Int. J. Med. Sci. 2020, Vol.

17 3039

Ivyspring
International Publisher International Journal of Medical Sciences
2020; 17(18): 3039-3048. doi: 10.7150/ijms.47807
Research Paper

Vitamin D Status and Pregnancy Complications: Serum

1,25-di-hydroxyl-Vitamin D and its Ratio to
25-hydroxy-Vitamin D are Superior Biomarkers than
25-hydroxy-Vitamin D
Ibrahim A. Albahlol1,2, Abdulrahman H. Almaeen3, Abdulrahman A. Alduraywish4, Umar F. Dar5, Tarek
H. El-Metwally6,7
1. Department of Obstetrics and Gynecology, College of Medicine, Jouf University, Sakaka, Saudi Arabia. [email protected]
2. Department of Obstetrics and Gynecology, Mansoura University, Mansoura, Egypt.
3. Department of Pathology, College of Medicine, Jouf University, Sakaka, Saudi Arabia. [email protected]
4. Department of Internal Medicine, College of Medicine, Jouf University, Sakaka, Saudi Arabia. [email protected]
5. Department of Family and Community Medicine, College of Medicine, Jouf University, Sakaka, Saudi Arabia. [email protected]
6. Department of Pathology, Biochemistry Division, College of Medicine, Jouf University, Sakaka, Saudi Arabia. [email protected].
7. Department of Medical Biochemistry, Faculty of Medicine, Assiut University, Assiut, Egypt.

 Corresponding author: Prof. Dr. Tarek H. El-Metwally, PhD, Department of Pathology, Biochemistry Division, College of Medicine, Jouf University, 2004
Sakaka 42421, Saudi Arabia. 00966-541860565. [email protected]. http://orcid.org/0000-0001-9040-6642

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/).
See http://ivyspring.com/terms for full terms and conditions.

Received: 2020.05.05; Accepted: 2020.09.14; Published: 2020.10.18

Abstract
Vitamin D (VitD) deficiency during pregnancy has been associated with adverse neonatal outcomes and
increased risk of late pregnancy complications. We planned to correlate serum VitD biomarkers;
25-hydroxyvitamin D (25-OH-VitD) and 1,25-dihydroxyvitamin D (1,25-diOH-VitD) levels; and their
ratio with the frequency of feto-maternal pregnancy complications. A prospective cross-sectional
case-control study was conducted at Aljouf Maternity and Children Hospital, Sakaka, Saudi Arabia, during
the period of September 1, 2017 to September 30, 2019. 322 pregnant women were stratified into 2
groups: controls (110 cases) and complicated group (212 cases). The later comprised severe
preeclamptic toxemia associated with intrauterine growth restriction (58 cases), gestational diabetes
mellitus (GDM; 82 cases), abortion (26 cases), undisturbed ectopic pregnancy (16 cases), premature
rupture of membranes (PROM; 14 cases), and, inevitable preterm labour (16 cases). After clinical
assessment, peripheral blood samples were collected. Serum biomarkers were measured using specific
immunoassays. The direct 1,25-diOH-VitD/25-OH-VitD ratio was calculated. Serum 25-OH-VitD
indicated widely spreading VitD deficiency among participants with significantly higher levels in controls
vs. GDM subgroup only. 1,25-diOH-VitD levels and the ratio were markedly reduced in the six
complicated subgroups vs. controls, with non-significant differences amongst the complicated subgroups.
ROC analysis showed very high sensitivity and specificity, to differentiate patients from controls, only for
1,25-diOH-VitD (AUC = 0.965; 0.947 - 0.983, p <0.001) followed by the ratio but not 25-OH-VitD. In
conclusions, 25-OH-VitD did not show significant changes except for GDM. 1,25-diOH-VitD levels and
the ratio showed strong associations with pregnancy complications. Serum 1,25-di-OH-VitD and its ratio
to 25-OH-VitD are more reliable and physiologically relevant biomarkers for VitD status in pregnancy.
Key words: Serum vitamin D biomarkers, pregnancy outcomes, pregnancy complications, 25-hydroxyvitamin D,
1,25-dihydroxyvitamin D

Introduction
Vitamin D (VitD) deficiency became a global Nuclear receptor-mediated, the dihydoxy active form
epidemic, particularly for women of reproductive age. of VitD, 1,25-diOH-VitD, controls cellular

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Int. J. Med. Sci. 2020, Vol. 17 3040

proliferation, differentiation and apoptosis through period between September 1, 2017 and September 30,
targeting ≥1000 genes. Nevertheless, pathogenetic role 2019. It was approved by the local bioethical
of hypovitaminosis D in a number of comorbidities is committees of Jouf University and Ministry of Health
still hotly debated [1,2]. With increased requirements (#6-16-4/40). Written informed consents were
of the conditional VitD, pregnancy is a high-risk factor obtained. The exclusion criteria were uncertain
for VitD deficiency since the fetus gains its VitD diagnosis, obesity, anemia, endocrinal disorders
requirements from mother [3]. Despite abundant (hypo-/hyper-parathyroidism and thyroidism, and
sunlight, published reports confirmed a wide spread diabetes other than GDM), immobilization for any
of hypovitaminosis D among Saudi citizens [4]. More reason, on anti-convulsion drug, acute infections,
than half of UK population is VitD deficient, i.e., malabsorption syndromes, comorbid chronic medical
having serum 25-hydroxy-VitD (25-OH-VitD) <20 disorder (renal or liver impairment, inflammatory
ng/mL - as defined by the Endocrine Society [5]. To and immunological disorders), multiple pregnancy,
maintain healthy musculoskeletal system, the antepartum hemorrhage and superimposed
Scientific Advisory Committee on Nutrition preeclampsia.
Department recommended a serum 25-OH-VitD
cut-off value of 10 ng/mL for all persons [6]. Examination and investigations
Linked to hypovitaminosis D, adverse fetal Among 450 pregnant women attending the
outcomes included abortion, intrauterine growth hospital, 322 voluntary participants fulfilling the
restriction, fetal death and congenital malformations. inclusion criteria were recruited. The 322 participants
Maternal adverse effects involved preeclampsia, were classified into 2 groups; control group of 110
gestational diabetes mellitus (GDM) and increased women who had normal feto-maternal pregnancy,
risk of preterm labor [7-9]. Although the causative and, complicated pregnancy group comprising 212
role of VitD deficiency in different pregnancy participants. The latter group was further subdivided
complications is still an open question - due to the into: severe preeclamptic toxemia (PET) associated
insufficient reports locally and globally, VitD with intrauterine growth restriction (IUGR; 58 cases),
supplementation may reduce GDM, preeclampsia, GDM (82 cases), abortion (26 cases), undisturbed
preterm labor, and subnormal neonatal ectopic pregnancy (16 cases), premature rupture of
anthropometric measures [10]. Meta-analyses of membranes (PROMs; 14 cases), and, inevitable
observational studies correlate hypovitaminosis D preterm labor (16 cases).
with pregnancy complications without asserting After history taking (for age, gravidity, parity,
causation [11]. The longitudinal pattern of changes in duration of pregnancy in weeks and significant
25-OH-VitD concentrations and its relationship with medical history) and general examination (for vital
1,25-diOH-VitD throughout pregnancy remains signs and weight and height to calculate body mass
largely unclear. Time-dependent decreases, increases index; BMI), the head, face, neck, chest, heart, back,
or no change in 25-OH-VitD levels were reported, lower limbs and abdomen were systematically
while 1,25-diOH-VitD levels mostly increase at term examined. The pregnancy was assessed
compared to non-pregnant women. Additionally, ultra-sonographically for viability and fetal biometry
serum VitD cutoffs that are defined for the general to determine pregnancy duration and the expected
adult population are inappropriately used for fetal weight, condition of amniotic fluid and for
pregnancy [3]. diagnosing fetal anomalies, if present. Women with
We planned to assess the relationship between routine laboratory findings that contradict the
hypovitaminosis D and feto-maternal pregnancy inclusion criteria were excluded.
complications and morbidities, using the more Serum was recovered from peripheral blood
patho-physiologically relevant biomarkers. We samples by centrifugation and was aliquotted and
measured serum 25-OH-VitD, as the established VitD stored till used at -80 oC. Specific quantitative ELISA
biomarker, and, 1,25-diOH-VitD as a functional VitD immunoassay kits (Sunlong Biotech Co. Ltd.,
biomarker, and their ratio, and, correlated them with Zhejiang, China) were used to measure total
the existing pregnancy feto-maternal status. 25-OH-VitD (in ng/mL; cat# SL2762Hu) and
1,25-DiOH-VitD (in pg/mL; cat# SL2845Hu). The
Materials and Methods direct 1,25-DiOH-VitD/25-OH-VitD ratio was
calculated for each patient. Participants were
Settings and participants stratified using the clinically relevant 25-OH-VitD
This prospective cross-sectional case control cutoff levels of high/toxic as ≥50/>80 ng/mL, normal
study was carried out at Aljouf Maternity and as ≥30-50 ng/mL, insufficient as ≥20 - <30 ng/mL,
Children Hospital, Sakaka, Saudi Arabia, during the deficient as ≥10 - <20 ng/mL and severely deficient as

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Int. J. Med. Sci. 2020, Vol. 17 3041

<10 ng/mL [5, 12-14]. Unfortunately, all of the cutoff PROMs (p <0.001); and preeclamptics vs. each of
recommendations do not refer to pregnancy and its ectopic pregnancy (p <0.028) and PROM (p = 0.045).
complication as distinct clinical entities. Gravidity showed nonsignificant difference among
the investigated groups except comparing GDM vs.
Statistical analysis each of controls (p <0.001), preeclamptics (p <0.001),
Data was analyzed using SPSS (Statistical abortion (p <0.036), ectopic pregnancy (p = 0.002) and
Package for Social Sciences, Version 23.0. Armonk, PROMs (p = 0.002). Parity showed nonsignificant
NY: IBM Corp). We expressed descriptive qualitative difference among the investigated groups except
data as frequency and percentage, and, quantitative comparing GDM vs. each of preeclamptics (p <0.001),
data as range and mean ± standard deviation (SD). abortion (p = 0.035), ectopic pregnancy (p = 0.002) and
Normal distribution was checked by PROMs (p <0.035). Duration of pregnancy showed
Kolmogorov-Sminov test. One-way ANOVA with very strong significant difference among the
Tukey's post-test was employed for multiple investigated groups with a few non-significant
comparisons. Correlation among variables was exceptions; it was significantly different comparing
assessed by Spearman correlation coefficient test. controls vs. all complication subgroups (p <0.01 -
Receiver Operating Characteristic (ROC) curve 0.001) except GDM, comparing preeclamptics vs. all
analysis was used to determine the area under curve other subgroups (p <0.05 - <0.001) except preterm
(AUC) for 25-OH-VitD, 1,25-DiOH-VitD and their labour, comparing GDM vs. the others (p <0.001),
ratio to check their sensitivity and specificity to comparing abortion vs. the others (p <0.001) except
differentiate the cases from controls. P value of <0.05 ectopic pregnancy, and, comparing ectopic pregnancy
at a confidence level of 95% was considered vs. the others (p <0.001) except PROMs.
significant. Serum 25-OH-VitD levels showed nonsignificant
difference among the investigated groups except
Results comparing GDM vs. each of controls (p <0.001) and
Demographics, anthropometrics, clinical ectopic pregnancy (p = 0.01). Serum 1,25-diOH-VitD
characteristics and vitamin D biomarkers levels showed nonsignificant difference among the
(Table 1) investigated groups of patients. However, controls
had markedly higher levels vs. each of the six
Age was nonsignificantly different among all
complicated groups (p <0.001). Similarly, the direct
groups except for preeclamptics vs. each of controls (p
1,25-diOH-VitD/25-OH-VitD ratio showed very
= 0.011) and PROMs (p = 0.014), and, PROMs vs.
marked significant reduction comparing controls vs.
GDM (p = 0.035). BMI showed nonsignificant
each of the six patients' groups (p <0.001), albeit, with
difference among the investigated groups except
nonsignificant difference among the complicated
comparing GDM vs. each of controls (p = 0.002),
groups.
abortion (p = 0.02), ectopic pregnancy (p <0.001) and

Table 1. Demographic, anthropometric, clinical characteristics, serum 25-hydoxyvitamin D (25-OH-VitD) and 1,25-dihydroxyvitamin D
(1,25-DiOH-VitD), and their ratio in Saudi women with and without pregnancy complications.
Parameter Controls Preeclampsia GDM Abortion EP PROMs PTL
n = 110 n = 58 n = 82 n = 26 n = 16 n = 14 n = 16
Age, Years 28.51 ± 5.538 31.52 ± 5.202 31.6 ± 5.7 31.2 ± 4.76 27.1 ± 6.27 26.1 ± 4.82 28.5 ± 4.41
(18 - 40) (19 - 42) (22 - 49) (23 - 42) (21 - 40) (19 - 32) (20 - 34)
BMI, kg/m2 22.21 ± 1.786 22.6 ± 1.706 23.3 ± 1.93 21.9 ± 2.06 20.9 ± 2.35 20.9 ± 2.35 22.5 ± 2.02
(17.8 - 25.1) (18.8 - 25.2) (18.2 - 25) (17.8 - 25.1) (17.6 - 25.4) (18.4 - 24.2) (18.6 - 24.6)
Gravidity, n 4.055 ± 2.365 3.345 ± 1.617 5.39 ± 2.07 3.92 ± 1.81 3.13 ± 1.67 3 ± 2.72 4 ± 2.42
(1 - 10) (1 - 6) (1 - 12) (1 - 8) (1 - 6) (1 - 7) (1 - 8)
Parity, n 2.691 ± 2.208 2.034 ± 1.578 3.37 ± 1.55 2.08 ± 1.57 1.38 ± 1.45 1.71 ± 2.27 2.13 ± 1.96
(0 - 8) (0 - 5) (0 - 8) (0 - 5) (0 - 4) (0 - 5) (0 - 6)
Pregnancy Duration, Weeks 35.45 ± 5.465 31.17 ± 4.849 25.1 ± 9.68 11.7 ± 4.09 6 ± 0.73 37.1 ± 2.74 33.6 ± 1.93
(20 - 41) (22 - 37) (6 - 40) (7 - 18) (5 - 7) (33 - 39) (29 - 35)
25-OH-VitD, ng/mL 28.46 ± 22.77 23.02 ± 3.674 17.7 ± 4.6 23.9 ± 19.5 33.6 ± 37 20 ± 2.59 20.8 ± 1.17
(13.48 - 105.6) (15.17 - 35.97) (11 - 35.5) (15.7 - 90.1) (17.5 - 128) (17 - 24.7) (18.5 - 22.6)
1,25-DiOH-VitD, pg/mL 111.3 ± 47.54 45.04 ± 9.178 42.4 ± 12.0 45.0 ± 22.4 35.8 ± 7.29 56.6 ± 52.3 34.0 ± 4.29
(49.2 – 281.3) (23.3 – 63.2) (15.9 – 84.0) (32.0 – 119.7) (27.3 – 50.8) (25.8 – 179.3) (27.8 – 41.1)
1,25-DiOH-VitD/25: OH-VitD Ratio 4.956 ± 2.650 1.998 ± 0.533 2.53 ± 0.994 2.34 ± 1.41 1.65 ± 0.72 2.73 ± 2.24 1.63 ± 0.2
(0.956 – 12.9) (1.215 – 3.582) (0.94 – 6.24) (0.485 – 6.71) (0.235 – 2.9) (1.52 – 7.96) (1.34 – 2.0)
Data shown are number of participants per group (n) and mean ± SDM and range. GDM = Gestational diabetes, EP= Ectopic pregnancy, PROMs = Premature rupture of
membranes, PTL = Preterm labor. For significance of differences, see the text.

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Int. J. Med. Sci. 2020, Vol. 17 3042

Group Toxic Normal Insufficiency Deficiency Insf./Defi.

Stratification of our participants on bases of (>80) (≥30 – <50) (≥20 - <30) (≥10 - <20)
the clinical 25-OH-VitD cutoff values (Table 2) Ectopic Pregnancy 2 (12.5) 0 (0) 6 (37.5) 8 (50.0) 14 (87.5)
(n = 16)
Using serum 25-OH-VitD as the biomarker for PROMs (n = 14) 0 (0) 0 (0) 4 (28.571) 10 (71.429) 14 (100)
VitD status, 43.636% of controls were deficient (≥10 Preterm labor (n = 0 (0) 0 (0) 12 (75.0) 4 (25.0) 16 (100)
16)
ng/mL), 38.181% were insufficient (≥20 ng/mL), and, Total (n = 322) 14 16 (4.969) 124 (38.509) 168 292
equal proportions (9.09%) were either normal (≥30 (4.508) (52.174) (90.683)
Data shown are frequency; n and (%). Insf. = Insufficiency, Defi. = Deficiency. For
ng/mL) or having toxic levels (>80 ng/mL). 82.759% significance of differences, see the text.
of preeclamptic cases were insufficient, 13.793% were
deficient and 3.448% had normal levels. 82.927% of
GDM cases were deficient, 12.195% were insufficient ROC curve analysis
and 4.878 had normal levels. 84.615% of abortion cases ROC curve analysis showed that
deficient, and equal proportions (7.692%) had either 1,25-DiOH-VitD level is the most sensitive and most
insufficient or toxic levels. 50.0% of ectopic pregnancy specific biomarker for differentiating between cases
cases were deficient, 37.5% were insufficient and the and controls; with an AUC of 0.965 (0.947 - 0.983, p
rest (12.5%/2) had toxic levels. 71.429%/ of PROMs <0.001). It is followed by 1,25-DiOH-VitD25-OH-VitD
cases were deficient and 28.571% were insufficient. ratio; with AUC of 0.843 (0.791 - 0.676, p <0.001).
75% of preterm labor cases were insufficient and 25-OH-VitD levels fell near the diagonal line with no
25.0% were deficient. Fortunately, severe deficiency effect, i.e., showing least sensitivity and least
(<10 ng/mL) was not observed in our cases. specificity (AUC = 0.61; 0.543 - 0.676, and, p = 0.001)
Therefore, VitD insufficiency/deficiency is widely (Figure 1 and Table 3).
spreading among the study participants (292 out of
322, i.e., 90.683%). Controls presented a better picture Table 3. Area Under the Curve (AUC) for differentiation
only considering insufficiency/deficiency proportion, between cases (n = 212) and controls (n = 110) using each of
and, % of normal/super-normal levels. 1,25-diOH-VitD, 25-OH-VitD: 1,25-diOH-VitD ratio, and, of
25-OH-VitD among Saudi women with and without pregnancy
complications.
Table 2. Serum 25-hydroxyvitamin D levels among Saudi women
Test Result Variable(s) AUC P value* Range of AUC
with and without pregnancy complications.
Lower Bound Upper Bound
Group Toxic Normal Insufficiency Deficiency Insf./Defi. 1,25-diOH-VitD 0.965 <0.001 0.947 0.983
(>80) (≥30 – <50) (≥20 - <30) (≥10 - <20) 1,25-diOH-VitD: 25-OH-VitD ratio 0.843 <0.001 0.791 0.895
Controls (n = 110) 10 (9.09) 10 (9.09) 42 (38.181) 48 (43.636) 90 (81.818) 25-OH-VitD 0.610 0.001 0.543 0.676
Preeclamptics (n = 0 (0) 2 (3.448) 48 (82.759) 8 (13.793) 56 (96.552) *Null hypothesis: true area = 0.5. Data shown are AUC, p values and AUC range at
58) 95% Confidence Interval.
Gestational 0 (0) 4 (4.878) 10 (12.195) 68 (82.927) 78 (95.122)
diabetes (n = 82)
Abortion (n = 26) 2 (7.692) 0 (0) 2 (7.692) 22 (84.615) 24 (92.308)

Figure 1. The ROC curve for sensitivity and specificity of each of 1,25-DiOH-VitD levels, 1,25-DiOH-VitD: 25-OH-VitD ratio, and, 25-OH-VitD levels for differentiation
between cases (n = 212) and controls (n = 110) among Saudi women with and without pregnancy complications. Diagonal segments were produced by ties.

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Int. J. Med. Sci. 2020, Vol. 17 3043

Results of the correlation analysis (presented 0.0013), and, negatively vs. each of 1,25-DiOH-VitD
as r/p values) (-0.333/= 0.048) and the ratio (-0.381/ = 0.027). Parity
related positively with duration of pregnancy
For controls, age showed positive correlations (0.358/= 0.036). BMI associated negatively with
vs. each of gravidity (0.514/<0.001), parity 1,25-DiOH-VitD (-0.385/= 0.026). Pregnancy duration
(0.468/<0.001) and BMI (0.837/<0.001), and negative
correlated negatively vs. 1.25-DiOH-VitD
relationship with the ratio (-0.155/<0.05). Gravidity
(-0.371/<0.031) and the ratio (-0.490/<0.006).
showed positive relationship vs. parity (0.958/<0.001)
25-OH-VitD correlated negatively vs. the ratio
and BMI (0.480/<0.001). Parity correlated positively
(-0.773/<0.001). 1,25-DiOH-VitD correlated positively
with BMI (0.448/<0.001). BMI correlated negatively vs. the ratio (0.665/<0.001).
vs. the ratio (-0.184/= 0.027). Pregnancy duration For the ectopic pregnancy cases, age correlated
correlated negatively with 1,25-DiOH-VitD positively vs. gravidity (0.884/<0.001), parity
(-0.241/<0.006) and ratio (-0.192/= 0.022). (0.927/<0.001), and BMI (0.922/<0.001), but,
25-OH-VitD correlated positively vs. 1,25-DiOH-VitD negatively vs. 1,25-DiOH-VitD (-0.639/<0.005) and
(0.157/<0.05) and negatively vs. the ratio
the ratio (-0.527/<0.02). Gravidity correlated
(-0.647/<0.001). 1,25-DiOH-VitD correlated positively
positively vs. parity (0.906/<0.001) and BMI
with the ratio (0.562/<0.001).
(0.854/<0.001), and, negatively vs. 1,25-DiOH-VitD
For preeclamptic cases, age had positive
(-0.565/<0.013). Parity correlated positively with BMI
correlation vs. gravidity (0.682/<0.001), parity (0.933/<0.001), and, negatively vs. 1,25-DiOH-VitD
(0.608/<0.001) and BMI (0.690/<0.001), and, negative (-0.624/<0.006) and the ratio (-0.437/<0.05). BMI
correlation vs. 1,25-DiOH-VitD (-0.267/= 0.021). correlated positively vs. duration of pregnancy
Gravidity related had positively with parity (0.433/<0.05), and, negatively vs. 1,25-DiOH-VitD
(0.962/<0.001) and BMI (0.497/<0.001), but, (-0.443/= 0.044). Pregnancy duration related
negatively vs. 1,25-DiOG-VitD (-0.282/= 0.016). Parity
negatively vs. 1,25-DiOH-VitD (-0.504/<0.027).
correlated positively vs. BMI (0.424/<0.001), and,
25-OH-VitD correlated negatively vs. the ratio
negatively with 1,25-DiOH-VitD (-0.218/<0.05). BMI
(-0.690/= 0.002). 1,25-DiOH-VitD correlated
correlated positively vs. duration of pregnancy
positively with the ratio (0.647/= 0.004).
(0.333/= 0.005) and negatively vs. 1,25-DiOH-VitD Among PROMs cases, age correlated positively
(-0.275/= 0.017). 25-OH-VitD correlated negatively vs. gravidity (0.578/<0.018), parity (0.578/<0.018),
with the ratio (-0.490/<0.001). 1,25-DiOH-VitD BMI (0.727/= 0.002), 1,25-DiOH-VitD (0.500/<0.038)
correlated positively vs. the ratio (0.853/<0.001). and the ratio (0.474/<0.05). Gravidity correlated
For GDM cases, age correlated positively vs. positively vs. parity (1.0/<0.001), BMI (0.517/= 0.03)
each of gravidity (0.680/<0.001), parity
and duration of pregnancy (0.459/<0.05), but,
(0.732/<0.001), BMI (0.657/<0.001) and 25-OH-VitD
negatively vs. 25-OH-VitD (-0.543/<0.026). Parity
(0.239/= 0.015), but, negatively vs. each of
associated positively vs. BMI (0.517/= 0.03) and
1,25-DiOH-VitD (-0.179/<0.05) and the ratio
pregnancy duration (0.467/<0.05), but, negatively vs.
(-0.320/<0.002). Gravidity correlated positively with 25-OH-VitD (-0.543/<0.026). BMI positively
parity (0.865/<0.001), BMI (0.671/<0.001) and correlated with 1,25-DiOH-VitD (0.800/<0.001) and
25-OH-VitD (0.216/<0.026), and, negatively vs. the ratio (0.655/= 0.007). 25-OH-VitD correlated
duration of pregnancy (-0.294/<0.004) and the ratio positively with 1,25-DiOH-VitD (0.468/<0.048).
(-0.183/<0.05). Parity correlated positively vs. BMI
1,25-DiOH-VitD correlated positively vs. the ratio
(0.725/<0.001) and 25-OH-VitD (0.187/0.047), and,
(0.786/<0.001).
negatively vs. duration of pregnancy (-0.179/<0.05).
Within the preterm labor cases, age correlated
BMI correlated positively vs. 25-OH-VitD
positively vs. gravidity (0.898/<0.001), parity
(0.412/<0.001) and negatively vs. the ratio
(0.908/<0.001), BMI (0.952/<0.001), and the ratio
(-0.225/<0.021). 25-OH-VitD correlated negatively (0.452/<0.042), but, negatively vs. 25-OH-VitD
with the ratio (-0.757/<0.001). 1,25-DiOH-VitD had (-0.439/<0.05). Gravidity correlated positively vs.
positive correlation vs. the ratio (0.592/<0.001). parity (0.933/<0.001), BMI (0.802/<0.001) and the
For abortion cases, age correlated positively with ratio (0.503/= 0.025). Parity correlated positively vs.
gravidity (0.406/<0.02), parity (0.535/= 0.0024), BMI
BMI (0.835/<0.001) and the ratio (0.454/= 0.04), but,
(0.757/<0.001), and pregnancy duration (0.337/=
negatively with 25-OH-VitD (-0.454/= 0.04). BMI
0.046), and, negatively vs. 1,25-DiOH-VitD
associated negatively with 25-OH-VitD
(-0.669/<0.001) and the ratio (-0.380/<0.028).
(-0.500/<0.027), but, positively vs. the ratio (0.548/=
Gravidity correlated positively with parity 0.016). 1,25-DiOH-VitD correlated positively vs. the
(0.815/<0.001) and duration of pregnancy (0.565/= ratio (0.786/<0.001).

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Int. J. Med. Sci. 2020, Vol. 17 3044

Discussion Therefore, the intracellular receptor supersaturating

concentration is not required, albeit, for potential
Age was insignificantly different among our
VDR-independent actions [34]. We resourced
complicated groups. It had significant difference only
1,25-diOH-VitD since several studies reported its
when controls are compared to preeclamptics.
superiority over 25-OH-VitD as an independent
Preeclampsia usually attacks extremely aged mothers;
clinical correlate [31, 35-37]. 25-OH-VitD is the local
mainly primigravida. Gravidity showed a significant
precursor and increases in its circulating levels
difference between the controls and GDM groups.
parallel consequent increases in 1,25-diOH-VitD
Repeated pregnancy is a definite risk factor for GDM
however, their relationship is not always
development. BMI was significantly different only
straightforward [30, 38]. For instance, adequate
comparing GDM and controls. GDM usually
circulating 1,25-diOH-VitD levels are necessary for
associates with higher BMI. As the gestational age is
sufficient 25-OH-VitD availability in VitD target cells,
unique for the occurring complication (e.g.,
e.g., monocytes [39]. Moreover, pregnant Brazilian
preeclampsia develops after 20 weeks of gestation,
women insufficient in VitD at baseline had higher
abortion frequently happens in the 1st trimester of
increases in 1,25-diOH-VitD concentrations, over
pregnancy, PROMs occurs after 37th weeks and before
pregnancy time, compared to women with sufficient
onset of labor and GDM is common at beginning of
VitD levels [3]. VitD supplementation do not increase
the last trimester), it showed significant differences
rate of sufficiency in all occasions [40]. However,
comparing all groups [15-19].
1,25-diOH-VitD levels had no seasonal variation like
Considering the association between VitD
that of 25-OH-VitD in healthy Canadians [41].
deficiency and risk of feto-maternal pregnancy
Moreover, a study found a strong correlation
complications, the bending and intriguing questions
between circulating 1,25-diOH-VitD and 25-OH-VitD
are: How to achieve and specify the optimal
levels throughout pregnancy and that 1,25-diOH-VitD
pregnancy VitD level? What optimum biomarker and
levels at 12 weeks’ gestation are approximately triple
cutoffs to measure?, and, how to determine the real
that of normal nonpregnant female [42].
benefits of VitD supplementation in pregnancy? [20].
Our controls were overwhelmingly VitD
Even implementing a personal physiological
insufficient which is in accord with another Saudi
response-dependent VitD signature turned confusing
study reporting 3.5% VitD sufficiency rate among
due to varying physiological responses at same
pregnant women [43]. However, significantly lower
supplementation dose and increment plasma
25-OH-VitD level vs. controls was recorded only for
25-OH-VitD levels [21]. Reports related VitD level to
our GDM women among other complications.
clinical morbidities using serum 25-OH-VitD as a
Ironically, those with ectopic pregnancy showed
VitD biomarker - based on its higher stability and
nonsignificant higher, while, other complications had
longer biological half-life. Such validity is
nonsignificantly lower levels than controls. Grouping
substantiated by the significant correlation of serum
deficient and insufficient subjects revealed decreasing
25-OH-VitD with non-calcemic clinical outcomes and
serum 25-OH-VitD rates as follows: Controls, ectopic
its existence at high cellular levels. Both assumptions
pregnancy, abortion, GDM, preeclampsia, preterm
are not unquestionably correct [22, 23]. Indeed, the
labour, and PROMs. Oppositely, 1,25-diOH-VitD
present study and a long list of previous studies,
levels showed marked reductions in each of 6
including ours, had shown that serum 25-OH-VitD
complicated groups vs. controls. The complicated
does not differentiate apparently healthy controls
women groups were nonsignificantly different.
from patients with different comorbidities including
Changes in 1,25-diOH-VitD/25-OH-VitD ratio
pregnancy, diabetes and infection [4, 8, 24-31]. This
mirrored 1,25-diOH-VitD pattern. While sensitivity
could be partially due to our and others observation
and specificity of both of 1,25-diOH-VitD and the
of widely spreading hypovitaminosis D among
ratio were very high, those of 25-OH-VitD were very
population [4, 11]. 25-OH-VitD is produced from
low in differentiating between our controls and
several sources and leak to serum without direct
complicated pregnancy. Pregnancy hypovitaminosis
reflection of cellular contents [32, 33].
D in developing countries and its associated adverse
Serum level of 1,25-diOH-VitD was considered a
feto-maternal hazards is a commonplace [10, 44].
less accurate predictor of intracellular VitD
Similar to our GDM women, studies had correlated
concentration. This came from the notion that
hypovitaminosis D higher risk of development of
1,25-diOH-VitD intracellular level exceeds its serum
GDM, miscarriage and still birth in the 2nd and 3rd
level by several folds along with extra-renal sources
trimester [23, 45-47]. Despite the wide spreading
for it [22]. Indeed, cells have concentrating modalities
25-OH-VitD deficiency in controls (71.2 vs. 83.3%),
for lipophilic ligands. Moreover, serum
women with GDM had a 2.66-fold increased risk of
1,25-diOH-VitD is already VDR receptor saturating.

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Int. J. Med. Sci. 2020, Vol. 17 3045

being deficient status [48]. women were correlated with increased production of
Other studies conducted in Saudi Arabia, USA proinflammatory cytokines [59]. Women with PROMs
and China did not find association between adverse and preterm labor were 100% VitD
pregnancy outcomes and low 25-OH-VitD level deficient/insufficient in our study. Mothers with
except for high prevalence of abortions among other preterm labor are three times more likely to have
outcomes [8, 24, 26]. Surprisingly, the Chinese study insufficiency when compared to full-term mothers
found higher prevalence of GDM and preterm [62]. Hypovitaminosis D is associated with high
delivery among pregnant women with high prevalence of infections, particularly Gardnerella
25-OH-VitD level despite older maternal age and vaginalis; commonly encountered in cases of PROMs
higher BMI [18]. Actually 5% of women with GDM of and preterm labor [63-65]. VitD deficiency increased
our study had normal VitD levels. Rodriguez et al susceptibility to microbial infection due to
reported no association of VitD concentration and macrophage dysfunction with defect in toll-like
GDM, preterm delivery and IUGR [25]. Also, mediated action of the antibacterial peptide
hypovitaminosis D in early pregnancy did not cathelicidin [66].
associate with adverse pregnancy outcomes [26]. BMI inversely correlates with 25-OH-VitD levels
GDM had no association with vitamin D deficiency at med-gestation and postpartum [45]. Our controls
[27, 28]. had negative correlations contrasting 1,25-diOH-VitD
Similar to the trend of our results, higher risks of and the ratio vs. pregnancy duration, BMI and
development of preeclampsia, GDM, preterm and 25-OH-VitD, while 25-OH-VitD had no such
cesarean delivery and IUGR were related to correlations. Among our preeclamptics, age, gravidity
hypovitaminosis D [49, 50]. Normal 25-OH-VitD and BMI correlated negatively with 1,25-diOH-VitD.
levels decrease the risk of preeclampsia and IUGR Our GDM group presented negative correlation
[51]. To establish a causal relationship, large VitD between age vs. 1,25-diOH-VitD and the ratio, while
supplementation RCTs must guarantee optimum gravidity and BMI had negative correlation with the
feto-maternal outcomes [52]. On the contrary, vitamin ratio. Our abortion cases had negative correlation
D supplementation did not affect the incidence of contrasting age, gravidity and BMI vs.
adverse pregnancy outcomes like preeclampsia, GDM 1,25-diOH-VitD and the ratio. 1,25-diOH-VitD
and IUGR [53]. Conflictingly, supplementation with negative correlation vs. age, gravidity, parity and BMI
VitD in early pregnants diminished GDM incidence in among our ectopics. In our PROMs cases, age and
pregnant with hypovitaminosis D [54]. Increased BMI had positive correlation vs. 1,25-diOH-VitD and
25-OH-VitD levels after VitD supplementation the ratio, while, gravidity and parity correlated
correlate with significant reduction in rate of preterm negatively vs. 25-OH-VitD. Our preterm labor cases
labour, pre-eclampsia, and GDM in pregnant Indian showed positive correlation between age, gravidity,
women [43]. Women with persistent 25-OH-VitD parity and BMI vs. the ratio but a negative correlation
deficiency up to 26th weeks of gestation had a between age, parity and BMI vs. 25-OH-VitD.
4.46-fold elevated risk for GDM [55]. Significant Reportedly, gestational age correlates positively with
reduction in GDM risk was observed with increasing 25-OH-VitD levels and had stronger association with
25-OH-VitD levels [56]. 1,25-diOH-VitD [3]. Disagreeable, serum 25-OH-VitD
Among our preeclampsia patients, 97% were level neither increased the risk of preterm birth nor
VitD deficient/insufficient. Hypovitaminosis D is a correlated with gestational age or BMI [67]. A big
risk factor for severe preeclampsia and VitD supplementation study conducted in
season-dependent variation in incidence of Bangladesh, with a widespread VitD deficiency and
preeclampsia seems to correlate with VitD level [57]. fetal-infant growth restriction, showed that
Low 25-OH-VitD levels and placental VitD receptor supplementation from mid-pregnancy to delivery or 6
expression negatively correlated with preeclampsia months postpartum had no significant effect on
[58]. 92% of our abortion patients had VitD pregnancy feto-maternal clinical outcomes, despite
deficiency/insufficiency. Relationship between low normalizing 25-OH-VitD levels [68].
VitD levels and miscarriage stems from VitD Our study is one of the few studies which
regulation of genes concerned with implantation, evaluated the association of variations in
trophoblastic invasion, angiogenesis, immunomo- 1,25-diOH-VitD and its ratio to 25-OH-VitD with
dulation, suppression of inflammation and protection pregnancy complications. Physiologically, 1,25-diOH-
from infection [31, 59, 60]. 25-OH-VitD levels were Vit-D levels increase with gestational age to reach a
lower in non-gravid women with history of maximum in 3rd trimester without increases in
pregnancy loss than normal non-gravid and pregnant 25-OH-VitD levels [30]. In our study, while
women [61]. Reduced VitD levels among pregnant 25-OH-VitD correlated negatively only with GDM,

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Int. J. Med. Sci. 2020, Vol. 17 3046

1,25-diOH-VitD and its ratio to 25-OH-Vit-D Dr Atef abdelmageed (Head, Obstetrics and
correlated negatively with all of pregnancy Gynecology Department) and Mr. Mohammed
complications investigated. This may help clearing Fahman (Manager of the Laboratory, Maternity and
the discrepancy among the previous studies utilizing Children Hospital, Sakaka, Aljouf, Saudi Arabia) for
25-OH-Vit-D as the sole VitD biomarker for adverse facilitating the collection of the samples and profiling
pregnancy complications. We claimed that addition of our patients.
1,25-diOH-VitD and the ratio in evaluating Other than clinical profiling of the patients and
implication in pregnancy complications are clinically collecting samples by IAB, all authors contributed
meaningful than 25-OH-VitD alone. They both equally to this article.
showed higher sensitivity and specificity in
differentiating healthy controls vs. complicated Funding
pregnancies. However, bigger multi-centric This study was generously funded by the
longitudinal and supplementation RCTs are strongly Deanship for Research, Jouf University, Sakaka, Saudi
warranted to validate and generalize these Arabia (#39/950).
assumptions. Several clinical outcomes, including
infection, kidney function, diabetes and inflammation, Competing Interests
were inversely correlated with circulating 1,25-diOH- The authors have declared that no competing
VitD levels but not 25-OH-VitD [29, 31, 37]. interest exists.
The norms and biomarkers for VitD levels have
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