Dr Jean-Paul Cervoni
email: jpcervoni@chu-besancon.fr.

Blandine Alby-Lepresle

Delphine Weil

Peng Zhong

François Aubin

Daniel Wendling

Eric Toussirot

Lucine Vuitton

Franck Carbonnel

Raphaële Blondet

Thierry Thévenot

Paul Calès

Elisabeth Monnet

Vincent Di

Vincent Di Martino

Cervoni

A pragmatic non-invasive assessment of liver fibrosis in patients with psoriasis, rheumatoid arthritis or Crohn's disease receiving methotrexate therapy

Keywords: Methotrexate, Liver fibrosis, Noninvasive fibrosis marker, Transient Elastography, Psoriasis, Crohn's disease, Rheumatoid arthritis, Metabolic syndrome, Non alcoholic steatohepatitis

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

Methotrexate (MTX) is an anti-folate chemotherapeutic agent and immunosuppressant used since the 50's. It is prescribed as a first-line therapy in various inflammatory disorders such as psoriasis (Pso) [START_REF] Roenigk | Methotrexate in psoriasis: consensus conference[END_REF], rheumatoid arthritis (RA) [START_REF] Mikuls | The changing face of rheumatoid arthritis therapy: results of serial surveys[END_REF] and inflammatory bowel diseases [START_REF] Mate-Jimenez | 6-mercaptopurine or methotrexate added to prednisone induces and maintains remission in steroid-dependent inflammatory bowel disease[END_REF] and can induce sustained remissions in more than two-third of patients [START_REF] Mate-Jimenez | 6-mercaptopurine or methotrexate added to prednisone induces and maintains remission in steroid-dependent inflammatory bowel disease[END_REF][START_REF] Griffiths | Medical resource use and costs among rheumatoid arthritis patients receiving disease-modifying antirheumatic drug therapy[END_REF][START_REF] Kinder | The treatment of inflammatory arthritis with methotrexate in clinical practice: treatment duration and incidence of adverse drug reactions[END_REF]. Its efficiency, low cost and good acceptance have been however balanced by concerns about its liver toxicity, resulting in limiting its use. Current guidelines to monitor liver fibrosis established in last decades by dermatologists [START_REF] Roenigk | Methotrexate in psoriasis: consensus conference[END_REF][START_REF] Menter | Guidelines of care for the management of psoriasis and psoriatic arthritis. Section 3. Guidelines of care for the management and treatment of psoriasis with topical therapies[END_REF] are no longer appropriate because they required too many unnecessary liver biopsies, an invasive procedure. The American College of Rheumatology suggests monitoring only transaminases levels, but it is established that persistently normal liver biochemistry cannot exclude cirrhosis [START_REF] Shergy | Methotrexate-associated hepatotoxicity: retrospective analysis of 210 patients with rheumatoid arthritis[END_REF][START_REF] Te | Hepatic effects of longterm methotrexate use in the treatment of inflammatory bowel disease[END_REF]. The development of non-invasive liver fibrosis markers in chronic viral hepatitis C and subsequently in a variety of chronic liver diseases renders liver biopsy unsuitable for prognostic purposes. In patients receiving MTX the most common histological feature is steatohepatitis [START_REF] Roenigk | Methotrexate in psoriasis: revised guidelines[END_REF], a condition that can be evaluated by non-invasive fibrosis tests [START_REF] De Ledinghen | Diagnosis of liver fibrosis and cirrhosis using liver stiffness measurement: comparison between M and XL probe of FibroScan(R)[END_REF][START_REF] Ratziu | Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease[END_REF][START_REF] Wong | Diagnosis of fibrosis and cirrhosis using liver stiffness measurement in nonalcoholic fatty liver disease[END_REF].

The more recent studies have focused on non-invasive screening of significant liver fibrosis in patients with Crohn's disease (CD) [START_REF] Barbero-Villares | Evaluation of liver fibrosis by transient elastography (Fibroscan(R)) in patients with inflammatory bowel disease treated with methotrexate: a multicentric trial[END_REF][START_REF] Gonzalez-Lama | Methotrexate in inflammatory bowel disease: a multicenter retrospective study focused on long-term efficacy and safety. The Madrid experience[END_REF][START_REF] Laharie | Assessment of liver fibrosis with transient elastography and FibroTest in patients treated with methotrexate for chronic inflammatory diseases: a case-control study[END_REF][START_REF] Laharie | Diagnosis of liver fibrosis by transient elastography (FibroScan) and non-invasive methods in Crohn's disease patients treated with methotrexate[END_REF], psoriasis [START_REF] Berends | Biochemical and biophysical assessment of MTX-induced liver fibrosis in psoriasis patients: Fibrotest predicts the presence and Fibroscan predicts the absence of significant liver fibrosis[END_REF][START_REF] Bray | Liver fibrosis screening for patients with psoriasis taking methotrexate: a cross-sectional study comparing transient elastography and liver biopsy[END_REF][START_REF] Chladek | Assessment of methotrexate hepatotoxicity in psoriasis patients: a prospective evaluation of four serum fibrosis markers[END_REF][START_REF] Rongngern | Diagnostic performance of transient elastography for detection of methotrexate-induced liver injury using Roenigk classification in Asian patients with psoriasis: a retrospective study[END_REF][START_REF] Talme | Transient Elastography May Improve Detection of Liver Fibrosis in Psoriasis Patients Treated with Methotrexate[END_REF][START_REF] Lynch | The use of transient elastography and FibroTest for monitoring hepatotoxicity in patients receiving methotrexate for psoriasis[END_REF] and rheumatoid arthritis [START_REF] Arena | Liver stiffness correlates with methotrexate cumulative dose in patients with rheumatoid arthritis[END_REF][START_REF] Park | Assessment of liver fibrosis by transient elastography in rheumatoid arthritis patients treated with methotrexate[END_REF][START_REF] Rouhi | Prevalence and risk factors for liver fibrosis detected by transient elastography or shear wave elastography in inflammatory arthritis: a systematic review[END_REF] treated by MTX. These studies indicated that significant liver fibrosis is a rare event in patient under MTX without comorbidities, underlying the futility of serial liver biopsy samples in this context. However, these studies were limited to one or two non-invasive tests and did not provide comparison between different strategies of non-invasive evaluation of fibrosis.

The purposes of the present study were therefore 1) to define an appropriate strategy (both specific and sensitive) to assess significant liver fibrosis exclusively based on non-invasive methods in three distinct inflammatory diseases (Pso, RA and CD); 2) to evaluate the prevalence of liver fibrosis in patients treated with MTX when compared with patients with the same inflammatory diseases without MTX (control group); and 3) to evaluate in this population the impact of risks factors (alcohol consumption, metabolic syndrome and other causes of liver disease) on liver fibrosis to investigate whether MTX remains an independent risk factors of liver fibrosis over the long term.

Patients and methods

Study design

We conducted a prospective observational study including consecutive patients with Pso, CD or RA seen between February 2008 and October 2010, in our University hospital. The recruitment of patients was performed by two hepatologists in collaboration with the dermatology, rheumatology and gastroenterology departments. The study protocol was approved by the ANSM (Agence Nationale de Sécurité du Médicament et des produits de santé) N°Eudact2008-001149-24 and by our local ethics committee (Comité de Protection des Personnes Est II) N°08/474, providing it did not require liver biopsy and did not change patients care. Every enrolled patient gave written informed consent.

Patients

Pso, RA or CD patients already treated by MTX (treated group) or not (control group) were either referred by their dermatologist, rheumatologist, and gastroenterologist, or recruited during a planned hospitalization for TNFα antagonist treatment. The only exclusion criterion was an age<18.

Collected data

During a medical visit in the hepatology department, 189 variables were collected for each patient using a questionnaire focused on chronic liver disease's risk factors and metabolic syndrome, MTX treatment's modalities, other associated therapies, and coffee drinking supposed to have antifibrosing effects [START_REF] Freedman | Coffee intake is associated with lower rates of liver disease progression in chronic hepatitis C[END_REF][START_REF] Ruhl | Coffee and tea consumption are associated with a lower incidence of chronic liver disease in the United States[END_REF]. Clinical data included age, weight, body mass index and waist circumference. Biochemical parameters of metabolic syndrome and liver disease risk factors were determined on blood samples and doppler ultrasonography and assessed for each patient.

Metabolic syndrome was defined according to the National Cholesterol Education Program Adult

Treatment III (NCEP-ATP III) [START_REF]Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III)[END_REF].

Non-invasive fibrosis scoring system and Definition of significant fibrosis

Liver stiffness was measured by Fibroscan® [START_REF] De Ledinghen | Diagnosis of liver fibrosis and cirrhosis using liver stiffness measurement: comparison between M and XL probe of FibroScan(R)[END_REF][START_REF] Sandrin | Transient elastography: a new noninvasive method for assessment of hepatic fibrosis[END_REF][START_REF] Yoneda | Noninvasive assessment of liver fibrosis by measurement of stiffness in patients with nonalcoholic fatty liver disease (NAFLD)[END_REF] performed by an experienced hepatologist (BA or JPC) with the M probe. Eight biochemical liver fibrosis scores: FibroTest® [START_REF] Ratziu | Diagnostic value of biochemical markers (FibroTest-FibroSURE) for the prediction of liver fibrosis in patients with non-alcoholic fatty liver disease[END_REF][START_REF] Imbert-Bismut | Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study[END_REF], Forns index [START_REF] Forns | Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model[END_REF], ASAT to Platelet Ratio Index (APRI) [START_REF] Wai | A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[END_REF], Fibrosis Probability Index (FPI) [START_REF] Sud | Improved prediction of fibrosis in chronic hepatitis C using measures of insulin resistance in a probability index[END_REF], Hepascore [START_REF] Adams | Hepascore: an accurate validated predictor of liver fibrosis in chronic hepatitis C infection[END_REF],

Fibrosis-4 (FIB-4) [START_REF] Sterling | Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection[END_REF], NAFLD fibrosis score (NFS) [START_REF] Angulo | The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD[END_REF] and FibroMeter NAFLD [START_REF] Cales | Comparison of blood tests for liver fibrosis specific or not to NAFLD[END_REF][START_REF] Cales | A novel panel of blood markers to assess the degree of liver fibrosis[END_REF] were also used in our study by combining appropriately the available biochemical variables.

We developed a pragmatic algorithm to assert a high probability of significant fibrosis without liver biopsy by using all the fibrosis tests available for each patient. We used a three-step approach summarized in figure 1. First, we excluded 'invalid' tests and tests with results ranged within the 'grey zone', i.e., unable to predict with sufficient accuracy if the fibrosis stage was lower or greater than F2. The relevant values for each score for predicting fibrosis stage F2 or higher were then: Fibroscan®>7.1kPa [START_REF] Berends | Biochemical and biophysical assessment of MTX-induced liver fibrosis in psoriasis patients: Fibrotest predicts the presence and Fibroscan predicts the absence of significant liver fibrosis[END_REF], Fibrotest®>0.49 [START_REF] Imbert-Bismut | Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study[END_REF], Forns index>6.9 [START_REF] Forns | Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model[END_REF], APRI score>1.5 [START_REF] Wai | A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C[END_REF], FPI>0.8 [START_REF] Sud | Improved prediction of fibrosis in chronic hepatitis C using measures of insulin resistance in a probability index[END_REF], Hepascore>0.5 [START_REF] Adams | Hepascore: an accurate validated predictor of liver fibrosis in chronic hepatitis C infection[END_REF], FIB-4 score>3.25 [START_REF] Sterling | Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection[END_REF], NFS>0.676 [START_REF] Angulo | The NAFLD fibrosis score: a noninvasive system that identifies liver fibrosis in patients with NAFLD[END_REF] and FibroMeter NAFLD>0.49 [START_REF] Cales | A novel panel of blood markers to assess the degree of liver fibrosis[END_REF]. The liver stiffness values which did not meet the quality criteria (i.e., inter-quartile range <30% of the median elasticity and success rate >50% [START_REF] Sandrin | Transient elastography: a new noninvasive method for assessment of hepatic fibrosis[END_REF]) were also excluded from our final assessment. Second, we identified, for each patient, the number of discordant results between each test. By combining all valid results, we then defined two detection methods of significant fibrosis: a "specific" method corresponding to the result given by the majority of the tests (this algorithm is called 'Fspec') and a "sensitive" method, when at least one score concluded to significant fibrosis (this algorithm is called 'Fsens'). If more than two conflicting results were identified for a patient, we controlled the patient's record and each score was recalculated.

Statistical analysis

The statistical analyzes were used to 1) compare the 3 populations (Pso, RA and CD), 2) compare the patients treated or not with methotrexate, 3) identify the factors associated with liver fibrosis as defined by our algorithm and 4) evaluate the diagnostic performance of each noninvasive score for the prediction of significant fibrosis. The distribution of patient demographic, clinical, biochemical and radiological characteristics at baseline was analyzed in the three patient populations (Pso, RA and CD) and then in the "treated" and "control" groups. Univariate analyzes used the chi-2 test for comparing qualitative variables, Student's t-test, ANOVA, or Mann-Whitney test for comparing quantitative variables, and linear regression to evaluate correlations between quantitative variables, as appropriate. Multivariate analyzes used the logistic regression model for the analysis of factors associated with significant fibrosis. The explanatory variables used in the different models were those whose degree of significance was considered sufficient in univariate analysis (p<0.10). The cumulative dose of methotrexate was systematically incorporated in each model.

Finally, the performance of the different non-invasive tests for the diagnosis of significant fibrosis was assessed by the area under the ROC curve (AUROC). For this specific analysis, Fspec and Fsens algorithms were revised to exclude the fibrosis test evaluated.

RESULTS

Study population

A total of 131 consecutive patients were included: 66 Pso, 40 RA and 25 CD. Eighty patients (63%) had received MTX for a median duration of 34 months (range 3-235 months) and with a median cumulative dose of 1575 mg (range: 280-15000mg). Demographics and clinical characteristics of the study population are summarized in table 1: median age was 52 years; 61% of patients had BMI≥25, 26% had BMI≥30 and 27% patients met the NCEP-ATP III criteria of metabolic syndrome [START_REF]Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III)[END_REF]; the mean homeostasis model assessment estimate of insulin resistance (HOMA-IR) index was 2.55.

Forty-four percent of patients were alcohol consumers, with a reported consumption of 35g per day.

Eight patients were anti-HBc antibody positive and one was anti-HCV positive but HBVDNA or HCVRNA were not detectable. One additional patient had a primary sclerosing cholangitis.

The Comparison between the 3 groups of patients is summarized in table 1. Patients with CD were younger (p<0.0001), had a lower BMI (p=0.02) and HOMA-IR index (p=0.01). Patients with RA received higher cumulated dose of MTX (mean dose of 3022mg vs. 971mg and 1047mg in the Pso group and the CD group, respectively). The comparison between patients receiving or not MTX is summarized in table 2. Males were more often treated with MTX whereas patients with CD were not. There was no difference in terms of prevalence of metabolic syndrome or fatty liver detected by ultrasonography. 3). The best combinations of two, three, or four non-patented fibrosis tests always included the Forns index and the APRI. By combining APRI, Forns and NFS or FPI, the proportion of failures was restricted to the minimum value of 2.4%.

In 98 patients (75%), there was a complete concordance between all the available tests. Conflicting values concerned one single test for 20 patients, two tests for eight patients, and three tests for five patients. Of the 33 patients with conflicting estimates of fibrosis stage, the Hepascore was involved 14 times, the Fibrotest 10 times, the Fibroscan® and the FibroMeter NAFLD 9 times, the APRI test 5 times, the FIB-4, the NFS and the FPI thrice, and the Forns index once. The prevalence of significant fibrosis as defined by the Fspec and by the Fsens algorithms was 10% (13 patients) and 28% (37 patients), respectively (table 4).

Risks factors associated with significant fibrosis

In univariate analyses, significant fibrosis according to Fspec, was associated with age (p<0.001), metabolic syndrome (p<0.001) and its different components, and high GGT levels (p<0.001).

Significant fibrosis defined by Fsens was still associated with age (p<0.001), metabolic syndrome (p=0.004), and also with the absence of Crohn's disease (p<0.001). The use of MTX (any dose) even with a cumulative dose >1500mg, did not impact on liver fibrosis as assessed by the Fspec or Fsens algorithms. A logistic regression analysis identified three predictors of a significant fibrosis defined by the Fsens method (table 5): old age (OR=1.06, p<0.001), male gender (OR=2.87, p=0.026) and presence of metabolic syndrome (OR=3.64, p=0.007). The cumulative dose of MTX had no effect.

The small number of patients diagnosed with significant fibrosis through the Fspec algorithm did not allow multivariate analysis.

DISCUSSION

The fear of liver fibrosis development limits the use of MTX and highlights the need of a reliable, non-invasive, routinely available and inexpensive sequential assessment of liver fibrosis. Until recently, only liver biopsy was recommended for this purpose, thus considerably hampering the use of MTX over the long term, because its acceptability is low. It was therefore appropriate to evaluate all the non-invasive alternatives to liver biopsy available that could detect significant fibrosis. We evaluated them in an original fashion, i.e., without imposing liver biopsy as a diagnostic reference tool, but by analyzing the non-invasive tests altogether and considering their concordance as the guarantee of true assessment. The non-use of liver biopsy was imposed by our local ethic committee and allowed a good adhesion of patients and their referring physicians to our study. It also permitted to prospectively include all consecutive patients without biasing the results by selecting patients with presumed severe liver disease, a well-known situation which encourages liver biopsy. Our approach allowed us to define algorithms evaluating risks of fibrosis according to a 'specific' method (Fspec) and a 'sensitive' method (Fsens). Using this original diagnostic strategy, we were able to compare patients with Pso, RA and CD and evaluate the real impact of MTX on liver fibrosis taking into account the confounding factors as much as possible. Moreover, we investigated from all the combination of tests available, the ability of obtaining at least two concordant fibrosis tests for predicting significant fibrosis (table 3). We found that by combining at least three nonpatented tests allowing a 'grey zone', only a minority of patients (i.e., 2.4%) could not be classified according to the presence of significant liver fibrosis. Such a finding may be useful for all patients with fatty liver disease, a condition largely represented in our population, and justify that our approach will be further investigated in the setting of NAFLD.

The main results of our work were the following: 1) In patients with Pso, RA or CD, MTX treatment was not associated with more frequent significant liver fibrosis (no cumulative dose effect); 2) significant fibrosis was associated with a metabolic syndrome, which was common in patients with RA or Pso, conversely to that observed in patients with CD; 3) some non-invasive fibrosis markers among the most popular ones, such as the PIIIP assay widely used by dermatologists, and the Fibroscan®, which enjoys significant promotional communication in the hepatology field, appeared to be disappointing and less contributive than the biochemical tests. In particular, the FibroMeter NAFLD, the Fibrotest, and the combination of the non-patented tests, even despite their grey zone, were contributive in more than 97% of patients, a proportion much higher than the proportion of Fibroscan® successes with the M probe.

The originality of the present study was the pragmatic use and integrated analysis of numerous non-invasive fibrosis tests in patients receiving MTX. In previous studies on the topic, only a limited number of non-invasive tests (i.e., PIIIP [START_REF] Chalmers | Replacement of routine liver biopsy by procollagen III aminopeptide for monitoring patients with psoriasis receiving longterm methotrexate: a multicentre audit and health economic analysis[END_REF][START_REF] Maurice | Monitoring patients on methotrexate: hepatic fibrosis not seen in patients with normal serum assays of aminoterminal peptide of type III procollagen[END_REF], and Fibroscan®, alone or combined with Fibrotest® [START_REF] Barbero-Villares | Evaluation of liver fibrosis by transient elastography (Fibroscan(R)) in patients with inflammatory bowel disease treated with methotrexate: a multicentric trial[END_REF][START_REF] Laharie | Assessment of liver fibrosis with transient elastography and FibroTest in patients treated with methotrexate for chronic inflammatory diseases: a case-control study[END_REF][START_REF] Laharie | Diagnosis of liver fibrosis by transient elastography (FibroScan) and non-invasive methods in Crohn's disease patients treated with methotrexate[END_REF][START_REF] Berends | Biochemical and biophysical assessment of MTX-induced liver fibrosis in psoriasis patients: Fibrotest predicts the presence and Fibroscan predicts the absence of significant liver fibrosis[END_REF][START_REF] Rongngern | Diagnostic performance of transient elastography for detection of methotrexate-induced liver injury using Roenigk classification in Asian patients with psoriasis: a retrospective study[END_REF][START_REF] Talme | Transient Elastography May Improve Detection of Liver Fibrosis in Psoriasis Patients Treated with Methotrexate[END_REF][START_REF] Arena | Liver stiffness correlates with methotrexate cumulative dose in patients with rheumatoid arthritis[END_REF][START_REF] Park | Assessment of liver fibrosis by transient elastography in rheumatoid arthritis patients treated with methotrexate[END_REF][START_REF] Rouhi | Prevalence and risk factors for liver fibrosis detected by transient elastography or shear wave elastography in inflammatory arthritis: a systematic review[END_REF]) were studied. Comparisons with liver biopsy examination were performed when available. In the study by Chalmers et al., the PIIIP monitoring reduced the number of liver biopsies by 7 fold and increased its cost-effectiveness [START_REF] Chalmers | Replacement of routine liver biopsy by procollagen III aminopeptide for monitoring patients with psoriasis receiving longterm methotrexate: a multicentre audit and health economic analysis[END_REF]. In our work, this marker seemed not effective for the diagnosis of significant fibrosis, since its AUROC did not significantly differ from 0.5 whatever the algorithm chosen for defining significant fibrosis (table 7). In our work, Fibroscan® was performed by two experimented operators with the standard M probe and provided disappointing results, conversely to that enthusiastically reported by Laharie et al. with the same probe [START_REF] Laharie | Assessment of liver fibrosis with transient elastography and FibroTest in patients treated with methotrexate for chronic inflammatory diseases: a case-control study[END_REF]. We collected only 61% interpretable liver stiffness measures. Most failures or inaccurate measures were observed in overweight subjects, who represented a high proportion of the study population (76%). Our failure rate was consistent with that published by Lynch et al. [START_REF] Lynch | The use of transient elastography and FibroTest for monitoring hepatotoxicity in patients receiving methotrexate for psoriasis[END_REF]. Moreover, even when considering interpretable results of Fibroscan®, we observed low predictive values for the diagnosis of significant fibrosis (Table 6). In the present study, the most efficient test was the FibroMeter NAFLD, which was always interpretable and provided the highest performances for predicting significant fibrosis (table 6). Interestingly, APRI provided excellent NPV.

According to our more specific algorithm (Fspec), the estimated prevalence of fibrosis, was 10 % in our population: 12% in Pso, 12% in RA and 0% in CD. This result, consistent with the literature [START_REF] Te | Hepatic effects of longterm methotrexate use in the treatment of inflammatory bowel disease[END_REF][START_REF] Laharie | Assessment of liver fibrosis with transient elastography and FibroTest in patients treated with methotrexate for chronic inflammatory diseases: a case-control study[END_REF][START_REF] Whiting-O'keefe | Methotrexate and histologic hepatic abnormalities: a meta-analysis[END_REF], may be explained in part by the inclusion of a population unselected by the availability of liver biopsy, which is rarely systematic and rather encouraged by the presumption of severe liver disease. The non-influence of MTX treatment on liver fibrosis progression reported in our study was also previously reported by Laharie et al. [START_REF] Laharie | Assessment of liver fibrosis with transient elastography and FibroTest in patients treated with methotrexate for chronic inflammatory diseases: a case-control study[END_REF] in similar populations of consecutive patients. One may hypothesize that such reassuring data may be explained by lower cumulative doses of MTX than those used in the historical series that incriminated MTX in liver fibrosis development [START_REF] Boffa | Sequential liver biopsies during long-term methotrexate treatment for psoriasis: a reappraisal[END_REF][START_REF] Zachariae | Liver biopsy in psoriasis. A controlled study[END_REF][START_REF] Zachariae | Methotrexate-induced liver cirrhosis. A follow-up[END_REF],

but our results do not support this hypothesis. Indeed, in the present study, only 49 patients had received a cumulative dose of MTX >1500 mg, but even with this dose considered to be risky, no trend towards advanced fibrosis was observed. Our results emphasize the preponderant role of confounding factors in patients receiving MTX treatment to explain the variability of liver fibrosis.

Those factors, especially the metabolic syndrome, were not studied in historical reports but were reported to have a detrimental impact in the more recent ones [START_REF] Belinsky | The contribution of methotrexate exposure and host factors on transcriptional variance in human liver[END_REF][START_REF] Langman | Role of non-alcoholic steatohepatitis in methotrexate-induced liver injury[END_REF][START_REF] Shetty | Methotrexate Hepatotoxicity and the Impact of Nonalcoholic Fatty Liver Disease[END_REF]. In the study by Langman et al, which included patients with repeated liver biopsies, the 13 patients with metabolic syndrome had histologically proven non-alcoholic steatohepatitis and subsequently developed liver fibrosis [START_REF] Langman | Role of non-alcoholic steatohepatitis in methotrexate-induced liver injury[END_REF]. In our study, the prevalence of the metabolic syndrome, defined by the most stringent criteria (NCEP-ATPIII) was twice as high as that described in European populations [START_REF] Balkau | Frequency of the WHO metabolic syndrome in European cohorts, and an alternative definition of an insulin resistance syndrome[END_REF], and higher than the prevalence of excessive alcohol consumption or viral hepatitis. In patients with RA, an increased risk of cardiovascular disease has been described recently [START_REF] Meune | Trends in cardiovascular mortality in patients with rheumatoid arthritis over 50 years: a systematic review and meta-analysis of cohort studies[END_REF], which is clearly influenced by the metabolic syndrome [START_REF] Zonana-Nacach | Prevalence and factors associated with metabolic syndrome in patients with rheumatoid arthritis and systemic lupus erythematosus[END_REF]. In patients with psoriasis, the high prevalence of metabolic syndrome (up to 30%) reported in the literature [START_REF] Gisondi | Prevalence of metabolic syndrome in patients with psoriasis: a hospital-based case-control study[END_REF] was similar to that observed in our patients (35%). It favors non-alcoholic steatohepatitis, often reported in this population [START_REF] Gisondi | Non-alcoholic fatty liver disease in patients with chronic plaque psoriasis[END_REF][START_REF] Miele | Prevalence, characteristics and severity of non-alcoholic fatty liver disease in patients with chronic plaque psoriasis[END_REF]. Moreover, steatohepatitis was reported to be associated with severe forms of psoriasis which require MTX therapy [START_REF] Miele | Prevalence, characteristics and severity of non-alcoholic fatty liver disease in patients with chronic plaque psoriasis[END_REF]. The low prevalence of significant fibrosis in patients with Crohn's disease was explained by the low prevalence of metabolic syndrome (12%), the predominance of females and the young age of the majority of patients, consistently to that observed in other liver diseases [START_REF] Martino | Progression of liver fibrosis in women infected with hepatitis C: long-term benefit of estrogen exposure[END_REF][START_REF] Poynard | Validation of liver fibrosis biomarker (FibroTest) for assessing liver fibrosis progression: proof of concept and first application in a large population[END_REF]. The question whether MTX may contribute to the development of metabolic syndrome and NAFLD remains unresolved but is not supported by our data. In our series, the prevalence of metabolic syndrome was not higher in treated patients (Table 2), and liver steatosis was less common in patients with CD, despite a similar proportion of CD and Pso patients receiving a cumulative dose of MTX>1500mg

(table 1).

Our study acknowledges several limitations. First, liver biopsy was not performed although it is considered as the gold standard for identifying liver fibrosis in patients receiving MTX. We dared conduct this study without liver biopsy, consistently with what is done in the more recent studies in patients with chronic hepatitis C and with the recommendations of our ethics committee, who was aware of the risks and sampling biases associated with this invasive procedure [START_REF] Poynard | Liver biopsy: the best standard...when everything else fails[END_REF]. As mentioned above, it allowed us to reduce selection biases and increase acceptability of the study. Second, we used an original strategy to combine non-invasive tests results and identify patients with significant fibrosis, that had not been validated before. Similar strategies, however, are encouraged by several guidelines devoted to the management of various chronic liver diseases, in order to restrict the use of liver biopsy to patients with discordant results according to non-invasive evaluations [START_REF] Castera | Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C[END_REF]. They are particularly important for the detection of fibrosing non-alcoholic steatohepatitis in the large population potentially concerned [START_REF] Xiao | Comparison of laboratory tests, ultrasound, or magnetic resonance elastography to detect fibrosis in patients with nonalcoholic fatty liver disease: A meta-analysis[END_REF]. We improved this approach by using the largest number of scores ever done and defining both a specific algorithm based on the majority of concordant tests, and a sensitive algorithm that did not omit any patient with significant fibrosis predicted by only one positive test. The excellent agreement between the tests used (Figure 1) for predicting significant fibrosis was a convincing finding and a pragmatic validation of our diagnostic approach.

In conclusion, our study suggests the safety of MTX regarding the risk of liver fibrosis or, in any case, its negligible effect in comparison with risk factors such as the metabolic syndrome. In this context, liver biopsy should be reduced as much as possible to render MTX, a very cost-effective treatment, widely acceptable. The use of non-invasive markers should be recommended, providing it integrates the analysis of discordant results. The use of a single direct marker such as PIIIP or hyaluronic acid should no longer be recommended, particularly hyaluronic acid in the context of rheumatoid arthritis. Our preference goes to biochemical tests dedicated to steatohepatitis such as the FibroMeter NAFLD or to a combination of at least three tests including APRI, Forns, and FPI or NFS.

Our experience of Fibroscan® with M probe in this population was disappointing. Further studies using XL probe would be useful in patients receiving MTX.

Figures legend:

( a )

 a MTX: methotrexate; (b) HCV: hepatitis C virus ; (c) ALT: alanine transaminase; (d) BMI: Body mass index; (e) NCEP-ATP III: National Cholesterol Education Program Adult Treatment III; (f) HOMA-IR: Homeostasis model assessment of insulin resistance; SE: standard error.

Figure 1 :

 1 Figure 1: Non-invasive liver fibrosis diagnosis strategy: results from used tests and significant

Table 1 : Main characteristics of the population: comparison of psoriasis, rheumatoid arthritis and Crohn disease population.

 1

		Population	Psoriasis	Rheumatoid	Crohn's	p-value
		N=131	N=66	arthritis	disease
				N=40	N=25
	age, years (mean±SE)	52±1	54±2	58±2	41±3	<0.0001
	Male gender (%)	48%	61%	35%	36%	0.015
	MTX treatment (a)	63%	71%	62%	44%	0.05
	median Cumulated
		37%	30%	55%	28%	0.02
	dose >1500mg, %
	Alcohol consumption (mean±SE)	15±3	19±5	7±6	17±8	NS
	Anti-HBc antibodies, %	6.3%	7.6%	7.7%	0%	NS
	HCV (b) antibodies	0.8%	1.5%	0	0	NS
	Number of cups of coffee per day
		2.6±0.2	2.8±0.3	2.8±4	1.7±0.5	0.07
	(mean±SE)
	ALT > ULN (c)	27%	27%	25%	28%	NS
	Hyaluronic acid (µg/L) (mean±SE)	36±4	29±6	58±7	18±9	0.004
	Presence of steatosis (ultrasound)	47%	56%	47%	19%	0.01
	Weight (kg) (mean±SE)	76±2	78±2	79±3	66±4	0.003
	BMI (d) (kg/m 2 ) (mean±SE)	26.6±0.5	27.2±0.7	27.2±0.9	23.9±1.1	0.02
	NCEP-ATP III (e) criteria:
	-Waist circumference	48%	50%	60%	24%	0.02
	-Dyslipidemia	43%	39%	55%	32%	NS
	-High blood pressure	64%	70%	75%	32%	<0.001
	-Hyperglycemia	17%	23%	15%	4%	NS
	-Number of criteria (mean±SE)	1.8±0.1	1.9±0.1	2.6±0.2	1.6±0.2	0.002
	HOMA-IR (f) (mean±SE)	2.55±0.3	2.86±0.4	2.75±0.5	1.29±0.7	0.01

Table 2 : Comparison of patients treated and untreated with methotrexate.

 2 Factors influencing the crude values of liver fibrosis testsOld age was correlated with high hyaluronic acid (R²=0.12; p<0.0001), and FibroMeter NAFLD values (R²=0.23; p<0.001). Male gender was associated with a higher Forns index (4.61 vs. 3.38, p<0.001), All patients underwent a Fibroscan® but liver stiffness measurements failed for 29 patients and were not interpretable in 22 additional patients. A total of 80 Fibroscan® results were thus contributing. Only 40% of overweight patients had interpretable Fibroscan® measures. Seven biochemical tests could be interpreted for each patient on average, with extremes ranging from three (for three patients) to nine (for 28 patients) tests. At least five tests could be interpreted for 92% of the patients (figure1). All the patented tests, i.e; FibroMeter®, Fibrotest®, and Hepascore could be interpreted. Regarding the non-patented fibrosis tests, because a substantial proportion of

		Untreated patients	Treated patients	p-value
		n=48	n=83
	Age (years) (mean±SE)	50 ±3	54 ±1	NS
	Male gender, n (%)	17 (35%)	46 (55%)	0.03
	SRAC (a) (g/day) (mean±SE)	17 ±5	14 ±5	NS
	Psoriasis	19 (40%)	47 (57%)	0.06
	Rheumatoid arthritis	15 (31%)	25 (30%)	NS
	Crohn's disease	14 (29%)	11 (13%)	0.03
	Metabolic syndrome	15 (31%)	20 (24%)	NS
	High blood pressure	30 (63%)	54 (65%)	NS
	Waist circumference (cm)	93 ±2	96 ±2	NS
	(mean±SE)
	Weight (kg) (mean±SE)	74 ±3	77 ±2	NS
	Insuline resistance (b)	8 (17%)	14 (17%)	NS
	Dyslipidemia (c)	23 (48%)	33 (40%)	NS
	Uricemia (mean±SE)	307 ±13	345 ±12	0.04
	Abnormal liver tests	19 (40%)	29 (35%)	NS
	Ultrasound steatosis	19 (40%)	40 (52%)	NS
	Fspec (d)	4 (8%)	9 (11%)	NS
	Fsens (e)	9 (19%)	28 (34%)	NS

(a) SRAC: Self-Reported Alcohol Consumption; (b) Insulin resistance defined by history of diabetes, fasting blood glucose >6,1mmol/L; (c) Dyslipidemia defined by triglycerides levels >1,5g/L and/or HDL-cholesterol <0,4g/L in men and 0,5g/L in women; (d) Fspec: Significant fibrosis >F2 defined by majority of tests; (e) Fsens: Significant fibrosis >F2 defined by at least one test.

higher Hepascore (0.17 vs. 0.11, p=0.028), FIB-4 (1.31 vs. 0.97, p=0.003), and NFS (-1.75 vs. -2.51, p=0.006). The Cumulative dose of MTX significantly correlated with the levels of hyaluronic acid (R²=0.048; p=0.01) and of Hepascore (R²=0.53; p=0.01), but not with the other fibrosis scores.

Alcohol consumption was associated with higher Forns index (4.49 vs. 3.44, p=0.003), APRI (0.31 vs. 0.24, p=0.03), FPI (0.27 vs. 0.06, p<0.001) and FibroMeter NAFLD (0.11 vs. 0.03, p=0.03). Coffee drinking did not influence the result of the fibrosis tests. Patients with metabolic syndrome had higher Forns index (4.97 vs. 3.69, p<0.001), APRI (0.34 vs. 0.25, p=0.009) , FPI (0.29 vs. 0.08, p<0.001), FIB-4 (1.32 vs. 1.00, p=0.01), NFS (-1.08 vs. -2.52, p<0.001), Fibrotest (0.18 vs. 0.13, p=0.006), FibroMeter (0.27 vs. 0.02, p<0.001) and Fibroscan ® (6.95 vs. 4.65 KPa; p<0.001). NFS (n=36), APRI (n=13), FIB-4 (n=35), FPI (n=44) and Forns (n=49) provided inconclusive results (i.e., within the 'grey zone'), we evaluated the interest of combining two, three, four, or five tests (table

Table 3 : contribution of the non-patented fibrosis tests for the diagnosis of significant fibrosis Combinations of available non patented fibrosis tests

 3 This table explores the ability of obtaining a majority of (and two or more) concordant fibrosis tests for predicting significant fibrosis. By combining all the available non patented fibrosis tests, the proportion of patients with noncontributive tests changed regarding the tests used. The best contribution was obtained by combining 3 tests: APRI, Forns, and either NFS or FPI, with no additional contribution obtained by the addition of the remaining two tests. By combining only two tests, the best contribution was given by APRI and Forns. NA: not applicable.

		nb patients	Nb with less
		with tests	than 2
	nb patients	within the	concordant	% patients with non-
	tested	grey zone	tests*	contributive testing**

Table 4 : Non-invasive significant liver fibrosis assessment according to the inflammatory disease.

 4

		Population	Psoriasis	Rheumatoid	Crohn's disease
		(+/-SD) a	(+/-SD)	arthritis	(+/-SD)	p-
				(+/-SD)		value
		n=131	n=66	n=40	n=25
	Fibroscan®	102	49	30	23
	Fibroscan®(mean in kPa)	6.3	7.4 ±0.7	5.4 ±0.9	4.9 ±1.1	NS
	Fibroscan® Failure rate	22%	26%	25%	8%
	Fibroscan® NI b	38.6%	39.7%	45%	25%
	Fibroscan® ≥F2 (%)	7.9%	9.5%	10%	0%	0.06
	Forns index (n)	127	63	40	24
	Mean Forns index	4.0 ±0.3	4.2 ±0.2	4.5 ±0.3	2.8 ±0.4	0.004
	Forns NI	38.6%	39.7%	45%	25%
	Forns ≥F2 (%)	9.5%	9.5%	10%	0%	NS
	APRI score (n)	130	65	40	25
	Mean APRI c score	0.38 ±0.04	0.41 ±0.06	0.41 ±0.07	0.26 ±0.09	0.06
	APRI NI	10%	14.1%	7.5%	4%
	APRI ≥F2 (%)	3.1%	3.1%	5%	0%	NS
	FPI (n)	124	64	40	20
	Mean FPI d	0.26 ±0.03	0.29 ±0.04	0.28 ±0.04	0.09 ±0.06	<0.001
	FPI NI	35.5%	39.1%	40%	15%
	FPI ≥F2 (%)	9.7%	12.5%	10%	0%	0.05
	Hepascore (n)	124	64	38	22
	Mean Hepascore	0.22 ±0.02	0.18 ±0.03	0.32 ±0.04	0.15 ±0.05	0.08
	Hepascore ≥F2 (%)	10.5%	7.8%	21%	0%	0.02
	FIB-4 (n)	130	65	40	25
	Mean FIB-4 e	1.37 ±0.11	1.44 ±0.15	1.60 ±0.19	0.83 ±0.24	<0.001
	FIB-4 NI	26.9%	26.2%	37.5%	12%
	FIB-4 ≥F3	3.8%	4.6%	5%	0%	NS
	NFS f (n)	128	64	39	25
	Mean NFS	-2.05 ±0.15	-1.98 ±0.21	-1.53 ±0.26	-3.02 ±0.33	<0.001
	NFS NI	28.1%	28.1%	35.9%	16%
	NFS ≥F2 (%)	5.5%	6.3%	7.7%	0%	NS
	Fibrotest®(n)	119	61	37	21
	Mean Fibrotest	0.21 ±0.02	0.22 ±0.02	0.24 ±0.03	0.14 ±0.04	0.06
	Fibrotest®≥F2 (%)	10.1%	11.4%	13.3%	0%	NS
	Fibrometer-NAFLD (n)	125	64	38	23
	Mean Fibrometer-NAFLD	0.19 ±0.02	0.24 ±0.03	0.21 ±0.04	0.05 ±0.05	<0.001
	Fibrometer-NAFLD ≥F2	15.5%	21.9%	13.2%	0%	0.04
	Number of tests	8.5 ±0.1	8.5 ±0.1	8.6 ±0.1	8. 3 ±0.2	NS
	Uninterpretable tests	7.1 ±0.1	7.0 ±0.2	6.9 ±0.3	7.5 ±0.3	NS
	Significant fibrosis ≥F2	0.7 ±0.1	0.9 ±0.2	0.9 ±0.3	0 ±0.3	0.003
	Complete concordance	74%	71.2%	62.5%	100%	0.01
	Number of conflicting results	0.4 ±0.1	0.4 ±0.1	0.6 ±0.1	0 ±0.1	0.03
	Fspec	9.9%	12.1%	12.5%	0%	NS
	Fsens	28.2%	33.3%	37.5%	0%	0.002
	(a)

SD: Standard deviation; (b) NI : Non interpretable ; (c) APRI: ASAT to Platelet Ratio Index; (d) FPI: Fibrosis Probability Index; (e) FIB-4: Fibrosis-4; (f) NFS : non-alcoholic fatty liver disease fibrosis score Cervoni et al. 17

Table 5 : Multivariate analysis of factor associated with significant liver fibrosis according to the 'Fsens' algorithm

 5

		Logistic regression
		N=131 ; R²=0,20
	OR	IC 95%	p

Table 6 : Positive and negative predictive value of significant liver fibrosis according to the 'Fspec' algorithm.

 6

		PPV a (%)	NPV b (%)	Well ranked tests (%)
	Fibroscan® (kPa)	46.2	56.8	55.7
	Forns index	69.2	57.6	58.8
	APRI c	30.8	91.5	85.5
	FPI d	76.9	57.6	59.5
	Hepascore	38.5	88.1	83.2
	FIB-4 e	30.8	74.6	70.2
	NAFLD f fibrosis score	46.2	71.2	68.7
	Fibrotest®	69.2	88.1	86.3
	Fibrometer-NAFLD	92.3	89.0	89.3

a PPV: Positive Predictive Value; b NPV: Negative Predictive Value; c APRI: ASAT to Platelet Ratio Index; d FPI: Fibrosis Probability Index; e FIB-4: Fibrosis-4; f NAFLD: Non alcoholic fatty liver disease

Table 7 : Non-invasive tests performance for the diagnosis of significant liver fibrosis.

 7 Because hyaluronic acid may increase in the event of inflammatory rheumatism regardless of liver fibrosis, we conducted several sensivity analyses by excluding 1) the 40 patients with RA and the 19 patients with identified psoriatic arthritis; 2) the six cases of liver fibrosis identified on the basis of

			AUROC (a) value
		« Fspec »	« Fsens »
		AUROC	IC 95%	AUROC	IC 95%
	Age	0.83	0.56-0.94 0.77	0.61-0.87
	Cumulated dose of	0.34	0.11-0.52 0.52	0.35-0.66
	MTX (b)
	Waist circumference	0.65	0.28-0.85 0.69	0.51-0.81
	BMI (c)	0.64	0.30-0.83 0.73	0.56-0.84
	GGT	0.87	0.57-0.97 0.66	0.50-0.78
	Fibroscan®(kPa)	0.65	0.27-0.85 0.67	0.48-0.80
	Hyaluronic acid (µg/l)	0.64	0.32-0.83 0.62	0.45-0.75
	PIIIP (kU/l) (d)	0.59	0.23-0.81 0.63	0.46-0.76
	Forns index	0.95	0.77-0.99 0.76	0.60-0.86
	APRI score (e)	0.90	0.76-0.96 0.72	0.54-0.84
	FPI (f)	0.97	0.84-1	0.81	0.65-0.90
	HEPASCORE	0.78	0.47-0.92 0.62	0.43-0.76
	FIB-4 (g)	0.95	0.67-0.99 0.78	0.61-0.88
	NFS (h)	0.90	0.72-0.97 0.84	0.68-0.92
	Fibrotest®	0.92	0.68-0.98 0.75	0.59-0.86
	Fibrometer-NAFLD	0.93	0.79-0.98 0.88	0.73-0.95

a AUROC: Area Under the Receiver Operating Characteristic Curve; b MTX: Methotrexate; c BMI: Body Mass Index; d PIIIP: Procollagen type 3; e APRI: ASAT to Platelet Ratio Index; f FPI: Fibrosis Probability Index; g FIB-4: Fibrosis-4; h NAFDL: Non-alcoholic fatty liver disease

the data that support the findings of this study are available from the corresponding author upon request.

Performance in liver fibrosis assessment

The diagnostic performances of the fibrosis tests are summarized in table 6 and 7. Six tests showed excellent diagnostic performances (AUROC≥0.9) for predicting significant fibrosis according to the Fspec algorithm: FibroMeter NAFLD, FPI, FIB-4 score, Fibrotest®, NFS, and Forns test. FibroMeter NAFLD obtained the best performance with a proportion of well categorized patients PPV and NPV of 89.3%, 92.3% and 89.0%, respectively. The FibroMeter NAFLD was also the best test for predicting significant fibrosis through the Fsens algorithm (AUROC=0.88).


Maëliss Peigné
email: maeliss.peigne@inserm.fr

Pascal Pigny

Michaël W Pankhurst

Elodie Drumez

Anne Loyens

Didier Dewailly

Sophie Catteau-Jonard

Paolo Giacobini
email: paolo.giacobini@inserm.fr

The proportion of cleaved anti-Müllerian hormone is higher in serum but not follicular fluid of obese women independently of polycystic ovary syndrome

Keywords: PCOS, AMH, molecular forms, BMI, obesity, follicular fluid

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

Introduction

Polycystic ovary syndrome (PCOS) is a complex reproductive endocrine disorder, affecting up to 20% of women of reproductive age and representing the primary cause of anovulatory infertility worldwide [START_REF] Teede | Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome[END_REF]. In about 50% of cases, PCOS associates with metabolic disturbances, such as obesity, type 2 diabetes mellitus, cardiovascular disease and other comorbidities [START_REF] Dumesic | Scientific Statement on the Diagnostic Criteria, Epidemiology, Pathophysiology, and Molecular Genetics of Polycystic Ovary Syndrome[END_REF][START_REF] Escobar-Morreale | Polycystic ovary syndrome: definition, aetiology, diagnosis and treatment[END_REF]. Moreover, obesity itself increases the symptoms of PCOS such as ovulation disorders or hyperandrogenism.

A frequent hallmark found in women with PCOS, is an elevation of Anti-Müllerian hormone (AMH) serum levels, which have been reported to be 2-3-fold higher than in women with healthy ovaries [START_REF] Cook | Relationship between serum müllerian-inhibiting substance and other reproductive hormones in untreated women with polycystic ovary syndrome and normal women[END_REF][START_REF] Pigny | Elevated Serum Level of Anti-Mullerian Hormone in Patients with Polycystic Ovary Syndrome: Relationship to the Ovarian Follicle Excess and to the Follicular Arrest[END_REF]. Interestingly, the severity of the PCOS phenotype correlates with AMH production, which is higher in anovulatory than in ovulatory PCOS patients [START_REF] Abbara | Anti-Müllerian hormone (AMH) in the Diagnosis of Menstrual Disturbance Due to Polycystic Ovarian Syndrome[END_REF][START_REF] Catteau-Jonard | Polycystic ovaries at ultrasound: normal variant or silent polycystic ovary syndrome?[END_REF][START_REF] Pellatt | Granulosa cell production of anti-Müllerian hormone is increased in polycystic ovaries[END_REF].

Clinically observed correlations between enhanced basal levels of AMH and PCOS have led to a potential physiological role for AMH in "follicular arrest" in women with PCOS [START_REF] Dewailly | The physiology and clinical utility of anti-Mullerian hormone in women[END_REF]. In this pathology, enhanced numbers of primary and small secondary follicles secrete abnormally high levels of AMH, that associates with excess androgen production/secretion and leading to arrested follicular maturation.

AMH is a glycoprotein synthesized as a homo-dimeric prohormone of 140 kDa (composed of two monomers of 70 kDa), which undergoes a proteolytic cleavage at monobasic sites to become biologically active [START_REF] Di Clemente | Processing of antimullerian hormone regulates receptor activation by a mechanism distinct from TGF-beta[END_REF][START_REF] Pepinsky | Proteolytic processing of mullerian inhibiting substance produces a transforming growth factor-beta-like fragment[END_REF]. The cleavage of AMH gives rise to a 110 kDa N-terminal homodimer formed by two 57 kDa subunits (AMHN) and a 25 kDa active C-homodimer composed of two identical 12.5 kDa (AMHC) subunits. These 2 homodimers remain associated in a non-covalent complex (AMHN,C) 6 [START_REF] Nachtigal | Bioactivation of Müllerian inhibiting substance during gonadal development by a kex2/subtilisin-like endoprotease[END_REF][START_REF] Pepinsky | Proteolytic processing of mullerian inhibiting substance produces a transforming growth factor-beta-like fragment[END_REF]. Although having no intrinsic activity, AMHN has the role of amplifying the biological activity of the AMHC [START_REF] Wilson | Mullerian inhibiting substance requires its N-terminal domain for maintenance of biological activity, a novel finding within the transforming growth factor-beta superfamily[END_REF].

AMHN,C and its pro-hormone (proAMH) are found in blood but there is little evidence for the presence of free AMHC [START_REF] Pankhurst | Human blood contains both the uncleaved precursor of anti-Mullerian hormone and a complex of the NH2-and COOHterminal peptides[END_REF]. This suggests that circulating cleaved AMHN,C levels represent the receptor-competent isoform available for a hormonal function in a variety of tissues and organs expressing the exclusive binding-receptor AMHR2 [START_REF] Barbotin | Emerging Roles of Anti-Müllerian Hormone in Hypothalamic-Pituitary Function[END_REF].

A number of studies point to AMH as a useful marker for the diagnosis of PCOS, however challenges to the use of AMH to diagnose PCOS have been fully reviewed in [START_REF] Teede | Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome[END_REF][START_REF] Teede | Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome[END_REF]. A recent study by Abbara and colleagues [START_REF] Abbara | Anti-Müllerian hormone (AMH) in the Diagnosis of Menstrual Disturbance Due to Polycystic Ovarian Syndrome[END_REF] has suggested that AMH has potential as a biomarker for diagnosis of PCOS, outperforming antral follicles count, and greater elevations in AMH were associated with a more certain diagnosis of PCOS. However, due to the variability in levels using first-generation and less reliable assays and the lack of an international standard, AMH has not yet been adopted as part of the diagnostic criteria for PCOS [START_REF] Teede | Recommendations from the international evidence-based guideline for the assessment and management of polycystic ovary syndrome[END_REF].

In recent years, studies have started to use novel approaches and analytical assays [START_REF] Pankhurst | Efficacy of predictive models for polycystic ovary syndrome using serum levels of two antimüllerian hormone isoforms (proAMH and AMHN,C)[END_REF][START_REF] Wissing | Associations of different molecular forms of antimüllerian hormone and biomarkers of polycystic ovary syndrome and normal women[END_REF] aiming at determining whether the ratios of proAMH or AMHN,C in serum are altered in women with PCOS relative to women without PCOS and to understand if different AMH-related isoforms can be used to predict PCOS. However, the results of those studies are conflicting, either reporting differences in the mean proAMH:AMHN,C ratio in the PCOS group versus the unaffected control group [START_REF] Pankhurst | Efficacy of predictive models for polycystic ovary syndrome using serum levels of two antimüllerian hormone isoforms (proAMH and AMHN,C)[END_REF] or reporting no differences in the relative distribution of the AMH isoforms between those groups [START_REF] Wissing | Associations of different molecular forms of antimüllerian hormone and biomarkers of polycystic ovary syndrome and normal women[END_REF]. We hypothesized that part of the discrepancies could be due to the heterogeneity of the disorder and that stratifying women with and without PCOS based on their metabolic phenotype (body mass index) could better help to pin-point eventual differences in AMH cleavage. These difference in AMH cleavage could lead to relative higher levels of active AMH in the serum which could participate to the existence of different PCOS phenotypes.

The aims of this study were thus to determine if the relative distribution of AMH isoforms may differ from one patient to another depending on their body mass index (BMI) and PCOS status and to investigate whether differences in the proportions of AMH isoforms are due to increased proAMH cleavage occurring in the follicular fluid or in the serum.

Materials and Methods

Population for early follicular phase serum study

From a database including clinical, hormonal and ultrasound features that were consecutively recorded between January 2014 and December 2018 in patients referred to our department in an academic hospital for hyperandrogenia (HA) and/or oligo-anovulation and/or infertility. 70 women with PCOS (42 obese; BMI ≥ 30 kg/m², and 28 normal weight; BMI ≤ 25kg/m²) and 37 controls (16 obese and 21 normal weight) were selected retrospectively, as described below. Patients were included in the PCOS group according to a modified Rotterdam classification (Rotterdam ESHRE/ASRM-Sponsored PCOS consensus workshop group, 2004), i.e., presence of at least 2 out of the 3 following items: -1) HA (clinical or biological; as previously described [START_REF] Peigné | The numbers of 2-5 and 6-9 mm ovarian follicles are inversely correlated in both normal women and in polycystic ovary syndrome patients: what is the missing link?[END_REF]; -2) oligo-anovulation, (i.e. oligomenorrhea or amenorrhea); -3) presence of Polycystic Ovarian Morphology (PCOM) at Ultrasound (U/S), with an ovarian area ≥ 5.5 cm 2 and/or a follicle number per ovary ≥ 19, according to our inhouse thresholds, as previously reported [START_REF] Dewailly | Diagnosis of polycystic ovary syndrome (PCOS): revisiting the threshold values of follicle count on ultrasound and of the serum AMH level for the definition of polycystic ovaries[END_REF] .

Controls were referred to our department for infertility due to tubal and/or sperm abnormality.

None of these women had any component of the Rotterdam classification: they had regular cycles, no hyperandrogenism and normal ovaries at U/S. All medications known to affect metabolism (e.g. metformin) or ovarian function (e.g. oral contraceptive pill) had to be stopped at least 3 months before blood tests and U/S.

Investigations

During the medical examination, age, height, weight and waist circumferences were registered. Blood sampling was performed in the early follicular phase (i.e. between cycle days 2 and 5; either spontaneous or induced by didrogesterone). Follicle Stimulating Hormone (FSH), Estradiol (E2), Luteinizing Hormone (LH), Total Testosterone (TT), Fasting Serum Insulin levels (I) were measured by immunoassay using an automatic analyzer (Architect, Abbott Laboratories, USA). Sex Hormone Binding Globulin (SHBG) levels were measured by immunoassay using the Immulite analyzer (Siemens, Germany). Androstenedione levels were determined by LC-MS-MS.

Biochemical tests (glucose, lipid levels) were performed in the central biochemistry department of the hospital using routine assays on Roche analyzers.

Population for follicular fluid and concomitant serum study

From October 2018 to January 2019, women undergoing IVF ovarian stimulation were included prospectively either in the obese (PCOS or control) or the normal weight group (PCOS or control). Selection criteria were identical to those described earlier. Follicular fluid collected after oocyte pick-up (as described below), and serum, retrieved at the last hormonal check-up (2 to 3 days before oocyte pick-up and frozen at -20° after hormonal analysis), were used for AMH measurements.

Follicular fluid samples

During the oocyte pick-up procedure, all follicles over 12 mm were retrieved. After oocyte isolation for IVF procedure, follicular fluid was collected, centrifuged at 2,000 rpm for 5 min to remove cells and frozen at -80° until analysis.

AMH measurements

Routinely, fully automated Access Dxi sandwich chemoluminescent immunoassays (Beckman Coulter) were used to measure serum AMH levels. The standard used in the Access Dxi assay is the human recombinant 140 kDa AMH. Its limit of detection is around 0.47 pmol/L (Pigny, unpublished) and its measurement range is 0.47-150 pmol/L (van Helden and Weiskirchen, 2015). The Access Dxi assay used the same antibodies pair than the AMH Gen II ELISA: a capture antibody binding C-term part of AMH part and a detection antibody initially thought to be directed against the C-term of AMH but which also required the Nterminal part of AMH for correct detection [START_REF] Kevenaar | Serum anti-mullerian hormone levels reflect the size of the primordial follicle pool in mice[END_REF][START_REF] Pankhurst | Enzyme-linked immunosorbent assay measurements of antimüllerian hormone (AMH) in human blood are a composite of the uncleaved and bioactive cleaved forms of AMH[END_REF].

Based on these features, it can be assumed that the Access Dxi assay recognizes in the biological samples both the pro-AMH (uncleaved) and the AMHN,C (receptor competentactive). To fix this point, four serums were spiked with 10 to 25 ng/ml of recombinant AMHC (25 kDa, R& D Systems, MIS 1737). Results showed the absence of cross-reactivity of AMHC in this assay (Supplementary Table 1). So we concluded that the only molecular AMH forms detected in the serum by Dxi Automatic analyzer were proAMH + AMHN,C that we defined as total AMH (Supplementary Figure 1A).

Specific measurement of AMH molecular forms in serum and follicular fluid

A specific assay for proAMH measurement was built for Beckman Coulter Dxi automatic analyzer based on previous protocol designed with the AMH Gen II ELISA (Pankhurst and 10 McLennan, 2016) and used in the left-over serum or follicular fluid of the patients. This experimental protocol included a deoxycholate (DOC) treatment step of the serum which leads to the dissociation of the non-covalent link between N-terminal and C-terminal parts of AMHN,C which prevents binding of the detection antibody. ProAMH is unaffected by the DOC treatment as the molecule is uncleaved (Supplementary Figure 1B).

With the Gen II ELISA the DOC treatment step was performed between the capture and detection steps which is feasible in a manual assay but not possible with an automatic assay.

Therefore, we decided to pre-treat the samples before assaying AMH with the analyzer. Based on Pankhurst's study [START_REF] Pankhurst | A specific immunoassay for proAMH, the uncleaved proprotein precursor of anti-Müllerian hormone[END_REF], we performed independent experiments to set up DOC concentration, incubation time and temperature. The final protocol is described hereafter.

Two 150 µL aliquots were made from each sample (bio-banked serum or follicular fluid, stored at -80°). Phosphate Buffer Saline (0.02 M, pH = 6.8, 37°C) was added in one aliquot (control) whereas Sodium Deoxycholate in PBS buffer at 37°C (final concentration 0.23%, i.e. 2333 µg/ml, corresponding to nearly 6000 times more than the endogenous serum DOC concentration, was added in the second aliquot. Aliquots were incubated at 37°C during 15 min before being spiked again with DOC or PBS solution and further incubated for 15 min at 37°C. Final volumes of both aliquots were 300 µL which allow a direct measurement with the Beckman Coulter Access Dxi automatic analyzer. Control aliquot with PBS allows the measurement of total AMH in the sample whereas aliquot with DOC treatment allows the measurement of proAMH only (Supplementary Figure 1).

The AMH prohormone index (API; [START_REF] Pankhurst | A specific immunoassay for proAMH, the uncleaved proprotein precursor of anti-Müllerian hormone[END_REF]) was calculated as a surrogate marker of conversion of proAMH to AMHN,C ; API = [proAMH]/[total AMH] x 100.

Validation of the specific proAMH assay

No difference in total AMH levels measured with the Dxi Analyzer was observed between proAMH (either standard of the Access Dxi assay or rhAMH provided by Dr. Michael Pankhurst [START_REF] Pankhurst | A specific immunoassay for proAMH, the uncleaved proprotein precursor of anti-Müllerian hormone[END_REF]) treated by DOC versus PBS buffer (Supplementary Table 2) thus confirming that proAMH is unaffected by the DOC treatment.

As previously described [START_REF] Pankhurst | Human blood contains both the uncleaved precursor of anti-Mullerian hormone and a complex of the NH2-and COOHterminal peptides[END_REF], an aliquot of rhAMH was treated with furin to convert proAMH to AMHN,C. By western blotting we observed that the untreated preparation (rhAMH) mainly contains proAMH (around 77% after densitometric analysis) whereas the furin-treated preparation mainly contains AMHN,C (around 60%) (Supplementary figure 2). Both preparations were measured after dilution using our specific assay for proAMH. As expected, addition of DOC induced a strong decrease of the AMH concentration in the furin -treated preparation. This demonstrated that DOC treatment reduces the assayed AMH value in a manner that is consistent with dissociation of the AMHN,C complex. (Supplementary figure 2). The assayed AMH values after DOC pretreatment were not modified by the lipid profile of the dilution medium (serum of menopausal women with or without dyslipidemia, or in the standard of the Access Dxi assay, which does not contain AMH nor lipids) (Supplementary figure 2).

Statistical analysis

All statistical analyses were performed using SPSS software 22.0 (SPSS Inc., Chicago, IL, USA). Results were expressed as medians, with 5 e -95 e or 10 e -90 e percentiles for continuous variables and by the frequencies and percentages for categorical variables. Comparisons between two independent groups were performed using the Mann-Whitney U test and between dependent groups (for paired comparison between follicular fluid and serum results) using the Wilcoxon signed rank test. Comparisons between four groups were performed using the ANOVA test with post-hoc pairwise comparisons between obese and normal weight controls, and obese and normal weight PCOS after applying Bonferroni correction. The correlations between the various parameters were expressed as Spearman's correlation coefficient (r). A p value of <0.05 was considered to be significant and a r absolute value ≥ 0.4 was considered to be strong and clinically relevant.

Ethics

For the first part of the study, all patients had previously given their informed consent for the use of their clinical, hormonal, ultrasound records and leftover serum. For the second part of the study, all patients gave their informed consent for the use of their follicular fluid and leftover serum. The Institutional Review Board of the University Hospital gave its unrestricted approval to the anonymous use of clinical, hormonal, ultrasound records and biologic collection from all patients (reference DC-2008-642).

Results

Molecular forms of AMH in early follicular phase serum

One hundred-seven women were selected for this first part of the study: 70 women with PCOS (42 obese and 28 normal weight) and 37 controls (16 obese and 21 normal weight).

Table 1 shows the main clinical, hormonal, metabolic and U/S characteristics of the study population.

Mean total AMH and proAMH levels were higher in women with PCOS, independently of their metabolic features (BMI and insulin resistance, indirectly evaluated by waist circumference, SHBG levels and fasting insulinemia), when compared with controls (28.7 -51.0); P = 0.8). However, when we split by BMI, we observed that obese women versus their normal weight counterparts in both control and PCOS groups had an API significantly lower, suggesting a greater conversion of proAMH to AMHN,C (Figure 1).

(
Serum total AMH and proAMH levels were not significantly different between obese and normal weight women in each group (Figure 1).

Possible interactions between the API and pathophysiological features of PCOS were then investigated. When we grouped PCOS patients and controls, a significantly lower API was found in obese compared to normal weight women (34.9 % vs 41.2% respectively; P <0.001), whereas total AMH and proAMH levels were not significantly different.

In the whole population (N = 107), the API negatively correlated to metabolic parameters: 2). Moreover, the API was found to be positively correlated with SHBG (r = 0.293), and HDL cholesterol level (r = 0.221) (Figure 2, Table 2) No significant correlation was found with serum androgens or LH levels (Table 2).

Molecular forms of AMH in follicular fluid and serum of patients at the end of ovarian stimulation

ProAMH is known to be secreted by granulosa cells and thus released into the follicular fluid before reaching the serum [START_REF] Campbell | The Role of Anti-Müllerian Hormone (AMH) During Follicle Development in a Monovulatory Species (Sheep)[END_REF]. We next tried to determine whether the cleavage of this protein occurs within the ovaries or in the serum by analysing the API in those samples.

Because the API in early follicular phase serum was only different between obese and normal weight women, this second part of the study focused on thirty-nine patients: 20 normal weight women (including 10 with PCOS) and 19 obese women (including 9 with PCOS). The proportion of patients with PCOS in both groups (obese and normal weight) was not different.

The main clinical, hormonal, metabolic, U/S and ovarian stimulation features of this second study population are shown in Supplementary Table 3.

We measured total AMH and proAMH in the follicular fluid (FF) retrieved after oocyte pickup as well as in the concomitant sampled serum of each woman. The total AMH and the proAMH were comparable in normal weight versus obese women when comparing the protein amount either in the FF or in the serum (Figure 3); respectively in the FF: for total AMH 18.9 pmol/L IC5-95 (7.2-56.4) vs. 21.4 pmol/L IC5-95 (6.6-86) and for proAMH 13.1 pmol/L IC5-95 (4.9-43.4) vs.15.4 pmol/L IC5-95 (3.6-76). When we analysed the API, we found no significant differences in the FF between the two study groups, while the API was significantly reduced in the concomitant serum of obese women as compared to normal weight women (Figure 3). Interestingly, in both normal weight and obese women, the API was significantly higher in the follicular fluid than in matched serum samples (Figure 3), thus suggesting that most of the cleavage of proAMH occurs in the general circulation rather than in the ovaries.

Lastly, no correlation was found between the proAMH ratio in follicular fluid and any clinical, hormonal, metabolic, ultrasonographic or ovarian stimulation parameters studied parameter (Table 2; univariate analysis in the whole population, N = 39).

Discussion

In this study, we found that the proportion of the inactive form of AMH in the serum is higher in normal weight versus obese women (greater API value), independently of PCOS.

Consistently with this, obese women have a lower API than women with normal weight, pointing to a more pronounced AMH cleavage in obesity. In contrast with our findings, Pankhurst et al. [START_REF] Pankhurst | Efficacy of predictive models for polycystic ovary syndrome using serum levels of two antimüllerian hormone isoforms (proAMH and AMHN,C)[END_REF] described a minor reduction in the ratio between pro-AMH (inactive) over total AMH in PCOS women. This study involved a heterogeneous study group where all patients were overweight but the PCOS group had a higher incidence of obesity. Our data is in keeping with the view that women with PCOS have a heterogenous spectrum of phenotypic presentation highly susceptible to several variables, such as age, BMI, diet composition, to name a few, and that future areas of research shall take the different clinical features of PCOS into account.

Our data confirm and expand a recent study showing that the ratio of AMH isoforms do not differ between PCOS and control subjects [START_REF] Wissing | Associations of different molecular forms of antimüllerian hormone and biomarkers of polycystic ovary syndrome and normal women[END_REF], thus implying a similar distribution of uncleaved (inactive) and cleaved AMH in the two study groups. Wissing and 16 colleagues analysed a study group composed by women with either a normal weight, slightly overweight and/or comprising some PCOS women that received metformin and they reported a negative correlation between metabolic parameters and the proportion of inactive AMH form in the serum. Here, we confirmed the correlation results found in Wissing et al., with an even greater coefficient of correlation between the API and BMI (r = -0.557) and waist circumference (r=-0.530) and demonstrated that a greater processing of AMH in the circulation does not associate with PCOS but rather with a severe metabolic disturbance such as obesity.

In this study, we found significantly less conversion of proAMH into the cleaved isoform (AMHN,C) in the follicular fluid when compared to the concomitant serum of the study subjects, that was independent of their weight and disease state (PCOS or not). In contrast with a previous study [START_REF] Pierre | Most Cleaved Anti-Müllerian Hormone Binds Its Receptor in Human Follicular Fluid but Little Is Competent in Serum[END_REF], which described a higher cleavage of AMH in the follicular fluid in women with PCOS compared to controls, we did not find a difference of Our data suggest that the cleavage of proAMH is only partly occurring at the follicular level and that most of the cleavage of proAMH is mainly occurring in extra-ovarian tissues.

Subtilisin/kexin-like proprotein convertases such as furin (PCSK3) or PCSK5 are thought to be responsible for AMH cleavage [START_REF] Nachtigal | Bioactivation of Müllerian inhibiting substance during gonadal development by a kex2/subtilisin-like endoprotease[END_REF]. Plasmin is also a candidate for proAMH cleavage because, in vitro, recombinant proAMH can be cleaved by plasmin [START_REF] Pepinsky | Proteolytic processing of mullerian inhibiting substance produces a transforming growth factor-beta-like fragment[END_REF]. Some authors described that proAMH cleaving proteases are found in vascular tissues (reviewed in (McLennan and Pankhurst, 2015). However, the in vivo cleavage of proAMH remains largely unknown and it likely occurs at several levels/tissues.

Interestingly, serum furin levels are known to be modulated by inflammatory conditions such as that one that can be found in metabolic or cardio-vascular diseases [START_REF] Fathy | Diagnostic utility of BNP, corin and furin as biomarkers for cardiovascular complications in type 2 diabetes mellitus patients[END_REF][START_REF] Ren | Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome (PCOS)[END_REF]. Further studies are required to assess the in vivo molecular nature of the AMH cleavage, the exact anatomical sites of AMH conversion and whether this could be influenced by the metabolic status of the individual.

An emerging hypothesis proposes that AMH may play several key extra-gonadal roles, acting across multiple sites along the hypothalamic-pituitary-gonadal axis [START_REF] Barbotin | Emerging Roles of Anti-Müllerian Hormone in Hypothalamic-Pituitary Function[END_REF].

This raises the hypothesis that AMH could act as an endocrine signal, virtually operating on any cell type expressing its exclusive binding receptor AMHR2 throughout the body. Indeed, AMH and AMHR2 expression have been reported in prostate, lungs [START_REF] Gustafson | Müllerian inhibiting substance in the diagnosis and management of intersex and gonadal abnormalities[END_REF] and several other organs, including the brain [START_REF] Cimino | Novel role for anti-Müllerian hormone in the regulation of GnRH neuron excitability and hormone secretion[END_REF][START_REF] Lebeurrier | Anti-Mullerian-hormone-dependent regulation of the brain serine-protease inhibitor neuroserpin[END_REF][START_REF] Malone | Defective AMH signaling disrupts GnRH neuron development and function and contributes to hypogonadotropic hypogonadism[END_REF][START_REF] Wang | Evidence for a Müllerian-inhibiting substance autocrine/paracrine system in adult human endometrium[END_REF][START_REF] Wang | Mullerian inhibiting substance acts as a motor neuron survival factor in vitro[END_REF] and the pituitary [START_REF] Garrel | Anti-Müllerian hormone: a new actor of sexual dimorphism in pituitary gonadotrope activity before puberty[END_REF].

Moreover, metabolic organs such as liver and pancreas as well as the hypothalamic arcuate nucleus also express AMHR2 [START_REF] Cimino | Novel role for anti-Müllerian hormone in the regulation of GnRH neuron excitability and hormone secretion[END_REF][START_REF] Segerstolpe | Single-Cell Transcriptome Profiling of Human Pancreatic Islets in Health and Type 2 Diabetes[END_REF]; "The human protein atlas," n.d.), suggesting that potentially AMH could act on those tissues.

The present finding should be interpreted with caution regarding the study sample size since no formal size calculation was performed to determine the appropriate number of patients and control. In a posterior power calculation, we calculated that, with our study sample size (37 controls and 70 PCOS), we could detect with a 80% power (two-tailed test at 5% significance level), an effect size of 0.57 (expressed as standardized mean difference and interpreted as medium effect size [START_REF] Cohen | A power primer[END_REF]).

18

In conclusion, this study showed that circulating AMH isoform concentrations cannot be used to improve the precision of PCOS phenotype diagnosis. However, the novelty of this study relies on the findings showing that: 1) a higher proportion of cleaved, most likely bioactive, AMH (AMHN,C) is detectable in the serum of obese women compared to women with normal weight and 2) a greater cleavage of proAMH to AMHN,C is likely occurring in extra-ovarian tissues and exacerbated in obese women. This study opens new avenues of investigation aimed at addressing novel roles of AMH in the context of metabolic disturbances associated with reproductive alterations.

  weight (r = -0.578), BMI (r = -0.557), waist circumference (r = -0.530), Triglycerides level (r = -0.191) and fasting insulinemia (r = -0.351; Figure 2, Table

  cleavage of AMH at the follicular fluid level between women with PCOS and controls. Part of this discrepancy could be explained by the smaller number of patients recruited in the study of Pierre et al. as compared to the present work and by the different analytical methods used to measure AMH isoforms in the two studies.

Figure 1 :

 1 Figure 1: AMH molecular forms in the early follicular phase (EFP) serum: women with

Figure 2 :

 2 Figure 2: Correlation of AMH prohormone index (API) in the early follicular phase serum with metabolic parameters. Spearman correlation; EFP: early follicular phase, BMI: body mass index; SHBG: Sex Hormone Binding Globulin; HDL: High density lipoprotein; API: (proAMH/totalAMH)x100 , ** p < 0.001, r = correlation coefficient.

Figure 3 :

 3 Figure 3: Proportion of AMH molecular forms in the follicular fluid (FF) and in the end of ovarian stimulation serum (Serum): normal weight versus obese women. API (API: AMH prohormone index (proAMH/totalAMH) x 100 ) values in follicular fluid (FF) (on the left) and serum at the end of ovarian stimulation for IVF (on the right) of the same obese or normal weight women. Light blue, normal weight women (n = 20); dark blue, obese women

Table 1 :

 1 Population characteristics in the early follicular phase serum study

Table 2 :

 2 Correlation of proAMH ratio in the serum and follicular fluid with clinical, hormonal, ultrasonographic, and metabolic parameters ±0,400 => clinically relevant), EPF: early follicular phase, FF: follicular fluid; BMI: body mass index; SHBG: Sex Hormone Binding Globulin; LH: luteinizing hormone; FSH: Follicle stimulating hormone; HDL: High density lipoprotein; LDL : Low density lipoprotein; AFC: antral follicular count, API: AMH prohormone index, *p<0.05, ** p<0.01

	Serum EFP API	FF API
	rho Spearman	rho Spearman

Acknowledgments

We would like to thank Dr Isabelle Loison, Dr Anne -Laure Barbotin, Dr Virgine Simon, Lydie Lombardo and Sylvie Vanoverschele for their precious help in collecting samples.

Fundings

This work was supported by the Institut National de la Sante ́ et de la Recherche Me dicale (INSERM), France [grant number U1172], by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (ERC-2016-CoG to P.G. grant agreement n° 725149/REPRODAMH), by the Centre Hospitalier Régional Universitaire, CHU de Lille, France (Bonus H to P.G.).

Conflict of interest

The authors have no conflict of interest to declare.

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