Abstract

BACKGROUND

Vitamin D plays a role in reproductive capacity. Recently, several investigators have demonstrated higher IVF pregnancy rates in vitamin D replete women. The objective of this study was to validate these findings and to further elucidate the role of vitamin D in reproduction among a diverse group of women.

METHODS

This was a retrospective cohort study in an academic tertiary care center of 188 infertile women undergoing IVF. Serum levels of vitamin D (25OH-D) were measured in previously frozen serum samples. The main outcome measure was clinical pregnancy, defined as sonographic presence of a heartbeat following IVF.

RESULTS

The relationship between vitamin D status and pregnancy rates differed by race (P < 0.01). Among non-Hispanic whites, pregnancy rates declined with progressively lower levels of vitamin D, while in Asians, the reverse was true. Adjusting for age and number and quality of embryos transferred among non-Hispanic whites, the odds of pregnancy were four times higher in vitamin D replete versus deficient patients. Live birth rates mirrored pregnancy rates. Vitamin D status was not associated with ovarian stimulation parameters or with markers of embryo quality.

CONCLUSIONS

Vitamin D deficiency is associated with lower pregnancy rates in non-Hispanic whites, but not in Asians, possibly due to their lower IVF success rates. Vitamin D deficiency was not correlated with ovarian stimulation parameters or with markers of embryo quality, suggesting its effect may be mediated through the endometrium.

Introduction

An epidemic of vitamin D deficiency has been emerging over the last decade among all racial groups in the USA, with the prevalence of vitamin D insufficiency nearly doubling from 1994 to 2004 (Looker et al., 2008). Vitamin D deficiency has been implicated in a host of chronic diseases, including diabetes, obesity, autoimmune disease, cardiovascular disease and cancer. More recently, poor vitamin D status has been implicated as a contributing factor to poor pregnancy outcomes (Bodnar et al., 2007) and infertility (Ozkan et al., 2010).

The importance of vitamin D in reproduction is evident from murine models. Vitamin D receptor knockout mice demonstrate uterine hypoplasia, gonadal insufficiency, reduced aromatase gene expression, impaired folliculogenesis and infertility (Yoshizawa et al., 1997; Kinuta et al., 2000). Rats deficient in vitamin D demonstrate compromised mating behavior, reduced fertility, decreased litter sizes and impaired neonatal growth (Halloran and DeLuca, 1980; Kwiecinski et al., 1989). Importantly, reproductive function can be normalized with vitamin D supplementation, but not with calcium alone, suggesting that vitamin D's role in reproduction lies outside of its classic endocrine role in regulating calcium homeostasis (Halloran and DeLuca, 1980; Kwiecinski et al., 1989; Johnson and DeLuca, 2001).

The presence of the vitamin D receptor (VDR) in many tissues along the female reproductive axis, including the pituitary, ovary, uterus and placenta (Johnson and DeLuca, 2001), suggests that vitamin D is an important regulator of the female reproductive system. The active form of vitamin D (1,25 dihydroxyvitamin D3 or calcitriol), when bound to its receptor, acts as a transcription factor to regulate the expression of the CYP19 gene, which encodes aromatase, an essential enzyme in the production of estrogen (Kinuta et al., 2000). Serum calcitriol and estradiol levels track together, both in the normal menstrual cycle (Gray et al., 1982) and in stimulated IVF cycles (Potashnik et al., 1992); however, the main circulating form of vitamin D (25-hydroxyvitamin D or 25(OH)D) does not fluctuate throughout the menstrual cycle (Johnson and DeLuca, 2001).

Calcitriol is produced by the decidua in response to IL-1B secreted by the blastocyst (Vigano et al., 2006). Calcitriol regulates decidual expression of HOXA10, calbindin (Daftary and Taylor, 2006) and osteopontin (Vigano et al., 2006) genes, all integrally involved in embryo implantation. The decidua and placenta continue to secrete large quantities of calcitriol throughout pregnancy, which is important for regulating the immune response at the maternal–fetal interface. The presence of the blastocyst up-regulates the production of the active form of vitamin D in the endometrium (Vigano et al., 2006). In turn, calcitriol may help to support successful implantation by attenuating decidual T-cell function (Evans et al., 2004). Decidual NK cells treated with calcitriol show decreased synthesis of cytokines, CSF2, IL-1 and IL-6 and TNFα (Evans et al., 2006).

An IVF population provides valuable insight into the role of vitamin D since it is possible to examine each aspect of a single conception cycle from follicular development to implantation. A recent study found that women with higher 25(OH)D levels in their serum and follicular fluid were significantly more likely to achieve pregnancy from IVF compared with women with lower levels of vitamin D (Ozkan et al., 2010). The primary objective of our study was to verify this relationship between vitamin D status and IVF outcomes, and additionally to isolate the effect of vitamin D either to ovarian stimulation, embryo quality or endometrium. As a secondary objective, we aimed to evaluate the relationship between vitamin D and IVF outcomes in the context of patient race.

Materials and Methods

This was a retrospective cohort study of 208 infertile women who underwent their first IVF cycle at University of Southern California (USC) Fertility Clinic from January 2006 to August of 2009. The study protocol was approved by the USC Institutional Review Board. Patients were excluded if they had previous IVF cycles at USC Fertility or if they underwent zygote intra-Fallopian tube transfer. Patient characteristics and cycle parameters were identified from patient medical records. Patient race was categorized according to self-identified race/ethnicity on their initial patient questionnaire.

All patients underwent IVF cycles using standardized regimens for pituitary down-regulation and controlled ovarian hyperstimulation. The particular protocol was chosen according to patient diagnosis and age. In general, good prognosis patients underwent a Leuprolide acetate down-regulation protocol (Lupron; TAP Pharmaceuticals, North Chicago, IL, USA; Porter et al., 1984), whereas those patients judged to have a poor prognosis underwent either an antagonist protocol with flexible ganirelix acetate start (Antagon; Organon, Inc., West Orange, NJ, USA) (Oliviennes et al., 1994), or a microdose flare protocol (Scott and Navot, 1994).

Controlled ovarian hyperstimulation was initiated with either recombinant FSH alone or in combination with human menopausal gonadotrophins (Menopur, Ferring, Inc., Suffern, NY, USA). Starting dose was selected on the basis of age, Day 3 FSH levels, and number of antral follicles, with adjustments made according to patient response. Serial monitoring of ovarian response was assessed by transvaginal ultrasound and serum estradiol (E2) assays. When two to three follicles reached or exceeded 17–18 mm, hCG (10 000 IU IM) was administered. Serum samples collected the day after hCG administration were stored at −20°C until assayed.

Transvaginal ultrasound guided oocyte retrieval was performed 34–35 h following hCG injection. Conventional insemination and/or ICSI was performed as indicated. Ultrasound-guided fresh embryo transfer was performed on Days 3 through 5 after egg retrieval. The number of embryos transferred depended upon embryo development and number of embryos available.

Luteal phase supplementation with vaginal micronized progesterone in capsules and oral estrace was started 3 days following egg retrieval. Clinical pregnancy was defined by the sonographic presence of a heartbeat at 7–8 weeks of gestation.

Vitamin D status

Vitamin D status was measured by assessing circulating levels of 25(OH)D in frozen, never previously thawed serum samples using radioimmunoassay (RIA; DiaSorin, Stillwater, MN, USA; Hollis et al., 1993). Intra-and inter-assay coefficients of variation were 10.5 and 8.2%, respectively. Serum 25(OH)D was categorized according to clinically accepted ranges for vitamin D deficiency (<20 ng/ml), insufficiency (20–30 ng/ml) and replete (>30 ng/ml; Holick, 2007).

Statistical analysis

Continuous data were summarized as the mean ± SD, or as median, 25th and 75th quartiles if highly skewed, and categorical data as percentage (%). Univariate analyses were carried out using the Kruskal–Wallis test for continuous outcomes and χ2 tests for categorical outcomes.

Multivariable logistic regression was used to evaluate predictors of clinical pregnancy and of live birth. Vitamin D status (deficient, sufficient, replete) was included in the model as an ordinal variable. Race/ethnicity was coded using dummy variables, and effect modification was evaluated by including race-by-vitamin D cross-product terms in the model and conducting a likelihood ratio test. Since race was found to be an effect modifier, race-specific results were obtained by evaluating appropriate linear combinations of regression coefficients in the interaction model. Adjusted results are presented as predicted probability of the outcome (clinical pregnancy or live birth), adjusted to mean levels of covariates included in the model.

All models were adjusted for maternal age. Additional covariates were included in the final model if they confounded the relationship between vitamin D and treatment outcome, as evidenced by a change in race-specific regression coefficients of at least 15%. Variables evaluated as potential confounders included age, BMI, obesity, parity, diagnosis, previous treatment failure, stimulation protocol, season of transfer, number of embryos transferred and markers of embryo quality. Mild confounding effects (10–15% change in at least one race-specific regression coefficient) were observed for number of embryos, embryo quality (the average number of blastomeres among transferred embryos) and diagnosis of diminished ovarian reserve. Taken jointly, these three covariates resulted in substantial confounding (>25% change in one or more race-specific regression coefficients) and thus they were retained in the final model. Model fit was evaluated using the Hosmer and Lemeshow test (Hosmer and Lemeshow, 1980).

Power was calculated based on results of the Ozkan study, in which patients with the highest levels of vitamin D had a clinical pregnancy rate of 47% and those with the lowest vitamin D levels had clinical pregnancy rates of 20%. Thus, in order to detect a 27% difference in clinical pregnancy rates, with 80% power and alpha of 0.05, we required at least 102 patients. All P values are two sided and statistical significance was established as P < 0.05.

All analyses were conducted using Stata 11.0 (StataCorp, College Station TX).

Results

Of 208 eligible patients, 18 did not have available serum for testing and 2 had missing outcome data, leaving 188 study participants in the final analysis. Of the 188 patients, 21% (39/188) were vitamin D deficient (25(OH)D <20 ng/ml), 37% (70/188) were vitamin D insufficient (20–30 ng/ml) and only 42% (79/188) were vitamin D replete (25(OH)D >30 ng/ml).

Race was categorized as non-Hispanic white (53%), Asian (26%), Hispanic white (14%) or other (7%), according to guidelines outlined in the 2010 USA Census Bureau (http://www.census.gov/geo/www/2010 census) Asian race included those patients whose origins were from Southeast Asia as well as from the Indian subcontinent. Serum 25(OH)D levels varied by race (P= 0.01), being highest in non-Hispanic whites (n= 100; mean: 30.6 ng/ml), intermediate in Asians (n= 49; mean: 27.1 ng/ml) and lowest in Hispanic whites (n= 26; mean: 25.6 ng/ml). Baseline patient characteristics including infertility diagnosis, age and BMI were compared among the different racial groups. Hispanic whites were on average younger with a higher BMI compared with other races. No other significant differences were noted.

Table I depicts patient and IVF cycle characteristics by vitamin D status. Vitamin D deficient women were, on average, younger (P= 0.03) and heavier (P= 0.03), and were less likely to have a diagnosis of diminished ovarian reserve (P= 0.01). Although vitamin D deficient women were heavier (P= 0.03), all patients had BMI values within the normal range. Vitamin D status was not associated with other infertility diagnoses, with parity, or previous IVF failure. Of all patients, 53% underwent long lupron protocol, 37% underwent microdose flare and 11% underwent an antagonist protocol. Women with vitamin D deficiency were more likely to have been treated with the long lupron protocol (P= 0.01). Vitamin D status was not associated with ovarian stimulation parameters (dose of medications required, peak estradiol levels, number of oocytes retrieved and number of mature oocytes) as noted in Table I. Although the number of embryos transferred differed significantly by vitamin D status, there was no trend (more embryos were transferred to women in the intermediate vitamin D group). Vitamin D status was not associated with fertilization rates or markers of embryo quality (the mean number of blastomeres or mean percent fragmentation).

Table I

Patient and IVF cycle characteristics by vitamin D status.

Characteristic Vitamin D >30 ng/ml, n= 79 (42%) Vitamin D 20–30 ng/ml, n= 70 (37%) Vitamin D <20 ng/ml, n= 39 (21%) P-valuea 
Race/ethnicity 
 Non-hispanic White 45 (57%) 39 (56%) 16 (41%) 0.38 
 Hispanic White 9 (11%) 7 (10%) 10 (26%)  
 Asian 20 (25%) 18 (26%) 11 (28%)  
 Other 5 (6%) 6 (9%) 2 (5%)  
Age (years) 36.5 ± 4.1 36.7 ± 3.7 34.7 ± 4.1 0.03 
BMI (kg/m222.7 ± 3.9 23.4 ± 4.3 24.8 ± 4.5 0.03 
 Obese (BMI ≥30) 3 (4%) 7 (10%) 8 (21%) 0.02 
Nulliparous 69 (87%) 60 (86%) 30 (77%) 0.32 
Diagnosis 
 DOR/ageb 38 (48%) 35 (50%) 9 (23%) 0.01 
 Tubal 11 (14%) 11 (16%) 7 (18%) 0.85 
 Endometriosis 11 (14%) 6 (9%) 8 (21%) 0.21 
 Uterine 5 (6%) 5 (7%) 5 (13%) 0.45 
 Anovulatory/endocrine 13 (16%) 9 (13%) 10 (26%) 0.23 
 Male factor 36 (46%) 25 (36%) 14 (36%) 0.40 
 Technologicalc 5 (6%) 10 (14%) 1 (3%) 0.10 
 Unexplained 14 (18%) 4 (6%) 4 (10%) 0.07 
Previous failed IVF 23 (29%) 15 (21%) 6 (15%) 0.22 
Stimulation protocol 
 Microdose flare 36 (45%) 24 (34%) 8 (20%) 0.04 
 Antagonist 10 (13%) 8 (11%) 3 (8%)  
 Long lupron 33 (42%) 38 (54%) 28 (72%)  
Peak estradiol (pg/ml) 3020 ± 1922 3168 ± 1646 3186 ± 1679 0.56 
Oocytes retrieved (n)d 11 (6.19) 14 (9.20) 17 (11.22) 0.10 
 Fertilization rate (%) 68 ± 25 71 ± 23 74 ± 17 0.79 
 Mean cells Day 3 (n6.1 ± 1.6 6.2 ± 1.1 6.4 ± 1.2 0.78 
 Mean fragmentation (%) 15.5 ± 13.8 16.9 ± 10.6 16.2 ± 11.8 0.44 
Embryos transferred (n3.2 ± 1.2 3.8 ± 1.3 3.1 ± 1.0 <0.01 
 Mean cells Day 3 (n6.8 ± 1.5 7.1 ± 1.9 7.1 ± 1.1 0.73 
 Mean fragmentation (%) 12.2 ± 9.9 12.0 ± 6.8 11.7 ± 9.0 0.79 
Characteristic Vitamin D >30 ng/ml, n= 79 (42%) Vitamin D 20–30 ng/ml, n= 70 (37%) Vitamin D <20 ng/ml, n= 39 (21%) P-valuea 
Race/ethnicity 
 Non-hispanic White 45 (57%) 39 (56%) 16 (41%) 0.38 
 Hispanic White 9 (11%) 7 (10%) 10 (26%)  
 Asian 20 (25%) 18 (26%) 11 (28%)  
 Other 5 (6%) 6 (9%) 2 (5%)  
Age (years) 36.5 ± 4.1 36.7 ± 3.7 34.7 ± 4.1 0.03 
BMI (kg/m222.7 ± 3.9 23.4 ± 4.3 24.8 ± 4.5 0.03 
 Obese (BMI ≥30) 3 (4%) 7 (10%) 8 (21%) 0.02 
Nulliparous 69 (87%) 60 (86%) 30 (77%) 0.32 
Diagnosis 
 DOR/ageb 38 (48%) 35 (50%) 9 (23%) 0.01 
 Tubal 11 (14%) 11 (16%) 7 (18%) 0.85 
 Endometriosis 11 (14%) 6 (9%) 8 (21%) 0.21 
 Uterine 5 (6%) 5 (7%) 5 (13%) 0.45 
 Anovulatory/endocrine 13 (16%) 9 (13%) 10 (26%) 0.23 
 Male factor 36 (46%) 25 (36%) 14 (36%) 0.40 
 Technologicalc 5 (6%) 10 (14%) 1 (3%) 0.10 
 Unexplained 14 (18%) 4 (6%) 4 (10%) 0.07 
Previous failed IVF 23 (29%) 15 (21%) 6 (15%) 0.22 
Stimulation protocol 
 Microdose flare 36 (45%) 24 (34%) 8 (20%) 0.04 
 Antagonist 10 (13%) 8 (11%) 3 (8%)  
 Long lupron 33 (42%) 38 (54%) 28 (72%)  
Peak estradiol (pg/ml) 3020 ± 1922 3168 ± 1646 3186 ± 1679 0.56 
Oocytes retrieved (n)d 11 (6.19) 14 (9.20) 17 (11.22) 0.10 
 Fertilization rate (%) 68 ± 25 71 ± 23 74 ± 17 0.79 
 Mean cells Day 3 (n6.1 ± 1.6 6.2 ± 1.1 6.4 ± 1.2 0.78 
 Mean fragmentation (%) 15.5 ± 13.8 16.9 ± 10.6 16.2 ± 11.8 0.44 
Embryos transferred (n3.2 ± 1.2 3.8 ± 1.3 3.1 ± 1.0 <0.01 
 Mean cells Day 3 (n6.8 ± 1.5 7.1 ± 1.9 7.1 ± 1.1 0.73 
 Mean fragmentation (%) 12.2 ± 9.9 12.0 ± 6.8 11.7 ± 9.0 0.79 

aContinuous data are presented as the mean ± standard deviation with P values obtained from Kruskal–Wallis test unless otherwise indicates. Categorical data are presented as n (%) with P values obtained from χ2 tests (the Pearson or Fisher exact, as appropriate).

bDecreased ovarian reserve or advanced reproductive age.

cIVF chosen for the purposes of sex selection or PGD.

dMedian (25th, 75th percentiles).

Table II depicts the patient and IVF cycle characteristics by pregnancy outcome. Clinical pregnancy rates were 43% in non-Hispanic whites, 38% in Hispanic whites and 35% in Asians. A similar pattern was seen for live birth rates: 35% in non-Hispanic whites, 27% in Hispanic whites and 26% in Asians. Clinical pregnancy was associated with lower dose of medication needed (P= 0.05), higher peak of E2 (P= 0.03), higher number of oocytes retrieved (P= 0.05) and higher mean number of cells on Day 3 both among the entire cohort of embryos (P= 0.03) and among the embryos that were selected for transfer (P= 0.01). The number of embryos transferred was greater in the clinical pregnancy group (3.6 versus 3.3, P= 0.02). Only dose of medication (P= 0.02) and the mean percent fragmentation among transferred embryos (P= 0.05) were significantly associated with live birth.

Table II

Patient and IVF cycle characteristics by pregnancy outcome.

Characteristic Pregnant, n= 77 (39%) Not pregnant, n= 111 (59%) P-value Live birth, n= 59 (31%) No live birth, n= 129 (69%) P-value 
Age (years) 36.2 ± 3.64 36.2 ± 4.30 0.74 35.9 ± 3.66 36.3 ± 4.19 0.50 
BMI (kg/m223.2 ± 4.35 23.5 ± 4.22 0.49 23.3 ± 4.57 23.4 ± 4.14 0.88 
 Obese (BMI ≥30) 9 (11%) 11 (9%) 0.65 7 (11%) 13 (9%) 0.68 
Nulliparous 69 (83%) 102 (84%) 0.93 56 (88%) 115 (82%) 0.29 
Diagnosis 
 DOR/agea 31 (37%) 59 (48%) 0.12 22 (34%) 68 (48%) 0.06 
 Tubal 12 (14%) 19 (16%) 0.83 11 (17%) 20 (14%) 0.58 
 Endometriosis 10 (12%) 17 (14%) 0.70 8 (13%) 19 (13%) 0.85 
 Uterine 5 (6%) 11 (9%) 0.43 4 (6%) 12 (9%) 0.58 
 Anovulatory/endocrine 14 (17%) 21 (17%) 0.95 11 (17%) 24 (17%) 0.98 
 Male factor 35 (42%) 48 (39%) 0.69 27 (24%) 56 (40%) 0.74 
 Technologicalb 9 (11%) 9 (7%) 0.39 5 (8%) 12 (9%) 0.74 
 Unexplained 12 (14%) 13 (11%) 0.41 11 (17%) 14 (10%) 0.14 
Previous failed IVF 21 (25%) 25 (20%) 0.42 16 (25%) 30 (21%) 0.55 
Race/ethnicitya 
 Non-Hispanic White 43 (56%) 57 (51%) 0.59 37 (58%) 74 (52%) 0.87 
 Hispanic White 10 (13%) 16 (14%)  9 (14%) 19 (13%)  
 Asian 17 (22%) 32 (29%)  14 (22%) 38 (27%)  
 Other 7 (9%) 6 (5%)  4 (6%) 10 (7%)  
Stimulation protocol 
 Microdose flair 27 (35%) 41 (37%) 0.18 21 (36%) 47 (36%) 0.17 
 Antagonist 5 (7%) 16 (14%)  3 (5%) 18 (14%)  
 Long lupron 45 (58%) 54 (49%)  35 (59%) 64 (50%)  
Peak estradiol (pg/mL) 3414 ± 1782 2897 ± 1730 0.03 3331 ± 1834 3008 ± 1730 0.27 
Oocytes retrieved (n16.9 ± 9.5 14.4 ± 9.0 0.05 16.8 ± 9.9 14.8 ± 9.0 0.18 
 Fertilization rate (%) 74.1 ± 16.8 68.0 ± 25.6 0.21 74.2 ± 16.1 68.6 ± 24.9 0.37 
 Mean cells Day 3 (n6.5 ± 1.1 6.0 ± 1.5 0.03 6.5 ± 1.2 6.1 ± 1.4 0.08 
 Mean fragmentation (%) 15.3 ± 9.3 16.8 ± 14.0 0.96 15.6 ± 9.9 16.5 ± 13.3 0.93 
Embryos transferred (n3.6 ± 1.2 3.3 ± 1.3 0.02 3.5 ± 1.2 3.4 ± 1.3 0.24 
 Mean cells Day 3 (n7.4 ± 1.5 6.7 ± 1.5 0.01 7.3 ± 1.7 6.9 ± 1.5 0.07 
 Mean fragmentation (%) 10.7 ± 6.6 13.0 ± 9.7 0.11 10.2 ± 6.4 12.9 ± 9.4 0.05 
Characteristic Pregnant, n= 77 (39%) Not pregnant, n= 111 (59%) P-value Live birth, n= 59 (31%) No live birth, n= 129 (69%) P-value 
Age (years) 36.2 ± 3.64 36.2 ± 4.30 0.74 35.9 ± 3.66 36.3 ± 4.19 0.50 
BMI (kg/m223.2 ± 4.35 23.5 ± 4.22 0.49 23.3 ± 4.57 23.4 ± 4.14 0.88 
 Obese (BMI ≥30) 9 (11%) 11 (9%) 0.65 7 (11%) 13 (9%) 0.68 
Nulliparous 69 (83%) 102 (84%) 0.93 56 (88%) 115 (82%) 0.29 
Diagnosis 
 DOR/agea 31 (37%) 59 (48%) 0.12 22 (34%) 68 (48%) 0.06 
 Tubal 12 (14%) 19 (16%) 0.83 11 (17%) 20 (14%) 0.58 
 Endometriosis 10 (12%) 17 (14%) 0.70 8 (13%) 19 (13%) 0.85 
 Uterine 5 (6%) 11 (9%) 0.43 4 (6%) 12 (9%) 0.58 
 Anovulatory/endocrine 14 (17%) 21 (17%) 0.95 11 (17%) 24 (17%) 0.98 
 Male factor 35 (42%) 48 (39%) 0.69 27 (24%) 56 (40%) 0.74 
 Technologicalb 9 (11%) 9 (7%) 0.39 5 (8%) 12 (9%) 0.74 
 Unexplained 12 (14%) 13 (11%) 0.41 11 (17%) 14 (10%) 0.14 
Previous failed IVF 21 (25%) 25 (20%) 0.42 16 (25%) 30 (21%) 0.55 
Race/ethnicitya 
 Non-Hispanic White 43 (56%) 57 (51%) 0.59 37 (58%) 74 (52%) 0.87 
 Hispanic White 10 (13%) 16 (14%)  9 (14%) 19 (13%)  
 Asian 17 (22%) 32 (29%)  14 (22%) 38 (27%)  
 Other 7 (9%) 6 (5%)  4 (6%) 10 (7%)  
Stimulation protocol 
 Microdose flair 27 (35%) 41 (37%) 0.18 21 (36%) 47 (36%) 0.17 
 Antagonist 5 (7%) 16 (14%)  3 (5%) 18 (14%)  
 Long lupron 45 (58%) 54 (49%)  35 (59%) 64 (50%)  
Peak estradiol (pg/mL) 3414 ± 1782 2897 ± 1730 0.03 3331 ± 1834 3008 ± 1730 0.27 
Oocytes retrieved (n16.9 ± 9.5 14.4 ± 9.0 0.05 16.8 ± 9.9 14.8 ± 9.0 0.18 
 Fertilization rate (%) 74.1 ± 16.8 68.0 ± 25.6 0.21 74.2 ± 16.1 68.6 ± 24.9 0.37 
 Mean cells Day 3 (n6.5 ± 1.1 6.0 ± 1.5 0.03 6.5 ± 1.2 6.1 ± 1.4 0.08 
 Mean fragmentation (%) 15.3 ± 9.3 16.8 ± 14.0 0.96 15.6 ± 9.9 16.5 ± 13.3 0.93 
Embryos transferred (n3.6 ± 1.2 3.3 ± 1.3 0.02 3.5 ± 1.2 3.4 ± 1.3 0.24 
 Mean cells Day 3 (n7.4 ± 1.5 6.7 ± 1.5 0.01 7.3 ± 1.7 6.9 ± 1.5 0.07 
 Mean fragmentation (%) 10.7 ± 6.6 13.0 ± 9.7 0.11 10.2 ± 6.4 12.9 ± 9.4 0.05 

Continuous data are presented as the mean ± standard deviation with P values obtained from Kruskal–Wallis test unless otherwise indicates. Categorical data are presented as n (%) with P values obtained from χ2 tests (the Pearson or Fisher exact, as appropriate).

aDecreased ovarian reserve or advanced reproductive age.

bIVF chosen for the purposes of sex selection or PGD.

As shown in Table III, race significantly modified the relationship between cycle outcome and vitamin D status (P < 0.01, for clinical pregnancy and live birth). Among non-Hispanic whites, clinical pregnancy rates progressively decreased with declining vitamin D status, from 51% in those who were vitamin D replete, to 44% in those who were insufficient, to 19% in those who were vitamin D deficient (P= 0.04). However, the opposite trend was seen among Asians, with pregnancy rates increasing with worsening vitamin D status (P= 0.01). Results were similar after fitting multivariable logistic models to adjust for maternal age, number of embryos transferred, embryo quality and diagnosis of diminished ovarian reserve (Table III). A similar relationship was observed between vitamin D status and the live birth rate, with worsening vitamin D status, live birth rates decreased among non-Hispanic whites (P= 0.03), but increased in Asians (P= 0.01).

Table III

Rates of clinical pregnancy, live birth and implantation by vitamin D status: unadjusted and multivariable adjusteda.

Outcomes Vitamin D replete (>30 ng/ml), n= 79 Vitamin D insufficient (20–30 ng/ml), n= 70 Vitamin D deficient (<20 ng/ml), n= 39 P-value 
Clinical pregnancy rate 
All subjects 34 (43%) 29 (41%) 14 (36%) 0.48 
 Non-Hispanic Whites 23 (51%) 17 (44%) 3 (19%) 0.04 
 Hispanic Whites 5 (56%) 3 (43%) 2 (20%) 0.12 
 Asians 3 (15%) 7 (39%) 7 (64%) 0.01 
Adjusted clinical pregnancy ratea 
 Non-Hispanic Whites 55% 36% 21% 0.01 
 Hispanic Whites 68% 38% 15% 0.03 
 Asians 14% 34% 64% 0.01 
Live birth rate 
 All subjects 26 (33%) 22 (31%) 11 (28%) 0.62 
  Non-Hispanic Whites 20 (44%) 13 (33%) 2 (13%) 0.03 
  Hispanic Whites 3 (33%) 2 (29%) 2 (20%) 0.51 
  Asians 2 (10%) 5 (28%) 6 (55%) 0.01 
Adjusted live birth ratea 
 Non-Hispanic Whites 47% 27% 14% 0.01 
 Hispanic Whites 42% 26% 14% 0.19 
 Asians 9% 25% 53% 0.02 
Implantation rate 
 All subjects 19 ± 26% 18 ± 27% 16 ± 27% 0.38 
  Non-Hispanic Whites 24 ± 28% 18 ± 24% 7 ± 16% 0.02 
  Hispanic Whites 26 ± 29% 24 ± 37% 13 ± 32% 0.17 
  Asians 7 ± 17% 18 ± 31% 30 ± 32% 0.01 
Adjusted implantation ratea 
 Non-Hispanic Whites 25% 15% 9% 0.01 
 Hispanic Whites 31% 16% 9% 0.07 
 Asians 7% 14% 28% 0.01 
Outcomes Vitamin D replete (>30 ng/ml), n= 79 Vitamin D insufficient (20–30 ng/ml), n= 70 Vitamin D deficient (<20 ng/ml), n= 39 P-value 
Clinical pregnancy rate 
All subjects 34 (43%) 29 (41%) 14 (36%) 0.48 
 Non-Hispanic Whites 23 (51%) 17 (44%) 3 (19%) 0.04 
 Hispanic Whites 5 (56%) 3 (43%) 2 (20%) 0.12 
 Asians 3 (15%) 7 (39%) 7 (64%) 0.01 
Adjusted clinical pregnancy ratea 
 Non-Hispanic Whites 55% 36% 21% 0.01 
 Hispanic Whites 68% 38% 15% 0.03 
 Asians 14% 34% 64% 0.01 
Live birth rate 
 All subjects 26 (33%) 22 (31%) 11 (28%) 0.62 
  Non-Hispanic Whites 20 (44%) 13 (33%) 2 (13%) 0.03 
  Hispanic Whites 3 (33%) 2 (29%) 2 (20%) 0.51 
  Asians 2 (10%) 5 (28%) 6 (55%) 0.01 
Adjusted live birth ratea 
 Non-Hispanic Whites 47% 27% 14% 0.01 
 Hispanic Whites 42% 26% 14% 0.19 
 Asians 9% 25% 53% 0.02 
Implantation rate 
 All subjects 19 ± 26% 18 ± 27% 16 ± 27% 0.38 
  Non-Hispanic Whites 24 ± 28% 18 ± 24% 7 ± 16% 0.02 
  Hispanic Whites 26 ± 29% 24 ± 37% 13 ± 32% 0.17 
  Asians 7 ± 17% 18 ± 31% 30 ± 32% 0.01 
Adjusted implantation ratea 
 Non-Hispanic Whites 25% 15% 9% 0.01 
 Hispanic Whites 31% 16% 9% 0.07 
 Asians 7% 14% 28% 0.01 

Pregnancy and birth rates expressed as n (%); implantations rates expressed as the mean ± SD; adjusted rates expressed as %.

aAdjusted for maternal age, number of embryos transferred, embryo quality (average number blastomeres among transferred embryos) and diagnosis of diminished ovarian reserve.

Discussion

In this ethnically diverse population, we were able to confirm that vitamin D status is related to IVF success in non-Hispanic white patients; pregnancy rates declined with progressively lower levels of vitamin D. The odds of pregnancy were four times higher in vitamin D replete compared with deficient patients, substantiating the findings of Ozkan et al. (2010) in a larger study population. However, among Asians the beneficial effect of sufficient levels of vitamin D was not observed and in fact, vitamin D status was inversely related to IVF success. The influence of race on the relationship between vitamin D and clinical pregnancy was statistically significant.

We believe that our diverse study population is representative of a typical IVF population; we observed the expected associations between typical predictors of successful IVF outcomes and pregnancy rates. Our observed prevalence of vitamin D insufficiency (37%) and deficiency (21%) is similar to the prevalence in Ozkan's study (36 and 27% respectively), but is slightly lower than in national studies in women of childbearing age (Holick, 2009). This may be due to IVF population demographics, which tends to be older, mostly Caucasian, higher in socioeconomic status and education level (Chandra, et al. 2005; Ginde et al., 2010), and more likely to be taking prenatal vitamin supplementation. These demographics are associated with higher vitamin D status in population-based US surveys among reproductive aged women (Holick, 2009).

It is unclear why we saw an inverse relationship between 25(OH)D levels and clinical pregnancy rates among Asians. It is possible that though statistically significant, this is a chance finding. The number of Asians in the study was relatively small (n= 49). We could not identify any clinically important racial differences in patient characteristics or cycle parameters (including embryo quality) contributing to their lower pregnancy rates. It is possible that the influence of vitamin D status on pregnancy outcomes was overshadowed by other factors that contribute to the lower pregnancy rate observed among Asian patients (35% in Asians, 43% in non-Asians). Previous studies have demonstrated significantly lower live birth rates after IVF in Asian ethnicities compared with Caucasians (Hammoud et al., 2009), despite a younger age among Asian subjects and similar embryo quality (Hammoud et al., 2009; Butts and Seifer, 2010). Others have found a higher prevalence of diminished ovarian function in Asian-Chinese versus Caucasian oocyte donors, suggesting that oocyte factors may be involved in the lower IVF success rates in Asian populations (Gleicher et al., 2007; Shaine et al., 2009). Ideally, vitamin D levels in the recipients of donor IVF cycles could be studied to further eliminate confounding due to oocyte quality.

Finally, numerous studies have shown racial differences in the metabolism of vitamin D. In particular, southern Asians have been reported to have increased activity of the enzyme responsible for deactivating both 25(OH)D and calcitriol (Awumey et al., 1998). There are also ethnic differences in VDR gene polymorphisms that may confound or modify the relationship between vitamin D levels and reproductive outcomes (Ingles et al., 1997; Ingles, 2007). Our finding of significant heterogeneity by Asian ancestry suggests that the relationship between vitamin D and reproduction should be considered within the context of ethnicity.

Although vitamin D status was not associated with ovarian stimulation parameters or with markers of embryo quality, vitamin D levels were correlated with pregnancy outcomes in our non-Hispanic white population. It is possible that vitamin D may have an impact on embryo quality not captured using the current methods available to assess embryo viability. However, more plausible, is that vitamin D may exert an effect on IVF cycle outcomes via the endometrium. The VDR is expressed in the endometrium and plays a vital role in activating the innate immune response (Evans et al., 2004; Vigano et al., 2006). Additionally, vitamin D may play an important autocrine role through its regulation of the transcription of genes such as HOXA10, critical for embryo implantation and placentation (Evans et al., 2006).

Recent studies demonstrate that vitamin D is important throughout gestation as well, not just at the time of implantation. There are varying levels of vitamin D metabolites and HOXA10 expression throughout pregnancy in the endometrium, decidua and placenta (Evans et al., 2006). In cultured syncytiotrophoblasts, calcitriol regulates hCG expression and secretion, and it stimulates estradiol and progesterone secretion from trophoblasts in a dose-dependent manner (Barrera et al., 2007; Barrera et al., 2008). Clinical studies demonstrate an association between lower vitamin D levels and increased risk of pre-eclampsia and gestational diabetes. Thus, vitamin D may have an important role in maintaining a healthy pregnancy (Bodnar et al., 2007).

These data add clinical support to the growing body of evidence that vitamin D may play an important role in IVF success as well, possibly via localized effects in the endometrium. Research is needed to further elucidate the mechanism by which vitamin D acts, the ethnic heterogeneity in vitamin D metabolism and its subsequent effects on IVF success. The prevalence of vitamin D deficiency and insufficiency is alarmingly high in infertile patients. A role for vitamin D supplementation may exist as a means of improving one's natural fertility both among the fertile and the infertile. Regardless of potential fertility benefits, patients can be counseled regarding appropriate vitamin D supplementation for overall health benefits, pregnancy health and chronic disease risk reduction.

Authors’ roles

All authors were involved in the final design of the study. K.B. was the principal investigator of the study. All authors applied for IRB for approval. B.J.R., K.B., K.C., R.J.P. and S.A.I. coordinated the study and with F.S., analyzed the data. F.S. also provided technical support in running the blood samples. S.A.I. provided primary statistical support. K.B., SA.I. and B.J.R. were involved in the many first drafts of this paper. All authors contributed to the final draft of this paper. SA.I and K.B. contributed equally to this paper.

Funding

No external funding was either sought or obtained for this study.

Conflict of interest

None declared.

References

Awumey
EM
Mitra
DA
Hollis
BW
Kumar
R
Bell
NH
Vitamin D metabolism is altered in Asian Indians in the southern United States: a clinical research center study
J Clin Endocrin Metab
 , 
1998
, vol. 
83
 (pg. 
169
-
173
)
Barrera
D
Avila
E
Hernandez
G
Halhali
A
Biruete
B
Larrea
F
Diaz
L
Estradiol and progesterone synthesis in human placenta is stimulated by calcitriol
J Steroid Biochem Mol Biol
 , 
2007
, vol. 
103
 (pg. 
529
-
532
)
Barrera
D
Avila
E
Hernández
G
Méndez
I
González
L
Halhali
A
Larrea
F
Morales
A
Díaz
L
Calcitriol affects hCG gene transcription in cultured human syncytiotrophoblasts
Reprod Biol Endocrinol
 , 
2008
, vol. 
6
 pg. 
3
 
Bodnar
LM
Catov
JM
Simhan
HN
Holick
MF
Powers
RW
Roberts
JM
Maternal vitamin D deficiency increases the risk of preeclampsia
J Clin Endocrinol Metab
 , 
2007
, vol. 
92
 (pg. 
3517
-
3522
)
Butts
SF
Seifer
DB
Racial and ethnic differences in reproductive potential across the life cycle
Fertil Steril
 , 
2010
, vol. 
93
 (pg. 
681
-
690
)
Chandra
A
Martinez
GM
Mosher
WD
Abma
JC
Jones
J
Fertility, family planning, and reproductive health of U.S. women: data from the 2002 National Survey of Family Growth
Vital Health Stat
 , 
2005
, vol. 
25
 (pg. 
1
-
160
Series 23
Daftary
GS
Taylor
HS
Endocrine regulation of HOX genes
Endoc Rev
 , 
2006
, vol. 
27
 (pg. 
331
-
355
)
Evans
KN
Bulmer
JN
Kilby
MD
Hewison
M
Vitamin D and placental-decidual function
J Soc Gynecol Investig
 , 
2004
, vol. 
11
 (pg. 
263
-
271
)
Evans
KN
Nguyen
L
Chan
J
Innes
BA
Bulmer
JN
Kilby
MD
Hewison
M
Effects of 24-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 on cytokine production by human decidual cells
Biol Reprod
 , 
2006
, vol. 
75
 (pg. 
816
-
822
)
Ginde
A
Sullivan
AF
Mansbach
JM
Camargo
CA
Jr
Vitamin D insufficiency in pregnant and nonpregnant women of childbearing age in the United States
Am J Obstet Gynecol
 , 
2010
, vol. 
202
 (pg. 
436.e1
-
436.e8
)
Gleicher
N
Weghofer
A
Li
J
Barad
D
Differences in ovarian function parameters between Chinese and Caucasian oocyte donors: do they offer an explanation for lower IVF pregnancy rates in Chinese women?
Hum Reprod
 , 
2007
, vol. 
22
 (pg. 
2879
-
2882
)
Gray
TK
McAdoo
T
Hatley
L
Lester
GE
Thierry
M
Fluctuation of serum concentration of 1,25-dihydroxyvitamin D3 during the menstrual cycle
Am J Obstet Gynecol
 , 
1982
, vol. 
144
 (pg. 
880
-
884
)
Halloran
BP
DeLuca
HF
Effect of vitamin D deficiency on fertility and reproductive capacity in the female rat
J Nutr
 , 
1980
, vol. 
110
 (pg. 
1573
-
1580
)
Hammoud
AO
Gibson
M
Stanford
J
White
G
Carrell
DT
Peterson
M
In vitro fertilization availability and utilization in the United States: a study of demographic, social, and economic factors
Fertil Steril
 , 
2009
, vol. 
91
 (pg. 
1630
-
1635
)
Holick
MF
Vitamin D deficiency: review
N Engl J Med
 , 
2007
, vol. 
357
 (pg. 
266
-
281
)
Holick
M
J Clin Endocrinol Metab
 , 
2009
, vol. 
94
 (pg. 
1092
-
1093
)
Hollis
BW
Kamerud
JQ
Selvaag
SR
Lorenz
JD
Napoli
JL
Determination of vitamin D status by radioimmunoassay with an 125I-labeled tracer
Clin Chem
 , 
1993
, vol. 
39
 (pg. 
529
-
533
)
Hosmer
DW
Jr
Lemeshow
S
Goodness-of-fit tests for the multiple logistic regression model
Commun Stat
 , 
1980
, vol. 
A9
 (pg. 
1043
-
1069
)
Ingles
SA
Can diet and/or sunlight modify the relationship between vitamin D receptor polymorphisms and prostate cancer risk?
Nutr Rev
 , 
2007
, vol. 
65
 (pg. 
S105
-
S107
)
Ingles
SA
Haile
RW
Henderson
BE
Kolonel
LN
Nakaichi
G
Shi
CY
Yu
MC
Ross
RK
Coetzee
GA
Strength of linkage disequilibrium between two vitamin D receptor markers in five ethnic groups: implications for association studies
Cancer Epidemiol Biomarkers Prev
 , 
1997
, vol. 
6
 (pg. 
93
-
98
)
Johnson
LE
DeLuca
HF
Vitamin D receptor null mutant mice fed high levels of calcium are fertile
J Nutr
 , 
2001
, vol. 
131
 (pg. 
1787
-
1791
)
Kinuta
K
Tanaka
H
Moriwake
T
Aya
K
Kato
S
Seino
Y
Vitamin D is an important factor in estrogen biosynthesis of both female and male gonads
Endocrinology
 , 
2000
, vol. 
141
 (pg. 
1317
-
1324
)
Kwiecinski
GG
Petrie
GI
DeLuca
HF
Vitamin D is necessary for reproductive functions of the male rat
J Nutr
 , 
1989
, vol. 
119
 (pg. 
741
-
744
)
Looker
AC
Pfeiffer
CM
Lacher
DA
Schleicher
RL
Picciano
MF
Yetley
EA
Serum 25-hydroxyvitamin D status of the US population: 1988–1994 compared with 2000–2004
Am J Clin Nutr
 , 
2008
, vol. 
88
 (pg. 
1519
-
1527
)
Oliviennes
F
Fanchin
R
Bouchard
P
de Ziegler
D
Taieb
J
Selva
J
Frydman
R
The single or dual administration of the gonadotropin-releasing hormone antagonist Cetrorelix in an in vitro fertilization-embryo transfer program
Fertil Steril
 , 
1994
, vol. 
62
 (pg. 
468
-
476
)
Ozkan
S
Jindal
S
Greenseid
K
Shu
J
Zeitlian
G
Hickmon
C
Pal
L
Replete vitamin D stores predict reproductive success following in vitro fertilization
Fertil Steril
 , 
2010
, vol. 
94
 (pg. 
1314
-
1319
)
Porter
RN
Smith
W
Craft
IL
Abdulwahid
NA
Jacobs
HS
Induction of ovulation for in vitro fertilization using buserelin and gonadotropins
Lancet
 , 
1984
, vol. 
324
 (pg. 
1284
-
1285
)
Potashnik
G
Lunenfeld
E
Levitas
E
Itskovitz
J
Albutiano
S
Yankowitz
N
Sonin
Y
Levy
J
Glezerman
M
Shany
S
The relationship between endogenous oestradiol and vitamin D3 metabolites in serum and follicular fluid during ovarian stimulation for in-vitro fertilization and embryo transfer
Hum Reprod
 , 
1992
, vol. 
7
 (pg. 
1357
-
1360
)
Scott
RT
Navot
D
Enhancement of ovarian responsiveness with microdoses of gonadotropin-releasing hormone agonist during ovulation induction for in vitro fertilization
Fertil Steril
 , 
1994
, vol. 
61
 (pg. 
880
-
885
)
Shaine
LK
Lamb
JD
Lathi
RB
Milki
AA
Langen
E
Westphal
LM
Poor prognosis with in vitro fertilization in Indian women compared to Caucasian women despite similar embryo quality
PLoS One
 , 
2009
, vol. 
4
 pg. 
e7599
 
Vigano
P
Lattuada
D
Mangioni
S
Ermellino
L
Vignali
M
Caporizzo
E
Panina-Bordignon
P
Besozzi
M
Di Blasio
AM
Cycling and early pregnant endometrium as a site of regulated expression of the vitamin D system
J Mol Endocrinol
 , 
2006
, vol. 
36
 (pg. 
415
-
424
)
Yoshizawa
T
Handa
Y
Uematsu
Y
Takeda
S
Sekine
K
Yoshihara
Y
Kawakami
T
Arioka
K
Sato
H
Uchiyama
Y
, et al.  . 
Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning
Nat Genet
 , 
1997
, vol. 
16
 (pg. 
391
-
396
)

Author notes

These authors contributed equally to this manuscript.