Infertility is multifactorial in its origin and is affected by
different factors including lifestyle, eating habits or nutrition.
Numerous studies have shown that reduce exposure to sunlight
and poor eating habits have led to Vitamin D insufficiency
and/or Vitamin D deficiency, even in sunny countries
among men and women of reproductive age (
Vitamin D is a fat-soluble vitamin and is considered as an essential nutrient required for our health. One of the main functions of Vitamin D is to help with the absorbance of calcium and phosphate, and helps building bones and keeps them strong and healthy. It also blocks the release of the parathyroid hormone involved in reabsorption of bone tissue, which makes bones thin and brittle. Considering these functions of Vitamin D, it plays a central role in calcium and phosphate hemostasis and in turn is-needed for the normal mineralization of bone, muscle contraction, nerve conduction, and general cellular function in all cells of the body including cell growth.
Vitamin D receptor (VDR) is a member of nuclear receptor
family of transcription factors. It forms a heterodimer
with a retinoid-X receptor and binds to hormone
response elements on DNA to regulate expression of specific
gene products. At post transcriptional level Vitamin
D regulates gene expression through microRNA-directed
According to the aforementioned role of Vitamin D
in reproduction, researchers have tried to assess the association
between serum Vitamin D concentrations and
assisted reproductive outcomes. In this regard, Pacis et
The clinical trial study was approved by Ethical Committee of Isfahan University of Medical Sciences and was registered in Iranian registry for the clinical trial (IRCT2015111124999N1) and was designed to be carried out at infertile couples that referred to Isfahan Fertility and Infertility Center from March 2016 to June 2016 and candidate of ICSI. Female with age ranging from 18 to 38 years who had Vitamin D level below 30 ng/ml without symptom of Vitamin D deficiency participated in the study.
Based on the ethical committee, initially individuals were questioned regarding clinical symptom of Vitamin D deficiency and individuals with these symptoms were excluded from the study, as Vitamin D treatment was mandatory for these individuals. Additionally, to roll out effects of male factor infertility and advance maternal age, couples with abnormal semen parameter based on WHO (2010) and/or female age greater than 38 were also excluded from the study. Other exclusion criteria were: secondary female infertility, polycystic ovarian disease, endometriosis, congenital or acquired uterine malformations, drugs consumption that would affect metabolism and Vitamin D absorption such as Carbamazepine and Phenobarbital Phenytoin, body mass index of lower 18 or higher 30 kg/m2 and hypothyroidism. Couples at risk of ovarian hyper stimulation syndrome or poor endometrium (less than 7 mm or grater 14 mm) were also excluded during the course of the study, since all the embryos for these case were vitrified
Initially 159 couples were interviewed based on Vitamin D level below 30 ng/ml. Fifty one couples were excluded based on exclusion criteria. All of the participants entered the study after giving written informed consent and were allowed to leave the study at any desired time.
Six couples were also excluded for other reasons including
declining to participate. The remaining 108 couples
were randomly divided into Vitamin D and placebo
groups based computer-generated or random allocation
software with one block (
Flow diagram of the progress through the phases of a 2-group parallel randomized trial.
Serum Vitamin D was assessed by high-performance liquid chromatography and defined based on couple’s information before starting the trial and also six to eight weeks after treatment, on the day of ovum pick. All the Vitamin D assessment was carried out at a single laboratory. The codes were unraveled after completion of data. Semen parameters, including volume, sperm density, percentage motility and normal morphology were also defined based on WHO (2010) manual.
Ovulation induction: all the participants received a combination
of recombinant follicle-stimulating hormone (FSH)
and human menopausal gonadotrophins (hMG) and were
followed by sequential vaginal ultrasound. Gonadotropin releasing
hormone (GnRH) antagonist was administered when
size of dominant follicles was around 12-14 mm and continued
until the day of hCG administration. On the day of hCG
administration, number of follicles greater than 12 mm and
type of endometrium were also defined and recorded. Type
of endometrium was defined according to study by Zhao et
Numbers of oocytes were recorded on the day of oocyte retrieval. All the couples underwent ICSI based Isfahan Fertility and Infertility policy. Fertilization rate was calculated based on the number of 2PN observed over the number of injected oocytes. On day 3, embryos were scored for the number of blastomeres, blastomere regularity and percentage cytoplasmic fragmentation. Embryos were considered as "good quality" that had between 6-8 blastomeres with even size and less than 25% fragmentation. These outcomes were taken as primary outcomes.
ß-hCG greater than 20 IU was considered as chemical pregnancy and clinical pregnancy was defined as pregnancy diagnosed by ultrasound through visualization of one or more gestational sac. Of note, multiple gestational sacs were considered as one clinical pregnancy. Therefore, clinical pregnancy rate was defined as the number of clinical pregnancy per 100 embryo transfer. These outcomes were considered as secondary outcomes.
Gathered data were analyzed using SPSS for Windows (version 16, SPSS Inc., Chicago, IL, USA). Continuous variables between two groups were compared with the independent t test, and categorical variables were compared with the chi-square test.
In the present study, the mean age of women in the
intervention group was 31.9 ± 4.2 years and in the control
group was 30.8 ± 4.4 years. The mean of body
mass index (BMI) in the intervention group was 23.9
± 2.1 and in the control group was 23.8 ± 1.9 and statistical
analysis showed no significant difference between
the demographic characteristics and the BMI
of the intervention and the control group (P>0.05,
Comparison of basal and clinical characteristics of couples in Vitamin D and Placebo groups
|Variable||Vitamin D||Placebo||P value|
|Female age (Y)*||31.9 ± 4.2||30.8 ± 4.4||0.29|
|Male age (Y)*||35.26 ± 5.2||35.1 ± 4.7||0.98|
|Duration of infertility (month)*||77.4 ± 22.1||68.1 ± 19.3||0.28|
|Number of previous ARTcycle*||1.9 ± 1.2||2.3 ± 1.5||0.37|
|Female education (%)|
|Body mass index (kg/m2)*||23.9 ± 2.1||23.8 ± 1.9||0.65|
ART; Assisted reproductive techniques and *; Data are presented as mean ± SD.
The primary serum Vitamin D levels of the intervention
and the control group were 14.4 ± 6.6 ng/ml and
12.7 ± 6.4 ng/ml, respectively. The differences between
the two groups were insignificant. Six weeks after treatment
with Vitamin D or placebo, the level of Vitamin D
significantly raised to 37.1 ± 7.7 ng/ml in the Vitamin D
group while it remained low (13.6 ± 6.6 ng/ml) in the
placebo group (
Regarding the ICSI primary outcomes, Table 2
showed that the mean number of retrieved oocytes
in the intervention or Vitamin D group was 9.42 ±
4.4 and in the control group was 8.72 ± 5, and their
difference was not statistically significant (P>0.05).
Percentage of type A endometrium on the day of hCG
injection was 81% and 55.8% in Vitamin D and placebo
groups, respectively and the difference between
the two groups was statistically significant (P<0.05).
The rate of fertilization in the Vitamin D group was
68.80% and in the control group was 68% and the difference
was not statistically significant. The rate of
good quality embryo on day3 in the Vitamin D group
was 59.9 and in the control group was 53.59% and the
difference was not statistically significant (P=0.36,
Comparison of ICSI outcomes in Vitamin D and placebo groups
|Variable||Vitamin D||Placebo||P value|
|Mean number of oocyte(Mean ± SD)||9.42 ± 4.4||8.72 ± 5||0.55|
|Fertilization rate (%)||68.8||68||0.88|
|Mean number of embryo(Mean ± SD)||5 ± 2.5||4.6 ± 3.3||0.53|
|Good quality embryo (%)||59.9||53.59||0.36|
ICSI; Intracytoplasmic sperm injection.
Comparison of serum Vitamin D levels in Vitamin D and placebo groups before and after intervention.
According to the results, chemical pregnancy was defined
by positive ß-hCG in the intervention and control
groups were 47.6 and 25.5%, respectively, and the difference
between both groups was statistically significant
Comparison of chemical pregnancy rate assessed by beta-human chorionic gonadotrophin (ß-hCG) in Vitamin D and placebo group.
Comparison of clinical pregnancy rate in Vitamin D and placebo groups.
Based on background studies Vitamin D plays an imperative
role in reproduction and therefore, assessment
of Vitamin D and thereby Vitamin D supplementation is
becoming part of daily practice. However, role of Vitamin
D supplementation during assisted reproductive management
remains controversial and there appear to be more
room for further study and to evaluate which parameters
are most affected by Vitamin D deficiency and thereby supplementation.
Part of these controversies may be related to
confounding factors affecting both Vitamin D levels and
assisted reproductive outcome. An example of these confounding
factors is the seasonal effect on Vitamin D level
The results of this study showed that despite similar
demographic and fertility characteristics between the
two groups, Vitamin D supplementation significantly improves
serum Vitamin D level in comparison to placebo
group and this observation is in line with previous studies
in this filed (
Comparing the mean value of serum Vitamin D between
both groups before intervention revealed no significant difference between the two groups and 50000
units of Vitamin D supplementation per week for 6
weeks based on the previous study by Diamond et al.
Our results also reveal that the improved Vitamin D level
is also associated with significant difference observed
in type of endometrium but no difference was observed
between other assessed parameters, including percentage
of mature oocytes, fertilization rate and embryo quality.
These observations are in concordance with previous report
by Asadi et al. (
Assessment of ICSI outcome in accordance with literature
showed that improved Vitamin D has no effect on fertilization
and embryo quality on day 3. In contrary to our
results and similar studies in this filed, only one study suggest
that high concentration Vitamin D reduces embryo
quality score following ICSI (
Another major finding of the present study was the difference
observed in rates of chemical and clinical pregnancies.
In this study rates of chemical and clinical pregnancy
rates relative to control group was improved by 10.7%
(47.6 vs. 25.5) and 82% (38.1 vs. 20.9), respectively. These
results are in accordance with several previous studies, suggests
that probably Vitamin D improves ICSI in term of
both chemical and clinical pregnancy rates (
These improved effect has been postulated to be related
to mechanisms including i. Miss regulation of NK cell
activity, ii. Immunomodulatory role during implantation
and recurrent miscarriage, iii. Regulation of cross talk
involved between embryos and endometrium which consequently
regulates of HOXA10 involved in embryo implantation.
It has been shown that endometrial HOX10A
expression increase in parallels that Vitamin D receptor
around time of implantation, at the time of maximal endometrial
Results of the present study showed that consuming Vitamin D supplementation could be effective in improving the clinical outcome of ICSI. Based on literature this effect is very likely to be attributed to local effect of Vitamin D on endometrium.