{"paper_id":"24092055-285d-414f-bd36-79c5ad253664","body_text":"Accelerated b iological aging six decad es after p renatal famine exposure  \n1\n \nAccelerated biological aging six decades after prenatal famine \nexposure \n \nMengling Cheng 1,2 , Dalton Conley 3 , Tom Kuipers 4 , Chihua Li 5,6 , Calen Ry an 2 , Jazmin Taubert 4 , \nShuang Wan g 7 , Tian Wang 7 , Jiayi Zho u 2 , Lauren  L. Schmitz 8 , Elmar W. Tobi 4 , Bas  Heijmans 4\n,\n L . H. \nLumey 4,6 , Daniel W. Bels ky 2,6*  \n \n1.  Swiss Centre of Expertise in Life Course Research, Universi ty of Lausanne, Lausanne, \nSwitzerland  \n2.  Robert N. Bu tler Columbia Aging Center, Columbia University, New York, N Y, US A  \n3.  Department of Socio logy, Princeton University, Princ eton, NJ, USA \n4.  Department of Biomedical Data Sci ences, Leiden Universi ty Medica l Center, Leiden,  \nNetherlands  \n5.  Institute for Socia l Resea rch, Universi ty of Mi chigan at Ann Arbor, Ann Arbor, MI, USA  \n6.  Department of Epidemiology, Colu mbia University Mailman School of Public Health, \nNew York, NY, U SA  \n7.  Department of Biosta tist ics, Columbia University Mailman School of Publi c Health, New  \nYork, NY, U SA  \n8.  Robert M. La Follet te School o f Public Affair s,  University o f Wisco nsin-Madison, \nMadison, WI, U SA  \n \nCorresponding author  \nDaniel W. Belsky, PhD, Columbia Univ ersity Mai lman School of Public Healt h, Rm 41 3, 72 2 West \n168th Street, New York, NY 10032 , U nited States. Email: db3275@cumc.c olumbia.edu\n. \n \n \nConflict of Interest \nDWB is listed as an invento r on th e D uke Univers ity and Unive rsit y of O tago Invention \nDunedinPACE , which is licensed to Tr uDiagnostic.  \n \n \nWord count: 3,597  \nReferenc es: 55 \nData elements: 1 table, 4 figures  \nSI appendix: supplemental methods, supplementary tabl es S1-9, suppleme ntary figure S1, \nsupplementary re ference s  \n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n2\n \nAbstract \n \nTo test the hyp othesi s that ea rly-lif e adversity a ccele rates th e pace o f biol ogical aging, we \nanalyzed data from the Dutch Hunger  Winter Families Study (DHWFS, N=95 1). DHW FS is a \nnatural-experiment bir th-cohort s tud y of surv ivors o f in-utero exposure to famine conditi ons \ncaused by the G erman occupation of the Western Neth erlands in Winter 1 944-5 , matched \ncontrols, and their s iblings. We condu cted DNA methylation ana lysis o f blo od samples collec ted \nwhen the survivo rs were aged 58 to q uantify bi ological aging using the Dun edinPACE , GrimA ge, \nand PhenoAge epigenetic clocks. Famine surviv ors had fas ter Duned inPACE , as compared with \ncontrols. This e ffec t was strongest a mong wo men. Results were similar fo r GrimAge, although \neffec t-sizes were smaller. We obse rv ed no diff erence s in PhenoAge between survivor s and \ncontrols. Famine ef fects wer e not ac c ounted fo r by blood-ce ll composit ion and were similar fo r \nindividuals exposed ear ly and lat er in gestation. Findings suggest in-utero u ndernutri tion may \naccelerat e biol ogical aging in later life .  \n \nKeywords \nearly-life adv ersi ty, biological aging, natural exper iment, prenatal exposu re , \nfamine/undernutrit ion  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n3\n \nSignificance Statement \n \nEnvironmental condit ions dur ing gestation are hyp othesi zed t o shape heal t h across the life  \ncourse. The Dutch Hunger Winter, a f amine caused by a German blockade of the Western  \nNetherlands in late  1944 and ended by the all ied li berati on of the Neth erla nds in Spring 1945, \nhas been studied  as a “natural exp eri ment” in which the t iming of a child’s  concepti on \ndetermined the ir exposur e to se vere under-nutriti on during gestation. We applied thi s natural-\nexperiment design to t est e ffec ts of i n-utero adversi ty on midli fe bio logical aging, as measured \nby epigenetic clock s. We found that i ndividuals with  in-utero famine expos ure had a faste r pace \nof biologica l aging six decades late r. The environmental c ondit ions surr oun ding pregnancy have \npotentia l to shape aging trajec torie s f or the next genera tion.   \n \n \n \n \n \n \n \n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n4\n \nIntroduction \nInsults to ea rly-lif e devel opment are predicted  by theo ry to impact trajec t ories of  \nhealthy aging (1-4). Consistent with t his hypothes is, longitudina l observa ti onal studies  have \nidentif ied assoc iation s between ea rly -life condi tions and later-li fe healt h o utcomes (5). But \nestablishi ng the causali ty of su ch asso ciations is dif ficu lt due to poten tial co nfounding effe cts of  \nfamily histor y and othe r fact ors tha t may affect both early-li fe devel opment and later aging \noutcomes (3, 6). Natural experiments , which seek t o over come this obstacl e to causal  inf erence ,  \nare study des igns that tak e advantage of his torica l event s beyond the cont rol of individua ls or \ntheir famil ies that  impact a subset o f otherwise comparable individua ls in a population. An \nestablished na tural-experiment des ign for inve stigating eff ects o f earl y-life  adversity on late r-\nlife hea lth is in-utero exposu re to famine (7). In studie s of th e Dutch Hunger Winter (1944-5), \nSiege of Leningrad (1941-4), Holodomor famine in Ukraine (1932-3), and Great Chinese Famine \n(1959-6 1), survivors of in-ute ro famin e exposure exhib it higher bu rdens of multiple aging-\nrelated d iseases and have sh orte r li fe spans  as compared to unexposed ind ividuals bo rn befo re \nor after  famine or in adjac ent, unaffe cted regions (8-13). Within the Fetal  Origins and \nDevelopmental Or igins of Health and Disease li teratu res, these observat ion s are oft en \ninterpre ted as r efle cting in-utero p ro gram ming of risk for car diovascu lar a nd metabolic diseas e \nlater in lif e (4, 14, 15). However, an al ternativ e hypoth esis is that famine-in duced insul t in ear ly \nlife impairs the deve lopment of more  general robustness  and resi lience  ca pacities of the  \norganism, resulting in accelera ted sys temic decline  with aging. \nTo explore this a lterna tive h ypothes is , we analyzed blood DNA methylat ion  (DNAm) \ndata colle cted in  a natural-experimen t study o f in-utero famine exposure t o test di ffe rences in \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n5\n \nthe pace and progress of bio logical aging between famine-survivors and matched cont rols. The \nDutch Hunger Winter Families Study ( DHW FS) enrolled a coho rt of  survivo r s of in-utero \nexposure to the Dutch  Famine (1944- 5), matched controls bo rn befo re or a fter th e famine in \nthe same hospitals as the surv ivors, a nd their same-sex siblings (16). We compared famine \nsurvivors to unexposed contr ols on th ree DNAm measures of biological agi ng linked in pr ior \nstudies with h isto ries o f early-li fe adv ersity, the  DunedinPACE, Grim Age , a nd PhenoAge DNA m \nclocks (17-19). Our analysis furth er ex plored di ffer ences in the e ffec ts of fa mine between \nwomen and men and by gestational ti ming of exposure, and tested co nsist ency of findings in \nboth between- and within-family co mparisons.  \n \n \nResults \nWe analyzed data for  N=951 cohort members with available DNAm data (N=487 famine \nsurvivors, N=159 time cont rols, N=30 5 sibling contro ls; Table 1 ). The characteris tics o f thi s \nanalysis sample were similar to the  D utch Hunger Winter Families Study in terview sample ( SI \nAppendix , Table S1 ).  \nOur analysis p roceeded  in thr ee step s . First, to tes t the hyp othesi s that in-utero famine \nexposure cont ribut ed to acc elera ted biological aging, we compared DNAm  measures of pace of  \naging (DunedinPA CE) and biological age (GrimAge and PhenoAge) between famine-survivors \nand controls. Sec ond, we conducted dose-response analysi s to test i f part i cipants who were \nexposed to th e famine for  more week s of gestation exhibit ed larger famine  effec ts as compared \nwith those expos ed for fewer weeks  of gestation. Third, to explore sp ecif ic ity of famine eff ects \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n6\n \nto exposure du ring specif ic per iods o f  gestation, we classi fied famine sur viv ors accord ing to \nwhen in gestation th ey were expos ed , as described in the Meth ods sec tion , and computed \neffec t estimates for each wind ow of e xposure. In each step, we conduc ted analysis (a) in the full \nDHW FS; (b) using a between-families comparison of famine-survivo rs to un exposed time \ncontrols  born immediately be fore or after th e famine; and (c) us ing a withi n-family comparison \nof famine-survivors to the ir unexpose d same-sex siblings. We also explored whether \nassociations  of famine exposu re with biological aging diffe red between me n and women.  \nIn-utero famine exposure was associated with faster biological aging as measured by \nDunedinPACE. Cohort members expo sed to famine in u tero had faster pac e of aging compared \nwith unexposed coho rt members (Du nedinPACE β=0.1 5, 95 % CI [0.0 3, 0.2 8 ], p =.018). \nDiffer ences be tween famine survi vor s and contro ls were of  smaller magnitude for Gr imAge and \nPhenoAge and not statistica lly di ffe re nt from zero a t the a lpha=0.05 level ( βs<0.10 , p>.099). \nResults were similar in ana lysis r estr ic ted to in clude on ly famine surv ivors a nd unrelated time \ncontrols. Results  are shown in Fig. 1 A and  reported  in SI Appendix , Table S2 ; full resu lts fo r all \nbiological aging measures are repor te d in SI Appendix , Table S3 ; a correlati on matrix is shown \nin SI Appendix , Fig. S1 .  \nLonger prenatal famine exposure was associated with faster biological aging as \nmeasured by DunedinPACE. In dose- response analys is, cohor t members w ho were exposed t o \nthe famine for more wee ks of gestat i on had faster b iological aging (per 10-weeks of exposu re \nDunedinPACE β=0.08 , 95 % CI [0.02 , 0. 14], p =.01 3). There were no dose res ponse eff ects for \nGrimAge and PhenoAge (βs=0. 04, p>. 160). Results are rep orted in SI Appendix , Table S4 ; full \nresults for al l biol ogical aging measur es are repo rted in SI Appendix , Table S5 . \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n7\n \nNo timing-specificity for famine exposure in predicting biological aging as measured by \nDunedinPACE . The effects of  in-utero  famine exposure may vary depend in g on when in \ngestation famine exposur e occurs. W e estimated assoc iations of famine ex posure with \nbiological aging at each of s ix time wi ndows from the precon cepti on peri o d through the end o f \ngestation. Because cohort  members could be exposed in multiple time win dows, we included \nindicator  variabl es for exposure in ea ch time window in the same regressi on. Effect-sizes for \nDunedinPACE ran ged from -0. 01 to 0. 18 and were somewhat larger for late r gestational \nexposure windows. Effect-si zes fo r Gr imA ge ranged from -0. 08 to 0.12. Effect-sizes fo r \nPhenoAge ranged from -0.1 9 to 0.28. For GrimAge and Pheno Age, there w ere no gestati onal \ntiming patterns in e ffec t-sizes. Effe ct-sizes are repor ted in SI Appendix , Table S6. \nSex differences in associations of in-utero famine exposure with biological aging.  \nWe conducted explora tory ana lysis o f  sex diffe rences in famine ef fects u sin g sex-stratified \nregressions and analys is of effe ct-measure modificat ion. In strat ifi ed analy sis, famine effec ts \nwere consis tentl y larger f or women and were near zero for men ( Fig. 1 B; SI Appendix , Table S2). \nFindings from sex-stratified d ose-resp onse analysis sh owed similar resu lts ( SI Appendix , Table \nS4). In sex-stratified analys is of gestat ional timing, results wer e dif ferent  fo r women and men \n( SI Appendix , Table S6 ). For wo men, DunedinPACE effect-sizes were simila r across gestat ional \ntime windows (effec t-sizes ranged f ro m β=0. 07- 0.25). In contrast, for men, DunedinPACE effect-\nsizes were largest for  late r-gestationa l exposures and smaller  for exposure in early gestat ion \n(effec t-sizes ranged f rom β=-0. 16- 0.1 9). This pattern was similar f or GrimAge. There was no \nconsisten t patte rn fo r PhenoAge. Results are shown in  Fig. 2 . Formal tests of eff ect-measure \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n8\n \nmodification found sex di ffe rences w ere stat istica lly d iffe rent from zero at  the p<0.05 level for \nDunedinPACE and Grim Age , but not PhenoAge.  \nSibling-comparison analysis. Finally, we repeated our ana lysis us ing a sibli ng-\ncomparison design. This design holds constant al l fact ors tha t are shar ed b y siblings in a family. \nIn the contex t of th e famine natura l e xperiment, the sibl ing comparison de sign aims to rule out \nthe possib ilit y that d iff erences b etwe en exposed and unexposed individua l s refle ct di ffer ences \nbetween families in thei r pre ferenc es  and/or abilit y to c onceive  and carry t o term a child unde r \nfamine conditi ons. In full-sample sibl i ng comparison analysis (n=227 pairs),  famine survivors  \ntended to b e aging faster than t heir u nexposed same-sex siblings; however, effect-size s were \nattenuated by  roughly half  as compared with the full-sample analysis and were not sta tist ically \ndiffer ent f rom zero. In sex-strati fied a nalysis, dif ference s between s isters  di scordant f or famine \nexposure (n=129 pairs) we re nearl y id entical to famine-effec t est imates fro m our original \nmodels whereas, among brothers (n=98 pairs), effec t est imates were near zero or in the \nopposite d irec tion o f our or iginal anal ysis. Resul ts are r eported  in SI Appendix , Table S2 and \nTable S3 .  \nSensitivity analysis . We repeated our  analysis with add itiona l covar iates f o r estimated \nproporti ons of whi te blood  cell  types.  In cell-count-adjusted analys is, effe ct -sizes were similar \nfor DunedinPACE, GrimA ge, and Phen oAge ( SI Appendix , Table S7-9 ).  \n \nDiscussion \nWe analyzed blood DNA methylat ion data from partic ipants in a na tural-ex periment \nstudy of the Dutch Famine to test the  hypothesis that in-ute ro famine expo sure would be \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n9\n \nassociated wit h accel erated b iologica l aging over six decades o f fol low-up. We found that \nsurvivors of in-uter o famine exposure  had faster pac e of bi ological aging as measured by the \nDunedinPACE clock. However, differe nces in bio logical age measured by th e GrimAge and \nPhenoAge clocks were smaller and le ss consiste nt. We did not obs erve e vi dence for a timing \nspecifi c eff ect o f famine exposur e on these measures. These find ings were robust to covaria te \nadjustment for ce ll coun ts and were s imilar in sibl ing-difference  analysis, al though estimates \nwere less pre cise.  \nAll three o f the DNAm clocks  we anal yzed show eviden ce of as sociat ion wi th morbidity \nand mortality in other studies (20, 21). A prior quasi-experimental anal ysis of early-li fe \neconomic adversi ty found evidenc e o f in-utero-exposure e ffec ts on la ter-lif e biological aging \nmeasured by both an earl ier ve rsion of the Duned inPACE clock and the Gri mA ge clock (19). \nHowever, only DunedinPACE showed consisten t eviden ce of as sociat ion wi th in-utero famine \nexposure in t he ful l DHWFS sa mple. It could be tha t DunedinPACE is somewhat more sensitive \nto precl inical  health changes occurr in g in famine survivors as compared with GrimAge. \nDunedinPACE was developed from an alysis of the ra te of ph ysiol ogical decl ine in midlif e adults \n(22). It is designed to measure t he Pace of Aging phenotype (23), defin ed a s the rate of dec line \nin system integri ty. GrimAge, in contr ast, was developed f rom analysis of mortality r isk in mid-\nlate li fe adul ts (24). It is des igned to measure biological age, or the cu rren t level of sys tem \nintegrity. These des ign diffe rences may have consequenc es fo r sensi tivi ty i n the cont ext of \nmidlife fo llow-up of in-utero famine e xposure. Alternat ively, th e eff ect-size  differ ences \nbetween DunedinPACE and GrimA ge were small and could re flec t stat isti ca l noise. Follow-up in \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n10\n \nother stud ies is n eeded t o clar ify t he significance o f the d iff erence in resu lt s for the  two \nmeasures.  \nPrior studies suggest that exposure d uring the early p hase of gesta tion may be more \nimpactful (11, 25-27). Our analysis of gestational t iming of famine exposur e did not find \nevidence for ear lier gesta tion as a se nsitive p eriod. Ov erall, res ults suggest that any in-utero \nexposure is asso ciated wi th a fast er p ace of bio logical aging six decades la t er.    \nOur analysis o f sex di ffer ences in  famine effec ts found  larger ef fects  of in-u tero famine \nexposure on DNAm measures of biol ogical aging in women as compared with men. This was \nobserved fo r all three epigenetic cloc ks, but was most pronounced f or the  DunedinPACE and \nGrimAge clocks. In non-human ani ma ls, there is evidenc e that males are  more vulnerab le to  \nearly-life insults  (28). However, prior studies o f in-utero famine exposure o ften repo rted larger \nfamine effec ts on card iovascula r and metabolic diseases  among wo men as compared with men \n(29-32). There is some evidence that select ive fe rti lity and/or  feta l loss res ult in f ewer male \nbirths dur ing periods o f famine (33). A result coul d be that the subse t of m ale babies born  are \nespeciall y robust. This would  be cons i stent with o ur resu lts. Bu t a reduc tio n in male births is not \ndocumented in the case of the Dutch  Famine (34, 3 5). There are not yet an alyses of s ex \ndiffer ences in morta lity  among survivors of in-ute ro exposure  to th e Dutch  Famine; the most \ncomprehensive stud y of mortal ity r eli ed on data fr om military consc ripts, who were all men \n(25). New models in population  scien ce suggest that envir onmental condi ti ons, such as in-utero \nexposure to famine, can induce subs t antial varia tion in sex dif feren ces in s urvival (36). \nReplicat ion of  the obs erved sex d iffe r ence in famine e ffec ts is ne eded.  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n11\n \nWe acknowledge limitations. There i s  no gold standard to measure b iologic al aging (37). \nWe analyzed the DNAm measures of aging with the best availabl e eviden c e for re liabi lity and \nvalidity. As new measures are introdu ced, follow-up will be n eeded. However, agreement \nbetween dif feren t biologica l aging m easures build confid ence tha t our fin dings do capture \naging processes. We were unable to c onduct dose-respons e analysis of fam ine-exposure \nseverit y. Because o f the lack o f family -level nutri tion da ta and the c onsiste ncy of ra tions acr oss \nthe affe cted areas  of th e Nethe rlands , analysis of exposu re seve rity wi ll ne ed to be condu cted \nin diff erent s ett ings, such as where fa mine severity was graded acro ss geographic loca tions o r \ntime (9). Survival bias c ould af fect the  population of famine surv ivors a live or in suf fici ently \ngood health to be su rveyed a t fo llow-up. However, characteris tics at  birth of the DHWFS \nparticipan ts are s imilar to th ose of fa mine-affected bir ths iden tifi ed in hos pital re cords but  not \nsuccessful ly enr olled in the cohort, in cluding birth weight, length, placen ta l weight, maternal \nage, and birth order (16). Excess deat hs among survivors of in-uter o famine exposure by the \ntime of our s tudy were <10% (13). Therefore, any b ias is l ike ly to b e modest. Moreover, i t is \nexpected tha t health y-participan ts an d survival b iases would bias effe ct-est imates toward the \nnull because n on-participa tion due to  ill heal th and death wou ld remove th e most affect ed \nfrom the populat ion. Therefo re, our e stimates of famine e ffec ts are expe ct ed to be \nconservati ve. Finally, becaus e the c oh ort so fa r lacks  fol low-up to determin e survival \ndiffer ences be tween famine-exposed  and control  parti cipants, the extent t o which diff erences  \nobserved in measures of bio logical aging will transla te int o dif ferenc es in h ealthspan and \nlifespan r emains to be de termined.  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n12\n \nWithin the con text o f these limitati on s, our findings prov ide evid ence f or lo ng-term \nimpacts of in-utero famine exposure that may extend to a wide range of a ging -related health  \noutcomes. Now that surv ivors o f in-utero famine exposu re are appr oachin g their ninth d ecade \nof lif e, furthe r study of famine bi rths i n administrative record data are ne e ded to clar ify the \nscope of famine effec ts on hea lthspa n and lifespan.  \n \n \nMaterials and Methods \n \nStudy setting: The Dutch Hunger Winter of 1944-1945 \nThe Dutch famine was initia ted by a f ood supply embargo imposed by the German occupying \nforces in early Octobe r 1944. The severity and wide spread natur e of the fa mine are well \ndocumented (16, 38, 39). Prior to the embargo, nutrition in the  Nethe rland s had generally been \nadequate. Off icial rations, which eve ntually con sisted  of l itt le more than b read and potato es, \nfell be low 900 kcal/day in la te November 1944, and were as low as 500 kcal/day by April 1945. \nThe macronutrient composit ion of  th e ration remained relat ively stable ov er this pe riod, but \nthe compositio n of non-rat ion foods  changed, with a reduction in the inta ke of fa t. The famine \nceased with l iberat ion in May 1945, after which Al lied f ood suppl ies were d istribu ted.  \n \nParticipants  \nFamine-Exposed individuals were id e ntified  from revi ew of arch ival obst et ric rec ords. \nWe selected  al l the 2,417 singleton b irths be tween 1 February 1945 and 31 March 1946 at \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n13\n \nthree ins titu tions in famine-exposed cities in the wes tern Ne therlands wh ose mothers were \nexposed to th e famine during or imm ediately p reced ing that pregnancy.  \nTime Controls were sele cted f rom birt hs at the same hospita ls and in the sa me months \nof the yea r as the famine-exposed group during 1943 and 1947 (two years before and two years \nafter th e famine). We sampled an equal number of bir ths in each month, a llocated a cross t he \nthree ins titu tions ac cording to their s i ze, to obtain  890 singleton bir ths.  \nOf the t otal famine-exposed and time-control bir ths, curren t addresse s wer e able to be  \ntraced fo r 70%. These individuals wer e invited  by mail to jo in the s tudy and  were additiona lly \nasked if a same-sex sibling  born b efor e or afte r the famine would be availa ble to par ticipa te. A  \ntotal o f N=547 of the famine-exposed group, N=176 of the time-control gro up, and N=308 \nsame-sex unexposed siblings consent ed to part icipat e and underwent a co mputer-assisted \nstructur ed inte rview by telephon e.  \nData Collect ion  was conduct ed in 2003-200 5, approximately six decades af ter the \nfamine. Of the N=1,031 participan ts who were interv iewed, N=971 also participa ted in a c lini c \nexam ( Fig. 3 ). Following the Helsinki g uidelines, we obta ined eth ical appr ov al both fr om the \nInstituti onal Rev iew Board o f Columbia Universi ty Medica l Center and fro m the Medical Ethical  \nCom mittee of th e Leiden Un iversi ty Medical Center (LUMC). The study participan ts prov ided \nverbal consen t in a telephon e inte rvi ew, and in case of cl inical  examinatio ns, a written in formed \nconsent was obtain ed and addit ional METC approval for epigenetic s tudies  was later con firmed \nby the METC of the LUMC.  \n \nMeasures \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n14\n \nFamine exposure. We defined the pe riod of famine from archival  reco rds of weekly ration \ndistribu tions, as desc ribed pr evious ly (16). Briefl y, the sta rt of the famine-e xposure period  was \ndefined as Nov ember 26, 1944 , based on the thr eshold <900 kca l/day of d istribu ted foo d \nrations. The end of  the famine-expos ure period was de fined as May  12, 19 45, one week \nfollowing the Ge rman surrender. Part icipants’ exposur e during gestation w as determined fr om \nthe date o f thei r mother’s last menstr ual period ( LMP) and their da te of b ir th. In cases where \nthe LMP date was missing or implausible (12% of birt hs), LMP was estimated from birth-record  \ndata on birth we ight and date of  birth  using tables of gender, par ity, and bi rth weight speci fic \ngestational ages from the c ombined birth re cords o f the Amsterdam Midwives School (1948-57) \nand the Univers ity o f Amsterdam Wil helmina Gasthuis Hospita l (1931-65).  \nFamine-exposed participants exper ie nced an average of 17 weeks o f gesta tion during \nwhich ration d istr ibutions  were <900kcal/day. Following prior wor k with t he cohort (29, 40), \nparticipan ts were c lassif ied as famine  exposed during each o f four 10-week  gestational per iods \non the basis o f rat ion dis tribut ions. F or each indi vidual, average rat ions w ere calcu lated f or \neach 10-week period of gestat ion and  periods with av erage rations  <900 kc al/day were \nclassifi ed as famine-exposed. Among the N=547 partic ipants re cruit ed as f amine exposed, \nN=403 met criteria for exposu re in at least one 10-week gestational  period.  A further N=82 had \nLMP dates prior t o the end  of th e famine, but fewer than 10 weeks of gesta tional exposu re to \nration dis tribu tions <900kca l/day. A fi nal N=62 had LMP dates after the en d of the famine. \nGestationa l peri ods for famine-exposed partic ipants and t ime-controls are shown in Fig. 4 . \n \nDNA methylation measures of biological aging.  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n15\n \nBiological aging is the p rogressive los s of syst em integrity with ad vancing c hronological \nage (41). Biological aging is thought t o arise f rom an accumulation of c ellu l ar-level changes that \nprogressively und ermine the robustn ess and resil ience capacity of ce lls, tis sues, and organ \nsystems (42-44). While there i s no gol d-standard measure of biologica l aging in humans (37), \nthe curren t-state o f the ar t are algori thms that combine info rmation from dozens or hund reds \nof DNA methylation (D NAm) marks, c hemical tags on the DNA sequence th at regulate gene \nexpression and ar e known to change with aging (45). These algorithms are often re ferr ed to as \n“epigenetic cl ocks” (46). We measured biological aging using epigenetic c lo cks computed fr om \nthe exist ing DNA methylation databa se for th e DHWFS.  \nBrief ly, DNA methylation (DNAm) was measured from blood col lect ed at t he clini c exam \nusing the Illumina Infinium Human M ethylat ion 450k BeadChip and prep ro cessed as prev iously \ndescribed (26). Furth er deta ils are p r ovided in the SI Appendix , Supplementary Methods .  \nOur primary analysis  focused  on thr e e epigenetic clock s for whi ch valida ti on data across \nmultiple studi es estab lish robu st asso ciations wi th health span and li fespan and sensitiv ity to \nexposures known t o hasten aging-related health d ecline: the Duned inPAC E clock, which \nmeasures pace of aging, and the Gri mA ge and PhenoA ge clocks, which measure biol ogical age. \nWe calculated Dunedi nPACE usin g the R code avai lable on G itHub \n( https://github.com/danbelsky/DunedinPAC E\n). We calculated high-technic al-reliabili ty “PC” \nversions o f the G rimAge and PhenoA ge clocks deve loped by the Levi ne La b (47) using the code \navailable f rom GitHub ( https://github .com/MorganLevineLab/P C- Clocks ). The clocks  are \ndescribed in detai l in th e SI Appendix , Supplementary Methods .  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n16\n \nThere are many other epigenet ic c loc ks, although none with comparable e vidence o f \nvalidity  to Duned inPACE , GrimA ge, an d PhenoAge. Most other cloc ks were developed to predi ct \ndiffer ences be tween indiv iduals in th eir chron ological age (sometimes ref e rred to as “ firs t-\ngeneration clo cks”). For comparison purposes, we report  resul ts in the SI Appendix , Table S3 \nand Table S5  for three o f the b est-kn own first-generation clock s, the Horv ath, Hannum , and \nSkin & Blood clo cks (46, 48, 49). We a lso repor t resu lts f or the original ve rsi ons of thr ee second-\ngeneration clo cks, the Zhang, GrimAg e, and PhenoAge clocks (24, 50, 5 1) . Original vers ions of \nthe cloc ks were computed us ing the methylclock R package  (52) and Pyth on code to calcula te \nGrimAge provided by Ake Lu.  \n \nAnalysis \nThe analysis sample for this stud y was formed from partic ipants in  the c lini c examination who \nprovided a bl ood sample from which DNA was extracted and sto red at LU MC. For our analysis, \nDNA were available fo r N=960 indivi d uals. After quali ty con trols, DNA met hylation da tasets \nwere available for N=951. These indiv iduals formed our ana lysis sample.  \nWe used regression analysi s to t est a ssociation s between in-utero famine exposure and \nDNAm measures of biological  aging. F irst- and second-generation ep igeneti c clock values have \nhigh correlat ions with ch ronol ogical a ge. For analysis and interp retat ion, th e standard approach \nis to regress clock  values on pa rtic ipa nts’ chronological age valu es and pre dict res idual valu es. \nThese values, often refer red to  as “age accelera tion r esiduals”, aim to quan tify the dif feren ce \nbetween how much aging a person has actually exp erienc ed rela tive to th e expectat ion based \non their chronol ogical age. No residu alizati on was perfo rmed for Dunedin PAC E, which is a rate \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n  \nAccelerated b iological aging six decad es after p renatal famine exposure  \n17\n \nmeasure and shows only moderate c orrelat ion with ch ronologica l age. To  account fo r the non-\nindependence o f measurements take n from siblings, we used generalize d estimating equation \n(GEE) regressions (53). Our models in cluded covar iates for par ticipan ts’ se x, age, and a ge-\nsquared at the time of th e clin ic exa m. We explored sex di fferen ces in fa mine effects b y \nrepeating analysis wi th inc lusion o f pr oduct te rm testing inte raction  betwe en famine exposure \nand sex. We repeated our anal ysis wi th a contro l group res tric ted to the \"ti me controls\" born \nimmediately before or aft er the famine. We tested consi stency of re sults i n within-family \ncomparisons of sib lings using sibling-f ixed-effects (FE) regressions (54). We tested th e \nsensitiv ity o f associa tions b etween fa mine exposure and biol ogical aging to diffe rences \nbetween parti cipants in leuk ocyte  co mposition of DNA samples by rep eati ng analysis with \nadditional covaria tes fo r DNAm estimates of l eukocy te prop orti ons est imated using the \nHouseman equations (55).  \n \n \nFunding sources \nThis research was suppor ted by Nat io nal Institut e on Aging grants ( R01AG0 66887; \nR01AG0421 90).  D WB is a fell ow of th e Canadian Institute for Advanced R e search Child Bra in \nDevelopment Networ k. LHL was supported by a NIDI/NIAS/UMCG Fellowship, Royal \nNetherlands Academy of Sci ences KN AW at the Netherlands Inst itut e for A dvanced Study. MC \nwas supported by th e Swiss National Centre of Competence in  Researc h “LIVES - Overcoming \nvulnerabil ity: Li fe cou rse persp ectiv es ” financed by the Swiss Nationa l Scie nce Foundation \n(51NF40 -1859 01) and the European Union Horizon 2020 Research and Inn ovation Programme \nunder the Mar ie Sklodowska-Curie Gr ant (801076). LLS was supported by National Inst itute on \nAging grant (R00AG056 599). E W T wa s supported by  a VENI grant from the Netherlands \nOrganization f or Scien tifi c Resear ch ( 916171 28) and by the Joint  Program ming Initiative (JPI \nHDHL proposal number 655) via ZonMw in The Netherlands (529051023).  The funders had no \nrole in s tudy des ign, data collec tion a nd analysis, decis ion to pub lish, or p r eparation o f the \nmanuscript.  \n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated b iological aging six decad es after p renatal famine exposure  \n18\n \nREFERENCES \n1. L. A. Gavrilov, N. S. Gavrilova, Early-lif e programmin g of aging and longevity: The idea of \nhigh initial damage load (the HIDL hy pothesis). Ann. N. Y. Acad. Sci.  1019, 496–501 \n(2004).  \n2. D. J. Barker, Fetal o rigins of coronary  heart disease. BMJ 311 , 171–174 (19 95). \n3. P. M. Brakefield  et al., What are the e ffects  of maternal and p re-adult envi r onments on \nageing in humans, and are there less ons from animal models? Mech. Ageing Dev.  126 , \n431–438 (200 5).  \n4. P. D. Gluckman, M. A. Hanson, C. Coo per, K. L. Thornburg, Effect of In u ter o and early-\nlife c onditi ons on adul t health  and dis ease. N. Engl. J. Med.  359 , 61–73 (20 08). \n5. Y. Ben-Shlomo , R. Cooper, D. Kuh, Th e last two decad es of life course epid emiology, and \nits rel evance f or res earch on ageing. Int. J. Epidemiol.  45, 973–988 (2016).  \n6. B. L. De Stavola, R. M. Daniel, Comme ntary: Incorpora ting concept s and methods from \ncausal infe rence in to l ife c ourse ep id emiology. Int. J. Epidemiol.  45, 1006– 1010 (2016).  \n7. A. Vaiserman, Early-life origin o f adult  disease: Evidence from natural expe r iments. Exp. \nGerontol.  46, 189–192 (2011).  \n8. K. Grey  et al., Severe malnutri tion o r famine exposure in ch ildhood and car diometabolic \nnon-com municable disease later in li f e: A systematic revi ew. BMJ Glob. Health  6 , \ne003161 (202 1).  \n9. C. Li, L. H. Lu mey, Exposure to the Chinese famine of 1959-61 in early lif e a nd long-term \nhealth cond itions: A sys tematic rev ie w and meta-analysis. Int. J. Epidemiol.  46, 115 7–\n1170 (2017).  \n10.  C. Li, L. H. Lu mey, Early-life exposure to the Chinese famine of 1959-1961 a nd type 2 \ndiabetes  in adu lthood:  A syst ematic r eview and meta-analysis.  Nutrients  14 , 2855 (2022).  \n11.  L. H. Lumey, A. D. Stein, E. Susser, Pre natal famine and adult h ealth. Annu. Rev. Public \nHealth 32, 237–262 (20 11).  \n12.  M. Cheng, N. Som met, M. Kerac, D. S.  Jopp, D. Spini, Exposure to the 1959– 1961 \nChinese famine and risk of non-communicable diseases in later  lif e: A life course \nperspecti ve. PLOS Glob. Public Health  3 , e00021 61 (2023).  \n13.  P. Eka mper, F. van Poppel, A. D. Stein, L. H. Lu mey, Independent and additi ve \nassociation of pre natal famine expos ure and inte rmediary li fe cond itions with adult \nmortality be tween age 18–63 years. Soc. Sci. Med.  119 , 23 2–239 (20 14).  \n14.  P. D. Gluckman, M. A. Hanson, Living with the past: Evolu tion, deve lopment, and \npatterns o f disease. Science  305 , 173 3–1736 (2004).  \n15.  R. C. Painter  et al., Early onset of co ro nary artery  disease a fter p renatal exposure to the \nDutch famine. Am. J. Clin. Nutr.  84 , 322-32 7 (2006).  \n16.\n L. Lumey  et al., Cohort profil e: The Dutch Hunger Winter Families Study. Int. J. Epidemiol.  \n36, 119 6–1204 (20 07).  \n17.  G. H.-J. Graf  et al., Social mobility and  biological aging among older adults i n the United \nStates. PNAS Nexus  1 , p gac029 (20 22) . \n18.  C. McCrory  et al., Early li fe advers ity and age acceleration  at mid-life and o lder ages \nindexed using the next-generati on Gr imA ge and Pace of Aging epigenetic clocks. \nPsychoneuroendocrinology  137 , 1056 43 (2022).  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated b iological aging six decad es after p renatal famine exposure  \n19\n \n19.  L. L. Schmitz, V. Duque, In utero exposure to th e Great D epress ion is r efle c ted in la te-life \nepigenetic aging signatures. Proc. Natl. Acad. Sci. U.S.A.  119 , e220 85301 19 (2022).  \n20.  E. Bernabeu  et al., Ref ining epigeneti c predic tion o f chrono logical and bio l ogical age. \nGenome Med.  15, 12 (20 23).  \n21.  R. F. Hillary  et al., Epigenetic measure s of ageing predict the preva lence an d incidence  of \nleading causes of dea th and diseas e b urden. Clin. Epigenetics  12, 115 (2020 ). \n22.  D. W. Belsky  et al., DunedinPACE, a D NA methylation biomarker o f the pac e of aging. \nElife 11, e73420 (20 22).  \n23.  D. W. Belsky  et al., Quanti ficat ion of biological aging in young adults. Proc. Natl. Acad. \nSci. U.S.A.  112 , E 4104–41 10 (2015).  \n24.  A. T. Lu  et al., DNA methylation Gr imAge strongly predicts lifespan  and he althspan. \nAging  11, 303–327 (201 9).  \n25.  P. Eka mper, F. van Poppel, A. D. Stein, G. E. Bijwaard, L. H . Lumey, Prenatal famine \nexposure and adult morta lity  from ca ncer, cardiovascu lar dis ease, and oth er causes \nthrough age 63 years. Am. J. Epidemiol.  181 , 271–27 9 (2015).  \n26.  E. W. Tobi  et al., Early gestation as th e crit ical t ime-window for changes in the prenata l \nenvironment to a ffec t the adu lt human blood methylome. Int. J. Epidemiol.  44, 121 1–\n1223 (2015).  \n27.  E. W. Tobi  et al., DNA methylation signatures lin k prena tal famine exposur e to growth \nand metabolism. Nat. Commun.  5 , 55 92 (2014).  \n28.  J. C. K. Wells, Natural sele ction  and se x differ ences in morbid ity and mortal i ty in ear ly \nlife. J. Theor. Biol.  202 , 65–76 (200 0).  \n29.  L. Lumey, A. D. Stein, H. S. Kahn, J. Ro mijn, Lipid profile s in middle-aged me n and \nwomen after famine exposure dur ing gestation: The Dutch Hunger Winter Families \nStudy. Am. J. Clin. Nutr.  89, 1737–1 74 3 (2009).  \n30.  J. Wang  et al., Exposure to the Chines e famine in chil dhood inc reases t ype 2 diabetes \nrisk in adu lts. J. Nutr.  146 , 2289 -22 95 (2016).  \n31.  C. Yu et al., Victims of Chinese famine  in early life have inc reased ri sk of me tabolic \nsyndrome in adulthood. Nutrition  53, 20-25 (2 018).  \n32.  H. Chen, W. N. Ne mbhard, H. G. Stockwell, Sex-specific e ffec ts of fetal exp osure to the \n1959- 1961 Chinese famine on risk of adult hyper tension. Matern. Child Health J.  18, \n527-5 33 (2014).  \n33.\n S. Song , Does famine influence  sex ra tio at bi rth? Evidenc e from the 1959-1961 Great \nLeap Forward Famine in China. Proc. Biol. Sci. 279 , 2883–2890 (2 012).  \n34.  A. D. Stein, P. A. Zybert, L. H. Lumey, Acute undernutr itio n is no t associa te d with excess \nof females at bi rth in  humans: The Dutch Hunger Winter. Proc. Biol. Sci.  271 , S13 8–141 \n(2004).  \n35.  J. S. Cramer, L. H. Lu mey, Maternal pr econcept ion die t and the sex ratio. Hum. Biol.  82, \n103–107 (201 0).  \n36.  V. Iannuzzi, M. G. Bacalini, C. Frances chi, C. Giuliani, The role o f genetics a nd epigenetics \nin sex dif ferenc es in human survival. Genus  79, 1 (2023).  \n37.  L. Ferrucci  et al. , Measuring biological  aging in humans: A quest. Aging Cell  19, e1308 0 \n(2020).  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated b iological aging six decad es after p renatal famine exposure  \n20\n \n38.  Z. Stein, M. Susser, G. Saenger, F. Ma rolla, Famine and Human Development: The Dutch \nHunger Winter of 1944-1945  (Oxford University  Press, 1975).  \n39.  I. de Zwarte, The Hunger Winter: Fighting Famine in the Occupied Netherlands, 1944–\n1945 (Ca mbridge University Press, 2020). \n40.  A. D. Stein  et al., Anthropometric me asures in middle age afte r exposure t o famine \nduring gestation: Evidence from the Dutch famine. Am. J. Clin. Nutr.  85, 869–876 (2007).  \n41.  T. B. L. Kirkwood, Understanding the odd science of aging. Cell 120 , 437–4 47 (2005).  \n42.  C. López-Otín, M. A. Blasco, L. Partridge, M. Serrano, G. Kroemer, The hallmarks of aging. \nCell 153 , 1194– 1217 (20 13).  \n43.  C. López-Otín, M. A. Blasco, L. Partridge, M. Serrano, G. Kroemer, Hallmark s of aging: An \nexpanding universe. Cell 186 , 243–27 8 (2023).  \n44.  J. Campisi  et al., From discoveries in a geing research to the rapeuti cs for  he althy ageing. \nNature  571 , 183–192 (2 019).  \n45.  J. Rutledge, H. Oh, T. Wyss-Coray, Me asuring biological age using omics dat a. Nat. Rev. \nGenet. 23, 715–72 7 (2022).  \n46.  S. Horvath, K. Raj, DN A methylation-based biomarkers and th e epigeneti c c lock th eory \nof ageing. Nat. Rev. Genet.  19, 371–3 84 (2018).  \n47.  A. T. Hig gins-Chen  et al., A computati onal solut ion fo r bols tering rel iabil ity of epigeneti c \nclocks: Implicati ons fo r clin ical trials a nd longitudinal track ing. Nat. Aging  2 , 644–661 \n(2022).  \n48.  G. Hannum  et al., Genome -wide met hylation p rofi les rev eal quant itati ve v iews of \nhuman agin g rates. Mol. Cell  49, 359–367 (2013).  \n49.  S. Horvath, DNA methylation age of h uman tissues and cell types. Genome Biol.  14, R115 \n(2013).  \n50.  M. E. Levine  et al., An epigenetic b io marker of aging for li fespan and heal t hspan. Aging  \n10, 573–5 91 (2018).  \n51.  Y. Zhang  et al., DNA methylation sign atures in pe riphera l blood s trongly pr edict al l-cause \nmortality. Nat. Commun.  8 , 14617 (2017).  \n52.  D. Pelegí-Sisó, P. de Prado, J. Ronkainen, M. Bustamante, J. R. González, M ethylcl ock: A \nbioconduct or package to estimate D NA methylation age. Bioinformatics  37 , 1759–1 760 \n(2020).  \n53.  J. A. Hanley, A. Ne gassa, M. D. d. Edw ardes, J. E. Forrester, Statis tical anal y sis of \ncorrela ted data using generalized est i mating equations: An orien tation.  Am. J. Epidemiol.  \n157 , 36 4–375 (200 3).  \n54.\n A. H. Petersen, T. Lange , What Is the causal inter pretat ion of sibling comparison designs ?  \nEpidemiology  31, 75–81 (2020).  \n55.  E. A. Houseman  et al., DNA methylati on arrays as sur rogate measures of c ell mixture \ndistribu tion. BMC Bioinformatics  13, 86 (2012).  \n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated b iological aging six decad es after p renatal famine exposure  \n21\n \nTable 1. Characteristics of the Dutch Hunger Winter Families Study DNA methylation sample.  \nThe table shows charact erist ics o f the  analysis sample overa ll (l eft column) and the famine-\nexposed and contr ol groups (middle a nd right columns).  \n \n \n \nD H W FS Fa m i n e - e xp o s e d\nTim e  co n t r o l s S ib lin g  c o n tr o ls\n( N = 9 51 ) ( N= 48 7) ( N = 1 59 )  ( N =305 )\nM e an / % ( SD ) M e an / % (SD ) M e an / % (S D ) M e a n / % (S D )\nA g e  ( y e a rs ) 58 ( 4) 59 ( 1 ) 5 9 ( 2 ) 57 ( 6 )\nMe n ( % ) 45 % 47 % 4 5% 42 %\nDu r a t i on of  e x pos ur e  ( w e e ks ) 1 7 ( 7 )\nD u n ed i n P A C E 0 .97 ( 0.11) 0 . 9 7 ( 0 .11) 0.95 ( 0. 11 ) 0. 9 7 ( 0.1 1)\nPC  G r i m A g e 6 9.92 ( 5.03) 7 0.42 ( 4 .20) 70 . 1 8 ( 4. 60 ) 68 . 98 ( 6.2 0)\nPC  P h en oA g e 5 0.18 ( 5.87) 5 0.61 ( 5 .18) 50 . 6 1 ( 5. 10 ) 49 . 27 ( 7.0 7)\nCon t ro l s\nP a ne l  I : D H W F S  s a m pl e  ( N = 95 1 )\n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated b iological aging six decad es after p renatal famine exposure  \n22\n \nFig. 1. Differences in biological aging between survivors of in-utero famine exposure and \nunexposed control participants in the Dutch Hunger Winter Families Study. The figure shows  \neffec t-sizes of in-utero famine exposu re associat ions with three DNA meth ylation (D NAm) \nmeasures of biologica l aging, DunedinPAC E, PC Grim Age , and PC PhenoA g e (N=951). Panel A \nshows effec t-sizes es timated in the  fu ll cohor t. Panel B shows ef fect-si zes e stimated for women \nand men separately. Effec t-sizes wer e estimated fr om generalized es timating equation \nregressions and are d enominated in s tandard-deviation un its o f the aging measures, \ninterpre table as Cohen`s d values. Err or bars show 95% confidenc e inte rval s.  \n \n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated b iological aging six decad es after p renatal famine exposure  \n23\n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated b iological aging six decad es after p renatal famine exposure  \n24\n \nFig. 2. Differences in biological aging between survivors of in-utero famine exposure and \nunexposed control participants in the Dutch Hunger Winter Families Study by gestational \ntiming of famine exposure. The figure shows effec t-sizes es timated fo r fa mine exposure during \nsix gestational time windows. Famine -exposed participan ts were exposed during up to two \nperiods. The devel opmental period s are ordered in the x-axis in chronol ogical orde r rela tive to \nthe famine. The left-most tic k shows effec t-sizes fo r late-gestati onal expos ure (def ined as \nexposure fo r the f inal 10 weeks o f gestation; N=139 exposed). The sec ond tick to the left  shows \neffec t-sizes fo r exposure  during the p enultimate 10 weeks of gesta tion (N = 146 exposed). The \nthird t ick shows e ffec t-sizes f or expos ure during the se cond 10 weeks of ge station (N =125 \nexposed). The fourth tic k shows ef fec t-sizes for  exposure du ring the f irst 1 0 weeks of gestation  \n(N=74 exposed). The fif th ti ck shows effec t-sizes fo r earl y gestational  expo sure with durat ion \n<10 weeks (N=94 exposed). The right-most tick shows e ffec t-sizes f or prec o nceptual exposu re, \ni.e., for exposure du ring the per iod pr eceding concept ion (N=52 exposed ). Numbers exposed do \nnot add up to the to tal exposed sample because many parti cipants wer e ex posed in two \nadjacent period s (N=143). Effect-si zes  are repor ted fo r DunedinPACE, PC Gr imA ge, and P C \nPhenoAge. Effect-sizes were estimate d from a multivariate regression in which indica tor \nvariables f or each expo sure window were included as p redic tor var iables along with covariat es \nfor sex, age, and age-squared. Effect- sizes are d enominated in s tandard-deviation un its of  the \naging measures, interpretab le as Coh en`s d values. Full resul ts are report e d in SI Appendix , \nTable S6 . Effect-sizes ar e plot ted sep arately f or women (circ les) and men ( triangles). The figure \nshows a consisten t sex-specif ic patt er n in DunedinPACE and P C GrimA ge effect-sizes. Women \nwho survived in-uter o famine exposu re, whether at ear ly or later gesta tion , tended to have \nfaster pace  of bi ological aging, as me asured by DunedinPACE, and older biological age, as \nmeasured by PC GrimA ge DNA m clock. In contrast, men who survived  later -gestational famine \nexposure tended  to have faster  pace of biologica l aging and older biol ogical age; whereas men \nwho survived ear ly-gestational famin e exposure te nded to hav e slower pa ce of bio logical aging \nand younger biological age, as measured by DunedinPACE and P C GrimA ge  DNAm clock, \nrespecti vely. There was no c onsiste nt  pattern in  PC PhenoA ge effect-sizes.  \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated b iological aging six decad es after p renatal famine exposure  \n25\n \n \n \n \n \n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated biological aging six decades after prenatal famine exposure \n26\n \nFig. 3. Flow diagram of the Dutch Hunger Winter Families Study. The figure shows how the \nanalysis sample size was arrived at in each step for survivors of in-utero famine exposure, time \ncontrols, and same-sex sibling controls. N=1,031 participants completed telephone interviews. \nOf this group, 971 participated in the clinic exam. DNA extracted from blood samples was \nanalyzed to determine DNA methylation and data passed quality controls for N=951 individuals.\nThe figure illustrates the number of individuals in each exposure and control group included in \nthe telephone interview, clinic examination, and analysis sample.  \n \n \n26\n \n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint \n\n \nAccelerated b iological aging six decad es after p renatal famine exposure  \n27\n \nFig. 4. Gestational timing of exposure to famine in the Dutch Hunger Winter Families Study. \nThe figure shows individua l gestation s of N=547 famine-exposed partic ipa nts (colo red l ines) \nand N=176 time controls (gray lines). Each gestation is plo tted as a s ingle h orizon tal lin e. The \nstart of  the l ine is the date of th e mother’s last menstrual pe riod (LMP). Th e end of th e line  is \nthe parti cipant’s date of bi rth. Individ ual gestations are  plot ted fr om the to p of the graph to the \nbottom, ordered by LMP date. For th e famine-exposed participan ts, the se gment of each line \nshowing the firs t 10 weeks of gestat i on is colo red gold. The segment showing the second 10 \nweeks is col ored o range. The segmen t showing the thi rd 10 weeks is co lor ed red. The segment \nshowing the last 10 weeks is color ed purple. For the time contr ols, 10-week gestational p eriods \nare color ed in gray, with lighte r shad es for th e earl ier gestat ional per iods. The x-axis shows the \ndate. The vertica l dashed l ines show t he start and end  of th e famine expos ure period \n(November 26, 1944 - May 12, 1 945).   \n \n  \n \n \n . CC-BY 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted November 4, 2023. ; https://doi.org/10.1101/2023.11.03.23298046doi: medRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}