 and 1U19 CA148112 -the GAME-ON initiative), the Department of Defence (W81XWH-10-1-0341), the Canadian Institutes of Health Research (CIHR) for the CIHR Team in Familial Risks of Breast Cancer, Komen Foundation for the Cure, the Breast Cancer Research Foundation, and the Ovarian Cancer Research Fund. This study would not have been possible without the contributions of the following: Per Hall (COGS); Douglas F. Easton, Paul Pharoah, Kyriaki Michailidou, Manjeet K. Bolla, Qin Wang (BCAC), Andrew Berchuck (OCAC), Rosalind A. Eeles, Douglas F. Easton, Ali Amin Al Olama, Zsofia Kote-Jarai, Sara Benlloch (PRACTICAL), Georgia Chenevix-Trench, Antonis Antoniou, Lesley McGuffog, Fergus Couch and Ken Offit (CIMBA), Joe Dennis, Alison M. Dunning, Andrew Lee, and Ed Dicks, Craig Luccarini and the staff of the Centre for Genetic Epidemiology Laboratory, Javier Benitez, Anna Gonzalez-Neira and the staff of the CNIO genotyping unit, Jacques Simard and Daniel C. Tessier, Francois Bacot, Daniel Vincent, Sylvie LaBoissière and Frederic Robidoux and the staff of the McGill University and Génome Québec Innovation Centre, Stig E. Bojesen, Sune F. Nielsen, Borge G. Nordestgaard, and the staff of the Copenhagen DNA laboratory, and Julie M. Cunningham, Sharon A. Windebank, Christopher A. Hilker, Jeffrey Meyer and the staff of Mayo Clinic Genotyping Core Facility Additional funding and acknowledgments from studies in PRACTICAL:

Data Availability Statement

The data used in this work were obtained from the Prostate Cancer Association Group to Investigate Cancer Associated Alterations in the Genome (PRACTICAL) consortium.

Readers who are interested in accessing the data must first submit a proposal to the Data Access Committee. If the reader is not a member of the consortium, their concept form must be sponsored by a principal investigator (PI) of one of the PRACTICAL consortium member studies. If approved by the Data Access Committee, PIs within the consortium, each of whom retains ownership of their data submitted to the consortium, can then choose to participate in the specific proposal. In addition, portions of the data are available for request from dbGaP (database of Genotypes and Phenotypes) which is maintained by the National Center for Biotechnology Information (NCBI): https://www.ncbi.nlm.nih.gov/gap/?term=Icogs+prostatehttps://www.ncbi.nlm.nih.gov/gap /?term=Icogs+prostate.

Anyone can apply to join the consortium. The eligibility requirements are listed here: http://practical.icr.ac.uk/blog/?page_id=9. Joining the consortium would not guarantee access, as a proposal for access would still be submitted to the Data Access Committee, but there would be no need for a separate member sponsor. Readers may find

Ethics Statement

All contributing studies were approved by the relevant ethics committees; written informed consent was obtained from the study participants [START_REF] Kote-Jarai | Multiple novel prostate cancer predisposition loci confirmed by an international study: The PRACTICAL consortium[END_REF] . The present analyses used de-identified data from the PRACTICAL consortium and have been approved by the review board at the corresponding authors' institution.

Conflict of Interest:

All authors declare no personal or financial conflicts of interest for the submitted work except as follows. CCF is a scientific consultant for CorTechs Labs, Inc. RE reports honorarium as a speaker for GU-ASCO meeting in San Francisco Jan 2016, support from Janssen, and honorarium as speaker for RMH-FR meeting Nov 2017. She reports honorarium as a speaker at the The authors thank the staff, advisory committees and research subjects participating in the PCaP and HCaP-NC studies for their important contributions.

PROtEuS

PROtEuS was supported financially through grants from the Canadian Cancer Society [13149, 19500, 19864, 19865]


Mathurin Fumery
email: mathurinfumery@gmail.com

Laurent Peyrin-Biroulet

Stéphane Nancey

Romain Altwegg

Cyrielle Gilletta

Pauline Veyrard

Guillaume Bouguen

Stephanie Viennot

Florian Poullenot

Jérôme Filippi

Stephane Nancey

Jerome Filippi

Anthony Buisson

Anne Bozon

Franck Brazier

Lieven Pouillon

Bernard Flourie

Lucile Boivineau

Laurent Siproudhis

David Laharie

Xavier Roblin

Momar Diouf

Xavier Treton

Stephane Nancey

Jerome Filippi

Biogen

Janssen

MSD Mayoly-Spindler Janssen

Roche, Takeda Norgine Pfizer

Tillots Anne Bozon

MSD Janssen Abbvie

Takeda Cilag

Momar

RA, CG, PV, GB, SV Sn

Effectiveness and safety of ustekinumab intensification at 90 Mg every four weeks In Crohn's disease: a multicenter study

Keywords: weeks; AEs, adverse events Crohn's disease, ustekinumab, intensification

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Ustekinumab (Janssen Biotech, Inc.), a fully human IgG1 monoclonal antibody targeting the IL-12/IL-23 shared p40 subunit, was recently approved for the treatment of Crohn's disease (CD). The UNITI program clearly demonstrated the efficacy of ustekinumab to induce and maintain clinical remission in moderate-to-severe CD, with continuous response for up to 4 years [START_REF] Feagan | Ustekinumab as Induction and Maintenance Therapy for Crohn's Disease[END_REF]. Patients receive a 6 mg/kg intravenous induction therapy, a 90 mg sub-cutaneous administration at 8 weeks and then a ustekinumab 90 mg every 12 weeks [q12w], or ustekinumab 90 mg every 8 weeks [q8w], for maintenance therapy, according to physician's judgement. As observed with anti-Tumor Necrosis Factors (TNFs), about 20 -35% of patients experienced loss of response to ustekinumab in clinical trials [START_REF] Feagan | Ustekinumab as Induction and Maintenance Therapy for Crohn's Disease[END_REF][START_REF] Wils | Long-term efficacy and safety of ustekinumab in 122 refractory Crohn's disease patients: a multicentre experience[END_REF][START_REF] Ma | Long-term Maintenance of Clinical, Endoscopic, and Radiographic Response to Ustekinumab in Moderate-to-Severe Crohn's Disease: Realworld Experience from a Multicenter Cohort Study[END_REF][START_REF] Ma | Clinical, endoscopic and radiographic outcomes with ustekinumab in medically-refractory Crohn's disease: real world experience from a multicentre cohort[END_REF]. In patients treated with anti-TNFs, a dose-response relationship has been demonstrated. An increase in dose or dosing frequency is recommended in patients with loss of response [START_REF] Roblin | Development of an algorithm incorporating pharmacokinetics of adalimumab in inflammatory bowel diseases[END_REF][START_REF] Torres | ECCO Guidelines on Therapeutics in Crohn's Disease: Medical Treatment[END_REF]. In the UNITI program, 20% of patients with loss of response during 90 mg q12W regimen recaptured response after escalation to 90 mg q8W [START_REF] Feagan | Ustekinumab as Induction and Maintenance Therapy for Crohn's Disease[END_REF].

Real-world effectiveness data provide valuable evidence to support the efficacy observed in randomized controlled trial. Real-world studies include patients representative of the realworld CD population and allow for a variable treatment regimen and optimisation [START_REF] Salleron | Effectiveness Research in Inflammatory Bowel Disease: A Necessity and a Methodological Challenge[END_REF]. Some real-word studies reported the experience with ustekinumab intensification from 90 mg q8W to q4W and even to q3W [START_REF] Wils | Long-term efficacy and safety of ustekinumab in 122 refractory Crohn's disease patients: a multicentre experience[END_REF][START_REF] Ma | Long-term Maintenance of Clinical, Endoscopic, and Radiographic Response to Ustekinumab in Moderate-to-Severe Crohn's Disease: Realworld Experience from a Multicenter Cohort Study[END_REF][START_REF] Ma | Clinical, endoscopic and radiographic outcomes with ustekinumab in medically-refractory Crohn's disease: real world experience from a multicentre cohort[END_REF][START_REF] Chateau | Two Cases of Inflammatory Bowel Disease Patients Treated With Ustekinumab 90 mg Every 3 Weeks[END_REF]. Recently, the University of Chicago group reported the effectiveness of ustekinumab dose interval shortening from 90 mg q8W to q4W in 51 patients with Harvey-Bradshaw score > 4 [START_REF] Ollech | Effectiveness of Ustekinumab Dose Escalation in Patients With Crohn's Disease[END_REF]. They showed that dose escalation resulted in improvement in clinical indices of disease activity.

We aimed to evaluate the effectiveness and safety of intensification from 90 mg q8w to q4W in patients with incomplete or loss of response to ustekinumab in CD in a retrospective multicenter study.

A c c e p t e d M a n u s c r i p t

Methods

Study population

All consecutive adult patients between October 2015 and December 2018 with active CD, as defined by Harvey-Bradshaw score ≥ 4 and at least one objective sign of inflammation (CRP ≥ 5 mg/L and/or fecal calprotectin ≥ 250 µg/g and/or radiologic and/or endoscopic evidence of disease activity) who required ustekinumab dose escalation to 90mg q4W for loss of response (as defined by a loss of response after initial improvement of symptoms, according to physician judgement) or incomplete response (lack of improvement of clinical symptoms) to ustekinumab 90mg q8W in eleven French university hospitals were included in a retrospective multicenter cohort study.

Data collection

The following data were recorded at baseline for each patient: gender, birth date, age at diagnosis, CD phenotype and behavior according to Montreal classification, previous and concomitant medications, smoking status, previous intestinal resection, Harvey-Bradshaw score, CRP, ustekinumab induction regimen, and indication of intensification (loss of response or incomplete response, and luminal and/or perianal CD). Harvey-Bradshaw score, CRP value, adverse events (AEs), concomitant medications, intervals of ustekinumab administration, ustekinumab discontinuation, CD-related hospitalization and intestinal resection were collected at the first clinic visit after dose escalation, at 6 months, at the last visit under ustekinumab, and at the date of the last follow-up. Endoscopic variables were also collected if available. This cohort was declared to the CNIL (Commission nationale de l'informatique et des libertés, declaration n°T196) as national recommendations.

A c c e p t e d M a n u s c r i p t

Outcome measures

Short-term clinical response response was defined by a decrease of Harvey-Bradshaw score ≥ 3 after dose intensification compared to baseline, with a decision to continue the same dose, and was evaluated at the first clinic visit after therapeutic escalation. Clinical remission was defined as a Harvey-Bradshaw score < 4. Endoscopic remission was defined by the absence of ulceration (exclusion of aphtae) and serious AE as an AE-related hospitalization.

Statistical analysis

Quantitative variables were described as medians and interquartile ranges (IQR).

Categorical variables were presented as counts and percentages of the cohort. Change from baseline in quantitative variables was evaluated with a paired Student test or a Wilcoxon signed-rank test. The distribution of the delay until ustekinumab withdrawal and surgery were estimated with the non-parametric method of Kaplan Meier. Risk factors for short-term response were assessed using a univariate logistic regression model with odds ratio and a 95% confidence interval; firth penalized likelihood was applied if necessary. Variables with a p <10% value were included in a multivariate logistic regression model. Statistical analyses were performed with SAS® software version 9.4 (SAS Institute, Cary, NC) and survival curves were built using RStudio software Version 1.0.143 -© 2009-2016 RStudio (R.3.4.0 software).

Results

Characteristics of patients

A total of one hundred patients were included. Demographic and baseline disease characteristics at ustekinumab intensification are listed in Table 1. Median duration of followup was 8.2 (Interquartile Range (IQR), 5.6 -12.4) months. Fifty-two (52%) of the patients A c c e p t e d M a n u s c r i p t were female, the median age was 34.9 years (IQR, 28.1 -46.3) and the median duration of CD was 11.6 years (IQR, 7.3 -20.1). According to Montreal classification, one third (n=31, 31%) had ileal location, one third colonic (n=29, 29%) and 40 (40%) had ileo-colonic disease. Most of patients had complicated behaviors (B2-stricturing, 28 (28%) or B3penetrating, 37 (37%)), 47 (47%) had perianal CD and 34 (34%) extra-intestinal manifestations. All but one were previously exposed to anti-TNF and 55 (55%) to vedolizumab. Half of patients had previous intestinal resection. The median duration of ustekinumab therapy before optimisation was 5.0 (IQR, 2.8 -9.0) months. At the time of therapeutic escalation, respectively 29 (29%) and 27 (27%) received co-treatment with steroids or immunosuppressants. Incomplete response (74, 74%) and luminal disease (77, 77%) were the main indications for ustekinumab intensification.

Short-term response and remission

After a median of 2.4 (IQR, 1.3 -3 .0) months following ustekinumab intensification, 61 patients (61%) experienced a clinical response. In addition, 31 (31%) and 27 (27%) of the patients achieved clinical remission and steroid-free clinical remission, respectively (Figure 1). The median value of Harvey-Bradshaw index significantly dropped from 8.0 at baseline (IQR, 5.0 -11.2) to 5.0 (IQR, 3.0-7.0) (p = 0.001). Ten patients of 29 (35.5%) receiving steroids at inclusion were weaned before the first visit after ustekinumab escalation. In general, systemic steroid was tapered according to ECCO guidelines. The median CRP level decreased from 12.3 mg/L (IQR, 5.0 -30.5) to 9.6 mg/dL (IQR, 3.2-18.0) (p =0.2). Among the baseline factors evaluated in the univariate analysis (Table 2), loss of response to ustekinumab (vs. incomplete response) was associated with short-term clinical response (odds Ratio (OR), 3.03, CI95% [1.03 ; 8.91], p=0.044) as well as duration of ustekinumab therapy before dose intensification (OR, 1.11, CI95% [1.00 ; 1.22], p=0.051). However, no After a median follow-up of 8.2 (5.6 -12.4) months, 61 (61%) were still treated with ustekinumab. The cumulative probabilities of ustekinumab persistence were 81% at 6 months and 51% at 12 months (Figure 2). At the end of follow-up, 26 (26%) patients were in steroid-free clinical remission. Reasons for ustekinumab withdrawal were the absence of response to optimisation, loss of response and pregnancy in 23 (38%), 13 (21%) and 3 (5%) patients, respectively. Nine patients could de-escalate ustekinumab, seven to 90 mg q8W and two to 90 mg q6W after a median time of 7.4 (IQR, 5.5 -13.2) months since optimisation. Thirty-nine patients had colonoscopy after a median interval of 5.9 (2.4 -7.8) months after ustekinumab intensification. Endoscopic remission was observed in 14 patients (35.9%).

A c c e p t e d M a n u s c r i p t

Sixteen (16%) patients had ustekinumab intensification for perianal disease and 7 (7%) for both perianal and luminal CD. Among them, 14/23 (61%) experienced an immediate response according to physician judgment. Closure of perianal fistula was observed in 5/23 (22%) patients at last news. Conversely, four patients experienced perianal CD worsening needing perianal surgery during follow-up.

Surgery and hospitalization

After a median follow-up of 8.2 (IQR, 5.6-12.5) months, 27 (27%) patients needed CDrelated hospitalization. Major abdominal surgery was required for 19 (19%) patients. Of these, 11 did not respond to ustekinumab intensification, whereas eight had a secondary loss of response to ustekinumab 90 mg q4W. Cumulative risks of hospitalization and surgery are highlighted in the Supplementary Figures 1 and2.

Adverse event

During the follow-up period, there were no death or malignancy. Thirteen adverse events were reported in 12 patients (12%), including eight infections (Table 3). Five patients experienced severe adverse events including two pyelonephritis, one nasopharyngitis with epiglottitis, one pneumonitis, and one infectious colitis. None of these AEs had led to definitive ustekinumab withdrawal.

A c c e p t e d M a n u s c r i p t

Discussion

This national retrospective multicenter observational cohort study assessed the effectiveness and safety of ustekinumab dose escalation in CD. Two-thirds of patients recaptured response and about half of them had steroid-free clinical remission at 6 months.

Importantly, most patients were candidates for surgery or inclusion in a clinical trial, as many had a previous exposure to anti-TNFs therapy and vedolizumab, with active disease as indicated by objective signs of inflammation despite ustekinumab therapy. In this refractory population, 20% of patient needed major abdominal surgery at the end of follow-up.

Secondary loss of response is frequent in patients treated by ustekinumab, ranging from 20% of patients in IM-UNITI to 35% in real world observational studies [START_REF] Feagan | Ustekinumab as Induction and Maintenance Therapy for Crohn's Disease[END_REF][START_REF] Wils | Long-term efficacy and safety of ustekinumab in 122 refractory Crohn's disease patients: a multicentre experience[END_REF][START_REF] Ma | Long-term Maintenance of Clinical, Endoscopic, and Radiographic Response to Ustekinumab in Moderate-to-Severe Crohn's Disease: Realworld Experience from a Multicenter Cohort Study[END_REF][START_REF] Ma | Clinical, endoscopic and radiographic outcomes with ustekinumab in medically-refractory Crohn's disease: real world experience from a multicentre cohort[END_REF]. To date, no prospective study has evaluated the management of loss of response during ustekinumab therapy. Randomized prospective studies evaluating the efficacy and safety of ustekinumab therapy in CD have not specifically evaluated the response to ustekinumab dose escalation.

In the IM-UNITI trial, among 29 patients randomized to the ustekinumab 90mg q12w group who lose response with therapeutic escalation to 90mg q8w, 41% achieved clinical remission and 55% clinical response [START_REF] Feagan | Ustekinumab as Induction and Maintenance Therapy for Crohn's Disease[END_REF]. Some real-world retrospective studies reported optimisation to ustekinumab 90 mg q4W. Dose escalation (90 mg q4W or q6W) effectively recaptured clinical response in 9/17 (53%) patients in the Canadian study of Ma and A c c e p t e d M a n u s c r i p t colleagues (4). The GETAID reported the long-term outcome in 88 patients treated by 10 various regimens of ustekinumab (2). Thirty-two patients required a dose intensification of ustekinumab during follow-up, among them 12 patients received 90 mg q4W. This strategy was effective in 18 (56%) including a dose increase in two patients, interval reduction in seven, and both in nine. In a Dutch study including 221 patients treated with ustekinumab, 11 patients were on a q4w interval and 70% were in corticosteroid-free clinical remission [START_REF] Biemans | Ustekinumab for Crohn's Disease: Results of the ICC Registry, a Nationwide Prospective Observational Cohort Study[END_REF]. Another Canadian cohort recruited 62 patients with CD treated with 90 mg of ustekinumab subcutaneously at weeks 0, 1, and 2 during induction and 90 mg every 4 (n=48) or 8 (n=14) weeks during maintenance (3). Clinical and endoscopic outcomes were not different between the two maintenance regimens. Some studies reported outcomes after a combination of intravenous reinduction with a dose escalation strategy, with an effectiveness in about half of patients [START_REF] Ma | Long-term Maintenance of Clinical, Endoscopic, and Radiographic Response to Ustekinumab in Moderate-to-Severe Crohn's Disease: Realworld Experience from a Multicenter Cohort Study[END_REF][START_REF] Biemans | Ustekinumab for Crohn's Disease: Results of the ICC Registry, a Nationwide Prospective Observational Cohort Study[END_REF]. More recently, the Chicago group had reported the effectiveness of ustekinumab dose escalation to q4W in 110 patients with CD followed with a median time of 9 months [START_REF] Ollech | Effectiveness of Ustekinumab Dose Escalation in Patients With Crohn's Disease[END_REF]. Clinical response as defined by Harvey-Bradshaw score improvement was observed in 42% of the 78 patients with available data. During the followup period, clinical remission following interval shortening was achieved in 28% of the 51 patients with Harvey-Bradshaw score > 4 before interval shortening. In this single-center center only a limited subset (n=11) of patients had endoscopic evaluation following dose escalation. In the study reported here, we included only patients with clinically active CD, and at least one objective sign of inflammation, with a standardized follow-up in 11 different centers. Our results are in line with those from Chicago, reporting clinical remission in one third of patients. In our cohort, endoscopic data were available in 39 patients and endoscopic remission was observed in 14 of them.

Currently, no data exists to identify which patients are more likely to benefit from dose escalation, and management of attenuated response is based on clinical judgment. As for anti-TNF therapy, therapeutic drug monitoring with incorporation of pharmacokinetic data in developing a management algorithm for primary and secondary failure will likely become a A c c e p t e d M a n u s c r i p t mainstay of therapy, identifying appropriate patients needing a rescue dose [START_REF] Vermeire | How, When, and for Whom Should We Perform Therapeutic Drug Monitoring?[END_REF][START_REF] Feuerstein | American Gastroenterological Association Institute Guideline on Therapeutic Drug Monitoring in Inflammatory Bowel Disease[END_REF][START_REF] Sparrow | Therapeutic Drug Monitoring of Biologics During Induction to Prevent Primary Non-Response[END_REF]. In the UNITI program, serum concentrations of ustekinumab were proportional to dose, and associated with clinical efficacy [START_REF] Feagan | Ustekinumab as Induction and Maintenance Therapy for Crohn's Disease[END_REF][START_REF] Adedokun | Pharmacokinetics and Exposure Response Relationships of Ustekinumab in Patients With Crohn's Disease[END_REF]. Also limited real-life data suggested better outcomes with higher exposure. Verstockt et al. demonstrated that a clear exposure-response relationship exist, both during induction as during maintenance therapy, with different thresholds depending on the targeted outcome [START_REF] Verstockt | Ustekinumab Exposure-outcome Analysis in Crohn's Disease Only in Part Explains Limited Endoscopic Remission Rates[END_REF]. Ustekinumab serum concentrations were higher in endoscopic responders at every time point. At week 8, ustekinumab through level lower than 5.0 µg/mL at W8 almost ruled out endoscopic response later. During maintenance, thresholds of 2.3 µg/mL at W16 and 1.9 µg/m at W24 were identified as the minimal exposure needed to achieve endoscopic response after 6 months. In a retrospective study, Battat et al. reported that maintenance trough concentrations of ustekinumab above 4.5 µg/mL were associated with biomarker reduction and endoscopic response [START_REF] Battat | Association Between Ustekinumab Trough Concentrations and Clinical, Biomarker, and Endoscopic Outcomes in Patients With Crohn's Disease[END_REF]. However, 42 out of their 56 included patients had been dose-escalated to q4w maintenance in order to achieve these high maintenance levels. Three ongoing randomized clinical trial (Rescue trial, EudraCT number 2018-004269-14, The Power trial NCT03782376 and STARDUST trial, NCT03107793) will address this issue and assess whether ustekinumab dose-optimisation may improve endoscopic remission rates by rescuing patients who quickly lost response after sub-cutaneous maintenance and if a treat to target approach on endoscopic remission and ustekinumab trough level may improve long-term outcomes.

Regarding safety, no death was observed. Five patients required hospitalization for infectious AEs. The interpretation of these results is limited by the relatively small sample and short follow-up period of the cohort. The ongoing I-CARE study, European prospective cohort study assessing long-term safety of inflammatory bowel diseases therapies (NCT02377258), should answer this question.

Our study has some limitations. The pharmacokinetics of ustekinumab could not be assessed, since this is not routinely performed in France. However, we used objective In conclusion, ustekinumab 90 mg q4W was effective to recapture response and to induce clinical remission in a subset of patients with CD with loss of response or incomplete response under 90 mg q8W therapy. Our findings suggest that ustekinumab intensification may be considered in routine practice in CD patients who experience loss of response or insufficient response. Large prospective studies with ustekinumab serum monitoring are warranted to elucidate the best approach for optimizing drug therapy and to confirm the longterm safety and efficacy of this strategy.

A

  c c e p t e d M a n u s c r i p t factors were independently associated with short-term clinical response in multivariate analysis probabily due to multicollinearity.Long-term outcomesFollow-up information at 6 months was available for 69 of 100 patients. Of those, 34 (49%) were in steroid-free clinical remission. The median Harvey-Bradshaw score was 4 (2.5 -6.0) and the median CRP level was 8.5 (2.25 -17.3) mg/L. Among the 35 non-responders to ustekinumab 90 mg q4W in the short term, 6 (17.1%) achieved clinical remission at 6 months.The median Harvey-Bradshaw score of the 65 short-term responders decreased from 7 (IQR, 5 -10) at baseline to 3.5 (IQR, 2 -5) at month 6 (P < 0.001), and the CRP level dropped from 9.3 mg/L (IQR, 3.2 -21.1) at baseline to 4 mg/L (IQR, 1.2 -10.2) at month 6 (P = 0.02).

A

  c c e p t e d M a n u s c r i p t measures of effectiveness as steroid-free clinical remission and mucosal healing. Strengths of the study include its multicenter national study design including all consecutive CD patients treated with ustekinumab 90mg q4W in 11 academic centers. Only patients with objective signs of inflammation and with a standardized follow-up, including Harvey-Bradshaw index and CRP levels measurements, were included.

FiguresFigure 1 .

 1 Figures

  Figure 1. Short-term effectiveness of ustekinumab intensificationfrom 90mg q8W to q4W

Figure 2 .

 2 Figure 2. Kaplan-Meier analysis of persistence of ustekinumab after intensification.

  A c c e p t e d M a n u s c r i p t

Figure 1 A

 1 Figure 1

Figure 2

 2 Figure 2

A c c e p t e d M a n u s c r i p t


Marie-José Adam

Isabelle Enderle

Gwenaëlle Le Bouar

Anne-Sophie Cabaret-Dufour

Cécile Tardif

Laurence Contin

Alexis Arnaud

Maïa Proisy

Sylvie Jaillard

Laurent Pasquier

Dr Maela Le Lous
email: maela.lelous@chu-rennes.fr

Performance of diagnostic ultrasound to identify causes of hydramnios

Keywords: Fetal Ultrasound, General cytogenetics, Fetal MRI

What's already known about this topic?

• -Hydramnios can be the consequence of a fetal abnormality (e.g. gastrointestinal obstruction), a maternal pathology (e.g. diabetes), or a placental pathology (e.g. chorioangioma). The diagnostic yield of ultrasonography in the presence of hydramnios is unclear.

What does this study add?

• A targeted ultrasound scan can detect the cause of hydramnios in the majority of cases

• Amniocentesis and MRI have modest added benefit.

Introduction

The prevalence of hydramnios is estimated to be between 0.7% and 2% of all pregnancies depending on the study [START_REF] Magann | A review of idiopathic hydramnios and pregnancy outcomes[END_REF][2][START_REF] Bundgaard | Prevalence of polyhydramnios at a Danish hospital--a population-based study[END_REF] . Hydramnios is defined by a volume of amniotic fluid (AF) >2 L regardless of the gestational age [START_REF] Magann | Comparative efficacy of two sonographic measurements for the detection of aberrations in the amniotic fluid volume and the effect of amniotic fluid volume on pregnancy outcome[END_REF] . On ultrasound, hydramnios is defined by an AF index (AFI) ≥25 cm [START_REF] Phelan | Amniotic fluid volume assessment with the four-quadrant technique at 36-42 weeks' gestation[END_REF][START_REF] Alley | Four-quadrant assessment of gestational age-specific values of amniotic fluid volume in uncomplicated pregnancies[END_REF] or by a deepest pocket (DP) greater than or equal to 10 cm, although some authors apply a DP cut-off of ≥8 cm [START_REF] Chamberlain | Ultrasound evaluation of amniotic fluid volume. II. The relationship of increased amniotic fluid volume to perinatal outcome[END_REF][START_REF] Manning | Antepartum fetal evaluation: development of a fetal biophysical profile[END_REF] .

Hydramnios can reveal a wide variety of pathologies. Conventionally, three causes of hydramnios are possible: fetal malformations (in particular digestive atresia and cardiopathies ), maternal pathologies (e.g. diabetes, alloimmunization), and placental pathologies (e.g. chorioangioma) [START_REF] Sandlin | Clinical relevance of sonographically estimated amniotic fluid volume: polyhydramnios[END_REF] . However, no cause is found in as many as 40 to 60% of cases [START_REF] Magann | A review of idiopathic hydramnios and pregnancy outcomes[END_REF][START_REF] Khan | Outcome of pregnancy in women diagnosed with idiopathic polyhydramnios[END_REF] . The risk of maternofetal anomalies increases with the severity of hydramnios [START_REF] Magann | A review of idiopathic hydramnios and pregnancy outcomes[END_REF][START_REF] Bundgaard | Prevalence of polyhydramnios at a Danish hospital--a population-based study[END_REF][START_REF] Lazebnik | The severity of polyhydramnios, estimated fetal weight and preterm delivery are independent risk factors for the presence of congenital malformations[END_REF] . Consequently, any woman presenting hydramnios, particularly if moderate or severe, requires comprehens ive assessment and appropriate monitoring.

On discovering hydramnios, a diagnostic ultrasound is performed to identify a potential fetal defect. The work-up is completed by biological maternal tests to detect alloimmunization, diabetes, or infection (parvovirus B19, cytomegalovirus). Symptoms suggestive of myasthenia or myotonic dystrophy type I congenital (Steinert's disease) can be revealed during a genetic consultation. Additionally, MRI is often carried out. Finally, depending on the couple's wishes, an amniocentesis can be performed to determine the karyotype, detect any AF infection, and for biochemical analysis for the diagnosis of esophageal atresia or mutations such as Steinert's disease or Prader-Willi syndrome [START_REF] Grangé | Guide Pratique de l'échographie Obstétricale et Gynécologique[END_REF][START_REF] Kollmann | Etiology and perinatal outcome of polyhydramnios[END_REF] .

However, there are no official recommendations defining the additional examinations to be carried out in this setting.

Our main objective was to assess the performance of diagnostic ultrasound to identify the cause of hydramnios and the added value of MRI or amniocentesis. Our secondary objective was to list the pathologies revealed by hydramnios.

Material and methods

Design

We carried out a single-center retrospective study at Rennes University Hospital, a tertiary maternofetal reference center. We included all patients referred to our center between January 2013 and May 2017 for a suspicion of hydramnios, with a DP of ≥8 cm [START_REF] Chamberlain | Ultrasound evaluation of amniotic fluid volume. II. The relationship of increased amniotic fluid volume to perinatal outcome[END_REF][START_REF] Manning | Antepartum fetal evaluation: development of a fetal biophysical profile[END_REF] and for which the neonatal outcome was known (delivery at Rennes University Hospital).

The exclusion criteria were multiple pregnancies, known fetal anomalies for which hydramnios appeared secondarily during the follow-up of the causal pathology.

The study received the approval of the local ethics committee (N°20-27).

Protocol

The protocol for routine ultrasound included a first trimester ultrasound scan between 11 and 13+6 WG, a second trimester ultrasound scan between 20 and 25 WG, and a third trimester ultrasound between 30 and 35 WG. In case of suspected hydramnios, the patient was referred for a targeted scan.

Local protocol for targeted scan included in addition to regular routine scan: particular attention to the placenta, nasal flow, neck, thyroid, thymus, esophagus, mobility and trophicity of limbs, four cavity chambers, interventricular septum, right cardiac outlet, left cardiac outlet, aortic arch, pulmonary venous drainage, head profile, choanes, corpus callosum, brain ventricles and gyration, cerebellum, vermis, choroid plexus, velocity in midcerebral artery.

If hydramnios was confirmed, the patients were screened for diabetes (oral glucose tolerance test of 75g), maternofetal alloimmunization (irregular agglutinins), and cytomegalovirus (CMV) and parvovirus B19 (maternal serology). Toxoplasmosis serology is already routinely performed every month in France.

Amniocentesis was then discussed with the couple for karyotyping, array-CGH, biochemical indexing for Bartter syndrome, enzymatic profiling for esophageal atresia. Total protein, alphafeto protein, digestives enzymes (γ-glutamyl transpeptidase, l-leucineaminopeptidase, and alkaline phosphatase) and electrolytes were assessed. An esophagus atresia index was calculated, as well as a Bartter index [START_REF] Muller | Amniotic fluid digestive enzymes: Diagnostic value in fetal gastrointestinal obstructions[END_REF] . Detection of CMV or parvovirus B19 was assessed in the amniotic fluid depending on maternal serology. The interest of investigat i ng for other genetic abnormalities was discussed during a genetic consultation.

Patients were monitored monthly until delivery. MRI was performed around 32 weeks of gestation when further exploration was needed, according to the opinion of the multidisciplinary staff. Amniotic fluid drainage was performed only if the patients were symptomatic (premature labor, respiratory or abdominal discomfort, dyspnea).

Collected Data

Data were collected from the ultrasound software "Xplore" with the following keywords: "hydramnios" and "excess of amniotic fluid". The following maternal clinical data were collected from the patient records: maternal age, height, weight, body mass index, parity, gestation, history of gestational diabetes, diagnosis of diabetes for the current pregnancy, maternal complications of hydramnios.

The following ultrasound data were collected: the severity of the hydramnio s according to the DP measurement (DP between 8-10, 10-12, 12-16, >16cm), gestational age at hydramnios onset, mode of discovery of hydramnios, the data from the first trimester ultrasound, the first trimester screening for trisomy 21, data from second and third or any additional ultrasounds, the diagnosis retained. Macrosomia was defined as a fetal estimated weight above the 90 th percentile.

Additional biological assessment data were collected: diabetes screening with an oral glucose tolerance test of 75g, irregular agglutinins, CMV and parvovirus B19 serology, amniocentesis, and MRI.

Neonatal data were also collected: gestational age at birth, delivery mode, birth weight, Apgar score at 1, 5 and 10 minutes, pH and lactates, transfer to intensive care unit, and the presence of neonatal pathology in connection with hydramnios.

Primary Endpoint

The primary endpoint was the rate of detection of an underlying fetal or neonatal pathology. The prevalence of fetal anomalies was stratified according to the severity of hydramnios at the initial scan (DP between 8-12, 12-16, ≥16cm).

Additionally, we compared "pathological" hydramnios, defined by the presence of a fetal anomaly or maternal diabetes, and "non-pathological" hydramnios by grouping the cases of idiopathic hydramnios with isolated macrosomia.

Statistical analysis

Data were compared using a χ2 test, or Fisher's if appropriate, for the qualitative parameters, and a Student's test for the quantitative parameters. The results were considered significant if p was <0.05. The diagnostic yields of ultrasonography, MRI and amniocentes is was calculated and expressed with their 95% confidence intervals (95% CI).

Results

During the study period, 402 patients underwent diagnostic ultrasound for suspected hydramnios. Among them, 225 cases of hydramnios were confirmed for a total of 17 460 births over the period studied, representing a prevalence of 1.3%. Among the 225 cases of confir med hydramnios, 17 were excluded because they were twin pregnancies, 27 because hydramnio s appeared during the monitoring of an identified causal pathology, and 23 whose neonatal outcome was not known. A total of 158 patients were therefore included (Figure 1).

The clinical and ultrasound characteristics of these 158 patients are described in Table 1. The causes of hydramnios were: a fetal or neonatal pathology in 37 cases (23.4%); diabetes in 39 (24.6%); isolated macrosomia in 16 (10.1%); and hydramnios was considered idiopathic in 66 (41.7%).

Fetal or neonatal pathologies:

The most frequent fetal or neonatal pathologies involved the gastrointestinal tract (n = 10, 31.3%) including six cases of esophageal atresia (18.8%) (Figure 2 3.

There were 7 terminations of pregnancies, at a mean gestational age of 32 weeks, 1 fetal death at 35 + 1 weeks gestation, and 3 neonatal deaths.

Diagnostic yield of ultrasonography

Targeted ultrasound identified abnormal findings diagnostic or suggestive of the etiology of hydramnios in 73% of cases (27/37) (Table 3). Certain missed diagnoses could have been amenable to prenatal detection or suspicion, such as anti D allo-immunization, bilatera l choanal atresia, Trisomy 18 and esophageal atresia. The fetus with trisomy 18 presented with fetal growth restriction and choroid plexus cysts but the parents declined amniocentesis. For 6 other anomalies the ability of ultrasonography to detect or suspect the condition was limited (one case each of congenital myotonic dystrophy type I Steinert's disease, Prader-Willi syndrome, Bartter syndrome, deletion of the short arm of chromosome 4, and Alagille syndrome). There were 5 unconfirmed suspicions of pathologies on ultrasound : 2 small for gestational age and one macrosomia who were eutrophic at birth, one suspicion of esophageal atresia with a healthy baby at birth, and one cases of pyelectasy with normal urinary tract at birth.

Additional diagnostic yield of amniocentesis

Among the 10 anomalies missed at targeted ultrasonography, five were detected antenatally by amniocentesis (one case each of congenital myotonic dystrophy type I Steinert's disease, Prader-Willi syndrome, Bartter syndrome, trisomy 18 and esophageal atresia), the diagnostic yield of ultrasound coupled with amniocentesis was 86.5% (32/37) [95% CI ] The only false positive of amniocentesis was a healthy newborn whose prenatal enzyme profile on AF analysis suggested esophageal atresia.

Additional diagnostic yield of fetal MRI (thoracoabdominal and cerebral)

Only one case of esophageal atresia not diagnosed by ultrasound was found with MRI. One potential missed diagnosis of MRI was represented by normal MRI findings in a fetus diagnosed at birth with unilateral choanal atresia and posterior cleft palate. There were no false positive MRI diagnoses.

Five malformations were diagnosed only at birth (one case each of congenital myopathy, bilateral choanal atresia, anti-D alloimmunization, deletion of the short arm of chromosome 4, and Alagille syndrome).

Characteristics of pathological versus non-pathological hydramnios

Table 4 displays a comparison of the antenatal and postnatal characteristics of pregnancies affected by "pathological hydramnios" (maternal diabetes and maternofeta l pathologies) with those of pregnancies with "non-pathological" hydramnios (isolated fetal macrosomia and idiopathic hydramnios). Patients with pathological hydramnios had a significantly higher BMI than patients with non-pathological hydramnios, had diagnosis of hydramnios made significantly earlier in pregnancy, and had more severe hydramnios than those in the non-pathological group .

The number of live births was significantly lower in the pathological group than in the non-pathological group, and babies had lower birth weight and were significantly more likely to require admission to an intensive care unit.

Delivery Mode:

A total of 11(6.9%) patient underwent cesarean-sections during labor (4 for failed induction of labor, 5 for non-reassuring fetal heart rate monitoring, 1 for a suspicion of placental abruption, 1 for arrest of labor), and 4 (2.5%) had elective c-sections (2 because of a scarred uterus, one for perineal protection, one for placenta praevia).

Prevalence of fetal anomalies according to the severity of hydramnios

The distribution of causes of hydramnios according to the AF volume is detailed in Table 5. The likelihood of some underlying fetal or neonatal pathology was significantly related to the severity of hydramnios. Among the four cases of severe hydramnios, with a DP >16 cm 3 had a fetal pathology and 1 was related to maternal diabetes. The risk of undetected anomalies at birth in the event of normal antenatal follow-up in our study was 4.1% (5 anomalies found at birth/(158-32 = 126 fetuses with normal follow-up) and only observed in cases where the DP was ≥10 cm.

The special cases of patients with diabetes

In our series, 40 patients with hydramnios were known to be diabetic: 13 of these had pre-existing diabetes and 27 had gestational diabetes. Four cases of diabetes were discovered during the evaluation of hydramnios. In one patient with well-controlled gestational diabetes, no obstetric history, normal nuchal translucency but absent first trimester aneuploidy screening, the ultrasound scan at 33 weeks showed a DP of 12 cm and an AFI of 32cm. The fetus was estimated eutrophic at 22 WG and the targeted scan was remarkable only for the presence of choroid plexus cysts. Amniocentesis was performed and revealed trisomy 18 resulting in a medical termination of pregnancy. In another patient with gestational diabetes well controlled by diet, ultrasound scan at 32 weeks revealed hydramnios, with a DP of 13 cm and an AFI of 32cm. The targeted ultrasound scan was considered normal. An amniocentesis was performed with an enzymatic profile suggestive of esophageal atresia. A fetal thoraco-abdominal MRI did not find a blind upper esophageal sac and the newborn was healthy. For two patients, diabetes was not diagnosed until the birth of their child. One of the children was born at 40 + 4 weeks weighing 3830g and presented a convulsive encephalopathy in the first days of life, at which time a deletion on the short arm of chromosome 4 was detected (amniocentesis had been refused by the couple). The other child had persistent severe icterus in the first days of life, revealing chronic cholestasis, ultimately leading to diagnosis of Alagille syndrome by gene testing. The pregnancy was marked by type 2 diabetes, well controlled with insulin therapy. The fetus was assessed as being eutrophic, with no ultrasound abnormalities apart from hydramnios with a DP of 11cm. Finally, one patient with a well-controlled type 1 diabetes experienced a fetal death at 35 WG. The ultrasound found a DP of 10 cm and an AFI of 29 cm from 26 WG, without fetal anomalies. The etiological assessment performed after birth (autopsy + karyotype) was negative.

Discussion

Targeted ultrasound scan can identify or suggest the etiology of hydramnios in 73.0% of cases with an underlying fetal or maternal pathology. In our series, the most frequent malformation causing hydramnios was esophageal atresia (n = 6), with five of the cases diagnosed by ultrasound, one by visualization of a distended hypopharynx, and the other suspected by a persistent non-visualization of the fetal stomach associated with the hydramnio s.

However, a "small stomach" is a subjective sign and can be associated with a wide range of pathologies [START_REF] Brumfield | Pregnancy outcomes following sonographic nonvisualization of the fetal stomach[END_REF][START_REF] Mckelvey | Persistent nonvisualisation of the fetal stomach: diagnostic and prognostic implications[END_REF] . Only the presence of a blind esophageal pouch is a certain diagnosis. A distended hypopharynx is a novel and sensitive sign that can lead to diagnosis of esophageal atresia (once in our series) [START_REF] Tracy | The Distended Fetal Hypopharynx: A Sensitive and Novel Sign for the Prenatal Diagnosis of Esophageal Atresia[END_REF] . It is worth noting that we did not find any cases of esophagus atresia in patients with a DP <10cm. Duodenal atresia was also common and all were diagnosed by ultrasound with the presence a double bubble image of the stomach. Other pathologie s diagnosed in our series were heart disease, cleft lip, chylothorax, diaphragmatic hernia, and Beckwith-Wiedmann syndrome. Choanal atresia was more difficult to diagnose with ultrasound -only one of the two cases in our series was detected -mainly because this is not a usual ultrasound plane of the systematic scan. This highlights the importance of examining the nasal flow by doppler ultrasound in patients with hydramnios. Other fetal malformations that may be responsible for hydramnios can be found on ultrasound. In 2014, in a series of 860 patients with hydramnios, Kollman et al reported that 8.5% of the fetuses had congenital anomalies, the most common being cardiac anomalies (32.9%) [START_REF] Kollmann | Etiology and perinatal outcome of polyhydramnios[END_REF] . There is a relatively high rate of anomalies in our series especially a high rate of esophageal atresia, compared to previous epidemiologica l studies. It is to notice that none of the pathologies in this series were cardiac, which is one of the most common pathologies to develop hydramnios [START_REF] Dashe | Hydramnios: anomaly prevalence and sonographic detection[END_REF] . Other fetal malformations have been described, such as hyperechoic kidneys associated with a mutation in the HNF-1-β gene [START_REF] Gondra | Hyperechogenic kidneys and polyhydramnios associated with HNF1B gene mutation[END_REF] .

Placental anomalies are relatively common and should be investigated by ultrasound. In our series, we found one case of chorioangioma (benign tumor of the placenta) which can cause fetal hydrops if large [START_REF] Rodríguez-Ayala | Chorioangioma: an uncommon cause of hydramnios and consequent preterm labor in second trimester of pregnancy[END_REF][START_REF] Willis | Placental chorioangioma associated with polyhydramnios and hydrops fetalis[END_REF] . Finally, some cases of hydramnios were due to a myopathy causing impaired fetal swallowing [START_REF] Palmerio | A rare cause of polyhydramnios: Steinert's syndrome. A clinical case report[END_REF][START_REF] Robyr | Familial diseases revealed by a fetal anomaly[END_REF] . It is therefore interesting to examine fetal mobility and poor fetal muscle tone on ultrasound. In our series, one case of myotonic dystrophy Type I congenita l (Steinert) was diagnosed by amniocentesis but it had been overlooked by ultrasound.

According to the data in the literature, the anomalies most frequently missed by ultrasound are ultimately cardiac and digestive abnormalities [START_REF] Dashe | Hydramnios: anomaly prevalence and sonographic detection[END_REF][START_REF] Abele | Idiopathic polyhydramnios and postnatal abnormalities[END_REF][START_REF] Yefet | Outcomes From Polyhydramnios With Normal Ultrasound[END_REF] . We did not notice major cardiac anomalies in our series, possibly owing to the detailed evaluation of fetal cardiac anomaly which is routinely performed at the 20-25 WG fetal anatomy scan.

Finally, we found ultrasound of poor predictive value for diagnosing fetal macrosomia (PPV of 36.7%). In all of our cases where macrosomia alone was responsible for hydramnio s, the DP was always <16 cm. The prevalence of fetal macrosomia alone to explain hydramnio s at birth was 10.1% in our series (n = 16). Other authors have shown an association between idiopathic hydramnios and fetal macrosomia [START_REF] Sohaey | Idiopathic polyhydramnios: association with fetal macrosomia[END_REF][START_REF] Wiegand | Idiopathic Polyhydramnios: Severity and Perinatal Morbidity[END_REF] .

In our study, the severity and precocity of hydramnios appeared to be associated with the presence of underlying fetal pathologies. This is in agreement with another French series [START_REF] Bertholdt | Postnatal outcome from polyhydramnios without sonographic abnormalities[END_REF] and other studies [START_REF] Bundgaard | Prevalence of polyhydramnios at a Danish hospital--a population-based study[END_REF][START_REF] Lazebnik | The severity of polyhydramnios, estimated fetal weight and preterm delivery are independent risk factors for the presence of congenital malformations[END_REF][START_REF] Magann | A review of idiopathic hydramnios and pregnancy outcomes[END_REF][START_REF] Barkin | Severe polyhydramnios: incidence of anomalies[END_REF][START_REF] Amitai | The association between pregnancies complicated with isolated polyhydramnios or oligohydramnios and offspring long-term gastrointestinal morbidity[END_REF] .

In our study, MRI only modestly increased the diagnostic yield: a missed ultrasound diagnosis of esophageal atresia was detected on MRI. Another study also demonstrates the usefulness of MRI in the diagnosis of brain abnormalities associated with hydramnios [START_REF] Fishel-Bartal | Fetal brain MRI in polyhydramnios: is it justified?[END_REF] . Amniocentesis plays a critical role in the evaluation of hydramnios, and it increased substantially the diagnostic yield (from 73% based on targeted ultrasonography to 86.5%. Aneuploidy was found in 10% of fetuses with an abnormal ultrasound and in 1% of fetuses with normal ultrasound. This is consistent with the data in the literature reporting an association between chromosomal anomalies and hydramnios with 3.2% of chromosomal anomalies being found after a normal ultrasound 3334 . Hydramnios can also signal the presence of microdeletional syndromes like DiGeorge or Prader-Willi syndromes [START_REF] Devriendt | Polyhydramnios as a prenatal symptom of the digeorge/velo-cardio-facial syndrome[END_REF] . Array-CGH may be useful to detect these. Studies have shown that the detection rate of esophageal atresia approaches 100%, with a specificity of 98%, when biochemical analysis of the amniotic fluid is performed measuring the total protein level reveals a γ-glutamyl transpeptidase index multiplied by alphafeto protein [START_REF] Muller | Specific biochemical amniotic fluid pattern of fetal isolated esophageal atresia[END_REF][START_REF] Allaf | Amniotic fluid biochemistry in isolated polyhydramnios: a series of 464 cases[END_REF][START_REF] Czerkiewicz | Biochemical amniotic fluid pattern for prenatal diagnosis of esophageal atresia[END_REF] . In our series, one case of esophageal atresia which was not diagnosed by ultrasound was detected at amniocentesis. Analysis of the AF can also point towards an antenatal diagnosis of Bartter's syndrome (severe polyuria, renal sodium loss and postnatal dehydration). If the total protein level and Bartter index (total protein level multiplied by the alpha-fetoprotein level) are low, the sensitivity and specificity of a positive diagnosis of Bartter syndrome are 93% and 100%, respectively [START_REF] Garnier | Bartter syndrome prenatal diagnosis based on amniotic fluid biochemical analysis[END_REF] . Aldosterone dosage would appear to be less relevant for diagnosis [START_REF] Rachid | Prenatal diagnosis of Bartter syndrome: amniotic fluid aldosterone[END_REF] .

During our study period the incidence of pregnancies presenting with hydramnios was 1.3% at the Rennes University Hospital of which 119 (75.3%) were mild (DP <12), 35 In our series we discovered four cases of gestational diabetes. In 2017%, Frank et al studied isolated hydramnios appearing in the 3rd trimester of pregnancy and found 4.8% of the patients had late onset gestational diabetes (all patients had screened negative for diabetes in the 2nd trimester) confirming the importance of screening patients with hydramnios for diabetes [START_REF] Wolf | Isolated polyhydramnios in the third trimester: is a gestational diabetes evaluation of value?()[END_REF] . However, presence of maternal diabetes does not exclude other causes for hydramnios, as was the case for 3 of our patients; therefore, additional testing (e.g. MRI or amniocentes is ) should also be considered.

Infectious assessment

All the women presenting with hydramnios in our center are assessed for infectio n, including CMV and parvovirus B19 serologies. Toxoplamosis serology is already routine ly performed on all pregnancies in France at monthly follow-up obstetric visits. In our series, only one case of suspected CMV was diagnosed. The diagnostic benefit of maternal serologic testing for viral infections is therefore debatable: the association between hydramnios and CMV, for example, has never really been demonstrated [START_REF] Yefet | The association between polyhydramnios and CMV infection -retrospective cohort study[END_REF] . Two other studies also seem to show that screening for parvovirus B19 and TORCH serologies is not useful for pregnancies with isolated hydramnios on ultrasound, especially if it is discovered in the 3rd trimester [START_REF] Pasquini | Prevalence of a positive TORCH and parvovirus B19 screening in pregnancies complicated by polyhydramnios[END_REF][START_REF] Fayyaz | TORCH screening in polyhydramnios: an observational study[END_REF] .

Idiopathic hydramnios

The prevalence of idiopathic hydramnios in our series was 41.8% which is in agreement with the rates reported in the literature at between 40 and 60% 1,10,28 . Idiopathic hydramnios is a diagnosis of exclusion, which can only be retained after an exhaust ive assessment for other causes of hydramnios. In our series, no diagnoses of idiopathic hydramnio s was made for a DP >16 cm. Dashe et al showed that the risk of abnormalities discovered postnatally is 1% for mild, 2% for moderate and 11% for severe hydramnios in cases with normal fetal ultrasound 2 . Other studies, such as that of Drelin et al (39) in 2009, found up to 28.4% of anomalies detected during the first year of life in newborns with idiopathic hydramnios, while Abele et al (40) found 9.3% of anomalies in newborns with hydramnio s considered as idiopathic, with the main abnormality being gastrointestinal atresia 4546 . In our series, 5(3.1%) anomalies were detected in the postnatal period. A diagnosis of idiopathic hydramnios is not without implication for outcome. Even in the absence of a pathology explaining hydramnios, Asadi et al. reported a higher risk of low birth weight (<2500 g), macrosomia (>4000 g), but also admission to intensive care, fetal distress at birth, fetal death, and poor adaptation to extra-uterine life, premature birth and neonatal death in cases of idiopathic hydramnios compared to normal fluid pregnancies [START_REF] Asadi | Perinatal outcome in pregnancy with polyhydramnios in comparison with normal pregnancy in department of obstetrics at Shiraz University of Medical Sciences[END_REF] . A 2020 French study also found 24.1% of unfavorable outcomes in cases of idiopathic hydramnios [START_REF] Bertholdt | Postnatal outcome from polyhydramnios without sonographic abnormalities[END_REF] . A meta-analys is combining 10 studies and nearly 200,000 patients with hydramnios also found a two-fold risk of retro-placental hematoma in pregnancies complicated by hydramnios [START_REF] Khazaei | The association between polyhydramnios and the risk of placenta abruption: a meta-analysis[END_REF] . Similarly, another study demonstrated a higher risk of gastro-intestinal morbidity in children with a history of hydramnios [START_REF] Amitai | The association between pregnancies complicated with isolated polyhydramnios or oligohydramnios and offspring long-term gastrointestinal morbidity[END_REF] . These data suggest that all women with hydramnios should be referred to a tertiary maternofetal care center for the diagnostic workup including a targeted diagnost ic ultrasound scan, and offered an MRI and an amniocentesis for genetic and biochemica l assessments.

Strengths and weaknesses

Our study has some obvious limitations inherent to its retrospective design includ i ng data loss, especially for those women who did not finally give birth in our center and who were excluded from the study. This could also present a selection bias because all the included patients gave birth in our tertiary referral maternofetal care center and were possibly more severe. At variance with other series, we did not find a high prevalence of CNS anomalie s, which have been reported in up to 32% of cases of isolated polyhydramnios [START_REF] Fishel-Bartal | Fetal brain MRI in polyhydramnios: is it justified?[END_REF] . This could be due to the fact that fetal MRI was performed in only 9.5% of cases in our series. Similar ly, amniocentesis was performed in only 20% of our cases: the yield of microarray in the presence of isolated hydramnios has been reported as 3.8% [START_REF] Sagi-Dain | Chromosomal Microarray Analysis Results From Pregnancies With Various Ultrasonographic Anomalies[END_REF] . In our series, 2 microdeletion syndromes were present (Prader-Willi and Beckwith-Wiedemann syndrome). Moreover, the absence of long-term follow-up might have masked some pathologies (e.g. myopathies or CNS pathologies) which could have become manifested later in life. However, the size of our sample of 158 patients makes it one of the largest series to date. Furthermore, the variety of pathologie s described in our series revealed by hydramnios is of interest both for sonographers and for prenatal counselling.

Conclusion

Patients should be informed that appropriate work-up of hydramnios can reveal a wide variety of pathologies. Diagnostic ultrasound offers the most yield to identify or suspect the underlying cause (73%). Amniocentesis offers an important complementary assessment looking for aneuploidies, microdeletion syndrome, Bartter syndrome, esophageal atresia and congenital myopathies. MRI had a limited role in our series: it may marginally improve the detection of esophagus atresia. Despite a meticulous prenatal diagnostic work-up, a small proportion of pathologies can only be diagnosed postnatally.

Funding: none

Conflicts of interest: none

  .a) and four of duodenal stenosis (12.5%) (Figure 2.b). Other fetal and neonatal pathologies are detailed in Table 2, including 3 cases of myopathies (Figure 2.c), 2 of choanal atresia (Figure 2.d and 2.e), and 3 of anemia (Figure 2.f). Five fetuses had aneuploidy (3 with trisomy 21, and 2 with trisomy 18). The mode of discovery of these pathologies is shown in Table

  (22.2%) moderate (DP 10-16), and four (2.5%) severe (DP ≥16). These findings are in agreement with data from two large retrospective studies: a 2002 study carried out by Dashe et al 2 , includ i ng 672 cases of hydramnios, found a prevalence of 0.7% with 446 mild (66%), 145 moderate (22%) and 81 severe (12%) cases; and a 2007 study carried out by Bundgaard et al 3 includ i ng 168 hydramnios, reported a prevalence of 2% of which 66.7% were moderate and 33.3% severe.

Figure 1

 1 Figure 1. Flow Chart

Figure 2 .

 2 Figure 2.

Fig 2 -

 2 Fig 2-b. Double bubble : duodenum atresia

Table 1 .

 1 Population characteristics Values expressed in mean ± SD for quantitative variables and effectives and percentages n(%) for qualitative variables.DP: Deepest pocket, WG: weeks of gestation, T3: third trimester, MRI: Magnetic resonance imaging, P: percentile.

	Accepted Article

Table 2 .

 2 Types of Pathologies detected.

		Pathology N=37	N(%)
		Fetal defects
		Oesophagus atresia	6(18.8)
		Duodenal atresia	4(12.5)
		Chylothorax	2(6.3)
	Accepted Article	Fetal Supraventricular Tachycardia Choanal atresia Polymalformative syndrome Left diaphragmatic hernia Cleft lip Cardiopathy Hydrops Myopathy Undocumented Myopathy RYR1 Mutation Steinert's disease Anemia Anemia with fetal transfusion RH alloimmunization Cytomegalovirus infection Genetic syndromes Beckwith-Wiedemann syndrome Prader-Willi syndrome Bartter's syndrome Alagille syndrome Deletion 4p-Aneuploidies Trisomy 21 Trisomy 18 Placental abnormality Chorioangioma	2(6.3) 2(6.3) 2(6.3) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 1(3.1) 2(6.3) 1(3.1) 1(3.1)

Table 3 .

 3 Fetal or neonatal pathologies and discovery mode, n = 37(32 antenatal and 5 postnatal)

				Normal
		Hydramnios range	Pathologies suspected on diagnostic ultrasound n = 27	ultrasound pathology found amniocentesis, with	Normal ultrasound pathology found n = 1 with MRI	Found at birth n = 5
	Accepted Article	n = 5 -Steinert's disease -Esophageal atresia -Prader-Willi Syndrome Eesophageal atresia -Trisomy 18 -Bartter 's syndrome 0 DP : Deepest pocket of amniotic fluid ; CMV cytomegalovirus DP 8 -11 (n = 18) -Cleft lip -Duodenal Stenosis -Trisomy 21 -Beckwith-Wiedemann -Anemia -Chorioangioma DP 12 -15 (n = 16) -Unilateral choanal atresia -3 esophageal atresia / 2 duodenum atresia -Hydrops (chylothorax) -Left diaphragmatic hernia -Myopathy -Trisomy 21 -Polymalformative sequence DP ≥ 16 (n = 3) -Suspected CMV -Fetal tachycardia	-Anti-D alloimmunization -Choanal atresia -Alagille Syndrome -Myopathy -Deletion chromosome 4p-0

Table 4 .

 4 Pathological versus non-pathological hydramnios.

	Pathological hydramnios	Non-pathological hydramnios
	N=76	N=82	p

Table 5 .

 5 Etiologies by polyhydramnios severity

	Hydramnios range Deepest pocket (cm)	Total N(%) N=158	Diabetes n = 39	n = 32 +5 pathology Fetal or neonatal	n = 16 Macrosomia	n = 66 Idiopathic Hydramnios	p
	8,0 -11.9	118(74.7)	30 (25.4)	15 + 3 (15.2)	14 (11.9)	56 (47.5)	0.002
	12 -15.9	36(22.8)	8 (22.2)	14 + 2 (44.4)	2 (5.6)	10 (27.8)
	≥ 16.0	4(2.5)	1 (25.0)	3 (75.0)	0 (0%)	0 (0%)
	Bold: post-natal diagnosis

Research data are available from the corresponding author upon request.