A Cross-Sectional Evaluation of Vitamin D Status and Ovarian Reserve Markers in Subfertile Women: A Single-Center Experience From Pakistan

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Abstract

Objectives

This study was conceived with the objective of assessing the correlation between ovarian reserve markers
and vitamin D deficiency (VDD) in a selected group of Pakistani subfertile women presenting at a
specialized subfertility treatment centre. The measurements of antral follicle count (AFC), serum antiMüllerian hormone (AMH), serum follicle-stimulating hormone (FSH), and serum vitamin D (VD) levels were
the main tools used for the ass djessment of ovarian reserve.

Materials and methods

All female patients aged 18 to 45 years presenting with primary and/or secondary subfertility at the
Australian Concept Medical Centre in Karachi, Pakistan from August 2016 to July 2021 were included in the
study. The data of all eligible patients were recorded in the pre-defined Performa designed for this study.
The Kruskal-Wallis test was applied to report the distribution of the data. The correlation between the
categorical variables (25-hydroxyvitamin D [25-OHD] levels with AFC and AMH) was assessed using the
chi-square test and Spearman correlation. The comparison was based on vitamin D levels grouped into
three categories: deficiency (<20 ng/ml), insufficiency (21-29 ng/ml), and sufficiency (>30 ng/ml).

Results

One hundred ninety-nine cases were evaluated for AFC and hormone analysis. The mean age and BMI
were 32.87±5.49 years and 28.27±4.97 kg/m
2
. VDD was noted in 127 (68.4%) cases. No significant
difference was noted across BMI, age, duration of subfertility, AMH, and FSH across the VD categories.
Moreover, a poor correlation was noted between VD, AMH and FSH on the scatter plot, between VD and
FSH (r = −0.003, p = 0.966) and between VD and AMH (r = −0.068, p = 0.342), respectively.

Conclusions

This study showed a high frequency of VDD in Pakistani subfertility women, from a specialized subfertility
center in the largest metropolis in the country. However, a statistically significant association was not found
between the markers of ovarian reserve and VD, showing no ethnic differences in the native Pakistani
population. Hence, VD supplementation is unlikely to have an impact on correcting the ovarian reserve
status in subfertility women in Pakistan. However, this is a potential area of interest, and evaluation of other
indices of reproduction/ovarian reserve and the effect of confounders is required to test this hypothesis
longitudinally

Categories: Endocrinology/Diabetes/Metabolism, Obstetrics/Gynecology, Pathology
Keywords: anti-Mullerian hormone, Pakistan, subfertility, ovarian reserve, vitamin d
Introduction

25-Hydroxyvitamin D (25-OHD) is a steroid hormone, rendering an upfront role in regulating the calcium and
phosphorous levels in the body. Vitamin D (VD) exists in five different forms, i.e., D1, D2 (ergocalciferol), D3
(cholecalciferol), D4 (dihydroergocalciferol), and D5 (sit calciferol). In humans, D2 and D3 are more
prevalent [1]. Vitamin D deficiency (VDD) is highly prevalent in South Asia, especially in children and
pregnant women [2]. The causes of this include limited exposure to sunlight, insufficient levels of VD in the
diet, and a higher prevalence of naturally occurring dark skin, which requires a relatively long exposure time
for VD synthesis [2,3].
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Open Access Original
Article DOI: 10.7759/cureus.21757
How to cite this article
Ahmed S, Siddiqui A, Qureshi A, et al. (January 31, 2022) A Cross-Sectional Evaluation of Vitamin D Status and Ovarian Reserve Markers in
Subfertile Women: A Single-Center Experience From Pakistan. Cureus 14(1): e21757. DOI 10.7759/cureus.21757

Introduction

25-Hydroxyvitamin D (25-OHD) is a steroid hormone, rendering an upfront role in regulating the calcium and
phosphorous levels in the body. Vitamin D (VD) exists in five different forms, i.e., D1, D2 (ergocalciferol), D3
(cholecalciferol), D4 (dihydroergocalciferol), and D5 (sitocalciferol). In humans, D2 and D3 are more
prevalent [1]. Vitamin D deficiency (VDD) is highly prevalent in South Asia, especially in children and
pregnant women [2]. The causes of this include limited exposure to sunlight, insufficient levels of VD in the
diet, and a higher prevalence of naturally occurring dark skin, which requires a relatively long exposure time
for VD synthesis [2,3].

VDD is common in women of childbearing age and is often linked to adverse maternal and neonatal
outcomes [4]. According to Hogan et al., around 14 million pregnant women are subjected to acute maternal
complications globally each year [5]. From a pathophysiological perspective, VD maintains the homeostasis
of calcium and phosphate by regulating calcium absorption from the intestine and promoting skeletal
utilization. Moreover, VDD is often associated with higher rates of small for gestational age (SGA) and low
birth weight neonates and certain subsequent complications that can last into adulthood [6].

Subfertility is estimated to be prevalent at a rate of 12-14%, with VDD considered to be one of the causative
factors [7]. Antral follicle count (AFC), anti-Mullerian hormone (AMH), and follicle-stimulating hormone
(FSH) serve as established markers of ovarian reserves. However, AFC values can be subject to possible
inter-observer or intra-observer variation inherent to sonographic measurements. Therefore, the results
might be biased and inaccurately estimated.

On the other hand, AMH, a protein hormone secreted by granulosa cells of the ovaries which regulates
early follicle development, is considered a better marker for ovarian reserve due to its standardization and
convenience of testing [8]. It is expressed from the onset of puberty until menopause; however, it highly
varies in each individual for unidentified reasons. To date, AMH has been found to be unaffected by the
stage of the ovarian cycle, unlike FSH [9].

The AMH receptor-II gene promoter is linked with VD triggered stimulation. In its active form, VD can upregulate AMH production. Hence, VD serves as aregulator for AMH concentrations in the blood [10].

Several studies [11,12] have advocated the impact of race and ethnicity on the evaluation of VDD and
subfertility. Even though VDD is highly prevalent in Pakistan, there exists a substantial gap in the scientific
literature regarding its significance in the female subfertile cohort. Longitudinal studies are required in
subfertile women to elucidate the association between VDD and subfertility further. To address gaps in local
data, this study was conducted to explore the association between VDD and ovarian reserve via
measurements of AFC, AMH, FSH, and 25-OHD in Pakistani women.

Results

A total of 199 cases fulfilled the predefined inclusion and exclusion criteria. VDD was noted in 127 (68.4%),
insufficiency in 39 (19.6%), and sufficiency in 33 (16.6%) cases. The mean age and BMI of the group were
32.87±5.49 years and 28.27±4.97 kg/m
2
respectively. No significant difference was noted across the BMI,
age, duration of subfertility, AMH, and FSH across the 25-OHD categories as shown in Table 1.

Discussion

Our results illustrate that the majority of the study participants were in the deficiency zone (68.4%). This, in
accordance with published literature, demonstrates the high frequency of VDD in Pakistani women in the
reproductive age group [15]. A study from Pakistan demonstrated that despite being a year-round sunny
environment, VDD has been reported to be alarmingly high among women. A local study group has
recorded that more than 90% of the pre-menopausal women had VDD with values lower than 20 ng/ml [16].

Similarly, another cross-sectional study from Karachi by Sheikh et al. showed a median concentration of VD
as low as 18.8 ng/ml [17]. VDD in Pakistan has been associated with a lack of sun exposure, especially for
women, due to religious obligations, poor dietary intake, and environmental factors, especially pollution [18].
With the growing frequency of subfertility in the metropolis, VDD as a potential contributor can be the
missing link in the puzzle. However, a thorough literature review revealed substantial gaps in the literature.
Therefore, this study was planned to collect baseline data from a specialized fertility treatment center and
assess the relationship between VDD and indicators of ovarian reserve in subfertile women. The AMH
levels undertaken in 199 cases correlated well with the AFC measurements.

The regression analysis of the study cohort revealed a poor correlation between subgroups of ovarian
reserve biomarkers like AMH and/or AFC and different subgroups of VDD. Moreover, the evaluation of
different ethnic groups in Pakistan in relation to VDD was also not able to show any significant correlation.
A study by Alavi et al. evaluated 305 subfertile women, referred for in vitro fertilization, showed almost
similar results, even after adjusting for baseline factors, authenticating our study findings and consistent
with global literature for our part of the world [19]. At this point, regardless of fertility status, finding the VDD
so prevalent in our cohort, the authors also felt, on prima facie, that there may be a need to relook and
revise the normal reference limit for VD levels among Pakistani women.

The findings can postulate that VD supplementation is unlikely to improve AMH production. As proposed by
various molecular characterization studies, VD receptor polymorphism did not lead to subfertility [20-21].
Hence, labelling VDD as a cause of subfertility is unjustifiable. There were certain limitations to this study.
First, various environmental and biological factors contributing to subfertility were not considered.

Furthermore, only the ovarian reserve markers were evaluated, while the status of other biochemical
markers, like thyroid profile, serum prolactin or serum insulin, and complete hormonal status were not
considered. Additionally, the impact of seasonal changes on VD levels was not accounted for. Moreover, the
retrospective nature of the study and the small sample size may be factors limiting the power of the study.
Hence, there is a likelihood that a significant correlation may arise with a larger sample size, emphasizing
the need for prospective large-scale multi-center studies.

Conclusions

This study showed a high frequency of VDD in Pakistani subfertile women, from a specialized subfertility
center in the largest metropolis in the country. However, a statistically significant association was not found
between the markers of ovarian reserve and VD, showing no ethnic differences in the native Pakistani
population. Hence, VD supplementation is unlikely to have an impact on correcting the ovarian reserve
status in subfertile women in Pakistan. However, this is a potential area of interest, and evaluation of other
indices of reproduction/ovarian reserve and the effect of confounders is required to test this hypothesis
longitudinally.

Additional Information

Disclosures

Human subjects: Consent was obtained or waived by all participants in this study. Institutional Ethical
Review Committee of the Australian Concept Medical Centre issued approval ACIMC-UI-07-2021. The
study was approved by the institutional ethical review committee of the Australian Concept Medical Centre
(ACIMC-UI-07-2021). The need for informed consent was waived off as no patient intervention was
undertaken and all information was kept anonymized.

Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.
Conflicts of interest: In compliance with the ICMJE uniform
disclosure form, all authors declare the following: Payment/services info: All authors have declared that no
financial support was received from any organization for the submitted work. Financial relationships: All
authors have declared that they have no financial relationships at present or within the previous three years
with any organizations that might have an interest in the submitted work. Other relationships: All authors
have declared that there are no other relationships or activities that could appear to have influenced the
submitted work.

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