1. Funded by: W.K. Kellogg Foundation NIH Grant UL1TR0000062
Immune Mediators and Vitamin D Status in the Development of Comorbidities of Pregnancy Rebecca Moore, MD
Jennifer Mulligan, PhD: Dept of Otolaryngology
Howell Harmon: Dept of Otolaryngology
Judith Shary: Dept of Pediatrics
Myla Ebeling: Dept of Pediatrics
Carol Wagner, MD: Dept of Pediatrics, Division of Neonatology
Funded by:
W.K. Kellogg Foundation
NIH Grant UL1TR

2. Disclosure Statement Rebecca Moore, MD
Has no financial relationships to disclose or conflicts of interest to resolve.

3. Pregnancy and Maternal Immunity
During pregnancy, the maternal immune system undergoes remarkable changes:
Transitions from pro-inflammatory to anti-inflammatory pathways1
Continues to protect the fetus from immune destruction
Guards the mother and child from infectious pathogens2
1Chaouat G, et al. Am J Reprod Immunol Somerset DA, et al. Immunology

4. Importance of Maternal Immunity
Disruption of this delicate balance:
Places mother and child at greater risk for infection, including chorioamnionitis
Increases likelihood of pregnancy complications, such as pregnancy loss, preeclampsia, gestational diabetes and preterm labor & birth
Somerset DA, et al. Immunology

5. 2016 Premature Birth Report Card

6. Role of Vitamin D in Immune Function
Vitamin D is a known immune modulator of both the innate and adaptive immune systems:
Induces antibacterial responses via toll-like receptors 1
Expression of the vitamin D receptor is associated with activated human T and B lymphocytes 2
1Liu PT, et al. Science Chun RF, et al. Expert Rev Clin Pharmacol

7. Role of Vitamin D in Immune Function
Vitamin D has the ability to control T-cell phenotypes:
Regulatory T cells 1
T-helper 1 vs T-helper 2 cells 2
Vitamin D actions
1Chambers ES, et al. Curr Allergy Asthma Rep Cantorna MT, et al. Mol Aspects Med

8. Vitamin D in Pregnancy
Increased risk of vitamin D insufficiency (serum 25(OH)D levels <32ng/mL) at delivery:1
83% African American
47% Caucasian
Total circulating vitamin D level (25(OH)D) definitions:2
Deficiency: <50 nmol/L (20 ng/mL)
Insufficiency: 50 nmol/L to 79 nmol/L (≥ 20 to <32 ng/mL)
Sufficiency: 80 nmol/L or greater (≥32 ng/mL)
Optimized sufficiency: 100 nmol/L or greater (40 ng/mL)
1Roberts JM, et al. J Nutrition Wagner C, et al. Am J Obstet Gynecol

9. Risk of Insufficiency
Associations between low maternal 25(OH)D levels and pregnancy complications have been established.1
Our previous NICHD trial yielded evidence to support a dose-dependent trend toward reduced premature labor & birth, as well as infection.2,3
Interim analysis showed an inverse association of serum circulating 25(OH)D and comorbidities of pregnancy.
1Robinson, et al. Am J Perinatol Hollis BW, Wagner CL. Calcified tissue international Wagner CL, et al. Am J Obstet Gynecol

10. Specific Aim
To further investigate maternal vitamin D levels in relation to immune mediator changes during pregnancy and the development of comorbidities of pregnancy (COP):
Preterm labor & birth
Preeclampsia
Chorioamnionitis

11. Hypothesis
Higher levels of maternal vitamin D during pregnancy are associated with decreased COP
Increased TGF-β and IL-10
Decreased L-2, IL-6 and TNF-α

12. Study Design Using the existing cohort of now 270 mothers:
Case-control study
Hematologic samples from current RCT
25(OH)D & immune mediator concentrations
Comorbidities of Pregnancy
Preterm labor & birth, Preeclampsia, Chorioamnionitis
Kellogg Foundation Vitamin D in Pregnancy Project RCT enrolled subjects:
18-45 years of age
8-14 weeks gestation
Singleton pregnancy
Subjects were randomized into one of two treatment regimens:
Control group: 400 IU/day vitamin D3
Study group: 4,400 IU/day vitamin D3

13. Study Design
Hematologic samples from pregnant women enrolled in an existing RCT were analyzed from each trimester, correlating with study visit 1, visit 4 and visit 7.
1st study visit
(10-14 weeks gestation)
4th study visit
(24 weeks gestation)
7th study visit
(36 weeks gestation)
1st Trimester
2nd Trimester
3rd Trimester

14. Methods Maternal 25(OH)D concentrations assayed using RIA.
TGF-β, TNF-α, IL-2, IL-6 and IL-10 concentrations measured by ELISA.
Comorbidities of Pregnancy (COP):
Preterm labor & birth <37 weeks gestation
Preeclampsia
Chorioamnionitis
Associations between maternal 25(OH)D and COP, and between immune mediator concentrations and COP, were analyzed using Wilcoxon rank-sum tests.
Associations between maternal immune mediator concentrations and 25(OH)D levels were examined using a Spearman correlation analysis.

15. Results: Maternal Demographics
COP = Comorbidities of pregnancy
Preterm labor& birth (n=21)
Preeclampsia (n=10)
Chorioamnionitis (n=3)
*6 subjects with preeclampsia & preterm labor & birth
No statistically significant differences between groups.

16. Demographics and 25(OH)D levels
COP = Comorbidities of pregnancy
Preterm labor& birth (n=21)
Preeclampsia (n=10)
Chorioamnionitis (n=3)
*6 subjects with preeclampsia & preterm labor & birth
No statistically significant differences between groups.
1st Trimester:
3rd Trimester:

17. 25(OH)D levels and Chorioamnionitis
No statistically significant differences between groups.

18. 25(OH)D levels and Preeclampsia
No statistically significant differences between groups.

19. 25(OH)D levels and Preterm Labor & Birth
No statistically significant differences between groups.

20. 25(OH)D levels and all COP
COP = Comorbidities of pregnancy
Preterm labor& birth (n=21)
Preeclampsia (n=10)
Chorioamnionitis (n=3)
*6 subjects with preeclampsia & preterm labor & birth
No statistically significant differences between groups.
(n=28)
(n=242)

21. Results
No significant correlations between maternal immune mediator concentrations and 25(OH)D levels were found using a Spearman correlation analysis.
No differences when comparing groups of 25(OH)D sufficient (≥32 ng/mL) vs insufficient and the development of preterm labor & birth, preeclampsia or chorioamnionitis.

22. Results Immune Mediators and Comorbidities Of Pregnancy:
No significant relationships were found in the preeclampsia (n=10) or chorioamnionitis (n=3) groups when compared to controls
Significant alterations were found in preterm labor & birth group (n=21), compared to controls

23. Decreased TGF-β in Preterm Labor & Birth
1st Trimester
2nd Trimester
p=0.017
p<0.0001 *

24. Increased TNF-α in Preterm Labor & Birth
2nd Trimester
3rd Trimester *

25. Decreased IL-6 in Preterm Labor & Birth
2nd Trimester
3rd Trimester *

26. Summary
Mothers in this study who developed preterm labor & birth were found to have decreased TGF-β and IL-6, in addition to increased TNF-α.
No significant correlations between maternal immune mediator concentrations and 25(OH)D levels were found in this cohort.

27. Discussion
Lower than expected prevalence of chorioamnionitis, preeclampsia and preterm labor & birth lead to inadequate power to detect a relationship between 25(OH)D levels and the selected COP.
Further analyses are underway to more accurately delineate the relationship between maternal immune mediator concentrations and preterm labor & birth.

28. Questions?

30. Study Design
Hematologic samples from pregnant women enrolled in an existing RCT were analyzed from each trimester, correlating with visit 1, visit 4 and visit 7.
6th visit
1st visit
(10-14 weeks gestation)
4th visit
(24 weeks gestation)
7th visit
(36 weeks gestation)
1st Trimester
2nd Trimester
3rd Trimester

31. Future Directions
Maternal hematologic samples collected nearer preterm birth/delivery will be analyzed and compared to age and race matched controls of the same gestation.
This data will hopefully incite further investigation into maternal immune mediator concentration changes as a possible predictor of preterm labor/birth.
6th visit
1st visit
(10-14 weeks gestation)
4th visit
(24 weeks gestation)
7th visit
(36 weeks gestation)
1st Trimester
2nd Trimester
3rd Trimester

32. Future Directions
Further analysis of maternal serum obtained at visit 6, which was just prior delivery for most women who developed COP, is underway.
This forthcoming data will allow us to more accurately determine the immune mediator concentrations and 25(OH)D levels of women who delivered prior to visit 7.
Placental samples from each group will be analyzed to determine the immunological landscape of women with COP vs those without, in relation to maternal 25(OH)D levels.

33. Maternal Serum 25(OH)D and COP
No statistically significant differences between groups.

34. CD4+ T helper cell subsets and functions
CD4+ T helper cell subsets and functions. The black arrows (→) indicate cytokines that induce differentiation and the black lines ( ) indicate cytokines that inhibit Th cell development. The grey arrows and lines indicate induction ( ) or suppression ( ) by 1,25(OH)2D3 treatment. Th1 immune responses are inhibited by Th2 and T reg cells and are important for protection from intracellular infections, cancer immunity and participate in autoimmune disease. Th2 cell immune responses are also important for fighting infections, can be suppressed by T reg cells and are pathogenic in asthma and allergies. Regulatory NKT cells can be either pathogenic or protective for autoimmune responses and are required for experimental asthma disease induction. All T cells that have been tested express the VDR. 1,25(OH)2D3 suppresses Th1 driven cytokine responses, induces T reg cells, induces IL-4 production and enhances NKT cell function.
1Chambers ES, Hawrylowicz CM. Curr Allergy Asthma Rep Cantorna MT, Yu S, Bruce D. Mol Aspects Med

35. Model of the effects of vitamin D status and 1,25(OH)2D3 treatment on Chron’s disease. When vitamin D is low NKT cells and T reg cells fail to develop, Th1 cells are overactive and the incidence of Th1 mediated diseases increase. Vitamin D regulates the APC such that Th1 cell responses are inhibited, T reg cell function is induced and NKT cells develop. The net result is decreased Chron’s disease and normalization of the Th cell response.