Camille Gay
email: camille.gay@ap-hm.fr

Naima Hamdaoui

Vanessa Pauly

Marie-Christine Rojat Habib

Amina Djemli

Marion Carmassi

Cécile Chau

Florence Bretelle

Marie-Christine Rojat Habib

Dr Gay Camille

Impact of antibiotic treatment for chronic endometritis on unexplained recurrent pregnancy loss

Keywords: Recurrent Miscarriage, Chronic Endometritis, Early Pregnancy Loss, Late Pregnancy Loss, Antibiotic treatment, Implantation Failure Recurrent Pregnancy Loss, Chronic Endometritis, Early Pregnancy Loss, Late Pregnancy Loss, Antibiotic treatment, Implantation Failure

Introduction

Recurrent Pregnancy Loss (RPL) affects about 1% of all couples and is likely to cause therapeutic vagrancy and psychological distress. Multiple origins can explain RPL, and recent studies suggest the influence of chronic endometritis. The aim of our study is to evaluate the impact of antibiotic treatment on obstetrical prognosis among patients consulting for RPL with isolated chronic endometritis.

Material and methods

We conducted a monocentric retrospective comparative study. Patients consulting for RPL, with normal etiologic examinations (except for chronic endometritis), were included.

Introduction

Miscarriage is defined as a pregnancy loss before the gestational age of 24 weeks of gestation [START_REF] The | ESHRE guideline: recurrent pregnancy loss[END_REF]. It includes early pregnancy losses (early miscarriages EM), before 12 weeks of gestation, and late pregnancy losses (late miscarriage LM), 12 to 24 weeks. It excludes ectopic pregnancy and molar pregnancy. Unrepeated miscarriages affect 15 to 25% of pregnancies, getting more frequent with advanced maternal age and number of previous episodes [START_REF] Ogasawara | Embryonic karyotype of abortuses in relation to the number of previous miscarriages[END_REF].

Recurrent Pregnancy Loss (RPL) affects about 1% of all couples and is likely to cause therapeutic vagrancy and psychological distress. It is defined by a history of at least 2 consecutive early pregnancy losses [START_REF] The | ESHRE guideline: recurrent pregnancy loss[END_REF].

Multiple origins can explain RPL (3), among which are: genetic abnormalities (boosted by maternal age, so as balanced or Robertsonian translocation (4)), uterine defect (malformation, intracavitary process… [START_REF] Patton | Anatomic uterine defects[END_REF], thrombophilia (antiphospholipid antibody syndrome, S or C protein deficit, factor II or V Leiden mutation, essential thrombocythemia ( 6)), auto-immune disease (lupus, inflammatory bowel disease, celiac disease, autoimmune thyroiditis (7-9)), endocrinal diseases (hypo or hyperthyroidism, hyperprolactinemia, polycystic ovary syndrome, unbalanced diabetes,…), environmental factors (pollutants, endocrine disruptor, coffee, alcohol, tobacco, cocaine, psychological stress,…(7,10)), ovarian insufficiency, vaginal infection. According to Collège National des Gynécologues et Obstétriciens de France (CNGOF), the current recommended etiologic examinations are parental karyotypes (possibly completed by other genetic analyses), biological examinations (hormonal analyses, ovarian reserve, hyperandrogenism, thyroid, prolactin, blood sugar), hereditary thrombophilia and immunological examinations (anticardiolipin antibody, antiphospholipid, lupus anticoagulant, thyroid antibodies, antithrombin III mutation, factor II and V Leiden, S and C protein), uterine examinations (pelvic ultrasound or diagnostic hysteroscopy), infectious examinations (vaginal sample), male examinations (sperm analysis), and environmental examinations [START_REF]Recommandations pour la Pratique Clinique ; Bilan à Réaliser devant des fausses couches à répétition du premier trimestre[END_REF]. Jaslow et al. proved that abnormalities in etiological examinations for miscarriage are as frequent in couples who underwent 2 miscarriages as those who experienced more [START_REF] Jaslow | Diagnostic factors identified in 1020 women with two versus three or more recurrent pregnancy losses[END_REF]. In another study, Cardinale et al. brought to light that the prevalence of abnormalities in etiologic examinations were the same between couples undergoing consecutive and non-consecutive miscarriages [START_REF] Cardinale | Two miscarriages, consecutive or non-consecutive, does it change something?[END_REF]. Thus, examinations of RPL can be reasonably considered after the occurrence of 2 early miscarriages.

However, despite these examinations, 50% of RPL cases remain unexplained [START_REF] Stephenson | Frequency of factors associated with habitual abortion in 197 couples[END_REF].

Recent studies suggest the influence of chronic endometritis on implantation troubles [START_REF] Chen | Analysis of the diagnostic value of CD138 for chronic endometritis, the risk factors for the pathogenesis of chronic endometritis and the effect of chronic endometritis on pregnancy: a cohort study[END_REF][START_REF] Achilles | Endometrial plasma cells: do they indicate subclinical pelvic inflammatory disease?[END_REF][START_REF] Kitaya | Prevalence of chronic endometritis in recurrent miscarriages[END_REF].

Bouet et al. found that this chronic endometrial inflammation is frequent among patients suffering from repeated implantation failure (prevalence of 14%) or RPL (27%) [START_REF] Bouet | Chronic endometritis in women with recurrent pregnancy loss and recurrent implantation failure: prevalence and role of office hysteroscopy and immunohistochemistry in diagnosis[END_REF]. Chronic endometritis is suspected during hysteroscopy facing [START_REF] Cicinelli | Endometrial micropolyps at fluid hysteroscopy suggest the existence of chronic endometritis[END_REF] endometrial stromal edema, strawberry aspect of mucosa (congested and red endometrium, with white spots), endometrial micro-polyps (<1 mm), and endometrial irregularities. Hysteroscopy can suggest the diagnosis of chronic endometritis, although with limited sensibility and sensitivity, and anatomopathological analysis of endometrial biopsy is necessary to confirm the diagnosis [START_REF] Song | Correlation between hysteroscopy findings and chronic endometritis[END_REF]. Chronic endometritis is defined by the existence of plasma cells in endometrial stroma.

Immunostaining (antiCD138/Syndecan-1) improves diagnosis sensitivity [START_REF] Bayer-Garner | Routine syndecan-1 immunohistochemistry aids in the diagnosis of chronic endometritis[END_REF]. Bouet et al. [START_REF] Bouet | Chronic endometritis in women with recurrent pregnancy loss and recurrent implantation failure: prevalence and role of office hysteroscopy and immunohistochemistry in diagnosis[END_REF] showed that endometrial biopsy with immunohistochemistry is more specific and sensitive than office hysteroscopy, and should be realized systematically for the diagnosis of chronic endometritis.

The aim of our study is to evaluate the interest of antibiotic treatment on obstetrical prognosis among patients consulting for RPL, for whom etiologic examinations were negative, except for chronic endometritis.

Materials and Methods

We conducted a monocentric retrospective comparative study in the Gynecology-Obstetrics service of Hospital Nord, Marseille, France, between January 1 st , 2013, and January 1 st , 2018.

All patients aged between 18 and 42 years consulting for repeated pregnancy losses (including early or late miscarriages) were included. Early spontaneous pregnancy loss was defined as loss of a embryo before 12 weeks of gestation. Late pregnancy loss was defined as loss of a fetus between 12 and 24 weeks of gestation. Recurrent pregnancy loss was defined as the loss of two or more pregnancies, consecutive or not, according to ESHRE guidelines [START_REF] The | ESHRE guideline: recurrent pregnancy loss[END_REF]. Patients refusing to participate in the study and those with abnormalities in their miscarriage etiologic examinations were excluded.

The etiologic examinations were parental karyotypes, hormonal tests (FSH, AMH, TSH, prolactin on the 3rd day of the menstrual cycle), immunologic tests (antib2GP1, anticardiolipin, lupus anticoagulant, antinuclear antibodies, thyroid antibodies), thrombophilia examinations (ATIII, PS, PC, Factor II, and V Leiden mutation, homocysteinemia), blood sugar, sperm analyses (spermiogram, culture), uterine morphological examinations (pelvic sonography and/or hysteroscopy), and infectious examinations (vaginal sample with culture and PCR tests for STI).

Moreover, endometrial biopsy was done during diagnostic hysteroscopy or with Pipelle de

Cornier to evaluate the presence of chronic endometritis. The diagnosis criterion was the existence of at least one plasma cell per field on endometrial biopsy, which is the often used threshold in the literature [START_REF] Zolghadri | The value of hysteroscopy in diagnosis of chronic endometritis in patients with unexplained recurrent spontaneous abortion[END_REF][START_REF] Kasius | The impact of chronic endometritis on reproductive outcome[END_REF]. Plasma cells were identified by means of traditional hematoxylin and eosin staining and by immunohistochemistry for Syndecan-1 (CD138). In case of chronic endometritis, according to French recommendations about pelvic inflammatory diseases [START_REF] Verdon | Prise en charge thérapeutique des infections génitales hautes non compliquées. RPC infections génitales hautes CNGOF et SPILF [Treatment of uncomplicated pelvic inflammatory disease: CNGOF and SPILF Pelvic Inflammatory Diseases Guidelines[END_REF], patients received antibiotic treatment, after excluding any related allergy: with an antibiotic adapted to antibiogram if germ was isolated, or with doxycycline (100mgx2/day) and metronidazole (500mgX2/day) by mouth for 14 days in other cases.

Data regarding the clinical parameters and etiologic examinations of the patients were extracted. Data concerning BMI, age, and tobacco consumption were analyzed as well.

Finally, obstetrical outcomes were collected one year after the inclusion as pregnancy wish, pregnancy occurrence, and pregnancy outcome. Pregnancy outcome was compared between 3 groups: patients without chronic endometritis, patients with treated chronic endometritis, and patients with untreated chronic endometritis (corresponding to patients who got pregnant before antibiotic treatment or those refusing to undergo the treatment). LBR was defined by the delivery of viable children (according to the WHO).

Qualitative variables are evaluated as number and percentage, and continuous variables are evaluated as median and interquartile range. The characteristics of each groups were compared according to conditions required, with Fisher's exact test for qualitative values, and Kruskal Wallis-H test for quantitative values. Post hoc analyses were performed when overall p value was less than 0.05. Also, a multiple logistic regression analysis was done with a backward elimination strategy. Variables significant at p-value <0.20 in univariate analysis were introduced into the multivariate model and step by step eliminated until the model conserve variables with a p value <0.05. Results are presented with Odds Ratios (OR) and their 95% Confidence Interval (95%CI). The significance level was chosen as 5%. Statistical analyses were performed using the SPSS software.

Our study received a favorable opinion from the Aix-Marseille University Ethics Committee (2019-17-10-006).

Results

364 patients consulted our service for examination for RPL or late miscarriage during the specified period. Among them, 208 had abnormal etiologic examinations and were thus excluded from the study. Some examinations were missing for 92 patients (notably no endometrial biopsy when the patient got pregnant or the hysteroscopy was done outside of the service), and these patients were excluded as well. 64 patients were included and corresponded to those with negative etiologic examinations, among whom 37 patients had chronic endometritis (60%). 6 patients were excluded because they no longer had wishes to get pregnant (new partner, life project, abortion). 5 patients did not get pregnant during the 12-month follow-up. 14 patients dropped out of the study. Finally, 42 patients were analyzed; 22 of whom had chronic endometritis (figure 1).

Among untreated patients, 7 got pregnant before the prescription, and 2 did not receive the prescription (because it was forgotten to be sent).

Groups were comparable in terms of age, BMI, the number of miscarriages, AMH, and FSH levels on day 2 (Table 1). The delay before pregnancy was lower in the untreated endometritis group in comparison to the no endometritis and treated endometritis groups (3.0, 7.8, and 5.8 months, respectively, p=0.02).

A germ was identified among 5 out of 13 (38.5%) patients with treated chronic endometritis (Streptococcus agalactiae and Gardnerella vaginalis), and among 3 out of 9 (33.3%) patients with untreated chronic endometritis (Streptococcus agalactiae, Gardnerella vaginalis, and

Escherichia coli).

A significant improvement of live birth rate (LBR) was observed among patients with treated chronic endometritis compared to the untreated and no endometritis groups (85%, 44%, and 40%, respectively, p=0.032). There was a significant reduction of miscarriages in the treated group in comparison to the untreated and no endometritis groups (15%, 56%, and 60%, respectively, p=0.032; Table 2).

In multivariate analysis, a significant improvement of LBR was observed among patients treated for chronic endometritis compared to the no endometritis group (OR 21.4 [1.93-236.70] p=0.013), and the untreated endometritis group (OR 24.90 [1.64-376.93] p=0.020).

Discussion

In our study, there was an improvement of LBR among patients treated by antibiotics for chronic endometritis in comparison to those with untreated chronic endometritis.

So far, only a small number of studies have evaluated the impact of antibiotic treatment for chronic endometritis on pregnancy outcomes among patients consulting for RPL. Cicinelli et al. [START_REF] Cicinelli | Chronic endometritis due to common bacteria is prevalent in women with recurrent miscarriage as confirmed by improved pregnancy outcome after antibiotic treatment[END_REF] showed that LBR was higher after treatment and hysteroscopic endometritis recovery than after treatment with persistent hysteroscopic signs of endometritis. McQueen et al. [START_REF] Mcqueen | Chronic endometritis in women with recurrent early pregnancy loss and/or fetal demise[END_REF] observed improved LBR after antibiotic treatment among patients consulting for RPL in comparison to LBR before treatment. These findings support the effects of antibiotic treatment (adapted to antibiogram, if available, or empirical). Three other studies are available concerning antibiotic treatment, but these have evaluated implantion failure [START_REF] Kitaya | Live birth rate following oral antibiotic treatment for chronic endometritis in infertile women with repeated implantation failure[END_REF][START_REF] Cicinelli | Prevalence of chronic endometritis in repeated unexplained implantation failure and the IVF success rate after antibiotic therapy[END_REF] or unexplained infertility [START_REF] Cicinelli | Chronic endometritis in patients with unexplained infertility: Prevalence and effects of antibiotic treatment on spontaneous conception[END_REF]. Kitaya et al. (27) observed improved LBR after antibiotic treatment among patients with cured endometritis in comparison to those with no endometritis in a population of patients suffering from repeated implantation failure. Cicinelli et al. [START_REF] Cicinelli | Chronic endometritis in patients with unexplained infertility: Prevalence and effects of antibiotic treatment on spontaneous conception[END_REF] showed that antibiotic treatment for chronic endometritis among patients suffering from unexplained infertility improved LBR. Despite our small number of patients, our study is the first one to compare outcomes between treated and untreated patients among unexplained RPL cases.

Various germs were found in our population, including commensal germs of the vaginal flora; these results are close to those of Cicinelli [START_REF] Cicinelli | Chronic endometritis due to common bacteria is prevalent in women with recurrent miscarriage as confirmed by improved pregnancy outcome after antibiotic treatment[END_REF]. Concerning antibiotic treatment, doxycycline (100mg orally twice a day) and metronidazole (500 mg orally twice a day) for 14 days was the proposed antibiotic protocol in most available studies, according to CDC guidelines [START_REF] Workowski | Sexually transmitted diseases treatment guidelines, 2010[END_REF].

In addition to its action against germs, antibiotic use for treating chronic endometritis could be effective in modulating the immune response, thanks to doxycycline immunomodulating action [START_REF] Bonnetblanc | Doxycycline [Doxycycline[END_REF][START_REF] Labro | Immunomodulatory effects of antimicrobial agents. Part I: antibacterial and antiviral agents[END_REF].

Of course, our study presents several limits. The number of included patients was low, and 21 patients were excluded (33%). Indeed, the statistical power of our results was also limited.

Correct medication intake could not be checked and the treatment was not compared to placebo. Stress is a factor favoring miscarriage, and the placebo effect could play a role in the next pregnancy outcome [START_REF] Qu | The association between psychological stress and miscarriage: A systematic review and meta-analysis[END_REF]. However, Kitaya et al. [START_REF] Kitaya | Live birth rate following oral antibiotic treatment for chronic endometritis in infertile women with repeated implantation failure[END_REF] explained the impossibility to perform a randomized controlled trial, because 95% of infertile couples desired antibiotic treatment and refused to take a placebo.

Patients who did not have pregnancy during the 12-month follow-up represented 11% of our patients, which is close to the general population (18% according to INSERM data [START_REF]Les Troubles de la Fertilité[END_REF]).

These patients were excluded since they presented infertility as well, and the aim of our study was to evaluate the impact of chronic endometritis on miscarriage. We also chose to exclude any other pathology that could be a confounding factor.

In the current study, we did not analyze the result of control endometrial biopsy or hysteroscopy due to the small percentage of realization. The time necessary to perform the examinations and wait for conception was demotivating for several patients. However, the comparison of pregnancy outcomes would be interesting to investigate between patients with persistent endometritis and those with normal endometrial biopsy. Cicinelli et al. [START_REF] Cicinelli | Chronic endometritis due to common bacteria is prevalent in women with recurrent miscarriage as confirmed by improved pregnancy outcome after antibiotic treatment[END_REF][START_REF] Cicinelli | Chronic endometritis in patients with unexplained infertility: Prevalence and effects of antibiotic treatment on spontaneous conception[END_REF] showed that pregnancy outcomes were improved after antibiotic treatment and chronic endometritis recovery (normal hysteroscopy and endometrial biopsy).

The definition of anatomopathological chronic endometritis is unclear, and the plasma cell level by fields differs from one study to another. Most studies define endometritis as a level of 1 plasma cell per field in the endometrial stroma. The presence of pathogenicity in a few plasma cells is not established and would not necessarily be associated with an inflammatory process. The biopsy sampling site could change the diagnosis (deep or superficial sample, sample site, associated hysteroscopy…). Likewise, the day of the cycle during biopsy could also play a role but is rarely studied [START_REF] Liu | Comparison of the prevalence of chronic endometritis as determined by means of different diagnostic methods in women with and without reproductive failure[END_REF]. A consensual pathological definition would be necessary to correctly analyze chronic endometritis. In 2019, the International Working Group for the Standardization of Chronic Endometritis Diagnosis published a unified diagnostic criterion for chronic endometritis at fluid hysteroscopy [START_REF] Cicinelli | International Working Group for Standardization of Chronic Endometritis Diagnosis. Unified diagnostic criteria for chronic endometritis at fluid hysteroscopy: proposal and reliability evaluation through an international randomized-controlled observer study[END_REF]. This could lead to a consensus on anatomopathological diagnosis in the future.

Conclusion

In our patients examined for RPL, LBR could be improved after antibiotic treatment of chronic endometritis in comparison to patients with untreated chronic endometritis.

A randomized controlled trial with a wider population of patients, an analysis of control endometrial biopsy after treatment, and a strict anatomopathological definition for chronic endometritis would be necessary to precisely determine the action of antibiotic treatment in chronic endometritis and clinical outcomes. However, in case of a history of fetal losses and diagnosis of chronic endometritis, in the context of therapeutic dead-ends induced by repeated miscarriages and the low medical risk associated with short antibiotic treatment, the benefit-risk ratio seems to be in favor of systematic treatment (except for contraindications), which awaits stronger findings. This research did not receive any specific grant from funding agencies in public, commercial, or non-profit sectors.

Table 1 :

 1 Main included patient's characteristics: (BMI Body Mass Index ; EM Early Miscarriage ; LM Late Miscarriage, IQR Interquartile Range)

		No	Treated	Untreated	Total (n=42) Median
		endometritis	endometritis	endometritis (n=9)		(IQR)
		(n=20)	(n=13)
	Age (year)	33,9	30,5	31,2	32,3	33 (9)	p=0.211
	BMI (kg/m²)	24,3	23,4	26,3	24,5	24 (3)	p=0.867
	Tobacco (patients) 6 (30%)	6 (46%)	1 (11%)	13 (31%)		p=0.236
	Number of EM	4,2 (/19	3,4 (/11	4,2 (/6 patients)	3,9 (/36		p=0.210
	(number among	patients)	patients)		patients)
	concerned patients)
	Number of LM	1 (/1 patient) 1,5 (/2	1 (/3 patients)	1,2 (/6 patients)		p=0.11
	(number among		patients)
	concerned patients)
	AMH (ng/mL)	2,37	3,47	3,17	2,95	2 (2)	p=0.908
	FSH (UI/L)	7,0	7,7	5,2	6,7	7 (4)	p=0.521
	Delay before	7,8	5.8	3	6.2	5 (3)	P=0.02
	pregnancy (month)

Table 2 :

 2 Pregnancy outcome in multiple analysis

Conflicts of Interest: None


MD, PhD Anne-Sylvia Sacri
email: anne-sylvia.sacri@inserm.fr

MD Alain Bocquet

MD, PhD Mariane De Montalembert

MD, PhD Serge Hercberg

MD, PhD g ; Laurent Gouya

PhD Béatrice Blondel

Pharm D h ; Amandine Ganon

PhD i ; Pascale Hebel

MD j ; Catherine Vincelet

MD k ; Franck Thollot

MD l ; Massimiliano Rallo

MD m ; Piotr Gembara

MD Corinne Levy

MD, PhD Martin Chalumeau

Young children formula consumption and iron deficiency at 24 months in the general population: A national-level study

Keywords: Iron deficiency, primary prevention, young-child formula, socioeconomic factors, effectiveness, nationwide cohort study

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

Introduction

Iron deficiency (ID) 1 is considered the most frequent micronutrient deficiency worldwide, including in industrialized countries, and is strongly suspected to be associated with adverse short-and long-term neurocognitive effects when it occurs in neonates and infants [START_REF] Lozoff | Long-term developmental outcome of infants with iron deficiency[END_REF]. In this context, ID is a target of various primary preventive strategies supported by medical societies and public health authorities, based on advice to parents for consumption of naturally ironrich foods during complementary feeding, iron fortification (e.g., formula or cereals) or iron supplementation (drops) in their children [START_REF] Baker | Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age)[END_REF][START_REF] Efsa | European Food Security Authority. Panel on Dietetic Products, Nutrition and Allergies (NDA). Scientific Opinion on nutrient requirements and dietary intakes of infants and young children in the European Union[END_REF][START_REF] Domellof | Iron requirements of infants and toddlers[END_REF]. In some countries like France, the preventive strategy involves both parental education related to complementary feeding and the recommendation for consumption of iron-fortified formulas (IFFs) at breastfeeding cessation [START_REF] Ghisolfi | Lait de vache ou lait de croissance : quel lait recommander pour les enfants en bas age (1-3 ans) ? [French][END_REF]. IFFs after age 6 months include "follow-on formulas" and "young-child formulas" (YCFs, sometimes inadequately called "growing-up formula") between age 6 and 10-12 months and after 10-12 months, respectively, to help to ensure the recommended average requirement (AR) of 5 mg/day at this age [START_REF] Ghisolfi | Lait de vache ou lait de croissance : quel lait recommander pour les enfants en bas age (1-3 ans) ? [French][END_REF][START_REF] Efsa | Dietary reference values for nutrients: Summary report[END_REF]. The European Food Security and Authority (EFSA) does not recommend YCF as the main source of iron after age 12 months and has criticized YCF-based strategies given the lack of evidence supporting their superiority as compared with balanced diet strategies [START_REF]Growing-up' formula: No additional value to a balanced diet, says EFSA. Parma[END_REF][START_REF] Efsa Panel On Dietetic | Scientific Opinion on nutrient requirements and dietary intakes of infants and young children in the European Union[END_REF]. In some industrialized countries, balanced diets were recently shown to be rarely achieved at this age at the population level [START_REF] Chouraqui | Food, water, energy, and macronutrient intake of non-breastfed infants and young children (0-3 years)[END_REF][START_REF] Grimes | Food Sources of Total Energy and Nutrients among U.S. Infants and Toddlers: National Health and Nutrition Examination Survey 2005-2012[END_REF].

The efficacy of IFFs has been evaluated by eight clinical trials, seven of which found a statistically significant effect of IFFs use on iron status [START_REF] Fuchs | Iron status and intake of older infants fed formula vs cow milk with cereal[END_REF][START_REF] Moffatt | Prevention of iron deficiency and psychomotor decline in high-risk infants through use of iron-fortified infant formula: a randomized clinical trial[END_REF][START_REF] Daly | Prevention of anaemia in inner city toddlers by an iron supplemented cows' milk formula[END_REF][START_REF] Gill | Follow-on formula in the prevention of iron deficiency: a multicentre study[END_REF][START_REF] Morley | Iron fortified follow on formula from 9 to 18 months improves iron status but not development or growth: a randomised trial[END_REF][START_REF] Lozano | Intake of an iron-supplemented milk formula as a preventive measure to avoid low iron status in 1-3 year-olds][END_REF][START_REF] Akkermans | A micronutrient-fortified young-child formula improves the iron and vitamin D status of healthy young European children: a randomized, double-blind controlled trial[END_REF]. The efficacy of IFFs seems well established at the scale of randomized trials, but this conclusion cannot be extrapolated to their effectiveness, notably after age 12 months, at the population level. Indeed, the ability of IFF-based strategies to reach disadvantaged families (with low economic and/or education level) [START_REF] Attree | A critical analysis of UK public health policies in relation to diet and nutrition in low-income households[END_REF][START_REF] Barker | Developmental biology: Support mothers to secure future public health[END_REF][START_REF] Fleurbaey | Évaluation des politiques de santé : pour une prise en compte équitable des intérêts des populations[END_REF][START_REF]Méthodes d'évaluation des politiques publiques -actes du séminaire[END_REF], who are at greatest risk of ID [START_REF] Baker | Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age)[END_REF][START_REF] Efsa | European Food Security Authority. Panel on Dietetic Products, Nutrition and Allergies (NDA). Scientific Opinion on nutrient requirements and dietary intakes of infants and young children in the European Union[END_REF][START_REF] Domellof | Iron requirements of infants and toddlers[END_REF][START_REF] Sacri | Very low prevalence of iron deficiency among young French children: A national cross-sectional hospital-based survey[END_REF], is poorly known and should be evaluated. Moreover, to be reliable, such demonstration must take into consideration betweencountry and between-region variability in iron intake from non-dairy food. Thus, the evaluation of the effectiveness of IFF-based prevention strategies requires large populations with a diversified recruitment collecting sociodemographic, biological and dietary data. Only three studies with a closely approaching design are available and were performed in Ireland,

The Netherlands, and the United Kingdom [START_REF] Sidnell | Nutrient intakes and iron and vitamin D status differ depending on main milk consumed by UK children aged 12-18 months -secondary analysis from the Diet and Nutrition Survey of Infants and Young Children[END_REF][START_REF] Uijterschout | Prevalence and risk factors of iron deficiency in healthy young children in the southwestern Netherlands[END_REF][START_REF] Mccarthy | Iron intakes and status of 2-year-old children in the Cork BASELINE Birth Cohort Study[END_REF]. They all found a protective effect of YCF on iron status at age 12 to 24 months (e.g., adjusted odds ratio [OR] for ID was 5.5 for lack of use of YCF [START_REF] Uijterschout | Prevalence and risk factors of iron deficiency in healthy young children in the southwestern Netherlands[END_REF]). However, these studies had limitations that prevent definitive conclusions on the effectiveness of YCF-based strategies: selection bias (single-center recruitment [START_REF] Mccarthy | Iron intakes and status of 2-year-old children in the Cork BASELINE Birth Cohort Study[END_REF], children requiring surgery [START_REF] Uijterschout | Prevalence and risk factors of iron deficiency in healthy young children in the southwestern Netherlands[END_REF]), strong attrition [START_REF] Sidnell | Nutrient intakes and iron and vitamin D status differ depending on main milk consumed by UK children aged 12-18 months -secondary analysis from the Diet and Nutrition Survey of Infants and Young Children[END_REF][START_REF] Mccarthy | Iron intakes and status of 2-year-old children in the Cork BASELINE Birth Cohort Study[END_REF], dietary survey not following EFSA recommendations [START_REF] Uijterschout | Prevalence and risk factors of iron deficiency in healthy young children in the southwestern Netherlands[END_REF], and/or lack of multivariate analyses to take into consideration socioeconomic status or non-dairy iron intake [START_REF] Sidnell | Nutrient intakes and iron and vitamin D status differ depending on main milk consumed by UK children aged 12-18 months -secondary analysis from the Diet and Nutrition Survey of Infants and Young Children[END_REF].

A non-biased evaluation of the independent effect of YCF at the national level is needed to produce evidence to discourage or support YCF-based strategies after age 12 months versus other strategies for ID primary prevention. Here we aimed to evaluate the effectiveness of a strategy for ID prevention based on the recommendation of YCF use after age 12 months at a national level by analyzing its association with iron status in children aged 24 months, taking into account other sources of iron and the family's socio-economic status.

Methods

General methodology

We conducted a cross-sectional observational study in primary care paediatric private practices throughout France from January 2016 to December 2017, named the CARMA study. The signed consent from at least one parent was obtained before inclusion. The study protocol was approved by local ethics and administrative authorities (CPP IDF III no. 3295) and was registered (ClinicalTrials.gov identifier: NCT02484274). We used the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) guidelines to report this study (Supplemental Table 1).

Participant selection

Investigators were recruited from the directory of the French Ambulatory Pediatricians Association (the national association of primary care pediatricians, n≈1600). Invitations to participate were sent to all pediatricians. Among the respondents, a selected sample of about 120 pediatricians stratified on the 21 French continental regions was recruited to take in consideration the between-region variability. Investigator recruitment was secondarily extended to all members of the association given difficulties in recruiting participants in this complex study. This recruitment in primary care pediatrician offices and different regions of the country aimed at including infants from the general ambulatory population at a national level. Each pediatrician was asked to include 10 consecutive children, aged 22 to 26 months, usually living in France (Supplemental Figure 1), who had health insurance coverage, and were not known to be affected by at least one of the following diseases that may affect iron metabolism: transfusion(s) since birth, celiac disease, inflammatory bowel disease, cystic fibrosis, other enteropathy (except allergy to cow's milk protein), enteral nutrition of more than 15 days in the last 6 months, chronic hemolytic diseases, chronic renal failure, hemophilia, hemochromatosis, malignant diseases and lead poisoning.

Data collection

The study phases are described in detail in Supplemental Methods. In summary, current sociodemographic data, past and current main dietary habits and medical data were collected by the investigator at inclusion. During the month after inclusion, a dietary survey was performed according to the EFSA reference methodology. Parents recorded all ingesta per 24hr period over three non-consecutive days (two during the week and one during the weekend) by using paper diaries, as in previous studies [START_REF] Efsa | General principles for the collection of national food consumption data in the view of a pan-European dietary survey[END_REF]. Investigators and on-line research assistants helped parents complete the survey when needed. Dietary data from these diaries were double-entered, tested for consistency, and transformed by using the ANSES-Ciqual nutrient composition table for non-infant foods [START_REF]Table de composition nutritionnelle des aliments Ciqual[END_REF] and the infant-specific table designed for the French Children's Food Union [START_REF] Tavoularis | Nutri-Bebe 2013 Study Part 1. Presentation and methodological considerations[END_REF]. With this process, we obtained the ingesta and their source for each individual and each nutrient.

After the dietary survey, a fasting blood sample was taken; serum ferritin (SF) and Creactive protein (CRP) were measured centrally in a unique laboratory after refrigerated transport of blood samples at +2 to +8°C. SF was measured by immunochemiluminescence (COBAS -Roche Diagnostics-COBAS 6000 E601, measurement range: 0.5-2000 μg/L, intraand inter-series coefficients of variation <1.1% and <5.7%, respectively) and CRP was measured by immunoturbidimetric assay.

In case of fever or any other condition that could alter the ingesta or biological iron status (e.g., bronchiolitis, gastroenteritis), the dietary survey and blood sampling were postponed to 15 days after discontinuation of symptoms. Children for whom no blood sample was taken, whose parents did not complete the dietary survey, or with a CRP level ≥10 mg/L were excluded from the statistical analyses.

Statistical analyses

We first described the general characteristics of the studied and analyzed population and compared them to those of the 2016 French National Perinatal Survey, a national representative sample of births (n=13 148) [START_REF] Blondel | Trends in perinatal health in metropolitan France from 1995 to 2016: Results from the French National Perinatal Surveys[END_REF]. Second, we characterized iron intake specifically and its distribution considering all sources from the whole diet including YCF or excluding YCF. Third, we described the distribution of SF and used multivariable linear regression models (after logarithmic transformation of SF [START_REF] Bland | Transformations, means, and confidence intervals[END_REF]) to study the crude then adjusted association of SF with past dietary habits (including declared YCF consumption) and current ingesta (including declared and measured YCF consumption) and perinatal and sociodemographic characteristics. The specific sources of iron evaluated in the analyses are listed as dietary characteristics in Table 1. YCF consumption was defined in several ways: "any consumption after 10 months" and "current consumption at age 24 months" (two binary [no/yes] declarative variables), "daily volume consumed" in five categories (measured variable in the dietary record) to evaluate a "dose effect", "consumption duration" (declarative variable) used as continuous variable, then in three categories to evaluate a "time effect". We evaluated the duration of consumption of any IFF since age 6 months by a continuous variable corresponding to the sum of consumption duration for both follow-on formula and YCF. This variable represented the cumulative iron intake from dairy food at about age 6 months. The statistical handling of other variables is detailed in Supplemental Methods.

Finally, SF was dichotomized with a 12-µg/l threshold to define ID, as suggested [START_REF] Baker | Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age)[END_REF][START_REF] Domellof | Iron requirements of infants and toddlers[END_REF][START_REF] Zimmermann | Nutritional iron deficiency[END_REF].

The prevalence of ID and its crude associations with potential determinants were studied by univariate analysis. No multivariable analysis was used given the low number of ID cases.

No multi-level model was used in these analyses despite recruitment in different practices because the variance of a multilevel model with random intercept including only the center of recruitment was low, 7%. For all continuous variables, the lack of deviance to linearity was searched and a fractional polynomial of degree 2 at most was used if appropriate. Several sensitivity analyses were performed by repeating the univariate analyses with a 10-µg/l threshold of SF to define ID [START_REF] Baker | Diagnosis and prevention of iron deficiency and iron-deficiency anemia in infants and young children (0-3 years of age)[END_REF][START_REF] Domellof | Iron requirements of infants and toddlers[END_REF][START_REF] Dallman | Iron deficiency in infancy and childhood[END_REF], repeating regressions analyses without sociodemographic characteristics or studying specific associations of perinatal and sociodemographic variables with iron status. Missing data (≤2%) were not imputed. The number of participants required (n=1000) was calculated with a very conservative hypothesis (Supplemental Methods); the recruitment was stopped prematurely for financial reasons.

The analyses involved use of Stata/SE v13.1 (StataCorp, College Station, TX, USA).

Results

Participants

A total of 830 young children were included by 58 pediatricians from 16 French regions; 15 were excluded because of non-eligibility criteria, 212 (26%) because of no blood sample, 8 (1%) because of lack of dietary survey, 31 (5%) because of CRP level ≥10 mg/L, and 3 (0.5%) because of missing data for SF level (Supplemental Figures 1 and2), leading to an analyzed population of 561 infants. The analyzed population was less disadvantaged than the 2016 French National Perinatal Survey population but did not differ from the non-analyzed population (Supplemental Table 2).

In the analyzed population, the mean age of children was 24 months (SD 0.6), 49% were girls, 3.6% had cow's milk protein allergy, and 10% had received iron supplementation according to parents. YCF was currently used in 73% of the population (408/561, 95% confidence interval [CI] 69-76) and cow's milk in 33% (186/561, 95% CI 29-37). The mean and median total iron intake for the population was 8.2 mg/day (95% CI 7.8-8.6) and 7.6 mg/day (interquartile range [IQR] 5.6-9.8). The mean iron intake for the population from all food excluding YCF and from YCF only was 4.7 mg/day (95% CI 4.6-4.9) and 3.5 mg/day (95% CI 3.1-3.8). The median iron intake for the population from all food excluding YCF and from YCF only was 4.3 mg/day (IQR 3.4-5.7) and 3.1 mg/day (IQR 0-5.2). Daily iron intake for the population from all food excluding YCF and total daily iron intake including YCF was below the 5 mg/day AR for 63% (95% CI 59-67) and 18% (95% CI 15-22) of children (Figure 1).

Median SF was 26 µg/L (IQR: 20-37, mean: 31 [SD 21]) (Supplemental Figure 3).

After dichotomization of SF at thresholds <12 and <10 μg/L, ID prevalence was 6.6% (37/561, 95% CI 4.7-9.0) and 3.9% (22/561, 95% CI 2.5-5.9) (Table 1 andSupplemental Table 3).

Dietary factors associated with iron status

On univariate analysis, SF was significantly (p <0.05) lower with cow's milk use beginning before age 24 months (median 23 vs 28 μg/L if not) or its current use at 24 months (23 vs 28 μg/L if not) and with increased iron intake from all food excluding YCF (β coefficient = -0.03, 95% CI -0.05-[-0.01]) (Table 1). We found no significant association between SF and exclusive breastfeeding beyond 6 months or cow's milk consumption duration ( 1). For the 58 children with a high daily volume of YCF consumed (≥450 mL; i.e., the 90 th percentile), we found no significant increase in mean SF level (34 vs 31 µmol/L with daily YCF volume <450 mL, p=0.23) or median SF level [90 th percentile] (26 [START_REF] Chouraqui | The Role of Young Child Formula in Ensuring a Balanced Diet in Young Children (1-3 Years Old)[END_REF] vs 27 µmol/L [58] with daily YCF <450 mL).

In several multivariable linear models adjusting for co-variables of interest (Table 2) and after checking for no significant collinearity between YCF and cow's milk consumption, SF was positively associated with YCF consumption, whether described by a binary variable (any use after age 10 months), volume consumed or duration of consumption. SF was also associated with duration of IFFs consumption since age 6 months (Table 2). In all multivariable models, unfavorable iron status was associated with a medical history of prematurity, an elevated rank among siblings and disadvantaged social situation: low father's education level (Table 2, Supplemental Table 6) as in univariate analyses (Supplemental Tables 4 and5).

After dichotomizing SF at the 12-µg/L threshold, ID was significantly associated (p <0.05) with the same markers as SF used as a continuous variable (Table 1) except iron intake from all food excluding YCF. For example, ID was significantly decreased with YCF use since age 10 months (25% vs 5% with YCF use, OR 0.15, 95% CI 0.07-0.31).

Sensitivity analyses at the 10-μg/L threshold found a statistically significant association between ID (p <0.05) and the same variables as with the 12-µg/L threshold, except for infant formula use, which was significant at the 10-µg/L threshold only (Supplemental Table 3). Similar results were found in the sensitivity analyses for the multivariate models without adjustment for sociodemographic variables comparatively to those adjusted for sociodemographic variables (Supplemental Table 6).

Discussion

Main results

With a simultaneous collection of medical, biological and dietary data at the national scale, a design rarely achieved in the literature [START_REF] Sidnell | Nutrient intakes and iron and vitamin D status differ depending on main milk consumed by UK children aged 12-18 months -secondary analysis from the Diet and Nutrition Survey of Infants and Young Children[END_REF][START_REF] Uijterschout | Prevalence and risk factors of iron deficiency in healthy young children in the southwestern Netherlands[END_REF], we found a protective role of YCF use on ID in children at age two years in the general population. This protective role was strong with, for example, a reduction of one-fifth of ID with YCF use since age 10 months (OR 0.15, 95% CI 0.07-0.31). This protective role was consistent according to the different definitions of YCF consumption (amount, duration), measured either retrospectively by asking parents about the past consumption or prospectively by quantifying current consumption with a dietary survey. This protective role is consistent with results from clinical trials [START_REF] Fuchs | Iron status and intake of older infants fed formula vs cow milk with cereal[END_REF][START_REF] Moffatt | Prevention of iron deficiency and psychomotor decline in high-risk infants through use of iron-fortified infant formula: a randomized clinical trial[END_REF][START_REF] Daly | Prevention of anaemia in inner city toddlers by an iron supplemented cows' milk formula[END_REF][START_REF] Gill | Follow-on formula in the prevention of iron deficiency: a multicentre study[END_REF][START_REF] Morley | Iron fortified follow on formula from 9 to 18 months improves iron status but not development or growth: a randomised trial[END_REF][START_REF] Lozano | Intake of an iron-supplemented milk formula as a preventive measure to avoid low iron status in 1-3 year-olds][END_REF][START_REF] Akkermans | A micronutrient-fortified young-child formula improves the iron and vitamin D status of healthy young European children: a randomized, double-blind controlled trial[END_REF] and previous observational studies [START_REF] Sidnell | Nutrient intakes and iron and vitamin D status differ depending on main milk consumed by UK children aged 12-18 months -secondary analysis from the Diet and Nutrition Survey of Infants and Young Children[END_REF][START_REF] Uijterschout | Prevalence and risk factors of iron deficiency in healthy young children in the southwestern Netherlands[END_REF][START_REF] Mccarthy | Iron intakes and status of 2-year-old children in the Cork BASELINE Birth Cohort Study[END_REF][START_REF] Vincelet | Measuring iron levels relative to the type of milk consumed within a population of 16 to 18 month old French infants[END_REF]] and was independent of potential confounders such as disadvantaged social situation, non-dairy iron intake and iron supplementation, which were not always taken into account in previous studies [START_REF] Sidnell | Nutrient intakes and iron and vitamin D status differ depending on main milk consumed by UK children aged 12-18 months -secondary analysis from the Diet and Nutrition Survey of Infants and Young Children[END_REF][START_REF] Uijterschout | Prevalence and risk factors of iron deficiency in healthy young children in the southwestern Netherlands[END_REF]. This protective role is easy to conceive theoretically because iron given to young children in any form is supposed to increase their iron stores in the absence of concurrent loss or illness, given the almost closedcircuit metabolism of iron [START_REF] Domellof | Iron requirements of infants and toddlers[END_REF][START_REF] Zimmermann | Nutritional iron deficiency[END_REF][START_REF] De Montalembert | Diagnosis of hypochromic microcytic anemia in children][END_REF][START_REF] Loreal | Iron metabolism and tools for the iron status assessment[END_REF].

Our study provides information on how YCF-based strategies could help improve iron status in the general population. First, the mean total iron intake was 8.2 mg/day, higher than the 5 mg/day AR recommended in Europe by the EFSA, in 82% of children [START_REF] Efsa | Dietary reference values for nutrients: Summary report[END_REF]. The mean intake from all sources excluding YCF was 4.7 mg/day and from YCF only was 3.5 mg/day for the population analyzed. Added to the EFSA recommended strategy based on naturally iron-rich food, YCF use allowed for a reduction from 63% to 18% of children with below the 5-mg/day AR. Thus, YCF use added to the rest of the diet allowed 45% of children to reach the daily AR. Furthermore, the YCF impact on iron status was detectable even for a small daily volume consumed, but the increase >500 mL/day was not associated with a significant increase in SF. Results from several studies support that a small volume of YCF (100-300 mL/day) allows for reaching the recommended AR [START_REF] Walton | Nutritional adequacy of diets containing growing up milks or unfortified cow's milk in Irish children (aged 12-24 months)[END_REF][START_REF] Ghisolfi | Nutrient intakes of children aged 1-2 years as a function of milk consumption, cows' milk or growing-up milk[END_REF][START_REF] Eussen | Theoretical Impact of Replacing Whole Cow's Milk by Young-Child Formula on Nutrient Intakes of UK Young Children: Results of a Simulation Study[END_REF]. Iron bioavailability mechanisms are complex, and the amount of iron ingested is not equal to the amount of iron absorbed. This absorption depends notably on the origin of iron (haem or not), the type of other concomitant ingested food and the period of life, notably acceleration of growth [2-4, 31, 39]. Thus, even a small amount of iron with IFFs could improve iron status [START_REF] Fomon | Erythrocyte incorporation of iron is similar in infants fed formulas fortified with 12 mg/L or 8 mg/L of iron[END_REF][START_REF] Walter | Prevention of iron-deficiency anemia: comparison of high-and low-iron formulas in term healthy infants after six months of life[END_REF].

Second, another finding helps in understanding how YCF use could help improve iron status: the association observed between increased SF and consumption duration of IFFs since age 6 months (i.e., follow-on formula, then YCF). This "time effect" implying the duration of exposure to IFFs suggests a role of long-term, "cumulative", consumption of IFFs in iron status at age 24 months. This finding, as the previous one about a non-linear effect of YCF volume consumed on iron status, can probably be explained in part by the role of hepcidin in regulating iron absorption and the prevention of iron overload if iron reserves are already high. Indeed, this peptide synthetized by hepatocytes participates in regulating iron absorption by enterocytes: when iron reserves are high, hepcidin synthesis increases and induces the degradation of ferroportin on enterocytes and macrophage membranes, thus limiting iron release in blood circulation [START_REF] Loreal | Iron metabolism and tools for the iron status assessment[END_REF][START_REF] De Montalembert | Exploration d'une anemie microcytaire chez l'enfant[END_REF][START_REF] Ganz | Hepcidin and iron regulation, 10 years later[END_REF], whereas when iron reserves are low, hepcidin synthesis is downregulated, thereby increasing iron bioavailability. The potential risk of iron excess with fortification use can be raised. Use of IFFs has been found associated with iron intake above the tolerable upper intake level but not excess iron stores based on SF distribution in infants [START_REF] Eussen | Theoretical Impact of Replacing Whole Cow's Milk by Young-Child Formula on Nutrient Intakes of UK Young Children: Results of a Simulation Study[END_REF][START_REF] Van Der Merwe | Iron status of young children in Europe[END_REF]. In our analyzed population, we found no significant difference in SF distribution with high daily volume consumed. This study suggests that small amounts of iron ingested regularly over time could be more beneficial to prevent ID than high amounts over a short period without leading to iron excess stores.

In this study, we found other determinants of iron status at 24 months usually identified in the literature: medical history of prematurity and factors linked to deprived situation (low father's education level) or family situation (high rank in siblings). The independent association found between iron status and a history of prematurity, remaining even after taking into account iron supplementation, suggests that this sub-population at high risk of ID should benefit from more specific prevention and monitoring of iron status, as previously found [START_REF] Mccarthy | Iron supplementation in preterm and lowbirth-weight infants: a systematic review of intervention studies[END_REF][START_REF] Akkermans | Predictive factors of iron depletion in late preterm infants at the postnatal age of 6 weeks[END_REF]. "Exclusive" breastfeeding beyond 6 months is a classical risk factor for ID [START_REF] Atkins | Iron intakes of Australian infants and toddlers: findings from the Melbourne Infant Feeding, Activity and Nutrition Trial (InFANT) Program[END_REF], but it was not independently associated with iron status in our study, probably because assessment of iron status occurred late, at 24 months, and many other nutritional factors may have intervened between 7 and 24 months.

Strengths and limitations

Our study has several strengths. The measurements of iron biomarkers were standardized and centralized as recommended by the World Health Organization [START_REF]Iron deficiency anaemia. Assessment, prevention and control. A guide for programme managers[END_REF], as was the evaluation of nutritional intake with a dietary survey performed on three non-consecutive days to improve the accuracy of intra-individual variability, as recommended by the EFSA [START_REF] Efsa | General principles for the collection of national food consumption data in the view of a pan-European dietary survey[END_REF]. Biological and dietary data were measured independently, with blinding. A specific strength of this study is the consideration of all sources of iron besides YCF in multivariate analyses in contrast to some other national studies [START_REF] Sidnell | Nutrient intakes and iron and vitamin D status differ depending on main milk consumed by UK children aged 12-18 months -secondary analysis from the Diet and Nutrition Survey of Infants and Young Children[END_REF][START_REF] Uijterschout | Prevalence and risk factors of iron deficiency in healthy young children in the southwestern Netherlands[END_REF]. There were several limitations. First, the analyzed population presented a selection bias as shown by less frequent disadvantaged situations than in all births. This bias can be explained mainly by recruitment by pediatricians and the significant attrition due notably to failure to obtain a blood sample (32% of the studied population). Indeed, only 20% of French children at age 24 months are followed by a pediatrician [START_REF] Bocquet | Comparison of prescriptions by pediatricians and general practitioners: a population-based study in Franche-Comte from the database of Regional Health Insurance Fund[END_REF] and those who accepted to participate in this complex study were probably familiar with clinical research. The study possibly selected families interested in the topic of nutrition with good knowledge and practices regarding ID prevention. This selection bias probably led to underestimating the ID prevalence, which was regardless close to the most recent national estimations [START_REF] Sacri | Very low prevalence of iron deficiency among young French children: A national cross-sectional hospital-based survey[END_REF].

Furthermore, this potential bias could not have altered the associations between iron status and YCF consumption in the analyzed children. Second, the recruitment was stopped before the calculated sample size was achieved, and the attrition may have led to loss of statistical power. However, the sample size was calculated on the basis of a very conservative hypotheses on expected associations that were not confirmed by the study (associations were much stronger than expected). To manage the lack of power, the main analyses were finally based on ID defined by SF level as a continuous variable. Finally, as in other studies, some inaccuracies may have occurred during the declarative food intake data collected by the pediatrician and regarding the quantities assessed by the parents during the survey. However, they may have been limited by the checks of a well-trained dietician as detailed in Supplemental Methods. Besides, these inaccuracies occurred with blinding to iron status, so they may have led to only a non-differential bias and a loss of statistical power.

Implications

In this first national-level study taking into account non-dairy iron intake and iron supplementation and the family's socio-economic status, YCF consumption allowed for reaching the required iron AR in 45% of children who were not reaching it otherwise by dietary intake. YCF use was strongly and independently associated with better iron status than non-use. The biological benefit of YCF was obtained even for a limited daily amount, about 200 mL/day, especially when implemented over a long period. These findings support national strategies for ID prevention based on the recommendation for YCF consumption after age 12 months [START_REF]Growing-up' formula: No additional value to a balanced diet, says EFSA. Parma[END_REF][START_REF] Efsa Panel On Dietetic | Scientific Opinion on nutrient requirements and dietary intakes of infants and young children in the European Union[END_REF]. YCF consumption seems a simple way to supply young children with the recommended iron intake and reach the recommended AR added to the rest of the diet in practice, according to the family's and country's culture. Indeed, a well-balanced diet by solid food is possibly difficult to achieve at this age [START_REF] Chouraqui | Food, water, energy, and macronutrient intake of non-breastfed infants and young children (0-3 years)[END_REF][START_REF] Grimes | Food Sources of Total Energy and Nutrients among U.S. Infants and Toddlers: National Health and Nutrition Examination Survey 2005-2012[END_REF]. These findings should trigger measures to enhance YCF accessibility to at-risk families (i.e., those in disadvantaged situations). Deciles and quartiles were calculated separately by iron sources. At a consequence, for each decile or quantile, mean daily iron intake from all food is not equal to the addition of mean daily iron intake from all food excluding YCF + YCF only.

In children who did not reached the required iron average requirements recommended from all food without YCF, 45% reached this threshold if iron intake from YCF was considered.

Figure 1 .

 1 Figure 1. Distribution of mean daily iron intake (mg/day) according to deciles and quartiles of iron intake stratified by iron sources (n=561): from all food (red line), from all food excluding young-child formula (YCF, blue line), and YCF only (green line).

569f

  Abbreviations: IFF = iron fortified formula, YCF = young-child formula

Table 1 )

 1 . SF was significantly higher with follow-on formula use between age 6 and 12 months (27 vs 23 μg/l if not), YCF use since age 10 months (27 vs 17 μg/L if not) or current use at 24 months (29 vs 21 μg/L if not), daily volume of YCF consumed (29 µg/L if <100 mL/day vs 21 µg/L if none), increasing YCF consumption duration (28 if > 12 months vs 17 μg/L with no YCF), and increasing IFFs consumption duration (β coefficient = 0.05, 95% CI : 0.03-0.07; i.e., 28 if > 12 months vs 17 µg/L if no IFF, this last result is not shown in

Table 1 ) ( Table

 1(

Table 1 .

 1 Dietary characteristics of children and univariate associations with serum ferritin 558 level and iron deficiency (ID) at a threshold of 12 µg/L (by milk according to the age of

	Horizontal orange line: threshold of iron intake of 5 mg/day corresponding to the average
	requirements recommended by the European Food Security and Authority (EFSA) at age 24
	months [6]

: consumption (since age 6 months) duration (months) f

	15.3 (5.3)	0.05	0.03-0.07	<0.001	8.9 (7.3)	15.8 (4.8)	0.85	0.81-0.89
	Cow

's milk: consumption beginning before 24 months

	No	356 (64)	28	21-40	-	3	-	-
	Yes	201 (36)	23	15-34	<0.001	13	5.98	2.75-13.00
	Cow

's milk: current consumption at 24 months

	No	375 (67)	28	21-39	-	3		-	-
	Yes	186 (33)	23	16-35	<0.001	14		5.38	2.59-11.15
	Cow's milk: consumption duration (months) f
		7.4 (4.4)	-0.01	-0.04-0.01	0.15	9.4 (4.7)	7.1 (4.3)	1.13	1.02-1.25
	Dietary

iron intake from all food excluding YCF f (mg/day)

  or β coefficient of a simple linear regression after logarithmic transformation (instead of medians) and 95% confidence interval (CI, 564 instead of interquartile range [IQR]) if the « characteristics » variable is continuous 565 c Degree of significance of the Student test, Kruskal-Wallis test or of the β coefficient of a simple linear regression (univariable analyses 566 after a logarithmic transformation of the dependent variable: serum ferritin level)567d For cells with a zero value, 1 was added to all values of the contingency table as suggested[START_REF]Cochrane Handbook for Systematic Reviews of Interventions[END_REF] 568

		4.7 (2.2)	-0.03	-0.05-(-0.01)	0.01	5.2 (2.8)	4.7 (2.2)	1.10	0.96-1.25
	Iron supplementation since birth g
	No	503 (90)	26	20-36	-	7		-	-
	Yes	57 (10)	29	22-41	0.17	4		0.49	0.11-2.08
	562 563	a or mean (SD) if the "characteristics" variable is continuous

b e 95% CI of odds ratio (OR) in multivariable analyses

Abbreviations: AR: average requirements; EFSA: European Food and Security Authority; ID: iron deficiency; IFF: iron-fortified formula; SF: serum ferritin; YCF: young children formula.

Acknowledgments

The authors thank URC-CIC Paris Descartes Necker Cochin (project manager: Nelly Briand and clinical research associates: Camille Boucheny, Charlène Ambvouli and Emilie Le Roux) for the implementation and monitoring of the study. The authors thank ACTIV network clinical research associates: Claire Prieur, Deborah Kern. They thank the participant families and the pediatricians who recruited them: Jean-Marie André, Pierre Bakhache, Sophie Barrois, Bénédicte Blanc, Marie-Emmanuelle Bouloumié, Fabienne Cahn-Sellem, Marie-Hélène Cavert, Anne Cheve, Christian Copin,

Muriel Cornic, François Corrard, Armelle Courboin, Véronique Dagrenat, Caroline De Backer, Pascal Dechelette, Jean-François Delobbe, Michèle Dudreuilh, Corinne Dumas, Christine Duvant-Fay, Annie Elbez, Chantal Gayot, Nathalie Gelbert, Patricia Giraud-Escoffier, Geneviève Granat, Cécile Guiheneuf, Frédéric Hassid, Kai Kassmann, Sylvie Labarge, Sophie Langlais, Hélène Le Scornet, Dominique Lemarié, Claudia Litzler, Christine Magendie, Delphine Menet, Bénédicte Nold, Jean-Claude Oilleau, France Paratte, Brigitte Pinçant, Sophie Ravilly, Olivier Romain, Bechara Saade, Catherine Salinier, Claire Sarreau, Ruth-Caroline Schweitzer, Anne Sengier-Njinou, Philippe Simon, David Somerville, Joachim Stoller, Marie-Pierre Streicher, Catherine Tuberg-Romain, Jean-Christophe Vigreux, Pascale Vurpillat-Almanza, Andreas Werner, Alain Wollner, Morched Zouari. Sources of funding: This work was supported by Paris Diderot University -Sorbonne Paris Cité for a doctoral grant for Dr. AS Sacri, Secteur Français des Aliments de l'Enfance, and the French Ministry of Health ["DGOS PHRC régional 2014 no. AOR14053"]. The sponsor was Assistance Publique -Hôpitaux de Paris (Délégation à la Recherche Clinique et à l'Innovation [DRCI]).

Conflict of interest statement:

The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors have no patents, products in development or marketed products to declare. for dietary iron intake excluding YCF are presented by categories for ease of reading i no change in the results taking into account time interval between iron supplementation and serum ferritin level measure Perinatal and sociodemographic variables in these models are not shown: in all models a lower SF was independently associated with a medical history of prematurity, a high rank in siblings, and a low father's education level Abbreviations: CI = confidence interval, IFF = iron fortified formula


D Saadoun
email: david.saadoun@aphp.fr

A Ghembaza

S Riviere

A Mekinian

J Boutemy

G Leroux

F Domont

H Maillard

M Vautier

P Cacoub

David Pr

MD Saadoun

Rituximab plus Belimumab in non-infectious refractory cryoglobulinemia vasculitis: a pilot study. Running head: Belimumab in cryoglobulinemia vasculitis

Keywords: cryoglobulinemia, vasculitis, biologic, rituximab, belimumab

Objective: To report the efficacy of rituximab plus belimumab in patients with refractory cryoglobulinemia vasculitis (CV).

Methods: Belimumab was administered intravenously at a dose of 10 mg/kg on days 0, 14, 28 and then every month in association with rituximab in 4 patients with refractory CV.

Demographic, clinical and laboratory characteristics, treatment modalities and outcomes were recorded.

Results: All patients had type II IgM Kappa cryoglobulinemia, which was associated with primary Sjögren syndrome (n=1), hepatitis C virus infection (n=1), and essential (n=2). Main manifestations of CV included purpura (n=4), arthralgia and peripheral neuropathy (n=3), and glomerulonephritis and skin ulcers (n=1). In all cases, CV was refractory and/or relapse following rituximab. Intravenous belimumab infusion along with rituximab resulted in rapid clinical response in the four patients. Osteitis and recurrent urinary tract infections occurred in two patients.

Conclusion:

Belimumab along with rituximab showed promising results in refractory patients with CV.

Introduction

Mixed cryoglobulinaemic vasculitis (CV) can cause manifestations ranging from purpura, arthralgia and constitutional symptoms to more serious lesions with skin ulcers, neurological and renal involvement. Immunosuppression has been traditionally considered as the first line intervention in severe non-infectious CV. Rituximab is the most widely used biologic agent for the management of CV. It has a safer profile than conventional immunosuppressants [START_REF] Sneller | A randomized controlled trial of rituximab following failure of antiviral therapy for hepatitis C virus-associated cryoglobulinemic vasculitis[END_REF],

and its use has substantially changed the natural history of CV by inducing long term remission [START_REF] Quartuccio | Retreatment regimen of rituximab monotherapy given at the relapse of severe HCVrelated cryoglobulinemic vasculitis: Long-term follow up data of a randomized controlled multicentre study[END_REF]. However, CV remains a challenging disease to manage owing to frequent relapses and treatment failure [START_REF] Cacoub | Anti-CD20 monoclonal antibody (rituximab) treatment for cryoglobulinemic vasculitis: where do we stand?[END_REF]. Studies are needed to identify rescue treatments for refractory CV patients. B-Lymphocyte stimulator (BLYS), also known as B cell activating factor (BAFF), is overexpressed in patients with CV [START_REF] Fabris | B-Lymphocyte stimulator (BLyS) up-regulation in mixed cryoglobulinaemia syndrome and hepatitis-C virus infection[END_REF]. Thus, a combination of rituximab and belimumab may result in improvements in CV. Here we report our experience of rituximab plus belimumab in patients with refractory CV.

2.Methods

We reviewed the chart of four consecutive patients with CV seen in Paris (Pitié Salpêtrière hospital and Saint Antoine hospital), Caen and Lille, in France between 2017 and 2020. CV was defined as serum cryoglobulinemia associated with the triad of purpura-arthralgiaasthenia sometimes associated with renal and/or neurologic involvement. Refractory cryoglobulinaemic vasculitis was defined as a clinical picture that does not improve within 4-6 weeks after the initial treatment, or that provides a < 50 % improvement after 12 weeks, or that shows chronic persistent manifestations after > 12 weeks of treatment. Belimumab was administered as intravenous infusions at a dose of 10 mg/kg on days 0, 14, 28 and then every month in association with rituximab. Demographic, clinical, laboratory characteristics, treatment modalities and outcomes were recorded.

The study was conducted in compliance with the Declaration of Helsinki, no formal consent from participants was required according to local ethics committees. Belimumab was prescribed off-label after validation in a multidisciplinary meeting. All patients were orally informed before receiving belimumab.

3.Results

Table 1 and 2 summarizes main clinical and laboratory data and outcome of the four CV patients.

Case 1: A 71-year-old woman with a prior medical history of monoclonal gammopathy was diagnosed with CV in 2011 based on palpable purpura on lower extremities, polyneuropathy, type II monoclonal IgM Kappa serum cryoglobulinemia at 0.59 g/l and low complement C4 level at 0.08 g/l. The patient was successfully treated with four consecutive rituximab weekly infusions of 375 mg/m2 and achieved complete resolution of her symptoms. In August 2018, purpura relapsed and polyneuropathy worsened. She was then treated with another infusion of rituximab 500 mg followed by intravenous administration of belimumab at 10 mg/kg on days 0, 14, 28 and then every month thereafter. Eight weeks after treatment, the patient had improvement of her neurologic symptoms and complete resolution of the cutaneous purpura.

Laboratory test results showed normal complement C4 level (0.27 g/l), and negative cryoglobulinemia. This positive response was maintained during all treatment period. No side effect was reported.

Case 2: A 75-year-old woman with a medical history of primary Sjögren syndrome presented recurrent palpable purpura, arthralgia, myalgia, and polyneuropathy revealing CV. Laboratory investigations showed type II monoclonal IgM Kappa serum cryoglobulinemia, and low complement C4 level at 0.02 g/l. Underlying infections and lymphoproliferative disorders were excluded. Between 2008 and 2017, she received multiple therapeutic regimens, including corticosteroids (1 mg/kg/day), cyclophosphamide (six cycles), azathioprine and rituximab (two bi-weekly infusions of 1000 mg intravenously in December 2014 and in September 2018). In 2018, CV relapsed leading to reintroduction of four weekly infusions of 375 mg/m2 rituximab. Belimumab (at 10 mg/kg at days 0, 14, 28 and then every 28 days intravenously) was administered along with corticosteroids (20 mg/day) because of purpura and arthralgia recurrence. After ten infusions, belimumab yielded a good clinical response with complete resolution of arthralgia. However, some transient episodes of purpura were noted and the patient developed recurrent urinary tract infections.

Case 3: A 74-year-old woman was admitted to the emergency department because of palpable purpura with necrotic lesions on lower limbs, arthralgia and acute renal failure with nephritic range proteinuria (1.6 g/day) and microscopic hematuria. A renal biopsy was performed and showed membranoproliferative glomerulonephritis. The patient was diagnosed with CV. Type II monoclonal IgM Kappa cryoglobulinemia was positive and with low C4 complement level.

Infectious (hepatitis B, hepatitis C and HIV), autoimmune and lymphoproliferative causes were excluded. The patient underwent seven plasma exchange sessions combined with rituximab and corticosteroids. Maintenance Rituximab infusions at 500mg every 6 months were given. Her kidney function recovered as serum creatinine returned to baseline level. In January 2017, due to purpura and skin ulcerations relapse, intravenous Belimumab 10 mg/kg on days 0, 14, 28 and then every month was added along with rituximab 500mg leading to the complete resolution of the cutaneous purpura and arthralgia. Nevertheless, the patient developed osteitis of the left hallux sesamoid bones and urinary tract infection, which were successfully treated with antibiotics. Three years after belimumab initiation, the patient remained symptom-free, with negative cryoglobulinemia, normal serum creatinine and C4 level.

Case 4: A 65-year-old woman with a medical history of hepatitis C virus (HCV) infection successfully treated with sofosbuvir and daclatasvir was admitted because of purpura, arthralgia and polyneuropathy, which occurred despite a sustained virologic response for HCV. Electrophysiological studies demonstrated lower limbs sensorimotor axonal polyneuropathy. IgM-K type II cryoglobulinemia was positive at 5.2 g/l. Autoimmune and lymphoproliferative causes were excluded. CV was diagnosed and the patient received four weekly infusions of rituximab (375 mg/m²) and two cycles of rituximab 500mg as maintenance therapy every six months. However, purpura relapsed and belimumab was added at 10 mg/kg on days 0, 14, 28 and then every month along with rituximab 500mg. By the eighteenth month of treatment, the patient had sustained clinical remission, with resolution of purpura and arthralgia, clinical stabilization of the neuropathy, and decreased level of cryoglobulins. Belimumab was well tolerated without evidence of infectious complications.

4.Discussion

CV is a major cause of morbidity and mortality, especially in elderly patients with severe renal impairment [START_REF] Sansonno | Monoclonal antibody treatment of mixed cryoglobulinemia resistant to interferon alpha with an anti-CD20[END_REF]. Prompt treatment is mandatory to relieve patient's symptoms and avoid long-term complications. Treatments strategies are mainly based on the underlying cause and disease severity. To date, the available therapeutic options included antiviral therapy (in case of hepatitis C), rituximab, immunosuppressants, and plasmapheresis [START_REF] Muchtar | How I treat cryoglobulinemia[END_REF]. Despite these strategies, CV relapse is noted in up to 40% of patients [START_REF] Cacoub | Anti-CD20 monoclonal antibody (rituximab) treatment for cryoglobulinemic vasculitis: where do we stand?[END_REF]. Moreover, side effects, mainly infections and drug toxicity, are a serious concern. BAFF, a member of tumor necrosis factor superfamily, is expressed by monocytes, dendritic cells, T cells, and neutrophils. It promotes B lymphocytes differentiation, proliferation and survival and thus increases immunoglobulin secretion [START_REF] Nardelli | Synthesis and release of Blymphocyte stimulator from myeloid cells[END_REF]. BAFF up-regulation may favor both autoimmunity and B-cell lymphoproliferation. This finding has been strengthened by BAFF overexpression found in many autoimmune diseases such as systemic lupus erythematosus (SLE), Sjögren syndrome, and its related B-cell lymphoma [START_REF] Cheema | Elevated serum B lymphocyte stimulator levels in patients with systemic immune-based rheumatic diseases[END_REF]. In patients with hepatitis C, BAFF was significantly overexpressed in patients with mixed cryoglobulinemia than those without which lead to presume that suppression of BAFF may be a promising therapeutic option for patients with CV [START_REF] Sène | Hepatitis C virus-associated B-cell proliferation--the role of serum B lymphocyte stimulator (BLyS/BAFF)[END_REF][START_REF] Landau | The B lymphocyte stimulator receptor-ligand system in hepatitis C virus-induced B cell clonal disorders[END_REF]. In transgenic mouse strain that expresses the human CD20 antigen on B cells, while splenic B cells subset were resistant to anti-CD20 monotherapy, targeting both CD20 and BAFF resulted in a marked decrease in B cell number [START_REF] Gong | Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy[END_REF].

Belimumab is a human anti-BAFF monoclonal IgG antibody that inhibits BAFF binding to its receptor. In a case report, monotherapy with rituximab first then followed by belimumab infusions were ineffective in a patient with Sjögren syndrome complicated with low-grade parotid B-cell lymphoma and CV. However, healing of a refractory skin ulcer, normalization of C4 and disappearance of cryoglobulin were observed when rituximab was administered shortly (six weeks) after belimumab infusions [START_REF] Vita | Sequential therapy with belimumab followed by rituximab in Sjögren's syndrome associated with B-cell lymphoproliferation and overexpression of BAFF: evidence for long-term efficacy[END_REF]. An open label study, assessing the efficacy and the safety of belimumab in 30 patients with primary Sjögren syndrome, showed that in three patients with positive cryoglobulinemia at baseline, circulating cryoglobulins disappeared at the end of follow-up [START_REF] Mariette | Efficacy and safety of belimumab in primary Sjögren's syndrome: results of the BELISS open-label phase II study[END_REF].

The use of the association of rituximab plus belimumab in patients with CV is based on data from SLE patients with the hypothesis that decreasing the number of naïve B-cells after complete depletion could be an option in controlling disease activity.

In fact, BAFF levels have been consistently described as increased after B-cell depletion via anti-CD20 [START_REF] Carter | Elevated serum BAFF levels are associated with rising anti-double-stranded DNA antibody levels and disease flare following B cell depletion therapy in systemic lupus erythematosus[END_REF]. Furthermore, patients who relapsed after rituximab administration were more likely to have greater BAFF levels [START_REF] Carter | Elevated serum BAFF levels are associated with rising anti-double-stranded DNA antibody levels and disease flare following B cell depletion therapy in systemic lupus erythematosus[END_REF]. Thus, dual therapy with rituximab plus belimumab provides an opportunity to target the surge in circulating BAFF levels after B-cell depletion, thereby minimizing the survival of autoreactive B cells.

In a Phase 2, single-arm, open-label proof-of-concept, study including 15 patients with severe, refractory SLE, dual therapy with rituximab plus belimumab showed encouraging results. This effect was more prominent in patients with prolonged suppression of autoreactive B cells [START_REF] Kraaij | Long-term effects of combined B-cell immunomodulation with rituximab and belimumab in severe, refractory systemic lupus erythematosus: 2-year results[END_REF]. These findings support the potential benefit of dual B-cell immunomodulation, also known as "Synergetic B-cell immunomodulation" over individual anti-CD20 depletion or BAFF blockade and pave the way for the use of this dual therapy in patients with CV, especially those with refractory forms. Recently, Chevalier et al. reported a good response to rituximab followed by belimumab administered as a 200 mg subcutaneous injection every week, in three patients with primary Sjögren syndrome complicated by CV [START_REF] Chevalier | Efficacity of a sequential treatment by anti-CD 20 monoclonal antibody and belimumab in type II cryoglobulinaemia associated with primary Sjögren syndrome refractory to rituximab alone[END_REF].

In our case series, belimumab along with rituximab improved markedly the patient's symptoms after failure of several immunosuppressants including rituximab. This treatment combination led to complete resolution of cutaneous and/or articular manifestations in three out of four patients, polyneuropathy stabilized in two patients and improved in another.

However, two patients who previously received multiple lines of immunosuppressive therapy, and presented hypogammaglobulinemia, experienced recurrent infections. Patients two and three experienced recurrent urinary tract infections that were well controlled with adapted antibiotics. Patient three developed severe hypogammaglobulinemia with soft tissue infections that required hospitalization with intravenous antibiotic administration along with immunoglobulin replacement therapy. In a previous report of 15 SLE patients treated with dual B-cell immunomodulation, serious and minor infections occurred in three and eight patients, respectively [START_REF] Kraaij | Long-term effects of combined B-cell immunomodulation with rituximab and belimumab in severe, refractory systemic lupus erythematosus: 2-year results[END_REF]. More recently, a phase II clinical trial, including 43 SLE patients who were randomized to receive prednisone, cyclophosphamide and rituximab with or without additional belimumab, showed no significant differences between the two groups with respect to serious infections after a 48 week follow-up period [START_REF] Atisha-Fregoso | CALIBRATE: A Phase 2 Randomized Trial of Rituximab Plus Cyclophosphamide Followed by Belimumab for the Treatment of Lupus Nephritis[END_REF].

Well-conducted randomized controlled studies should be conducted to elucidate the effect of that combination therapy in patients with CV.

In refractory cryoglobulinemic vasculitis, belimumab could represent a promising option in combination with rituximab. This treatment showed a good efficacy in our experience.

However, patients should be closely monitored to avoid serious infections because of profound B cell depletion, especially in patients who have received multiple lines of immunosuppressive therapy.

Declaration of interest

Nothing to declare

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