Stéphane Supiot
email: stephane.supiot@ico-unicancer.fr

Loig Vaugier

David Pasquier

Xavier Buthaud

Nicolas Magné

Didier Peiffert

Paul Sargos

Gilles Crehange

Pascal Pommier

Genevieve Loos

Ali Hasbini

Igor Latorzeff

Marlon Silva

Fabrice Denis

Jean-Léon Lagrange

Cyrille Morvan

Loic Campion

Audrey Blanc-Lapierre

OLIGOPELVIS GETUG P07, a Multicenter Phase II Trial of Combined High-dose Salvage Radiotherapy and Hormone Therapy in Oligorecurrent Pelvic Node Relapses in Prostate Cancer

Keywords: Oligometastases, Oligorecurrent, Prostate cancer, Salvage, Pelvic radiotherapy, Intensity-modulated radiotherapy, Prostate bed irradiation, Lymphadenectomy, Positron-emission tomography prostate-specific membrane antigen, Choline, Fluciclovine, Stereotactic body radiotherapy, Stereotactic ablative radiotherapy, Stereotactic radiotherapy

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

3

Abstract

Background: Oligorecurrent pelvic nodal relapse in prostatic cancer is a challenge for regional salvage treatments. Androgen depriving therapies (ADTs) are a mainstay in metastatic prostate cancer, and salvage pelvic radiotherapy may offer long ADTfree intervals for patients harboring regional nodal relapses.

Objective: To assess the efficacy of the combination of ADT and salvage radiotherapy in men with oligorecurrent pelvic node relapses of prostate cancer.

Design, setting, and participants:

We performed an open-label, phase II trial of combined high-dose intensity-modulated radiotherapy and ADT (6 mo) in oligorecurrent (five or fewer) pelvic node relapses in prostate cancer, detected by fluorocholine positron-emission tomography computed tomography imaging.

Outcome measurements and statistical analysis:

The primary endpoint was 2-yr progression-free survival defined as two consecutive prostate-specific antigen levels above the level at inclusion and/or clinical evidence of progression as per RECIST 1.1 and/or death from any cause.

Results and limitations:

Between August 2014 and July 2016, 67 patients were recruited in 15 centers. Half of the patients had received prior prostatic irradiation.

The median age was 67.7 yr. After a median follow-up of 49.4 mo, 2-and 3-yr progression-free survival rates were 81% and 58%, respectively. Median progression-free survival was 45.3 mo. The median biochemical relapse-free survival (BRFS) was 25.9 mo. At 2 and 3 yr, the BRFS rates were 58% and 46%, respectively. Grade 2+ 2-yr genitourinary and gastrointestinal toxicities were 10% and 2%, respectively.

4

Conclusions: Combined high-dose salvage pelvic radiotherapy and ADT appeared to prolong tumor control in oligorecurrent pelvic node relapses in prostate cancer with limited toxicity. After 3 yr, nearly half of patients were in complete remission. Our study showed initial evidence of benefit, but a randomized trial is required to confirm this result.

Patient summary:

In this report, we looked at the outcomes of combined high-dose salvage pelvic radiotherapy and 6-mo-long hormone therapy in oligorecurrent pelvic nodal relapse in prostatic cancer. We found that 46% of patients presenting with oligorecurrent pelvic node relapses in prostate cancer were in complete remission after 3 yr following combined treatment at the cost of limited toxicity.

Introduction

The major cause of death among prostate cancer patients is the development of metastases. The natural history of the metastatic process has been revealed by recent imaging techniques based on nonspecific prostate cancer markers such as fluorocholine (FCH), prostate-specific membrane antigen (PSMA), or fluciclovine positron-emission tomography (PET) [START_REF] Colombie | Prognostic value of metabolic parameters and clinical impact of (1)(8)F-fluorocholine PET/CT in biochemical recurrent prostate cancer[END_REF][START_REF] Supiot | Oligometastatic prostate cancer: is it worth targeting the tip of the iceberg?[END_REF][START_REF] Calais | F-fluciclovine PET-CT and (68)Ga-PSMA-11 PET-CT in patients with early biochemical recurrence after prostatectomy: a prospective, singlecentre, single-arm, comparative imaging trial[END_REF]. These have made it possible to identify limited metastatic relapses in prostatic cancer, especially in small pelvic lymph nodes (PLNs), which occur early and at low prostate-specific antigen (PSA) levels, when conventional imaging is unable to reveal disease localization due to the small size (frequently <10 mm) [START_REF] Colombie | Prognostic value of metabolic parameters and clinical impact of (1)(8)F-fluorocholine PET/CT in biochemical recurrent prostate cancer[END_REF][START_REF] Lepinoy | Pattern of occult nodal relapse diagnosed with (18)F-fluoro-choline PET/CT in prostate cancer patients with biochemical failure after prostate-only radiotherapy[END_REF]. This oligometastatic state may be an apparent turning 5 point between still-controllable regional disease that might be managed with local intervention, and diffuse disease for which androgen depriving therapies (ADTs) are the mainstay [START_REF] Tosoian | Oligometastatic prostate cancer: definitions, clinical outcomes, and treatment considerations[END_REF][START_REF] Ost | Surveillance or metastasis-directed therapy for oligometastatic prostate cancer recurrence: a prospective, randomized, multicenter phase II trial[END_REF]. Oligorecurrent prostate cancer is now the preferred term for designating such relapses after primary curative-intent treatments [START_REF] Guckenberger | Characterisation and classification of oligometastatic disease: a European Society for Radiotherapy and Oncology and European Organisation for Research and Treatment of Cancer consensus recommendation[END_REF].

In locally advanced disease, the role of radiotherapy in the management of micrometastatic lymph nodes is highly debated [START_REF] Pommier | Is there a role for pelvic irradiation in localized prostate adenocarcinoma? Preliminary results of GETUG-01[END_REF][START_REF] Roach | Sequence of hormonal therapy and radiotherapy field size in unfavourable, localised prostate cancer (NRG/RTOG 9413): long-term results of a randomised, phase 3 trial[END_REF]. In the salvage postprostatectomy setting, there is a further lack of clear evidence regarding the role of radiotherapy in lymph nodes [START_REF] Spiotto | Radiotherapy after prostatectomy: improved biochemical relapse-free survival with whole pelvic compared with prostate bed only for high-risk patients[END_REF]. Metastasis-directed therapy, mostly using stereotactic radiotherapy to PLNs is one option, and it has been shown to decrease biochemical relapse and delay the need for ADT [START_REF] Ost | Surveillance or metastasis-directed therapy for oligometastatic prostate cancer recurrence: a prospective, randomized, multicenter phase II trial[END_REF][START_REF] Phillips | Outcomes of observation vs stereotactic ablative radiation for oligometastatic prostate cancer: the ORIOLE phase 2 randomized clinical trial[END_REF]. However, most patients relapse in the pelvic area [START_REF] Ost | Pattern of progression after stereotactic body radiotherapy for oligometastatic prostate cancer nodal recurrences[END_REF][START_REF] Deek | Patterns of recurrence and modes of progression after metastasis-directed therapy in oligometastatic castration-sensitive prostate cancer[END_REF]. Salvage elective node radiotherapy (ENRT) with an additional boost to any PET-positive PLN is another attractive option, but has the advantage of tackling the potential pelvic micrometastatic invasion. The best current evidence available for ENRT is derived from retrospective studies with varying doses and schedules of radiotherapy [START_REF] Picchio | C-choline PET/CT as a guide to radiation treatment planning of lymph-node relapses in prostate cancer patients[END_REF][START_REF] Fodor | Toxicity and efficacy of salvage carbon 11choline positron emission tomography/computed tomography-guided radiation therapy in patients with lymph node recurrence of prostate cancer[END_REF][START_REF] Tran | Oligorecurrent nodal prostate cancer: long-term results of an elective nodal irradiation approach[END_REF][START_REF] Ingrosso | Salvage radiotherapy in patients affected by oligorecurrent pelvic nodal prostate cancer[END_REF][START_REF] Sato | Long-term clinical outcomes of salvage pelvic radiation therapy for oligo-recurrent pelvic lymph nodes after definitive external-beam radiation therapy for non-metastatic prostate cancer[END_REF].

The main objective of this phase II trial was to assess the efficacy of high-dose salvage ENRT in a prospective manner in a well-defined population. We hypothesized that such ENRT combined with 6-mo of ADT would achieve a 2-yr progression-free survival rate of 70%. Here, we present the main objective of the trial after a minimum follow-up of 3 yr.

Patients and methods

Study design and participants 6

The complete trial design has been published previously [START_REF] Supiot | OLIGOPELVIS -GETUG P07: a multicentre phase II trial of combined salvage radiotherapy and hormone therapy in oligometastatic pelvic node relapses of prostate cancer[END_REF]. Pelvic (below the aortic bifurcation) oligorecurrent castration-sensitive prostate cancer patients with fewer than six metastatic PLNs detected using FCH-PET were included in the trial. FCH-PET/computed tomography (CT) images were scored as positive when focal tracer accumulation was greater than background activity or tracer physiological distribution. The diagnosis of malignant lymph nodes following a PET analysis was based on visual assessment of increased focal FCH uptake corresponding to lymph nodes on the CT image, even if they were <10 mm as described by Colombie et al [START_REF] Colombie | Prognostic value of metabolic parameters and clinical impact of (1)(8)F-fluorocholine PET/CT in biochemical recurrent prostate cancer[END_REF]. Images at relapse were centrally reviewed blindly by a nuclear oncologist (C.M.).

If dubious prostate or prostate bed FCH uptake was noted, pelvic magnetic resonance imaging was performed to assess for local relapse. If ADT had been administered previously to the patient, a minimum of 6-mo wash-out period was required, and serum testosterone had to be >6 nmol/l prior to inclusion. Previous irradiation of the prostate or the prostate bed was allowed, provided that there was a minimum of 1 cm gap between the prostate and salvage pelvic radiotherapy fields.

Patients with extrapelvic metastases or patients under active ADT were excluded from the trial.

The trial population was divided into four groups, each with a different treatment plan:

Procedures

Image-guided intensity-modulated radiation therapy was required to deliver 54 Gy in 1.8 Gy fractions to the whole pelvis, with a simultaneous integrated boost of 66 Gy in 2.2 Gy fractions to pathological PLNs. Patients who had not received previous irradiation received 66 Gy in 2 Gy fractions to the prostatic bed, with up to 72 Gy in 2 Gy fractions in the case of prostatic bed local relapse. PLNs were contoured according to the recommendations in the RTOG consensus statement [START_REF] Lawton | RTOG GU Radiation oncology specialists reach consensus on pelvic lymph node volumes for high-risk prostate cancer[END_REF] modified by the GETUG group [START_REF] Sargos | Definition of lymph node areas for radiotherapy of prostate cancer: a critical literature review by the French Genito-Urinary Group and the French Association of Urology (GETUG-AFU)[END_REF], including the common iliac, external iliac, internal iliac, presacral (S1-S3), and obturator regions. The upper limit was defined by the bifurcation of the abdominal aorta. Androgen blockade was achieved by luteinizing hormone releasing hormone agonist or antagonist injections for 6 mo, ideally administered either on the 1st day of radiation therapy or in the 3 mo before the 1st day of radiation therapy.

Outcomes

The primary objective of this study was to describe the 2-yr progression-free survival (PFS) rate in men with oligorecurrent pelvic node relapses of prostate cancer, receiving the combination of ADT with salvage radiotherapy. Progression was defined by a cluster of events, including PSA progression defined as two consecutive PSA levels above the level at inclusion and measured in the same laboratory and/or clinical evidence of progression as per RECIST 1.1 or death from any cause.

Secondary objectives included biochemical relapse-free survival (BRFS), overall survival (OS), time to the start of a second-line treatment (TTST), time to start palliative ADT (TTADT), acute and late toxicity, and quality of life assessed using the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 and PR25 questionnaires. Biochemical relapse was defined as two consecutive PSA levels of >0.2 ng/ml following the post-treatment nadir. For patients with prior organpreserving prostate treatment (external beam radiotherapy or brachytherapy), biochemical relapse was defined as (1) a PSA level of >0.2 ng/ml following the posttreatment nadir and (2) a PSA level of more than the nadir following the prior organpreserving prostate treatment. Toxicity (CTC-AE v4) and quality of life were evaluated prior to treatment and 1 mo after the completion of radiotherapy, and then every 6 mo for 2 yr. If a patient presented with the same toxic event several times, only the highest-grade event was reported. Toxicities were recorded until the progression, as defined above. PSA and testosterone levels were determined prior to radiotherapy, 1 mo after completion, every 6 mo for 2 yr, and then yearly until progression. Quality of life was evaluated prior to treatment and 1 mo the after completion of radiotherapy, and then every 6 mo for 2 yr using the EORTC QLQ C30 and PR25 questionnaires. PSA and testosterone levels were determined prior to radiotherapy, 1 mo after completion, and then every 6 mo. Repeat FCH-PET was performed at biochemical recurrence.

Statistical analysis

Patients with oligorecurrent pelvic node relapses detected by FCH-PET represent an intermediate group between patients with rising PSA alone and frank metastatic disease. To calculate the patient population, we relied on both studies relating to a rising PSA patient population [START_REF] Crook | Intermittent androgen suppression for rising PSA level after radiotherapy[END_REF] and first-line metastatic patients [START_REF] Hussain | Intermittent versus continuous androgen deprivation in prostate cancer[END_REF] observing 2-yr 9 PFS rates of ≤50%. We therefore estimated that a BRFS rate at 2 yr of 70% would be a significant result. The one-step phase 2 Fleming design was applied. Based on the hypothesis of a 2-yr PFS rate of 70% with salvage PLN IG-IMRT combined with 6-mo ADT, 63 evaluable patients were required to demonstrate with a power of 95% and a one-sided alpha risk of 5% that the 2-yr PFS rate was >50% [START_REF] Supiot | OLIGOPELVIS -GETUG P07: a multicentre phase II trial of combined salvage radiotherapy and hormone therapy in oligometastatic pelvic node relapses of prostate cancer[END_REF]. A target sample size of N = 70 was thus calculated to account for a 10% dropout rate. Data from all evaluable patients were analyzed. PFS, BRFS, TTST, TTADT, and OS were computed from the beginning of treatment. Patients without the outcome were censored on the date of the last time the patient was known to be free of this outcome. The Kaplan-Meier method was applied to estimate survival curves [START_REF] Schemper | A note on quantifying follow-up in studies of failure time[END_REF]. We used Cox regression, assuming proportional hazards, to run post hoc exploratory univariate analyses to investigate the prognostic value of PSA levels at baseline on PFS, Gleason score at diagnosis, number of nodes, PSA level 6 mo after treatment initiation, PSA doubling time, and the time from initial treatment to the initiation of the treatment being studied. We estimated hazard ratios (HRs) and their 95% confidence intervals (CIs). We did not impute missing data for covariates.

Quality of life scores at 18 and 24 mo were compared with baseline scores using a Wilcoxon signed test for matched pairs. All p values were corrected according to a Benjamini-Hochberg procedure to control for false discovery rates. Quality of life differences were considered clinically relevant when the value was >10.

All p values were based on two-sided tests and were considered significant if <0.05.

We used SAS version 9.4 for the analyses. This trial has been registered under number NCT02274779. 10

Results

Between August 2014 and July 2016, 74 patients were recruited in 15 centers (Fig. 1 and Table 1). Seven patients were excluded for various reasons (lost to follow-up immediately after randomization and before any treatment: one; more than five lymph nodes: one; time between ADT and radiotherapy >3 mo: two; and only one PSA level before randomization and unacceptable PSA follow-up: three). Of the patients who were analyzed, 35 had received previous prostatic irradiation, mostly to the prostate bed (29 patients) or to the prostate (three patients, each following external beam radiotherapy or brachytherapy to the prostate). Only four patients presented with a concurrent local relapse. The median age was 67.7 yr. The median number of PETpositive oligorecurrent PLNs was one (interquartile range [IQR]: 1-2). Of note, 61% of patients only had one node visualized.

Acute toxicity and 1-yr toxicity were reported earlier [START_REF] Vaugier | Early toxicity of a phase II trial of combined salvage radiotherapy and hormone therapy in oligometastatic pelvic node relapses of prostate cancer (OLIGOPELVIS GETUG P07)[END_REF]. At 2 yr, grade 2+ genitourinary and gastrointestinal toxicities were 10% and 2%, respectively (Table 2).

No clinical parameters, such as a prior history of prostatic irradiation, could predict for 2-yr toxicity. Two patients suffered severe grade 3 incontinence occurring after combined prostatectomy and prostate bed irradiation. The completion rates for the quality of life questionnaires were 67% and 54% at 18 and 24 mo, respectively. No significant alteration in urinary or intestinal quality of life was noted at 2 yr (Fig. 2, and Supplementary Tables 1 and2). Of note, no patient suffered from chronic grade 2 diarrhea or intestinal bleeding. Testosterone levels went back to normal levels in all patients except for one after a median time of 14.4 mo (Supplementary Fig. 1).

Hormonal treatment-related quality of life and sexual activity were maintained at 2 yr.

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Reasons for not completing the quality of life form were independent of the patients in one of four cases (omission from the investigator staff). In other cases, participation could be related to patient health status. We compared age and baseline clinical characteristics of both respondents and nonrespondents at 24 mo, but there was no significant difference except for median PSA doubling time: 5. The median follow-up for survivors was 49.4 mo (IQR: 42.3-53.1 mo). The 2-and 3yr PFS rates were 81% and 58%, respectively (Fig. 3A). Median PFS was 45.3 mo (95% CI: 31.8-48.5 mo). In all patients except for two, PSA decreased by >50% at 6 mo (Supplementary Fig. 2). We also performed a sensitivity analysis by applying the worst-case imputation method, where the four patients excluded from the analysis due to lack of follow-up were considered to have progressed 6 mo after treatment initiation. In this scenario, the median PFS was 41.8 mo and the 2-yr PFS was 76%.

At 2 and 3 yr, the BRFS rates were of 58% and 46%, respectively (Fig. 4). The 2and 3-yr time to secondary therapy initiation (TTST) was 84% and 63%, respectively (Fig. 3B). The median TTST was 49.8 mo. Median BRFS was 25.9 mo (Fig. 3C). The first secondary treatment at biochemical-clinical progression was stereotactic body radiotherapy (SBRT), ADT, or both in 22.5%, 42.5%, and 10% of patients, respectively. At relapse, 13%, 27%, and 25% of patients had progression with one metastasis, three or fewer metastases, and more than three metastases, respectively, while 7.5% had PSA progression only. Metastases were revealed by FCH-PET in 39% of patients, Ga-PSMA PET in 9%, and imaging methods other than PET in 4.5%.

The relapses occurred in the lymph nodes outside the pelvis (M1a), bones (M1b), PLNs (N1), and prostatic bed in 50%, 30%, 25%, and 15% of patients, respectively 12 (Table 3). Compared with groups A + B, progression for group C + D patients was as follows: M1a metastatic lymph nodes (39% vs 19%, p = 0.081), M1b bone metastases (28% vs 6.5%, p = 0.028), and prostatic bed local relapses (11% vs 6.5%, p = 0.7). The median TTADT was 51.9 mo (Supplementary Fig. 3). The 3-yr OS rate was 97% (two deaths), with one prostate cancer-related death at 33 mo after the beginning of treatment. No prognostic factor could predict for progression or biochemical complete response (Table 4 and Supplementary Table 3). We compared outcomes between groups based on prior therapy (Fig. 5 and Supplementary Fig. 4).

In patients with prior prostatectomy, the 2-yr PFS rate was 97% in groups A + B versus 66% in group C (p < 0.01). The 2-yr metastasis-free survival (MFS) was 87%, and the MFS median was 48.2 (Supplementary Fig. 5).

Discussion

This trial addressed the use of 6-mo ADT combined with salvage high-dose pelvic radiotherapy in prostate cancer patients whose oligometastatic relapse had been identified on FCH-PET imaging. Our findings provide evidence of prolonged PFS in these patients at the cost of limited toxicity, even in those with a past history of prostate bed radiotherapy. To our knowledge, this trial is the first to prospectively address the efficacy and toxicity of elective pelvic radiotherapy combined with shortterm ADT.

In the metastatic setting, ADT can be administered continuously or intermittently. In patients in whom metastases were diagnosed using conventional imaging (CT scan and total bone scan), continuous ADT is recommended, as the SWOG-9346 study was not able to rule out a 20% greater risk of death with intermittent therapy than with continuous therapy [START_REF] Hussain | Intermittent versus continuous androgen deprivation in prostate cancer[END_REF]. In patients with a rising PSA level and no visible metastases on conventional imaging, intermittent ADT was noninferior to continuous therapy [START_REF] Crook | Intermittent androgen suppression for rising PSA level after radiotherapy[END_REF]. In our situation, where conventional imaging would not have been able to detect metastatic lymph nodes, intermittent ADT could be considered a validated option. In this latter study, ADT was given for 8 mo and relapse was defined as clinical disease progression or PSA levels of >10 ng/ml [START_REF] Crook | Intermittent androgen suppression for rising PSA level after radiotherapy[END_REF]. Using this definition, the median time from initiation of ADT to progression was 28.1 mo. In our study, we chose 6 mo of ADT, as proposed by randomized trials in localized [START_REF] Jones | Radiotherapy and short-term androgen deprivation for localized prostate cancer[END_REF][START_REF] D'amico | Androgen suppression and radiation vs radiation alone for prostate cancer: a randomized trial[END_REF] or biochemically relapsing prostate cancer [START_REF] Carrie | Short-term androgen deprivation therapy combined with radiotherapy as salvage treatment after radical prostatectomy for prostate cancer (GETUG-AFU 16): a 112-month follow-up of a phase 3, randomised trial[END_REF]. The relapse was observed at a median PSA level of 3.9 ng/ml (IQR: 2.4-4.9 ng/ml), lower than the 10 ng/ml cutoff in the PR7 study [START_REF] Crook | Intermittent androgen suppression for rising PSA level after radiotherapy[END_REF]. However, we found median PFS of 45.3 mo in our study and time to palliative ADT of 51.9 mo. This means that salvage radiotherapy combined with 6 mo of ADT may increase PFS, while delaying the need for palliative ADT. At 3 yr, 45% achieved a persistent complete response while achieving normal testosterone levels.

Longer follow-up is needed to assess whether or not salvage radiotherapy combined with ADT is capable of perhaps even curing certain patients. A short PSA doubling time negatively predicted progression following ADT + ENRT. This patient population may benefit from prolonged ADT. Patients with a prior history of prostate bed irradiation had a worse outcome and presented with more bone metastases at relapse than patients with no prior radiotherapy. This might be explained by more frequent prior exposure to ADT in this group, which might have selected more biologically aggressive tumor clones. This further supports the early use of PLN irradiation in the RTOG 0534 SPPORT trial that showed improved MFS using combined salvage prostate bed and pelvic radiotherapy treatment [START_REF] Pollack | Short term androgen deprivation therapy without or with pelvic lymph node treatment added to prostate bed only salvage radiotherapy: the NRG Oncology/RTOG 0534 SPPORT trial[END_REF]. In the future, the discovery of oligorecurrent metastatic PLNs may be less common as combined salvage prostate bed and pelvic radiotherapy will increasingly be used. Importantly, tumor control was achieved at the cost of limited toxicity only. Despite high doses to lymph nodes close to the intestine or the bladder, grade 3 toxicity was infrequent, corroborating retrospective studies [START_REF] Picchio | C-choline PET/CT as a guide to radiation treatment planning of lymph-node relapses in prostate cancer patients[END_REF][START_REF] Fodor | Toxicity and efficacy of salvage carbon 11choline positron emission tomography/computed tomography-guided radiation therapy in patients with lymph node recurrence of prostate cancer[END_REF][START_REF] Tran | Oligorecurrent nodal prostate cancer: long-term results of an elective nodal irradiation approach[END_REF][START_REF] Ingrosso | Salvage radiotherapy in patients affected by oligorecurrent pelvic nodal prostate cancer[END_REF][START_REF] Sato | Long-term clinical outcomes of salvage pelvic radiation therapy for oligo-recurrent pelvic lymph nodes after definitive external-beam radiation therapy for non-metastatic prostate cancer[END_REF]. Despite a 1 cm gap between prostate bed and salvage pelvic radiation therapy fields, pelvic tissues were partially irradiated again, and this may have increased toxicity. Despite this, we did not observe increased toxicity between patients either with or without a past history of prostate radiotherapy. A similar tolerance profile has been reported in cases of repeated SBRT for pelvic oligorecurrences [START_REF] Jereczek-Fossa | Robotic image-guided stereotactic radiotherapy, for isolated recurrent primary, lymph node or metastatic prostate cancer[END_REF][START_REF] Decaestecker | Repeated stereotactic body radiotherapy for oligometastatic prostate cancer recurrence[END_REF].

Metastasis-directed therapy using radiotherapy is an active area of research the positive impact of which on OS has been demonstrated in various primary histologies [START_REF] Palma | Stereotactic ablative radiotherapy versus standard of care palliative treatment in patients with oligometastatic cancers (SABR-COMET): a randomised, phase 2, open-label trial[END_REF]. In pelvic nodal oligorecurrent prostate cancer specifically, radiotherapy can be administered either to the whole pelvis as proposed in our study or to the lymph nodes involved using SBRT. SBRT versus observation was shown to increase PFS and delay the need for ADT in both prospective randomized trials and retrospective analyses [START_REF] Ost | Surveillance or metastasis-directed therapy for oligometastatic prostate cancer recurrence: a prospective, randomized, multicenter phase II trial[END_REF][START_REF] Siva | Stereotactic ablative body radiotherapy (SABR) for oligometastatic prostate cancer: a prospective clinical trial[END_REF], with median distant PFS, TTST, and TTADT of around 20-28 mo, compared with 47-52 mo in our study. Similarly, median time to biochemical recurrence was 10 mo in the STOMP study and 25.9 mo in our study. We need to mention that our patient population comprised only those with oligorecurrence to PLNs and not to bone or abdominal lymph node metastases, the prognosis of which is worse. Lastly, in our study, PSA response was also longer because 6-mo-long ADT administration and testosterone recovered to noncastrate level after a median time of approximately 8 mo after the completion of treatment. However, this may also suggest that salvage high-dose elective pelvic radiotherapy may be superior to SBRT, as already suspected from retrospective studies [START_REF] Bleser | Metastasis-directed therapy in treating nodal oligorecurrent prostate cancer: a multi-institutional analysis comparing the outcome and toxicity of stereotactic body radiotherapy and elective nodal radiotherapy[END_REF][START_REF] Lepinoy | Salvage extended field or involved field nodal irradiation in (18)F-fluorocholine PET/CT oligorecurrent nodal failures from prostate cancer[END_REF]. Salvage lymphadenectomy has also been proposed for PLN oligorecurrences, at the cost of only limited toxicity.

A pooled analysis of multiple series showed that complete biochemical response rates ranged from 13% to 79% (mean 44%). The 2-yr BRFS rates ranged from 23% to 64% [START_REF] Ploussard | Salvage lymph node dissection for nodal recurrent prostate cancer: a systematic review[END_REF], yielding results comparable with those of our study. Whether high-dose pelvic radiotherapy compares favorably with extended lymphadenectomy or whether both treatments need to be added remains an open question. An international randomized phase II trial (PEACE 5 -STORM, NCT03569241) is currently comparing 6-mo ADT and SBRT or lymphadenectomy with or without elective whole pelvis radiotherapy. This trial has several limitations, the first being the limited number of patients inherent to the trial design. Second, FCH was the only radiotracer available when the study was initiated. Now, PSMA-based PET tracers are more widely available and may select patients with a more precise definition of the extension of the disease at biochemical relapse. However, despite the use of PSMA-based PET tracers, most patients relapse following stereotactic ablative radiotherapy to oligorecurrent disease [START_REF] Kneebone | Stereotactic body radiotherapy for oligometastatic prostate cancer detected via prostate-specific membrane antigen positron emission tomography[END_REF], suggesting a role for combining systemic and radiotherapy-based local treatment. Moreover, the toxicity profile was good despite high-dose irradiation to the pelvis, but completion rate of the quality of life form was fairly low, which is a limitation. Our study also lacks genomic characterization of the tumors. Tumor mutational profiles can provide a biological definition of oligometastasis and may help select patients who may benefit from metastasis-directed treatments [START_REF] Deek | The mutational landscape of metastatic castration-sensitive prostate cancer: the spectrum theory revisited[END_REF]. Lastly, this study was not randomized. The need for a randomized arm in phase 2 trials is highly debated. When the study started in 2014, very limited information on FCH-PET was available and the prevalence of oligorecurrent PLNs was unknown. We therefore 16 decided to perform a single-arm phase 2 trial based on experts' recommendations [START_REF] Gan | Randomized phase II trials: inevitable or inadvisable?[END_REF] for cases when limited patients are available for recruitment. The only possibility for validating the benefits of adding salvage pelvic radiotherapy to a standard arm (intermittent ADT) will be an ongoing phase 3 study (OLIGOPELVIS 2 GETUG P12, NCT03630666). A standard arm in this situation is highly debated, as discussed during the Advanced Prostate Cancer Consensus Conference 2019 [START_REF] Gillessen | Management of patients with advanced prostate cancer: report of the Advanced Prostate Cancer Consensus Conference 2019[END_REF]. Recent trials showed that patients with metastatic prostate cancer benefit from androgen receptor agents such as abiraterone, apalutamide, or enzalutamide [START_REF] James | Abiraterone for prostate cancer not previously treated with hormone therapy[END_REF][START_REF] Chi | Apalutamide for metastatic, castration-sensitive prostate cancer[END_REF][START_REF] Davis | Enzalutamide with standard first-line therapy in metastatic prostate cancer[END_REF]. Future trials could also consider intensified androgen-axis blockade as a means of increasing tumor control in oligometastatic relapse of prostate cancer, especially in patients with a higher risk of relapse (short PSA doubling time or a prior history of prostate/prostate bed radiotherapy).

Conclusions

Combined high-dose elective salvage pelvic radiotherapy and ADT appeared to prolong tumor control in oligorecurrent pelvic node relapses in prostate cancer, with a significant proportion of patients still in complete remission 3 yr after the procedure, at the cost of only limited toxicity. Our study showed initial evidence of benefit, but a randomized trial is required to confirm this result.

Author contributions: Stéphane Supiot had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. ADT = androgen depriving therapy; CI = confidence interval; HR = hazard ratio; ISUP = International Society of Urological Pathology; PSA = prostate-specific antigen.

( 1 )

 1 group A: previous radical prostatectomy and no previous prostate bed radiation, with fewer than six FCH-PET-positive PLNs; (2) group B: the same as group A, but with an FCH-PET-positive signal in the prostate bed, suggesting local relapse; (3) group C: both previous radical prostatectomy and salvage prostate bed radiation therapy; and (4) group D: previous irradiation of the prostate (brachytherapy or external beam radiotherapy).

7

 7

  3 mo (IQR: 3.5-10.3 mo) in respondents versus 4.3 mo (IQR: 2.2-7.0 mo) in nonrespondents.

Fig. 1 -Fig. 2 -

 12 Fig. 1 -Flow chart of the trial. ADT = androgen depriving therapy; BT =

Fig. 3 -Fig. 4 -Fig. 5 -

 345 Fig. 3 -(A) Biochemical-clinical progression-free survival. (B) Time to start of a

Table 1 -Patient characteristics, according to patient group

 1

	Patient and tumor characteristics	Group A + B	Group C + D	p value
		(n = 31)	(n = 36)
	Median age (yr)	68.0 (IQR: 64.0-	67.0 ± (IQR:	0.9 (WMW)
		72.0)	63.0-73.5)
	ISUP score, n (%)			0.028 (F)
	1	2 (6.4)	6 (17)
	2	7 (23)	16 (44)
	3	18 (58)	8 (22)
	>3	4 (13)	6 (17)
	Tumor stage, n (%)			0.11 (F)
	pT1	2 (6.5)	-
	pT2	12 (39)	12 (33)
	pT3	17 (55)	18 (50)
	cT1	-	3 (8.3)
	cT2	-	3 (8.3)
	Pathological node involvement, n			0.6 (F)
	(%)
	pN0	26 (84)	30 (83)
	pN1	1 (3.2)	-
	Nx	4 (13)	6 (17)
	Number of PET-positive PLN, n (%)			0.8 (F)
	1	19 (61)	22 (61)
	2	7 (23)	9 (25)
	3	2 (6.4)	3 (11)
	4	2 (6.4)	1 (2.8)
	5	1 (3.2)	-
	Median PSA at baseline (ng/ml)	3.9 (IQR: 1.0-5.6) 3.6 (IQR:1.7-5.3) 0.9 (WMW)
	Median PSA doubling time (mo)	4.6 (IQR: 3.2-9.2) 5.2 (IQR: 2.6-7.6) 0.9 (WMW)
	Patients with prior administration of	2 (6.4)	10 (28)	0.028 (F)
	ADT, n (%)

Table 3 -Pattern of relapses at progression as assessed by the TNM 2005 classification for patients with (groups C + D) and without (groups A + B) prior prostate/prostatic bed radiotherapy

 3 with metastases in separate anatomic regions were counted twice. The trial population groups were as follows: group A: previous radical prostatectomy and no previous prostate bed radiation, with fewer than six FCH-PET-positive PLNs; group B: the same as group A, but with an FCH-PET-positive signal in the prostate bed, suggesting local relapse; group C: both previous radical prostatectomy and salvage prostate bed radiation therapy; and group D: previous irradiation of the prostate (brachytherapy or external beam radiotherapy).

	Groups A + B	Groups C + D	p value
	(N = 31)		(N = 36)
	n	%	n	%

PSA = prostate-specific antigen; F = Fisher test; FCH = fluorocholine; PET = positronemission tomography; PLN = pelvic lymph node; TNM = tumor, node, metastasis. Patients

Table 4 -Predictors of progression

 4

	Parameter	p value	HR	95% HR CI
	PSA at baseline (4 ng/ml)	0.2	0.88	0.70 -1.09
	ISUP at diagnosis (>4 vs ≤3)	0.6	1.24	0.52 -2.96
	Time from diagnosis to initiation of the	0.7	1.09	0.72 -1.67
	treatment being studied (5 yr)
	Number of nodes (>1 vs 1)	0.7	0.87	0.45 -1.67
	PSA doubling time (3 mo)	0.3	0.88	0.70 -1.10
	History of previous ADT	0.2	1.65	0.76 -3.60
	Treatment group (AB vs CD)	<0.01	0.34	0.17 -0.68
	Time to testosterone recovery (1 yr)	0.5	1.25	0.62 -2.54

Study concept and design: Supiot, Campion.

Acquisition of data: Pasquier, Buthaud, Magné, Peiffert, Sargos, Crehange, Pommier, Loos, Hasbini, Latorzeff, Silva, Denis, Lagrange.

Analysis and interpretation of data: Supiot, Vaugier, Blanc-Lapierre.

Drafting of the manuscript: Supiot, Vaugier, Blanc-Lapierre.

Critical revision of the manuscript for important intellectual content: Supiot.

Statistical analysis: Blanc-Lapierre.

Obtaining funding: Supiot.

Administrative, technical, or material support: Vaugier. Supervision: Supiot.

Other: None.

Financial disclosures: Stéphane Supiot certifies that all conflicts of interest, including specific financial interests and relationships and affiliations relevant to the subject matter or materials discussed in the manuscript (eg, employment/affiliation, grants or funding, consultancies, honoraria, stock ownership or options, expert testimony, royalties, or patents filed, received, or pending), are the following: None.

Funding/Support and role of the sponsor:

This study received an educational grant from Astellas pharmaceuticals. The funder had no role in the study design, data collection, data analysis, data interpretation, or writing of the report.


Dr Hussain Munawar

Cara Nichole Soomro
email: munawar_soomro@hotmail.com

Barbara Maesano

Carl-Gustaf Heude

Isabella Bornehag

Annesi-Maesano

Soomro

Cara Nichole Maesano
email: cara.maesano@gmail.com

Barbara Heude
email: barbara.heude@inserm.fr

Carl-Gustaf Bornehag
email: carl-gustaf.bornehag@kau.se

Isabella Annesi-Maesano
email: isabella.annesi-maesano@inserm.fr

Munawar Hussain

Cara Nicole

The association between maternal urinary phthalate metabolites concentrations and pregnancy induced hypertension: Results from the EDEN Mother-Child Cohort

Keywords: Phthalate, prenatal exposure, endocrine disruptor, urinary biomarkers, mother-child cohort, pregnancy induced hypertension

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

Introduction

Phthalates are widely used in our daily life and are contained within products ranging from food wrappers, personal care products, cosmetics, toys, polyvinyl chloride flooring, building materials and medical devices. Despite their usefulness, however, phthalates can interfere and disrupt the endocrine system and might be related with raised blood pressure and increased risk of cardiovascular diseases [START_REF] Cantonwine | Urinary Concentrations of Bisphenol A and Phthalate Metabolites Measured during Pregnancy and Risk of Preeclampsia[END_REF][START_REF] Trasande | Urinary phthalates are associated with higher blood pressure in childhood[END_REF]. Phthalates are a broad class of chemical compounds with a wide range of consumer product and medical device applications that vary based on their molecular weight. Phthalates are classified into two main groups. Low molecular weight (LMW) phthalates are frequently added to cosmetic products, shampoos, lotions, and other personal care products to the preserve scent. Whereas high molecular weight (HMW) phthalates are used as plasticizers of polyvinyl chloride (PVC) because of their flexibility and resistant kinking properties.

Phthalates are ubiquitous in the environment. Exposure to phthalates can occur through inhalation, ingestion (including dietary ingestion and incidental ingestion), skin absorption (including air-to-skin transport), use of personal care products and/or contact with contaminated surfaces [START_REF] Soomro | Prenatal Exposure to Phthalates and the Development of Eczema Phenotypes in Male Children: Results from the EDEN Mother-Child Cohort Study[END_REF]. After exposure the phthalates are metabolized in monoester metabolites and conjugated before being excreted in the urine; hence urinary phthalate metabolites can be used as a biomarker of exposure [START_REF] Swan | Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans[END_REF]. Studies have revealed that phthalates can also cross the feto-placental barrier and enter the amniotic fluid and fetal circulation [START_REF] Swan | Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans[END_REF][START_REF] Ferguson | Associations between Maternal Biomarkers of Phthalate Exposure and Inflammation Using Repeated Measurements across Pregnancy[END_REF][START_REF] Li | Exposure levels of environmental endocrine disruptors in mother-newborn pairs in China and their placental transfer characteristics[END_REF].

Therefore, it is hypothesized that the prenatal and early years of life are very critical in the development of immune system and during this period the exposure to toxic pollutants can result in an increased risk of adverse health outcomes during pregnancy including preeclampsia (high blood pressure plus proteinuria after 20 th week of gestation) as well as later in life, which may also cause hypertension in children or adult population. However, the exact mechanism through which phthalate exposure can influence cardiovascular health effects is still unknown. However, it is hypothesized that as phthalates are endocrine disruptors, they affect the metabolism and actions of androgens, thyroid hormones and cortisol [START_REF] Boas | Thyroid effects of endocrine disrupting chemicals[END_REF][START_REF] Ye | Environmental pollutants and hydroxysteroid dehydrogenases[END_REF]. Another possible mechanism, by which endocrine disruptors may cause a proinflammatory response, vascular dysfunction and increased oxidative stress [START_REF] Rahmani | Prenatal Exposure to Phthalic Acid Induces Increased Blood Pressure, Oxidative Stress, and Markers of Endothelial Dysfunction in Rat Offspring[END_REF][START_REF] Mathieu-Denoncourt | Plasticizer endocrine disruption: Highlighting developmental and reproductive effects in mammals and non-mammalian aquatic species[END_REF].

A cross-sectional study in US children and adolescents aged 6-19 years, assessed LMW and HMW phthalates and suggested that the increased urinary phthalate concentrations of HMW phthalate metabolites were associated with higher blood pressure [START_REF] Trasande | Urinary phthalates are associated with higher blood pressure in childhood[END_REF]. Another cross-sectional study from Uppsala, Sweden in older adults (aged 70 years) showed that increased serum concentrations of monobenzyl phthalate (MBzP) were related to multiple cardiovascular risk factors [START_REF] Wiberg | Serum levels of monobenzylphthalate (MBzP) is related to carotid atherosclerosis in the elderly[END_REF]. However, there is only one prospective birth cohort study from Cincinnati, USA that examined the relationship between urinary phthalate metabolite concentrations (both LMW and HMW) during pregnancy (16 to 26 weeks of gestation) with maternal blood pressure and the risk of pregnancy induced hypertensive diseases, which needs to be confirmed [START_REF] Werner | The association between maternal urinary phthalate concentrations and blood pressure in pregnancy: The HOME Study[END_REF].

The main objective of our study was to evaluate the association between maternal exposure to phthalates during pregnancy and pregnancy induced hypertension in a population-based sample of pregnant mothers.

Methods and Materials

Study population and data collection

This is a nested cross-sectional study in design. The population included in this study is a subgroup of French EDEN (Etude des Déterminants pré et post natals du développement de la santé de l'Enfant) mother-child prospective birth cohort (https://eden.vjf.inserm.fr). The study was conducted in two cities of France i.e Nancy and Poitiers. The mothers' recruitment, follow-up procedures, and methodological assumptions have been published in previous studies [START_REF] Heude | Cohort Profile: The EDEN motherchild cohort on the prenatal and early postnatal determinants of child health and development[END_REF]. Women were given an appointment between the 24 th and 28 th gestational weeks, by a study midwife during which an interview on lifestyle factors were conducted and biological samples were collected. The information on the mothers and their newborns, including parity, mode of delivery, gestational age, season of birth, birth weight, behavioral and environmental data were collected by the use of questionnaires, interviews and from obstetric and pediatric records. The current study includes mothers of all male offspring for whom maternal urine samples were analyzed for phthalate metabolites as a part of earlier studies [START_REF] Soomro | Prenatal Exposure to Phthalates and the Development of Eczema Phenotypes in Male Children: Results from the EDEN Mother-Child Cohort Study[END_REF][START_REF] Chevrier | Maternal urinary phthalates and phenols and male genital anomalies[END_REF][START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: a 2-step standardization method based on regression residuals[END_REF][START_REF] Botton | Phthalate pregnancy exposure and male offspring growth from the intra-uterine period to five years of age[END_REF]. Furter, we also excluded all the mothers with previous history of hypertension, diabetes or kidney diseases before their pregnancy.

The blood pressure of the mothers was measured during their routine monthly visits, with the subject in supine position, using a standard sphygmomanometer. One set of measures was taken by the study midwife between 24 and 28 weeks using a different (semi-automated) device, with the two measurements averaged to determine the values for that visit.

Measurements taken within each gestational trimester (T1, T2, and T3) were averaged to determine the values for systolic blood pressure (SBP) and diastolic blood pressure (DBP) assigned to each period.

Women were classified as having pregnancy induced hypertension based on SBP ≥ 140 mmHg and /or DBP ≥ 90 mmHg measured during at least two visits after 22 nd week of gestation, such that the elevated measures could have occurred during or across any of the three periods, at any point after 22 weeks. Consequently, no diagnosis of pregnancy induced hypertension was made before 24 weeks. All cases of pregnancy induced hypertension were reviewed by a physician. Further, two devices were used to counter check the results.

Urine collection and analysis

Women were invited for a clinical examination and to provide a first morning urine at home before the hospital study visit between the 24 th and 28 th gestational weeks (second trimester of the pregnancy). If forgotten, the urine sample was collected at the hospital. A polypropylene Quantification of the phthalate metabolite concentrations was performed using online solid phase extraction high performance liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry Creatinine concentrations were also quantified following an identical analytical procedure as described previously [START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: a 2-step standardization method based on regression residuals[END_REF]. The concentrations of metabolites below the limit of detection (LOD) <3% were replaced by instrumental reading values or by the compound specific lowest instrumental reading value divided by the square root of two (LOD/√ -2) when the instrumental value was found missing. The vast majority of the samples/metabolites (>97%) were above the LOD. A two-step standardization was performed to reduce variability in the biomarker urinary concentrations due to sampling conditions as described previously [START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: a 2-step standardization method based on regression residuals[END_REF]. There was no significant difference in the concentrations of phthalates in the morning urine samples collected at the participants homes compared to those of the urine sampled collected later in the day at the hospital.

Other Variables

Information on potential confounders including: gestational age (two-class variable: < 37 gestational week, ≥ 37 gestational week), parity, number of siblings (0, 1,2 ≥ 3), maternal age at delivery (< 25 years, 25-29 years, 30 -34 years, ≥ 35 years), pre-pregnancy maternal body mass index (BMI, < 18.5, 18.5-24.9, 25.0-30.0, > 30 kg/m 2 ), maternal educational level (< 2 years after high school, High school +2 years, ≥ High school +3 years), household income, city of residence (Nancy / Poitiers), mode of delivery (vaginal, caesarean section), smoking during pregnancy (yes/no), maternal alcohol use during pregnancy (yes/no), maternal physician-diagnosis of pregnancy induced hypertension.

Statistical analysis

Urinary concentrations of phthalate metabolites were natural log (ln) transformed due to the non-normality of the distribution. For the purpose of present analysis, MECPP, MEHHP, MEOHP and MEHP were grouped together as sum ΣDEHP (DEHP being their parent compound). We calculated the molar sum of these DEHP metabolites (nanomoles per milliliter) by dividing each metabolite concentration by its molecular weight and then summing the individual concentrations.

The prevalence of pregnancy induced hypertension was computed. We applied multiple logistic regression analysis to examine the association between each phthalate metabolite and ΣDEHP and pregnancy induced hypertension outcomes adjusted for potential confounders.

The potential confounders were determined a priori from literature review [START_REF] Cantonwine | Urinary Concentrations of Bisphenol A and Phthalate Metabolites Measured during Pregnancy and Risk of Preeclampsia[END_REF][START_REF] Soomro | Prenatal Exposure to Phthalates and the Development of Eczema Phenotypes in Male Children: Results from the EDEN Mother-Child Cohort Study[END_REF][START_REF] Valvi | Prenatal Phthalate Exposure and Childhood Growth and Blood Pressure: Evidence from the Spanish INMA-Sabadell Birth Cohort Study[END_REF][START_REF] Trasande | Association of exposure to di-2-ethylhexylphthalate replacements with increased blood pressure in children and adolescents[END_REF].

Further, the Directed Acyclic Graph (http://www.dagitty.net/dags.html) was used to see the hypothesis relations between biomarkers concentration, confounders and pregnancy induced hypertension outcomes. The confounders included in our final model were maternal smoking, maternal age, maternal BMI, maternal education level, gestational age, and number of siblings. Models were additionally adjusted for recruitment center. We used continuous metabolite levels in our first analysis and then we used categorical levels of the phthalates metabolites in our second analysis. All statistical analyses were conducted using Stata statistical software 14 (Stata Corp LP, 4905 Lakeway Drive, College Station, Texas 77845, USA) and considered a p-value < 0.05 as statistically significant.

Ethical Approval

The study was approved by the relevant ethical committees in France (Comité Consultatif pour la Protection des Personnes dans la Recherche Biomédicale, Le Kremlin Bicêtre University Hospital, and Commission Nationale de l'Informatique et des Libertés), and all participating women gave informed written consent for their own participation and consent was obtained from both parents for each newborn child after delivery.

Results

Characteristics of study population

Of the 2002 women recruited into the EDEN mother child cohort, 998 gave birth to boys and 604 women with available data on both pregnancy induced hypertension diagnosis and urine analysis for phthalates were selected for this study (Figure 1). The majority (98.5%) of these women were educated, 43.5% had less than two years of education after high school while 54.8% had more than that and between 25-34 years of age (29.86 ± 4.8) (Table 1). The average BMI was 23.1 ± 4.5, while 26.4% of women were overweight or obese (BMI ≥25 kg/m 2 ). The active smokers were 13.7% and 25% declared some alcohol intake during pregnancy. Four percent gave birth pre-maturely, 15.7% had caesarean section at delivery. The prevalence of pregnancy induced hypertension was found 4.80%, while the prevalence of preeclampsia was 2.32%. Whereas, the prevalence of pregnancy induced hypertension in overall EDEN Cohort was also the same 4.80%. The participants included in this study did not differ in major demographic characteristics except for maternal age, smoking and gestational age (Table S1). However, results did not change when adjusted for them in the final models.

Prenatal exposure to phthalates

Phthalate metabolites were detected in the urine of more than 97% of the mothers. MEP had the highest geometric mean (110.7 µg/L), while MCNP had the lowest (1.5 µg/L). The limit of detection (LOD) ranged from 0.2 to 0.6 µg/L. A summary of detection frequency, as well as geometric means and select percentiles of urinary concentrations of phthalate metabolites are outlined in Table 2.

Spearman correlation coefficients between ln-transformed phthalate metabolites urinary concentrations are shown in Table S2. The majority of biomarkers were positively correlated, except for the correlation between MEP and MiBP, MECPP, MEHHP, MCOP, MCPP or MCNP. All other biomarkers were significantly correlated with each other (with correlation coefficients ranging from r = 0.08, p < 0.05 to r = 0.97, p < 0.05). We also observed that DEHP metabolites were highly correlated with each other (r ≥ 0.77, p < 0.05).

Associations between phthalate metabolites and pregnancy induced hypertension

Low molecular weight phthalates metabolites; MEP and MBP were positively associated with pregnancy induced hypertension in crude (OR: 1.43 95% CI: 1.04-1.96, p-value = 0.02 and OR: 1.48 95% CI: 1.10-2.01, p-value =0.01) and in adjusted (OR: 1.47, 95% CI: 1.01-2.14, pvalue = 0.04 and OR: 1.66, 95% CI: 1.11-2.47, p-value = 0.01) models respectively as shown in Table 3. We observed borderline association between one of the high molecular weight phthalate metabolite; Mono (3-carboxypropyl) phthalate in crude (OR: 1.46, 95% CI: 0.96-2.24, p-value = 0.08) as well as in adjusted model (OR: 1.56, 95% CI: 0.93-2.64, p-value = 0.09). We did not observe associations between other high molecular weight phthalate metabolites and pregnancy induced hypertension. The FigureS1 shows the scattered plot between levels of phthalates metabolites and occurrence of events during the gestational age of the mothers. Further, we did not find significant associations between the urinary concentrations of phthalates metabolites and preeclampsia in the mothers (data not shown).

While taking into account pregnancy induced hypertension quartiles, we did not observe any significant association. However, a significantly increased odds ratio for MBP metabolite was observed in the 4 th quartile in crude (OR: 3.46, CI: 1.10-10.87, p-value = 0.03) as well as when adjusted for confounders (OR: 5.91 CI: 0.93-37.79, p-value = 0.06) as shown in Table 4. An association for ΣDEHP (OR: 5.45, CI: 0.85-34.84, p-value = 0.07) was also observed for the second quartile.

Discussion

In this study, we report an association between maternal prenatal urinary concentration of low molecular weight phthalate metabolites including MEP and MBP and pregnancy induced hypertension. MEP is metabolite of Diethyl phthalate (DEP) used in plasticizers, automotive, cleaning and furnishing products, adhesives, sealants and in cosmetic products, while MBP is a metabolite of Di-n-butyl phthalate (DBP), used in diverse range of products such as PVC, paints, printing inks, adhesives, aerosols and cosmetic products including hair sprays, perfume solvents and fixatives, skin emollients and nail polish. The ubiquitous use of these phthalates in so many different types of products signals its relevance as an environmental stressor, and indeed, more than 97% of the mothers included in our study tested positive for phthalate metabolites. We observed that the MEP and MBP which are a LMW phthalates and are frequently added to our daily used cosmetic products, shampoos, lotions, and other personal care products.

Comparison of findings with other published work

To our knowledge, this is the second study to assess the relationship of phthalate concentrations in relation to induced hypertension during pregnancy. An earlier research focused on a prospective birth cohort study from Cincinnati, USA investigated the relationship between for 9 phthalate monoester metabolites reflecting exposure to 6 phthalate diesters and gestational hypertension, preeclampsia, eclampsia and HELLP syndrome [START_REF] Werner | The association between maternal urinary phthalate concentrations and blood pressure in pregnancy: The HOME Study[END_REF].

This study suggested that maternal urinary MBzP concentrations may be associated with increased diastolic blood pressure and risk of pregnancy induced hypertensive diseases. Our analyses, however, shows a significant association with MEP and MBP but not with MBzP phthalate metabolites, except for a partial significance in the 4 th quartile (OR: 2.75, CI: 0.95-7.91, p-value = 0.06).

Another study from Boston, USA, investigating the relationship between Bisphenol A and phthalate metabolites and the risk of preeclampsia [START_REF] Cantonwine | Urinary Concentrations of Bisphenol A and Phthalate Metabolites Measured during Pregnancy and Risk of Preeclampsia[END_REF] found a significant association between preeclampsia and MEP and DEHP. In our present study, we found a significant association between pregnancy induced hypertension and MEP (OR: 1.47, CI: 1.01-2.14, p-value = 0.04) or MBP (OR: 1.66, CI: 1.11-2.47, p-value = 0.01) and a borderline significance in MCPP (OR: 1.56, CI: 0.93-2.64, p-value = 0.09).

Other studies on phthalate exposure and blood pressure in children have also shown significant positive associations [START_REF] Trasande | Urinary phthalates are associated with higher blood pressure in childhood[END_REF][START_REF] Valvi | Prenatal Phthalate Exposure and Childhood Growth and Blood Pressure: Evidence from the Spanish INMA-Sabadell Birth Cohort Study[END_REF][START_REF] Trasande | Association of exposure to di-2-ethylhexylphthalate replacements with increased blood pressure in children and adolescents[END_REF]. In studies done in the United States, [START_REF] Trasande | Urinary phthalates are associated with higher blood pressure in childhood[END_REF][START_REF] Trasande | Association of exposure to di-2-ethylhexylphthalate replacements with increased blood pressure in children and adolescents[END_REF], cross-sectional analysis of a subsamples found increased urinary concentrations of MEP and DEHP were associated with increased blood pressure in children age 6-19 years. However, a study from Spain focused on a population based birth cohort observed that the sums of HMW phthalates as well as the sum of LMW phthalates were significantly related with lower systolic blood pressure in girls, but not in boys at the age of 4 and 7 years [START_REF] Valvi | Prenatal Phthalate Exposure and Childhood Growth and Blood Pressure: Evidence from the Spanish INMA-Sabadell Birth Cohort Study[END_REF]. A prospective study on elderly adults from Uppsala, Sweden showed inverse associations between MEP and systolic and diastolic blood pressure [START_REF] Olsen | Associations between circulating levels of bisphenol A and phthalate metabolites and coronary risk in the elderly[END_REF].Additionally, a cross-sectional study of older adults found increased serum concentrations of MBzP were related to several cardiovascular risk factors [START_REF] Wiberg | Serum levels of monobenzylphthalate (MBzP) is related to carotid atherosclerosis in the elderly[END_REF].

Potential mechanisms

The exact mechanisms describing phthalate interactions with pregnancy induced hypertension are still unclear; however they are likely to be multifactorial in nature and depend on diet, living style, genetic and environmental factors. Nutritional imbalances or environmental chemical exposures during pregnancy and development can increase the risk of disease and their effects may be share some common pathways [START_REF] Barouki | Developmental origins of noncommunicable disease: implications for research and public health[END_REF]. Indeed, hormones, cytokines and nutritional components can both directly activate receptors that stimulate gene expression and also activate or inhibit the enzymes and pathways that are responsible for DNA methylation, chromatin re-modelling and non-coding RNAs which ultimately control gene expression. Thus, epigenetic regulatory pathways are likely sites for effects of both nutrient and environmental toxicant effects during development and potentially also across the lifespan [START_REF] Barouki | Developmental origins of noncommunicable disease: implications for research and public health[END_REF]. There can be another possibility that, as phthalates particularly the LMW phthalate might be related to various cocktail of chemicals, including in cosmetics, it cannot be excluded that the phthalate might be a proxy of another exposure or behavior that is the true case of hypertension.

Studies have also suggested that exposure to phthalates during the early pregnancy may lead to early placental maladaptation, which increases the risk of higher blood pressure during the pregnancy and gestational hypertensive disorders [START_REF] Philips | Early pregnancy bisphenol and phthalate metabolite levels, maternal hemodynamics and gestational hypertensive disorders[END_REF]. Epidemiological evidence indicates that exposure to phthalates in utero is associated with increased blood pressure in childhood [START_REF] Trasande | Urinary phthalates are associated with higher blood pressure in childhood[END_REF]. Studies have revealed that the risk of pregnancy induced hypertension may increase through oxidative stress, up-regulation of inflammatory markers (tumor necrosis factor alpha and interleukin 6), and inhibition of peroxisome proliferator-activated receptor gamma [START_REF] Rahmani | Prenatal Exposure to Phthalic Acid Induces Increased Blood Pressure, Oxidative Stress, and Markers of Endothelial Dysfunction in Rat Offspring[END_REF][START_REF] Usuda | Peroxisome proliferator-activated receptors for hypertension[END_REF][START_REF] Sutherland | Preterm birth and hypertension risk: the oxidative stress paradigm[END_REF]. The possible mechanism here may involve phthalate-mediated deregulation of blood pressure and reduced endothelium-derived nitric oxide production, which is mainly produced by endothelial nitric oxide synthase, one of the key vessel relaxation factors in regulation of vascular tone [START_REF] Lee | Maternal exposure to di-(2ethylhexyl) phthalate exposure deregulates blood pressure, adiposity, cholesterol metabolism and social interaction in mouse offspring[END_REF]. In addition to the transcriptional regulation, endothelial nitric oxide synthase activity can be modulated by kinase-dependent phosphorylation at various sites.

Strengths and limitations

The nested cross-sectional design of our study is a sample of a population based prospective cohort that includes nutritional, environmental, biological and social factors starting in utero, which allowed us to control for potential confounders and to assess prenatal exposure before the expression of health outcomes. The proposed cohort design offers advantages over the traditional cross-sectional designs; the diagnosis of pregnancy induced hypertension was conducted by physicians, which ensures quality and we were able to further adjust for multiple potential confounders. The values of the exposure biomarkers were standardized for urine sampling conditions to reduce subject variability [START_REF] Mortamais | Correcting for the influence of sampling conditions on biomarkers of exposure to phenols and phthalates: a 2-step standardization method based on regression residuals[END_REF]. However, we were limited to one urine sample per mother during the second trimester of pregnancy, thus we were unable to assess the concentration variability of each metabolite across the pregnancy. Furthermore, it is not clearly known at which point(s) in gestation the mothers have a higher risk of exposure, or even if exposure is relatively constant over the course of the pregnancy. Former studies have also used second trimester maternal urinary concentration for biomarkers [START_REF] Gascon | Prenatal exposure to bisphenol A and phthalates and childhood respiratory tract infections and allergy[END_REF][START_REF] Whyatt | Asthma in inner-city children at 5-11 years of age and prenatal exposure to phthalates: the Columbia Center for Children's Environmental Health Cohort[END_REF].

Consistency in urinary concentrations during pregnancy has been observed for some environmental exposure biomarkers and other studies have suggested that phthalate biomarkers are relatively stable for a period of weeks to months [START_REF] Teitelbaum | Temporal variability in urinary concentrations of phthalate metabolites, phytoestrogens and phenols among minority children in the United States[END_REF][START_REF] Hauser | Temporal variability of urinary phthalate metabolite levels in men of reproductive age[END_REF]. Nonetheless, increasing the number of urine samples collected would have provided a more accurate estimate of average exposure during the gestation period. We also adjusted for many potential confounders; however residual confounding (if any) cannot be discarded.

In our study, we considered only male offspring because the initial purpose was to see the effects of phthalates on malformations of male genitalia [START_REF] Philippat | Exposure to phthalates and phenols during pregnancy and offspring size at birth[END_REF]. Considering only males limits the generalization of our results, it is not a source of bias. From a statistical point of view, focusing on one sex is another way to increase the study accuracy when the sample size is limited. Another limitation of our study is small number of cases, however we had limited number of mothers tested for phthalates metabolites during the second trimester of the pregnancy. While we also did not tested for multiple hypothesis testing is another limitation of our study. Further, we also excluded all the mothers with previous history or family history of hypertension, and/or medication for hypertension, diabetes or kidney diseases before their pregnancy.

Creatinine correction is most often used for urinary biomarkers of phthalates, phenols, and pesticides. There are limitations to the usage of creatinine to normalize for urine dilution; different investigators have used specific gravity as an alternative of creatinine to adjust phthalate urinary biomarkers for urine dilution, [START_REF] Whyatt | Asthma in inner-city children at 5-11 years of age and prenatal exposure to phthalates: the Columbia Center for Children's Environmental Health Cohort[END_REF][START_REF] Just | Prenatal exposure to butylbenzyl phthalate and early eczema in an urban cohort[END_REF] but we did not have specific gravity measurements. Nevertheless, specific gravity is extremely correlated with creatinine [START_REF] Barr | Urinary creatinine concentrations in the U.S. population: implications for urinary biologic monitoring measurements[END_REF] and consequently it is not doubtless that we overcorrected for urine dilution. Regarding exposure assessment, it might be important to explore other techniques of assessment, particularly those that may offer a more exhaustive and coordinated picture of the individual environment, for instance by assessing particular items used in conjunction with indoor air levels and additional biomarkers of exposure.

The exposures we studied are relatively prevalent, and some biomarker urinary concentrations approach those with significant effects in experimental models. In a healthy cohort such as ours, effects of hormonally active environmental exposures may be small, yet a continuous exposure can have adverse impacts.

A further dimension to consider in future research is multiple exposures of hormonally active agents such as these. In terms of prevention, exposure to these chemicals can be better avoided if the contents of products are already known or listed on the products. Unfortunately, many phthalates are often not listed on the labels because they are considered non-active ingredients.

Conclusion

We observed that prenatal exposure to certain phthalates, including MEP and MBP, were associated with pregnancy induced hypertension. Our data provide new suggestive evidence that prenatal exposure to certain phthalates may play a role in the development of pregnancy induced hypertension. participating women gave informed written consent for their own participation and consent was obtained from both parents for each newborn child after delivery.

Abbreviations:

BMI

Consent for publication

Consent for publication was taken from the relavent authority

  container (FP40VPS manufactured by Centre Européen de Biotechnologie SA (CEB) in Angers, France was used. The urine samples were aliquoted and then frozen at -80° C. The analyses of the samples were conducted at the National Center for Environment Health Laboratory at the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia (USA) for eleven metabolites of eight phthalates: Diethyl phthalate (DEP), Di-n-butyl phthalate (DBP), Di-isobutyl phthalate (DiBP), Di(2-ethylhexyl) phthalate (DEHP), Benzylbutyl phthalate (BBzP), Di-isononyl phthalate (DiNP), Di-n-octyl phthalate (DOP), and Di-isodecyl phthalate (DiDP). The targeted phthalate metabolites measured were monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-isobutyl phthalate (MiBP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), mono(2-ethylhexyl) phthalate (MEHP), monobenzyl phthalate (MBzP), monocarboxy-isooctyl phthalate (MCOP), mono(3-carboxypropyl) phthalate (MCPP) and monocarboxy-isononyl phthalate (MCNP).

  The study was approved by the relevant ethical committees in France (Comité Consultatif pour la Protection des Personnes dans la Recherche Biomédicale, Le Kremlin Bicêtre University Hospital, and Commission Nationale de l'Informatique et des Libertés), and all

	CI MCNP	Confidence Interval Monocarboxy-isononyl phthalate
	DEHP OR	di(2-ethylhexyl) phthalate Odds Ratio
	DBP SD	Di-n-butyl phthalate Standard Deviation
	DiBP	di-isobutyl phthalate
	DiDP	di-isodecyl phthalate
	DiNP	di-isononyl phthalate
	HMW	high molecular weight
	LOD	limit of detection
	LMW	low molecular weight
	MEP	Monoethyl phthalate
	MBP	Mono-n-butyl phthalate
	MiBP	Mono-isobutyl phthalate
	MECPP	Mono(2-ethyl-5-carboxypentyl) phthalate
	MEHHP	Mono (2-ethyl-5-hydroxyhexyl) phthalate
	MEOHP	Mono(2-ethyl-5-oxohexyl) phthalate
	MEHP	Mono(2-ethylhexyl) phthalate
	MBzP Declaration	Monobenzyl phthalate
	Body Mass Index Monocarboxy-isooctyl phthalate Ethics approval and consent to participate MCOP
	BBzP MCPP	Benzylbutyl phthalate Mono (3-carboxypropyl) phthalate

Table 1 .

 1 Characteristics of the study population and by centre.

	Characteristics	All n=604 Mean ± SD	Poitiers n= 347	Nancy n= 257	P value
		No. %	No. %	No. %
	Maternal age (years)	29.86±4.8
	<25	79 13.0	48 13.8	31 12.0
	25 -29 30 -34	218 36.1 205 34.0	123 35.5 120 34.6	95 37.0 85 33.1	0.8
	≥35	102 16.9	56 16.1	46 17.9
	Parity
	0	269 44.5	174 50.1	95 37.0
	1	222 36.8	117 33.7	110 42.8	0.005
	≥2	113 18.7	56 16.2	52 20.2
	BMI (kg/m 2 )	23.1±4.5
	<18.5	57 9.4	27 7.9	30 11.7
	18.5 -24.9	379 62.9	217 62.5	162 63.0
	25 -30	110 18.3	68 19.6	42 16.3	0.4
	>30	49 8.1	30 8.6	19 7.4
	Missing	9 1.5	5 1.4	4 1.6
	Maternal education
	<2 years after high school	263 43.5	172 49.6	91 35.4
	High school +2 years ≥ High school +3 years	132 21.9 199 32.9	72 20.7 98 28.3	60 23.3 101 39.4	0.004
	Missing	10 1.7	5 1.4	5 1.9
	Active smoking (cig/day)
	0	519 86.0	295 85.0	224 87.2	0.6

Table 2 .

 2 Distribution of phthalate metabolites concentration (µg/L) in maternal prenatal urine (n = 604).

	Parent compound	Abbrev.	Phthalate metabolites	Abbrev.	LOD (µg/L)	%>LOD	Geometric mean (GM)	Minimum			Percentile			Maximum
									5th	25th	50th	75th	95th
	Diethyl phthalate	(DEP)	Monoethyl phthalate (µg/L)	MEP	0.6	100	110.7	7.9	21	51	96	195	714	4093.4
	Di-n-butyl phthalate	(DBP)	Mono-n-butyl phthalate (µg/L)	MBP	0.2	100	51.6	2.8	12	28	43	73	438	2418.8
	Di-isobutyl phthalate	(DiBP)	Mono-isobutyl phthalate (µg/L)	MiBP	0.2	100	42.7	1.5	12	25	39	69	166	689.9
	Di(2-ethylhexyl) phthalate (DEHP)	Mono(2-ethyl-5-carboxypentyl) phthalate (µg/L)	MECPP	0.2	100	40.5	2.6	12	25	38	62	160	2640.5
	Di(2-ethylhexyl) phthalate (DEHP)	Mono(2-ethyl-5-hydroxyhexyl) phthalate (µg/L)	MEHHP	0.2	100	26.9	1.7	6.8	16	27	44	103	2103.8
	Di(2-ethylhexyl) phthalate (DEHP)	Mono(2-ethyl-5-oxohexyl) phthalate (µg/L)	MEOHP	0.2	99.7	22.1	0.6	5.6	14	23	36	84	1441.4
	Di(2-ethylhexyl) phthalate (DEHP)	Mono(2-ethylhexyl) phthalate (µg/L)	MEHP	0.5	98	7.2	0.1	1.6	4.4	7.7	15	35	292.3
	Benzylbutyl phthalate	(BBzP)	Monobenzyl phthalate (µg/L)	MBzP	0.3	100	19.7	1.6	4.5	11	18	33	104	970.4
	Di-isononyl phthalate	(DiNP) Monocarboxy-isooctyl phthalate (µg/L) MCOP	0.2	99	4.1	0.1	1.2	2.4	3.9	6.5	19	375.3
	Di-n-octyl phthalate	(DOP)	Mono(3-carboxypropyl) phthalate (µg/L)	MCPP	0.2	100	2.1	0.1	0.7	1.3	1.9	3.4	9.1	78.7
	Di-isodecyl phthalate	(DiDP)	Monocarboxy-isononyl phthalate (µg/L)	MCNP	0.2	97.5	1.5	0.1	0.5	0.8	1.2	2.2	9.5	686.8

Table 3 .

 3 Association (Odd Ratio and 95% CI) between maternal urinary concentrations of phthalate metabolites and pregnancy induced hypertension. OR adjusted for maternal smoking, maternal age, maternal BMI, maternal education level, gestational age, number of siblings, and recruitment centre MEP = Monoethyl phthalate; MBP = Mono-n-butyl phthalate; MiBP = Mono-isobutyl phthalate; MECPP = Mono (2-ethyl-5-carboxypentyl) phthalate; MEHHP = Mono (2-ethyl-5hydroxyhexyl) phthalate; MEOHP = Mono (2-ethyl-5-oxohexyl) phthalate; MEHP = Mono (2-ethylhexyl) phthalate; MBzP = Monobenzyl phthalate; MCOP = Monocarboxy-isooctyl phthalate; MCPP = Mono (3-carboxypropyl) phthalate; MCNP = Monocarboxy-isononyl phthalate; DEHP = di(2-ethylhexyl) phthalate.

	Phthalate	Unadjusted		Adjusted a
	metabolite	OR 95% CI P-Value	OR	95% CI	P-Value
	MEP	1.43 1.04-1.96* 0.02	1.47	1.01-2.14*	0.04
	MBP	1.48 1.10-2.01* 0.01	1.66	1.11-2.47*	0.01
	MiBP	1.27 0.81-1.99 0.28	1.22	0.69-2.16	0.49
	MECPP	1.26 0.82-1.95 0.29	1.22	0.68-2.21	0.49
	MEHHP	1.21 0.79-1.83 0.37	1.16	0.67-1.99	0.59
	MEOHP	1.19 0.78-1.83 0.41	1.12	0.64-1.95	0.69
	MEHP	0.94 0.66-1.33 0.72	0.84	0.54-1.34	0.48
	MBzP	1.31 0.91-1.89 0.15	1.08	0.66-1.78	0.75
	MCOP	0.81 0.51-1.29 0.38	0.99	0.58-1.69	0.98
	MCPP	1.46 0.96-2.24 † 0.08	1.56	0.93-2.64 †	0.09
	MCNP	1.05 0.72-1.52 0.81	1.24	0.80-1.92	0.33
	ΣDEHP	1.03 0.93-1.15 0.56	1.01	0.88-1.17	0.85
	*P<0.05	† P<0.10

a

Table 4 .

 4 Association (Odd Ratio and 95% CI) between maternal urinary concentrations of phthalate metabolites and pregnancy induced hypertension by quartiles.

		Quartiles
	Phthalate metabolites	Q1(Referen

ce) Q2 OR (95% CI) P-value Q3 OR (95% CI) P-value Q4 OR (95% CI) P-value MEP

		Crude	1	1.63 (0.52-5.11) 0.39	0.98 (0.28-3.52) 0.99	2.29 (0.78-6.77) 0.13
		Adjusted	1
				0.99 (0.19-5.12) 0.98	0.63 (0.09-4.24) 0.63	1.24 (0.26-5.92) 0.78
		Crude	1	1.25 (0.33-4.78) 0.73	1.78 (0.51-6.23) 0.36	3.46 (1.10-10.87) 0.03*
		Adjusted	1
	MBP
				1.78 (0.25-12.77) 0.56	2.76 (0.37-20.40) 0.31	5.91 (0.93-37.79) 0.06 †
		Crude	1	2.75 (0.72-10.61) 0.14	3.49 (0.94-12.97) 0.06 †	2.75 (0.72-10.61) 0.14
		Adjusted	1
	MiBP
				0.46 (0.07-3.08) 0.42	2.43 (0.49-11.98) 0.27	1.92 (0.35-10.46) 0.44
		Crude	1	1.41 (0.44-4.57) 0.55	2.07 (0.69-6.21) 0.19	1.41 (0.44-4.57) 0.55
		Adjusted	1
	MECPP
				7.90 (0.83-75.43) 0.07 †	6.09 (0.62-59.70) 0.12	6.47 (0.66-63.11) 0.11
		Crude	1	1.85 (0.60-5.65) 0.28	1.85 (0.60-5.65) 0.28	1.20 (0.36-4.04) 0.75
		Adjusted	1
	MEHHP
				2.95 (0.51-17.06) 0.22	2.46 (0.40-15.14) 0.33	2.29 (0.33-15.91) 0.39
		Crude	1	4.25 (1.17-15.41) 0.02*	2.75 (0.71-10.61) 0.14	2.04 (0.50-8.31) 0.31
		Adjusted	1
	MEOHP
				16.08 (1.63-158.12) 0.01*	3.33 (0.26-42.47) 0.35	5.72 (0.52-62.95) 0.15
		Crude	1	0.58 (0.21-1.65) 0.30	0.79 (0.30-2.05) 0.62	0.48 (0.16-1.44) 0.19
		Adjusted	1
	MEHP
				0.40 (0.07-2.22) 0.29	0.84 (0.19-3.62) 0.81	0.52 (0.09-3.08) 0.47
		Crude	1	1.42 (0.44-4.57) 0.55	0.79 (0.21-3.01) 0.73	2.75 (0.95-7.91) 0.06 †
		Adjusted	1
	MBzP
				0.81 (0.15-4.21) 0.80	0.50 (0.08-3.11) 0.46	1.79 (0.39-8.25) 0.45
		Crude	1	0.99 (0.36-2.74) 0.99	0.86 (0.31-2.45) 0.79	0.74 (0.25-2.18) 0.58
		Adjusted	1
	MCOP
				3.99 (0.70-22.68) 0.11	3.41 (0.49-23.49) 0.21	2.42 (0.36-16.17) 0.35
	MCPP	Crude	1	1.41 (0.44-4.57) 0.55	1.41 (0.44-4.57) 0.55	2.07 (0.69-6.21) 0.19

Acknowledgments

We acknowledge the participating families, midwife research assistants for data collection. The EDEN Mother-Child Cohort Study Group I. Annesi-Maesano, J.Y. Bernard, J. Botton, M.A. Charles, P. Dargent-Molina, B. de Lauzon-Guillain, P. Ducimetière, M. de Agostini, B. Foliguet, A. Forhan, X. Fritel, A. Germa, V. Goua, R. Hankard, B. Heude, M. Kaminski, B. Larroque †, N. Lelong, J. Lepeule, G. Magnin, L. Marchand, C. Nabet, F Pierre, R. Slama, M.J. Saurel-Cubizolles, M. Schweitzer, O.

Thiebaugeorges.

Availability of data and material

Data and Material was received from the PI (Professor Isabella Annesi-Maesano) of the EDEN Mother-Child Cohort

Competing interests none

Authors' contributions

MHS has participated in study design, data analysis and interpretation, draft writing, editing and submission. CNM, BH, CGB, and IAM participated in study design, data interpretation and editing. All authors read and approved the final manuscript

Conflict of interest: none

Fundings:

We acknowledge all funding sources for the EDEN study: