Document Type : Original Article
Authors
1 Department of Obstetrics and Gynaecology, Zekai Tahir Burak Women’s Healthcare Training and Research Hospital, Ankara, Turkey
2 Zekai Tahir Burak Women’s Healthcare Training and Research Hospital, Ankara, Turkey
Abstract
Keywords
Intrauterine insemination (IUI) is used to transport spermdirectly
into the uterus. It is a simple, non-invasive, andcost-effective
technique used for assisted reproduction. Themost common indication
for IUI is cervical infertility, and
it is also used in male subfertility, anovulation, endometriosis
cases in which at least one tube is healthy, as well as unexplained
infertility (
Recently, the effect of vitamin D (VD) has been investigated on not only the musculoskeletal system, but also inthe reproductive and other systems (
Calcitriol (1, 25 dihydroxyvitamin D3) which is the active
form of VD stimulates CYP19 expression (CYP19
encodes the aromatase enzyme) that results in increased
estrogen production, when it was bound to VDR (
There are several studies which presented controversial
results on the differences in 25-hydroxyvitamin D3[25 (OH) D3]
levels of the patients undergoing different
infertility treatment modalities (
This case-control study was conducted between March
2014 and June 2014 in the infertility outpatient clinics of
Zekai Tahir Burak Women’s Health Education and Research
Hospital. This is a government supported tertiary
level maternity hospital located in the capital city of Turkey.
The institutional review board approved the study and
informed consent was obtained from each patient (approval
number: 23.09.2013/9). All of the study protocols were carried
out in accordance with the Helsinki Decleration (
We defined the infertile patients as those reproductive age couples who were unable to become pregnant in the absence of contraception. For the women below 35 years of age, infertility was diagnosed as a minimum of 1 year of trying to become pregnant, whilst for the women above 35 years of age, the diagnosis was limited to 6 months of unprotected sexual intercourse. After we obtained detailed information about age, duration of infertility, infertility type, previous history of surgery, and any systemic disturbances (such as diabetes mellitus, hypertension, and thyroidal disease), a complete physical and gynaecological examination was performed on all of the women. We confirmed tubal patency in the women using hysterosalpingography (HSG) and if there was bilateral tubal occlusion detected with HSG, we applied laparoscopy and hysteroscopy to define any pathology such as pelvic adhesions or endometriosis. When we suspected an intracavitary lesion in the uterus after HSG, or transvaginal ultrasound, we performed hysteroscopy.
We included women with mild male factor infertility, unexplained infertility, and polycystic ovary syndrome (PCOS). We excluded patients who had advanced age (above 40 years of age), any systemic or endocrine diseases, stage 3-4 endometriosis, or intracavitary lesions in uterus (such as endometrial polyp, submucous myoma, and uterine septum), smokers and women who used of any kinds of drugs or substances likely to affect levels of VD. We also excluded patients whose partner had a motile sperm count lower than 5 million/mL. The fertile group consisted of patients who applied to the family planning unit of our hospital for contraceptions. These patients had given birth in the previous 12 months, has not breastfed their neonate, and had no history of infertility.
After initial clinical assessment, infertile patients were evaluated for clomiphene citrate (CC) or gonadotropins (Gn) and IUI use. Those patients who had used CC with IUI treatments for three times or were above 35 years of age were directed into the Gn with IUI regimen (n=63), whilst the other infertile patients were directed into the CC and IUI regimen (n=41). When 18-20 mm (dominant follicles) were found through ultrasound, 2 human chorionic gonadotropin (hCG, Pregnyl, MSD, Netherlands) ampoules containing 5,000 units each, were injected intramuscularly, and IUI applied 36 hours after the injection. When there were 3 or more dominant follicles, or endometrial thickness was less than 6 mm, hCG was not administered. Then 2 weeks later, a blood sample was obtained from patients for ß-hCG measurement. Clinical pregnancy was diagnosed 5 weeks after IUI, when the evidence of fetal heart activity or presence of the gestational sac in the uterine cavity was detected.
The concentration of serum 25 (OH) D3 was used to determine
the status of VD in the body for this study since it has
been proven to be the best biomarker for VD insufficiency. It
also reflects VD levels from both dietary intake and in-skin
synthesis (
The serum levels of 25 (OH) D3 levels and baseline
hormones including estradiol, follicle stimulating hormone
(FSH), luteinizing hormone, prolactin, and thyroid
stimulating hormone were measured on the third day of
the menstrual cycle when ovulation induction was started.
We performed the recruitment of study volunteers in a single
season, because the blood levels of VD have seasonal
variabilities (
After overnight fasting, venous blood samples were obtained early in the morning and transferred to the laboratory in a non-transparant box to avoid exposure to light, and then serum was separated by centrifugation at 5,000 rpm (2,236 g) for 10 minutes. The serum 25 (OH) D3 levels were measured using an enzyme linked immunosorbent assay kit (Immunodiagnostic AG, Leverkusen, Germany), and presented in ng/mL. The intra-assay and inter-assay coefficients of variation for serum 25 (OH) D3, were 8.9 and 10.6% respectively. Serum 25 (OH) D3 concentrations <20 ng/mL was considered as VD deficiency. Types of VD deficiency were also classified as mild (10-20 ng/ mL), moderate (5-10 ng/mL), and severe (<5 ng/mL). Serum 25 (OH) D3 concentrations between 20 and 30 ng/ mL was accepted as VD insufficiency whereas a threshold value of =30 ng/mL was considered sufficient serum VD levels. Basal hormone levels were measured using an Immulite 2000 analyzer (EURO/DPC Ltd., Gwynedd, UK). Body mass index (BMI) was defined as the weight in kilograms divided by the square of the height in meters.
We examined the women who had a positive result for ß-hCG using transvaginal ultrasound at at least weeks 6-7 of gestation to detect fetal cardiac activity. The difference between the two subgroups (pregnant and non-pregnant) of infertile patients in terms of 25 (OH) D3 levels was the primary outcome measured of this study. The secondary outcome was the comparison of serum 25 (OH) D3 levels between infertile and fertile groups.
Data were recorded and analysed using the Statistical
Package for the Social Sciences program for Windows
version 17.0 (SPSS Inc, Chicago, IL, USA). The normal
distribution of the variables was assessed using the Shapiro-
Wilk’s test. Continuous variables were presented as
the mean with standard deviation (SD) or median (range),
and categorical variables were presented as the number
(percentage) of subjects. Continuous variables were
compared using independent samples t-test if they were
normally distributed or with the Mann-Whitney U test if
they were non-normally distributed. Categorical variables
were analyzed using the Chi-square (χ2) test or Fisher’s
exact test. Correlations were calculated using Spearman's
correlation analysis. In all analyses, two-tailed P<0.05
were considered as statistically significant. Post-hoc power
analysis demonstrated that we achieved a power of 0.95
with a 5% level of significance and a 0.5 effect size by
using a two sample comparison (
One hundred and four infertile and one hundred and three fertile women were included into this cross-sectional, case-control study. Examination of the infertile and fertile patients showed that there was no statistically significant difference between the groups regarding their mean age and BMI. Obstetric history characteristics were statistically significantly different between the two groups (P<0.001 for all). The mean FSH levels were higher in the infertile patients than in the fertile patients (7.4 ± 2.1 mU/mL vs 6.2 ± 1.6 mU/mL, P=0.001), but it was within the normal range in either group. Mean prolactin levels of the fertile group (14.4 ± 5.4 ng/mL) were higher than the infertile group’s (12.2 ± 4.6 ng/mL). This difference was statistically meaningful (P=0.002), but those values were in the normal range as with mean FSH levels. There were no statistically significant differences in 25 (OH) D3 levels between the 2 groups [7.3 (3-25.5) ng/mL vs. 6.8 (3.4-37.1) ng/mL, P=0.512], as seen in Table 1. No significant correlation between serum 25 (OH) D3 and FSH levels was observed either in the entire study population (Spearman’s r=0.051, P=0.466).
Descriptive characteristics and serum 25 (OH) D3 levels of infertile and fertile patients
Characteristic | Infertile group n=104 | Fertile group n=103 | P value |
---|---|---|---|
Age (Y)** | 28.1 (4.7) | 29.4 (5.4) | 0.088a |
BMI (kg/m2)** | 25.1 (3.6) | 25.7 (3.8) | 0.234a |
Gravida* | 0 (0-6) | 2 (1-6) | <0.001b |
Parity* | 0 (0-1) | 2 (0-5) | <0.001b |
Alive* | 0 (0-1) | 2 (0-5) | <0.001b |
Abortion* | 0 (0-5) | 0 (0-4) | <0.001b |
FSH (mIU/mL)** | 7.4 (2.1) | 6.2 (1.6) | 0.001a |
LH (mIU/mL)** | 5.3 (2.5) | 4.9 (1.9) | 0.154a |
Estradiol (pg/mL)* | 44.0 (12-148) | 42.7 (21-99) | 0.791b |
TSH (µlU/mL)** | 1.9 (0.8) | 2 (0.9) | 0.859a |
Prolactin (ng/mL)** | 12.2 (4.6) | 14.4 (5.4) | 0.002a |
25 (OH) D3 (ng/mL)* | 7.3 (3-25.5) | 6.8 (3.4-37.1) | 0.512b |
BMI; Body mass index, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, TSH; Thyroid stimulating hormone, *; Median (minimum-maximum), **; Mean (SD), a; Student t Test, and b; Mann Whitney U test. P<0.05 is considered as statistically significant.
The severity of VD deficiency in the infertile and fertile groups showed that most of the participants were deficient for VD (96.2 vs. 97.1%) and only 1 (1%) participant in the fertile group had 25 (OH) D3 levels =30 ng/mL. 19 (18.3%) patients were in the severe deficiency group, 56 (53.8%) cases were in the moderate deficiency group, and lastly 25 (24%) women were in the mild deficiency group among the infertile group. The number of fertile patients in the same groups was 19 (18.4%), 58 (56.3%), and 23 (22.3%), respectively (P=0.776).
After IUI treatment, the numbers of clinical pregnancies
and live births among 104 infertile patients were 14
(13.3%) and 10 (9.61%), respectively. When infertile patients
were divided into two subgroups (pregnant and non-
pregnant), there was no statistically significant difference
between these subgroups regarding age, BMI, obstetrical
history, baseline hormone levels, or ovulation induction
agent used. Similarly, no significant difference was observed
between the pregnant and non-pregnant subgroups
of infertile patients in terms of serum 25 (OH) D3 levels
(P=0.267). Ten (71.4%) patients out of the 14 clinical
pregnancies had moderately deficient VD levels. The only
significant parameter that may predict pregnancy was the
age of the patients, namely the pregnant group was statistically
significantly younger than the non-pregnant group
(
Individual characteristics, ovulation induction type and vitamin D levels in pregnant and non-pregnant patients after IUI
Characteristic | Non-pregnantgroup n=90 | Pregnant groupn=14 | P value | |
---|---|---|---|---|
Age (Y)** | 28.5 (4.7) | 25.5 (4.4) | 0.027a | |
BMI (kg/m2)** | 25 (3.5) | 25.3 (4) | 0.784a | |
Gravida* | 0 (0-6) | 0 (0-2) | 0.745b | |
Parity* | 0 (0-1) | 0 (0-1) | 0.459b | |
Alive* | 0 (0-1) | 0 (0-1) | 0.459b | |
Miscarriage* | 0 (0-5) | 0 (0-2) | 0.335b | |
FSH (mIU/ml)** | 7.1 (3.5-13.6) | 6.8 (3.4-13.5) | 0.378a | |
LH (mIU/ml)** | 4.9 (2.1-14) | 5.9 (2.2-10.3) | 0.247a | |
Estradiol (pg/ml)** | 44.5 (12-148) | 42.5 (20-82) | 0.398a | |
TSH (µlU/ml)** | 1.8 (0.4-5.3) | 2.0 (1.3-3.5) | 0.160a | |
Prolactin (ng/ml)** | 12.3 (4.9-28.4) | 12.9 (7.6-20.9) | 0.788a | |
25 (OH) D3 (ng/mL)* | 7.3 (3-25.5) | 8.1 (4.7-22.1) | 0.267b | |
Ovulation induction type*** | 0.777c | |||
CC | 55 (61.1) | 8 (57.1) | ||
Gn | 35 (38.9) | 6 (42.9) | ||
IUI; Intrauterine insemination, BMI; Body mass index, FSH; Follicle-stimulating hormone, LH; Luteinizing hormone, TSH; Thyroid stimulating hormone, CC; Ovulation induction with clomiphene citrate, Gn; Ovulation induction with gonadotropin, *; Median (minimum-maximum), **; Mean (SD), ***; n (%), a; Student’s t test, b; Mann-Whitney U test, and c; Fisher’s exact test. P<0.05 is considered as statistically significant.
Our study showed that infertile and fertile patients had similar serum VD levels and that there was no statistically significant difference in serum VD measurements between the pregnant and non-pregnant groups after IUI.
VD has an essential role in both male and female reproductive
system (
Ovulation induction with IUI is the most utilized method
of infertility treatment in our unit. The success of IUI
treatment is multifactorial, and pregnancy rates per cycle
have been estimated as 10.2% in a IUI cycle with controlled
ovarian stimulation (
A study by Ott et al. (
In two rat studies, VD deficiency was shown to significantly
increase infertility, decrease probability of viable
births and healthy full-grown individuals (
VD has been found to be related with the activation of
key enzymes in steroidogenesis such as 3-beta-hydroxysteroid
dehydrogenase, and it has been shown to induce
the production of progesterone that consequently leads to
uterine quiescence (
A recent randomized controlled trial by Asadi et al. (
The strength of this study is that our data is single-centered
and reliable. The data were obtained prospectively
from the patients living in the same geographical region
during the same season. There is a limitation to our study;
VD deficiency was wide-spread in our study population,
consistent with the results of previous studies on this issue
(
No significant difference was observed between pregnant and nonpregnant women who underwent ovulation induction with IUI treatment with regard to serum 25 (OH) D3 levels. No association was found between infertility and serum 25 (OH) D3 levels either. Further research which compares women who have deficient and sufficient serum VD levels is warranted.