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Home , Calcitriol, Steroid hormone, Vitamin D

European Review for Medical and Pharmacological Sciences 2018; 22: 2502-2512 D: a with -like activity

G. MONASTRA1, S. DE GRAZIA2, L. DE LUCA3, S. VITTORIO3, V. UNFER4

1Department of Experimental Medicine, Sapienza University of Rome, Rome, Italy 2Department of Research and Development, Lo.Li. Pharma, Rome, Italy 3Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, Polo Universitario SS. Annunziata, University of Messina, Messina, Italy 4Faculty of Medicine and Psychology, Department of Developmental and Social Psychology, Sapienza University of Rome, Rome, Italy

Abstract. – OBJECTIVE: This review is aimed Abbreviations at demonstrating the progesterone-like activity 1,25(OH)2D = 1α,25-dihydroxyvitamin D; 1α-OHase: exerted by the active form of , or cal- 1α-hydroxylase; CL = ; CRF = corticotro- citriol (1,25(OH)2D). To achieve this outcome, we pin-releasing factor; CSF = colony-stimulating factor; compared the effects in vivo and in vitro exert- CYP24A1 = family 24 subfamily A ed by progesterone and vitamin D, with a spe- member 1; CYP27A1 = Cytochrome P450 Family 27 cial focus on the female reproductive system Subfamily A Member 1; CYP27B1 = Cytochrome P450 and . Family 27 Subfamily B Member 1; DBP = vitamin D MATERIALS AND METHODS: This is a litera- Binding ; FGF = Fibroblast ; IL = ture review of the most important articles pub- interleukin; TNF-α = tumour necrosis factor alpha; Treg lished in English on vitamin D as a hormone, cells = T regulatory cells; VDR = vitamin D ; mainly found by MEDLINE. Furthermore, a sec- VEGF = Vascular endothelial growth factor. tion of our review contains some unpublished data, concerning the analysis in silico of the sim- ilarities between the steric structure of proges- terone and , based on the availability of the experimental structures of progesterone and Introduction vitamin D3 receptors in complex with their phys- iological ligands in the RCSB Protein Data Bank. A Story Coming from the Thirties RESULTS: Vitamin D was shown to exert ma- Between the end of sixties and the beginning ny physiological activities during the very ear- ly stages of gestation in perfect synchrony with of seventies some advanced researches and stu- progesterone. Both the molecules mutually help dies brought out to understand much better the and reinforce the activity exerted by each one. A activity of calcitriol or 1,25(OH)2D, two names little bit later than progesterone is released, vita- referred to the active form of vitamin D (in our min D secretion rises, but only if pregnancy oc- paper “vitamin D” will be used with the same curs. Calcitriol contributes to prepare the endo- meaning of them). They offered a wider picture metrium to be receptive. Moreover, it supports of this very intriguing molecule, until then consi- the implantation process and the course of preg- nancy through different but similar pathways to dered only an essential vitamin for growth those used by progesterone, giving rise to a sig- and for fighting . In that period vitamin D nificant synergy of action. It is increasingly ev- was defined, for the first time, as a new steroid ident that vitamin D gives an essential support hormone and its chemical structure was characte- from the luteal phase onwards. rized1,2. In 1971 Norman et al3 published a semi- CONCLUSIONS: Based on the evidence dis- nal paper where they stated that “It is possible that played in this review we may define appropriate- ly vitamin D as a with proges- 1,25- dihydroxycholecalciferol should be reclassi- terone-like activity. fied as a steroid hormone. Certainly, its secretion by the followed by its selective accumula- Key Words: tion in the target intestinal mucosa where it exerts Calcitriol, CYP24A1, CYP27A1, CYP27B1, Embryo its characteristic physiological effect on implantation, , , Pregnancy, satisfies the minimal definitions of Progesterone, Progesterone receptor, Vitamin D, Vita- min D receptor. a hormone”. Furthermore, in the same years, its was identified4,5. Vitamin D

2502 Corresponding Author: Vittorio Unfer, MD; e-mail: [email protected] Vitamin D: a steroid hormone with progesterone-like activity started to be considered a hormone, even thou- photoconversion of 7-dehydrocholesterol and gh many years had to pass before this concept subsequent thermosensitive but noncatalytic iso- became established in the scientific community, merization to . Then, cholecalci- as stated in 1997 by the Nutritional Committee ferol is hydroxylated by mitochondrial and Report for both North America and Europe6. De- microsomal 25-OHase CYP27A1. The resultant spite this and other authoritative opinions and de- 25-hydroxycholecalciferol (calcidiol) is 1α-hyd- clarations7, some scholars still do not accept this roxylated in the kidney by CYP27B1 (a mito- classification8, but they are a minority. It is worth chondrial 1α-OHase). This reaction yields the of note that a couple of decades before the six- secosteroid 1α,25(OH)2D (calcitriol). Therefore, ties, some studies (1934) demonstrated the hor- CYP27A1 and CYP27B1 are key invol- monal properties exerted by a group of molecules ved in the synthesis of vitamin D, whereas the having the cyclopentanoperhydrophenanthrene inactivating CYP24A1 determines the ring, with seventeen-carbon tetracyclic hydro- rate-limiting step for calcitriol catabolism12. In carbon. Ovariectomized rats (Wistar strain) were physiologic conditions at least the 80% of this injected with 100 mg of each compound dissolved vitamin is produced by the , whereas the re- in sesame oil and the treatment was able to induce maining 20% or less is taken up from dietary estrus in these animals, showing that such effect sources. In areas rich in , this endoge- is due to the presence of the classic steroid skele- nous production provides enough quantity of ton9. A marked activity was shown by vitamin D, at least in healthy young subjects. and its irradiation product calciferol (crystalline However, among people with darker skin, a se- vitamin D), and still greater activity by neo-ergo- vere and widespread deficiency may be found sterol9. Later, Feyel-Cabanes (1949) and Chambon as seen in the US especially among blacks and (1951) highlighted that ergosterol and 7-dehydro- Hispanics13. Moreover, subjects who are obese, , administered at non-estrogenic nor have , low high-density lipoprotein progestogenic doses, act synergically with proge- cholesterol level, do not drink daily, are all sterone in sustaining the pregnancy10 or obtaining significantly, independently associated with vi- implantation of ovariectomized rabbits11. These tamin D deficiency13. interesting results were then forgotten. The times Besides the chemical structure, there are other were not yet ripe to go ahead in that direction of very convincing connections between vitamin D research. and the class of (steroid) . These last ones are bound to transport in plasma, howe- Vitamin D and Steroid Hormones ver only the free molecule can spread into its target In fairly recent years, the studies on calci- cells. These hormones are chemical messengers in triol had a revival, even under the impulse of a wide number of species and tissues to transmit the discovery of areas, in developed countries, signals. Obviously, the ability of steroid hormones with deficiency and widespread rickets. Another to give rise to biological message depends on the cause for this renewed interest in the field came presence of their receptors on their specific targets. from the finding that many vitamin D receptors Unlike the growth factors and hormones (VDR) in tissues and cells are not connected which bind only to receptors on cell membrane, with its effects on bone, being linked to other, the -soluble steroid hormones can pass through before unknown, benefits for health. Nowa- the bilayer surrounding the cell and interact days, vitamin D and its metabolites, from the also with their related receptors on the , structural point of view, are called secosteroids, inducing a genomic response. The receptor-hor- i.e., compounds whose structure shows the abo- mone complex binds to DNA and triggers or re- ve-cited steroid skeleton with seventeen-carbon presses the transcription of one or more genes14. tetracyclic hydrocarbon, that is not joined at the Furthermore, steroid hormones bind to a variety of level of the bond located in position 9-10 of the receptor types located near or associated with the ring B. Therefore, chemically they are like the plasma membrane15-17. In this way, they activate sy- , , testoste- stems of second messengers, with rapid cellular re- rone, progesterone, , chorionic gona- sponses that may range from seconds (e.g., opening dotropin. By now, both calcidiol and calcitriol of ion channels) to 10-60 min (e.g., activation of are considered respectively pre-hormone and phosphatidylinositol-3’-kinase or endothelial nitric hormone. Indeed, they are synthetized endoge- oxide synthase). Instead, the genomic responses nously in the skin by means of UVB stimulated commonly need from some hours to days before

2503 G. Monastra, S. De Grazia, L. De Luca, S. Vittorio, V. Unfer becoming entirely manifest; they may be stopped evolutionary process. Progesterone and vitamin D by transcription or translation inhibitors16. There- receptors belong to two different classes of the abo- fore, many steroid hormone receptors are in the ve-mentioned superfamily23. This relationship has cytoplasm and/or nucleus. to be keep in mind, being in perfect agreement with The parallelism between the above picture and the core of our review. In this context, we aimed the behaviour of calcitriol, its receptor and the re- at determining which are the similarities between sulting downstream activity, is striking. In fact, the steric structure of progesterone and vitamin D, as for steroid hormones, vitamin D and its meta- in consideration of their common phylogenesis, by bolites circulate in plasma linked to a transport using in silico methods. protein produced by the liver: vitamin D Binding The crystal structures of progesterone in complex Protein (DBP). Additionally, as for the classic ste- with the -binding domain (LBD) of the roid hormones, VDR is a lo- progesterone receptor (PDB code 1A28)24 and of vi- cated both in the nucleus of cells and on the cell tamin D in complex with the LBD of the vitamin membrane. The complex between vitamin D and D nuclear receptor (VDR) (PDB code 1DB1)25 have its nuclear receptor, once activated, binds to DNA been reported in the RCSB Protein Data Bank. By and, in this way, modifies the expression of nu- structure-based pharmacophore model tool of Li- merous (genomic response). On the other gandScout software26, we highlighted the binding side, the interaction with the VDR located on the requirements for these two ligands that are schema- cell membrane induces the formation of second tically represented in Figure 1. cellular messengers (such as cAMP, diacylglyce- Progesterone showed the ability to bind its re- rol, triphosphate, arachidonic acid) or the ceptor establishing i) hydrogen bond interaction phosphorylation of some cellular proteins. The- with Gln725 and ii) hydrophobic contacts through refore, calcitriol, as progesterone, estrogens, etc., the methyl groups with Met909, Met759, Met756 exerts genomic and nongenomic actions. On the and Trp755 (Figure 1A). whole, this picture further confirms the hormonal On the other hand, vitamin D interacts with nature of vitamin D16. its receptor establishing three different hydrogen causes /oste- bonds interactions: i) with Ser237 and Arg274 oporosis, disorders, metabolic through the hydroxyl group in position 1, ii) with syndrome ( and ), , kidney Tyr143 and Ser278 through the hydroxyl group in disease, neurological and cardiovascular diseases, position 3, and iii) with His305 e His397 through pregnancy complications, and its administration the hydroxyl group in position 25. Furthermore, proved to be useful in these pathologies. As well there are some hydrophobic contacts with the dif- known, only cells having the specific receptor for ferent hydrophobic residues present in the binding a given hormone, respond to its stimuli; therefo- site (Figure 1B). re, it is important to highlight that the distribution To obtain more insight on the structural simila- of VDR is almost ubiquitous, involving various rity between progesterone and vitamin D, the two organs and tissues. VDR was found in hema- ligands have been aligned in their binding confor- topoietic tissues, immune system, / mation creating a shared feature pharmacophore , lymphocytes, skin, muscle, smo- model by LigandScout software (Figure 2). oth muscle cells, myoblasts, cardiac mu- The alignment shows an interesting overlap- scle cells and atrial myocytes, β cells, ping of vitamin D (stick grey) and progesterone , medullary cells, (stick green) highlighting three convergent points , hippocampus/selected neurons, that have been translated in three pharmacophore cartilage chondrocytes, liver parenchymal cells, features describing chemical functionalities: lung, Sertoli/seminiferus tubule, pituitary gland, – one hydrophobic sphere (yellow), mapped by , parathyroid, ovarian, myometrial and en- the methylene group in position 19 of vitamin dometrial cells, placenta18-22. D and the methyl group in position 19 of proge- sterone; Progesterone and Vitamin D – another hydrophobic sphere overlapped by the Structural Similarities methyl group in position 18 of vitamin D and It was discovered that all the nuclear receptors the methyl group in position 18 of progesterone of steroid hormones show common structure/fun- – one hydrogen bond acceptor (red sphere) occu- ction domains and are named as “nuclear receptors pied by hydroxyl group in position 3 of vitamin superfamily” including four classes with a shared D and the carbonyl group of progesterone.

2504 Vitamin D: a steroid hormone with progesterone-like activity

Figure 1. Interaction patterns of progesterone-LBD of progesterone receptor (A) and vitamin D-LBD of VDR (B). The labels refer to the residues involved in the interaction with the receptors: green and red arrows represent H-bond inte- ractions, yellow areas highlight the groups involved in the hydrophobic contacts.

These information reveal that vitamin D con- start their development to prepare ovulation. This tains some chemical functionalities useful for the process occurs from the 1st to the 14th day of the binding to progesterone receptor. menstrual cycle. The next luteal phase begins after In Figure 3 is represented a possible interaction of ovulation and lasts normally from the 15th to the 28th vitamin D with progesterone receptor obtained by day. It needs, on one hand, satisfactory secretions of superimposition of the two proteins 1A28 and 1DB1. progesterone by the corpus luteum (for this reason The results (Figure 3) showed that vitamin D it is also termed secretory phase), and, on the other could hypothetically interact with the proges- hand, a prompt endometrial response to its stimu- terone receptor by establishing a H bond with lus. In the luteal phase, the corpus luteum (CL), a Gln725 and hydrophobic interactions with the transient endocrine gland which develops in this same hydrophobic amino acid residues involved specific period, undergoes a prompt neovasculariza- in the interaction with progesterone. Clearly, this tion. Such process is principally under the control is only a hypothesis that has to be carefully tested of Vascular endothelial growth factor (VEGF) and in depth. However, this interaction, even if it is Fibroblast growth factor (FGF), both upregulated real, does not necessarily imply the induction of a in the luteinized granulosa cells and then absolute- physiological effect.

Progesterone Release in the Luteal Phase for Preparing the Scenario The female reproductive system plays very com- plex roles, with the participation of a huge number of actors whose activity is finely regulated. In this pro- cess, progesterone and calcitriol play pivotal func- tions, and without their appropriate levels, reached in well-determined periods, gestation cannot occur. The menstrual cycle determines an essential tim- ing for and it is divided into two main phases, the follicular and luteal phases, that must Figure 2. Shared-feature pharmacophore model. Progeste- work in series, in an integrated manner, to allow rone and vitamin D are represented in green and grey stick pregnancy. During the follicular phase, also called respectively. Yellow spheres represent hydrophobic contacts proliferative phase, the follicles inside the while red sphere represents hydrogen bond acceptor.

2505 G. Monastra, S. De Grazia, L. De Luca, S. Vittorio, V. Unfer

els of vitamin D were detected through human pregnancy, independently of calcium homeosta- sis34-36. In addition, the above-cited presence of VDR on ovaries, uterus, endometrium and pla- centa should be emphasized. It is noteworthy that the number of VDR increases continuously during the first and second trimester of pregnancy, mean- ing that this vitamin plays a key physiological role in pregnancy. Especially in the first months of gestation adequate levels of vitamin D are even more essential, affecting not only the well-being of the mother but also that one of the fetus. VDR was detected in the endometrium throughout all Figure 3. Superimposition of the crystal structures 1A28 the phases of the menstrual cycle and also later, and 1DB1. Amino acid residues present in the progestero- ne receptor, involved in the interaction with the ligand, are namely in proliferative phase endometrial stromal represented by green stick. Progesterone is represented by cells, in secretory phase endometrial stromal cells green stick while vitamin D is represented by grey stick. and in early pregnant decidual cells37. The levels of 1α-OHase mRNA were comparable in endo- metrial stromal cells without any influence due to ly necessary for the embryo implantation27,28. CL the phase of the cycle. Instead, such concentra- produces several different hormones; among them, tions were found significantly higher in decidual progesterone is the most important since its activity cells37. As a side note, we observe that vitamin D, is necessary and sufficient to hinder the proliferation detected in the seminal fluid38, could also func- of endometrium and promote its receptive state for tion as a trigger molecule to provide additional blastocyst implantation to maintain early pregnancy. levels to further support certain processes under- Progesterone is produced by the luteal cells in a pul- lying a successful onset of pregnancy. It was ob- satile way with levels that may vary up to sevenfold served that, in response to IL-1β secreted by the in a range of a few hours. Its synthesis depends on blastocyst, the dendritic cells of the endometrium the availability of appropriate levels of circulating and the macrophages produce 1α-OHase (the en- cholesterol as substrate and on low stimulation by zyme responsible for the last step of the synthesis (LH), produced by gonado- process) and, consequently, calcitriol37. On the tropic cells of the gland. Therefore, other hand, the stimulation of the endometrial progesterone prepares the environment conducive to 1α-OHase expression due to TNF is not as potent pregnancy success and it is categorically necessary, as that one achieved using very low concentra- during the secretory phase, to induce the endometri- tions of IL-1 β37. This evidence means that be- um receptivity to the embryo implantation, even if tween the two main pro-inflammatory , moderate concentrations are sufficient29. In this sce- at least in this case, the central role is played by nario, vitamin D plays the role of second main actor IL-1 β, whereas TNF exerts a secondary function, and its function comes to light from several exam- despite its strong effects in many contexts. Thus, ples that we present below. Overall, it modulates the there is not only an increased calcitriol production innate and , controls cell in the first stages of pregnancy, but also a wide proliferation and functions as a regulator of various array of new synthetized receptors in the endome- and important metabolic processes, with effects on trium, ready for binding. the implant, the production of cytokines and the im- Using blood cells of women with a history of mune response to infections30-33. recurrent pregnancy losses (RPL) Vitamin D was reported to hinder in vitro the proliferation of T Activities in Common Between Vitamin helper 1 (Th1) cells and to decrease the produc- D and Progesterone in the inception of tion of their cytokines, such as IFN-γ, IL-2 and Pregnancy TNF-α. On the other hand, vitamin D stimu- lated the secretion of T helper 2 (Th2) cytokines; Vitamin D among them, IL-10 is the most important anti-in- The metabolism of 1,25(OH)2D in pregnancy flammatory released under the effect of differs drastically from what normally occurs in calcitriol39. Furthermore, in vitro vitamin D sig- the non-pregnant state. Increased circulating lev- nificantly decreased in a dose-dependent manner

2506 Vitamin D: a steroid hormone with progesterone-like activity the cytotoxicity of natural killer (NK) cells39 and stimulates the production of progesterone in cul- it was able to suppress in the same cells the secre- tures of human ovarian cells50. It also provokes the tion of proinflammatory cytokines, such as IFN-γ synthesis or blocks the release of key molecules and TNF-α, and to increase IL-10 release39, as that have, respectively, beneficial (such as activin well as CSF2, IL-1, IL-6 too, thus modulating the A, CRF, VEGF)51-53 or harmful effects (such as inflammation that normally develops in the initial estrogens, besides the already cited TNFα and phase of pregnancy40. other inflammatory cytokines)53-56 in the period Accordingly to that, vitamin D significantly between the luteal phase and the 12th week of lowers the frequency of T helper 17 (Th17) cells pregnancy (decidualization and so on). in addition to reducing the Th17/Treg ratio in Vitamin D production and VDR expression pro- peripheral blood of patients with recurrent mis- mote an increase in the level of two target genes carriage compared with the control group41. The in both cycling and early pregnant endometrium, same modulatory effect of inflammation was CYP24 and the already cited osteopontin. CYP24, seen in the induction and upregulation of the sup- the rate-limiting step for calcitriol catabolism, is pressive activity of T regulatory (Treg) cells in one of the most potent 1,25(OH)2D-responding skin-draining lymph nodes42. genes and its protein product is responsible for Vitamin D by binding to VDR on the endo- the reaction that deactivates the vi- metrium, upregulates the target genes, such as tamin D, by a mechanism57,58. and osteopontin, also called secreted phosphoprotein one (SPP1)37. Development of re- Progesterone ceptivity requires changes in endometrial Progesterone receptor, as VDR, is expressed expression; endometrial expression of calbindin in the glandular epithelium and stroma of the hu- mRNA was shown to be highly regulated during man endometrium during the menstrual cycle59. implantation. Indeed, calbindin participates to In experiments with human and mouse T cells the regulation of endometrial receptivity acting progesterone was seen to induce the expression as calcium binding protein: it is well known that of the VDR gene, which is one of the major genes the function of reproductive organs and its mus- responding to progesterone. It was able to elicit the culature is depending on the influx of extracellu- VDR mRNA expression in human Treg, Th1, and lar calcium or the release of intracellular stores43. Th17 cells with various cytokine conditions, and Osteopontin is an adhesion protein involved in in spleen CD4+ T cells and uterus from pregnant implantation and decidualization44. Its action has or progestin-injected mice60. The specificity of this a direct influence as at the effect is demonstrated by its suppression due to uterine-placental interface throughout pregnancy, the receptor antagonist of progesterone, RU48660. including the regulation of immune cells behavior Therefore, progesterone promotes VDR gene ex- and cytokine production. Both calbindin and oste- pression in T cells in heterogeneous conditions and opontin are essential for the embryo implantation different species. The expression of VDR due to and for the development of the . Their progesterone rises the sensitivity of T cells to cal- effects on the endometrium favor the success of citriol and facilitates a more efficient regulation of pregnancy37,45-47. Therefore, 1,25(OH)2D is reput- T cells by calcitriol with an increase of Tregs and ed to exert an autocrine/paracrine action aimed suppression of effector T cells which exert a poten- at regulating both acquired and innate immune tial pro-inflammatory activity60. Progesterone-in- responses at the fetal-maternal interface, as con- duced VDR in T cells allows to prevent adverse firmed by the presence of the VDR in the endo- immune responses, causing complications during metrium. This hypothesis, in general terms, was gestation60. Of note, if VDR gene is knocked down born about twenty years ago and is based on the with siRNA, the inhibition of differentiation widespread co-expression of VDR and 1α-OHase into effector T cells caused by progesterone and in many tissues where vitamin D plays various calcitriol significantly decreases60. physiological functions48. Furthermore, progesterone promoted Th2 cells Notably, vitamin D in human cumulus granu- formation and the release of their anti-inflamma- losa cells significantly increases the expression of tory cytokines61 and regulated the differentiation 3β-HSD, necessary for conversion from pregnen- of human naive cord blood fetal T cells into Treg olone to progesterone49 and in this way it signifi- cells62. It also suppressed Th1 and Th17-relat- cantly increases the production of such hormone ed genes and enhanced Th2-related genes in the by granulosa cells. Moreover, vitamin D directly lymphocytes obtained from pregnant cows63. It is

2507 G. Monastra, S. De Grazia, L. De Luca, S. Vittorio, V. Unfer remarkable that progesterone inhibited IFN-γ and progesterone induced a greater sensitivity in T cell IL-17 release in dose-dependent manner in both toward calcitriol to up-regulate the Treg-associated pregnant and non-pregnant cows63. In addition, molecules (FoxP3, CD8, LAP-TGF-β1). progesterone neutralized the cytotoxicity of NK The efficacy of Treg cells in immune suppres- cells by hindering their degranulation and perfo- sion is higher when they are produced under the rin release64. Moreover, in mononuclear cells iso- combined effect of calcitriol and progesterone lated from human placental blood progesterone than when there is only one of these effector mo- was able to antagonize the effects of lipopolysac- lecules60. All these data demonstrate that proge- charide (LPS) significantly reducing TNF-α, IL- sterone potentiates the anti-inflammatory effects 1β, IL-6, IL-8, and MIP-1α release. Instead, the of vitamin D, and vice versa. levels of IL-10 grew in a remarkable way when LPS was added in vitro with progesterone65. As Studies on Vitamin D in Pregnancy and calcitriol, progesterone stimulates the synthesis or Assisted reproductive technology (ART) suppresses the release of pivotal molecules exert- The relevance of vitamin D activity for pre- ing, respectively, helpful (such as activin A, CRF, gnancy, from the luteal phases onwards, is con- VEGF) or detrimental effects (such as estrogens firmed by the findings of several studies. Insuffi- or TNF)64,66-69 in the period between the luteal cient levels of vitamin D have been related to the phase and the 12th week of pregnancy. Finally, pathogenesis and the abnormal development of progesterone induced the release of osteopontin luteal phase. Vitamin D deficiency is prevalent in in human decidua70 and calbindin in the uterus of pregnant women with a major risk of miscarria- Egyptian buffalo46. These effects are briefly de- ge and recurrent pregnancy losses. Furthermore, picted in Figure 4. the treatment with vitamin D or its precursor was Vitamin D effect to promote Treg activity in found effective in women looking for gestation presence of progesterone appears strongly redu- and in expectant mothers71. Several investigations ced when VDR gene is knocked down60. Of note, confirm this picture. A study analyzed 1072 wo-

Figure 4. Progesterone and vitamin D share several biological activities, as an inducer of anti-inflammatory pathways (Th2 and Treg cells). In addition, they stimulate the release of corticotropin-releasing factor (CRF), activin A, vascular endothe- lial growth factor (VEGF), osteopontin, calbindin. Moreover, both progesterone and vitamin D inhibit the proinflammatory pathways (NK, Th1 and Th17 cells). All these effects contribute in supporting pregnancy since its inception. It is noteworthy that their action is synergic.

2508 Vitamin D: a steroid hormone with progesterone-like activity men attending an academic infertility centre: a gest that vitamin D, whose metabolism is stri- high rate of hypovitaminosis D among childbea- ctly regulated, contributes to prepare the endo- ring women was demonstrated to be related with metrium to be receptive. Moreover, it supports infertility72. In another work, the estimation of the implantation process and the course of pre- 25-hydroxy cholecalciferol (25-OHD) in patients gnancy through different, but similar pathways was made before the treatment protocol for ICSI. to those used by progesterone, giving rise to a The mean value of 25-OHD, number of oocytes, significant synergy of action79. A little bit later fertilized oocytes and endometrial thickness than progesterone is released, vitamin D enters were significantly higher in pregnant women. A the scene, and at endometrium level, its con- significant positive association of 25-OHD with centration strongly increases only if pregnancy clinical pregnancy and thickness of endometrium occurs (i.e., after fertilization). It is increasingly was observed. Deficiency of 25-OHD in females evident that calcitriol gives an essential support hinders the accomplishment of optimal endome- from the luteal phase onwards, as glimpsed for trial thickness required for implantation of the the first time by the pioneering studies carried embryo after ICSI. The improvement in vitamin out by Feyel-Cabanes10 and Chambon11 between D status positively affected the success results in the thirties and the fifties of the past century. In ART73. Also, Ozkan et al74 found that women with conclusion, based on the evidence displayed in higher vitamin D level in their serum are signifi- this review we may define appropriately vitamin cantly more likely to achieve clinical pregnancy D as a steroid hormone with progesterone-like following IVF-embryo transfer. The same group activity. highlighted that another favourable parameter for ART outcome is the higher content of vitamin D in follicular fluids. As stated by Hollis and Wa- Conflict of Interest gner75, there is growing evidence of the important Vittorio Unfer and Sara De Grazia are employees at Lo.Li. role exerted by vitamin D supplementation during Pharma, Rome, Italy. The other authors declare that they pregnancy to achieve satisfying circulating levels, have no conflict of interests. associated to a lower risk of comorbidities of pre- gnancy and better outcomes. Concerning the sa- fety in pregnant women, vitamin D supplementa- tion was demonstrated to be completely safe at the References dose of 4,000 IU/day for 24-28 weeks76,77. Finally, a recent systematic review78 showed clearly an 1) Haussler MR MJ, Norman AW. The association of a association between vitamin D levels in women metabolite of vitamin D3 with intestinal mucosa chro- undergoing ART and their reproductive outco- matin in vivo. J Biol Chem 1968; 243: 4055-4064. Myrtle JF HM, Norman AW mes. Vitamin D deficiency and insufficiency re- 2) . Evidence for the biolo- gically active form of cholecalciferol in the intesti- present a crucial key point to consider in women ne. J Biol Chem 1970; 245: 1190-1196. following ART procedures. 3) Norman AW MJ, Midgett RJ, Nowicki HG, Williams V, Popjak G. 1,25-dihydroxycholecalciferol: identi- fication of the proposed active form of vitamin D3 Conclusions in the intestine. Science 1971; 173: 51-54. 4) Haussler MR, NORMAN AW. Chromosomal recep- Vitamin D and progesterone modulate nume- tor for a vitamin D metabolite. Proc Natl Acad Sci rous components of the immune system in the USA 1969; 62: 155-162. whole organism and at fetoplacental level too. 5) Tsai HC, NORMAN AW. Studies on the mode of action of calciferol. VI. Effect of 1,25-dihydroxy-vi- Their action involves both the immune cells tamin D3 on RNA synthesis in the intestinal mu- (macrophagic, dendritic, uterine natural killer, cosa. Biochem Biophys Res Commun 1973; 54 and lymphocytic cells) and non-immune cel- 622-627. ls (trophoblast cells), shifting the equilibrium 6) Institute of Medicine (US) Standing Committee. Die- toward a Th2 profile. Vitamin D shows several tary reference intakes: calcium, phosphorus, ma- physiological activities during gestation in per- gnesium, Vitamin D, and fluoride. 1997, Washin- fect synchrony with progesterone or also acting, gton (DC): National Academy Press. in some cases, as a substitute. The molecules 7) Norman AW. From vitamin D to hormone D: fun- damentals of the vitamin D es- mutually help and reinforce the activity exerted sential for good health. Am J Clin Nutr 2008; 88: by each one. Overall, the data so far known sug- 491S-499S.

2509 G. Monastra, S. De Grazia, L. De Luca, S. Vittorio, V. Unfer

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