Vitamin D – a novel role in pregnancy

Vitamin D regulates placental development and function. It is a potent regulator of the immune systemstimulating antimicrobial responses while suppressing inflammation. Its deficiency has been linked to increased risk of serious chronic and inflammatory diseases. Vitamin D deficiency during pregnancy increases susceptibility to infection and inflammation, leading, in turn, to outcome like preterm birth or preeclampsia. Pregnant women with darker skin pigmentation are more likely to be vitamin D deficient, particularly when living in regions with low exposure to sunlight. It is possible that during pregnancy, a primary non-infectious inflammatory process is activated by vitamin D deficiency. Combined assessment of vitamin D deficiency and inflammatory markers in early pregnancy or during different stages of pregnancy may facilitate the recognition of the risk of complications.

In tro duc tion.Be yond its clas si cal ef fects on bone and cal cium ho meo sta sis caus ing rick ets and os teo po ro sis, vi ta min D has be come in creas ingly rec og nized as a potent reg u la tor of mul ti ple phys i o log i cal func tions above.Vi ta min D can be ob tained from lim ited food sources, such as fatty fish and fish oil, and, to day, from dietary sup ple ments or we can also make it our selves, through a chem i cal re ac tion that hap pens in the skin when it is ex posed to ul tra vi o let B (UVB) light es pecially to meet the increased demands of pregnancy.
The vi ta min D de fi ciency ep i demic dur ing pregnancy is caused by a lack of ad e quate sun light ex po sure needed to syn the size vi ta min D 3 (cholecalciferol) in the skin, cou pled with oral in takes that are too low even with reg u lar use of pre na tal vi ta mins con tain ing 400 IU vi ta min D 3 [1] Vi ta min D de fi ciency dur ing preg nancy has been linked with a num ber of se ri ous short-and long-term health prob lems in off spring, in clud ing im -paired growth, skel e tal prob lems, type 1 di a be tes, asthma, and schizo phre nia [2].
Func tional as pects of vi ta min D. Vi ta min D in hu mans, re fers to Vi ta min D 3 , which is also known as cholecalciferol.In re sponse to UVB light, it is cre ated by skin cells called keratinocytes uti liz ing 7-dehydrocho les terol, a prod uct of cho les terol.This ver sion has no bi o log i cal ac tiv ity in the body till this mol e cule is mod i fied pri mar ily in the liver, by a se ries of re lated enzymes for hydroxylation, to gen er ate 25-hydroxyvitamin D3 (25(OH)D 3 ).The 25(OH)D 3 made by the liver is none the less the ma jor cir cu lat ing form of vi ta min D. When it is needed in the body, a fi nal con ver sion to the bi o log i cally ac tive form by the en zyme 1a-hydroxylase (CYP27b1), is re quired to be come 1,25-dihydroxyvi ta min D (1,25(OH) 2 D 3 ).This was first dis cov ered in the kid ney, and re nal pro cess ing is re spon si ble for gener at ing much of the body's cir cu lat ing 1,25(OH) 2 D 3 sup ply [3] (Fig. 1).
The ac tive form of vi ta min D, the 1,25(OH) 2 D 3 molecule vir tu ally func tions as an «on/off» switch for genes of ev ery tis sue in the hu man body.This form of vita min D acts by at tach ing to the vi ta min D re cep tor (VDR), which serves as a tran scrip tion fac tor in side a cell's nu cleus.Once bound to 1,25(OH) 2 D 3 , the VDR pro tein makes a com plex with retinoid-x re cep tor (RXR).This com plex now binds to a spe cific re gion of the cell's DNA ad ja cent to a tar get gene.Their at tachment to the DNA in duces cel lu lar ma chin ery to be gin tran scrib ing the neigh bor ing gene to a spe cific pro tein.
Re cently, many other tis sues, in clud ing cells of the im mune sys tem and the skin, have been iden ti fied to per form the con ver sion of 25(OH)D 3 .Skin is there fore unique among or gans in that it is ca pa ble of manufacturing bi o log i cally ac tive 1,25(OH) 2 D 3 in the pres ence of UVB light from start to fin ish, al though lo cal pro duction of 1,25(OH) 2 D 3 from cir cu lat ing in other tis sues is a sub stan tial source of vi ta min D's bi o log i cal ac tiv ity.
By in duc ing a cell to make a par tic u lar pro tein, 1,25(OH) 2 D 3 af fects a range of cel lu lar func tion, and this abil ity to trig ger gene ac tiv ity in dif fer ent tis sues is the ba sis of vi ta min D's enor mous phys i o log i cal ef -fects.Pre cisely, vi ta min D is man u fac tured in one tissue and cir cu lates through the body in flu enc ing many other tis sues, and VDR is a nu clear re cep tor that responds to pow er ful steroidal hor mones such as es trogen and tes tos ter one [4].
Al though at least 1,000 dif fer ent genes are be lieved to be reg u lated by 1,25(OH) 2 D 3 , in clud ing sev eral involved in the body's cal cium pro cess ing that ac count for D's well-known role in bone for ma tion as well as genes with crit i cal roles in a va ri ety of cel lu lar defenses.
Cir cu lat ing lev els of 25(OH)D 3 -the biomarker of vi ta min D sta tus.Cir cu lat ing lev els of 25(OH)D 3 are a di rect re flec tion of vi ta min D sta tus, which depends on ac cess to vi ta min D ei ther through ex po sure to sun light or through di etary in take whereas se rum con cen tra tions of 1,25(OH) 2 D 3 , are pri mar ily de fined by the en do crine reg u la tors of re nal CYP27b1 ac tiv ity.The net ef fect of this is that vi ta min D sta tus can vary sig nif i cantly in pop u la tions as a con se quence of geograph ical, so cial or eco nomic fac tors.Un til re cently, rick ets was con sid ered to be the only sig nif i cant clin i -2 CHANDER P. ARORA, HOBEL C. J. How ever, an en tirely new per spec tive on vi ta min D de fi ciency has arisen from the ob ser va tion that se rum 25(OH)D 3 levels cor re late in versely with se rum parathyroid hormone be low a thresh old of 80 nmol/l 25(OH)D 3 [5].This has led to a com plete re-eval u a tion of the op ti mal se rum level of 25(OH)D 3 so that se rum con cen tra tions of 25(OH)D 3 up to 75 nmol/l are now con sid ered to be in ad e quate and are more com monly re ferred to as vi tamin D «in suf fi ciency» as op posed to «de fi ciency» [6].A 25(OH)D 3 level of 30 ng per mil li li ter (75 nmol/l) or higher pro vides ad e quate sub strate for 1-OHase to convert 25(OH)D 3 to its ac tive form, 1,25(OH) 2 D 3 [6].We cat e go rized the se rum lev els of 25(OH)D 3 as: less than 50 nmol/l (< 20 ng per mil li li ter) as de fi cient, 50-75 nmol/l (< 20-30 ng per mil li li ter) as in suf fi cient, and 75-150 nmol/l (< 30-60 ng per mil li li ter) as suf ficient (Fig. 2).Once 1,25(OH) 2 D 3 com pletes the task of main taining nor mal cel lu lar pro lif er a tion and dif fer en ti a tion, it in duces ex pres sion of the en zyme 25-hydroxyvitamin D-24-hy drox y lase (24-OHase), which en hances the catab o lism of 1,25(OH) 2 D 3 to bi o log i cally in ert 1,24,25trihydroxyvitamin D.

Skin (Keratinocytes)
Lo cally pro duced 1,25(OH) 2 D 3 does not en ter the cir cu la tion and has no in flu ence on cal cium me tab olism.There fore cir cu lat ing lev els of 25(OH)D 3 are a direct re flec tion of vi ta min D sta tus, which de pends on ac cess to vi ta min D ei ther through ex po sure to sun light or through di etary in take.
Vi ta min D re quire ments dur ing preg nancy.In ad di tion to caus ing poor global min er al iza tion of the skel e ton, vi ta min D de fi ciency has im pli ca tions for numer ous other nonskeletal health out comes.In utero or early life vi ta min D de fi ciency has been linked to an increased risk of type 1 di a be tes [7], asthma [8], and schizo phre nia [9,10].
Fas ci nat ing new data also show that vi ta min D regulates pla cen tal de vel op ment and func tion [11], which sug gests that ma ter nal vi ta min D sta tus may be as so ci ated with ad verse out comes of preg nancy, such as mis car riage, preeclampsia, and preterm birth.
Preterm de liv ery in the en vi ron ment of in fec tion is be lieved to re sult from the ac tions of pro-in flam ma tory cytokines se creted as part of the ma ter nal and/or fe tal host re sponse to mi cro bial in va sion [12,13].En hanced ex pres sion of the pro-in flam ma tory cytokines IL-1, IL-6 and TNFa are as so ci ated with preterm de liv ery [14].Such cytokines have been de tected in el e vated con cen tra tions in the amniotic fluid and plasma of women with preterm la bor and hu man ges ta tional tis sues are po ten tially rich sources of in flam ma tory cytokines [15].These cytokines can be in duced by a num ber of stim uli, in clud ing bac te rial endo tox ins, and they have been shown to pro mote spon ta ne ous la bor and rup ture of mem branes via their ac tions on the ges ta tional tissues [16][17][18].
Sev eral stud ies have re ported al tered lev els of CYP27b1 in pla cen tas from preeclampsia preg nan cies [19,20].Fur ther more, in a re cent nested case-con trol study of preg nant women Bodnar and col leagues showed that vi ta min D de fi ciency sig nif i cantly increases the risk of preeclampsia [1].This cou pled with ev i dence of the prev a lence of vi ta min D in suf fi ciency in preg nant moth ers -par tic u larly Af ri can-Amer i can moth ers [1] -has sup ported a role for vi ta min D sufficiency in pro tect ing against this prev a lent com pli ca tion of preg nancy [21].There was no sig nif i cantly al tered ex pres sion of vi ta min D re cep tor (VDR) or CYP27b1 in preg nan cies with intrauterine growth re stric tion (IUGR) [22].More re cent stud ies of vi ta min D in suf ficiency dur ing preg nancy and lac ta tion have served to underline the mag ni tude of the prob lem [1,3,4,23,24].
Our re search group re cently re ported vi ta min de ficiency in co hort study of spon ta ne ous preterm pregnan cies (N = 27) com pared to matched case con trols (N = 32) in an NIH Be hav ior In Preg nancy Study (BIPS).In the study, se rum sam ples were col lected in 528 eth ni cally di verse women at 18-20 weeks (T1), 28-30 weeks (T2) and 34-36 weeks (T3).Cir cu lat ing lev els of vi ta min D (25(OH)D 3 ) were sig nif i cantly lower at each visit in the cases who sub se quently delivered preterm.The lev els in di cated de fi cient in controls.Sub se quent vis its also showed lower lev els in the PTB cases com pared to the con trol group.We as sessed the in volve ment of vi ta min D in the oc cur rence of IL-6 lev els in women with spon ta ne ous preterm birth.At all three time in ter vals, sig nif i cantly higher lev els of IL-6 were as so ci ated with the preterm birth cases as compared to the con trols [25].Sig nif i cantly higher lev els of this pro-in flam ma tory cytokine, IL-6 were found in sub jects with IUGR as com pared with con trols.This also cor re lated with lower se rum vi ta min D lev els in this co hort.It is pos si ble that a non-in fec tious in flamma tory re sponse is ac ti vated by vi ta min D de fi ciency that is a marker of risk for or caus ative in preterm birth or de vel op ment of IUGR [26].
Ra cial dis par ity in vi ta min D syn the sis.Since the most im por tant source of vi ta min D is the skin's synthe sis of the vi ta min from UVB so lar ra di a tion [27].Any pro cess that re duces UVB pho tons from en ter ing the epi der mis will di min ish cholecalciferol (vi ta min D 3 ) pro duc tion.The skin pig ment mel a nin ab sorbs UVB pho tons and can re duce vi ta min D 3 syn the sis by .90% [28].Con se quently, Af ri can Amer i cans are at high risk of vi ta min D de fi ciency.
Ac cord ing to CDC, 42 % of Af ri can-Amer i can women in US be tween the ages of 15-49 years are de ficient in vi ta min D (less than 20 ng per mil li li ter of serum lev els) [29].Nev er the less, it is also clear that some groups are more at risk of vi ta min D in suf fi ciency than oth ers.The 1988-1994 Na tional Health and Nu tri tion Ex am i na tion Sur vey re vealed that 42 % of Af ri can-Amer i can women of child-bear ing age had 25(OH)D 3 lev els that were lower than 50 nmol/l, half the cur rent op ti mal tar get level.This com pares with only 4 % of white women [23].
We re cently re ported vi ta min de fi ciency in co hort study of Af ri can-Amer i can preg nan cies com pared to Cau ca sians.Al though the co hort used had women who de liv ered at term, the lev els of 25(OH)D 3 in Cau ca sians were sig nif i cantly lower in the sub jects with in fec tion than the ones with out (p < .001).Women with vi ta min D in suf fi ciency in the sec ond tri mes ter were more likely to de velop in fec tion dur ing preg nancy but not sub jects with suf fi cient vi ta min D at T1.The pro portion of in fec tion per cent in the Cau ca sian and Af ri can-Amer i can groups was sig nif i cantly dif fer ent at all the time pe ri ods as well as their lev els of 25(OH)D 3 .Af rican Amer i cans show a trend to have higher pro por tion of in fec tions than the Cau ca sian group at all three vis its and had sig nif i cantly lower 25(OH)D 3 (p < .001) at these vis its [30].
Our re sults re veal a pos i tive as so ci a tion be tween 25(OH)D 3 con cen tra tions and el e vated risk of in fec tion [31].Vi ta min D in suf fi ciency may, there fore be involved in the pathogenesis of ma ter nal in fec tion dur ing preg nancy.Vi ta min D lev els could mod u late this in fection sus cep ti bil ity dur ing preg nancy.
In an other study, ap prox i mately 29 % of Black pregnant women and 5 % of white preg nant women re siding in the north east ern United States had vi ta min D deficiency, i. e. se rum 25(OH)D 3 of less than 50 nmol/l, whereas 54 % of Black women and 47 % of white women had se rum 25(OH)D 3 lev els in dic a tive of vi ta min D in suf fi ciency, i. e. 25(OH)D 3 of 50-75 nmol/l [1].
Immunomodulation by vi ta min D. Re cently vitamin D de fi ciency and even in suf fi ciency has be come a world wide is sue af fect ing the pop u la tions across the globe.The broader im pli ca tions of vi ta min D re strictions es pe cially with the grow ing trend of sun-blocks, have be come more ev i dent.Vi ta min D sta tus has been found to be a ma jor con trib ut ing fac tor to im mune response [32].Pre cisely, the ac tive form of vi ta min D, 1,25(OH) 2 D 3 has been shown to be the key mod u la tor of im mune re sponses [33,34].The de gree to which inflam ma tory path ways play a role in la bors that are not as so ci ated with clin i cal signs of in fec tion is un known, how ever even nor mal la bor shares cer tain char ac ter istics with in flam ma tory pro cesses.
Liu [35] sub stan tially ad vanced this line of in ves tiga tion by show ing that hu man im mune cells re spond to in fec tion by man u fac tur ing both vi ta min D re cep tor proteins and the en zyme that con verts cir cu lat ing 25(OH)D 3 into the bi o log i cally ac tive 1,25(OH) 2 D 3 and to in duce the im mune cells to start pro duc ing cathelicidin.Cathelicidin, the nat u rally pro duced antimicrobial agent that acts against a va ri ety of bac te ria, ex plaining the tu ber cu lo sis sun shine cure: the sun-soaked skin could boost their im mune cells to gen er ate the nat u ral antibiotic that fought of the tu ber cu lo sis bacteria bac teria (TB) (Fig. 3).
In par tic u lar, the ac tive form of vi ta min D, 1,25(OH) 2 D 3 has been shown to be a key mod u la tor of im mune re sponses [36,37].Both the re cep tor for 1,25(OH) 2 D 3 , VDR [36,38] and the en zyme that cat alyzes the syn the sis of 1,25(OH) 2 D 3 from precursor 25(OH)D, CYP27b1 [39,40] are abun dantly ex pressed by cells from the im mune sys tem.The pres ence of CYP27b1 in macrophages [41] and den dritic cells [42] in di cates that lo cal (autocrine 25(OH)D 3 or paracrine) syn the sis of 1,25(OH) 2 D 3 is a piv otal fea ture of vi ta min D ac tion within the im mune sys tem.
Pla cen tal pro duc tion of vi ta min D. The pla centa was one of the first ex tra-re nal sites shown to syn thesize 1,25(OH) 2 D 3 from 25(OH)D 3 [43,44].Or ga ni za -tion of 1a-hy drox y lase, the en zyme needed to con vert 25(OH)D 3 to the ac tive form is lo cal ized both in ma ternal decidua and fe tal tropho blasts and is more abun dant in first and sec ond tri mes ter.We de tected high est lev els of 25(OH)D 3 at 18-20 weeks of preg nancy in the study as well as con trol group.This could also be due to the pres ence of VDR in the pla centa as has been sug gested by [45].
This means that vi ta min D func tions in an autocrine fash ion at the fe tal-ma ter nal in ter face [45].One pos sible ex pla na tion is that 1,25(OH) 2 D 3 func tions as a reg ula tor of pla cen tal cal cium trans port in ad di tion to an immunomodulatory func tion [46].
From the stud ies high light ing im mu no log i cal function of the het er o ge neous cells that make up the placenta, ma ter nal and fe tal cells are able to me di ate in nate [47,48] and adap tive [49,50] im mune re sponses.Further more, a se ries of stud ies us ing hu man pla cen tas [11,22] have dem on strated the ex pres sion of CYP27b1 (gene for pro duc ing the en zyme 1a-hy drox y lase) in decidua (ma ter nal tis sue) and trophoblast (fe tal tis sue) [11,22].Within de cid uas, CYP27b1 is ex pressed by both stromal cells and macrophages, while in trophoblasts CYP27b1 is lo cal ized pre dom i nantly in the syncytiotrophoblast (the cells that form the bar rier between ma ter nal and fe tal blood) [22].In pri mary cultures of hu man decidual cells, both 1,25(OH) 2 D 3 and 25(OH)D 3 (the lat ter me tab o lized en dog e nously to 1,25(OH) 2 D 3 ) in duced ex pres sion of antimicrobial cathelcidin, but sup pressed the in flam ma tory cytokine production [32].
Within the decidua, the gene ex pres sion for this enzyme (CYP27b1) was iden ti fied in decidual stromal cells as well as the decidual macrophages, sug gest ing a pos si ble immunomodulatory func tion for lo cal ized syn the sis of 1,25(OH) 2 D 3 [11,22].Vi ta min D -the trig ger for an ti bac te rial/anti-in flam ma tory re sponses in decidual and trophoblastic cells.
Anal y sis of decidual cells in vi tro has en dorsed a pos si ble role for 25(OH)D 3 and 1,25(OH) 2 D 3 as mod ula tors of im mune re sponses in ma ter nal tis sue [32].Human decidual cells from 1 st tri mes ter pla cen tas incubated for 24 hrs with 25(OH)D 3 or 1,25(OH) 2 D 3 showed in duc tion of cathelicidin and sup pres sion of cytokines such as IL-1, TNFa and IL-4 [32].The fact that a physiological dose of 25(OH)D 3 (100 nmol/l) was as ef-Fig.3. Macrophage cells respond to bacterial cell walls by manufacturing both VDR proteins and the enzyme (1-OHase) that converts circulating 25(OH)D 3 into the biologically active 1,25(OH) 2 D 3 .These events induced the immune cells to start producing cathelicidin that demonstrates antimicrobial activity against a variety of bacteria fective as a phar ma co log i cal dose of 1,25(OH) 2 D 3 (100 nmol/l), un der lines the im por tance of lo cal ized (placental) ver sus en do crine (sys temic) syn the sis of 1,25(OH) 2 D 3 .
Us ing pri mary hu man pla cen tal tis sue and trophoblastic cells it has been shown that the fe tal side of the pla centa also in duces cathelicidin-me di ated an ti bac terial re sponse to treat ment with vi ta min D me tab o lites.Col lec tively these data in di cate that ex og e nous 1,25(OH) 2 D 3 or lo cally me tab o lized 25(OH)D 3 are capa ble of in duc ing anti-bac te rial and anti-in flam ma tory re sponses in both the ma ter nal and fe tal com po nents of the pla centa [22].
In ad di tion to stromal and trophoblastic cells, the pla centa is made up of an ar ray of leu ko cytes that are fun da men tal to the im mu nol ogy of ges ta tion.We have shown that the most abun dant of the decidual im mune cells, uNK, do not ex press CYP27b1 [22] but are poten tial tar gets for paracrine re sponses to 1,25(OH) 2 D 3 syn the sized by stromal cells or macrophages.By contrast, decidual macrophages ex press abun dant lev els of CYP27b1 and may there fore sup port the same autocrine vi ta min D-in duced in nate im mu nity demonstrated in pe riph eral blood-de rived macrophages.In the case of the lat ter we have shown that spe cific an ti bac terial re sponses such as toll-like re cep tor (TLR)-me diated in duc tion of cathelicidin are highly de pend ent on vi ta min D sta tus [51].
When a macrophage or monocyte is stim u lated through its toll-like re cep tor 2/1 (TLR2/1) by an in fectious agent such as My co bac te rium tu ber cu lo sis or patho gen-as so ci ated mo lec u lar pat terns such as lipopolysaccharide, the sig nal up-reg u lates ex pres sion of the VDR and 25-hydroxyvitamin, the en zyme D-1ahy drox y lase (1-OHase).1,25 (OH) 2 D 3 trav els to the nucleus, where it in creases the ex pres sion of cathelicidin, a pep tide ca pa ble of pro mot ing in nate im mu nity and induc ing the de struc tion of infectious agents such as M. tu ber cu lo sis.
Hu man cathelicidin antimicrobial pep tide (CAMP) gene is a di rect tar get of the VDR and is strongly upreg u lated in myeloid cells by 1,25-dihydroxyvitamin D 3 [51].It is also likely that the 1,25(OH) 2 D 3 pro duced in monocytes or macrophages is re leased to act lo cally on ac ti vated T lym pho cytes, which reg u late cytokine syn the sis, and ac ti vated B lym pho cytes, which reg u late im mu no glob u lin syn the sis.The autocrine syn the sis of 1,25(OH) 2 D 3 that drives in nate im mune re sponses is de pend ent on the avail abil ity of sub strate 25(OH)D 3 (Fig. 3).It has been shown that Cau ca sian lev els of cathelicidin pro duc tion were 3-times higher than those in Af ri can Amer i cans [51].In vi tro supplementation of 25(OH)D 3 to the Af ri can-Amer i can se rum was able to re store cathelicidin ex pres sion to lev els sim i lar to the ones for Cau ca sians es tab lish ing that vi ta min D de ficiency com pro mises in nate im mune re sponses to in fection [52].
The pla centa is one of the first tis sues shown to be ca pa ble of syn the siz ing 1,25 (OH) 2 D 3 with 1a-hy droxylase re cep tor ac tiv ity de tected both in ma ter nal decidua and fe tal trophoblast [19].
The net ef fect is that vi ta min D sta tus can vary signif i cantly in pop u la tions as a con se quence of stress from so cial, be hav ioral of eco nomic fac tors.Sen si tivity of parathyroid hor mone to the thresh old of 80 nmol/l of 25(OH)D 3 has led to re-eval u a tion of the op ti mal level up to 75 nmol/l are now con sid ered to be in ad e quate and are more com monly re ferred to as vi tamin D in suf fi ciency as op posed to de fi ciency [53].
A new born's vi ta min D stores are com pletely re liant on vi ta min D from the mother [13].Not sur prisingly, poor ma ter nal vi ta min D sta tus dur ing preg nancy is a ma jor risk fac tor for in fant rick ets [54].This could be in dic a tive of the fact that fe tal pro gram ming is as soci ated with the in creased risk of com mon dis eases in adult life.

Fig. 1 .
Fig. 1.Skin keratinocytes convert 7-dehydrocholesterol to vitamin D in response to sunlight exposure.This is converted to the circulating form 25(OH)D 3 in liver and to 1,25(OH) 2 D 3 in the target tissues