Articles. Inverse association between maternal 25OHD level and cord GLP-1/GIP concentrations. Clinical Investigation

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1 Clinicl Investigtion nture publishing group Inverse ssocition between mternl 25OHD level nd cord GLP-1/GIP concentrtions Shimpei Niw 1,2, Hidetoshi Mezw 1,3, Noki Kobyshi 3,4, Hiroyuki Id 3 nd Mitsuyoshi Urshim 1,3 Bckground: Becuse vitmin D my hve beneficil effects on glucose metbolism in pregnnt women with gesttionl dibetes mellitus, we explored whether mternl 25-hydroxyvitmin D (25OHD) levels in norml pregnncy hve ssocition with dibetes-relted hormone levels nd glycted lbumin (GA). Methods: A prospective cohort study ws performed to collect serum smples from 612 pirs of pregnnt women nd cord blood of their offspring. Levels of 25OHD nd GA in mternl nd cord blood were mesured by rdioimmunossy nd enzyme ssy, respectively. Using cord serum, 12 dibetes-relted hormones were ssyed. Spermn s rnk correltion coefficient ws used to quntify the strength of ssocition between biomrkers. Results: A prominent ssocition between mternl nd cord 25OHD levels (r = 0.76, 95% confidence intervls (CIs): , P < ) nd wek ssocition between mternl nd cord GA (r = 0.22, 95% CIs: , P < ) were shown. Among the 12 dibetes-relted hormones, both mternl nd cord 25OHD levels showed prominent negtive ssocitions with glucgon-like peptide 1 (GLP-1) nd glucose-dependent insulinotropic polypeptide (GIP). Conclusions: These results suggest tht decresed mternl 25OHD my be ssocited with decresed cord 25OHD nd incresed cord GLP-1 nd GIP levels, which my be involved with the trnsfer of mternl glucose to the fetus. INTRODUCTION Vitmin D is mde primrily under the skin by exposure to sunlight nd cn be obtined to lesser extent in foods such s mushrooms, oily fish, nd egg yolks. Vitmin D is first hydroxylted in the liver to form 25-hydroxyvitmin D (25OHD), then very smll mount of 25OHD is ctivted in the kidney to ply importnt roles in the body, including in bone integrity nd clcium metbolism (1). Indeed, vitmin D supplementtion with clcium cn prevent bone frcture nd bone loss in the elderly (2). In ddition to clcium metbolism, vitmin D repletion for 12 wk not only increses serum vitmin D concentrtions but lso improves β-cell ctivity (3). Moreover, mternl vitmin D deficiency during pregnncy hs been suggested to be ssocited with n incresed risk of gesttionl dibetes mellitus (4,5). In fct, recent rndomized, double-blind, plcebo-controlled clinicl trils hve suggested tht vitmin D supplementtion cn decrese fsting glucose s well s insulin levels in gesttionl dibetes (6,7), lthough the results hve been inconsistent (8). In ddition, ssocitions between serum 25OHD nd glucose levels in pregnnt women without gesttionl dibetes remin unknown. Therefore, using Bio-Plex MAGPIX Suspension Arry System (Bio-Rd, Hercules, CA), we performed screening of cord blood for 12 well-known dibetes-relted hormones: glucgon-like peptide 1 (GLP-1); glucose-dependent insulinotropic polypeptide (GIP); ghrelin; insulin; c-peptide; glucgon; leptin; plsminogen ctivtor inhibitor-1 (PAI-1); resistin; visftin; dipsin; nd diponectin. We then nlyzed ssocitions between levels of these hormones nd 25OHD nd glycted lbumin (GA), n independent mrker of nemi sometimes observed in pregnnt women in mternl nd cord blood (9). RESULTS Prticipnt Chrcteristics Among the 650 pregnnt women who fulfilled inclusion criteri, totl of 612 mother/infnt pirs prticipted in this study. Mternl chrcteristics nd lifestyles divided by three 25OHD levels of mothers (deficient, insufficient, nd sufficient) re shown in Tble 1. More thn hlf of the prticipnts were 25OHD deficient. Prticipnts with lower 25OHD levels tended to spend less time engged in outdoor ctivities, to hve fewer siblings, nd to show lower GA levels thn those with higher 25OHD levels. However, no significnt differences in mternl nd neontl nthropometric mesures, other mternl lifestyles including smoking, lcohol consumption, nd dietry hbits, or neontl complictions t birth were identified. Mternl nd Cord Blood 25OHD Levels Levels of 25OHD in cord blood showed strong positive ssocitions with those in mternl blood t 34 gesttionl weeks 1 Division of Moleculr Epidemiology, Jikei University School of Medicine, Tokyo, Jpn; 2 Sfety nd Risk Mngement Deprtment, Diichi Snkyo Co., Ltd., Tokyo, Jpn; 3 Deprtment of Peditrics, Jikei University School of Medicine, Tokyo, Jpn; 4 Deprtment of Peditrics, Shiomidi Hospitl, Yokohm, Jpn. Correspondence: Mitsuyoshi Urshim (urshim@jikei.c.jp) Received 29 April 2015; ccepted 14 September 2015; dvnce online publiction 30 December doi:.38/pr Peditric Reserch Volume 79 Number 4 April 2016

2 Assocition between 25OHD nd incretin Articles Tble 1. Mternl nd offspring chrcteristics strtified by mternl 25OHD levels Deficient (0 <20 ng/ml), n = 337 Mternl 25OHD Insufficient (20 <30 ng/ml), n = 181 Sufficient (30 <80 ng/ml), n = 94 Totl, Vribles n = 612 P Mternl ge, yers (SD) 31.5 (5.0) 31.2 (5.2) 31.7 (4.7) 31.9 (4.9) 0.34 d Mternl body mss index b, kg/m 2 (SD) 21.0 (2.9) 21.1 (3.0) 20.9 (2.8) 21.0 (3.0) 0.83 d Vitmin D supplementtion c, n (%) 46 (7.8) 18 (5.7) 18 (.3) (21.7) e Time spent on outdoor ctivity < 0.001* e Almost none, n (%) 43 (7.4) 31 (9.8) 8 (4.7) 4 (4.4) Averge < 0.5 h/d, n (%) 116 (20.0) 72 (22.6) 37 (21.5) 7 (7.7) Averge h/d, n (%) 178 (30.6) 99 (31.1) 61 (35.5) 18 (19.8) Averge h/d, n (%) 162 (27.9) 77 (24.2) 45 (26.2) 40 (44.0) Averge > 2.0 h/d, n (%) 82 (14.1) 39 (12.3) 21 (12.2) 22 (24.2) Number of siblings, 0/1/2/3/ 4, n 268/223/93/20/8 170/5/46/9/7 73/71/29/7/1 25/47/18/4/ * e Glycted lbumin, % (SD) 13.9 (1.4) 13.7 (1.3) 14.1 (1.5) 14.2 (1.2) * f Gesttionl ge, weeks (SD) 38.8 (1.2) 38.9 (1.3) 38.9 (1.1) 38.7 (1.3) 0.52 f Mle gender, n (%) 314 (51) 181 (54) 90 (50) 43 (46) 0.35 e Anthropometry t birth Birth weight, g (SD) 3,065 (396) 3,080 (419) 3,047 (353) 3,043 (393) 0.56 d Birth height, cm (SD) 48.7 (1.9) 48.8 (2.0) 48.8 (1.7) 48.6 (1.9) 0.89 f Birth hed circumference, cm (SD) 33.2 (1.4) 33.3 (1.4) 33.2 (1.3) 33.1 (1.5) 0.50 f Birth chest circumference, cm (SD) 31.9 (1.7) 31.9 (1.7) 31.9 (1.6) 31.9 (1.6) 0.99 f Kup index, g/cm 2 (SD) 12.9 (1.1) 12.9 (1.1) 12.8 (1.0) 12.8 (1.1) 0.72 f Apgr score At 1 min, men (SD) 8.6 (0.7) 8.5 (0.8) 8.6 (0.7) 8.5 (0.7) 0.53 f At 5 min, men (SD) 9.2 (0.6) 9.1 (0.6) 9.2 (0.5) 9.2 (0.5) 0.65 f Complictions t birth Neontl jundice, n (%) 33 (5.4) 19 (5.6) 12 (6.6) 2 (2.1) 0.28 e Trnsient tchypne of newborn, n (%) 24 (3.9) 18 (5.3) 4 (2.2) 2 (2.1) 0.13 e 25OHD, 25-hydroxyvitmin D. All other vribles in questionnires listed in the Methods section did not show significnt differences mong the three levels of 25OHD (dt not shown). b Body mss index ws clculted using the following formul: weight before pregnncy divided by squre of height. c Mothers tking supplementl vitmin D from 40 to 1,000 IU/d for 1 mo were considered s showing positive intke. d P vlue ws clculted by ANOVA. e P vlue ws clculted by the χ 2 test. f P vlue ws clculted by the Kruskl Wllis equlity-of-popultions rnk test. *P < (Spermn s rnk correltion coefficient: 0.76, 95% confidence intervls (CIs): , P < ). Medin 25OHD level in cord blood ws ng/ml, pproximtely hlf the men of 19 ng/ml in mternl serum (Figure 1). Mternl nd Cord Blood GA Levels Medin (25th 75th percentile) GA ws 9.8% (rnge: 9.2.3%) in cord blood nd 13.7% (rnge: %) in mternl blood. Norml rnge of GA in helthy dults ccording to SRL dt is considered to be between 12.4 nd 16.3%. Sixty-five (11%) nd 30 (5%) pregnnt women were below nd bove this norml rnge, respectively. In contrst, ll GA vlues in cord blood except one cse were below the lower limit of the norml rnge in helthy dults. Levels of GA in cord blood were positively ssocited with GA in mternl blood t 34 gesttionl weeks (Spermn s rnk correltion coefficient: 0.22, 95% CIs: , P < ; Figure 2). Associtions Between 25OHD nd GA Levels Levels of 25OHD in mternl blood were positively ssocited with GA in mternl blood t 34 gesttionl weeks (Spermn s rnk correltion coefficient: 0.21, 95% CIs: , P < ; Figure 3). On the other hnd, no significnt ssocitions were seen between cord 25OHD nd cord GA, between cord 25OHD nd mternl GA, or between mternl 25OHD nd cord GA. Associtions Between 12 Kinds of Dibetes-Relted Hormones nd 25OHD in Either Cord Blood or Mternl Blood Compred with levels in helthy dults for the 12 kinds of hormones mesured with the sme Bio-Plex suspension rry system, men GLP-1 level ws ~0-fold higher, ghrelin, glucgon, visftin, nd diponectin were ~-fold higher, dipsin, PAI-1, nd resistin were severl times higher, nd GIP ws couple of times higher. In contrst, c-peptide, insulin, nd Volume 79 Number 4 April 2016 Peditric Reserch 537

3 Niw et l Cord blood 25OHD (ng/ml) Mternl glycted lbumin (%) Mternl 25OHD (ng/ml) Mternl 25OHD (ng/ml) Figure 1. Assocition between mternl nd cord blood 25OHD levels. We used liner regression models to ssess the ssocition between mternl nd cord blood 25OHD levels. Spermn s rnk correltion coefficient ws used to quntify the strength of the ssocition. 25OHD, 25-hydroxyvitmin D. Figure 3. Assocition between mternl 25OHD nd glycted lbumin levels. We used liner regression models to ssess the ssocition between mternl 25OHD nd glycted lbumin levels. Spermn s rnk correltion coefficient ws used to quntify the strength of the ssocition. 25OHD, 25-hydroxyvitmin D. Cord blood glycted lbumin (%) (Tble 5). Insulin, c-peptide, GIP, nd ghrelin levels were positively ssocited nd resistin, visftin, nd dipsin were negtively ssocited with GA in cord blood. In contrst to GA in cord blood, GIP nd GLP-1 were negtively ssocited with GA in mternl blood. As we were interested in this conversion of negtive nd positive correltions, we creted cordmternl GA rtio s new prmeter. Cord-mternl GA rtio showed wek positive ssocition with GIP (r = 0.22), GLP-1 (r = 0.22), nd ghrelin (r = 0.18) (P < ech) Mternl glycted lbumin (%) Figure 2. Assocition between mternl nd cord blood glycted lbumin levels. We used liner regression models to ssess the ssocition between mternl nd cord blood glycted lbumin levels. Spermn s rnk correltion coefficient ws used to quntify the strength of the ssocition. leptin did not differ mrkedly from those in helthy dults (). Among these 12 kinds of hormones, both GLP-1 nd GIP were prominently nd negtively ssocited with 25OHD in both cord blood nd mternl blood in both mono nd multiple vrint nlyses (Tbles 2 nd 3). No other hormones showed significnt ssocitions with both cord nd mternl blood. Associtions Between the 12 Dibetes-Relted Hormones or GA in Cord Blood, Mternl Blood, or Cord-Mternl GA Rtio Among the 12 kinds of dibetes-relted hormones, ssocitions of GA either in cord blood or in mternl blood s well s cord-mternl GA rtio (clculted s cord GA divided by mternl GA (%)) were nlyzed by Spermn s rnk correltion coefficient (Tble 4) nd multiple liner regression DISCUSSION Using the Bio-Plex suspension rry system, we conducted prospective cohort study to screen 12 kinds of dibetes-relted hormones for ssocitions with cord nd mternl 25OHD, s well s GA. To the best of our knowledge, this study is the first to find tht GLP-1 nd GIP re ssocited with 25OHD nd GA. First, we demonstrted strong liner reltionship between serum levels of mternl nd cord 25OHD (r = 0.76, 95% CIs: , P < ), supporting previous findings (11 14). 25OHD ws reported to be trnsferred from mother to fetus cross the plcent (15 18). Our result lso suggests tht 25OHD my be pssively trnsferred from mother to fetus through the plcent. We lso found positive ssocition between GA levels of mternl nd cord blood, s in previous study (19), but the ssocition ws not s strong s tht observed between mternl nd cord 25OHD. Albumin ws reported not to significntly pss through the plcent (20), but fetl liver cn synthesize lbumin t high rte t two-thirds of gesttion (21,22). These results suggest tht glucose my be not only pssively, but lso ctively, trnsferred from mother to fetus through the plcent through regultion by unknown fctors. Second, we found positive ssocition between mternl 25OHD nd mternl GA in norml pregnncy. In contrst to our results, most previous studies hve focused on pregnnt 538 Peditric Reserch Volume 79 Number 4 April 2016

4 Assocition between 25OHD nd incretin Articles Tble 2. Hormones ssocited with 25OHD in cord blood nd mternl blood Mesures in cord blood r b 95% CI P c r b 95% CI P c Correltion with 25OHD in cord blood Correltion with 25OHD in mternl blood Insulin (pg/ml) 0. ( 0.18, 0.02) ( 0.14, 0.02) 0.16 C-peptide (pg/ml) 0.02 ( 0.06, 0.) ( 0.02, 0.14) 0.16 GIP (pg/ml) 0.21 ( 0.29, 0.13) <0.0001* 0.12 ( 0.20, 0.04) * GLP1 (pg/ml) 0.35 ( 0.42, 0.28) <0.0001* 0.23 ( 0.30, 0.15) <0.0001* Ghrelin (pg/ml) 0. ( 0.18, 0.02) ( 0.16, 0.00) Glucgon (pg/ml) 0.11 ( 0.19, 0.03) ( 0.12, 0.04) 0.33 Leptin (pg/ml) 0.02 ( 0., 0.06) ( 0.13, 0.04) 0.27 PAI (pg/ml) 0.02 ( 0.06, 0.) ( 0.06, 0.) 0.65 Resistin (pg/ml) 0.03 ( 0.05, 0.16) ( 0.06, 0.11) 0.55 Visftin (pg/ml) 0.07 ( 0.15, 0.01) ( 0.14, 0.02) 0.17 Adipsin (pg/ml) 0.05 ( 0.03, 0.13) ( 0.16, 0.00) 0.05 Adiponectin (pg/ml) 0.05 ( 0.04, 0.13) ( 0.15, 0.01) OHD, 25-hydroxyvitmin D; CI, confidence intervl. Dt were trnsformed by nturl logrithm. b Spermn s rnk correltion coefficient ws represented by r. c Since levels of 12 dibetes-relted hormones were mesured, vlues of P < (= 0.05/12) were considered significnt. *P < Tble 3. Incretins ssocited with 25OHD in cord blood nd mternl blood by multiple liner regression blood b Coefficiency t 95% CI P Coefficiency t 95% CI P Mesures in cord Correltion with 25OHD in cord blood Correltion with 25OHD in mternl blood GIP (pg/ml) to 0.20 <0.0001* to * GLP1 (pg/ml) to 0.35 <0.0001* to * 25OHD, 25-hydroxyvitmin D; CI, confidence intervl; GIP, glucose-dependent insulinotropic polypeptide; GLP, glucgon-like peptide. Multivrite nlysis ws performed by liner regression djusting for potentil confounders such s mternl ge, mternl BMI, intke of vitmin D supplement, time spent for outdoor ctivity, number of sibling, nd month of the birth. b Dt were trnsformed by nturl logrithm. *P < women with gesttionl dibetes mellitus (23), type 1 dibetes (24) or type 2 dibetes (25) nd hve shown negtive correltions between 25OHD nd glucose control levels. However, these inverse ssocitions between 25OHD nd glucose levels hve not been studied in helthy dults or pregnnt women without gesttionl dibetes mellitus, nd cusl reltionships hve not lwys been shown (25,26). For exmple, obesity cn represent confounder for both 25OHD nd bnorml glucose metbolism (27). Third, we identified GLP-1 nd GIP s significnt dibetesrelted hormones ssocited with 25OHD in both mternl nd cord blood mong the 12 dibetes-relted hormones. To the best of our knowledge, this represents novel finding. Both GLP-1 nd GIP re gut-derived incretin hormones tht stimulte insulin nd suppress glucgon secretion, inhibit gstric emptying, nd reduce ppetite nd food intke (28). Plcentl trnsfer of both incretin hormones might be considered negligible (29), nd both hormones re secreted in the fetus (30 33). Eting provokes the secretion of multiple gstrointestinl hormones involved in the regultion of gut motility, secretion of gstric cid nd pncretic enzymes, gll bldder contrction, nd nutrient bsorption s well s insulin secretion. In contrst to dults, nutrition is bsiclly supplied through the plcent to fetuses, not through eting. Moreover, GLP-1 seems to be higher thn in helthy dults using the sme Bio-Rd suspension rry system (,34). However, the ctive form of GLP-1 in cord blood ws reported to be close to the lower limit of detection (35). Thus, even the higher levels of GLP-1 nd GIP in cord blood observed in this study do not necessry to ffect glucose levels decresing by incresing insulin nd decresing glucgon secretion through the pncres s observed in dults. The roles nd mechnisms of GLP-1 nd GIP secretion in the fetus wrrnt further investigtion in the future. Fourth, mternl GA levels were negtively ssocited with GLP-1 nd GIP (r = 0.13), wheres cord GA levels showed positive ssocition (r = 0.13). Cord-mternl GA rtio ws positively ssocited with GLP-1 (r = 0.22), GIP (r = 0.22), nd ghrelin (r = 0.18). These results imply tht GLP-1, GIP, nd ghrelin my fcilitte ctive trnsfer of glucose from mternl blood to fetl blood through the plcent. This study hs severl limittions. First, the study ws designed s prospective cohort to investigte 25OHD nd GA levels in mternl blood t 34 gesttionl weeks s exposures nd dibetes-relted hormones in cord blood t birth s outcomes. Although time gp existed between exposures nd outcomes, this study ws close in nture to cross-sectionl design. We thus cnnot discuss cusl reltionships between 25OHD nd GLP-1/GIP levels or between cord-mternl GA rtio nd GIP/GLP-1/ghrelin levels, only the existence of Volume 79 Number 4 April 2016 Peditric Reserch 539

5 Niw et l. Tble 4. Hormones ssocited with GA in cord blood nd mternl blood Hormones in cord blood Correltion with GA in cord blood Correltion with GA in mternl blood Correltion with cord-mternl GA rtio c r b P d r b P d r b P d Insulin (pg/ml) * C-peptide (pg/ml) 0.19 <0.0001* GIP (pg/ml) * * 0.22 <0.0001* GLP1 (pg/ml) * 0.22 <0.0001* Ghrelin (pg/ml) 0.22 <0.0001* <0.0001* Glucgon (pg/ml) Leptin (pg/ml) PAI (pg/ml) Resistin (pg/ml) 0.18 <0.0001* Visftin (pg/ml) * Adipsin (pg/ml) Adiponectin (pg/ml) GA, glycted lbumin; GIP, glucose-dependent insulinotropic polypeptide; GLP, glucgon-like peptide. Dt were trnsformed by nturl logrithm. b Spermn s rnk correltion coefficient ws represented by r. c Rtio ws simply clculted s GA in cord blood divided by GA in mternl blood. d Since levels of 12 dibetes-relted hormones were mesured, vlues of P < (=0.05/12) were considered significnt. *P < Tble 5. Hormones ssocited with GA in cord blood nd mternl blood by multiple liner regression Correltion with GA in cord blood Correltion with GA in mternl blood Correltion with cord-mternl GA Mesures in cord rtioc blood b Coefficiency t 95% CI P Coefficiency t 95% CI P Coefficiency t 95% CI P Insulin (pg/ml) C-peptide (pg/ml) GIP (pg/ml) to 0. GLP1 (pg/ml) Ghrelin (pg/ml) to 0.08 Resistin (pg/ml) to 0.01 Visftin (pg/ml) to * to 0.04 <0.0001* to 0.03 <0.0001* to to 0.02 <0.0001* * to * * * to to to to GA, glycted lbumin; GIP, glucose-dependent insulinotropic polypeptide; GLP, glucgon-like peptide. Multivrite nlysis ws performed by liner regression djusting for potentil confounders such s mternl ge, mternl BMI, intke of vitmin D supplement, time spent for outdoor ctivity, number of sibling, nd month of the birth. b Dt were trnsformed by nturl logrithm. c Rtio ws simply clculted s GA in cord blood divided by GA in mternl blood. *P < * <0.0001* <0.0001* <0.0001* * significnt ssocitions. Second, we pplied the BioPlex ssy for simultneous quntifiction of multiple nlytics, in process termed multiplexing. However, this technique is not considered optiml for mesuring levels of gut hormones (36). With other methods, levels of some of these hormones might differ mrkedly (,34). We therefore compred dibetesrelted hormone levels s mesured by BioPlex within smples of this study popultion, nd not with results from other studies. Third, we compred hormone levels in cord blood with those in helthy dults s provided by Bio-Rd Lbortories. However, the number of smples used for their dt ws only or 11 (,34). We cn therefore mke only rough estimte of whether hormone levels seem higher in cord blood thn in dults. In conclusion, mong the 12 dibetes-relted hormones, both cord nd mternl 25OHD levels showed prominent negtive ssocitions with GLP-1 nd GIP. Moreover, GLP-1, GIP, nd ghrelin showed positive ssocitions with cord-mternl GA rtio. METHODS Study Design This prospective cohort study ws conducted t Shiomidi Hospitl in Kngw prefecture, generl hospitl in rurl re of Jpn locted t 35 degrees 24 min north ltitude, nd 139 degrees 36 min est longitude. Inclusion criteri were pregnnt women 20 y old 540 Peditric Reserch Volume 79 Number 4 April 2016

6 Assocition between 25OHD nd incretin Articles t entry nd independent of vitmin D supplement intke. Exclusion criteri were pregnnt women who: (i) showed mjor complictions such s gesttionl dibetes mellitus or toxemi of pregnncy; (ii) needed emergent cesren section; (iii) showed multiple fetuses such s twins; (iv) hd fetus with cler evidence of intruterine growth retrdtion or congenitl mlformtion; (v) did not hve smples vilble; nd (vi) hd other difficulties judged by the obstetricin or peditricin in chrge. Enrollment ws performed by the collborting peditricin (N.K.). Pregnnt women were enrolled from June 2011 to September Ethics Sttement The study protocol ws developed by ll uthors nd pproved by the ethics committee t Jikei University School of Medicine nd the clinicl study committee t Jikei Hospitl, s well s the institutionl review bord t Shiomidi Hospitl. The dt monitoring center ws in the Division of Epidemiology t Jikei University School of Medicine, nd ll dt were monitored nd fixed by H.M., who did not prticipte in sttisticl nlyses. All women provided written, informed consent to prticipte in the study. Questionnires About Lifestyle During the third trimester of pregnncy, prticipnts were sked to send bck questionnires contining: (i) bsic dt such s ge, weight before pregnncy nd recent weight, nd height for pregnnt women, long with ge, weight, nd height for husbnd (or prtner); (ii) smoking sttus (current, pst, or nonsmoker), nd exposure to pssive smoking; (iii) men frequency, mount, nd kind of lcohol consumption per week over the preceding month of pregnncy; (iv) men frequency of consumed food items (dried shiitke, mushroom, slmon, srdines, mckerel, sury, tun, egg) per week during the preceding month of pregnncy; (v) vitmin D supplementtion, nd timing nd dosge if used; (vi) men dily exposure to sunlight; (vii) fmily structure; (viii) medicl history of llergic diseses (bronchil sthm, topic dermtitis, food llergy, drug llergy, metl llergy, solr eczem, llergic rhinitis, chemicl sensitivity, nd others); (ix) skin rection to sun exposure s evluted with modified Fitzptrick scle (type 1 = lwys burns, never tns; type 2 = usully burns, tns minimlly; type 3 = sometimes mild burn, tns uniformly; or type 4 = rrely burns, lwys tns well); nd (x) number of siblings. Clinicl Informtion t Birth The following clinicl informtion were collected: (i) plnned cesren section or not; (ii) birth dte; (iii) weight, height, hed circumference, nd chest circumference t birth; (iv) Apgr score t 1 nd 5 min; nd (v) complictions such s neontl jundice nd trnsient tchypne of the newborn. Smples nd 25OHD Mesurements Serum smples from prticipting pregnnt women were collected t 34 wk of gesttion. Umbilicl cord blood (5 ml) ws smpled from the plcent side fter plcentl delivery t birth. Soon fter blood smpling without freezing, levels of 25OHD were mesured by rdioimmunossy t SRL (Hchioji, Tokyo, Jpn) (37), which hs prticipted in the Lbortory Accredittion Progrm of College of Americn Pthologist. Miniml detection level ws 5 ng/ml. The Institute of Medicine hs defined dequte vitmin D sttus s serum 25OHD level 20 ng/ml for the generl popultion, including pregnnt women (38). When levels re 30 ng/ml, bone frcture cn be prevented (39). We therefore divided prticipting pregnnt women into three groups ccording to 25OHD levels: deficient, <20 ng/ml; insufficient, 20 but <30 ng/ml; nd sufficient, 30 ng/ml. Multiplex Immunossy Anlysis After freezing t 80 C, series of hormones relted to metbolism (GLP-1, GIP, ghrelin, insulin, c-peptide, glucgon, leptin, PAI- 1, resistin, visftin, dipsin, nd diponectin) were ssyed in cord blood using the Bio-Plex MAGPIX Suspension Arry System (Bio- Rd, Hercules, CA), ccording to the instructions from the mnufcturer. A single opertor blinded to clinicl informtion performed ll mesurements using humn dibetes ssy kits. Identicl positive nd negtive qulity controls re included on ech ssy in duplicte. Assys were performed in one btch, with smples mixed rndomly. The lower limit of detection rnged from 2.4 pg/ml for resistin to 3 pg/ml for visftin, while intr-ssy vribility ws less thn %. Men recovery rtio represented by observed dt/expected dt ws 99.4%. Glycted Albumin After mximum of 2.5 y freezing t 80 C, serum smples obtined from prticipting pregnnt women nd cord blood of offspring were mesured for GA using n enzymtic method by SRL (40). Sttisticl Anlysis Continuous dt were compred mong the three mternl groups by mens of ANOVA for fctors showing norml distribution nd by Kruskl Wllis equlity-of-popultions rnk test for fctors not showing norml distribution. The χ 2 test ws used for nlysis of binry or ctegoricl dt. Spermn s rnk correltion coefficient ws used to quntify the strength of ssocition between 25OHD/GA nd dibetes-relted hormones not showing norml distributions. All reported P vlues were two-sided. Vlues of P < 0.05 were considered significnt in the nlyses of prticipnt chrcteristics. On the other hnd, since 12 dibetes-relted hormones were mesured, vlues of P < (= 0.05/12) were considered significnt using Spermn s rnk correltion. Liner regression model ws pplied to obtin coefficiency. For significnt vribles in Spermn s rnk correltion nlysis, multivrite nlysis ws performed by liner regression djusting for potentil confounders such s mternl ge, mternl BMI, intke of vitmin D supplement, time spent for outdoor ctivity, number of sibling, nd month of the birth. All sttisticl nlyses were independently performed by M.U. nd S.N., who were not involved in dt collection. Stt version 13.1 softwre (SttCorp, College Sttion, TX) ws used for ll nlyses. ACKNOWLEDGMENTS We thnk the ptients who provided blood smples for this reserch project. We re lso grteful to Chikko Sknshi for technicl support with Bio- Plex. STATEMENT OF FINANCIAL SUPPORT This work ws supported by the Ministry of Eduction, Culture, Sports, Science nd Technology in the Jpn-Supported Progrm for the Strtegic Reserch Foundtion t Privte Universities nd the Jikei University School of Medicine. Disclosure: The uthors declre tht they hve no competing interest. References 1. Holick MF. Vitmin D deficiency. 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