Association between maternal folate concentrations during pregnancy and insulin resistance in Indian children

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1 Dibetologi (2014) 57: DOI /s ARTICLE Assocition between mternl folte concentrtions during pregnncy nd insulin resistnce in Indin children Ghttu V. Krishnveni & Srgoor R. Veen & Smuel C. Krt & Chittrnjn S. Yjnik & Croline H. D. Fll Received: 30 June 2013 /Accepted: 30 September 2013 /Published online: 26 October 2013 # The Author(s) This rticle is published with open ccess t Springerlink.com Abstrct Aims/hypothesis In n Indin birth cohort, higher mternl homocysteine concentrtion in pregnncy ws ssocited with lower birthweight of the offspring. Lower mternl vitmin B12 nd higher folte concentrtions were ssocited with higher offspring insulin resistnce. Disordered one-crbon metbolism during erly development my increse lter metbolic risk. We explored these ssocitions in nother birth cohort in Indi t three ge points. Methods We mesured plsm vitmin B12, folte nd homocysteine concentrtions t 30±2 weeks gesttion in 654 women who delivered t one hospitl. Neontl nthropometry ws recorded, nd the children s glucose nd insulin concentrtions were mesured t 5, 9.5 nd 13.5 yers of ge. Insulin resistnce ws estimted using HOMA of insulin resistnce (HOMA-IR). Results Mternl homocysteine concentrtions were inversely ssocited with ll neontl nthropometric mesurements (p <0.05), nd positively ssocited with glucose concentrtions in the children t 5 (30 min; p =0.007) nd 9.5 yers of ge (120 min; p =0.02). Higher mternl folte concentrtions were ssocited with higher HOMA-IR in the children t 9.5 (p =0.03) nd 13.5 yers of ge (p =0.03). Mternl vitmin B12 concentrtions were unrelted to offspring outcomes. Electronic supplementry mteril The online version of this rticle (doi: /s ) contins peer-reviewed but unedited supplementry mteril, which is vilble to uthorised users. G. V. Krishnveni (*): S. R. Veen : S. C. Krt Epidemiology Reserch Unit, CSI Holdsworth Memoril Hospitl, P.O. Box 38, Mndi Mohll, Mysore , Indi e-mil: gv.krishnveni@gmil.com C. S. Yjnik Dibetes Unit, KEM Hospitl, Pune, Indi C. H. D. Fll MRC Lifecourse Epidemiology Unit, Southmpton Generl Hospitl, Southmpton, UK Conclusions/interprettion Mternl vitmin B12 sttus did not predict insulin resistnce in our cohort. However, ssocitions of mternl homocysteine nd folte concentrtions with birth size, nd with childhood insulin resistnce nd glycemi in the offspring, suggest role for nutritionlly driven disturbnces in one-crbon metbolism in fetl progrmming of dibetes. Keywords Child. Folte. Homocysteine. Insulin resistnce. Pregnncy. Progrmming. Vitmin B12 Abbrevitions Ft% Percentge body ft GDM Gesttionl dibetes mellitus HMH Holdsworth Memoril Hospitl HOMA-IR HOMA of insulin resistnce MUAC Mid upper rm circumference SDS SD score SES Socioeconomic sttus Introduction Nutrition during fetl development hs long-term helth consequences [1]. Fetl nutrition is influenced by mternl nutritionl sttus, nd experiments in niml models hve shown tht both mternl undernutrition nd overnutrition cn cuse obesity, insulin resistnce nd dibetes in the dult offspring [2, 3]. Vitmin B12 nd folte re importnt nutrients required for nucleic cid synthesis, DNA methyltion nd cellulr growth nd differentition. Severl studies hve reported tht low mternl folte nd vitmin B12 concentrtions nd high homocysteine concentrtions ( mrker of dernged onecrbon metbolism) predict smller newborn size [4 7]. However, dt linking mternl diet nd nutrient sttus to long-term offspring outcomes in humns re limited.

2 Dibetologi (2014) 57: Recently it ws observed in rurl popultion in Pune, Indi, tht low mternl vitmin B12 concentrtions nd high erythrocyte folte concentrtions during pregnncy were ssocited with higher insulin resistnce in 6-yer-old offspring [8]. In nother study, mong children in Nepl born to mothers who took prt in rndomised controlled tril of micronutrients in pregnncy, mternl vitmin B12 deficiency t bseline ws ssocited with higher offspring insulin resistnce t the ge of 6 8 yers [9], though there ws no pprent folte effect. The Prthenon Study in Mysore, Indi, is n observtionl study to investigte mternl nd fetl fctors relted to offspring crdiovsculr nd dibetes risk [10, 11]. We hve previously shown tht low mternl vitmin B12 sttus during pregnncy ws relted to higher incidence of gesttionl dibetes mellitus (GDM) [12]. Mternl GDM ws mjor predictor of the children s diposity nd insulin resistnce t 9.5 yers of ge [11]. In the current nlysis we imed to replicte the Pune nlysis, to exmine whether low mternl vitmin B12 nd high plsm folte concentrtions predicted higher insulin resistnce nd other crdiometbolic risk fctors in children. Our seril follow-up llowed us to investigte these ssocitions t 5, 9.5 nd 13.5 yers of ge. Methods Study popultion During , 830 women booking consecutively into the ntentl clinic of the CSI Holdsworth Memoril Hospitl (HMH) in Mysore, Indi, nd mtching our eligibility criteri (<32 weeks gesttion t recruitment, no known history of dibetes, intention to deliver t HMH, singleton pregnncy) hd 100 g, 3 h OGTT t 30±2 weeks gesttion (Fig. 1) [10]. GDM ws dignosed in 49 women of the 785 women who completed n OGTT (6.2%) using the Crpenter nd Coustn criteri [13]. Vitmin B12 nd folic cid supplements It ws routine for generl prctitioners nd obstetricins to prescribe folic cid nd/or multivitmin supplements to women throughout pregnncy. Supplement use ws recorded t recruitment, but not subsequently, nd no informtion ws therefore vilble on their use t 30 weeks gesttion, when blood smples were tken, or t term. Follow-up of the offspring At HMH, 663 women delivered live bbies without mjor congenitl nomlies. The bbies were mesured for hed nd mid upper rm circumference (MUAC), weight, length, nd subscpulr nd triceps skinfold thicknesses within 72 h of birth s described before [10]. On further follow-up, 25 children hd died nd eight hd mjor medicl conditions. Of the remining children, detiled nthropometry ws performed nd percentge body ft (ft%) ws mesured using bioimpednce in ll vilble children t 5yer(n =585), 9.5 yer (n =539) nd 13.5 yer follow-ups (n =545), nd systolic nd distolic BP were mesured s described elsewhere [11, 14]. Pubertl sttus ws ssessed using Tnner s method t 9.5 nd 13.5 yers of ge [15], nd ws clssified s the stge of brest development (in girls) or genitl development (in boys). The socioeconomic sttus (SES) of the fmily ws determined t 9.5 yers using the Stndrd of Living Index designed by the Ntionl Fmily Helth Survey-2 [16]. Glucose, insulin nd fsting lipid concentrtions were mesured t 5 nd 9.5 yers from 2 h OGTT [11, 14]. At 13.5 yers of ge, glucose, insulin nd lipid concentrtions were mesured using fsting blood smples. The lbortory ssys were crried out t the Dibetes Unit, KEM Hospitl, Pune, Indi, whose lbortory is member of the UK Ntionl Externl Qulity Assessment Service (NEQAS) qulity control progrmme for insulin ssys. Lbortory stff were blind to the identity of the smples nd the vitmin B12 nd folte sttus of the mothers. Plsm glucose nd lipid concentrtions were mesured by stndrd enzymtic methods (Hitchi-902; Roche Dignostics, Mnnheim, Germny). Insulin ws mesured by ELISA (Mercodi Ultrsensitive; Mercodi, Uppsl, Sweden). Inter- nd intrssy CV ws <7.0%. Insulin resistnce ws estimted using the HOMA of insulin resistnce (HOMA-IR) t ll three time points [17]. Vitmin B12, folte nd homocysteine mesurements Mternl vitmin B12, folte nd homocysteine concentrtions were nlysed using plsm smples stored t 80 C for 8 yers; dequte smples were vilble for 654 mothers who were included in the nlysis. Mesurements were crried out t the Dibetes Unit, KEM Hospitl, Pune, using microbiologicl ssys for vitmin B12 nd folte nd fluorescence polristion immunossy (Abbott Lbortories, Abbott Prk, IL, USA) for homocysteine [18 20]. The children s vitmin B12 nd folte concentrtions were mesured t 9.5 yers (n =528), nd homocysteine concentrtions were nlysed t 5 yers of ge (n =567). Intr- nd inter-ssy CV were <8% for these ssys. Mternl vitmin B12 deficiency ws defined s concentrtion <150 pmol/l, nd folte deficiency concentrtion <7 nmol/l. Mternl hyperhomocysteinemi ws defined s homocysteine concentrtion >10 μmol/l. These cut-offs were bsed on the levels used for dult popultions elsewhere [21 23]. The HMH reserch ethics committee pproved the study. Informed written consent ws obtined from the prents nd ssent from the children. Sttisticl methods The distributions of the children s skinfold thickness, insulin concentrtions, HOMA-IR, nd vitmin B12 nd homocysteine concentrtions were skewed;

3 112 Dibetologi (2014) 57: Fig. 1 Flow chrt of the study prticipnts 830 women prticipted OGTT, nthropometry t 30±2 weeks gesttion (49 GDM) vitmin B12, folte, homocysteine Did not deliver t HMH, no detils (n=156) 674 births t HMH 11 excluded (7 still births + 4 mjor nomlies) 663 live births without mjor nomlies (41 GDM) nthropometry within 72 h of birth Mternl nutrient sttus not vilble (n=9) 654 bbies included t birth 578 children included t 5 yers Anthropometry, insulin resistnce (HOMA-IR) 533 children included t 9.5 yers Anthropometry, insulin resistnce (HOMA-IR) 539 children included t 13.5 yers Anthropometry, insulin resistnce (HOMA-IR) these dt were nturl log-trnsformed (ln). Differences between groups were nlysed using independent t tests. To fcilitte comprison of ssocitions, exposure nd outcome vribles were converted into SD scores (SDS). The dt represent SD chnge in offspring outcome per SD chnge in mternl nutrient sttus. Associtions between mternl vitmin B12, folte nd homocysteine concentrtions nd offspring outcomes were exmined using multivrite liner regression, djusting for gesttionl ge (for birth vribles) or current ge (for childhood vribles) nd sex. Additionl djustments were mde for mternl prity, religion, BMI, GDM nd SES, nd the children s vitmin B12, folte, current BMI nd pubertl stge where relevnt; p vlues of <0.05 were considered significnt. All nlyses were performed using IBM SPSS sttistics version 19 (IBM, Armonk, NY, USA). Results Chrcteristics of the 654 mothers with informtion on gesttionl nutrient sttus, nd their children t birth nd t 5 (n = 578), 9.5 (n =533) nd 13.5 yers of ge (n =539) re presented in Tble 1. Vitmin B12 deficiency ws present in 41.4% of the women, while only 4% hd folte deficiency nd hyperhomocysteinemi. The prevlence of vitmin B12 deficiency ws highest mong Hindu women (n =183, 49.1%, medin vitmin B pmol/l; Muslim women: n =67, 29.8%, pmol/l; others: n =21, 37.5%, pmol/l). Plsm folte concentrtions were lowest mong Muslim women (25.8 nmol/l; Hindu women: 40.0 mmol/l; others: 45.7 nmol/l). Homocysteine concentrtions were highest mong Hindu women (6.3 μmol/l; Muslim women: 5.6 μmol/l; others: 5.6 μmol/l). Mternl homocysteine concentrtions were inversely ssocited with both vitmin B12 (r = 0.15, p <0.001) nd plsm folte concentrtions (r = 0.24, p <0.001). Vitmin B12 concentrtions were unrelted to SES or prity. Higher SES (p <0.001) nd lower prity (p <0.001) were ssocited with higher plsm folte concentrtions. Supplement use t recruitment (men [SD] 20 [6] weeks gesttion) ws not significntly ssocited with vitmin B12 nd folte concentrtions t 30±2 weeks gesttion. At 9.5 yers of ge, vitmin B12 deficiency ws present in 2.7% of children, nd folte deficiency in only one child. Offspring vitmin B12 (r =0.17, p <0.001) nd folte concentrtions (r =0.19, p <0.001) were positively correlted with mternl vitmin B12 nd folte concentrtions, respectively. Offspring homocysteine concentrtions t 5 yers were positively correlted with mternl homocysteine concentrtions (r =0.14, p <0.001). Higher mternl folte concentrtions were ssocited with more dvnced pubertl stge in the offspring t 13.5 yers of ge (p =0.04). There ws no ssocition between mternl vitmin B12 nd homocysteine concentrtions nd offspring pubertl sttus.

4 Dibetologi (2014) 57: Tble 1 Generl chrcteristics of the mothers during pregnncy nd their offspring Chrcteristic n Vlue Mother Age, yers (20.0, 26.0) BMI, kg/m (21.0, 26.0) Vitmin B12, pmol/l (123.0, 224.0) B12 deficiency, n (%) (41.4) Plsm folte, nmol/l (17.9, 51.2) Folte deficiency, n (%) (4.0) Homocysteine, μmol/l (5.1, 7.0) Hyperhomocysteinemi, n (%) (3.9) Offspring Birth Birthweight, kg (2.575, 3.165) Crown heel length, cm (47.5, 50.1) Birthweight SDS ( 1.6, 0.3) Birth length SDS ( 1.1, 0.3) Hed circumference, cm (32.9, 34.7) MUAC, cm (9.6, 10.9) Subscpulr skinfold thickness, mm (3.9, 4.9) 5yers Height, cm (102.8, 108.3) BMI, kg/m (12.9, 14.2) Height SDS ( 1.5, 0.3) BMI SDS ( 1.9, 0.7) Insulin resistnce (HOMA-IR) (0.5, 1.1) Insulin increment (14.8, 41.9) 9.5 yers Height, cm (127.0, 134.4) BMI, kg/m (13.4, 15.5) Height SDS ( 1.3, 0.2) BMI SDS ( 2.0, 0.4 ) Insulin resistnce (HOMA-IR) (0.5, 1.2) Vitmin B12, pmol /l (249.8, 404.8) Plsm folte, nmol /l (18.0, 35.0) 13.5 yers Height, cm (148.9, 158.2) BMI, kg/m (15.5, 19.3) Height SDS ( 1.4, 0.1) BMI SDS ( 1.8, 0.06) Insulin resistnce (HOMA-IR) (1.0, 2.0) Vlues given re medin (interqurtile rnge) or n (%) SDSs re bsed on WHO growth stndrds Mternl nutrients nd neontl nthropometry Newborn weight nd MUAC tended to increse with mternl plsm folte concentrtions, though these ssocitions were mrkedly ttenuted nd none were sttisticlly significnt following djustment for mternl covrites (Tble 2). Higher mternl homocysteine concentrtions were ssocited with smller neontl weight, MUAC nd skinfold mesurements (Tble 2). The prevlence of low birthweight (32.0% vs 19.1%; p =0.1) nd preterm (12.0% vs 7.6%; p =0.4) births tended to be higher mong those born to mothers with hyperhomocysteinemi compred with bbies born to mothers with norml homocysteine levels. Mternl vitmin B12 concentrtions were unrelted to nthropometry t birth (dt not shown). Mternl nutrients nd children s nthropometry There were positive ssocitions between mternl folte concentrtions nd offspring weight, height nd MUAC t 5 yers of ge. These ssocitions were not significnt fter djusting for mternl covrites nd offspring nutrient sttus (Tble 3). Mternl folte concentrtions were positively ssocited with the children s nthropometric mesurements t 9.5 nd 13.5 yers, except for subscpulr skinfold thickness nd ft% (Tble 3). The strength of these ssocitions diminished fter full djustment, but remined sttisticlly significnt for height (Tble 3). There were no ssocitions of mternl homocysteine or vitmin B12 concentrtions with the children s nthropometry. Mternl nutrients nd children s insulin resistnce nd risk fctors At 5 yers of ge, fter djusting for mternl nd offspring covrites including current BMI, mternl folte concentrtions were positively ssocited with fsting glucose concentrtions in their offspring (Tble 3). Mternl folte concentrtions were positively ssocited with fsting insulin nd HOMA-IR in the offspring t both 9.5 nd 13.5 yers of ge, nd this ssocition remined significnt fter full djustment including mternl vitmin B12 nd children s current BMI nd pubertl stge (Tble 3). There were lso positive ssocitions between mternl folte nd offspring 30 min insulin concentrtions t 9.5 yers of ge; however, this ssocition becme non-significnt fter full djustment (Tble 3). There were no differences in the ssocitions between mternl plsm folte nd offspring outcomes between children whose mothers hd low or norml vitmin B12 concentrtions, nd there were no significnt interctions between mternl folte nd vitmin B12 deficiency (electronic supplementry mteril [ESM] Tble 1). Higher mternl homocysteine concentrtions were ssocited with higher 30 min glucose nd insulin concentrtions t 5 yers of ge (Tble 4). Higher mternl homocysteine concentrtions were ssocited with higher 120 min glucose concentrtions in the children t 9.5 yers (Tble 4). These ssocitions remined significnt fter further djusting for birthweight. There ws lso borderline positive ssocition between mternl homocysteine nd HOMA-IR t 9.5 yers of ge. There ws no ssocition between mternl homocysteine nd offspring HOMA-IR t 13.5 yers of ge.

5 114 Dibetologi (2014) 57: Tble 2 Men (SD) nthropometry t birth ccording to fourths of mternl folte nd homocysteine concentrtions Vrible n Weight, kg Length, cm Hed circumference, cm MUAC, cm Subscpulr skinfold thickness,mm Triceps skinfold thickness,mm Plsm folte concentrtion, nmol/l < (0.488) 48.3 (2.5) 33.6 (1.6) 10.1 (1.1) 4.4 (3.9, 5.0) 4.0 (3.5, 5.2) (0.472) 48.6 (2.5) 33.7 (1.5) 10.3 (1.0) 4.3 (3.9, 4.9) 4. 1 (3.5, 4.8) (0.492) 48.7 (2.6) 33.7 (1.7) 10.4 (1.0) 4.4 (3.9, 4.9) 4.2 (3.7, 5.7) > (0.440) 48.8 (2.0) 33.9 (1.4) 10.3 (1.0) 4.4 (3.9, 4.9) 4.1 (3.6, 4.6) Model 1 β (95% CI) 0.07 ( 0.007, 0.14) 0.06 ( 0.02, 0.14) 0.05 ( 0.02, 0.13) 0.08 (0.003, 0.15) 0.01 ( 0.07, 0.09) p vlue Model 2 b β (95% CI) 0.02 ( 0.06, 0.10) ( 0.09, 0.10) 0.04 ( 0.05, 0.12) 0.02 ( 0.06, 0.10) 0.03 ( 0.12, 0.05) 0.03 ( 0.05, 0.11) 0.03 ( 0.11, 0.06) p vlue Homocysteine concentrtion, μmol/l < (0.446) 48.7 (2.3) 33.9 (1.4) 10.4 (0.9) 4.5 (3.9, 5.0) 4.2 (3.8, 4.9) (0.417) 48.7 (2.2) 33.8 (1.5) 10.4 (1.0) 4.4 (3.9, 4.9) 4.2 (3.7, 4.8) (0.481) 48.5 (2.7) 33.6 (1.7) 10.1 (1.1) 4.3 (3.9, 4.9) 3.9 (3.5, 4.6) > (0.539) 48.4 (2.6) (1.6) 10.1 (1.2) 4.3 (3.7, 5.0) 4.0 (3.5, 4.8) Model 1 β (95% CI) 0.12 ( 0.19, 0.05) 0.06 ( 0.15, 0.02) 0.04 ( 0.12, 0.04) 0.12 ( 0.19, 0.04) 0.07 ( 0.15, 0.01) p vlue Model 2 β (95% CI) 0.13 ( 0.21, 0.05) 0.09 ( 0.18, 0.002) 0.06 ( 0.14, 0.02) 0.15 ( 0.23, 0.07) 0.12 ( 0.20, 0.04) p vlue < ( 0.17, 0.01) 0.12 ( 0.20, 0.03) β nd p vlues derived by liner regression using mternl folte nd homocysteine concentrtions, nd newborn mesurements s continuous SDS; β represents SDS chnge in outcome vrible per SDS chnge in exposure vrible Log-trnsformed vribles, vlues given re geometric men (interqurtile rnge) Model 1, djusted for bby s sex nd gesttionl ge Model 2, model 1 + mternl BMI, GDM sttus, SES, prity, religion nd b vitmin B12 Mternl vitmin B12 concentrtions were not ssocited with ny of the offspring risk fctors (Tble 4). Mternl folte, vitmin B12 nd homocysteine concentrtions were not ssocited with offspring blood pressure or fsting lipid concentrtions (dt not shown). All ssocitions remined unchnged fter excluding the offspring of GDM mothers. Discussion In this well-chrcterised cohort of urbn Indin children, higher mternl homocysteine concentrtion ws ssocited with smller size t birth, nd higher postlod glucose concentrtions t 5 nd 9.5 yers. Higher mternl folte concentrtion ws ssocited with higher insulin resistnce (HOMA- IR) t 9.5 nd 13.5 yers. There were no ssocitions of mternl vitmin B12 concentrtion with size t birth or with insulin resistnce nd other crdiometbolic risk mrkers. Erlier studies hve observed high prevlence of vitmin B12 deficiency nd elevted levels of homocysteine but not folte deficiency mong Indins [4, 8]. Consistent with these findings, more thn 40% of our study women were vitmin B12-deficient, while only 4% hd low serum folte concentrtions. The homocysteine concentrtions were, however, lower thn those reported in other studies in the Indin subcontinent (Tble 5). A recent systemtic review nd metnlysis found tht higher mternl homocysteine concentrtion in pregnncy is consistently ssocited with lower birthweight, in different popultions [24]. Overll, it showed 31 g reduction in birthweight per SD increse in homocysteine, nd hyperhomocysteinemi (>90th percentile) ws ssocited with n OR of 1.25 (95% CI 1.09, 1.44) for smll-for-gesttionl-ge bby. Our results re consistent with

6 Dibetologi (2014) 57: Tble 3 Associtions between mternl folte concentrtions (SDS) nd offspring outcomes t 9.5 nd 13.5 yers Vrible 5 yers 9.5 yers 13.5 yers Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 β 95% CI p vlue β 95% CI p vlue β 95% CI p vlue β 95% CI p vlue β 95% CI p vlue β 95% CI p vlue Anthropometry (SDS) Weight 0.13 (0.05, 0.2) ( 0.05, 0.1) (0.09, 0.3) < ( 0.01, 0.2) (0.09, 0.3) < ( 0.003, 0.2) 0.06 Height 0.17 (0.09, 0.3) < ( 0.02, 0.2) (0.1, 0.3) < (0.02, 0.2) (0.1, 0.3) < (0.02, 1.2) 0.01 BMI 0.04 ( 0.03, 0.1) ( 0.08, 0.1) (0.02, 0.2) ( 0.04, 0.1) (0.02, 0.2) ( 0.04, 0.1) 0.3 MUAC 0.04 (0.02, 0.2) ( 0.05, 0.1) (0.05, 0.2) ( 0.03, 0.2) (0.03, 0.2) ( 0.03, 0.1) 0.2 Subscpulr 0.02 ( 0.1, 0.06) ( 0.1, 0.07) ( 0.005, 0.2) ( 0.05, 0.1) ( 0.02, 0.1) ( 0.05, 0.1) 0.3 skinfold thickness Ft% 0.03 ( 0.1, 0.04) ( 0.1, 0.05) ( 0.03, 0.1) ( 0.07, 0.1) ( 0.01, 0.1) ( 0.05, 0.1) 0.5 Risk fctors (SDS) b Glucose ( 0.01, 0.2) (0.03, 0.2) ( 0.04, 0.1) ( 0.02, 0.2) ( 0.09, 0.09) ( 0.07, 0.1) 0.6 Glucose ( 0.1, 0.07) ( 0.005, 0.005) ( 0.03, 0.2) ( 0.07, 0.1) 0.5 Glucose ( 0.1, 0.05) ( 0.005, 0.005) ( 0.06, 0.1) ( 0.1, 0.09) 0.9 Insulin ( 0.07, 0.09) ( 0.005, 0.005) ( 0.004, 0.2) (0.0003, 0.2) (0.04, 0.2) (0.01, 0.2) 0.02 Insulin ( 0.05, 0.1) ( 0.005, 0.005) (0.04, 0.2) ( 0.02, 0.2) 0.1 Insulin ( 0.06, 0.1) ( 0.004, 0.006) ( 0.07, 0.1) ( 0.1, 0.07) 0.6 HOMA-IR 0.02 ( 0.06, 0.1) ( 0.08, 0.1) ( 0.001, 0.2) (0.01, 0.2) (0.04, 0.2) ( 0.01, 0.2) 0.03 β nd p vlues derived by liner regression using mternl folte nd offspring mesurements s continuous SDS; β represents SDS chnge in outcome vrible per SDS chnge in exposure vrible Log-trnsformed vribles Model 1, djusted for child s sex nd ge Model 2, model 1 + mternl BMI, GDM sttus, SES, prity, religion, vitmin B12 concentrtion, nd children s 9.5 yer vitmin B12 nd folte concentrtions, nd b pubertl stge nd current BMI

7 116 Dibetologi (2014) 57: Tble 4 Associtions between mternl vitmin B12 nd homocysteine concentrtions (SDS) nd offspring risk fctors Risk fctor (SDS) 5 yers 9.5 yers 13.5 yers Model 1 Model 2 Model 1 Model 2 Model 1 Model 2 β 95% CI p vlue β 95% CI p vlue β 95% CI p vlue β 95% CI p vlue β 95% CI p vlue β 95% CI p vlue Mternl B12 concentrtion Glucose ( 0.15, 0.01) ( 0.19, 0.02) ( 0.11, 0.06) ( 0.16, 0.06) ( 0.08, 0.09) ( 0.12, 0.07) 0.6 Glucose ( 0.12, 0.04) ( 0.12, 0.06) ( 0.13, 0.04) ( 0.19, 0.03) 0.1 Glucose ( 0.13, 0.03) ( 0.13, 0.05) ( 0.06, 0.10) ( 0.09, 0.13) 0.7 Insulin ( 0.10, 0.06) ( 0.11, 0.06) ( 0.07, 0.09) ( 0.09, 0.10) ( 0.07, 0.09) ( 0.08, 0.08) 0.9 Insulin ( 0.07, 0.09) ( 0.07, 0.10) ( 0.10, 0.07) ( 0.16, 0.04) 0.3 Insulin ( 0.10, 0.06) ( 0.10, 0.07) ( 0.04, 0.12) ( 0.07, 0.1) 0.6 HOMA IR 0.03 ( 0.11, 0.05) ( 0.12, 0.05) ( 0.08, 0.08) ( 0.10, 0.09) ( 0.07, 0.09) ( 0.08, 0.08) 0.99 Mternl homocysteine concentrtion Glucose ( 0.06, 0.11) ( 0.07, 0.11) ( 0.03, 0.15) ( 0.007, 0.17) ( 0.07, 0.10) ( 0.03, 0.15) 0.2 Glucose (0.02, 0.19) (0.05, 0.30) ( 0.03, 0.15) ( 0.03, 0.15) 0.2 Glucose ( 0.02, 0.05) ( 0.04, 0.14) (0.01, 0.19) (0.02, 0.20) 0.02 Insulin ( 0.03, 0.05) ( 0.06, 0.12) ( 0.10, 0.16) ( 0.02, 0.14) ( 0.13, 0.03) ( 0.10, 0.05) 0.5 Insulin ( 0.01, 0.15) (0.01, 0.18) ( 0.08, 0.10) ( 0.07, 0.10) 0.7 Insulin ( 0.05, 0.12) ( 0.04, 0.14) ( 0.05, 0.13) ( 0.03, 0.13) 0.2 HOMA-IR 0.03 ( 0.05, 0.11) ( 0.05, 0.12) ( 0.007, 0.16) ( 0.003, 0.04) ( 0.12, 0.04) ( 0.09, 0.06) 0.7 β nd p vlues derived by liner regression using mternl vitmin B12 nd homocysteine concentrtions nd offspring mesurements s continuous SDS; β represents SDS chnge in the outcome vrible per SDS chnge in the exposure vrible Model 1, djusted for child s sex nd ge Model 2, model 1 + mternl BMI, GDM sttus, SES, prity nd religion, vitmin B12 concentrtions, nd children s 9.5 yer vitmin B12 nd folte concentrtions, pubertl stge nd current BMI Log-trnsformed vribles

8 Dibetologi (2014) 57: Tble 5 Comprison of mternl nutrients nd offspring outcomes in Mysore nd studies from other settings Vrible Mysore Pune [8, 42] Nepl [9, 43] Bnglore [4] Pkistn [44] UK [6] Netherlnds [30] Mternl mesurements Age, yers 23.9 (4.3) 21.4 (3.5) 19.3 (2.1) 24.6 (4.1) 26.0 (0.5) 27.8 (5.8) 33.3 (30.7, 35.5) Pregnncy stge t mesurement 3rd trimester 2nd trimester 3rd trimester 1st trimester 1st trimester At delivery 1st trimester 3rd trimester BMI, kg/m (3.6) 18.9 (1.8) 20.5 (1.7) 23.1 (5.4) 22.0 (4.0) Vitmin B12, pmol/l 187 (100) 151 (80) (99) 247 (198) 232 (87) 162 (23, 317) 324 (132) 179 (134, 219) B12 deficiency (<150 pmol/l), n (%) 271 (41.5) 380 (60) 423 (71) 147 (27) 177 (47) Serum folte, nmol/l 35.5 (19.4) 17.7 (20.2) 14 (2, 91) 9.1 (6.1, 16.4) Erythrocyte folte, nmol/l 941 (352) 1048 (408) 418 (178) Folte deficiency (<7 nmol/l), n (%) 26 (4.0) 1 (0.2) 1(0.2) 57 (10.5) b Homocysteine, μmol/l 6.4 (2.3) 8.8 (3.1) 9.1 (3.2) 10.3 (4.3, 23) 5.5 (4.5, 6.7) Hyperhomocysteinemi (>10 μmol/l), n (%) 25 (3.9) 177 (28) 193 (33) 179 (34.1) c Newborn mesurements Birthweight, g 2,854 (473) 2,618 (388) 2,850 (450) 3,430 (470) 3,425 (3,075, 3,855) Low birthweight d, n (%) 129 (19.5) 177 (28) 180 (33) Child mesurements Childhood HOMA-IR [17] 0.8 (0.5, 1.2) 0.81 (0.60) 0.4 (0.2, 0.7) Mternl vitmin B12 newborn size None None None Positive None None None Mternl folte newborn size None None Positive None Positive Positive None Mternl homocysteine newborn size Negtive Negtive None Negtive None Mternl vitmin B12 child HOMA-IR None Negtive None Negtive Mternl folte child HOMA-IR Positive None Positive None Mternl homocysteine child HOMA-IR Borderline positive None None Vlues given re men (SD), medin (interqurtile rnge) or n (%) Erythrocyte folte <283 nmol/l b Serum folte <6.8 nmol/l c Homocysteine concentrtions >12 μmol/l d Birthweight <2,500 g

9 118 Dibetologi (2014) 57: this, showing 60 g fll in birthweight per SD of mternl homocysteine concentrtion, which ws echoed in reduction in ll birth mesurements. We did not find ssocitions between mternl vitmin B12 or folte sttus nd newborn size. Evidence from other popultions hs been inconsistent on this point, especilly for vitmin B12. The Pune study showed no ssocition between mternl B12 sttus nd birthweight [8], while nother Indin study, from Bnglore, showed positive ssocition [4]. Severl studies, including the Pune study, hve shown positive ssocitions between mternl folte sttus nd birthweight [5, 6, 8]. The protective effect of mternl folte repletion ginst neurl tube defects is well known. This hs led to the recommendtion for folte supplementtion in the preconceptionl period, nd, in number of countries, folte fortifiction of stple foods, to chieve wide coverge of mothers in the crucil erly stges of pregnncy [25]. Studies linking mternl folte sttus with long-term outcomes in the children re, however, few nd re minly bsed on observtionl dt [8, 26 28]. In the only dt from rndomised controlled tril, children of women in Nepl who were supplemented with vitmin A plus folic cid from erly gesttion hd less microlbuminuri nd lower prevlence of metbolic syndrome t 6 8 yers thn children of mothers supplemented with vitmin A lone [29]. Interestingly, these reductions in microlbuminuri nd metbolic syndrome were not observed in children of mothers who received the sme dose of folic cid s prt of multiple micronutrient supplement. In the UK, observtionl studies hve shown higher bone minerl content in children of mothers with higher dietry folte intkes [26]; in the Pune (Indi) study, higher bone minerl content nd density in children of mothers with higher folte sttus in pregnncy [27]; nd in our study reported here, tller height nd better cognitive function [28] in children of mothers with higher folte sttus. By contrst, the Pune study linked higher mternl folte sttus with potentilly dverse outcome: higher childhood insulin resistnce [8]. Our study in Mysore hs now confirmed this, nd, for the first time, showed tht this ssocition trcks into erly dolescence. This is smll effect: 1 SD higher mternl folte concentrtion ws ssocited with 0.09 SD increse in childhood HOMA-IR t both 9.5 nd 13.5 yers of ge in our children. In Pune children, there ws similr, lbeit bigger, effect: 0.18 SD increse in HOMA-IR for 1 SD increse in erythrocyte folte. In Pune, lower mternl vitmin B12 sttus ws lso ssocited with higher offspring insulin resistnce, nd the highest men HOMA-IR ws in children of mothers with B12 deficiency nd high folte sttus. The only other study to exmine this question is the Nepl rndomised tril [9]. This tril showed tht mternl vitmin B12 deficiency t bseline ws ssocited with higher HOMA-IR in the children, but there ws no ssocition with mternl folte sttus, nd no difference in HOMA-IR between children of mothers who were or were not supplemented with folic cid. In Mysore, there ws lso n ssocition between higher mternl homocysteine concentrtion nd postlod glucose concentrtion in the children. This ws not observed in the Pune study, nd it ws not investigted in the Nepl study. These differences between studies my rise from differences in mternl micronutrient sttus nd differences in fetl growth (Tble 5). Mternl plsm folte levels in Mysore were considerbly higher thn those reported in Western studies [30, 31]. A predominntly vegetrin diet mong these women, nd tendency to use high-dose folic cid supplements throughout pregnncy, my explin this finding. The prevlence of folte deficiency, though uncommon in ll three studies, ws reltively higher mong the Nepl women [9]. The prevlence of vitmin B12 deficiency (which ws extremely high in Pune) ws high in ll studies but lowest in Nepl (27%). Surprisingly, the prevlence of hyperhomocysteinemi nd men homocysteine concentrtions were much lower in Mysore thn in Pune nd Nepl, which hd similr prevlence. HOMA-IR vlues in Mysore nd Pune were similr, but lower in Nepl. In the Pune nd Nepl studies, it ws the vitmin B12 sttus during erly pregnncy which ws ssocited with offspring HOMA-IR; however, there ws no ssocition with the lte-gesttion concentrtions, similr to the sitution in Mysore. Both in our study nd in Pune, offspring HOMA-IR ws ssocited with mternl folte t 28 weeks gesttion. While optimum micronutrients during the erly stges of pregnncy hve beneficil effects on fetl development, our findings suggest tht exposure to high folte levels in lte pregnncy my confer long-term metbolic risk. The mechnism underlying our findings my be specultive. There is evidence tht the blnce between folte nd vitmin B12 sttus is importnt for helth. In n elderly popultion in the USA, higher serum folte concentrtion presumbly due to higher intke, especilly in the presence of vitmin B12 deficiency, ws ssocited with impired cognitive function nd nemi [32]. There is suggestion tht the risk of cncer, crdiovsculr disese nd neuropsychitric disorders is incresed t both high nd low levels of folte sttus [33]. Enzymes tht require folte s cofctor my be inhibited by high levels of folte [34]. Higher folte in our mothers my reflect higher dietry intke or the folte trp phenomenon, considering the low vitmin B12 sttus. In B12 deficiency, the ctivity of vitmin B12-dependent methionine synthse is reduced nd cellulr folte is trpped s inctive 5-methyltetrhydrofolte, which my lek into plsm, thus elevting serum folte levels [35, 36]. Impired methionine synthesis from homocysteine, nd thus protein synthesis, my hinder len tissue deposition. There my lso be effects on DNA synthesis or on DNA methyltion tht my hve long-term effects on gene expression nd metbolism

10 Dibetologi (2014) 57: [36, 37]. The ccumultion of homocysteine in the body my lso hve dverse outcomes. Our dt do not give definitive evidence for the folte trp s mechnism: first, becuse there ws no interction between mternl folte nd vitmin B12 in reltion to the child s insulin resistnce (the ssocition between mternl folte nd insulin resistnce in the child ws not restricted to children of B12-deficient mothers); nd, second, becuse the inverse ssocition between folte nd homocysteine ws minly in mothers with low folte concentrtions (ESM Fig. 1). The Pune study findings did not support the folte trp either [8]: lthough there ws n interction between folte nd vitmin B12, the folte mesurement in Pune ws erythrocyte folte, which does not increse in the folte trp. The folte trp mechnism therefore seems unlikely, though it cnnot be ruled out from the type of dt tht we hve. Mternl homocysteine level ws reltively low despite high prevlence of B12 deficiency, which my be relted to the ction of other B vitmins, such s vitmin B2 nd B6, nd proteins on homocysteine conversion [23]. In our study the ssocition between mternl folte nd offspring HOMA-IR ws not present t 5 yers of ge, but emerged t 9.5 yers nd persisted through to 13.5 yers of ge. We hve previously shown tht the ssocition between mternl vitmin D deficiency [38] nd glucose intolernce [11, 14] with offspring HOMA-IR ws present t 9.5 yers of ge, but not t 5 yers of ge. At younger ges, high bet cell ctivity in children my msk the insulin resistnce. Our study, tken together with the other studies from Indi nd Nepl, suggests tht the folte vitmin B12 homocysteine pthwy my hve role in regulting fetl growth nd in the intruterine progrmming of lter dibetes. The proof of principle for this comes from niml studies showing tht folte nd other methyl donor nutrients in the mternl diet influence diposity nd insulin resistnce in the dult offspring nd tht this is ssocited with epigenetic chnges, for exmple ltered DNA methyltion [37, 39]. The initil findings from humn studies re complex, nd more dt, especilly from rndomised trils of folte nd/or vitmin B12 in pregnncy, would be helpful. A mjor strength of our study ws the mesurement of circulting mternl nutrients during pregnncy, which is more robust indictor of mternl nutritionl sttus thn dietry or supplement intke levels. Detiled neontl nd childhood nthropometry, nd mesurement of n rry of crdiovsculr risk fctors were other strengths. Offspring outcomes were vilble t three time points during childhood nd erly dolescence, which llowed us to exmine whether the observed ssocitions were persistent. The plsm smples hd been stored for 8 yers before nlysing mternl nutrients. These nutrients re thought to be stble over long periods, however, when frozen t 80 C [40]. A comprehensive rnge of mesurements both during pregnncy in the mother nd lter follow-up in the offspring enbled relevnt djustments. It is likely tht plsm folte levels were influenced by folic cid supplements. Unfortuntely, we did not collect dt on supplement use t 30 weeks gesttion when mternl folte sttus ws mesured. Supplementtion could confound the ssocition between mternl folte concentrtions nd insulin resistnce in the child. For exmple, if mothers of higher SES took more supplements, their children could lso hve higher insulin resistnce becuse of greter diposity. The ssocition ws unchnged, however, by djusting for mternl SES nd the child s BMI. In conclusion, our study replictes the Pune finding of higher insulin resistnce in children born to mothers with higher gesttionl folte levels. It lso showed tht this ssocition persists through childhood into dolescence. We did not see n ssocition with mternl vitmin B12 sttus. In the context of other studies, our study suggests tht dernged mternl one-crbon pthwy reltes to impired fetl growth nd crdiometbolic risks in lter life. These re novel ssocitions which my help to elucidte the complex mechnisms underlying the development of type 2 dibetes. These findings re suggestive of nutrient-medited tertogenesis, where micronutrient deficiencies during intruterine development promote incresed risks in the offspring [41], cptured under the brod mbit of developmentl origins of helth nd disese. It is not cler whether these smll chnges observed in reltion to mternl B vitmins represent n incresed risk of lter disese. Follow-up of children in different cohorts nd rndomised controlled trils could help clrify these issues. Acknowledgements We re grteful to the prticipting fmilies, the stff t CSI Holdsworth Memoril Hospitl nd the MRC Lifecourse Epidemiology Unit for their contributions, D. S. Bht (KEM Hospitl, Pune, Indi) for lbortory ssys nd Sneh-Indi for its support. We cknowledge the substntil contributions mde by J.C. Hill (MRC Lifecourse Epidemiology Unit, Southmpton, UK). Funding The Prthenon Cohort ws initilly funded by the Prthenon Trust (Switzerlnd) nd is now supported by the Medicl Reserch Council (UK), DFID nd the Wellcome Trust (079877/Z/06/Z nd /Z/10/Z). G. V. Krishnveni ws mentored in non-communicble disese epidemiology supported by Fogrty Interntionl Center nd the Eunice Kennedy Shriver Ntionl Institute of Child Helth & Humn Development t the Ntionl Institutes of Helth, USA (grnt no. 1 D43 HD065249). Access to reserch mterils The Prthenon Cohort tem is open to dt shring with bon fide reserchers, nd subject to Government of Indi regultions; plese contct the corresponding uthor Dr G. V. Krishnveni with description of your reserch pln. Dulity of interest The uthors declre tht there is no dulity of interest ssocited with this mnuscript. Contribution sttement GVK, CSYnd CHDF designed the reserch; GVK, SRV, SCK nd CSY conducted the reserch; GVK nd CHDF

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