This document presents a thesis protocol investigating vitamin D levels between fertile and infertile women. It will be a prospective comparative study conducted at Bokaro General Hospital, Jharkhand, India. The study aims to assess and compare serum 25-hydroxyvitamin D levels in 60 infertile women and 60 fertile women between ages 21-40 years. Exclusion criteria include women with medical illnesses or using medications affecting vitamin D. Blood samples will be collected and 25-hydroxyvitamin D levels measured using ELISA to classify vitamin D status. Results will be statistically analyzed to compare vitamin D deficiency between the two groups and assess any associations with factors. The study aims to help evaluate prevalence of vitamin D deficiency in infertile women and potential benefits of

1. THESIS PROTOCOL FOR THE DEGREE OF DIPLOMATE OF
NATIONAL BOARD IN OBSTETRICS & GYNAECOLOGY
SESSION: 2021-2024
TOPIC OF THE STUDY
“A COMPARATIVE STUDY OF VITAMIN D LEVELS BETWEEN
FERTILE AND INFERTILE WOMEN ”
BY
DR. JUHI SHARMA
DNB Resident (PRIMARY),
DEPARTMENT OF OBSTETRICS & GYNAECOLOGY,
BOKARO GENERAL HOSPITAL, BOKARO

2. GUIDE
DR. SHAMA PARWEEN (DGO, DNB)
Sr. Consultant
DEPARTMENT OF OBSTETRICS & GYNAECOLOGY,
BOKARO GENERAL HOSPITAL, BOKARO
CO-GUIDE
DR. FARHAT MAZHARI
Consultant
DEPARTMENT OF OBSTETRICS & GYNAECOLOGY,
BOKARO GENERAL HOSPITAL, BOKARO
THESIS PROTOCOL INVESTIGATING TEAM
HEAD OF DEPARTMENT
DR. ASHA KUMARI
ADDL. CMO (M&HS) & HOD
DEPARTMENT OF OBSTETRICS & GYNAECOLOGY,
BOKARO GENERAL HOSPITAL, BOKARO
HEAD OF INSTITUTION
DR. PANKAJ SHARMA,
CHIEF MEDICAL OFFICER (M & HS)
BOKARO GENERAL HOSPITAL,
BOKARO STEEL CITY

3. INTRODUCTION
• The role of vitamin D in bone health and calcium homeostasis has been long
recognized. However, in the past decade, the focus is gradually shifting to the non-
skeletal benefits of vitamin D. There is increasing evidence that in addition to sex
steroid hormones, the classic regulators of human reproduction, vitamin D also plays a
role in modulating the reproductive processes in women and men.1
• Vitamin D is a steroid hormone, mainly produced in the skin after sunlight exposure.
Diet and dietary supplements constitute alternative sources of vitamin D for humans.2
There are 2 distinct forms of vitamin D - ergocalciferol (D₂) and cholecalciferol (D₃).
Cholecalciferol is formed in the human skin from 7- dehydrocholesterol (7DHC). UV
radiation converts 7-DHC to previtamin D₃ which is rapidly converted to D₃. Green
plants, mushroom, fish fat and Cod liver oil are rich sources of ergocalciferol.3,4
• Vitamin D₃ is provided either by UV radiation of the skin or from diet and is biologically
inactive and requires hydroxylation in the liver and kidneys to produce its active form-
1,25,dihydroxy vitamin D or calcitriol.2 Vitamin D status of the body is best indicated by
the circulating levels of 25(OH) vitamin D due to its longer half-life and higher serum
concentration as compared to 1, 25-Dihydroxycholecalciferol.2 Calcitriol acts through
binding to specific nuclear Vitamin D receptor, which acts in concert with the retinoid X
receptor (RXR), forming a heterodimer.5 The VDR-RXR heterodimer binds to vitamin D
responsive elements (VDRE) located in the promoter region of the target genes, thus
regulating transcription.6

4. • Although the main role of vitamin D is considered to be the absorption of calcium
and phosphorus from the gut, the wide distribution of VDR in almost all human
tissues and the fact that 3% of the human genome is regulated by the vitamin D
endocrine system points to extra skeletal role of vitamin D in various systems and
organs, reproduction being the very important one amongst them.2,7
• Calcitriol is produced by the decidua in response to IL-B secreted by the
blastocyst. Calcitriol regulates the Decidual expression of the genes involved in
embryo implantation. The presence of blastocyst up regulates the production of
the active form of vitamin D in the endometrium.8 A study also found that
women with higher 25(OH) vitamin D levels in the serum and follicular fluid were
significantly more likely to achieve pregnancy as compared to women with lower
levels of vitamin D.⁹
• Infertility is defined as failure to conceive for more than 1 year and affects about
48.5 million couples worldwide with significant psychological, medical and
economic consequences.¹⁰ Prevalence of vitamin D insufficiency has doubled from
1994 to 2004.¹¹ All these facts point towards a crucial role of vitamin D in
reproduction.

5. REVIEW OF LITERATURE
• Saran S et al (2020) conducted a study to compare the levels of vitamin D in infertile and fertile
women. On 150 women on infertility and same number of control at Government Medical
College, Badaun and Magadh Maternity and infertility clinic, Badaun. They found that significant
difference in serum vitamin D levels in fertile and infertile women could not be established.12
• Kokanali D et al (2019) conducted a retrospective case-control study in Zekai Tahir Burak
Womenʼs Health Education and Research Hospital, in Ankara, the capital city of Turkey over a 2
year-period. 274 infertile and 111 fertile women with polycystic ovary syndrome were included in
this retrospective study. Infertile and fertile groups were matched by age, body mass index and
homeostasis model assessment of insulin resistance. Anthropometric, clinical and laboratory
characteristics of the women were recorded. Serum 25(OH)D 3 levels were used to assess serum
vitamin D levels. They found No significant differences between groups in terms of
anthropometric, clinical and laboratory features except for serum 25(OH)D 3 levels and the
incidence of vitamin D deficiency. They concluded that Serum vitamin D levels are lower in
infertile women with polycystic ovary syndrome compared to fertile women.13
• Abodunrin ON et al (2018) conducted a comparative cross-sectional study to assess the
prevalence of vitamin D deficiency and insufficiency among women with infertility compared to
fertile women, at the Lagos University Teaching Hospital (LUTH). There was a statistically
significant difference in the mean ages of infertile (35.4+5.5 years) and fertile (33.5 + 4.8years)
women, p=0.008. Women with infertility had a significantly lower median serum vitamin D
concentration compared to fertile women (37.6 ng/mL versus 51.9ng/mL; p=0.001). A
significantly higher proportion of women with vitamin D deficiency and insufficiency were
infertile, compared to fertile women (20.0% versus 10.0%; p=0.048). However, there was no
identifiable association in this study between multiple variables and vitamin D status. They
concluded that Vitamin D deficiency and insufficiency are more prevalent amongst women with
infertility.14

6. • Fung JL et al (2017) conducted a prospective cohort study to evaluate the role of vitamin D intake
and serum levels on conception of clinical pregnancy and live birth. Clinical pregnancy and live
birth were compared between those who did or did not meet the vitamin D estimated average
requirement (EAR) intake (10 μg/d) and with serum 25-hydroxyvitamin D (25(OH)D) considered at
risk for inadequacy or deficiency (<50 nmol/L) or sufficient (≥50 nmol/L). They found that among
132 women, 37.1% did not meet the vitamin D EAR and 13.9% had serum levels at risk for
inadequacy or deficiency. Clinical pregnancies were significantly higher among women who met
the vitamin D EAR (67.5% vs. 49.0%) and with sufficient serum 25(OH)D (64.3% vs. 38.9%)
compared with those who did not. Live births were higher among those who met the vitamin D
EAR (59.0% vs. 40.0%). The adjusted odds ratio (AOR) of conceiving a clinical pregnancy was
significantly higher among those who met the EAR (AOR = 2.26; 95% confidence interval [CI],
1.05–4.86) and had sufficient serum 25(OH)D (AOR = 3.37; 95% CI, 1.06–10.70). They concluded
that women with vitamin D intake below EAR and serum 25(OH)D levels at risk for inadequacy or
deficiency may be less likely to conceive and might benefit from increased vitamin D intake to
achieve adequacy.15
• Lata I et al (2017) conducted a prospective study of vitamin D levels in infertile females and to
know the correlation of vitamin D deficiency (VDD) with serum AMH in infertile females compare
to fertile females in Department of Maternal and Reproductive Health, Sanjay Gandhi
Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India. The study included 35
patients with Vitamin D deficiency as cases and 35 fertile normal females as control. In both
groups, correlation of VDD with AMH was studied. They found that The VDD was present in
64.28% of infertile females. In vitamin D deficient cases, the mean for vitamin D was 6.18 ± 2.09
and AMH was 1.94 ± 1.30. In vitamin D deficient controls, the mean for vitamin D was 4.85 ± 3.02
and AMH was 3.47 ± 2.59. On comparison, the vitamin D levels were lower in fertile than infertile
females, which was significant (P = 0.04), and AMH levels were lower in cases than control group
(P = 0.003). They concluded that the VDD was present in 64.28% of infertile females. No
significant correlation was found in between VDD and AMH levels in both the groups.16

7. • Behbahani BM et al (2016) conducted a study on vitamin D condition on infertile women during
their reproductive ages, between 20 and 40 in Shiraz and determining their relation with
demographic and social characteristics. 180 infertile women admitted in Qadir Mother and Child
Hospital were selected through simple random sampling and studied variables were tested in
them. Also, blood sample was taken from them and their serum 25-hydroxyvitamin D levels were
measured through ELISA. In this research, the normal level for vitamin D was appointed to be
greater than or equal to 30 ng/ml and the serum level lower than 30 ng/ml was considered to be
the deficit level. They suggested that among the 180 studied women, 95 (52.79%) of them had a
vitamin D deficiency and 85 (47.22%) of them had normal level of vitamin D. The lowest level of
vitamin D in studied women was 21.4 ng/ml and the highest level of that was 43.80 ng/ml. There
was no significant difference observed between vitamin D condition and indices of age, education
background, birthplace, job, physical exercise and income. Also, there was no significant
difference found between vitamin D condition and weight, height and body mass index. They
concluded that more than half of the infertile women had a vitamin deficiency and this could be
considered as a problem for their fertility health and the deficiency is not due to the individuals’
socioeconomic situation. Hence, focusing on preventive measures and early treatment seems to
be of significance.17

8. Need for Study
• In order to assess the vitamin D status in infertile women.
• This evaluation would provide an estimate of the prevalence
of vit D deficiency in infertile women.
• Vit D deficiency can be easily corrected by its
supplementation.
• This is the very easy and cost-effective way of improving
fertility and also the pregnancy rates.
• Vitamin D supplementation doesnot show any significant side
effects.
• Vitamin D status checkup can be done in rural population
also.This investigation is accessible to them and can be
corrected by vitamin D intake.

9. AIM AND OBJECTIVES
AIM
To assess the prevalence of vitamin D deficiency and insufficiency
among women with infertility compared to fertile women
OBJECTIVES
primary objectives-
• To determine the serum concentration of 25-hydroxyvitamin D in women
with infertility
• To determine the serum concentration of 25-hydroxyvitamin D in fertile
women
secondary objectives-
• To compare proportions of low serum 25-hydroxyvitamin D levels between
women with infertility and fertile women
• To assess the factors associated with serum 25-hydroxyvitamin D status
among women with infertility and fertile women

10. MATERIAL AND METHODS
Study location : The study will be conducted on patients
attending Gynae OPD & ANC Clinic in the Department of
Obstetrics and Gynaecology at Bokaro General Hospital, Bokaro.
Study design : It is a prospective, comparative and observational
study.
Study duration : 18 Months
Study population : All the patients of reproductive age group (21-
40yrs) attending Gynae OPD with history of infertility and all the
spontaneously conceived patients attending ANC clinic on their
first booking visit in Department of obstetrics and gynaecology at
Bokaro General Hospital, Bokaro will be included in this study. It
will be ensured that the patients don’t have comorbidities like
Hypertension, Diabetes, Heart diseases during the period of study
after taking consent.

11. sample size-
In a study with research hypothesis viz.
Null hypothesis H0: m1 = m2 vs.
Alternative hypothesis Ha: m1 = m2 + d
(Where d is the difference between two means and n1 and n2 are the sample size of each group)
For: Group: Control group (vitamin D levels in fertile women)
Group: Case group (vitamin D levels in infertile women)
Such that, N = n1 + n2, the ration, r = n1 / n2
Taking the α at 5% and desired power of study as 80%
Confidence level = 95%, Confidence interval = 5%
Zα is the normal deviate at a level of significance ( Zα is 1.96 for 5% level of significance )
Z1 – β is the normal deviate at (1- β)% power with β% of type II error (0.84 at 80% power of study)
r = n1 / n2 is the ratio of sample size required for 2 groups =1
δ is standard deviation (Difference between two groups standard deviation) = 9.36 (app),
d is (Difference between two groups mean ) = 19.10 – 14.32=4.78 (app) .
Therefore,
n = {(r+1) (Zα/2 + Z1-β)2 δ2} /r d2
n = (1+1) (1.96+0.84)2 (9.36)2 /1* (19.10 – 14.32)2 = 1373.7248 / 22.85 = 60.12 ≈ 60
The total sample size required for the study 120, each group contain 60 patients.

12. Inclusion Criteria:
Group A ( cases)
. Female patients age between 21-40 years who are not able to conceive since last
12 months after regular intercourse.
. Known cases of infertility without any other medical issues like
Hypertension,Diabetes Mellitus, coronary artery disease or any other systemic
disorders.
Group B ( controls)
. (Pregnant women presenting to the OPD at first antenatal visit)
1. The patients must have been spontaneously conceived.
2. Patients who will give an informed written consent.
Exclusion criteria
• Female infertility cases with structural abnormality of reproductive organs like
uterus, fallopian tube etc. example fibroid, septate uterus, polyps.
• Patients with pre-existing chronic medical conditions of the kidneys, liver, bone
and parathyroid gland
• Patients on medications which could affect vitamin D metabolism, such as
anticonvulsants, hydroxychloroquine and prednisolone.
• Patients on vitamin D or calcium supplements
• Patients of age <21 and >40.

13. Methods
This study will be carried out at the Department of Obstetrics & Gynaecology, Bokaro
General Hospital, a tertiary health institution in Jharkhand State. Participants will be recruited from
the hospital’s Gynaecology OPD and Antenatal Clinic. General examination will be done. The
information collected will be included like socio-demographic data, past gynaecology, obstetric,
medical, drug, food, social and other relevant history. Height, weight and BMI will be measured with
standardized protocols. Subsequently, 5 ml blood will be taken from them and their serum 25-
hydroxyvitamin D levels will be measured using ELISA.
As per Endocrine Society 25-hydroxyvitamin D levels lower than equal to 20 ng/ml will
be considered to be the deficiency level, 21-29 ng/ml will be considered to be insufficiency level and
levels greater than 30 ng/ml will be considered to be normal¹⁸.

14. STATISTICALANALYSIS:
All the data would be selected randomly, tabulated, and then analyzed with appropriate statistical
tools “SPSS vs 24”. Data will be presented as mean with standard deviation or proportions as
appropriate. Mean, standard deviation and variance would be calculated and following statistical
significance tests were applied.
For Qualitative data following statistical test applied
1. “Chi – square Test” would be used for statistical significance test.
2. “Fisher’s exact test” would be used for statistical significance test.
3. Test of Significance for Difference of Proportions.
For Quantitative data following statistical test applied
4. Karl Pearson’s Correlation Coefficient and regression would be used for correlate two different
parametric data at a time
5. Student’s |t|-test will be used as the statistical tool to test for significance of observed mean
differences.
Finally the calculated value should be compared with the tabulated value at particular degree of
freedom and finds the level of significance.
A “p-value” should be considered to be non-significant if > 0.05 and significant if <0.05.
Statistical methods would be used to find the significance of homogeneity of study characteristics
between the two groups of patients. Their inference will be as follows-
P > 0.05 statistically insignificant
P < 0.05 statistically significant
P < 0.01 statistically highly insignificant
P < 0.001 statistically very highly significant

15. REFERENCES
1. Lerchbaum E, Obermayer-Pietschet B. Vitamin D and fertility: a systematic review. Eur J
Endocrinol. 2012;166(5):765-78
2. Holick M F. Vitamin D deficiency: review. N Engl J Med. 2007; 357: 266-81.
3. Holick MF, Chen TC. Vitamin D deficiency: a worldwide problem with health consequences. Am J
Clin Nutr. 2008; 87:1080s-6s
4. Holick MF. The Vitamin D deficiency pandemic and consequences for nonskeletal health:
mechanisms of action. Mol aspects Med.2008; 29(6): 361-8.
5. Rosen CJ, Adams JS, Bikle DD. The non-skeletal effects of Vitamin D: an Endocrine Society
scientific statement. Endocr Rev. 2012; 33(3): 456-92
6. Haussler MR, Jurutka PW, Mizwicki M, Norman AW. Vitamin D receptor (VDR)-mediated actions
of 1 α, 25(OH) ₂ Vitamin D₃: genomic and non-genomic mechanisms. Best Pract Res Clin
Endocrinol Metab. 2011; 25(4): 543-59
7. Bouillon R, Carmeliet G, Verlinden L. Vitamin D and human health: lessons from Vitamin D
receptor null mice. Endocr Rev. 2008; 29(6): 726-76.
8. Vigano P, Lattuada D, Mangioni S, Ermellino L, Vignali M, Caporizzo E, et al. Cycling and early
pregnant endometrium as a site of regulated expression of the Vitamin D system. J Mol
Endocrinol. 2006; 36: 415-24.

16. 9. Ozkan S, Jindal S, Greenseid K, Shu J, Zeitlian G, Hickmon C, et al. Replete Vitamin D stores predict reproductive
success following in vitro fertilization. Fertil Steril. 2010; 94: 1314-9
10. Mascarenhas MN, Flaxmann SR, Boerma T, Vanderpoel S, Stevens GA. National, regional and global trends in
infertility prevalence since 1990: a systematic analysis of 277 health surveys. PLoS Med. 2012; 9(12): e1001356
11. Looker AC, Pfeiffer CM, Lacher DA, Schleicher RL, Picciano MF, Yetley EA. Serum 25-hydroxyvitamin D status of
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12. Saran S. A comparative study of vitamin D levels between infertile and fertile women in India. Journal of
OBGYN. 2020;7(1):16-9.
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14. Abodunrin ON; Comparative Study of Serum 25-Hydroxyvitamin D Levels between women with infertility and
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Issue; Pub-2019-04-09
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16. Lata I, Tiwari S, Gupta A, Yadav S, Yadav S. To study the Vitamin D levels in infertile females and correlation of
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17. STUDY PROFORMA
Name: Age: Address: Hospital number:
Occupation: Education status: Marital status:
Religion: Presenting complaints:
GYNAECOLOGICAL HISTORY
Duration of infertility (in months): ..................................................
Type of infertility: Primary [ ] Secondary [ ]
Cause of infertility:
Hormonal [ ] Tubal [ ] Uterine [ ] Cervical [ ] unexplained [ ]
Combined male & female factors [ ]
Others, specify .........................
Specify gynaecological conditions identified, if any:
Endometriosis [ ] Fibroid [ ] PCOS [ ] Asherman's syndrome [ ]
Premature ovarian failure [ ] others, specify………………….........
MENSTRUAL HISTORY
OBSTETRIC HISTORY
Parity (no. alive) ............……………
Was the last pregnancy conceived spontaneously: Yes [ ] No [ ]
Mode of delivery: NVD [ ] CS [ ]
Are you breastfeeding: Yes [ ] No [ ]
MEDICAL & DRUG HISTORY
Any chronic medical conditions: Kidneys Yes[ ] No[ ] Liver Yes[ ] No[ ]
Parathyroid gland Yes[ ] No[ ] Bone Yes[ ] No[ ] Others, specify ................................
Any routine use of prescribed medications: Yes [ ] No[ ]
If Yes, specify: ......................................................
Any use of vitamin D supplements: Yes [ ] No[ ]
Any use of calcium supplements: Yes[ ] No[ ]

18. FAMILY AND SOCIAL HISTORY
Do you smoke cigarettes: Yes[ ] No[ ]
Do people around you smoke cigarettes: Yes[ ] No[ ]
Do you take alcohol in any form Yes[ ] No[ ]
Estimated number of hours spent outdoors per day ……………………..
Are you involved in extreme/rigorous physical activity: Yes[ ] No[ ]
Husband age
Husband occupation
Staying with husband or not
Its duration
Contraception history
EXAMINATION
Height (m):
Weight (kg):
BMI:
WAIST TO HIP RATIO:
ACNE
MALE PATTERN BALDNESS
DEEPENING OF VOICE
BUILT
BREAST
B.P.

19. LABORATORY INVESTIGATION:
ANC ROUTINE INVESTIGATIONS-
BLOOD GROUP HB TLC DLC
PLATELET COUNT VIRAL MARKERS-HIV HBSAG
ANTIHCV VDRL FBS URINE-RE
SERUM 25-HYDROXYVITAMIN D CONCENTRATION (ng/ml)
INFERTILITY INVESTIGATIONS-
CBC ESR FBS PPBS
S.TSH S.PROLACTIN S.LH S.FSH
LH/FSH RATIO
TOTAL TESTESTERONE (ng/ml)
SERUM 25-HYDROXYVITAMIN D CONCENTRATION (ng/ml)
TRANSVAGINAL SONOGRAPHY
HSG
HUSBAND SEMEN ANALYSIS