MD, DMD, PhD Neukam

MD Emeka Nkenke
email: emeka.nkenke@uk-erlangen.de

MD Falk Wehrhan
email: falk.wehrhan@uk-erlangen.de

Keywords: Craniofacial, Pathology, ONJ, Msx-1, aminobisphosphonate, bone, jaw

Objectives:

Bone-destructive disease treatments include bisphosphonates and antibodies against RANKL (aRANKL). Osteonecrosis of the jaw (ONJ) is a side-effect. Etiopathology models failed to explain their restriction to the jaw. The osteoproliferative transcription factor Msx-1 is expressed constitutively only in mature jaw bone. Msx-1 expression might be impaired in bisphosphonaterelated ONJ. This study compared the expression of Msx-1, BMP-2 and RANKL, in ONJ-affected and healthy jaw bone.

Material and Methods:

An automated immunohistochemistry-based alkaline phosphatase-anti-alkaline phosphatase method was used on ONJ-affected and healthy jaw bone samples (n=20 each): cell-number ratio (labelling index, Bonferroni adjustment). Real-time RT-PCR was performed to quantitatively compare Msx-1, BMP-2, RANKL, and GAPDH mRNA levels.

Results:

Labeling indices were significantly lower for Msx-1 (p<0.03) and RANKL (p<0.003), and significantly higher (p<0.02) for BMP-2 in ONJ compared to healthy bone. Expression was 7-fold lower (p<0.03) for Msx-1, 22-fold lower (p<0.001) for RANKL, and 8-fold higher (p<0.02) for BMP-2 in ONJ bone.

Conclusions:

Msx-1, RANKL suppression and BMP-2 induction were consistent with the bisphosphonateassociated osteopetrosis and impaired bone remodelling in BP-and aRANKL-induced ONJ. Msx-1 suppression suggested a possible explaination of the exclusivity of ONJ in jaw bone. Functional analyzes of Msx-1-RANKL interaction during bone remodeling should be performed in the future.

Msx-1 is suppressed in Bisphosphonate exposed jaw bone-analysis of bone turnover related cell signalling

Falk Wehrhan MD, DMD 1 ; Peter Hyckel MD, DMD, PhD 2 ; Kerstin Amann MD,PhD 3 ; Jutta Ries PhD 1 ; Phillip Stockmann,MD, DMD 1 ; Karl A. Schlegel MD, DMD, PhD 1 ,Friedrich W.

Introduction

Bisphosphonates (BP) ameliorate bone-destructive disorders by inhibiting bone resorption. During the past decade, amino-BP treatments have increased by more than 100-fold, due to their excellent efficacy in preventing pain and skeletal events due to bone-invading malignancies [START_REF] Schwabe | Mineralstoffpräparate und OSteoporosemittel[END_REF]. In 2003, the first case of BP-associated osteonecrosis of the jaw (ONJ) was described. That report has been substantiated by an increasing number of case reports and studies that described ONJ as a critical complication associated with long-term intravenous amino-BP treatments [START_REF] Marx | Pamidronate (Aredia) and zoledronate (Zometa) induced avascular necrosis of the jaws: a growing epidemic[END_REF]. Thus, the incidence of ONJ appears to be increasing in proportion to the increasing application of BP treatment [START_REF] Abu-Id | Bis-phossy jaws" -high and low risk factors for bisphosphonate-induced osteonecrosis of the jaw[END_REF]. A related therapy is based on inhibiting the receptor activator for nuclear factor κB ligand (RANKL), an osteoclast differentiation factor that stimulates bone resorption, with the humanized antibody, anti-RANKL (aRANKL). Recent clinical studies have shown that the occurrence of ONJ was also associated with aRANKL treatment; thus, both amino-BP and aRANKL treatments are associated with the development of ONJ [START_REF] Taylor | Osteonecrosis of the jaws induced by anti-RANK ligand therapy[END_REF].

Numerous studies have attempted to describe the pathology that might explain the restriction of ONJ to the jaws, but currently, no generally accepted etiology of ONJ is available [START_REF] Reid | Osteonecrosis of the jaw: who gets it, and why?[END_REF].

Early descriptions of ONJ as an avascular necrosis have been revised due to recent demonstrations of patent vascularisation in ONJ (Hansen et al., 2006b). Indeed, amino-BP treatment was found to improve vascularisation of avascular bone in cases of femur head necrosis and to accelerate fracture repair in extracranial skeletal bone [START_REF] Agarwala | Alendronate in the treatment of avascular necrosis of the hip[END_REF][START_REF] Matos | The effect of zoledronate on bone remodeling during the healing process[END_REF][START_REF] Amanat | Optimal timing of a single dose of zoledronic acid to increase strength in rat fracture repair[END_REF].

Several authors have reported an association between ONJ and local inflammation and infection; moreover, ubiquitous, intraoral actinomyces are commonly found in ONJ (Hansen et al., 2006a[START_REF] Bisdas | Biphosphonateinduced osteonecrosis of the jaws: CT and MRI spectrum of findings in 32 patients[END_REF][START_REF] Reid | Osteonecrosis of the jaw: who gets it, and why?[END_REF]. However, there is no evidence of any specific flora associated with [START_REF] Reid | Osteonecrosis of the jaw: who gets it, and why?[END_REF][START_REF] Belibasakis | Regulation of RANKL and OPG gene expression in human gingival fibroblasts and periodontal ligament cells by Porphyromonas gingivalis: a putative role of the Arg-gingipains[END_REF].

Another assumption was that low bone turnover following BP therapy was associated with the clinical appearance of ONJ [START_REF] Marx | Bisphosphonate-induced exposed bone (osteonecrosis/osteopetrosis) of the jaws: risk factors, recognition, prevention, and treatment[END_REF]. Histomorphological changes similar to osteopetrosis were found to be restricted to the jaw bone following BP-therapy consistent to the first description of ONJ as local osteopetrosis of the jaw bone [START_REF] Favia | Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: An analysis of 31 cases with confocal laser scanning microscopy[END_REF]. Moreover, BP therapy-related generalised osteopetrosis of the axial skeleton has only been found in pre-adolescent patients [START_REF] Whyte | Bisphosphonate-induced osteopetrosis: novel bone modeling defects, metaphyseal osteopenia, and osteosclerosis fractures after drug exposure ceases[END_REF].

Previous research that assumed ONJ was restricted to the jaws had not considered a potential differential interaction between amino-BP and extracranial skeletal and maxillofacial bone. In vitro studies have shown that jaw-derived and extracranial osteoblasts responded differentially to pamidronate [START_REF] Stefanik | Disparate osteogenic response of mandible and iliac crest bone marrow stromal cells to pamidronate[END_REF] and presented different patterns of proliferation and osseous differentiation marker expression [START_REF] Matsubara | Alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells[END_REF]. The jaw bone is derived from cranial neural crest cells (CNC); in contrast, extracranial bone is derived from mesenchymal stem cells (MsC); thus, the biologically unique features of CNC-derived bone should be considered in the etiopathology of ONJ [START_REF] Chung | Stem cell property of postmigratory cranial neural crest cells and their utility in alveolar bone regeneration and tooth development[END_REF]. Msx-1 is a transcription factor that induces proliferation and inhibits terminal differentiation of osteoblasts (Roybal et al.). Related to their embryonic origin in CNC, osteoblasts derived from the first branchial arch (jaw bone) express Msx-1 throughout adolescence; in contrast, Msx-1 is downregulated in MsC-derived bone after maturation [START_REF] Blin-Wakkach | Endogenous Msx1 antisense transcript: in vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals[END_REF][START_REF] Orestes-Cardoso | Msx1 is a regulator of bone formation during development and postnatal growth: in vivo investigations in a transgenic mouse model[END_REF]. In jaw bone, Msx-1 was shown to be expressed permanently, with the highest concentration in the periodontal ligament; however, in long extracranial bones, Msx-1 was shown to be only transiently reactivated during fracture repair [START_REF] Orestes-Cardoso | Msx1 is a regulator of bone formation during development and postnatal growth: in vivo investigations in a transgenic mouse model[END_REF][START_REF] Chung | Stem cell property of postmigratory cranial neural crest cells and their utility in alveolar bone regeneration and tooth development[END_REF][START_REF] Babajko | Msx1 expression regulation by its own antisense RNA: consequence on tooth development and bone regeneration[END_REF][START_REF] Ruhin-Poncet | Msx and dlx homeogene expression in epithelial odontogenic tumors[END_REF].

Several studies have shown that Msx-1 regulation plays roles in both tooth development and adult [START_REF] Babajko | Msx1 expression regulation by its own antisense RNA: consequence on tooth development and bone regeneration[END_REF][START_REF] Maxson | The Bmp pathway in skull vault development[END_REF]. In CNC-derived bone Msx-1 was also coexpressed with RANKL in CNC-derived osteoblasts and chondroblast progenitors (Houpis et al., Idowu et al., 2008[START_REF] Miah | Point mutations of 3BP2 identified in human-inherited disease cherubism result in the loss of function[END_REF]. Overexpression of Msx-1 has been postulated to cause cherubism, characterised by hyperproliferation and a honeycomb-like, cystic bone structure, which occurs exclusively in jaw bone [START_REF] Hyckel | Cherubism -new hypotheses on pathogenesis and therapeutic consequences[END_REF][START_REF] Ueki | Mutations in the gene encoding c-Abl-binding protein SH3BP2 cause cherubism[END_REF]. In giant cell granuloma of the jaw bone, presenting identical patho-histologic appearance to cherubism overexpression of Msx-1 has been demonstrated (Houpis et al.). Considering the restriction of ONJ to the jaw bone, we hypothesised that the etiology of ONJ might be related to a BP-induced impairment of Msx-1 expression. Moreover, Msx-1 was shown to play a pivotal role in the modulation of bone morphogenetic protein-2 (BMP-2)-related bone homeostasis. This data has spurred interest in investigating the role of Msx-1 in other homeostatic disorders involving bone structures that express Msx-1, such as alveolar bone [START_REF] Ruhin-Poncet | Msx and dlx homeogene expression in epithelial odontogenic tumors[END_REF][START_REF] Gersch | Reactivation of Hox gene expression during bone regeneration[END_REF].

In this study, based on the facts that BP suppresses RANKL expression and BMP-2/4 and Msx-1 interact during bone homeostasis, we performed a comparative analysis of Msx-1, BMP-2/4, and RANKL gene and protein expression in samples of BP-induced ONJ and healthy jaw bones [START_REF] Viereck | Bisphosphonates pamidronate and zoledronic acid stimulate osteoprotegerin production by primary human osteoblasts[END_REF][START_REF] Nishida | Apoptosisinducing effect of a new bisphosphonate, YM529, on various hematopoietic tumor cell lines[END_REF][START_REF] Nishida | Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2[END_REF][START_REF] Tsubaki | Nitrogen-containing bisphosphonate, YM529/ONO-5920, inhibits macrophage inflammatory protein 1 alpha expression and secretion in mouse myeloma cells[END_REF]. We hypothesized that Msx-1 expression would be diminished in the ONJ-affected jaw bones.

Material and Methods:

Patients and material sampling Jaw bone specimens from 40 patients were included in this study. ). The surgical procedure and the patient data were documented previously [START_REF] Stockmann | Osteotomy and primary wound closure in bisphosphonate-associated osteonecrosis of the jaw: a prospective clinical study with 12 months follow-up[END_REF]. The specimens used in this study were part of the tissue samples provided for routine histopathological diagnostics, and each displayed the histopathological characteristics of ONJ. In addition, our criteria for including the specimen in this investigation were: prior intravenous application of either pamidronate or zoledronate for at least 12 months and clinical evidence of exposed jaw bone for at least 8 weeks. Any specimens from patients with former radiotherapy were excluded. All specimens were obtained during clinically routine procedures and harvested from the same tissues needed for standard diagnostics; therefore, no surgical procedure specific to this study was performed and no additional material was harvested from the patients. In the ONJ group bone tissue, adjacent to the necrotic, exposed jaw bone surface was used. The bone specimens were part of the material which was resected. The necrotic tissue itself was excluded from the analysis, only solid bone pieces were included in the investigation.

Attached gingiva tissue was removed from the specimens.

The controls comprised 20 alveolar bone specimens, harvested during intraoral surgery procedures, from patients that did not have any BP-history and did not present any clinical signs of intraoral inflammatory processes or periodontitis. In the group of healthy jaw bone samples were harvested from bone material which was removed when bone surface was smoothened after dental extraction.

The average size of the 40 specimens used in this study was 5 × 3 × 3 mm. Each group of 20 specimens was divided into 2 equal parts. One part was immediately shock frozen at -80°C, and the other was immersed in RNA preserving reagent (RNALater, Quiagen, Hilden, Germany) for 24 h at 4°C, and then frozen and stored at -80°C.

Immunohistochemical staining

In preparation for immunohistochemical staining, tissue samples were fixed in 4% formalin; then, samples were decalcified (10% EDTA, pH 7.4), paraffin-embedded, cut into serial sections with a (Leica, Nussloch, Germany), and dewaxed in graded alcohol. Immunohistochemical staining was performed with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) method and an automated staining device (Autostainer plus, DakoCytomation, Hamburg, Germany), according to the manufacturer's instructions (Dako Real, Cat. K5005, DakoCytomation). Msx-1 was targeted with a polyclonal, rabbit-IgG, anti-human Msx-1 antibody (anti-Msx-1; M0944-100G, Sigma-Aldrich, Taufkirchen, Germany; dilution 1:100). BMP-2/4 was detected by a polyclonal, rabbit-IgG, anti-human BMP antibody (anti-human BMP 2/4, sc-9003, Santa Cruz, Santa Cruz, USA; dilution: 1:100). RANKL was targeted with a polyclonal, rabbit-anti-human RANKL antibody (sc-9073, Santa Cruz, dilution 1:100). Antibodies were incubated with tissue sections in the autostainer (20°C, 1h). The secondary antibody used for all primary antibodies, a biotinylated polyclonal, goat-anti-rabbit IgG (E 0466, DAKO, dilution 1:100), was applied according to the staining kit instructions. Visualisation of stained sections was performed with Fast Red solution, localised with biotin-associated activation (ChemMate-Kit, Dako), followed by incubation in hematoxylin for nuclear counterstaining. Two consecutive tissue samples were processed per immunohistochemical staining. One served as a negative control in each case (identical treatment, but replacement of the primary antibody with an istotype-IgG of the primary antibody). A known positive staining sample was also included in each series as a positive control.

Semiquantitative immunohistochemical analysis

Bone tissue sections were qualitatively evaluated under a bright-field microscope (Axioskop, Zeiss, Jena, Germany) at 100-400× magnification. We compared the numbers and localisations of stained osteocytes, osteoblasts, osteoblast progenitors, and fibroblasts in samples of ONJ-affected and healthy bone. In healthy bone, we observed bone trabecular, periosteal, and endosteal structures. In ONJ samples, we examined bone tissue attached to the necrotic bone defect. Within these areas, 3 visual fields per section for each sample were digitized at 200 × magnification with a CCD camera Randomised systematic subsampling was performed according to the method described by Weibel [START_REF] Weibel | Measuring through the microscope: development and evolution of stereological methods[END_REF]. We performed semiquantitative analyses of the cytoplasmic expression of Msx-1, BMP-2/4, and RANKL to determine the labelling index; i.e., the ratio of positively stained cells to the total number of cells per visual field, multiplied by 100.

Quantitative mRNA analysis and real-time, reverse transcription, polymerase chain reaction

Frozen tissues were agitated (Mixer Mill, Quiagen, Hilden, Germany) in lysis buffer (RNeasy Kit, QIAGEN, Hilden, Germany), and total RNA was extracted from tissues with the RNeasy Kit according to the manufacturer's protocol. The mRNA detected with each probe was quantitatively measured with a commercial microfluid Lab-on-a-Chip kit (Agilent RNA 6000 Pico Kit Agilent, Waldbronn, Germany) and the Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany). We BMP-2, and RANKL mRNA were analysed in duplicate, and normalised against GAPDH as an internal control gene. The mRNA from BP-affected tissue was expressed relative to the mRNA isolated from tissues from normal patients without ONJ. Relative gene expression was determined with the ∆∆Ct method. In order to exclude influences from other disorders that might be associated with the normal group of patients, RNA was isolated from healthy oral periosteum (pool of 15 patients) for calibration.

Statistical analysis

The labelling index per visual field of positively stained cells was used to analyse the immunohistochemical cytoplasmic staining and the spatial pattern of expression. To assess the relative gene expressions from the real-time RT-PCR products, we set the median gene expression levels of Msx-1, BMP-2, and RANKL observed in the pool of healthy oral periosteum as a calibrator. Then, the gene expressions in both the ONJ-affected and unaffected groups were expressed relative to the calibrator. Multiple measurements per study group were aggregated prior to analysis. The labelling indices and relative gene expression data were expressed as the median (ME) and the interquartile range (IQR). Graphical diagrams represented the median, the interquartile range, minimum (Min), and maximum (Max) values. Confirmatory comparisons between treatment and control groups were performed with generalised estimating equations (GEE) that used the "treatment modality", and the "subject id" as independent factors for appropriate

Results

Histochemical assessments revealed that all examined ONJ-affected samples displayed necrotic bone, represented by empty osteocyte lacunae, and vital bone zones. (Figs. 1C,2C,3C). The necrotic lesions consistently showed partial confluency, as described by other authors (6, 36).

Inflammatory infiltrates, including multinucleated cells, were present in all ONJ-affected samples.

Capillaries were observed in both ONJ-affected and normal bone samples.

Osteoblast Msx-1 expression and Msx-1-mRNA were reduced in ONJ-affected jaw bone

In the ONJ-affected bone, the density of cells that expressed Msx-1 was reduced compared to that observed in normal jaw bone (Fig. 1). However, the fact that Msx-1 protein was detected in both ONJ-affected and normal osteoblasts indicated that Msx-1 expression persisted in the jaw bone during adulthood, as described by other groups [START_REF] Orestes-Cardoso | Postnatal Msx1 expression pattern in craniofacial, axial, and appendicular skeleton of transgenic mice from the first week until the second year[END_REF][START_REF] Babajko | Msx1 expression regulation by its own antisense RNA: consequence on tooth development and bone regeneration[END_REF].

In normal jaw bone, the Msx-1 staining density was higher in periosteal and endosteal cells compared to trabecular cells. Also, the fibrous tissue surrounding the bone trabeculae displayed nuclear and cytoplasmic Msx-1 staining. In the ONJ group, Msx-1 staining was rare in endosteal cells, and the fibroblasts in the fibrous and inflammatory tissue surrounding the bone trabeculae displayed reduced Msx-1 expression. Next, we assessed the number of osteoblasts, osteocytes, and endosteal cells that expressed Msx-1 compared to the total number of bone-related cells. This labeling index (ratio of Msx-1 expressing cells:total number of bone related cells) showed that the relative cellular Msx-1 expression in the ONJ group was significantly diminished (p <0.03; ME: 19.01; IQR: 16.2) compared to that of normal bone (ME: 74.21; IQR: 32.0) (Fig. 1E). Next, we evaluated the relative levels of gene expression for Msx-1 (Fig. 1F). We used the mRNA of 15 healthy oral mucoperiosteal specimens as a calibrator, because Msx-1, BMP-2/4, and RANKL are constitutively expressed in oral mucoperiosteum [START_REF] Babajko | Msx1 expression regulation by its own antisense RNA: consequence on tooth development and bone regeneration[END_REF][START_REF] Teot | Extraosseous primary and recurrent giant cell tumors: transforming growth factor-beta1 and -beta2 expression may explain metaplastic bone formation[END_REF][START_REF] Spencer | Wnt signalling in osteoblasts regulates expression of the receptor activator of NFkappaB ligand and inhibits osteoclastogenesis in vitro[END_REF][START_REF] Leucht | Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration[END_REF][START_REF] Gersch | Reactivation of Hox gene expression during bone regeneration[END_REF]. We found that Msx-1 mRNA-expression was : 12.39, range: 3.15). When the Msx-1-mRNA expression level in oral mucoperiosteum was set to unity (ME: 1.00, IQR: 1.72), we found that the mean Msx-1 mRNA level was 7-fold lower in ONJ-affected than in healthy jaw bone.

BMP-2/4 expression and the density of connective tissue-related cells that expressed BMP-2/4 were increased in ONJ-affected bone

BMP-2/4 expression was detected in osteoblasts from both the healthy (Fig. 2A) and the ONJ-affected samples (Fig. 2C). The ONJ-affected jaw bone displayed a higher density of osteocytes that expressed BMP-2/4 compared to normal jaw bone. In addition, in ONJ-affected jaw bone, the periosteum and connective tissue adjacent to the bone margins displayed a higher density of cellular BMP-2/4 staining than samples of the extracellular matrix that covered the normal jaw bone. The median labeling index of osteoblasts and osteocytes that expressed BMP-2/4 was significantly higher in the ONJ-affected bone (p<0.02; ME: 60.43; IQR: 24.0) than in the control jaw bone (ME: 26.32; IQR: 16.0) (Fig. 2E). Moreover, BMP-2/4 mRNA expression was significantly elevated in the ONJ-bone (p<0.02; ME: 8.9; IQR: 6.1) compared to the control jaw bone (ME: 1.14; IQR: 1.07) (Fig. 2F). The BMP-2/4 mRNA expression was not significantly different between the control mucoperiosteum calibrator (ME: 0.84; IQR: 0.78) and normal jaw bone. The level of BMP-2 mRNA expression in ONJ-affected jaw bone was approximately 8-fold higher than in normal jaw bone.

Number of cells that expressed RANKL and RANKL-mRNA levels was dramatically reduced in the ONJ-affected bone

In normal jaw bone, RANKL expression was detected in the bone sections, including the periosteal soft tissue (Fig. 3A). However, in ONJ-affected jaw bone, RANKL was only sparsely expressed in endosteal and periosteal cells (Fig. 3C). In the soft tissues of ONJ-affected samples, the spatial distribution of RANKL expressing cells was inhomogeneous compared to that observed in the normal jaw bone. Local concentrations of multinucleated cells that expressed RANKL were detected only at zones of osseous resorption in ONJ samples (Fig. 3C,D). The overall density of cells that expressed RANKL was significantly lower in the ONJ-affected compared to normal jaw bone. This was reflected in the significantly reduced labeling index for cytoplasmic RANKL in ONJ-affected (p<0.003; ME: 22.63; IQR: 14) compared to normal jaw bone (ME: 53.45; IQR: 26) (Fig. 3E). RANKL mRNA expression was significantly lower (p<0.001) in ONJ-affected samples (ME: 69.8; IQR: 135.3) than that detected in healthy jaw bone (ME: 1538.80; IQR: 1322.34) and mucoperiosteal tissue samples (ME: 1.00; IQR: 147.33) (Fig. 3F). The levels of RANKL mRNA were 22-fold lower in the ONJ-affected jaw bone compared to normal jaw bone.

Discussion

This study was the first to investigate the influence of BP on jaw bone specific osseous differentiation in ONJ. The results showed that expression of the osteoproliferative transcription factor Msx-1 was significantly diminished in ONJ-affected jaw bone at both the protein (p<0.03) and mRNA (p<0.03) levels. Furthermore, we found that the expression of RANKL was significantly reduced in the bone surrounding the ONJ lesion, and the expression of BMP-2/4 was significantly elevated. These results indicated that the remodeling is suppressed in BP-compromised jaw bone. The immunohistochemical and molecularbiological findings in this study were consistent with those found in osteopetrotic bone [START_REF] Cohen | The new bone biology: pathologic, molecular, and clinical correlates[END_REF]. ONJ has previously been described to present histomorphology of local osteopetrosis [START_REF] Favia | Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: An analysis of 31 cases with confocal laser scanning microscopy[END_REF][START_REF] Marx | Bisphosphonate-induced exposed bone (osteonecrosis/osteopetrosis) of the jaws: risk factors, recognition, prevention, and treatment[END_REF] [START_REF] Lesclous | Bisphosphonate-associated osteonecrosis of the jaw: a key role of inflammation? Bone[END_REF]. The reduction of osteoclastic activity and overwhelming mineralization without adequate increase of blood supply was suggested to predispose to ischemic bone necrosis resulting in secondary inflammation and bacterial superinfection as seen in ONJ [START_REF] Favia | Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: An analysis of 31 cases with confocal laser scanning microscopy[END_REF] [START_REF] Barry | Osteomyelitis of the maxilla secondary to osteopetrosis: report of a case[END_REF]. Indeed, osteopetrosis is a known endogenous disorder associated with exposed bone and osteomyelitis of the jaw bone [START_REF] Barry | Osteomyelitis of the maxilla secondary to osteopetrosis: report of a case[END_REF][START_REF] Reid | Osteonecrosis of the jaw: who gets it, and why?[END_REF]. Osteopetrosis-like lesions have been reported following oral application of white phosphorus [START_REF] Abu-Id | Bis-phossy jaws" -high and low risk factors for bisphosphonate-induced osteonecrosis of the jaw[END_REF].

It has been suggested that a BP-related loss of Msx-1, especially in the periodontal ligament, might be associated with some ONJ characteristics, including a sclerotic, non-healing extraction socket and periapical hypermineralised thin lines around dental roots [START_REF] Chung | Stem cell property of postmigratory cranial neural crest cells and their utility in alveolar bone regeneration and tooth development[END_REF][START_REF] Babajko | Msx1 expression regulation by its own antisense RNA: consequence on tooth development and bone regeneration[END_REF]. The peridontal ligament has been found to harbour the highest endogenous concentration of Msx-1 in the jaw bone [START_REF] Ishii | Combined deficiencies of Msx1 and Msx2 cause impaired patterning and survival of the cranial neural crest[END_REF][START_REF] Orestes-Cardoso | Msx1 is a regulator of bone formation during development and postnatal growth: in vivo investigations in a transgenic mouse model[END_REF], Orestes-Cardoso et al., 2001). In rat periodontal ligamentum cells, BP treatment caused recruitment of bone forming cells, induction of osseous differentiation markers osteopontin and bone sialoprotein, and reduction of periodontal ligamentum cell numbers [START_REF] Lekic | Bisphosphonate modulates proliferation and differentiation of rat periodontal ligament cells during wound healing[END_REF]. Msx-1 has been reported to be critically involved in preventing terminal differentiation and stimulating proliferation of osteoblast progenitors; thus, the loss of Msx-1 is likely to be associated with poor osteoblast proliferation and overwhelming mineralisation in jaw bone [START_REF] Newberry | Stimulus-selective inhibition of rat osteocalcin promoter induction and protein-DNA interactions by the homeodomain repressor Msx2[END_REF][START_REF] Dodig | Identification of a TAATcontaining motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice[END_REF]. Msx-1 and Msx-2 functions have been shown to overlap in the developing jaw, but only Msx-1 expression continues into adulthood [START_REF] Berdal | Differential impact of MSX1 and MSX2 homeogenes on mouse maxillofacial skeleton[END_REF]. Msx-1 represses transcription of the terminal differentiation mediator osteocalcin by inhibiting the osteogenesis regulator, DLX5 [START_REF] Hoffmann | Transcriptional control of the tissue-specific, developmentally regulated osteocalcin gene requires a binding motif for the Msx family of homeodomain proteins[END_REF]. Msx-1 has been shown to be overexpressed in giant cell granuloma (Houpis et al.). Msx-1 has been postulated to be causative involved in the development and progression of cherubism.

Cherubism and giant cellular granuloma share both the histologic phenotype of hyperproliferation of non-mineralized bone tissue and their restriction to jaw bone [START_REF] Hyckel | Cherubism -new hypotheses on pathogenesis and therapeutic consequences[END_REF][START_REF] Ueki | Mutations in the gene encoding c-Abl-binding protein SH3BP2 cause cherubism[END_REF]. BP-induced sclerotic changes and hypermineralisation of exposed alveolar bone are consistent with the loss of Msx-1 dependent osteocalcin repression [START_REF] Favia | Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: An analysis of 31 cases with confocal laser scanning microscopy[END_REF] [START_REF] Shirakabe | Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5[END_REF]. Furthermore, systemic osteopetrosis following Amino-BP therapy has only been described in the premature, growing skeleton, consistent with the downregulation of Msx-1 in the extracranial skeleton after the maturation of bones [START_REF] Whyte | Bisphosphonate-induced osteopetrosis: novel bone modeling defects, metaphyseal osteopenia, and osteosclerosis fractures after drug exposure ceases[END_REF][START_REF] Whyte | Bisphosphonateinduced osteopetrosis[END_REF]. BMP-2 is an essential osteoinductor and inducer of bone mineralisation. Our finding that BMP-2 expression was significantly higher at both the protein (p<0.02) and mRNA (p<0.02) levels in ONJ-affected bone is consistent with the clinical and radiologic observations that ONJ-affected jaw bone appears osteopetrotic [START_REF] Marx | Bisphosphonate-induced exposed bone (osteonecrosis/osteopetrosis) of the jaws: risk factors, recognition, prevention, and treatment[END_REF]. Experimental and clinical findings have shown that BMP-2 and Msx-1 expression are partially antagonistic during jaw bone and dental development [START_REF] Dodig | Identification of a TAATcontaining motif required for high level expression of the COL1A1 promoter in differentiated osteoblasts of transgenic mice[END_REF][START_REF] Newberry | Stimulus-selective inhibition of rat osteocalcin promoter induction and protein-DNA interactions by the homeodomain repressor Msx2[END_REF]. BMP-2 is a morphogen that inhibits proliferation and mediates osseous differentiation and mineralisation. Msx-1 has been found to modulate signalling downstream of BMP-2 by antagonising the expression of the osteogenesis regulator, DLX5 [START_REF] Cohen | The new bone biology: pathologic, molecular, and clinical correlates[END_REF] [START_REF] Ryoo | Stage-specific expression of Dlx-5 during osteoblast differentiation: involvement in regulation of osteocalcin gene expression[END_REF]. Msx knockout animals showed unaltered BMP-2 expression [START_REF] Aioub | Msx2 -/-transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodental osteopetrosis[END_REF].

BP treatment has been shown to prevent osteoclast activation and bone resorption in malignancies and osteoporosis [START_REF] Nishida | Nitrogen-containing bisphosphonate, YM529/ONO-5920 (a novel minodronic acid), inhibits RANKL expression in a cultured bone marrow stromal cell line ST2[END_REF][START_REF] Viereck | Bisphosphonates pamidronate and zoledronic acid stimulate osteoprotegerin production by primary human osteoblasts[END_REF] primarily by suppressing RANKL activity. Thus, we were not surprised to find that the expression of RANKL was significantly reduced in ONJ-affected bone at both the protein (p<0.003) and mRNA (p<0.001) levels Clinically, BP was also shown to enhance fracture repair of extracranial skeletal bone; this suggested that BP could differentially impact RANKL-mediated bone remodelling in jaw and extracranial bone structures, even though the RANK/RANKL-OPG system regulates both CNC-derived bone and MsC-derived osteoblasts [START_REF] Agarwala | Alendronate in the treatment of avascular necrosis of the hip[END_REF][START_REF] Amanat | Optimal timing of a single dose of zoledronic acid to increase strength in rat fracture repair[END_REF][START_REF] Matos | The effect of zoledronate on bone remodeling during the healing process[END_REF]. Experimentally application of zoledronic acid induced inflammation of the jaw bone, but did not affect the axial skeleton (Senel et al.). The disparate effects of BP could be due to the parallel regulation of RANKL and Msx-1 expression in CNC-derived Msx-1 progenitors (Houpis et al., Idowu et al., 2008[START_REF] Miah | Point mutations of 3BP2 identified in human-inherited disease cherubism result in the loss of function[END_REF]. Experimental disruption of Msx resulted in significantly reduced RANKL expression and osteopetrosis in the jaw bone of a mouse model (Aioub et ). Despite our finding that RANKL expression was reduced throughout the ONJ-affected jaw bone, we observed histological evidence of osteoclast activity in the ONJ zone; this might be due to direct RANKL activation in osteoclasts by intraoral gram-negative bacteria, consistent with the findings from other groups [START_REF] Bisdas | Biphosphonateinduced osteonecrosis of the jaws: CT and MRI spectrum of findings in 32 patients[END_REF][START_REF] Reid | Osteonecrosis of the jaw: who gets it, and why?[END_REF][START_REF] Belibasakis | Regulation of RANKL and OPG gene expression in human gingival fibroblasts and periodontal ligament cells by Porphyromonas gingivalis: a putative role of the Arg-gingipains[END_REF]. The notion that local intraoral gram-negative bacteria might have induced bone resorption by the local activation of RANKL is supported by the clinical findings that periodontitis was reduced with application of the BP, alendronate [START_REF] Menezes | Effect of sodium alendronate on alveolar bone resorption in experimental periodontitis in rats[END_REF][START_REF] Reddy | Formulation and evaluation of Alendronate Sodium gel for the treatment of bone resorptive lesions in Periodontitis[END_REF].

The findings of this study may contribute to elucidate the differential impact of BP on CNCderived jaw bone remodelling compared to mesoderm-derived extracranial bone. ONJ development is associated with impaired jaw bone turnover and local osteopetrosis; both these processes have been linked to the loss of Msx-1 in alveolar bone [START_REF] Favia | Histologic and histomorphometric features of bisphosphonate-related osteonecrosis of the jaws: An analysis of 31 cases with confocal laser scanning microscopy[END_REF][START_REF] Aioub | Msx2 -/-transgenic mice develop compound amelogenesis imperfecta, dentinogenesis imperfecta and periodental osteopetrosis[END_REF]. To our knowledge, this is the first study to investigate unique features of CNC-derived bone in connection with BP treatment. Msx-1 is also known to be a key regulator of the Vitamin D receptor. Therefore, understanding the clinical impact of Msx-1 could be of therapeutic relevance in other diseases associated with impaired bone homeostasis [START_REF] Lezot | Does Vitamin D play a role on Msx1 homeoprotein expression involving an endogenous antisense mRNA?[END_REF]. The association of ONJ with aRANKL therapy supports the suggestion that the mechanism underlying the restriction of ONJ to the jaws may be CNC-specific cell signalling alterations [START_REF] Taylor | Osteonecrosis of the jaws induced by anti-RANK ligand therapy[END_REF][START_REF] Stopeck | Denosumab versus zoledronic acid for the treatment of breast cancer patients with bone metastases: rusults of a randomized phase 3 study[END_REF][START_REF] Henry | A double-blind, randomized study of denosumab versus zoledronic acid for the treatment of bone metastases in patients with advanced cancer (excluding breast and prostate cancer) or multiple myeloma[END_REF]. In agreement with leading international experts in the field of ONJ, we concluded that targeting the unique features of the jaw bone is a promising approach to elucidate the underlying pathologic mechanisms of ONJ [START_REF] Khosla | Bisphosphonate-associated osteonecrosis of the jaw: report of a task force of the American Society for Bone and Mineral Research[END_REF]. Of note, BP and aRANKL had differential impacts on proliferation, vascularisation, and surface marker expression jaw bone compared to extracranial bone derived structures [START_REF] Stefanik | Disparate osteogenic response of mandible and iliac crest bone marrow stromal cells to pamidronate[END_REF][START_REF] Matsubara | Alveolar bone marrow as a cell source for regenerative medicine: differences between alveolar and iliac bone marrow stromal cells[END_REF].

This suggests that BP effects on Msx-and RANKL-related interactions in CNC-and MsC-derived osteoblasts, osteoclasts, and bone structures should be investigated in more detail in the future. The relative level of Msx-1 mRNA in ONJ-affected bone was 7-fold lower than that observed in normal bone samples (p<0.03) (Fig. 1F). All samples were normalized to GAPDH expression, and the relative expression was calibrated to the Msx-1 mRNA concentrations found in healthy oral mucoperiostal tissue samples. In Fig. 2F the relative level of BMP-2 mRNA expression in ONJ-affected samples was 8-fold higher than that observed in controls (p<0.02); this substantiated the observation of overwhelming mineralisation. All samples were normalized to GAPDH expression, and the relative expression was calibrated to the BMP-2 mRNA concentrations found in healthy oral mucoperiostal tissue samples. The relative level of cellular RANKL expression (labeling index) was significantly diminished in ONJ-affected samples compared to controls (p< 0.003) (Fig. 3E).

The mean RANKL mRNA level detected in ONJ-affected bone samples were nearly 22-fold lower than that of control bone (p < 0.001) (Fig. 3F). All samples were normalized to GAPDH expression, and the relative expression was calibrated to the RANKL mRNA concentrations found in healthy oral mucoperiostal tissue samples.

  ONJ, even though ONJ progression and superinfection are promoted by gramnegative bacteria

  Zeiss, Jena, Germany) and Axiovision software (Axiovison, Zeiss, Jena, Germany).

  synthesised cDNAs from total RNA with the High Capacity cDNA Archive Kit (Cat. 4322171; Applied Biosystems, CA, USA) according to the manufacturer's protocol. Real-time reverse transcription (RT) quantitative polymerase chain reaction (qPCR) analyses were performed with the QIAGEN kits: Hs_BMP2_1_SG QuantiTect Primer Assay (200) (Cat. GT00012544) to amplify BMP-2, Hs_MSX1_SG QuantiTect Primer Assay (200) (Cat. GT00224350) to amplify Msx-1, and Hs_TNFSF11_va.1_SG QuantiTect Primer Assay (200) (Cat. QT01011381) to amplify RANKL;for normalisation, we used the Hs_GAPDH_1_SG QuantiTect Primer Assay (200) (Cat. QT00079247), to amplify GAPDH. We performed relative quantifications of mRNA with the ABI Prism 7300 Sequence Detection System (Applied Biosystems). We performed PCR amplification with the QuantiTect TM SYBR ® green PCR kit(Cat. 204143; QIAGEN). In total, 40 ng of cDNA was used for each PCR reaction in a total volume of 25 µl. Each PCR run included a 15 min activation time at 95°C, followed by 40 cycles of: denaturing at 94°C for 15 s, annealing at 55°C for 30 s, and extension at 72°C for 34 s. Undesired PCR side-products that might contribute to the assessed by melting curve analysis after PCR. The quantities of MSX-1,

  analysis of repeated measurements per individual. Multiple p values were adjusted according to Bonferroni by multiplying each p value obtained by the number of confirmatory tests performed (n=10). Two-sided adjusted p-values ≤0.05 were considered significant. All calculations were performed with SPSS 17.0 for Windows (SPSS Inc, Chicago, USA).

Figure legends Figure 1

 1 Figure legends

Figures

  Figures 1A and 1B (detail of 1A in black rectangle) demonstrate Msx-1, accentuated at the

(

  detail of 1C in black rectangle) show reduced expression of Msx-1 in ONJ-affected jaw bone.

Figure

  Figure1Eshows the relative number of cells that expressed the Msx-1 protein. Labeling index was

Figure 2

 2 Figure2

Figures

  Figures 2A and 2B (detail of 2A in black rectangle) show pronounced BMP-2/4 staining of

Figure 3 RANKL

 3 Figure 3

Figures

  Figures 3A and 3B (detail of 3A in black rectangle) represent cytoplasmic RANKL-staining at the

  Figure 1

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	alveolar bone regeneration
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	significantly reduced in ONJ-affected bone samples (p<0.03; ME: 1.81; range: 3.82) compared to
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  al.,

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Acknowledgements

The authors thank Heidemarie Heider, Andrea Kosel, and Miriam Ramming for operating the immunohistochemistry autostainer. In addition, we would like to thank Andrea Krautheim-Zenk for help with the mRNA and RT-PCR processing. All authors declare no conflict of interests.

Funding: This investigation was funded by the ELAN-Fonds of the University of Erlangen-Nuremberg, Germany Disclosure statement: All authors declare no disclosures.


Daniela Josa

Angeles Peña-Gallego
email: angeles.pena@usc.es

Jesús Rodríguez-Otero

Enrique M Cabaleiro- Lago

A MP2 and DFT study of the aromatic character of polyphosphaphospholes. Is the pyramidality the only

Keywords:

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

Introduction

The backbone of classical heterocyclic chemistry is fundamentally formed by five-membered heterocycles such as pyrroles, furans, and thiophenes together with six-membered pyridines.

Their chemistry has been continuously developed reaching huge proportions. These molecules have an essential role in biological chemistry and a recent interest in applied chemistry (as example, doped films of polypyrrole have electroconducting properties). However, the chemistry of phospholes has been underdeveloped when compared to its nitrogen, oxygen, and sulfur counterparts. In fact, the first phosphole was discovered as late as 1959 [1,2].

The aromaticity of the phosphole and its derivatives has been one of their most analysed properties [3]. The potential aromaticity of phosphole was discussed after its first practically applicable synthesis [4,5] was reported in the 1960s. In different reviews the nonaromatic behaviour of phospole was indicated [6][START_REF] Hughes | Phospholes and related Compounds[END_REF][START_REF] Quin | Cyclic Phosphines[END_REF]. This fact was explained in basis to the pyramidal preference of tricoordinate phosphorous in its compounds [START_REF] Coggon | [END_REF].

The planarity of the tricoordinate phosphorous can be influenced by substituents. In this sense, several cases have been studied: -acceptor groups either at phosphorus or at the neighbouring carbon [10,11], bulky substituents at phosphorus [12][13][14], etc. Some studies have analysed the replacement of -CH= units in the phosphole by -P=, resulting polyphosphaphospholes [15,16]. The main consequence of this replacement is the decrease of pyramidality and an enhancement of the aromatic character [15]. The recent interest in compounds with planar or partially planar tricoordinate phosphorous is focused in the relationship between aromaticity and pyramidality. In this article we discuss this fact but also introduce new important effects.

Aromaticity is a confused term due to it is not a physical observable [17]. The criterions to decide if a molecule is or not aromatic and the ways to quantify this aromaticity are innumerable [17][18][19][20][21][22][23].

It is well know that aromatization affects magnetic properties such as magnetic susceptibility and its anisotropy, leading to especially negative values for such properties [24][25][26]. These magnitudes are global properties, which can be affected by parts of the molecule not directly implicated in the aromaticity. To avoid this problem, the Nucleus Independent Chemical Shift (NICS) proposed by Schleyer et al. is very useful. The NICS is defined as the negative of the magnetic shielding [27] and it can be evaluated at any point of the molecule, exhibiting very negatives values in the center of aromatic rings.

The ACID (anisotropy of the current-induced density) is a new method based on magnetic properties and developed by Herges and Geuenich [28]. This method allows the visualization of the ring current formed when a magnetic field is applied and allows us to study the electronic delocalization in molecules [29,30].

Computational details

The geometry of each structure was optimized with the 6-311+G(d,p) basis set and the density functional theory (specifically, the Becke3LYP functional) [31,32] or Møller-Plesset perturbation level with the inclusion of energy corrections through second-order (MP2). All structures were minima as frequency calculations at same level have shown. In order to evaluate the relationship of planarity with aromaticity, geometries of 1-4b structures (Fig. 1) with planarity constraints were optimized at the B3LYP and MP2 level with 6-311+G(d,p) basis set. These planar structures were transition states.

The anisotropy of magnetic susceptibility values was calculated at the B3LYP/6-311+G(d,p) level using the IGAIM (Individual Gauges for Atoms in Molecules) method [33,34] on the B3LYP and MP2 optimized structures. In the NICS calculations, B3LYP/6-311+G(d,p) level with GIAO (Gauge-independent Atomic Orbital) method [35] were employed.

Finally, CSGT (Continuous Set of Gauge Transformations) method [33,34,36] at B3LYP/6-31+G(d) level of theory was employed in ACID calculations, carried out with the program supplied by Herges [28].

Quantum chemical calculations were carried out with the Gaussian98 [START_REF] Frisch | Gaussian Inc[END_REF] and Gaussian03

[38] program packages.

Results and discussion

<Figure 1>

Fig. 1 shows the polyphospholes studied in the present work. These structures, except for the pentaphosphole (5), are no planar. Table 1, collects the pyramidality data of these structures.

The values for different magnetic properties: anisotropy of the susceptibility (we have chosen this property instead of magnetic susceptibility due to the inherent problems of this property [39]) and NICS are shown in the same Table 1. The NICS criterion is based on the negative of the magnetic shielding computed at the centre of the ring. In general [40][41][42][43][44][45][46], negative values imply aromaticity (diatropic ring current) and positive values imply antiaromaticity (paratropic ring current). The NICS values have been obtained at ring critical point as defined by Bader [START_REF] Bader | Atoms in Molecules-A Quantum Theory[END_REF]48]. NICS values at points in the ring plane (NICS(0)) contain important spurious contributions from the in-plane tensor components that are not related to aromaticity as Schleyer has indicated [49]. For this reason, NICS(1) and NICS(-1) values (1 Å above/below the plane of the ring, Fig. 2) are showed in Table 1. These values reflect  effects and they are a better indicator of the ring current than values in ring plane, because at 1 Å the effects of the local -bonding contributions are diminished. The result obtained by the analysis of the values for these magnetic properties is the aromatic character of all these polyphosphaphospholes.

<Table 1>

The different values of NICS(1) and NICS(-1) indicate the different aromatization in each face of the molecules and the higher aromatization in the opposite side to the hydrogen atom joined to the phosphorous atom.

<Figure 2>

Different studies have looked for a direct connection between planarity and aromaticity in these compounds. In this sense, the search of more aromatic derivatives of the phosphole was reduced many times by the search of substituents that produced more planar structures. In this work we are interested in investigating this connection between planarity and aromaticity and if other factors should be considered. For this purpose the NICS values of all compounds are presented in Table 1 together with pyramidality data. The pyramidality at the tricoordinate phosphorous atom was measured by the sum of the bond angles around the P-H group, . At first sight a good correlation between planarity and aromaticity is observed. Even so, some details attract attention: the structures of 2a and 2b at B3LYP/6-31++G(d,p) level have very similar planarity values but its NICS(0) values differ in nearly two ppm, or, in the case of structures with three phosphorous atoms, 3a and 3c have similar  but very different NICS(0) values. In order to visualize the correlation between pyramidality and aromaticity, the anisotropy and NICS(1) and NICS(-1) values for all polyphospholes are plotted against the bond angle sums (Fig. 3). A rough relation between pyramidality and aromaticity is present but some facts seem indicate that a deeper analysis is recommended. If the four compounds with three phosphorous atoms are analyzed, we can observe that NICS(1) and NICS(-1) values for 3a are more negative than expected values if a perfect correlation between pyramidality and aromaticity exists at both DFT and MP2 method. This result seems to indicate that the extension of conjugation is greater when the phosphorous atoms are connected and the P-H group is an end of this connection. In this sense, 3d, structure where the three P atoms are not connected, is less aromatic than 3a, presenting a similar pyramidality.

<Figure 3>

Similar behavior is observed if compounds with two atoms of phosphorous are analyzed. So, the compound 2a with the phosphorous atom linked to P-H group is more aromatic than compound 2b, even though at B3LYP level the planarity of their structures is very similar.

We should remember that the evaluation of the absolute aromaticity of a compound remains a controversial issue [START_REF]See special issue 5 entirely dedicated to aromaticity[END_REF]. The main reason to this affirmation with regard to the magnetic properties as indication of aromatic character is the lack of a reference. For this reason, we have thought in the use of some system as reference value. We have chosen the planar structures corresponding to each system as reference structure. These planar structures are transition states. Table 2 shows the differences (NICS = NICS (pyramidal compound) -NICS (planar compound)) of the magnetic properties between the planar and pyramidal structures. As for the absolute values of magnetic properties, a direct relationship between pyramidality and aromaticity is not perfect. So, one of the structures with a smaller pyramidality ( = 337.8), the compound 4a, presents values even bigger than 3c, a structure with  = 328.0. But, when the distance between phosphorous atoms is analyzed, the patron is the same indicated for the absolute values of NICS and even the differences are more significant (for example, the difference between NICS for structure 3c and 3d is more than 3 ppm). The increase of this effect may indicate that the interaction between phosphorous atoms is larger in pyramidal structures than in planar structures.

<Table 2>

In order to carry out a deeper study of the aromaticity of the phosphole and these derivatives, the ACID method was employed. This is a relatively recently published method to investigate the delocalization and conjugation effects in molecules. It provides a powerful way to visualize the density of delocalized electrons and quantify conjugation effects. The ACID approach has several advantages: it is a scalar field which is invariant with respect to the relative orientation of the magnetic field and the molecule, it has the same symmetry as the wave function, and it can be plotted as an isosurface. In our group this method has been extensively employed in order to distinguish between pericyclic/pseudopericyclic and coarctate/pseudocoarctate reactivity, differentiation where the aromaticity has a crucial paper [51,52]. Fig. 4 presents the ACID isosurface of the transition states for these reactions at an isosurface value of 0.03 au. Current density vectors are plotted onto the ACID isosurface. The first point to emphasize is that Fig. 4 shows a strong diatropic ring current for all these compounds, indicating their aromatic character. This affirmation is opposite to other studies that have indicated that the pyramidality of phosphole prevented the aromaticity and it is in agreement with the previously calculated NICS values. In this figure the importance of the position of the -P= units with regard to the PH group is clearly shown. In this sense, the most obvious case is the comparison between 3c and 3d. In the last case the ring current in the P=P zone is smaller than in other area of the structure. This point may be more clearly observed if the critical isosurface values, CIV, are compared. The CIV indicates the isosurface value at which the topology changes from cyclic to noncyclic. Large CIV denotes aromaticity or antiaromaticity and a small CIV denotes disconnections. So, these values are a good tool to quantify the extent of conjugation. In this case and as an example, the structure 3d exhibits the smallest CIV (0.034) indicating a smaller aromaticity than other structures with similar  (the CIV for the 3a structure is 0.041). This result is in agreement with result obtained in the analysis of the NICS values and it confirms the important role of the position of P atoms.

<Figure 4>

Conclusions

This work allows to us to indicate the aromatic character of the phosphole and the studied polyphospholes. This aromaticity is shown by the negative values of the magnetic properties, and the ACID figures.

It is also interesting to notice the qualitative agreement between MP2 and DFT results.

In order to avoid the problem of lack of reference of the magnetic properties as indicator of the aromaticity, a model was employed: the magnetic properties of the structures are compared with the magnetic properties of these planar structures.

The relationship between aromaticity and pyramidality is showed through graphics of magnetic properties versus . But the main conclusion of this work is the importance for the aromaticity of these compounds of other aspects besides the pyramidality. In this sense, the influence of the position of -P= units has been clearly indicated.

Tables

Figure captions

Fig. 1 The polyphosphaphospholes object of the study

Fig. 2 Fig. 3

 23 Fig. 2 Figure indicating the position where NICS is evaluated

Fig. 4

 4 Fig.4ACID figures for the studied structures

Figure 1

 1 Figure 1Click here to download high resolution image

Figure 2

 2 Figure 2 Click here to download high resolution image

Figure 3

 3 Figure 3 Click here to download line figure: Figure3.doc

Figure 4

 4 Figure 4Click here to download high resolution image

Table 1

 1 Values of  and magnetic properties for the optimized structures at B3LYP and

	MP2 level
		(º)	 anis /cgs-ppm	NICS (0)/ppm	NICS (1)/ppm	NICS (-1)/ppm
		DFT	MP2	DFT	MP2	DFT	MP2	DFT	MP2	DFT	MP2
	1	292.4	294.1	-42.5	-44.0	-5.9	-6.2	-5.6	-5.9	-6.1	-6.5
	2a	300.7	308.8	-53.2	-54.6	-7.8	-8.7	-7.2	-7.8	-7.4	-8.4
	2b	300.4	302.6	-50.8	-52.5	-5.6	-6.0	-6.7	-7.1	-7.2	-7.6
	3a	309.1	318.0	-65.9	-67.9	-8.3	-9.6	-8.2	-9.0	-8.9	-9.9
	3b	312.6	321.4	-63.5	-66.0	-7.8	-9.2	-8.3	-9.2	-8.8	-9.9
	3c	316.8	328.0	-67.3	-68.7	-9.6	-11.1	-9.0	-9.9	-9.1	-10.4
	3d	310.9	317.2	-64.4	-67.6	-6.7	-7.8	-7.9	-8.7	-8.6	-9.4
	4a	324.6	337.8	-83.1	-87.4	-10.2	-12.7	-10.0	-11.3	-11.0	-12.5
	4b	329.0	339.7	-83.7	-85.8	-11.3	-13.0	-10.7	-11.6	-11.2	-12.4
	5	360.0	359.9	-113.0	-111.9	-17.7	-17.6	-15.1	-15.0	-15.1	-15.2

Table 2

 2 Difference between NICS for planar and pyramidal structures

		NICS (1)/ppm NICS (-1)/ppm
		DFT	MP2	DFT	MP2
	1	5.0	4.8	4.5	4.2
	2a	4.6	4.0	4.4	3.4
	2b	4.4	4.1	3.9	3.5
	3a	4.6	3.8	3.9	2.9
	3b	3.9	3.1	3.4	2.4
	3c	3.5	2.6	3.4	2.2
	3d	4.3	3.8	3.6	3.1
	4a	3.8	2.5	2.8	1.3
	4b	2.9	1.9	2.4	1.2

Acknowledgments

The authors thank the Xunta de Galicia for financial support "Axuda para a Consolidación e Estructuración de unidades de investigación competitivas do Sistema Universitario de Galicia, 2007/50, cofinanciada polo FEDER 2007-2013". The authors want to express their gratitude to the CESGA (Centro de Supercomputación de Galicia).


Manon ; Van Eijsden

Tanja Vrijkotte

Reinoud Gemke

Marcel F Van Der Wal

Tgm Vrijkotte

Rjbj Gemke

The Amsterdam Born Children and their Development (ABCD) study

Keywords: Pregnancy, Infant, Child pregnancy, pregnancy outcome, prenatal exposure delayed effects, prenatal nutritional physiological phenomena, lifestyle, eating, micronutrients, ethnic groups, socioeconomic factors, prospective studies, infant, child, child development, child behavior, Netherlands

come   L'archive ouverte pluridisciplinaire

2 pregnancy outcomes (fetal origins of disease hypothesis) [START_REF] Barker | Mothers, babies and health in later life[END_REF] formed the basis of the Amsterdam Born Children and their Development (ABCD) study, which was established in 2003. This large prospective population-based cohort study examines the association between maternal lifestyle, medical, psychosocial, and environmental conditions during pregnancy and children's health at birth as well as in later life. Specific attention is paid to ethnic disparities.

Consequently, the study has detailed measurements of ethnic background, including country of birth of the pregnant woman herself, country of birth of her parents, and ethnic identity.

The study is a collaboration between the Public Health Service of Amsterdam and the two university medical centers in Amsterdam (Academic Medical Center (AMC) and VU University Medical Center (VUmc)), and has been implemented in cooperation with hospitals and midwives, Youth Health Care (YHC) centers, primary schools, and the University of Tilburg.

What does the study cover -and how has this changed?

The main objectives of the ABCD study are:

1) to investigate children's health at birth as well as in later life, and ethnic disparities therein;

2) to investigate maternal lifestyle, medical, psychosocial, and environmental conditions during pregnancy, and ethnic disparities therein;

3) to investigate the extent to which (ethnic disparities in) maternal conditions during pregnancy explain (ethnic disparities in) children's health at birth as well as in later life.

However, a fourth objective has been added over the past few years, following mounting evidence that also postnatal conditions may have important consequences for health and disease in later life: 9 ) to investigate early-life conditions, and ethnic disparities therein, and the extent to which these conditions explain children's health in later life.

(Figure 1 here)

The pregnancy and early-life conditions and outcomes of interest in the ABCD study are broadly defined into the research areas shown in Figure 1. The main maternal and environmental risk factors concern nutrition and metabolism, psychosocial stress, substance abuse, medical conditions, and air pollution. The main outcome parameters concern birth outcomes and infant growth and, when the children are older, obesity and metabolic syndrome, cognitive development, psychosocial health, and nutrition. More information is available on our website: www.abcd-study.nl.

Results of the study will be used to improve prenatal care and to develop culture-specific intervention strategies to promote the health of all children from the earliest age possible.

(Table 1 here)

Who is in the sample?

Between January 2003 and March 2004, all pregnant women living in Amsterdam were asked to participate in the ABCD study during their first prenatal visit to an obstetric care provider (general practitioner, midwife, or gynecologist).Altogether, 12 373 women were approachedby estimate, ≥ 99% of the target population. According to Dutch law, all pregnant women, including illegal immigrants and asylumseekers, are entitled to receive prenatal care, which is free of charge if costs are a problem. For all of the women approached, the care provider Service by prepaid mail. A reminder was sent two weeks later. The questionnaire included an informed consent sheet the women could use to grant permission for follow-up of their infants at the age of 3 months and every 5 years thereafter, and for the perusal of their medical files.

In addition, women were invited to participate in the ABCD biomarker study. For this, an extra blood sample (10 ml EDTA and 9 ml serum) was taken during routine blood collection for screening purposes following the first antenatal check-up.

Of the 12 373 women approached, 8266 women filled out the pregnancy questionnaire (response rate: 67%). Of this group, 7050 women granted permission for follow-up (85%) and 7043 women granted permission for perusal of her and her child's medical files (85%). A total of 4389 women (53%) participated in the biomarker study. To enhance participation among foreign-born women, two supportive measures were taken: (1) A Turkish, Arabic, or English translation was provided to women born in Turkey, Morocco, or other non-Dutchspeaking countries, and (2) the possibility of completing the questionnaire orally was offered to women who were illiterate or had reading difficulties. Although the participation rates among foreign-born women (42% to 64%) were still lower than the participation rate among Dutch-born women (77%), they were comparable with response rates observed in other population-based multi-ethnic studies in the Netherlands. [START_REF] Fassaert | Acculturation and use of health care services by Turkish and Moroccan migrants: a cross-sectional population-based study[END_REF]11,[START_REF] Van Der Meer | Fatty fish and supplements are the greatest modifiable contributors to the serum 25-hydroxyvitamin D concentration in a multiethnic population[END_REF] To investigate the degree of selection bias resulting from this ethnic nonresponse, we conducted a nonresponse analysis by probabilistic medical record linkage with the Netherlands Perinatal Registry; results indicated that selection bias was minimal. [START_REF] Tromp | Anonymous non-response analysis in the ABCD-cohort study enabled by probabilistic record linkage[END_REF] Table 1 shows the sociodemographic characteristics of the participants according to country of birth. The questionnaire focused primarily on the mother, however paternal information was available for country of birth (58.0% Dutch, 6.5% Surinamese, 4.6% Turkish, 8.5% Moroccan, 13.1% other non-Western, 6.8% other Western, and 2.5% unknown), employment status (81.2% employed) and height (mean 180.9 cm, SD 11.5).

Approval of the study was obtained from the Central Committee on Research Involving Human Subjects in the Netherlands, the medical ethics review committees of the participating hospitals, and the Registration Committee of the Municipality of Amsterdam.

(Figure 2 here) (Figure 3 here)

How often have they been followed-up and what is attrition like?

Figure 2 shows the structure of the ABCD study. Of the 8266 respondents, 7863 women gave birth to viable singleton infants and 132 women gave birth to viable multiples. The remaining mothers either experienced a miscarriage or fetal death (n=92) or were lost-to-follow-up (n=179) with no registered birth and no information on miscarriage or fetal death available from the care provider. By law, all children born in Amsterdam after 24 weeks' gestation (either stillborn or liveborn) must be registered at the municipality's registry office, after which a 'mutation report' is sent to the Public Health Service's YHC department. Between the 4th and 7th day after delivery, YHC nurses visit the liveborn infants and their parents to obtain a blood sample to screen for congenital metabolic disorders. At this time they also record the date of delivery, infant sex, birth weight, and gestational age (based on ultrasound or, if unavailable (< 10%), on the timing of the last menstrual period) as provided by the obstetric care provider. Three months after giving birth, the mothers who gave permission for follow-up (6735 mothers of singletons, 119 mothers of multiples) received a questionnaire concerning the course of their pregnancy and delivery, the baby's health, development, and growth, and the mother's lifestyle during and after pregnancy (phase II). Again, the questionnaire was available in multiple languages to increase the response in specific ethnic groups. A total of 5218 mothers (5131 mothers of singletons, 87 mothers of multiples) filled out this questionnaire (response rate: 76%). Compared with the initial sample, this group was older, more highly educated, of lower parity, and more often of Dutch origin. However, we found no effect of attrition bias in our first follow-up study, which investigated the association of maternal stress and emotional problems with infant crying. [START_REF] Van Der Wal | Stress and emotional problems during pregnancy and excessive infant crying[END_REF] After the 3-month questionnaire, follow-up measurements were planned for singleton infants only. Until the children are 4 years old, specially trained YHC nurses conduct an average of 14 standardized routine measurements to monitor their growth and feeding patterns. Of the 6735 women who gave birth to liveborn singleton infants, 6575 women gave permission to collect growth data. Data collection is ongoing, and so far, growth data (including feeding type and duration) of 5273 ABCD children have been digitized.

At the current stage, 6161 of the 6735 mothers who gave permission for follow-up are being approached for the 5-year follow-up measurement of their child (phase III, 2008-2010).

Attrition in this follow-up number is due to withdrawal, infant or maternal death, and loss-tofollow-up as a result of an unknown address or emigration. Details of the phase III follow-up are given below ('What has been measured?'). In short, mothers receive two questionnaires about the child's health, development, and behavior, with one of them to be filled out by the child's teacher. In addition, the children are invited to participate in the ABCD health check, which takes place at school. Efforts to enhance participation among all women and children, regardless of ethnicity and education, include the use of translated questionnaires, an

F o r R e v i e w O n l y
information leaflet, and a specially developed cartoon that explains the health check measurements to the children (Figure 3). Also, women from ethnic minority groups who do not respond within a month are approached by phone by trained students who explain the study in the women's own language. Data collection is ongoing, and preliminary results show a response rate of 66% for the general questionnaire (n=4060 out of the 6161 mothers approached) and a response rate of 58% for the teacher questionnaire. Of the participating mothers, 94% gave permission for the health check. Further follow-up measurements will take place every five years (in 2013 at age 10, in 2018 at age 15, etc) until adulthood.

(Table 2 here)

What has been measured?

Table 2 provides an overview of the measurements in the three subsequent phases of the study (details are available from the authors upon request). In phase I, the maternal micronutrient status (including vitamins A, D, B 12 , folate, iron, Zn, Mg, Ca, and n-3 and n-6 fatty acids), metabolic function (including cholesterol, triglycerides, and thyroid hormone levels), and cortisol levels were measured in blood samples collected at a median of 13 weeks' gestation (interquartile range (IQR): 12-14 weeks). The pregnancy questionnaire was filled out at an average of 16 weeks' gestation (IQR: 14-18 weeks) and included ethnicity-related and sociodemographic variables, lifestyle, psychosocial conditions, and obstetric/medical information. Whenever possible, validated questionnaires were included, such as the CES-D questionnaire for depression [START_REF] Radloff | The CES-D scale: a self-report depression scale for research in the general population[END_REF] and the Job Content Questionnaire for job strain. [START_REF] Karasek | The Job Content Questionnaire (JCQ): an instrument for internationally comparative assessments of psychosocial job characteristics[END_REF] Furthermore, residential information was used to model individual exposure to traffic-related air pollution (in collaboration with the Institute of Risk Assessment Sciences at Utrecht University). Information on birth outcomes was obtained from YHC, as mentioned previously. Supplementary information concerning complications during pregnancy and neonatal outcomes was obtained by probabilistic medical record linkage with the Netherlands Perinatal Registry. [START_REF] Tromp | Anonymous non-response analysis in the ABCD-cohort study enabled by probabilistic record linkage[END_REF] The questionnaire filled out by mothers in phase II concerned the course of their pregnancy and delivery, their physical and psychosocial health, and the development and health of their child. The questionnaire was filled out at the average infant age of 13 weeks (IQR: 12-13 weeks). Follow-up data from YHC included infant nutrition (ie, duration of breast-feeding and/or bottle-feeding; time of introduction of solid foods), and infant and child weight, length/height, and head circumference at an average of 8 time points in the first 14 months, and 6 time points between the ages of 14 months and 4 years.

In the current phase (phase III), participating mothers are sent a questionnaire approximately two weeks after their ABCD child's 5th birthday. In addition to items about the child's health, development, and behavior, this general questionnaire contains items about family sociodemographics, maternal lifestyle and psychosocial conditions, and family history of medical conditions. The questionnaire is accompanied by a questionnaire about the child's performance, behavior, and CITO (Central Institute for Test Development) index for elementary education, to be filled out by the child's teacher. In both questionnaires, validated questions and scales were used wherever possible (eg, the Strengths and Difficulties Questionnaire for behavior). [START_REF] Goodman | The Strengths and Difficulties Questionnaire: a research note[END_REF] The general questionnaire includes an informed consent sheet for granting permission for the child's participation in the ABCD health check. Two weeks before the health check, mothers are notified by letter and receive an additional self-administered food frequency questionnaire (FFQ). The FFQ was developed by TNO Food (Zeist, the Netherlands). It consists of 71 food items for which the frequency of consumption and portion size are to be reported. A validation study comparing the FFQ with the gold standard of doubly labeled water showed that the questionnaire is a valid instrument for estimating mean energy intake in a group of 4-to 6-year-old children and for ranking subjects according to their energy intake; the Pearson correlation coefficient between energy intake and energy expenditure was 0.62. [START_REF] Dutman | Validation of a food frequency questionnaire and options for data processing using the doubly labeled water method in children[END_REF] For children living in Amsterdam, the health check is held at the child's primary school. Children and parents who have moved outside of Amsterdam and children enrolled in small schools with no space available for the health check are invited to a central location during the weekend and holidays. The health check itself consists of a fasting capillary blood sample for assessing glycaemic control and lipid profile (glucose, C-peptide, total cholesterol, HDL, LDL, triglycerides) as well as fatty acid profile; physical measurements; and a cognitive test battery. The physical measurements include anthropometric measurements (height, weight, waist and hip circumference), body composition measures (fat mass and fatfree mass) by bioelectrical impedance analysis, blood pressure in supine and sitting positions, and cardiovascular function measures (heart rate, heart rate variability, and pre-ejection period) in supine and sitting positions using the VU Ambulatory Monitoring System. [START_REF] De Beer | Early growth patterns and cardiometabolic function at the age of 5 in a multiethnic birth cohort: the ABCD study[END_REF] Cognitive function is examined using four tasks from the Amsterdam Neuropsychological Tasks (ANT) program. These tasks estimate sensory-motor speed, visuomotor skills, and response selection. 20

What has it found ?

A full list of publications is available on our website (www.abcd-study.nl). The main findings to date are summarized below. Data collection for phase III is ongoing, and papers are in preparation. Factors found to be related to adverse pregnancy outcomes (stillbirth or miscarriage, low birth weight, or preterm birth) include having high levels of thyroid-stimulating hormone, [START_REF] Benhadi | High maternal TSH levels in pregnancy are associated with increased risk for miscarriage, fetal or neonatal death[END_REF] folate depletion, [START_REF] Van Eijsden | Association between short interpregnancy intervals and term birth weight: the role of folate depletion[END_REF] an adverse fatty acid profile, [START_REF] Van Eijsden | Maternal n-3, n-6, and trans fatty acid profile early in pregnancy and term birth weight: a prospective cohort study[END_REF] smoking, overweight/obesity, high blood pressure and a low educational level, [START_REF] Goedhart | Ethnic differences in term birthweight: the role of constitutional and environmental factors[END_REF][START_REF] Goedhart | Ethnic differences in preterm birth and its subtypes: the effect of a cumulative risk profile[END_REF] living in a poor neighborhood, [START_REF] Agyemang | The effect of neighbourhood income and deprivation on pregnancy outcomes in Amsterdam, the Netherlands[END_REF] and having work-related stress. [START_REF] Vrijkotte | First trimester working conditions and birth weight: a prospective cohort study[END_REF] Maternal stress and mood disorders, including work-related stress as well as pregnancy-related anxiety or depression, were associated with continuing to smoke during pregnancy [START_REF] Goedhart | Psychosocial problems and continued smoking during pregnancy[END_REF] and an increased risk of having an excessive crying infant. [START_REF] Van Der Wal | Stress and emotional problems during pregnancy and excessive infant crying[END_REF] No association was found between maternal stress and mood disorders and preeclampsia or gestational hypertension, nor between maternal physical activity and these outcomes. [START_REF] Vollebregt | Is psychosocial stress in first ongoing pregnancies associated with pre-eclampsia and gestational hypertension?[END_REF][START_REF] Vollebregt | Does physical activity in leisure time early in pregnancy reduce the incidence of preeclampsia or gestational hypertension?[END_REF] Maternal prepregnancy BMI was associated with infant BMI at age 14 months. [START_REF] Mesman | Maternal pre-pregnancy body mass index explains infant's weight and BMI at 14 months: results from a multi-ethnic birth cohort study[END_REF]

Risk factor-outcome associations

Ethnic disparities

In the above-mentioned pregnancy outcomes, large differences were observed between ethnic groups. Particularly among women from Surinamese, Antillean, and Ghanaian background, low birth weights and preterm births were more prevalent. [START_REF] Goedhart | Ethnic differences in term birthweight: the role of constitutional and environmental factors[END_REF][START_REF] Goedhart | Ethnic differences in preterm birth and its subtypes: the effect of a cumulative risk profile[END_REF] Ethnic disparities were also observed in the start of prenatal care (ie, the timing of the first visit to a care provider), [START_REF] Alderliesten | Late start of antenatal care among ethnic minorities in a large cohort of pregnant women[END_REF] use of folic acid supplements, [START_REF] Van Eijsden | Folic acid knowledge and use in a multi-ethnic pregnancy cohort: the role of language proficiency[END_REF] maternal thyroid function, [START_REF] Benhadi | Ethnic differences in TSH but not in free T4 concentrations or TPO antibodies during pregnancy[END_REF] and the previously mentioned risk factors. [START_REF] Goedhart | Ethnic differences in term birthweight: the role of constitutional and environmental factors[END_REF][START_REF] Goedhart | Ethnic differences in preterm birth and its subtypes: the effect of a cumulative risk profile[END_REF][START_REF] Vrijkotte | First trimester working conditions and birth weight: a prospective cohort study[END_REF][START_REF] Van Eijsden | Ethnic differences in early pregnancy maternal n-3 and n-6 fatty acid concentrations: an explorative analysis[END_REF] The observed disparities in birth weight and preterm birth were explained in part by these risk factors and their accumulation. [START_REF] Goedhart | Ethnic differences in term birthweight: the role of constitutional and environmental factors[END_REF][START_REF] Goedhart | Ethnic differences in preterm birth and its subtypes: the effect of a cumulative risk profile[END_REF] What are the main strengths and weaknesses of the study?

The study's main strengths relate to its unique design and mode of operation. It was implemented during routine care, which allowed for detailed measurement in early pregnancy and, currently, for detailed follow-up of children's growth and development. Collecting maternal blood samples in conjunction with routine blood collection for screening purposes Biochemical analyses generally allow for a more objective measure of dietary intake [START_REF] Wild | A critical evaluation of the application of biomarkers in epidemiological studies on diet and health[END_REF][START_REF] Hunter | Biochemical indicators of dietary intake[END_REF] and have the advantage of not suffering from culture-specific bias, a desirable feature for our multiethnic cohort. A drawback of this design relates to the potential changes in exposure occurring in late pregnancy, which we did not measure. However, our choice to focus on early pregnancy was based on existing evidence that the trajectory of human development is set at this stage. [START_REF] Godfrey | Fetal programming and adult health[END_REF] In addition, changes are most likely to be attenuations (eg, women with the most stressful working conditions going on leave earlier). [START_REF] Croteau | Work activity in pregnancy, preventive measures, and the risk of delivering a small-for-gestational-age infant[END_REF] The response rate of the study (initial participation, phase I) was 67%, which is lower than, for example, large population-based pregnancy cohorts in the UK, such as the Southampton Women's Survey (75%) [START_REF] Inskip | Cohort profile: the Southampton Women's Survey[END_REF] and ALSPAC (85%) 41 but higher than, for example, the Generation R study in Rotterdam, the Netherlands (61%). [START_REF] Jaddoe | The Generation R Study: design and cohort profile[END_REF] Particularly in an era where response rates to large epidemiological studies are decreasing, [START_REF] Galea | Participation rates in epidemiological studies[END_REF] our response rate may be considered adequate. Supportive measures were taken to enhance enrolment of ethnic minority women in particular (translated questionnaires and completing them orally with trained female interviewers). As mentioned earlier, although selective participation could not be prevented, this resulted in a minimal degree of selection bias. [START_REF] Tromp | Anonymous non-response analysis in the ABCD-cohort study enabled by probabilistic record linkage[END_REF] Even though the current response rate is 66% (phase III, preliminary results), we need to make even more of an effort to include all ethnic minority groups in the cohort. Ideally, the study would once more provide female trained interviewers who can help the ethnic minority women with the questionnaires in their own language. However, the costs to do so are substantial, and in the Netherlands funding agencies are not yet prepared to finance longitudinal studies at the level that is necessary. So far, the study has continued with a minimum of funds, thanks to a small group of dedicated researchers and technical support staff. In this context, the help of the Because the study has been a collaborative effort between the Public Health Service and the university medical centers from the very start, it integrates both clinical and public health relevance. This is an important strength in terms of the study's contribution to health care and prevention. Results of the study are disseminated to health care workers, obstetric care providers, pediatricians, and policymakers on a regular basis, and have, for instance, led to a special prenatal educational program for pregnant Turkish women that focuses on smoking and psychosocial health. [START_REF] Jurg | Draaiboek Blije Moeders, Blije Baby's: interventie voor Turkse zwangeren [Handbook Happy Mothers, Happy Babies: an intervention for pregnant Turkish women[END_REF]

Can I get hold of the data?

Where can I find out more?

The ABCD study welcomes collaboration and the interest of colleagues. A set of guidelines for researchers interested in using data for research purposes is available at www.abcdstudy.nl. In brief, we request a short research proposal that should include information on the background, research questions and methods, a plan for publication, timetable, and budget.

Approval of a proposal depends upon the topic and the quality of the proposal. If topics are already the subject of research in progress, then access may be refused or subject to constraints. Collaborations are established through a formal contract, which includes mutual obligations (data delivery, rules for publication and presentation, contribution to the ABCD infrastructure, etc).

For more information, please see our website or contact one of the principal investigators at abcd@ggd.amsterdam.nl. -----------------------------------------------PHASE I ---------------------------------------------------------------------------------------------------------PHASE II ---------------------------------------------------------

- -----------------------------------------------PHASE III AND BEYOND-------------------------------------

  form which included personal data such as name, address, and date of birth. Based on this information, a questionnaire covering sociodemographic characteristics, obstetric history, lifestyles, and psychosocial conditions was sent to the pregnant women within two weeks, to be filled out at home and returned to the Public Health

  to use biomarkers to measure nutritional status in early pregnancy.

  providers, YHC, and currently also the primary schools has been indispensable to the data collection.

Figure 2 Figure 3 F o r R e v i e w O n l y 22

 2322 Figure 2 Structure of the ABCD study * attrition at this stage due to withdrawal, infant or maternal death, and unknown address or emigration

F o r R e v i e w O n l y

Table 1

 1 Response rates and maternal characteristics in the study population according to country of birth*

			Total	Dutch	Surinamese	Turkish	Moroccan	Other NW	Other W
	N		8266	5082	462	343	607	1040	732
	Response rates (%)
	biomarker study	53.1	59.0	44.8	41.4	36.7	41.4	53.0
	permission medical files	85.2	89.4	77.1	74.3	68.9	80.5	86.7
	permission follow-up	85.3	91.3	76.7	70.8	69.9	78.6	88.8
	Maternal age (%)
	<25 y		13.8	10.4	17.5	40.8	25.5	17.7	7.5
	25-29 y		22.0	18.1	21.0	28.9	35.6	31.8	20.8
	30-34 y		40.4	45.8	35.7	19.0	24.2	31.7	41.8
	≥35 y		23.8	25.7	25.8	11.4	14.7	18.8	29.9
	Maternal education (%)
	<6 y		23.0	11.4	41.8	63.8	59.0	41.6	16.4
	6-10 y		37.9	37.6	44.4	29.4	33.3	38.5	42.8
	≥11 y		38.1	50.7	11.0	5.8	5.3	17.0	39.9
	unknown		1.0	0.3	2.8	0.9	2.5	2.9	1.0
	Parity (%)
	0		55.4	60.6	39.4	37.3	40.2	45.5	64.9
	1		31.4	31.9	30.7	35.9	27.2	32.6	28.1
	≥2		13.2	7.5	29.9	26.8	32.6	21.9	7.0
	BMI (%) <18.5 kg/m 2 18.5-24.9 kg/m 25.0-29.9 kg/m ≥30.0 kg/m 2	2 2	5.0 71.4 17.3 6.3	4.7 77.5 13.6 4.2	6.5 55.0 23.4 15.2	5.0 61.2 22.2 11.1	2.6 51.4 33.3 12.5	5.7 59.8 25.2 9.3	7.2 77.5 11.7 3.6
	unknown		0.04	0.0	0.0	0.6	0.2	0.0	0.0
	Smoking (%)
	no		75.5	71.1	73.4	75.2	96.0	87.8	72.8
	quit		14.9	17.7	15.2	6.4	1.0	8.8	19.8
	yes		9.4	11.2	11.5	17.5	2.5	2.8	7.2
	unknown		0.2	0.0	0.0	0.9	0.5	0.7	0.1
	* NW: non-Western, W: Western

Table 1

 1 Response rates and maternal characteristics in the study population according to country of birth* (continued)

		Total	Dutch	Surinamese	Turkish	Moroccan	Other NW	Other W
	Gestational age at blood sampling
	(start prenatal care) (%)
	<12 weeks	27.9	30.7	21.2	19.2	16.5	24.5	31.1
	12-14 weeks	37.5	41.4	28.1	33.2	29.8	29.2	36.6
	14-16 weeks	16.7	16.1	21.0	16.6	20.1	16.5	15.3
	16-18 weeks	7.7	6.1	10.8	12.2	11.9	10.5	7.4
	18-20 weeks	3.2	2.1	7.4	5.0	5.4	4.9	3.1
	≥20 weeks	6.6	3.2	11.3	13.1	16.3	14.1	6.1
	unknown	0.3	0.4	0.2	0.6	0.0	0.2	0.3
	Gestational age at completion
	pregnancy questionnaire (%)
	<12 weeks	5.1	5.7	3.5	3.2	2.8	4.0	6.7
	12-14 weeks	12.6	13.9	7.4	11.4	8.4	11.1	13.4
	14-16 weeks	26.2	29.6	19.0	21.6	18.9	19.6	25.3
	16-18 weeks	25.2	26.6	23.8	23.6	23.7	21.6	23.8
	18-20 weeks	11.9	11.3	15.8	12.8	13.2	10.6	13.3
	≥20 weeks	18.8	12.8	30.1	27.1	32.8	32.8	17.5
	unknown	0.1	0.1	0.4	0.3	0.2	0.3	0.1

* NW: non-Western, W: Western

Agyemang C, Bindraban N, Mairuhu G, van Montfrans G, Koopmans R, Stronks K.

Acknowledgements

We thank all participating mothers and their children for their time and involvement, and are grateful to all obstetric care providers, YHC centers, and primary schools for assisting in the implementation of the ABCD study. We especially thank our data manager Hans Reurs for his continued technical support. The ABCD study has been supported by grants from the Netherlands Organisation for Health Research and Development (ZonMW), the Netherlands Heart Foundation, Nutricia NL and Danone Research, Center for Specialized Nutrition. We gratefully acknowledge the additional support provided by the University of Tilburg (a research assistant for the 5-year measurement), the National Institute for Public Health and the Environment (RIVM) (maternal blood measurements), LabAnywhere BV (children's blood measurements), and science center NEMO (location and facility ABCD health check for children living outside of Amsterdam).

Perinatal risk factors

Perinatal outcomes Child outcomes (and ethnic disparities therein) (and ethnic disparities therein) (and ethnic disparities therein)