Kateryna Fal
email: kateryna.fal@cea.fr

Niklas Korsbo

Juan Alonso-Serra

Jose Teles

Mengying Liu

Yassin Refahi

M-E Chabouté

Henrik Jönsson
email: henrik.jonsson@slcu.cam.ac.uk

Olivier Hamant
email: olivier.hamant@ens-lyon.fr

Marie-Edith Chabouté

Tissue folding at the organ-meristem boundary results in nuclear compression and chromatin compaction

Keywords: Biological sciences, Plant biology, biophysics and computational biology nucleus, mechanical forces, meristem, organogenesis, chromatin, Arabidopsis 2

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

Introduction

Forces act as instructive mechanical signals that affect all aspects of cell and developmental biology (1) [START_REF] Lecuit | Force generation, transmission, and integration during cell and tissue morphogenesis[END_REF]. Although much attention has been paid to the role of forces at the cell cortex, forces can also propagate to the nucleus, and affect chromatin and gene expression (3)(4) [START_REF] Le | Mechanical regulation of transcription controls Polycomb-mediated gene silencing during lineage commitment[END_REF] [START_REF] Tajik | Transcription upregulation via force-induced direct stretching of chromatin[END_REF].

In turn, chromatin changes can also affect nucleus stiffness and shape [START_REF] Pagliara | Auxetic nuclei in embryonic stem cells exiting pluripotency[END_REF]. However, the impact of forces on nuclei has mainly been studied in single cell systems using artificial micromechanical manipulations so far. Patterns of forces are naturally present in multicellular tissues. In particular, mechanical conflicts result from differential growth and tissue morphogenesis (e.g. [START_REF] Aigouy | Cell flow reorients the axis of planar polarity in the wing epithelium of Drosophila[END_REF] [START_REF] Hervieux | A Mechanical Feedback Restricts Sepal Growth and Shape in Arabidopsis[END_REF]). In plants, because cells are encaged in a wall, cell movement is hindered; when tissues grow and change their shape, cells have to cope with the resulting mechanical stress, without resolving to cell intercalation (nor cell death in young tissues). In Arabidopsis shoot apical meristems, tissue folding occurs at the boundary between organs and meristem. Finite element model simulations suggest that this domain experiences highly anisotropic tension in the circumferential direction or radial compression [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF]. Radial contraction of the outer cell wall has been reported in that domain in previous studies and would be consistent with compression at the boundary [START_REF] Burian | A correlative microscopy approach relates microtubule behaviour, local organ geometry, and cell growth at the Arabidopsis shoot apical meristem[END_REF]. Here, we characterized the nuclear compression at the organ boundary correlating with changes of chromatin organization and linker histone H1.3 activity.

Results

Nuclei become elongated as the organ-meristem boundary folds

Using pPDF1::CFP-N7 marker line, which marks nuclei (N7 nuclear localization signal) in the epidermis (PDF1 promoter), we observed that some nuclei at the boundary often display an elongated shape (Fig. 1A). These elongated nuclei appeared late in boundary formation, i.e. when tissue folding is pronounced (i.e. beyond flower stage 2, [START_REF] Smyth | Early flower development in Arabidopsis[END_REF]) and when major supracellular alignment of cortical microtubules is observed (Fig. 1A). When staining with FM4-64, we observed that these nuclei might be physically constrained by cell geometry (as the nuclear-cytoplasmic ratio is high in meristematic cells, Fig. 1A), consistent with predicted compression in that domain [START_REF] Kwiatkowska | Growth and morphogenesis at the vegetative shoot apex of Anagallis arvensis L[END_REF] [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF].

Because cell division follows the direction of maximal tension, cells at the boundary divide along one of their long axes [START_REF] Louveaux | Cell division plane orientation based on tensile stress in Arabidopsis thaliana[END_REF]. Therefore, cells appear more elongated in that domain. In principle, this may be sufficient to constrain nucleus shape. However, in that scenario, the presence of elongated nuclei would not be the result of progressive compression emerging from neighboring tissue growth. To check whether organ growth can indeed actively compress nuclei at the boundary, we used the p35S::SUN1-YFP line (with SUN1 for SAD1/UNC-84 DOMAIN PROTEIN 1) that marks the nuclear envelope (15) and followed nuclear behavior in non-dividing cells every 12 hours. We could identify a population of non-dividing cells that exhibit progressive nucleus deformation (Fig. 1B,C). The magnitude of nucleus deformation increased with tissue curvature, consistent with organ growth being the main cause for nuclear compression in these cells (Fig. 1B,C). In fact, we could mimic such nucleus deformation by mechanically compressing shoot apices laterally in a microvice with a force of ca. 0.1N (see SI Appendix, Fig. S1). Altogether, this suggests that nuclear deformation in the boundary region is the result of compressive stresses caused by organ outgrowth.

To quantify these nuclear phenotypes, we used images obtained with the p35S::SUN1-YFP line and developed a computational pipeline to track cells and nuclei and quantify nuclear shapes (see SI Appendix, Fig. S2). In brief, MorphoGraphX [START_REF] Barbier De Reuille | MorphoGraphX: A platform for quantifying morphogenesis in 4D[END_REF] was used to extract the meristem surface, and segment (2D) cells on which the curvature was mapped. In addition, 3D cell segmentation of the epidermal layer followed by cell tracking was performed using MARS/ALT [START_REF] Willis | Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche[END_REF]. The nuclei were segmented in 3D using the MARS watershed algorithm. Finally, meristem regions and nuclear shapes were extracted and quantified using MATLAB (The MathWorks, Inc., Natick, Massachusetts, United States) scripts, after projecting the nuclear signal in 2D.

A subpopulation of nuclei at the boundary region exhibited a significant increase in their aspectratio. In each boundary we could find a group of at least 10 nuclei with an aspect ratio superior to 1.55, whereas meristematic nuclei typically have an aspect ratio of 1.2 +/-0.15 (n=132) (Fig. 2A, see SI Appendix, Fig. S3,S4). When backtracking these most affected nuclei via their cell lineages, we also found that they increased their aspect ratio following the increased growth of the primordia, correlating with the increased (negative) curvature of the boundary domain (Fig. 2B-C, see SI Appendix, Fig. S3,S4). Altogether, these quantifications demonstrate the existence of active nucleus compression operating at the meristem boundary during organogenesis and highlight the presence of a subset of boundary cells with increasing negative curvature in time, indicative of cellular compression.

Linker histone H1.3 is induced by compression

The boundary domain has a specific gene expression profile [START_REF] Tian | An organ boundary-enriched gene regulatory network uncovers regulatory hierarchies underlying axillary meristem initiation[END_REF]. This pattern relies on hormones, microRNA and mechanical stress [START_REF] Aida | Genetic control of shoot organ boundaries[END_REF] [START_REF] Landrein | Mechanical stress contributes to the expression of the STM homeobox gene in Arabidopsis shoot meristems[END_REF] [START_REF] Fal | Interplay between miRNA regulation and mechanical stress for CUC gene expression at the shoot apical meristem[END_REF]. Nucleus compression is likely to have a more global effect on chromatin, than on specific gene expression, as shown in animal systems [START_REF] Miroshnikova | Emerging roles of mechanical forces in chromatin regulation[END_REF] [START_REF] Booth-Gauthier | Force-Induced Changes in Subnuclear Movement and Rheology[END_REF](5) [START_REF] Tajik | Transcription upregulation via force-induced direct stretching of chromatin[END_REF]. Furthermore, changes in global chromatin state have been associated with changes in chromatin compaction, which relies on linker histones (H1 isoforms). In plants, at least 3 isoforms of H1 have been studied. Their expression and competitive binding to DNA depend on cell cycle as well as on external stress factors. H1.3 has been associated with the cell's response to abiotic stresses in past work [START_REF] Rutowicz | A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis[END_REF]. In particular, H1.3 is induced by drought stress [START_REF] Rutowicz | A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis[END_REF], which, based on data from animals (e.g. [START_REF] Ascenzi | Molecular genetic analysis of the drought-inducible linker histone variant in Arabidopsis thaliana[END_REF] [START_REF] Ascenzi | A drought-stress-inducible histone gene in Arabidopsis thaliana is a member of a distinct class of plant linker histone variants[END_REF]), might also drive nucleus compaction.

When analyzing the expression pattern of three variants of histone H1 in the meristem, we found that H1.1 and H1.2 were expressed in the entire meristem, with a higher expression level in organs. Interestingly, H1.3 exhibited a patchy pattern, with the strongest signal deep in meristem boundaries (Fig. 3A). To go beyond this correlation, we next applied compression on meristem to check whether H1.3 expression would be induced. To do so, we used NPA-treated seedlings and placed the naked stems between two blades of the microvice (see SI Appendix, Fig. S1A, see material and methods). When the blades were touching the meristem without deforming it, no clear H1.3 expression could be detected in the meristem (Fig. 3B, see SI Appendix, Fig. S5B, n=6/6 meristems with less than 3 nuclei exhibiting H1.3 induction at t=8h). However, when the meristem was deformed upon compression, most meristems expressed H1.3 ectopically (Fig. 3B, see SI Appendix, Fig. S5B, n=10/11 meristems with more than 3 nuclei exhibiting H1.3 induction at t=8h). Our finding suggests that cells at the meristem boundary are experiencing high and long enough compressive stress to induce such expression.

Global chromatin changes occur at the organ-meristem boundary

Our results suggest that nuclear compression, naturally occurring during organogenesis, generates iterative chromatin changes each time a new organ emerges. To further test this hypothesis, we investigated whether nucleus compression is strong enough to affect chromatin architecture. To do so, we used whole-mount fixed meristems and stained them with DAPI, which displays brighter signal in heterochromatin regions corresponding to chromocenters (e.g. ( 27)). We analyzed their distribution in successive optical sections (see SI Appendix, Fig. S6A).

To check whether nucleus compression affects the distribution of these chromocenters, we compared nuclei with an aspect ratio superior or inferior to 1.55, which corresponds to the threshold identifying the significantly most deformed nuclei (cf. Fig. 2B). We observed that the number of chromocenters decreased in more compressed nuclei (see SI Appendix, Fig. S6B,pvalue = 8.2×10 -10 , two-tailed Student's t-test). To further confirm this result, we used a pHTR5::mCG-MBD-GFP line (MBD for Methyl-CpG Binding Domain, (28)(29)(30)), which marks CG methylation (a marker of heterochromatin, Fig. 4A,B). Consistent with our previous results on the DAPI stained meristems, we detected a decrease in the number of mCG regions within the nuclei with the higher aspect ratio (Fig. 4C,D, p-value = 7.3×10 -5 , two-tailed Student's t-test). These results indicate that nuclei compression at the organ boundary colocalizes with modifications of chromatin architecture.

In isolated human umbilical vein endothelial cells, the average nuclear signal density changes as a function of the nuclear shape and is higher in the most elongated nuclei [START_REF] Versaevel | Spatial coordination between cell and nuclear shape within micropatterned endothelial cells[END_REF]. Therefore, we tested the correlation of the average nuclear signal density and the nuclear aspect ratio.

Consistently with the reported data on human cells, we detected an increase in average nuclear signal density in the nuclei with the higher aspect ratio (Fig. 4E, p-value = 4.5×10 -8 , two-tailed Student's t-test; see SI Appendix, Fig. S6C).

Last, to check whether the induction of H1.3 in deformed nuclei could be associated with such global chromatin changes, we analyzed the nuclei of the h1.3 mutant. The size and shapes of the h1.3 nuclei in the boundary domain were not significantly affected (Fig. 5A,B). Similarly, the number of chromocenters appeared comparable in wild type and h1.3 mutant at the boundary (see SI Appendix, Fig. S7A). In line with previous analyses of whole plant CHH DNA methylation in h1.3 [START_REF] Rutowicz | A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis[END_REF], we introgressed the pHTR5::mCHH-GFP reporter into h1.3 plants to further analyze the role of H1.3 in the nuclei at the organ boundary. We detected induction of the signal density (normalized signal of the CHH) in the WT nuclei with the aspect ratio above 1.55 but not in those of h1.3 (Fig. 5C, p-value wt = 1.35×10 -5 ; p-value h1.3 = 0.1, two-tailed Student's ttest; see SI Appendix, Fig. S7B andS7C), thereby suggesting that H1.3 is required for DNA methylation changes at the organ boundary.

Discussion

We show that organogenesis at the shoot apical meristem leads to nucleus compression in a specific set of cells, which is accompanied by major chromatin changes. This demonstrates that forces have a strong and direct impact on nuclei in a tissue context. The role of forces on chromatin is now well established, notably through experiments with artificial deformations of single cells in culture (4)(5)(6). Our results not only show that such effects also occur in plants, but they also demonstrate that these effects occur in the natural context of a developing organ. In fact, several reports have proposed that the main role of forces in the nucleus is to affect the physics of chromatin, which in turn would affect gene expression (instead of affecting specific genes that would then affect chromatin). For instance, nuclei have been found to exhibit an auxetic behavior, because of the decompaction of chromatin upon nucleus stretching [START_REF] Pagliara | Auxetic nuclei in embryonic stem cells exiting pluripotency[END_REF]. Our results are consistent with such conceptual framework and suggest that forces may primarily have global effects on chromatin, which would thus subsequently affect the expression of specific genes.

In previous work, we had already shown that STM and CUC3 are induced by mechanical stress at the organ-meristem boundary (20) [START_REF] Fal | Interplay between miRNA regulation and mechanical stress for CUC gene expression at the shoot apical meristem[END_REF]. However, both of these genes are induced in shallow boundaries, i.e. before we can detect significant nucleus deformation. We thus believe that nucleus deformation may contribute to their expression, but it is unlikely that we would find a clear-cut contribution of nucleus deformation on the expression of these genes, since they are already highly expressed when nuclei become more deformed. Instead, the analysis of chromatin status appears more convincing, and also better fits with the idea that mechanical stress would have a more global role on the structural component of the nucleus, i.e. nucleoskeleton and chromatin, rather than an instructive role on specific genes. These new data are important as they establish a link between growth-derived patterns of compression in tissue to nucleus shape and chromatin status.

Further studies would be required to elucidate the structural role of histones, in response to growth-derived forces, in plants. Here we reveal the induction of linker histone H1.3 at the organ-meristem boundary. One may speculate whether H1.3 expression could scale with mechanical stress intensity in different tissues. Interestingly, H1.3 expression has never been detected in the female megaspore mother cell, i.e. in one of the most mechanically protected cells, arguably [START_REF] She | Chromatin reprogramming during the somatic-to-reproductive cell fate transition in plants[END_REF]. Yet, we could not detect major phenotypic defects in the H1.3 mutant meristem, at least in control growth conditions (see SI Appendix, Fig. S8). The situation might actually be more complex. Many cross-talks between linker histones have been identified [START_REF] Rutowicz | Linker histones are fine-scale chromatin architects modulating developmental decisions in Arabidopsis[END_REF], and unsurprisingly, chromatin changes are associated with many pathways, from drought (26) to light [START_REF] Bourbousse | Plant Chromatin Catches the Sun[END_REF]. Integrating the contribution of mechanical forces in this network is thus likely going to be a long endeavor, but also an exciting one. In particular, this may compel us to see the structural side of chromatin, and analyze the role of the relevant regulators with this lens, also echoing results obtained in animal cells in culture (e.g. ( 35)). This work also opens the path to the investigation of the mechanisms leading to such nucleus compression and chromatin compaction. For instance, nuclei may be simply squeezed by pressure, meaning that nuclear density would increase, resulting in chromatin changes.

Interestingly, hyperosmotic stress leads to nucleus deformation and chromatin changes in animal cells in culture as well as in isolated nuclei [START_REF] Finan | The effects of osmotic stress on the structure and function of the cell nucleus[END_REF]. The behavior of nuclei in the cells at the organmeristem boundary may recapitulate such effects, albeit with mechanically-driven, instead of osmotically-driven, nucleus deformation, and in a growing tissue context. The role of the nuclear envelope is also likely to be important in that pathway (e.g. (37)(38)). In particular, chromatin organization depends on proteins from the Linker of Nucleoskeleton to Cytoskeleton (LINC) complex in plants [START_REF] Poulet | The LINC complex contributes to heterochromatin organisation and transcriptional gene silencing in plants[END_REF].

In Mammalian cells, stretching was recently proposed to soften chromatin, through a reduction in heterochromatin formation, thereby insulating the genetic material from cellular and supracellular mechanical stress [START_REF] Nava | Heterochromatin-Driven Nuclear Softening Protects the Genome against Mechanical Stress-Induced Damage[END_REF]. Interestingly, nuclei become stiffer in plant cells through heterochromatin clustering when exposed to hyper-osmotic stress, i.e. when tensile stress level at the cell cortex decreases [START_REF] Goswami | Mechanical Shielding in Plant Nuclei[END_REF]. This could suggest that cortical tension might positively correlate with softer nuclei across kingdoms. Whether this applies to our system remains an open question.

The observed reduction in the number of chromocenters at the meristem boundary, both via DAPI staining and mCG-MBD-GFP signal quantification, echoes the behavior of human stem cells placed on stiff environment (42). In our system, such reorganization of chromatin may involve a decrease in individual heterochromatin clusters as well as the spreading of more transient CHH methylation. The accumulation of H1.3 histones would compete with other linker histones [START_REF] Rutowicz | A Specialized Histone H1 Variant Is Required for Adaptive Responses to Complex Abiotic Stress and Related DNA Methylation in Arabidopsis[END_REF], but it remains to be shown whether this would also affect nuclear stiffness ultimately.

From a developmental point of view, our work adds another layer of complexity to the definition of the boundary domain in plant shoot apices [START_REF] Aida | Genetic control of shoot organ boundaries[END_REF]. Beyond morphology [START_REF] Kwiatkowska | Growth and morphogenesis at the vegetative shoot apex of Anagallis arvensis L[END_REF], hormone signatures [START_REF] Brunoud | A novel sensor to map auxin response and distribution at high spatiotemporal resolution[END_REF], gene expression [START_REF] Tian | An organ boundary-enriched gene regulatory network uncovers regulatory hierarchies underlying axillary meristem initiation[END_REF], miRNA regulation [START_REF] Sieber | Redundancy and specialization among plant microRNAs: role of the MIR164 family in developmental robustness[END_REF], and stress pattern [START_REF] Hamant | Developmental patterning by mechanical signals in Arabidopsis[END_REF], boundary domains may indeed be also defined by nucleus compression and associated chromatin changes. This likely contributes to its cadastral role and may be conserved beyond the plant kingdom.

Materials and methods

Plant materials and growth conditions

All procedures were performed on plants from the Col-0 ecotype, grown in "long day" conditions, with16hr/8hr light/dark period at 21°C. The pPDF1::CFP-N7 (20),

Meristem dissection

Stems were cut when the SAM switched to an inflorescence meristem identity, i.e. between the appearance of the first flower to the appearance of the first silique (stages 13 to 17, ( 12)) and transferred on a half MS medium with vitamins and 0.125 μg/μL of BAP for imaging as described in [START_REF] Burian | A correlative microscopy approach relates microtubule behaviour, local organ geometry, and cell growth at the Arabidopsis shoot apical meristem[END_REF]. Young floral buds were dissected out to access the SAM.

Meristem compression

p35S::SUN1-YFP and pH1.3::H1.3-GFP line were grown in vitro on a medium containing 10 μM napthylphthalamic acid (NPA, Sigma) as described in [START_REF] Hamant | Time-lapse imaging of developing meristems using confocal laser scanning microscope[END_REF]. As soon as naked inflorescences had formed, the plants were transferred to medium without inhibitor. Within 48 hours, plants were placed in a microvice (see SI Appendix, Fig. S1A). The two blades of the microvice were brought closer so as to induce a strong deformation of the shoot apex. Images were acquired with an upright Leica SP8 or Zeiss LSM700 microscope, as described below (n>5).

DAPI staining of the whole-mount meristems

Prior to staining, the dissected SAM samples were fixed 10 min on ice and then under vacuum (3 times 20 min at room temperature) in a fixing solution containing PBS 1x, EGTA 0.5M, DMSO 10% Formaldehyde 4%. The samples were then dehydrated in ethanol-methanol series as follows: 1x ethanol 70% for 5min, 1x ethanol 100% for 5min, 2x methanol 100% for 5min, 2x ethanol 100% for 5min, and incubated overnight in ethanol 100% at 4°C. Rehydration was performed as follows: 1x ethanol 100% for 5min, 1x ethanol 70% for 5min in PBSt (1% Tween20 in PBS), 1x ethanol 50% for 5min in PBSt, 1x ethanol 30% for 5min in PBSt, and 3x PBSt for 5 mins. Then, the samples were incubated for 30 minutes at 37°C with the mix of cell wall digesting enzymes [START_REF] Rozier | Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization[END_REF] and RNAse A (0.1mg/ml) and refixed in 4% formaldehyde in PBS.

After washing with PBS buffer, the samples were incubated with the DAPI solution (5µg/ml).

The mounting of the samples was performed as described in [START_REF] Rozier | Analysis of 3D gene expression patterns in plants using whole-mount RNA in situ hybridization[END_REF].

Confocal laser scanning microscopy and image analysis

Dissected shoot apical meristems from greenhouse plants or naked meristems from in vitrogrown NPA-treated seedlings were imaged with a water-dipping lens (x25, NA = 0.8) using an Cell labels resulting from the two (MGX and MARS) segmentations were aligned by importing the MARS segmented stack into MGX and applying the manual label transfer followed by application of the Lineage Tracking function.

Cell lineage tracking

To compute cell lineages, affine transformation and subsequently nonlinear transformation were computed to register consecutive confocal images of the membrane-marked channel using the Block-matching framework (50) [START_REF] Michelin | Spatio-temporal registration of 3D microscopy image sequences of Arabidopsis floral meristems[END_REF]. To compute cell lineages, the computed nonlinear transformation was applied to the corresponding segmented images and optimal cell pairings between registered segmented image and reference segmented image were computed by identifying maximally overlapping cell labels from the two images.

Tissue region extraction

The analysis was performed on epidermal nuclei that were all classified as belonging to the meristem, the organ, or the boundary region in between. The classification was based on the quantified surface curvature of the cells, where the boundary region was defined by cells having negative curvature, as in [START_REF] Stanislas | A phosphoinositide map at the shoot apical meristem in Arabidopsis thaliana[END_REF]. The rest of the nuclei were classified as meristem or organ depending on which side they were found of a fitted curve along the boundary (Fig. S2A).

Cell and nuclei size and shape quantification

For each segmented object (cell or nucleus), the z-slice with the largest area was extracted for further analysis. The aspect ratio was calculated as the length of the main axis divided by the length of the minor axis of the ellipse representing the covariance matrix of the pixel positions for the region. All properties were extracted using the Matlab function regionprops.

Software/protocol

Scripts required to do the segmentation and quantitative analysis are provided via the Sainsbury Laboratory GitLab repository https://gitlab.com/slcu/teamhj/publications/fal_et_al_2021 [publically available upon acceptance], where also a more detailed protocol for executing the steps of the pipeline is provided. All scripts have been developed in Python (https://python.org) and Matlab (MathWorks Inc., Natick, Massachusetts, United States), and the protocol is

  pPDF1::mCitrine-MBD (45), p35S::SUN1-YFP (15), pH1.1::H1.1-EGFP, pH1.2::H1.2-EGFP (32), pH1.3::H1.3-EGFP (24), pHTR5::HTR5-GFP (46) pHTR5::mCG-MBD-GFP and pHTR5::mCHH-GFP (30) reporter lines were used for this study.

  dependent on the MorphoGraphX ((16) https://www.mpipz.mpg.de/MorphoGraphX) and MARS/ALT (50) software. boundary domain; the bottom panel displays close-ups in the boundary region where the nuclei (marked with an asterisk) become more elongated following the 36 hours. Green: SUN1-YFP, grey: cell membranes stained with the FM4-64. Scale bars, 10µm, for nuclei at the boundary -5µm.

Fig. 2 .

 2 Fig. 2. Quantitative nucleus morphometry at the organ-meristem boundary (A) Quantification of nuclear aspect ratio (upper panel) and local tissue curvature (lower panel) at the boundary region of a growing organ. The nuclear segmentation is performed on the confocal stack images of a representative organ at the meristem, acquired through the 36-hour period. The spatial heatmap plots illustrate the convex hulls of each nucleus, filled with colors to represent the indicated data. The highest values of the nuclear aspect ratio (upper panel) and the local tissue curvature (lower panel) are shown in black; the lowest values of the nuclear aspect ratio the local tissue curvature -in yellow. (B) Nuclei at the boundary domain with the highest aspect ratio changes within the acquisition period. (C) Plots illustrate the changes in aspect ratio (left), and local tissue curvature (right) of the selected nuclei, highlighted in (B) during the 36 hours observation.

Fig. 3 .

 3 Fig. 3. Expression of linker histones at the SAM (A) Maximal projections of the confocal stacks, showing the expression patterns of the linker histone reporter lines for H1.1, H1.2 and H1.3 at the shoot apex (upper panel) and a close-up in the organ boundary area (lower panel). Scale bar, 20µm. (B) Representative naked meristems from in vitro NPA-grown pH1.3::H1.3-GFP seedlings, as viewed from the top, with or withoutlateral compression with a microvice. t=0h is the timepoint when the meristem is placed between the blades of the microvice with (bottom) or without (top) compression. Note that the blades of the microvice appear partly fluorescent in the green channel, while a debris can be spotted on top of the meristem in the autofluorescence channel (bottom panels). Scale bar, 50µm.

Fig. 4 .

 4 Fig. 4. Chromatin changes in compressed nuclei at the organ-meristem boundary (A,B) Maximal projection of the confocal stacks showing the expression of the pHTR5::HTR5-GFP (A) and pHTR5::mCG-MBD-GFP (B) reporter lines with the global top view (left panels) and close-up in the organ boundary area (right panels). Scale bar, 20µm. (C) Representative

Fig. 5 .

 5 Fig. 5. Chromatin changes in compressed nuclei at the organ-meristem boundary in h1.3 mutant

Acknowledgments:

We thank Célia Baroux, Fred Berger, Katja Graumann, and Daniel Grimanelli for providing material used in this article. We are grateful to Grégoire Malandain for the use of Blockmatching software. We thank Platim for help with imaging. We also thank Adrienne Roeder for insightful suggestions on this manuscript.

Funding: This work was supported by the European Research Council (ERC-2013-CoG-615739 ''MechanoDevo''), Human Frontier Science Program grant RGP0009/2018, the Schlumberger Foundation for Education and Research, CNRS (mécanobiologie « NEstress ») and the Gatsby Charitable Foundation (GAT3395-PR4B).

upright SP8 confocal microscope (Leica, Germany) to generate stack of optical sections with an interval of 0.15 μm between slices. The membranes were stained with FM4-64. All original confocal data is available via the University of Cambridge Data Repository (https://doi.org/xxx link available upon acceptance). Images are presented using the Fire lookup table (ImageJ).

Image analysis for chromocenter and CG-methylated regions detection was performed using the 3D object counter option in Fiji software (http://fiji.sc/wiki/index.php/Fiji). The nuclear average signal density was calculated following along the line of the protocol, described in [START_REF] Versaevel | Spatial coordination between cell and nuclear shape within micropatterned endothelial cells[END_REF].

In this case, the mean signal values were normalized to the maximal projected area of the corresponding nuclei and then correlated with their aspect ratio. Statistical analysis was performed using either Microsoft Excel or R software. The two-tailed Student test was performed to compare the means of independent biological sample groups.

Computational Methods

Segmentation

Surface extraction, 2D cell segmentation and Gaussian curvature quantification were performed for the membrane channel (FM 4-64) of the confocal stack images, using the level set method

https://www.mpipz.mpg.de/MorphoGraphX).

For 3D segmentation of the epidermal layer of cells and corresponding nuclei, an in house script using the MARS/ALT algorithms was developed [START_REF] Fernandez | Imaging plant growth in 4D: robust tissue reconstruction and lineaging at cell resolution[END_REF]. The confocal images were processed by cropping and applying Gaussian smoothing and alternating sequential filter (ASF). The resulting images were segmented using a watershed algorithm with automatic seeding (50) [START_REF] Willis | Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche[END_REF]. The segmentation was done for both the membrane channel (FM 4-64) and the nuclear marker channel (p35S::SUN1-YFP) of each image. Computed seeds from the membrane channels were used also for the segmentation of the nuclei signal. Cells of the epidermal cell layer were extracted by identifying cells neighboring the image background (the region outside the tissue), which itself was defined as the largest segmented region of the image [START_REF] Willis | Cell size and growth regulation in the Arabidopsis thaliana apical stem cell niche[END_REF].

Author contributions:

K.F., H.J and O.H. conceived and designed the experiments. K.F., and M.L. performed the experiments. N.K., J.T. and Y.R. developed the image analysis pipeline and performed the analysis of nucleus shapes. M-E.C. contributed important protocols and tools. K.F., H.J. and O.H. wrote the manuscript with inputs from all co-authors.

Competing interests:

Authors declare no competing interests.

Data and materials availability:

All original confocal data is available via the University of Cambridge Data Repository (https://doi.org/xxx link available upon acceptance). Scripts for the analysis pipeline are available via the Sainsbury Laboratory GitLab repository (https://gitlab.com/slcu/teamhj/fal_etal_2020 link publically available upon acceptance).


Eléna Coiplet
email: elena.coiplet@gmail.com

Marine Freuchet
email: marinefreuchet@hotmail.fr

Claire Sunyach
email: c.sunyach@gmail.com

Julien Mancini
email: julien.mancini@ap-hm.fr

Jeanne Perrin
email: jeanne.perrin@ap-hm.fr

Blandine Courbiere
email: blandine.courbiere@ap-hm.fr

Hélène Heckenroth
email: helene.heckenroth@ap-hm.fr

Christel Pissier
email: christel.pissier@ap-hm.fr

Naima Hamdaoui
email: hamdaouinaima2611@gmail.com

Florence Bretelle
email: florence.bretelle@ap-hm.fr

Assessment of a Screening Questionnaire to Identify Exposure to Lead in Pregnant Women

Keywords: lead exposure, pregnancy, risk factors, environment

Lead readily crosses the placenta and displays adverse effects on birth outcomes and neurodevelopment. Systematic identification of the risk of exposure during pregnancy is essential but rarely performed, probably due to hospital staff's workload and their lack of awareness. We aimed to evaluate the relevance of a questionnaire to screen pregnant women for lead exposure. A cross-sectional, multicentre study was carried out on a population of 792 pregnant women from February 2018 to May 2020. A total of 596 women had a blood lead test: 68.5% had blood lead levels below 10 µg/L. The estimated prevalence above 25 µg/L was 4% (95% confidence interval (CI) [2.6-5.9]) and 1.3% had levels above 50 µg/L (95% CI [0.6-2.6]). Multivariate analysis showed that three risk factors significantly increased the probability of blood lead levels above 25 µg/L: the use of traditional cosmetics (adjusted odds ratio [aOR]: 3.90; 95% CI [1.65-9.21]; p = 0.002), degraded old housing (aOR: 2.67; 95% ]; p = 0.018), and (marginally) eating bread more than twice a day (aOR: 2.40; 95% ]; p = 0.060). Our study reveals that a three-question tool can be used to quickly screen for the risk of lead exposure in our population and to trigger lead blood tests and special vigilance during pregnancy follow-up.

Introduction

Lead has been intensively used since antiquity, causing high dispersion in the environment. Long considered an occupational disease in France, lead poisoning was only recognised in the 1980s as a public health issue with the discovery of several cases of severely poisoned children. From the 1990s onwards, preventive measures taken by public authorities contributed to a sharp reduction in the prevalence of lead poisoning. Nevertheless, the question of the toxic effects of lead on pregnant women and their children remains. In France, major sources of exposure are housing, tap water and industrial pollution. White lead was used in buildings until the middle of the 20th century [START_REF] Klein | Current diagnosis and treatment of lead poisoning[END_REF]. Other sources are occupational (industry, public works) and recreational activities (pottery, hunting, fishing). Poisoning can also arise from one's diet, smoking, traditional medicines and cosmetics (Kohl), and cookware (handcrafted ceramics, tagine dishes). Massive lead poisoning has become rare. However, exposure to low doses is not without consequences. Lead has no known physiological role in humans, and no 'non-toxic' threshold could be defined. In France, the High Council of Public Health (HCSP) has recommended intervention for lead concentrations ≥50 µg/L and vigilance for ≥25 µg/L since 2017 [START_REF] Conseil De La | Expositions au Plomb chez L'enfant Mineur et la Femme Enceinte[END_REF]. The toxic effects of lead, even at low doses, justify keeping blood lead levels in pregnant women as low as possible. In 2011, less than 1% of women exceeded the threshold of 100 µg/L [START_REF] Dereumeaux | Surveillance biologique de l'exposition des femmes enceintes françaises aux polluants de l'environnement: Résultats du volet périnatal du programme national de biosurveillance mis en oeuvre au sein de la cohorte Elfe[END_REF].

Health professionals can rely on guidelines (updated in 2017) to manage lead exposure during pregnancy based on a screening questionnaire [START_REF] Conseil De La | Expositions au Plomb chez L'enfant Mineur et la Femme Enceinte[END_REF][START_REF]et des Solidarités L'intoxication par le Plomb de L'enfant et de la Femme Enceinte[END_REF]. Symptomatology is unspecific; the systematic identification of the risk of exposure is therefore essential. Diagnosis relies on blood lead level measurement. Hormonal and calcium metabolism modifications during pregnancy cause a release into blood from bone reserves, increasing the biologically active lead pool [START_REF] Gulson | Pregnancy increases mobilization of lead from maternal skeleton[END_REF][START_REF] Silbergeld | Lead in bone: Implications for toxicology during pregnancy and lactation[END_REF]. Lead readily crosses the placental barrier and is fetotoxic [START_REF] Gardella | Lead exposure in pregnancy: A review of the literature and argument for routine prenatal screening[END_REF]. Studies have shown that prenatal lead exposure increases the risk of abortion or premature delivery, intrauterine growth retardation, low birth weight, high blood pressure and cognitive impairment in children [START_REF] Yazbeck | Maternal blood lead levels and the risk of pregnancy-induced hypertension: The EDEN cohort study[END_REF][START_REF] Poropat | Blood lead and preeclampsia: A meta-analysis and review of implications[END_REF][START_REF] Xie | The effects of low-level prenatal lead exposure on birth outcomes[END_REF][START_REF] Rahman | Infant and mother related outcomes from exposure to metals with endocrine disrupting properties during pregnancy[END_REF][START_REF] Wasserman | The Yugoslavia prospective lead study: Contributions of prenatal and postnatal lead exposure to early intelligence[END_REF][START_REF] Schnaas | Reduced intellectual development in children with prenatal lead exposure[END_REF][14][START_REF] Hu | Fetal lead exposure at each stage of pregnancy as a predictor of infant mental development[END_REF][START_REF] Klein | Lead poisoning in pregnancy [SATURNISME AU COURS DE LA GROSSESSE[END_REF][START_REF] Ernhart | A critical review of low-level prenatal lead exposure in the human: 1. Effects on the fetus and newborn[END_REF][START_REF] Jelliffe-Pawlowski | Effect of magnitude and timing of maternal pregnancy blood lead (Pb) levels on birth outcomes[END_REF]. Data during pregnancy remain limited, and prenatal lead exposure from cord blood samples has been analysed [START_REF]NTP Monograph: Health Effects of Low-Level Lead[END_REF][START_REF] Yazbeck | Lead exposure in pregnant women and newborns: A screening update[END_REF][START_REF] Audrey | A comparative study of manganese and lead levels in human umbilical cords and maternal blood from two urban centers exposed to different gasoline additives[END_REF]. In our practice, blood lead tests are rarely prescribed during pregnancy follow-up. Time-starved obstetricians must dedicate consultation to medical aspects at the expense of environmental risk factors. To the best of our knowledge, no study has evaluated the use of a screening questionnaire to screen the risk of lead exposure during pregnancy. Thus, we assessed the relevance of the HCSP questionnaire compared to blood testing to save practitioners time. The main objective of our study was to determine the relevance of such a questionnaire to quickly identify lead exposure in pregnant women. The secondary objectives were to estimate the current prevalence of high blood lead levels (≥50 µg/L) in the Marseille urban area and the association with potential obstetric complications associated with lead poisoning.

Materials and Methods

Study Design

A multicentre, cross-sectional study was carried out from 1 February 2018 to 31 May 2020 on a population of pregnant women, who were followed in the maternity wards of university hospitals in Marseille in a mother infant care centre (MICC) and in a gynaecologist's private practice in Marseille. A standardised lead exposure screening questionnaire consisting of 18 questions was completed by pregnant women during their first follow-up visit with a gynaecologist or a midwife (Appendix A). The risk factors investigated were a personal or family history of lead poisoning, birth in a country with high lead use (African, Middle Eastern, Southeast Asian, Central American and Eastern European nations), living in dilapidated old dwellings or in proximity to an industrial site, recent renovation work in one's home, the use of traditional products (medicines, cosmetics, cookware), pica behaviour, the consumption of tap water, alcohol and vegetables, exposure to tobacco, and engaging in a high-risk leisure activity or occupation. A list of occupational and recreational activities was joined to the questionnaire. Our questionnaire was based on the HCSP [START_REF] Conseil De La | Expositions au Plomb chez L'enfant Mineur et la Femme Enceinte[END_REF] except for 2 questions regarding diet: eating shellfish and crustaceans, and the consumption of bread more than twice a day, based on the results of 'L'Étude de l'Alimentation Totale Française 2' [START_REF] Saoudi | Prenatal exposure to lead in France: Cord-blood levels and associated factors: Results from the perinatal component of the French Longitudinal Study since Childhood (Elfe)[END_REF] and 'L'Étude de Santé sur l'Environnement, la Biosurveillance, l'Activité Physique et la Nutrition' (ESTEBAN) [START_REF]Étude de L'alimentation Totale Française 2 (EAT 2) Tome 1-Contaminants Inorganiques, Minéraux, Polluants Organiques Persistants[END_REF], which reported that bread, vegetables, or crustaceans and molluscs are major contributors to lead exposure [START_REF] Yazbeck | Lead exposure in pregnant women and newborns: A screening update[END_REF][START_REF] Audrey | A comparative study of manganese and lead levels in human umbilical cords and maternal blood from two urban centers exposed to different gasoline additives[END_REF][START_REF] Dereumeaux | Biomarkers of exposure to environmental contaminants in French pregnant women from the Elfe cohort in 2011[END_REF]. A blood lead level was prescribed systematically. The assay was performed at the hospital sampling centre or in a private laboratory at the patient's convenience. When the blood lead level was ≥25 µg/L, a control was realised three months later. When the blood lead level was ≥50 µg/L, the lead level was determined on cord blood at delivery, and an environmental survey at the family home was conducted.

Inclusion Criteria

Pregnant women over 18 years old, regardless of their term, who signed informed consent were recruited. They were provided with an information notice.

Data Collection

Questionnaire and lead level results were collected weekly. When the blood lead level was not recorded in the medical file, patients were contacted by telephone. Obstetric history and course of pregnancy information were retrieved from electronic patient records. All data were anonymised and entered into Excel files.

Determining Blood Lead Levels

Blood samples were collected in vacutainer EDTA K2 tubes (Becton Dickinson and Company, Franklin Lakes, NJ, USA). Calibration was based on the standard addition method. One hundred microlitres of whole blood samples were introduced into 15 mL polypropylene tubes along with 100 µL of whole blood provided by the French blood agency (Etablissement Français du Sang; EFS, Marseille, France) and 10 mL of nitric acid 0.5% containing the internal standard (10 µg/L bismuth). The samples were handled using an SPS 4 autosampler (Agilent Technologies, Santa Clara, CA, USA). The mass spectrometer was a Determination by mass spectrometry coupled to an induced plasma (ICP-MS 7800; Agilent Technologies). The operating conditions were checked daily using a 10 µg/L tuning solution (Agilent Technologies). Linearity was validated in the 20-500 µg/L range. Data were processed with Mass Hunter software 4.4 (Agilent Technologies). The method was validated according to European Medicines Agency's guidelines and accredited by the French committee of accreditation (COFRAC).

Statistical Analyses

The blood lead level was described by its mean (± standard deviation). The prevalence of high blood lead levels (≥25 µg/L and ≥50 µg/L) was estimated with the calculation of an exact 95% confidence interval. The relationship between blood lead levels ≥25 µg/L and each response to the screening questionnaire was analysed with the Wald test from univariable binary logistic regression models. The same test was employed to study the relationship between blood lead levels ≥25 µg/L and obstetrical history. Student's t-test was used for mean comparisons. A multivariate binary logistic regression analysis was then run to study the variables independently associated with blood lead levels ≥25 µg/L. Only the variables significant in univariate analysis were entered in this model. Additional stepwise analyses did not identify any other significant variables. Receiver Operating Characteristic (ROC) curves were used to estimate the ability of different scoring methods (using all the questions or only those selected in the logistic regression) to distinguish women with blood lead levels ≥25 µg/L using the area under the curve (AUC). All analyses were performed using IBM SPSS Statistics 20.0 software (IBM Inc., New York, NY, USA). A value of p ≤ 0.05 was considered statistically significant.

Authorizations

This study has been declared to the 'Commission de l'Informatique et des Libertés' (CIL) (2018-13) and was approved by the local ethics committee (2018-27-09-004 and 2018-18-10-005). Informed consent was obtained from the participants.

Results

During the inclusion time, 11% of the women consulting for pregnancy in the hospital ward completed the questionnaire. Patients in our sample had their pregnancy follow-up performed in university hospitals (93.8%), in private practice (5.4%) and in an MICC (5.4%). The screening questionnaire was completed by 792 patients; 596 (75.3%) women had their blood lead levels measured. The country of birth was France for 58.3% of the women. Among immigrant women, 41.5% were born in a country with high lead use, mainly Algeria (13.2%) and Comoros (7.5%). Maternal age was 30.46 ± 5.88 years. Blood tests were performed in the course of the 1st, 2nd or 3rd trimester for 46.9%, 21.7% and 31.4% of the patients, respectively.

The prevalence of blood lead levels above the 25 µg/L HCSP vigilance threshold and above the 50 µg/L intervention threshold was 4% (95% confidence interval (CI) [2.6-5.9], n = 25) and 1.3% (95% CI [0.6-2.6], n = 8), respectively. Seven patients (0.9%) reported no risk factors for lead exposure, and 3 of them had a blood lead level below 10 µg/L. The other four did not do the test. Out of 596 samples taken, 408 (68.5%) revealed lead levels strictly below 10 µg/L, and 188 (31.5%) women had lead levels above 10 µg/L, with an average of 18.8 ± 20.3 µg/L (Figure 1). The risk factors for lead exposure, according to their frequency, are summarised in Table 1. The most common sources of exposure were drinking tap water (75.5%), the consumption of bread more than twice a day (51.3%), and birth in a country with high lead use (43.4%). The 596 lead tests and the analysis of the questionnaires allowed us to identify risk factors associated with lead poisoning (Table 2). The risk of a high blood lead level (≥25 µg/L) was significantly increased by the use of traditional cosmetics (p = 0.001), degraded old housing (p = 0.019), and the consumption of bread more than twice a day (p = 0.045). However, the results were at the limit of significance for birth in a country with high lead use (p = 0.076) and living close to an industrial site (p = 0.102). Despite being not statistically significant, other factors were rather strongly associated with lead poisoning (odds ratio >2): living close to an industrial site, a high-risk country of birth, and history of lead poisoning (Table 3). Women poisoned reported an average of 4.9 ± 1.8 potential sources of lead exposure, which was significantly higher than in the blood lead level group < 25 µg/L (4.9 ± 1.8 vs. 3.9 ± 1.9; p = 0.009). Univariate analysis did not show a significant association of clinical characteristics according to the level of lead impregnation (Table 4). At the end of inclusion, 52.5% of patients had given birth; 83.6% gave birth after 37 weeks of gestation (WG), with an average of 38.6 ± 3.3 WG. The gender distribution was equal (199 girls and 207 boys), and the average birth weight was 3077 ± 739 g. There was no significant difference between the 2 groups in the risk of intrauterine growth restriction (IUGR) (p = 0.177), anaemia (p = 0.701), preterm delivery (p = 1.0), hypertension (p = 1.0) or birth weight (p = 1.0) (Table 4). According to the multivariate analysis, the three risk factors identified in univariate analysis independently increased the risk of blood lead levels above 25 µg/L (Table 3). Use of traditional cosmetics (OR: 3.90; 95% CI [1.65-9.21]; p = 0.002), living in degraded old housing (odds ratio (OR): 0.67; 95% CI [1.19-6.038]; p = 0.018), and the consumption of bread more than twice a day (OR: 2.40; 95% CI [0.96-6.11]; p = 0.060). The ROC curve shows that considering only the three risk factors significantly associated with high blood lead levels tended to be more discriminating for the detection of a high blood lead level than summing up the 18 questions (Figure 2). When patients mentioned only one of the three questions, the risk reached 2.7%, which increased to 10.3% with two positive questions and to 17.6% with three positive questions (Table 5). a Q3, Q7 and Q14 refer to the questions of the questionnaire, i.e., old or degraded housing, use of traditional cosmetics, and consumption of bread more than twice a day. For example, a score of 3 indicates a positive response to all three questions.

Eight women had a blood lead level ≥50 µg/L. The four newborns tested had a blood lead level above 25 µg/L, with an average of 95.5 µg/L (55 to 194 µg/L). Their average age was 38 (27 to 46). The patients mainly originated from North Africa (one from Morocco, five from Algeria). The highest level of lead impregnation tested was 194 µg/L in a 30-year-old Algerian-born woman who arrived in France 3 months before her first visit to the maternity yard. She had completed three pregnancies without particularities and was 12 WG. She reported 5 risk factors for lead exposure: country of origin, old habitat, use of Kohl and the consumption of tap water and bread. The pregnancy was marked by gestational diabetes mellitus. She gave birth to a full-term eutrophic boy. The cord blood lead level was 264 µg/L. The environmental survey at home revealed peeling paint and the use of a stove imported from Algeria.

Discussion

Reducing child lead poisoning and preventing impacts on health during infancy and adulthood involves identifying pregnant women at risk. As clinical signs are not specific, only a strong action to investigate exposure risk factors, followed by the prescription of a blood test if factors are identified, can detect poisoned patients. However, in France, this is not systematically performed during pregnancy, probably due to hospital staff's workload and their lack of awareness. Our study shows that three questions are enough to screen for risk of lead exposure in our population. This is particularly important since maternity wards of university hospitals and the MICC in southern France provide pregnancy care for pregnant women born in countries with a high level of lead use and/or living in precarious housing conditions, a population potentially at risk of lead poisoning.

Towards an Efficient and Simple Screening Lead Poisoning Questionnaire

The French HCSP proposed an 18-item questionnaire and recommended prescribing a blood lead test if the answer to at least one question indicated possible lead exposure. Almost all women tested reported at least one risk factor. According to the HCSP recommendations, blood lead levels should be prescribed to almost all pregnant women. This can be discussed with regard to the workload of health professionals and to health costs. We found that only 3 of the 408 women with blood lead levels below 10 µg/L reported no potential lead exposure factor. This suggests that the questionnaire proposed by the HCSP cannot fully distinguish women reporting one lead exposure risk factor from those with lead poisoning attested by high blood lead levels. On the other hand, women with blood lead levels >25 µg/L reported 4.9 ± 1.8 potential lead exposure sources. Hence, lead poisoning appears complex and cannot be traced to a single source. Almost all our patients reported at least one risk factor for lead exposure in the questionnaire; however, the analysis revealed that the use of traditional cosmetics, degraded housing and the consumption of bread more than twice a day significantly increased the risk of high blood lead levels. A history of lead poisoning showed a high odds ratio, but due to the rarity of the event, this risk was not significant. Tobacco smoking, alcohol consumption, age and old housing were the chief factors associated with increased blood lead levels reported in the ESTEBAN study performed among the general population [START_REF]Étude de L'alimentation Totale Française 2 (EAT 2) Tome 1-Contaminants Inorganiques, Minéraux, Polluants Organiques Persistants[END_REF]. By contrast, it reported no association between elevated blood lead levels and the use of traditional cosmetics.

A snapshot of Lead Poisoning in a Pregnant Population Consulted at University Hospital in Marseille

In our population, almost all women (95.1%) lived in an area of Marseille where 32% of the housing units were built before 1949. We found a prevalence of lead poisoning ≥50 µg/L of 1.3% (95% CI [0.6-2.6]), which is slightly higher than in recent studies [START_REF] Oleko | Imprégnation de la Population Française par le Plomb. Programme National de Biosurveillance, Esteban 2014-2016[END_REF]. Differences in biological matrices (venous blood vs. umbilical cord blood) could account for these results. Importantly, we confirmed a significant decrease in lead exposure as previously stated [START_REF] Dereumeaux | Biomarkers of exposure to environmental contaminants in French pregnant women from the Elfe cohort in 2011[END_REF][START_REF] Bierkens | Predicting blood lead levels from current and past environmental data in Europe[END_REF]. The mean blood lead level above 10 µg/L was 18.8 µg/L, as reported before [START_REF]Étude de L'alimentation Totale Française 2 (EAT 2) Tome 1-Contaminants Inorganiques, Minéraux, Polluants Organiques Persistants[END_REF]. The HCSP's 2017 updated guidelines do not recommend particular pregnancy follow-up when blood lead levels are below 50 µg/L. Even if we did not find any link between elevated blood lead levels and obstetric complications, unlike recent studies [START_REF] Jelliffe-Pawlowski | Effect of magnitude and timing of maternal pregnancy blood lead (Pb) levels on birth outcomes[END_REF][START_REF] Etchevers | Imprégnation des enfants par le plomb en France en 2008-2009[END_REF][START_REF] Efsa | Scientific Opinion on Lead in Food[END_REF], we recommend controlling the blood lead level when the first test is above 25 µg/L. Efforts should be made to reduce lead exposure in these patients.

Implications for Health Professionals and Patients

Lead poisoning is rare but probably underdiagnosed due to heterogeneous screening strategies. Health professionals should inform patients about lead poisoning and promote the screening process. The under-diagnosis of lead exposure is probably worse in our population. In our sample, 41.5% of the women came from high-risk countries. If all patients were prescribed a lead test, 196 (24.7%) did not take it, indicating that the public is likely poorly informed about lead poisoning. In our practice, women also often miss their pregnancy follow-up visits. This could be explained by precarious social, language and cultural barriers, as well as poor accessibility to means of transport. Even if questionnaires appear to be an easy way to screen for risk factors, obstacles probably remain due to hospital staff's high workload and lack of awareness. Apart from saving time for practitioners, this 3-question screening could be simpler to understand for our non-French speaking patients. It is easy to carry out for pregnancy follow-up visits and should help to address the crucial public health issue of lead exposure. Our 3-question tool could implement a lead intoxication prevention policy and thereby help reduce health inequalities.

Strengths and Weaknesses of the Study

To our knowledge, we have provided the first evaluation of a lead poisoning screening questionnaire. We are nevertheless aware of a selection bias arising from inclusion in university hospital maternity wards and the MICC in Marseille, which recruit a large number of patients in precarious situations. In addition, the questionnaire and lead blood test were only proposed to a small number of patients (11%). This likely reflects the fact that obstetricians and midwives are poorly informed of lead exposure consequences for pregnancy complications, issues and child health. Consequently, neither inquiring nor blood testing are included in their clinical approach. Even if the population of pregnant women consulting in our hospital is rather socially homogeneous, we cannot rule out selection bias. However, despite the small size of our sample, which underpowered our study, our results are consistent with previously published studies [START_REF] Saoudi | Prenatal exposure to lead in France: Cord-blood levels and associated factors: Results from the perinatal component of the French Longitudinal Study since Childhood (Elfe)[END_REF].

Conclusions

The health impact of environmental pollution is a critical public health issue. Our study confirms a decrease in lead poisoning in southern France. However, this remains a concern given the potential effects on the course of pregnancy and child neurodevelopment. We showed that a simple three-question tool can be used to quickly screen for the risk of lead exposure during pregnancy and trigger special vigilance and blood lead tests during follow-up. We strongly believe that our study will allow time-starved clinicians to screen for this environmental toxic exposure and deliver appropriate individual healthcare to lead exposed pregnant women. Importantly, our study could help to achieve the UN's 2030 Sustainable Development Goals (SDGs) 3 ('good health and well-being') and 10 ('reduced inequalities') [START_REF]The 2030 Agenda for Sustainable Development and the SDGs[END_REF].

Figure 1 .

 1 Figure 1. Distribution of blood lead levels in the sample (A) Presents categorical distribution of blood lead level values (n = 596). Note that laboratories sometimes reported "below 10 μg/L" rather than accurate value. (B) Distribution of blood lead levels above 10μg/L.

Figure 1 .

 1 Figure 1. Distribution of blood lead levels in the sample (A) Presents categorical distribution of blood lead level values (n = 596). Note that laboratories sometimes reported "below 10 µg/L" rather than accurate value. (B) Distribution of blood lead levels above 10 µg/L.

13 Figure 2 .Table 5 .

 1325 Figure 2. Comparison of ROC curves considering Q3, Q7, Q14 individually, Q3 + Q7 + Q14 or all 18 questions. When all 18 questions were considered, the area under the curve was 0.654 (95% confidence interval (CI) [0.538-0.771], p = 0.010). When only the three significant questions were taken into account the area under the curve is 0.731 (95% CI [0.626-0.836], p < 0.001). Individual areas under the curve (AUCs) for those teach three questions are 0.614 (95% CI [0.493-0.734]) for Q3, 0.638 (95% CI [0.511-0.764]) for Q7, and 0.608 (95% CI [0.497-0.718]) for Q14. Table 5. Calculation of an overall score from exposure factors significantly associated with high blood lead levels (n = 596). Blood Lead Level ≥ 25 µg/L Score Q3 + Q7 + Q14 a N % 0 (n = 179) 2 1.1

Figure 2 .

 2 Figure 2. Comparison of ROC curves considering Q3, Q7, Q14 individually, Q3 + Q7 + Q14 or all 18 questions. When all 18 questions were considered, the area under the curve was 0.654 (95% confidence interval (CI) [0.538-0.771], p = 0.010). When only the three significant questions were taken into account the area under the curve is 0.731 (95% CI [0.626-0.836], p < 0.001). Individual areas under the curve (AUCs) for those teach three questions are 0.614 (95% CI [0.493-0.734]) for Q3, 0.638 (95% CI [0.511-0.764]) for Q7, and 0.608 (95% CI [0.497-0.718]) for Q14.

Q1:

  Have you ever had a personal health history with lead poisoning (acute and chronic absorption of lead into the body)? Or has someone in your surroundings? YES NO Q2: Were you born in a country in Africa (including Comoros, Madagascar), the Middle East, Southeast Asia, the West Indies or Eastern Europe? YES NO Q3: Do you live in an old and degraded apartment (built before 1975)? YES NO Q4: Over the last 6 months, have you been in contact with someone working in construction, in an industrial site, or have you been living in an old apartment or in an apartment in construction (spread of dust, including stripping or sanding old paint)? YES NO Q5: Do you ingest traditional food supplements (clays, medicinal herbs?) YES NO Q6: Have you ever eaten non-food substances such as clay, soil, plaster, paint flakes? YES NO Q7: Do you use traditional cosmetics? (kohl, surma)? YES NO Q8: For the preparation/preservation of food, do you often use traditional containers (tajine dishes), enamelled ceramic pottery, pewter, or crystal dishes? YES NO Q9: Do you drink tap water? YES NO Q10: if yes, are there any lead pipes? YES NO Q11: Do you smoke? YES NO Q12 Are you passively exposed to the smoke of cigarettes? YES NO Q13: Have you been drinking alcohol while pregnant? YES NO Q14: Do you eat bread more than twice a day? YES NO Q15: Do you eat vegetables grown close to an industrial site more than twice a day? YES NO Q16: Have you been eating seafood while pregnant? YES NO Q17: Do you live or spend much time in places close to an industrial site releasing lead into the atmosphere? YES NO Q18: Do you practice (or have you practiced) shooting, hunting, fishing, pottery or a risky professional activity (in the industrial sector, construction work, metallurgy, in contact with objects containing lead)? Or is there in your surroundings someone practicing such leisure activities or working in those areas? YES NO

Table 3 .

 3 Multivariate analysis of factors significantly associated with lead poisoning.

		Univariable		Multivariable
		Analysis		Analysis
		Crude Odds Ratio	95% CI	Adjusted Odds Ratio a	95% CI	p
	Living close to an industrial site	3.62	[0.78-16.92]	NE b
	High risk country of birth c	2.15	[0.92-4.99]	NE
	Consumption of bread more than twice per day	2.50	[1.02-6.11]	2.40	[0.96-5.99]	0.060

Table 1 .

 1 Frequency of lead exposure risk factors mentioned in the questionnaire.

		YES	NO	I Don't Know
		Number of Patients (n)	Number of Patients (n)	Number of Patients (n)
		Frequency (%)	Frequency (%)	Frequency (%)
	Tap water consumption (Q9) a	598 75.5	193 24.4	1 0.1
	Bread consumption > 2 times/day (Q14)	406 51.3	384 48.5	2 0.3
	Birth in a high-risk country b (Q2)	344 43.4	448 56.6	0 0
	Active or passive smoking (Q12)	310 39.1	486 61.4	28 3.5
	Vegetables consumption > 2 times/day (Q15)	305 38.5	470 59.3	17 2.1
	Living in an old and deteriorated dwelling (Q3)	217 27.4	571 72.1	4 0.5
	Use of traditional containers (tagine dish),	177	609	6
	handmade ceramic, tin or crystal containers (Q8)	22.3	76.9	0.8
	Exercise of a hobby or profession at risk c (Q18)	155 19.6	634 80.1	3 0.4
	Consumption of shellfish and crustaceans (Q16)	152 19.2	639 80.7	1 0.1
	Use of traditional cosmetics (kohl) (Q7)	115 14.5	676 85.4	1 0.1
	Carrying out recent renovation work in an old	105	686	1
	home (Q4)	13.3	86.6	0.1
	Consumption of traditional medicines or food	53	736	3
	supplements (Q5)	6.7	92.9	0.4
	Living close to an industrial site (Q17)	33 4.2	740 93.4	3 0.4
	Alcohol consumption (Q13)	25 3.2	761 96.1	1 0.1
	Pica behaviour (Q6)	23 2.9	766 96.7	3 0.4
	Personal or family history of lead poisoning (Q1)	7 0.9	778 98.2	7 0.9

a Q refers to questions of the questionnaire (Appendix A). b Blood lead levels in the general population are often lower in France than in some countries in Africa, the Middle East, from Southeast Asia, the West Indies and Eastern Europe. c These activities include crafts (print making, stained glass, ceramics), outdoor sports (hunting and fishing), liquor distillation, creating stained glass, enamelling copper, casting bronze, pottery with certain leaded glazes and paints; casting ammunition, jewellery making and electronics (with lead solder), glassblowing with leaded glass.

Table 2 .

 2 Univariate analysis of exposure sources according to the level of lead impregnation (n = 596).

	Blood Lead Level
	<25 µg/L (n = 572)	≥25 µg/L (n = 24)	p a
	n (%)	n (%)

Table 2 .

 2 Cont. p-value from Wald test (binary logistic regression model). b Blood lead levels in the general population are often lower in France than in some countries in Africa, the Middle East, from Southeast Asia, the West Indies and Eastern Europe. c These activities include crafts (print making, stained glass, ceramics), outdoor sports (hunting and fishing), liquor distillation, creating stained glass, enamelling copper, casting bronze, pottery with certain leaded glazes and paints; casting ammunition, jewellery making and electronics (with lead solder), glassblowing with leaded glass.

	Blood Lead Level
	<25 µg/L (n = 572)	≥25 µg/L (n = 24)	p a
	n (%)	n (%)

a

Table 3 .

 3 Multivariate analysis of factors significantly associated with lead poisoning.

		Univariable		Multivariable
		Analysis		Analysis
		Crude Odds Ratio	95% CI	Adjusted Odds Ratio a	95% CI	p
	Living close to an industrial site	3.62	[0.78-16.92]	NE b
	High risk country of birth c	2.15	[0.92-4.99]	NE
	Consumption of bread more than twice per day	2.50	[1.02-6.11]	2.40	[0.96-5.99]	0.060
	Use of traditional cosmetics	4.30	[1.86-10.08]	3.90	[1.65-9.2]	0.002
	Degraded old housing	2.67	[1.17-6.06]	2.76	[1.19-6.38]	0.018
	History of lead poisoning	4.93	[0.55-43.9]	NE
	Renovation work in an old dwelling	1.70	[0.61-4.66]	NE
	Use of traditional medicines or food supplements	1.31	[0.30-5.81]	NE
	Pica behaviour	1.51	[0.19-11.88]	NE
	Use of traditional food containers (tagine dish), handmade ceramic, tin or crystal containers	0.96	[0.35-2.62]	NE
	Tap water consumption	0.99	[0.38-2.54]	NE
	Active or passive smoking	0.62	[0.18-2.12]	NE
	Alcohol consumption	1.2	[0.15-9.33]	NE
	Consumption of selfish or crustaceans	1.20	[0.43-3.29]	NE
	Vegetable consumption more than twice per day	0.97	[0.42-2.27]	NE
	Exercise of a hobby or profession at risk d	0.43	[0.10-1.87]	NE

a Each odds ratio is adjusted for the two other variables entered in the model. b Not Entered in the multivariate binary logistic regression model. c Blood lead levels in the general population are often lower in France than in some countries in Africa, the Middle East, from Southeast Asia, the West Indies and Eastern Europe. d These activities include crafts (print making, stained glass, ceramics), outdoor sports (hunting and fishing), liquor distillation, creating stained glass, enamelling copper, casting bronze, pottery with certain leaded glazes and paints; casting ammunition, jewellery making and electronics (with lead solder), glassblowing with leaded glass.

Table 4 .

 4 Univariate analysis of clinical characteristics according to the level of lead impregnation (n = 596).

		Blood Lead Level
		<25 µg/L (n = 572)	≥25 µg/L (n = 24)	p a
		n (%)	n (%)
	Age
	>35 years old	112 (19.6)	7 (29.2)	0.294
	Obstetrical history
	Spontaneous miscarriage	134 (26.4)	5 (21.7)	0.617
	Prematurity (<37 WG)	24 (10.2)	1 (4.3)	0.451
	IUGR	19 (3.8)	1 (4.3)	0.596
	Pregnancy hypertension	22 (4.4)	0 (0.0)	1
	Pregnancy course
	Anemia (Hb < 11 g/L)	137 (32.6)	7 (36.1)	0.701
	IUGR	28 (7.4)	3 (15.8)	0.177
	Pregnancy hypertension	15 (4)	0 (0)	1
	Prematurity (<37 WG)	37 (9.8)	1 (5.6)	1
	Low birth weight (<2500 g)	48 (14)	2 (14.3)	1

IUGR: Intrauterine Growth Restriction, a p-value from Wald test (binary logistic regression model).

Table 5 .

 5 Calculation of an overall score from exposure factors significantly associated with high blood lead levels (n = 596).

		Blood Lead Level ≥25 µg/L
	Score Q3 + Q7 + Q14 a	N	%
	0 (n = 179)	2	1.1
	1 (n = 293)	8	2.7
	2 (n = 107)	11	10.3
	3 (n = 17)	3	17.6

Acknowledgments:

The authors wish to thank the patients for their participation and the full staff of the maternity ward for their help with the study. Authors wish to specially thank N. Fabresse from the pole biologie-pathologie-APHM Timone's Hospital for his help with biological data collection.

Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. is supported by ARS PACA (Regional Agency for Health, Provence-Alpes-Cote d'Azur C2017-000265) and Region Sud Provence-Alpes-Cote d'Azur (C2017-1348) through funding of the Plateforme CREER (Couple reproduction Enfant Environnement et Risque).

Author Contributions: E.C., M.F. and N.H. contributed to patient recruitment, medical visits and data analysis. Questionnaires and lead level results were collected by E.C., M.F.; and C.S., E.C. and M.F. collated and analysed the data and wrote the final manuscript through collaboration. J.P., B.C. and H.H. conceived of and managed the study work. F.B. supervised the conceptualisation, design and methodology of the study. J.M. performed statistical analysis. C.P. supervised and performed biological analysis. All authors have read and agreed to the published version of the manuscript.

Conflicts of Interest:

The authors report no competing interests.