Women’s fertility and vitamin D: Could hypovitaminosis D biomarkers correlate with

the disease, and explain the unexplained female factor infertility?

Albahlol Ibrahim A1, Alshaikh Ahmed Baker1, Almaeen Abdulrahman H2,

Alduraywish Abdulrahman A3, Dar Umar Farooq4, El-Metwally Tarek H2,5

1. Departments of Obstetrics and Gynecology.

2. Pathology.
3. Internal Medicine.
4. Community and Family Medicine, College of Medicine, Jouf University, Sakaka, Saudi Arabia.
5. Department of Medical Biochemistry, Faculty of Medicine, Assiut University, Assiut, Egypt.

Ibrahim A. Albahlol: [email protected]. 00966 0565702858; Ahmed Baker Alshaikh: [email protected].

00966532182255; Abdulrahman H. Almaeen: [email protected]. 00966 0506396076; Abdulrahman A. Al-
duraywish: [email protected]. 00966504286289; Umar Farooq Dar: [email protected]. 009660582461655.

Abstract
Objective: Vitamin D (Vit D) deficiency correlates women reproductive pathophysiology. We analyzed Vit D deficiency
biomarkers in infertile women.
Patients & method: This case-control study enrolled 80 infertile women polycystic ovary syndrome (PCOS), and other
etiologies; anovulation and unexplained and 25 controls. Serum calcidiol and calcitriol were determined by ELISA and their
direct ratio was calculated.
Results: 72% of controls and 92.5% of patients had Vit D deficiency/insufficiency (calcidiol mean ± SDM of 33.50±22.10
vs. 20.26±5.226 ng/mL and an AUC of 0.808±0.048). Calcitriol had an AUC of 0.909±0.031 that more effectively distin-
guished patients and etiologies (53.49±23.30 pg/mL) from controls (114.0±43.20 pg/mL; P<0.001). 4 other etiology cases
and 17 controls had calcitriol levels ≥100 pg/mL. 64% of controls (4.090±0.020) and 16.25% of patients [2.634±0.855,
P<0.04; 5 PCOS (3 primary/2 secondary), 3 secondary unexplained, and 5 others (one primary tubal, one primary/one sec-
ondary peritoneal, one primary/one cervical and one primary tubal had a normal ratio ≥3.333 at an AUC of 0.740±0.065.
All biomarkers revealed patient levels ~50% lower than controls; lowest in PCOS and unexplained etiologies.
Conclusion: Vit D levels are significantly reduced in infertile women; lowest in PCOS and unexplained etiology, for all bio-
markers, where calcitriol was the optimal predictor of both infertility and etiology.
Keywords: Female infertility, Polycystic ovary syndrome; unexplained infertility; Vitamin D; 25-Hydroxy-cholecalceferol, 1;
25-Dihydroxyl-cholecalceferol.
DOI: https://dx.doi.org/10.4314/ahs.v24i3.30
Cite as: Ibrahim AA, Baker AA, Abdulrahman AH, Abdulrahman AA, Farooq DU, Tarek E-MH. Women’s fertility and vitamin D:
Could hypovitaminosis D biomarkers correlate with the disease, and explain the unexplained female factor infertility? African Health Sciences.
2024;24(3). 259-273. https://dx.doi.org/10.4314/ahs.v24i3.30

Introduction the uterus and cervix, tubal issues, and endometriosis

Female infertility places a substantial psychological, so- and pelvic adhesions. However, 30% of cases are of
cial, medical and economic burden on the couple and unknown etiology 1,2. Vitamin D deficiency is prevalent
community. Over 70% of female infertility cases results in reproductive age women, and seasonal reproductive
from low ovarian reserve or dysfunction, issues with capacity may be in part a consequence of variations in
Vit D levels. However, the literature to date is incon-
Corresponding author: clusive and does not confirm a role for Vit D in fe-
Tarek H El-Metwally, male infertility 3-6. The total fertility rate in Saudi Ara-
Department of Medical Biochemistry, bia has dropped from 7.175 in 1950 to 2.148 in 2023;
Biochemistry Division, Pathology Department, with a steady decline starting from 1980 (https://
College of Medicine, Jouf University, Sakaka, www.macrotrends.net/countries/SAU/saudi-arabia/
Saudi Arabia. 00966541860565. fertility-rate, accessed May 20, 2023). To the
Email: [email protected] Saudi ministry of health, causes of women infertility
African © 2024 Ibrahim AA et al. Licensee African Health Sciences. This is an Open Access article distributed under the terms of the Creative commons Attribution
Health Sciences License (https://creativecommons.org/licenses/BY/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.

259 African Health Sciences, Vol 24 Issue 3, September, 2024

include ovulation disorders, polycystic ovary syndrome sequence of low levels obtained by consumption of
(PCOS), amenorrhea, pituitary dysfunction, endometri- unfortified foods and insufficient production via con-
osis, fallopian tube blockage due to salpingitis as in pel- ditional synthesis from 7-dehydocholesterol upon ex-
vic inflammatory disease, previous infection with gon- posure of epidermal keratinocytes to UVB rays. Blood
orrhea and chlamydia, or any abdominal surgery, and levels vary depending on a number of individual fac-
uterine abnormalities such as uterine fibroids (https:// tors, including genetic polymorphisms, skin color and
www.moh.gov.sa/en/HealthAwareness/Educational- ethnicity, body mass index (BMI), and supplement use,
Content/wh/Pages/036.aspx, accessed May 20, 2023). as well as by environmental, life-style, food fortification
In a Saudi nation-wide representative sample of 15810 policies, drug and pollutant exposure, and seasonal vari-
general population women aged 18-44 years, rate of in- ations. Conditions that affect absorption, digestion and
voluntary infertility ranged from 25 to 50% within one transport of the vitamin also lead to variations in blood
year 7,8. In Arar, Saudi Arabia, the prevalence of infer- levels. In the circulation, calcitriol is approximately 1000
tility among followed up women was 65.3%; 19.8% of times lower than calcidiol 4,5,13,21-23. Discrepancy among
them were primary and 80.2% were secondary infertili- published literature considering Vit D role in female in-
ty; ovulation defect constituted 24.6%, followed by the fertility may also originate from variance in the pre- and
polycystic ovary (21.8%), tubal adhesions or obstruc- analytical factors.
tion (6.7%), endometriosis (3.2%), and uterine fibroid
(3.0%) 9. In Najran, Saudi Arabia, 65% of followed up The putative role for Vit D in female reproduction is
women infertility was primary and 35% was second- indicated by uterine hypoplasia and anovulation in Vit
ary infertility, with PCO disease as the most common D receptor (VDR) and 1α-hydroxylase knockout mice,
(56%), followed by fibroids (22%), endometrial polyps (reviewed by Lerchbaum and Obermayer-Pietsch)24.
(9%), adenomyosis (5%), hydrosalpinx (4%), congeni- Expression of HOXA10, required for multiple aspects
tal abnormality (2%), and other causes (1%) 10. Among of fertility - including the maintenance of a healthy
Saudi women followed up in Riyadh city, 67.0% had pregnancy, is upregulated by Vit D. The VDR and me-
secondary infertility, while 33.0% had primary infertility. tabolizing enzymes are expressed in female reproduc-
Defective ovulation constituted 28.9%, tubal adhesion tive and endocrine tissues. Vit D deficiency is shown
or obstruction was 7.4%, and male factors was 12.6% in the clinic and lab to result in poorer fertility and im-
11
. To Khadawardi prevalence of infertility was 23.3% paired functionality of the reproductive system, PCOS,
among followed up Saudi women, with endometriosis endometriosis and pre-term birth, along with many
constituting 10.7% 12. systemic conditions that include increased insulin resis-
tance4,5,25-27. Vit D controls the expression of aromatase
Vit D is a steroidal preprohormone with extensive im- and estrogen production, and increase sensitivity to
munomodulatory, anti-inflammatory, and metabolic FSH and human chorionic gonadotropin secretion,
functionality, including cell cycle control, insulin secre- and estrogen and progesterone synthesis in human pla-
tion, and anti-autoimmunity. The subsequent hydrox- centa 28. Vit D supplementation resulted in significant
ylation of Vit D to its prohormone 25-hydoxy-form reductions in total testosterone, free androgen index,
(calcidiol) occurs in the liver and other tissues, and then hirsutism, and C-reactive protein (CRP) levels, as well
further to the hormone form 1,25-dihydroxy-chole/er- as a significant increase in sex hormone binding glob-
go-calciferol form (calcitriol) in the kidney and other ulin and total antioxidant capacity 29,30. Serum calcidiol
tissues (including epithelial and immune cells) is con- concentration significantly correlated with female infer-
trolled by its own level and by various hormones, sterols tility with insignificant difference in its level between
and growth factors 4,5,13. Among 1000 genes controlled primary and secondary causes 6. Low Vit D is correlated
by Vit D receptor-mediated transcription and mem- with poorer in vitro fertilization (IVF) outcomes, follic-
brane-receptor signaling, those implicated in wom- ulogenesis and reduced ovarian reserves 5,27,31,32. In IVF,
en infertility include insulin secretion and action, and women with normal serum Vit D levels (comparable
expression of Homeobox 10, miR222, antimüllerian with follicular levels) have a significantly higher chance
hormone, immunomodulatory and anti-inflammatory of euploid blastocyst production than deficient pa-
cytokines/chemokines, steroidogenesis, angiogenesis tients33. In healthy reproductive-aged Caucasian women,
and antioxidant defense 14-20. Deficiency of Vit D, an Vit D levels are significantly negatively correlated with
essential micronutrient, is a global epidemic, as a con- multiple sex hormones 34. Polymorphisms in the Vit D

African Health Sciences, Vol 24 Issue 3, September, 2024 260

binding protein (VDBP) that reduce its expression are infertile women, there were three groups based on eti-
associated with metabolic syndrome and low Vit D in ology: 30 with PCOS, 27 with other etiologies (incor-
patients and controls; however, this is not directly cor- porating tubal n = 6, tubo-peritoneal n = 5, cervical n
related with PCOS 35. In Pakistani women, one VDBP = 6 and uterine n = 5 issues and anovulation n = 5),
polymorphism, which activates 1α-hydroxylase, and in- and 23 of unexplained etiology. Unexplained classifi-
activates 24-hydroxylase, exhibits an inverse correlation cation only occurred after full investigations, including
between Vit D levels and PCOS 36. Consequently, many laparoscopy. These groupings were further defined by
fertility clinics advise screening for (generally as calcidi- primary (n = 35)/secondary (n = 45) infertility status.
ol levels) and supplementation of Vit D, most critical A complete medical history was taken for all patients,
for women who are not at risk of ovarian hyper-stimu- and weight, height and vital signs were recorded and
lation but suffer from PCOS or similar. Supplementa- a complete examination was conducted systematically.
tion of Vit D led to higher rates of positive pregnan- Following laboratory and infertility investigations, indi-
cy tests than in unsupplemented cases 4,5,25,27,37. Almost viduals with abnormal findings were excluded from the
96% of women in IVF treatment were Vit D deficient/ study.
insufficient, and this negatively correlates with duration
of infertility and BMI 38. Blood Sampling and Investigations
Proposing an inverse relationship, herein we determine Serum was recovered from 5 mL peripheral blood
the correlation between Vit D biomarkers, calcidiol, samples via clotting and centrifugation. Aliquots were
calcitriol and their direct ratio, with multiple causes of stored at -80 oC. Calcidiol (ng/mL; cat# SL2762Hu)
infertility in a local population of healthy and infertile and calcitriol (pg/mL; cat# SL2845Hu) were measured
Saudi women. using ELISA kits in accordance with instructions (Sun-
long Biotech Co. Ltd., Zhejiang, China). Participants
Patients and method were grouped according to clinically designated levels,
Setting and Patients from severely deficient < 10 ng/mL to high/toxic at
This case-controlled study was conducted between Sep- 50/> 80 ng/mL 39-42. However, the clinical cutoff levels
tember 12, 2018 and September 30, 2019 at Aljouf Ma- do not pertain to female infertility. We also determined
ternity and Children’s Hospital and College of Medicine, the direct calcitriol/calcidiol ratio for all participants.
Jouf University, Sakaka, Saudi Arabia. The bioethical
protocols (#6-16-4/40) were approved by committees Data Analysis Procedure
at the university and Ministry of Health, and informed Data analysis used SPSS (Statistics package for social
consent was provided by all participants included - in sciences, Version 23.0, IBM Corp, Armonk, NY) and
adherence to the provisions of the Declaration of Hel- Prism-7.0 Package (GraphPad Software, San Diego,
sinki. Participants were excluded if they demonstrated CA). Qualitative statistics were described as frequen-
unwillingness, have previous pelvic surgery, male factor cies/percentages, while quantitative as mean ± standard
infertility, or other co-morbidities include thyroid dis- deviation (SD)/median ± interquartile range (IQR).
orders and chronic conditions (i.e., liver, kidney, auto- Variable distribution was determined by the Kolmog-
immune diseases and diabetes). Patients prescribed glu- orov-Sminov test, non-normal variable distribution was
cocorticoids, antifungal, antiepileptic and antiretroviral identified by a significant p-value. Kruskal-Wallis test
drugs were also excluded, due to effects of these drugs was used followed by Mann-Whitney test for multiple
for Vit D metabolism. Infertility (post-male factor ex- comparisons of Vit D levels in the groups. The area un-
clusion) results in no conception after one year of un- der the curve (AUC) was determined by ROC for both
protected intercourse 1. No prior pregnancy is termed biomarkers and their ratio to discriminate the cases and
primary infertility. controls, where higher AUC represent greater sensitivi-
ty and specificity for the biomarker. A p-value of ≤0.05
Clinical Examination and Assessment at a confidence level of 95% was considered significant.
105 participants were included in the study, of which
25 were healthy (previously pregnant; aged 27.6 ± 5.3 Results
years) controls and 80 were infertile (aged 27.76 ± 5.052 Comparisons of the characteristics and investigations
years with a duration of 3.538 ± 1.807 years). Of the among participants (Table 1)

261 African Health Sciences, Vol 24 Issue 3, September, 2024

Age: infertility subgroups only other and secondary etiol-
Participant age was not a significant factor between ogies vs. PCOS and primary PCOS were significantly
controls and infertile group/subgroups, with the excep- different (P < 0.05). For all healthy and infertile groups
tion of secondary unexplained infertility vs. PCOS (P < the levels of calcidiol was not clinically severely defi-
0.01) and primary PCOS patients (P < 0.01). cient. For the controls, 16 (64%) were insufficient, 4
(16%) were normal, 2 (8%) deficient, and 3 (12%) had
BMI: toxic levels (> 80 ng/mL). For the patients, over a half
PCOS individuals exhibited highest BMIs, while un- (44, 55%) were deficient, 30 (37.5%) insufficient, and
explained infertility group were the lowest, specifically 6 (7.5%) had normal levels. Over 92% of patients and
primary conditions. Controls vs. infertility group and 72% of controls exhibited insufficient/deficient levels.
subgroups were significantly different P < 0.01 and P <
0.001, respectively, as were the infertile group vs. PCOS Calcitriol
(P < 0.001), primary PCOS (P < 0.001), unexplained With the exception of infertile primary other etiology
(P < 0.01) and primary unexplained cases (P < 0.01). cases, healthy controls exhibited higher levels of cal-
Further, PCOS group and sub-groups were significant- citriol than all other infertile groups (P < 0.001). Within
ly different from unexplained and subgroups, and other the infertile subgroups, only other- and secondary other
and secondary other etiologies (P < 0.001). etiologies vs. PCOS and primary PCOS exhibited a sig-
nificant difference (P < 0.05). At a cutoff of 100 pg/
Gravidity: mL, 17 control (68%) but only 4 (5%) infertile women
Naturally, healthy control showed significantly higher (specifically other etiologies) had normal levels.
gravidity than the infertile group and subgroups (P <
0.001), while primary infertility had zero gravidity, and Direct Calcitriol/Calcidiol Ratio
within the infertile group, subgroups PCOS and its Controls showed a higher ratio than all infertile sub-
groups had the lowest gravidity (P < 0.001). groups (P < 0.001 overall, P < 0.05 vs secondary PCOS
and primary unexplained, P < 0.01 vs. secondary unex-
Parity: plained, and no significant difference vs. primary other
Healthy participants had significantly higher parity than etiologies). Among infertile subgroups there were no
the infertile group and subgroups (P < 0.001), primary significant differences. At a cutoff of 3.333 (100 pg/
infertile group had zero parity, while in the infertile co- mL calcitriol vs. 30 ng/mL calcidiol), 64% (16) controls
hort PCOS and subgroups exhibited the lowest parity and 16.25% (13) infertile patients had normal ratios. Of
(P < 0.001). Primary vs. secondary groups were also the infertile cases, five were PCOS (3 primary, 2 second-
significantly different (P < 0.05). ary), five other etiologies (3 primary, 2 secondary), and
four unexplained (all secondary).
Disease Duration:
Patients with unexplained infertility had the longest du- Given that BMI is higher for the infertile patients than
ration (P < 0.05), with other etiologies next, and PCOS the controls, normalization of the levels of biomarkers
individuals the shortest. for BMI (ng/mL / kg/m2) demonstrated greater levels
of deficiency in the infertile group and subgroups for
Calcidiol: all markers.
With the exception of primary other etiology cases,
healthy controls exhibited significantly higher levels of Comparing the patients with primary vs. secondary
calcidiol than all infertile groups (P < 0.001). However, causes showed nonsignificant difference due to the big
the difference for other and secondary etiologies were differences among primary subgroups, and among sec-
less, with P< 0.01 and P< 0.05, respectively. Within the ondary subgroups, for the three biomarkers.

African Health Sciences, Vol 24 Issue 3, September, 2024 262

 Table 1. Variations in serum calcidiol, calcitriol and their ratio as biomarkers for vitamin D status when investigating infertile
(n = 80) and fertile (n = 25) women, described as range, mean ± SDM and number.

Parameter Group/Subgroup Range Mean ± SDM n

Age, Years Controls 19-38 27.600 ± 5.300 25
All 20-42 27.760 ± 5.052 80
PCOS 20-34 25.500 ± 3.203 30
PCOS-Primary 21-34 25.380 ± 3.074 21
PCOS-Secondary 20-33 25.780 ± 3.667 9
Infertile Unexplained infertility 22-42 29.780 ± 5.815 23
Unexplained-Primary 22-32 25.140 ±3.532 7
Unexplained-Secondary 25-43 31.810 ± 5.492 16
Other infertility 21-40 28.560 ± 5.228 27
Others-Primary 21-27 25.000 ± 2.517 7
Others-Secondary 24-40 29.800 ± 5.396 20
BMI, kg/m 2
Controls 18.2-25.2 22.200 ± 2.000 25
All 17.8-30.4 24.390 ± 2.804 80
PCOS 24.2-30.4 26.820 ± 1.736 30
PCOS-Primary 24.2-30.4 27.010 ± 1.962 21
PCOS-Secondary 24.8-27.6 26.370 ± 0.985 9
Infertile Unexplained infertility 17.8-26.8 22.220 ± 2.427 23
Unexplained-Primary 17.8-24.2 20.540 ± 2.435 7
Unexplained-Secondary 18.4-26.8 22.960 ± 2.093 16
Other infertility 19.4-26.9 23.530 ± 1.951 27
Others-Primary 22.6-26.1 24.270 ± 1.424 7
Others-Secondary 19.4-26.9 23.270 ± 2.072 20
Gravidity Controls 1-9 3.880 ± 2.030 25
All 0-4 0.989 ± 1.097 80
PCOS 0-2 0.367 ± 0.615 30
PCOS-Primary 0 0±0 21
PCOS-Secondary 1-2 1.222 ± 0.441 9
Infertile Unexplained infertility 0-3 1.261 ± 1.054 23
Unexplained-Primary 0 0±0 7
Unexplained-Secondary 1-3 1.813 ± 0.750 16
Other infertility 0-4 1.444 ± 1.251 27
Others-Primary 0 0±0 7
Others-Secondary 1-4 1.950 ± 1.050 20
Parity Controls 0-8 3.520 ± 1.980 25
All 0-3 0.675 ± 0.925 80
PCOS 0-1 0.167 ± 0.379 30
PCOS-Primary 0 0±0 21
PCOS-Secondary 0-1 0.556 ± 0.527 9
Infertile Unexplained infertility 0-3 1.087 ± 1.041 23
Unexplained-Primary 0 0±0 7
Unexplained-Secondary 0-3 1.563 ± 0.892 16
Other infertility 0-3 0.889 ± 1.013 27
Others-Primary 0 0±0 7
Others-Secondary 0-3 1.2 ± 1.005 20

Disease Duration, Years Controls - - 25

All 1-11 3.538 ± 1.807 80
PCOS 1-5 2.800 ± 1.157 30
PCOS-Primary 1-5 2.667 ± 1.111 21
PCOS-Secondary 1-5 3.111 ± 1.269 9
Infertile Unexplained infertility 2-9 4.348 ± 2.102 23
Unexplained-Primary 2-5 3.286 ± 0.951 7
Unexplained-Secondary 2-9 4.813 ± 2.316 16
Other infertility 2-11 3.667 ± 1.861 27
Others-Primary 2-5 3.286 ± 1.113 7
Others-Secondary 2-11 3.800 ± 2.067 20
Calcidiol, ng/mL Controls 16.1-92.5 33.500 ± 22.100 25
All 13.1-34.4 20.260 ± 5.226 80
PCOS 13.1-29.25 17.310 ± 3.944 30
PCOS-Primary 13.1-21.8 16.760 ± 3.074 21
PCOS-Secondary 13.22-29.25 18.600 ± 5.488 9
Infertile Unexplained infertility 13.8-23.7 18.670 ± 2.816 23
Unexplained-Primary 13.8-22.4 18.130 ± 3.425 7
Unexplained-Secondary 13.8-23.7 18.900 ± 2.597 16
Other infertility 17.99-34.4 24.900 ± 4.931 27
Others-Primary 20.4-30.2 24.490 ± 3.229 7
Others-Secondary 17.99-34.4 25.040 ± 5.467 20
Calcitriol, pg/mL Controls 49.8-221 114.00 ± 43.200 25
All 30.2-122.2 53.490 ± 23.300 80
PCOS 30.2-92.5 44.950 ± 17.500 30
PCOS-Primary 30.7-92.2 43.520 ± 14.940 21
PCOS-Secondary 30.2-82.2 48.270 ± 23.10 9
Infertile Unexplained infertility 30.4-88.8 48.500 ± 15.640 23
Unexplained-Primary 31.8-62.6 43.910 ± 9.423 7
Unexplained-Secondary 30.4-88.8 50.510 ± 17.590 16
Other infertility 33.2-122.2 67.230 ± 28.260 27
Others-Primary 38.2-122.2 79.630 ± 31.460 7
Others-Secondary 33.2-111.8 62.900 ± 26.530 20
Calcitriol/Calcidiol Ratio Controls 1.14-9.96 4.090 ± 2.020 25
All 1.255-5.454 2.634 ± 0.855 80
PCOS 1.255-5.456 2.616 ± 0.858 30
PCOS-Primary 1.76-5.456 2.624 ± 0.851 21
PCOS-Secondary 1.255-4.031 2.599 ± 0.926 9
Infertile Unexplained infertility 1.65-4.311 2.612 ± 0.777 23
Unexplained-Primary 1.893-2.953 2.454 ± 0.457 7
Unexplained-Secondary 1.65-4.311 2.681 ± 0.886 16
Other infertility 1.34-4.921 2.674 ± 0.942 27
Others-Primary 1.675-4.921 3.221 ± 1.145 7
Others-Secondary 1.34-4.175 2.482 ± 0.807 20
Mann-Whitney U test and ROC curve analysis of vitamin D parameters

263 African Health Sciences, Vol 24 Issue 3, September, 2024

Mann-Whitney U test and ROC curve analysis of and their ratio and found significant differences across
vitamin D parameters the categories of P < 0.0001, <0.001, and < 0.040, re-
BMI was the only normally distributed independent spectively (Table 2). From ROC curve analysis, calcitri-
variable (P < 0.001). Using the Mann Whitney U test, ol was the best indicator of fertility AUC = 0.909 ±
data for calcidiol, calcitriol and their ratios were cross 0.031 (0.714-0.9.3), using the nonparametric approach,
tabulated and identified a significant difference between at asymptotic 95% CI (P < 0.001), followed by calcidiol
cases and controls (2-sided Test asymptotic significance AUC = 0.808 ± 0.048 (0.714-0.903); P < 0.001, and the
= 0.000; Figure 1). For categorical data, we used the ratio AUC = 0.740 ± 0.065 (0.612-0.869); P < 0.001
Kruskal-Wallis test to cross-tabulate calcidiol, calcitriol (Figure 2).

Figure 1. Mann-Whitney U test for cross tabulation of calcitriol, calcidiol and their ratio for infertile (n
= 80) and fertile (n = 25) women. Significant differences noted across cases and controls for all biomarkers.

African Health Sciences, Vol 24 Issue 3, September, 2024 264

 Table 2. Kruskal Wallis Test for cross tabulation of calcitriol, calcidiol and their ratio for infertile (n = 80)
and fertile (n = 25) women, stratified for infertility. Data presented as mean ± standard deviation from the

mean (SDM), median ± interquartile range (IQR), and P values.

Fertile controls Polycystic ovary Unexplained Other factors (n = P

disease (n = 30) etiology (n = 23) 27)
Calcitriol Mean ± SDM 113.99 ± 43.19 44.95 ± 17.5 48.50 ± 15.64 67.23 ± 28.26 <0.001
Median ± IQR 119.80 ± 42.8 36.75 ± 20.4 42.40 ± 27 69.80 ± 50
Calcidiol Mean ± SDM 33.47 ± 22.07 17.31 ± 3.94 18.67 ± 2.82 24.89 ± 4.93 <0.001
Median ± IQR 25.12 ± 6.36 16.35 ± 6.4 18.20 ± 4.6 24.73 ± 9.71
Their Ratio Mean ± SDM 4.09 ± 2.02 2.62 ± 0.86 2.61 ± 0.78 2.67 ± 0.94 0.040
Median ± IQR 3.68 ± 2.49 2.36 ± 1.02 2.55 ± 0.99 2.67 ± 1.28

Figure 2. ROC curve analysis of AUC to determine ability of calcitriol, calcidiol, and their
ratio to distinguish fertile (n = 25) from infertile (n = 80) women. Calcitriol exhibited the highest
sensitivity and specificity; however calcidiol and the ratio were also predictive.

Results of the correlation analysis biomarkers exhibited a negative correlation with these
Healthy fertile control women: characteristics, although was only significant for BMI
In healthy controls, the selected test characteristics were vs. ratio. The ratio correlated negatively with calcidiol
significantly positively correlated with each other. Vit D and positively with calcitriol (Table 3).

265 African Health Sciences, Vol 24 Issue 3, September, 2024

 Table 3. Correlation of test characteristics in fertile women (n = 25). Presented data are r/P values of non-parametric spearman correction analysis

Gravidity Parity BMI Calcidiol Calcitriol Ratio

Age 0.614/0.0006 0.688/<0.0001 0.862/<0.0001 0.047/0.413 -0.192/0.179 -0.302/0.071
Gravidity 0.958/<0.0001 0.540/0.003 -0.195/0.175 -0.045/0.416 -0.060/0.387
Parity 0.586/0.001 -0.203/0.166 -0.096/0.324 -0.021/0.461
BMI 0.146/0.243 -0.111/0.299 -0.368/0.035
Calcidiol 0.092/0.332 -0.689/<0.0001
Calcitriol 0.540/0.0039

Infertile women: calcitriol was negatively associated with the character-

Infertile patient characteristics were significantly posi- istics, achieving significance vs. age and duration. The
tively correlated with each other, with BMI significantly ratio exhibited a positive (nonsignificant) correlation
correlated with all characteristics. Calcidiol showed only with BMI, and negative association with the other char-
significant positive correlation with gravidity, while BMI acteristics. The ratio was positively correlated with cal-
was nonsignificantly negatively correlated. Conversely, cidiol and calcitriol, with vs. calcitriol being significant
(Table 4).

Table 4. Correlation between characteristics and Vit D biomarkers in infertile cases (n = 80). Data shown are r/P values of non-parametric Spearman correction analysis.

Gravidity Parity BMI Duration Calcidiol Calcitriol Ratio

Age 0.579/<0.0001 0.701/<0.0001 -0.251/0.0122 0.776/<0.0001 0.090/0.215 -0.194/0.042 -0.376/0.0003
Gravidity 0.828/<0.0001 -0.322/0.002 0.311/0.003 0.252/0.012 -0.033/0.385 -0.224/0.023
Parity -0.352/0.0007 0.441/<0.0001 0.153/0.087 -0.129/0.126 -0.311/0.003
BMI -0.235/0.016 -0.165/0.072 -0.029/0.400 0.141/0.106
Duration 0.0151/0.447 -0.188/0.047 -0.304/0.003
Calcidiol 0.634/<0.0001 0.0124/0.457
Calcitriol 0.750/<0.0001

Infertile women with PCOS: vs. age, parity and BMI; BMI vs. age; parity vs. age and
Overall, fewer significant correlations were identified. gravidity. For biomarkers, calcitriol was significantly
Significant positively correlated data included, duration positively associated with only calcidiol and the ratio.
(Table 5).

Table 5. PCOS-associated infertility patients (n = 30) and vitamin D biomarkers. Correlations are shown as r/P values of non-parametric Spearman correction analysis.

Gravidity Parity BMI Duration Calcidiol Calcitriol Ratio

Age 0.140/0.230 0.454/0.006 0.576/0.0004 0.877/<0.0001 0.040/0.416 -0.001/0.499 -0.180/0.171
Gravidity 0.727/<0.0001 -0.058/0.381 0.194/0.153 0.082/0.333 -0.069/0.360 -0.072/0.353
Parity 0.181/0.169 0.474/0.004 0.016/0.468 -0.129/0.248 -0.160/0.199
BMI 0.443/0.007 -0.055/0.387 0.001/0.498 -0.100/0.300
Duration 0.082/0.333 0.023/0.453 -0.165/0.191
Calcidiol 0.635/<0.0001 -0.112/0.2786
Calcitriol 0.615/0.0002

Infertile women with PCOS of primary causes (n = 21) markers only calcitriol was significantly correlated with
Significant positive correlations were observed for du- calcidiol (r = 0.820; P = 0.005).
ration vs. age (r = 0.850; P < 0.0001), and vs. BMI (r = Calcitriol
0.443; P = 0.022); BMI vs. age (r = 0.593; P = 0.0023). 0.615/0.0002
For biomarkers, calcitriol was significantly positively as-
sociated with only calcidiol and the ratio (r = 0.518; P = Infertile women with Unexplained Etiologies:
0.008, and r = 0.638; P = 0.009, respectively). Significant positive correlations were observed for age
Infertile women with PCOS of secondary causes (n = 9) vs. gravidity, parity, and duration; gravidity vs. pari-
Significant positive correlations were observed for du- ty, and BMI; and parity vs. BMI. For biomarkers, cal-
ration vs. age (r = 0.836; P = 0.004), parity (r = 0.844; cidiol was significantly positive vs. calcitriol and BMI.
P = 0.016), and BMI (r = 0.647; P = 0.033). For bio- Calcitriol significantly correlated negatively vs. age and
duration, and positively vs. ratio. Ratio showed similar
correlations vs. age and duration (Table 6).

African Health Sciences, Vol 24 Issue 3, September, 2024 266

 Table 6. Infertility of unexplained etiology (n = 23) and vitamin D biomarkers. Correlations are shown as r/P
values of non-parametric Spearman correction analysis.

Gravidity Parity BMI Duration Calcidiol Calcitriol Ratio

Age 0.620/0.0008 0.612/0.0009 0.058/0.397 0.779/<0.0001 0.036/0.435 -0.403/0.028 -0.415/0.025
Gravidity 0.875/<0.0001 0.450/0.016 0.169/0.220 0.130/0.277 -0.060/0.393 -0.150/0.247
Parity 0.447/0.016 0.164/0.227 0.116/0.299 -0.117/0.298 -0.181/0.205
BMI -0.231/0.145 0.535/0.004 0.167/0.223 -0.115/0.300
Duration -0.160/0.233 -0.566/0.0024 -0.471/0.012
Calcidiol 0.459/0.014 -0.171/0.218
Calcitriol 0.770/<0.0001

Infertile women with primary Unexplained Etiologies (n = 7) 0.0023). While calcitriol correlated positively with the
Significant positive correlation was observed only for ratio (r = 0.849; P < 0.0001).
duration vs. age (r = 0.873; P < 0.010), and calcidiol vs.
BMI (r = 0.883; P = 0.008). Infertile women with Other Etiologies:
Infertile women with secondary Unexplained Etiologies (n = 16) Significant positive correlations were noted for age
Significant positive correlations were observed for age vs. gravidity, parity and duration; gravidity vs. parity,
vs. duration (r = 0.801; P = 0.0002), gravidity vs. parity and BMI; and parity vs. BMI. For biomarkers, signifi-
(r = 0.735; P = 0.0008). For biomarkers, significant neg- cant negative correlations were noted for calcidiol vs.
ative correlations were noted for calcitriol and the ratio duration; calcitriol vs. all characteristic, except with
vs. age (r = -0.640; P = 0.0045; r = -0.771; P = 0.0004) BMI which was positive. Inter-biomarker relationships
and duration (r = -0.638; P = 0.0046; r = -0.683; P = showed positive correlation for calcidiol vs. calcitriol,
and calcitriol vs. ratio. The ratio showed negative cor-
relation with age, gravidity and parity, but positive vs.
BMI (Table 7).

Table 7. Other etiology infertility cases (n = 27) correlation with vitamin D biomarkers, described as r/P values of
non-parametric Spearman correction analysis.
Gravidity Parity BMI Duration Calcidiol Calcitriol Ratio
Age 0.685/<0.0001 0.812/<0.0001 -0.441/0.0106 0.665/<0.0001 -0.24/0.114 -0.599/0.0005 -0.595/0.0005
Gravidity 0.788/<0.0001 -0.378/<0.026 0.304/0.062 -0.061/0.381 -0.503/<0.004 -0.519/<0.003
Parity -0.551/<0.002 0.471/<0.007 -0.123/0.271 -0.600/0.0005 -0.631/0.0002
BMI -0.209/0.148 0.130/0.259 0.628/0.0002 0.692/<0.0001
Duration -0.363/0.031 -0.389/0.022 -0.288/0.073
Calcidiol 0.599/0.0005 0.190/0.172
Calcitriol 0.871/<0.0001

Infertile women with primary Other Etiologies (n = 7) and parity vs. BMI (r = -0.603; P < 0.003). For biomark-
Significant positive correlations were observed only for ers and characteristics, significant negative correlations
BMI vs. calcitriol and vs. ratio (r = 0.786; P = 0.024 for were noted for calcidiol vs. duration (r = -0.462; P =
both), and calcitriol and ratio (r = 0.893; P = 0.006). 0.020); calcitriol vs. all (except BMI) (r = -0.598; P =
Infertile women with secondary Other Etiologies (n = 20) 0.0006 with age, r = -0.560; P = 0.005 with gravidity, r
Significant positive correlations were observed for age = -0.693; P = 0.0004 with parity, and r = -0.588; P =
vs. gravidity and BMI (r = 0.845; P < 0.0001; r = 0.731; 0.003 with duration); ratio vs. gravidity (r = -0.616; P <
P = 0.030, respectively); gravidity vs. parity and dura- 0.002), parity (r = -0.740; P < 0.0001), and duration (r =
tion (r = 0.719; P = 0.0002; r = 0.476; P = 0.017, re- -0.434; P = 0.028). Positive correlations were observed
spectively); parity vs. duration (r = 0.627; P < 0.002). for calcitriol vs. BMI (r = 0.529; P = 0.008); ratio vs. age
Negative correlations were noted for age vs. parity and (r = 0.845; P < 0.003) and BMI (r = 0.617; P < 0.002).
duration (r = -0.426; P < 0.0001; r = -0.382; P = 0.0001, Inter-biomarker analysis revealed positive associations
respectively); gravidity vs. BMI (r = -0.407; P = 0.037); for calcidiol vs. calcitriol (r = 0.599; P = 0.0005); cal-
citriol vs. ratio (r = 0.871; P < 0.0001).

267 African Health Sciences, Vol 24 Issue 3, September, 2024

Discussion nonsignificantly different from healthy controls 49,59.
While our study is not unique, it offers new insight into Calcidiol exhibits significant seasonal variation while
infertility and association with vitamin D in our area, as calcitriol does not 60-62.
Vit D levels in reproductive-aged Aljouf Saudi women
have not been published. It also shows the applicability The pathogenesis of endometriosis may be associated
of the biomarkers. We show that Vit D is insufficient/ with high levels of Vit D resulting in impaired elimina-
deficient in 82% of healthy participants, while cases tion of endometrial cells in the peritoneal cavity 63. Our
expressed a rate of 90 – 100%. We found that calcitri- findings were contrary to these, we noted low calcidiol
ol was most sensitive in differentiating between these and consistent and lower levels of calcitriol in the oth-
groups. er etiology patients, and calcidiol correlated negative-
ly with only the duration, while calcitriol and the ratio
We describe the situation where calcidiol is the prohor- were negatively correlated with all characteristics except
mone and calcitriol the active form, in which calcitri- BMI. This may be related to a “change point issue”,
ol is the more precise discriminant factor 26. Similar to wherein serum and follicular fluid levels of AMH are
the current literature, including meta-analyses 6,25,26,43-47,
negatively correlated with Vit D to ~30 ng/mL and
we found wide-spread Vit D deficiency/insufficiency thereafter show a positive insignificant relationship with
among healthy controls, and significantly worse sce- tubal factor infertility patients. Further, significant neg-
narios in infertile women. While the three biomarkers ative seasonal variations were noted for both 61, which
exhibited significant difference between controls and may be indicative of the critical nature of lower rather
cases, we found calcitriol > ratio > calcidiol for dis- than higher Vit D levels. The impact of Vit D in IVF
crimination. For individual disease categories, we found and associated parameters including resulting preg-
biomarker performance was worse for PCOS, then un- nancy is controversial 28. However, investigating IVF
explained- and other etiologies. On the whole, a posi- outcomes in an egg-donor model, which separates the
tive correlation was observed for healthy control char- effect of Vit D on eggs and endometrium, data indi-
acteristics, while only the ratio was negatively correlated cates that Vit D effect may occur via the endometrium.
with BMI. Similarly for infertile patients, positive cor- Deficiency and insufficiency levels of Vit D exhibited
relations were observed for all with the exception of similar negative consequences 64.
BMI which was negatively correlated with the three bio-
markers and calcidiol vs gravidity which was positively A study of Iranian women reported similar data to
correlated. our PCOS cases, whereby the lowest Vit D levels were
noted but did not exhibit any correlation with patient
All of the other etiology infertility patients (cervical, characteristics (or between the biomarkers). Vit D lev-
uterine, tubal or peritoneal origin or anovulation) ex- els in serum and follicular fluid were lower in PCOS
hibited low Vit D levels. Low Vit D is significantly cor- patients compared to controls and correlated negatively
related with endometriosis risk and severity, as well as with BMI. Vit D controls insulin expression and sen-
a number of other gynecological conditions including sitivity, and in turn α1-hydoxylase expression and cal-
leiomyoma, uterine myoma, and dysmenorrhea 30,34,48-54. citriol level 65. Low levels of Vit D were linked to both
For severe cases of endometriosis, calcidiol levels were PCOS and associated risk factors such as obesity and
half of those in healthy controls or in women with less insulin resistance 31,37,45,66. In the Iranian study, lower Vit
severe endometriosis. Contrary to our findings, calcidi- D was noted among PCOS-infertile women, specifical-
ol was positively associated with gravidity and parity 55, ly those who were obese, than controls 67. Ovulation is
while we noted negative associations between both of significantly improved in PCOS cases where low Vit
calcitriol and ratio vs. gravidity and parity. Some in vi- D is treated 68,69, and cases with higher serum and fol-
tro research noted an effect of calcitriol on reduction licular levels had significantly increased IVF pregnan-
of inflammatory markers and factors associated with cy rate than others 70. Later in the reproductive period,
endometrial stroma cells 49, others purported that the Vit D and ovarian reserve are significantly positively
data is insufficient to definitively indicate a Vit D-en- correlated 71. Ovarian stimulation outcome in PCOS is
dometriosis relationship 31,56,57. However, we do know significantly associated with Vit D deficiency 72. PCOS
that estradiol and progesterone are inversely correlated patients show a negative correlation with Vit D vs tes-
with calcidiol levels 58. Endometriosis patients showed tosterone, SHBG, free androgen index, DHEAS levels,
significantly higher calcidiol levels but calcitriol was and LH/FSH ratio, which is worse in obese patients73,74.

African Health Sciences, Vol 24 Issue 3, September, 2024 268

However, pregnancy outcome was not significantly as- The study was limited by the small case numbers, espe-
sociated with Vit D levels 75. Post-confounder normal- cially in the subgroups, a year round sample collection
ization, PCOS patients showed association with higher period could have allowed seasonal variation, and, since
Vit D levels; however, this did not lead to improved we did not estimate reproductive hormones levels, we
metabolic function 62. Serum Vit D was not associat- did not analyze their correlations with Vit D biomark-
ed with PCOS or AMH 76, and there was no significant ers.
correlation between conceiving or miscarriage risk with
pre-pregnancy Vit D levels 3. These studies may be con- Conclusion
founded by the fact that both patients and controls ex- Over 90% of the infertile women in the study exhibited
hibit insufficient/deficient levels of Vit D62, or that a Vit D deficit as determined by the three biomarkers.
low cutoff level (20 ng/mL) is used 6,76-78. This is a crit- Lowest levels were reported for PCOS and unexplained
ical point as the recommended reproductive thresholds reasons, and then other etiologies. Calcitriol was the
for serum calcidiol is higher (≥ 45 ng/mL) than for the optimal predictor of infertility, and the ratio was dis-
non-pregnant population 64,79. criminatory for healthy vs. infertile participants. Given
the seasonal nature of calcidiol, we recommend the use
Our study found a significant reduction in Vit D in of calcitriol and their ratio as the more precise mark-
unexplained/known etiology cases, with positive cor- ers. It is vital that standardized no-skeletal Vit D levels
relation noted between calcidiol vs. BMI, and negative are fully characterized in the female reproductive-aged
between calcitriol/ratio vs. duration and age. Contrary populations. Our data corroborate a role for Vit D in
to this, there was no noted association between Vit D unexplained infertility, and indicate a therapeutic role
deficiency and ovarian stimulation outcome 72. How- for Vit D supplementation.
ever, in one African study, incorporating all etiologies,
with a major unexplained, infertile patients did not ex- Declaration of competing interest
hibit lower Vit D than the controls 80. Nor was correla- The authors declared no confect of interests.
tion observed with Vit D and follicular count/AMH
77,78
. While follicular and serum Vit D levels are putative Funding
markers of egg and embryo quality, and higher levels The authors extend their appreciation to the Deanship
result in higher pregnancy chance, a positive association of Scientific Research at Jouf University, Sakaka, Saudi
has only been observed and no significant correlations Arabia for funding this work through research grant no
reported 81,82. (DSR2020-04-472).
Given the seasonal variation in calcidiol, we assessed
the efficacy of calcidiol, calcitriol and their ratio as pre- Acknowledgement
dictive markers for infertility vs. healthy controls. Cal- We highly appreciate the generous support from Dr
cidiol was inferior to calcitriol in predicting infertility Atef Abdelmageed (Head, Obstetrics and Gynecology
and negatively correlated with the clinical characteris- Department) and Mr. Mohammed Fahman (Manager
tics of all studies etiologies. This study provides insight of the Laboratory, Maternity and Children Hospital,
into the variation of these three makers as they relate Sakaka, Aljouf, Saudi Arabia) for facilitating the collec-
to infertility. In summary, the lowest rate of Vit D defi- tion of the samples and profiling our patients. We deep-
ciency was in PCOS cases and is similar to unexplained ly thank Dr. Ashok Thirunavukkarasu (Department of
cases, while other etiology preformed marginally bet- Family and Community Medicine, College of Medicine,
ter. Calcidiol showed negative correlation with BMI in Jouf Univ., Sakaka, Saudi Arabia) for double checking
patients, while calcitriol and ratio exhibited a positive on our statistical analysis.
correlation with BMI of healthy controls. All biomark-
ers were negatively correlated with disease duration in Conflict of interest
all patients. Consequently, it is reasonable to predict an None to declare.
association for Vit D deficiency and unexplained fac-
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