Abstract
Objective
To examine the association between serum 25-hydroxyvitamin D (25(OH)D) and menstrual cycle length and regularity.
Design
Community-based, cross-sectional study of serum 25(OH)D (adjusted for seasonal differences in timing of blood draw) and menstrual cycle length. Women ages 23-34 reported their gynecologic history. Menstrual cycles were described with four independent categories (normal, short, long, irregular). We used polytomous logistic regression to estimate the association between a doubling of seasonally-adjusted 25(OH)D and the odds of each cycle category.
Setting
Women from the Detroit, Michigan area attended a study clinic visit.
Participants
1102 African-American women ages 23-34.
Intervention
None
Main Outcome Measure
Self-reported menstrual cycle length over the previous 12 months excluding women who were using cycle-regulating medications over the entire year. Women who reported that their cycles were “too irregular to estimate” were classified as having irregular cycles. A typical cycle length of <27 days was considered “short,” >34 days was “long,” and 27-34 days was “normal”.
Results
The median 25(OH)D level was 14.7 ng/ml (interquartile range: 10.9, 19.6). A doubling of 25(OH)D was associated with half the odds of having long menstrual cycles (adjusted odds ratio (aOR) (95% Confidence interval (CI): 0.54 (0.32, 0.89)). 25(OH)D was not associated with the occurrence of short (aOR(CI): 1.03 (0.82, 1.29)) or irregular (aOR(CI): 1.46 (0.88, 2.41) menstrual cycles. Results were robust to several sensitivity analyses.
Conclusions
These findings suggest that vitamin D status may influence the menstrual cycle and play a role in ovarian function. Further investigation of 25(OH)D and ovarian hormones, and prospective studies of 25(OH)D and cycle length, are needed.
Keywords: vitamin D, ovarian function, ovulation, PCOS
Background
Vitamin D is important for bone health (1), and its importance for reproduction is a burgeoning area of research (2-4). Vitamin D receptor is expressed in several reproductive tissues including the ovary, placenta, and the uterus (2-4). Recent data suggest that lower vitamin D may be associated with several reproductive endpoints including uterine fibroids (5) and early menarche (4), but data are limited.
Diet-induced vitamin D deficiency reduces fertility in both rats and mice. The probability of becoming pregnant is reduced 45-70% with a concomitant 67-100% reduction in the number of viable pups (2). Knock-out mice lacking the enzyme for converting the circulating form of vitamin D to its active form show estrus cycle disturbances including arrested follicular development, prolonged estrous cycles, and anovulation (6, 7).
Vitamin D deficiency has been associated with polycystic ovarian syndrome (PCOS) in multiple studies (reviewed in (8)). Moreover, among women with PCOS, supplementation with vitamin D has been reported to normalize menstrual cycles and improve ovarian folliculogenesis and ovulation (9, 10). Despite the growing PCOS literature, data regarding vitamin D and menstrual cycle function in healthy women are sparse. Only one previous study has examined the association between concentrations of 25-hydroxyvitamin D (25(OH)D), the accepted biomarker of vitamin D status, and menstrual cycle length and regularity (11). This previous study, in 35-44 year olds (N=636), reported that decreasing 25(OH)D was associated with an increase in the odds of irregular menstrual cycles.
Our primary objective was to examine the association of vitamin D with menstrual cycle length and regularity in a population of African-American women from the Detroit area.
Methods
Study sample
The Study of Environment, Lifestyle, & Fibroids (SELF) was designed to prospectively measure the incidence and growth of uterine fibroids (14). Vitamin D was one of the primary environmental exposures of interest. A complete description of the eligibility requirements, recruitment and other study methods has been published (14). Briefly, in 2010-2012 African-American women ages 23-34 were recruited from the Detroit, Michigan area through letters from the collaborating health system, local radio, television, newspaper and magazine advertisements, fliers and brochures at health clinics throughout the community, and information booths at community events. Women were not eligible for the study if they reported a prior clinical diagnosis of fibroids. Enrollment involved completion of several steps including a computer-assisted telephone interview (CATI), a computer-assisted web-based interview (CAWI), a self-administered hard copy questionnaire, and a clinic visit.
Information was collected on demographics, alcohol, smoking, physical activity, hormone use, gynecologic and reproductive history, and history of medical diagnoses including vitamin D deficiency. At enrollment, the participants attended a clinic visit where height and weight were measured and a blood sample was collected.
All of the enrolled women (N=1696) provided informed consent, and the study protocol was approved by the institutional review boards at NIEHS and Henry Ford Health System in Detroit.
Menstrual cycle characteristics
Women were excluded from the menstrual cycle analysis if they reported that their last menses was more than one year prior to the interview (N=90) or they had only had one period in the last year (N=37) (the majority of both groups lacked periods due to pregnancy/breastfeeding). Women were also excluded if they reported currently using hormonal contraception (N=403) or any other medication that regulated their menstrual cycles for “all” of the year prior to interview (N=33). (Women who reported using a medication that regulated their menstrual cycles for “some” of the year prior to interview were included, and investigated in a sensitivity analysis that is described in the “statistical analysis” section.) After these exclusions, 1133 women were eligible for our menstrual cycle analyses.
Women were asked to report their typical menstrual cycle length in the past year during the CATI. The CATI was completed close in time to the clinic visit, and most women completed the CATI the month before, or during, the clinic visit. Menstrual cycle length and regularity was described with four mutually exclusive categories. Women who reported that their cycles were “too irregular to estimate” were classified as having irregular cycles (N=53). Women who reported a typical cycle length of ≤26 days were classified as having “short” cycles (N=380) and ≥35 days were classified as having “long” cycles (N=55). “Normal” cycle length was 27-34 days (N=632). Eight women said they did not know their typical cycle length and one woman refused to answer, these women were excluded from the analysis. Four women reported a typical cycle length of less than 12 days and were also excluded. After these exclusions, 1120 women remained eligible for our analyses.
Use of contraceptive methods to treat menstrual pain or irregularity
The analysis of menstrual cycle characteristics was limited to women not using hormonal contraception. This exclusion could potentially bias the analysis: if lower vitamin D causes menstrual problems, and women tend to use hormonal contraception to treat menstrual problems, we would be excluding the very women who are showing the physiologic effects of low vitamin D. We do not have data on current use of a hormonal contraception for treatment of menstrual problems, but we do have ever/never data. Therefore, as a secondary analysis, we examined whether women with a previous diagnosis of vitamin D deficiency were also more likely to have ever used a hormonal contraceptive for irregular cycles or for menstrual pain.
Women were asked about their use of several different hormonal contraceptive methods. They were also asked, for each method, if they had used that method to treat menstrual pain or to treat irregular menstrual cycles. We created two composite variables: one to summarize whether she had ever used any contraceptive method to treat menstrual pain (yes/no), and another to describe whether she had ever used a contraceptive method to treat irregular menstrual cycles (yes/no). All of the 1696 participants provided enough information to be classified as yes or no for both of these variables (i.e. none of the women were “missing”).
Vitamin D status
Vitamin D status was quantified in stored serum samples through the measurement of 25-hydroxyvitamin D (25(OH)D), the accepted biomarker of vitamin D status (15). Eighteen women had an unsuccessful blood draw and thus did not have a measure of 25(OH)D, leaving 1102 women in our analyses of 25(OH)D and menstrual cycles.
25(OH)D was quantified with an FDA approved direct, competitive chemiluminescence immunoassay (CLIA) using the DiaSorin LIAISON 25-OH Vitamin D Total assay (16, 17). This assay is co-specific for 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2. The sensitivity of the assay is 2.5 ng/ml and the recovery of endogenous 25(OH)D is 100% (18). The intra- and inter-assay CVs based on blind replicates were 2.9% and 8.6%, respectively. Despite the acceptable inter-assay CV, we found that the second, smaller batch sent for assay showed somewhat lower values for the blind controls than the first batch. To account for any potential batch-effects, all primary analyses were rerun using only women whose samples were assayed in the first shipment. Our results did not change, and only the full-sample results are shown.
Because 25(OH)D varies with season or temporal changes in supplement use, and because our menstrual cycle questions asked about cycles in the last 12 months, we estimated an annual mean 25(OH)D using a cosinor model (19) (Supplemental Text). Data collected on supplement use was incorporated into this model because there was less seasonal variation for supplement users. For simplicity, this estimated annual mean 25(OH)D will be referred to as “25(OH)D”.
History of vitamin D deficiency
Participants were asked, “Has a doctor or other health professional ever told you that you had any of the following conditions…Vitamin D deficiency?” If she said yes, she was further asked at what age (in years) she received the diagnosis. Eighteen women were missing data for this question, which left 1678 women in our analysis of vitamin D deficiency and the use of contraceptive methods for menstrual pain or irregularity.
Potential covariates
Potential covariates were selected from the literature due to their associations with menstrual cycle length and/or vitamin D status and included: age (20, 21), education (22, 23), income, body mass index (19, 23-26), alcohol intake (21), cigarette smoking (20, 21, 23, 27), and physical activity (19, 21, 23, 26). Most data on covariates were collected through the CATI, CAWI, and clinic interviews. A 5-level physical activity variable was created based on data from several questions about walking, sports, exercise routines/classes, recreational activities like dancing, household chores, and time spent doing heavy manual labor at work (see supplemental text for details). BMI was calculated from weight and height measured during the clinic visit.
Statistical analysis
Analyses of 25(OH)D and menstrual cycle characteristics
To achieve normality, and for ease of interpretation (see below) 25(OH)D was modeled on the log2 scale. We examined the univariable associations between 25(OH)D and the covariates of interest using medians and the corresponding interquartile ranges.
We used polytomous logistic regression (also known as multinomial regression) to simultaneously estimate the associations between 25(OH)D and short, long and irregular cycles. Women who reported a typical cycle length of 27-34 days were considered the referent group. We estimated two separate models, one with a dichotomous 25(OH)D measure and one with a continuous measure (on the log2 scale). The dichotomous 25(OH)D variable was defined as “sufficient” versus “deficient” based on the Institute of Medicine cutpoint of 20 ng/mL (28). The regression coefficient from the continuous model was exponentiated to obtain the association between a doubling of 25(OH)D and the odds of each cycle category. The levels of 25(OH)D in this population are low (median: 14.8 ng/ml), so a doubling of 25(OH)D can be thought of as changing a woman from the range of deficiency to sufficiency. This model has more power than the dichotomous exposure measure because it is continuous, and because the majority of participants are deficient (76%). The multivariable model was adjusted for age (continuous), education (three categories), income (three categories), BMI (four categories), smoking (never, former, <10 cig/day, ≥10 cig/day), alcohol (three categories), and physical activity (five categories). Nine women were missing information for income, one woman was missing education, and three women were missing physical activity information. These women were excluded from the multivariable model, leaving 1089 women in the analysis.
We performed several sensitivity analyses. First, we excluded women with a recent diagnosis of vitamin D deficiency. We did this because once diagnosed, women are likely to begin supplements. Thus, a current 25(OH)D measurement would reflect the recent supplement use, but her reporting of menstrual cycles over the past 12 months would likely reflect, at least in part, her pre-supplement cycle characteristics. Because we wanted to identify diagnoses of vitamin D deficiency that occurred within the past 12 months, and we only asked about age at diagnosis, we conservatively excluded women with a vitamin D deficiency at an age that was within 2 years of her age at enrollment (N=81).
We performed three additional, independent, sensitivity analyses in which we removed women whose cycles might be influenced by other medical conditions or experiences: 1) Women who reported a history of PCOS (N=36), 2) women who reported that, over the past year, they used a medication that regulated their menstrual cycles “some of the time” (N=155), and 3) women who had been pregnant during the past year but were cycling at the interview (N=100).
As a final sensitivity analysis, we adjusted for the presence of fibroids detected at the enrollment ultrasound because prior studies have reported an association between vitamin D deficiency and fibroids (reviewed in (29)).
Analyses of vitamin D deficiency and use of contraceptive methods to treat menstrual pain or irregularity
The associations of a history of vitamin D deficiency with ever/never use of a contraceptive method to treat either 1) menstrual pain, or 2) menstrual irregularity, were examined initially with frequency tables. Two multivariable logistic regression models were used to estimate the associations between a history of vitamin D deficiency and ever/never contraceptive use to treat 1) menstrual pain or 2) menstrual irregularity. Due to the retrospective nature of the data for this analysis, we did not adjust for current behaviors or characteristics as these do not reflect the relevant time period for this analysis, with the exception of BMI. Because current BMI tends to be highly correlated with a past BMI measure (30), we adjusted for current BMI as a surrogate for BMI during the relevant time period. We also adjusted for smoking history (ever/never) as smoking is a suspected risk factor for menstrual pain (31-33). We adjusted for education as a potential marker of a woman's access to healthcare and treatment-seeking behavior. We also adjusted for age at enrollment because older women had more years during which they could have been diagnosed with a vitamin D deficiency or have used hormonal contraception to manage menstrual cycle problems.
Results
25(OH)D and menstrual cycle characteristics
The median cycle length was 28 days (interquartile range: 25, 30). The median 25(OH)D level was 14.7 ng/ml (interquartile range: 10.9, 19.6). The majority of women were vitamin D deficient, with 76% of the women (N=838) having a 25(OH)D of 20 ng/ml or less. 25(OH)D was lower in women who reported long menstrual cycles (Table 1). Average 25(OH)D was also lower among women who reported lower education, lower income, higher BMI, current smoking, high alcohol use, and low physical activity.
Table 1. Participant characteristics SELF cohort (N=1102).
| N (%) | Median 25(OH)D (ng/ml) (interquartile range) | |
|---|---|---|
| Menstrual cycle length | ||
| Short (< 27 days) | 373 (34) | 14.8 (11.1, 19.2) |
| Long (≥ 35 days) | 55 (5) | 11.8 (9.8, 14.5) |
| Irregular | 51 (5) | 16.6 (10.7, 21.5) |
| Normal (27 – 34 days) | 623 (57) | 14.8 (11.0, 20.3) |
| Seasona | ||
| Winter | 225 (20) | 13.3 (9.8, 17.7) |
| Spring | 253 (23) | 13.4 (9.9, 19.4) |
| Summer | 326 (30) | 17.8 (12.9, 22.6) |
| Fall | 298 (27) | 14.1 (10.9, 19.2) |
| Age | ||
| 23 – 25 | 227 (21) | 14.3 (11.0, 19.4) |
| 26 – 30 | 460 (42) | 14.6 (10.7, 18.8) |
| 31 – 35 | 415 (38) | 15.1 (11.0, 20.7) |
| Education | ||
| High school graduate or less | 269 (24) | 13.3 (9.9, 17.8) |
| Some college/technical | 548 (50) | 14.7 (11.1, 19.4) |
| College graduate or more | 284 (26) | 16.4 (11.4, 22.7) |
| Missing | 1 | |
| Annual income | ||
| < 20K | 534 (49) | 13.8 (10.5, 18.5) |
| 20 – 50K | 392 (36) | 15.1 (10.9, 19.9) |
| > 50K | 167 (15) | 16.6 (12.4, 21.9) |
| Missing | 9 | |
| Body mass index | ||
| < 25 kg/m2 | 208 (19) | 17.2 (12.3, 21.8) |
| 25 - < 30 | 208 (19) | 15.2 (11.2, 20.2) |
| 30 - <35 | 208 (19) | 16.2 (11.2, 22.3) |
| ≥ 35 | 478 (43) | 13.5 (10.3, 17.6) |
| Smoker | ||
| Non-smoker | 776 (70) | 14.9 (11.1, 20.7) |
| Former | 86 (8) | 16.1 (12.5, 19.2) |
| Current | ||
| < 10 cig/day | 174 (16) | 13.3 (9.8, 17.0) |
| ≥ 10 cig/day | 66 (6) | 12.9 (9.3, 17.4) |
| Alcoholb | ||
| Low | 343 (31) | 14.8 (11.3, 20.2) |
| Medium | 530 (48) | 15.0 (11.0, 19.8) |
| High | 229 (21) | 13.5 (9.9, 18.3) |
| Physical activity | ||
| Low | 181 (16) | 13.3 (10.2, 18.1) |
| Low/Moderate | 257 (23) | 14.3 (10.7, 18.7) |
| Moderate | 280 (25) | 14.9 (10.9, 19.5) |
| High | 212 (19) | 15.8 (11.3, 21.9) |
| Very high | 169 (15) | 15.9 (11.1, 20.7) |
| Missing | 3 | |
25(OH)D levels in this row are the unadjusted, measured values. The other rows of the table show the average annual mean 25(OH)D (see Methods).
Medium: 1 – 5 drinks/day or ≥ 4 drinks on a single occasion once per month or less, High: ≥ 6 drinks/day or ≥ 4 drinks on a single occasion more than once per month.
The overall adjusted association between continuous 25(OH)D and cycle category (normal cycle length of 27-34 days, short, long, irregular) was statistically significant by a 3 degree-of-freedom likelihood ratio test (p=0.04). A doubling of 25(OH)D was associated with about half the odds of having long menstrual cycles (Adjusted odds ratio (aOR) (95% Confidence interval (CI): 0.54 (0.32, 0.89)) (Table 2). 25(OH)D was not statistically significantly associated with the occurrence of short or irregular menstrual cycles. Women who were sufficient in their 25(OH)D (>20 ng/ml) had about half the odds of long cycles compared with women who were deficient (aOR(CI): 0.42 (0.17, 1.0)) (Table 2). Sufficiency was not associated with short or irregular menstrual cycles (Table 2).
Table 2.
The association between 25(OH)D and menstrual cycle length and regularity among African-American women in the SELF study (N=1089).
| N | Adjusted* OR (95% CI) for a doubling† of 25(OH)D | |
|---|---|---|
| Short cycles (≤26 days) | 367 | 1.03 (0.82, 1.29) |
| Long cycles (≥35 days) | 54 | 0.54 (0.32, 0.89) |
| Irregular cycles | 51 | 1.46 (0.88, 2.41) |
| Sufficient 25(OH)D (compared with deficient) | ||
|
|
||
| Short cycles (≤26 days) | 367 | 0.82 (0.60, 1.13) |
| Long cycles (≥35 days) | 54 | 0.42 (0.17, 1.02) |
| Irregular cycles | 51 | 1.39 (0.72, 2.69) |
Adjusted for: age, BMI, education, income current smoking, alcohol use, and physical activity. Referent category: 27 -34 days (N=617)
Given the low levels of 25(OH)D in this population, a “doubling” of 25(OH)D can be thought of as changing most of the women from the range of deficiency to sufficiency.
The association between continuous 25(OH)D and long menstrual cycles persisted with sensitivity analyses (Supplemental Table 1). The aOR did not change, but the confidence interval was even smaller when the sample was limited to women who were not recently diagnosed with vitamin D deficiency (long aOR(CI): 0.48 (0.28, 0.83)). Removing women who reported a history of PCOS had only minor effects (long aOR(CI): 0.57 (0.33, 0.97)). Nor was there substantial change in the results with removal of women who reported using a medication that regulated their menstrual cycles “some of the time” or women who had been pregnant during the past year or after adjustment for fibroid status at enrollment (Supplemental Table 1).
History of vitamin D deficiency and ever use of contraception for menstrual pain or irregularity
Of 1678 women, 223 (13%) reported that a doctor or other health care professional had told them they were vitamin D deficient. The timing of diagnosis of the deficiency ranged from their current age to 28 years earlier, but most were diagnosed within 4 years of enrollment (91%). Fifty (23%) were the same age at baseline as they were at their diagnosis and 77 (35%) had been diagnosed one year earlier. Of the 1678, 251 women reported ever use of hormonal contraceptives for menstrual pain and 579 reported ever use for irregular menstrual cycles. Of the 223 women with a history of vitamin D deficiency, 17 (7.6%) also reported being told by a health care professional that they had PCOS. This was a higher proportion than among women who did not report a history of vitamin D deficiency (2.5%), thus history of vitamin D deficiency was also associated with PCOS.
Women with a history of vitamin D deficiency were more likely to report ever using a contraceptive method for either menstrual pain or for irregular cycles (Table 3). The results were unchanged when we excluded women who were first diagnosed with vitamin D deficiency at less than 15 years of age (N=4) (data not shown). Given the observed associations between history of vitamin D deficiency and PCOS, we performed a sensitivity analysis excluding women with PCOS. The estimate for menstrual pain was slightly stronger (aOR(CI): 1.63 (1.12, 2.37)), while the estimate for ever use of a method to treat irregular cycles was slightly weaker (aOR(CI): 1.33 (0.98, 1.81)).
Table 3.
Associations between a history of clinically-recognized vitamin D deficiency and the use of contraceptive methods to treat menstrual pain or irregularity among African-American women in the SELF study (N=1678).
| History of clinically-recognized vitamin D deficiency | |||
|---|---|---|---|
|
| |||
| Yes (N=223) N (%) | No (N=1455) N (%) | Adjusted* OR (95% CI) for history of vitamin D deficiency | |
| Ever used a contraceptive method to treat menstrual pain | 46 (21) | 205 (14) | 1.53 (1.06, 2.21) |
| Ever used a contraceptive method to treat irregular menstrual cycles | 94 (42) | 485 (33) | 1.37 (1.04, 1.84) |
Adjusted for age, BMI, education, and ever smoking, all measured at enrollment.
Discussion
Our study is the first to examine the association between 25(OH)D and menstrual cycle length and regularity in reproductive aged African-American women. We observed that an increase in 25(OH)D was associated with a reduced odds of long menstrual cycles (but not short or irregular cycles). This association remained robust after several sensitivity analyses. Only 24% of our participants were sufficient in vitamin D, which reduced our power to detect an effect of sufficiency. However, the analysis comparing women with sufficient and deficient 25(OH)D showed the same direction of effect and borderline significance, i.e., women with sufficient 25(OH)D had a reduced likelihood of long cycles.
One previous study has reported a shortening of long (30-38 days) menstrual cycles among women exposed to phototherapy in a crossover design (it is unclear if the participants received any ultraviolet light exposure) (34). In the only previous study of 25(OH)D and menstrual cycles, increasing 25(OH)D was associated with a reduced odds of irregular cycles (but not with long menstrual cycles)(11). The differences between these two studies could be the result of the disparate study populations, as the women in the previous analysis were older (35-44 years). Long and irregular cycles may be different in younger versus older women. For example, the irregular cycles of PCOS may resolve with age (35), while the irregular cycles associated with perimenopause will be rare in young women.
Our menstrual cycle analysis required the exclusion of women using hormonal contraceptives because they were not having natural cycles. Because some women use these to manage menstrual problems, this analysis could be biased by selective removal of women with menstrual problems. Therefore, we used an indirect approach to investigate the association between vitamin D and menstrual cycle problems for which our whole sample could be analyzed. We looked at the association between having a history of vitamin D deficiency and having ever used a contraceptive for menstrual pain or for irregular menstrual cycles, and found positive associations. These findings further support our primary analysis, again suggesting that vitamin D influences menstrual cycle characteristics. These results are also consistent with one recent randomized trial that found that treatment with vitamin D reduced the symptoms of dysmenorrhea (13). Vitamin D may influence menstrual pain through its hypothesized roles as an immune modulator or a prostaglandin inhibitor (12).
25(OH)D is converted to its active form 1,25(OH)2D by the enzyme 1α-hydroxylase, coded by the CYP27B1 gene. Mice that lack either Cyp27b1 or the vitamin D receptor have shown hypogonadism, arrested follicular development, prolonged estrous cycles, and hypoplastic uteri (6, 7, 36, 37). The reproductive phenotypes in these studies may be the result of either suboptimal gonadotropin secretion from the pituitary or hypothalamus, or defects in the ovarian response to gonadotropins (6). In one study, the prolonged estrous cycles were reversed with vitamin D3 supplementation and occurred independently of calcium (6). Although in another study the effects appeared to be calcium dependent (38).
In humans, long menstrual cycles are typically the result of a long follicular phase (22, 39, 40). Long menstrual cycles can arise from several mechanisms including decreased ovarian responsiveness to gonadotrophin stimulation(39) and hypoestrogenic intervals during the follicular phase (also known as “inactive phases”) (41). These inactive phases have mostly been described for perimenopausal women, however, they do occur in premenopausal women (41), but the reasons for their occurrence in younger women are unknown. The hormonal milieu of long cycles is variable (42). Some long cycles show a delay in the follicular rise of estrogen (42, 43), while others show typical early follicular increases in estrogen followed by either episodic rises and falls or by prolonged high estrogen {Harlow, 2000 #259}. A SNP in the follicle stimulating hormone receptor that induces a higher ovarian threshold to FSH has been related to longer menstrual cycle length (44). Anovulation appears to occur in both short and long menstrual cycles and therefore does not explain consistently long cycles (43).
The associations between vitamin D and cycle length may involve Anti-Müllerian hormone (AMH), which helps regulate follicle recruitment in the ovary (4) (also reviewed in (8)). AMH is produced in the granulosa cells of early-developing follicles, and its hypothesized actions include inhibition of primordial follicle recruitment, slowing of follicular growth and thus delaying or preventing atresia, and inhibiting granulosa cell differentiation (8, 45). AMH, antral follicle count and ovarian volume have been positively associated with menstrual cycle length in 200 healthy women and several lines of evidence support a role for AMH in menstrual cycle function (46). The promoter region for the human gene encoding AMH contains a domain for the vitamin D response element, suggesting that vitamin D may regulate AMH expression (47). Vitamin D has also been shown to modulate AMH signaling in human luteinized granulosa cells (48).
This study has several limitations. First, this analysis is limited by the use of self-reported cycle length and by the relatively small number of women with extreme cycle lengths, particularly long or irregular cycles. Second, this study is based on a cross-sectional design: women gave a blood sample around the time that they retrospectively reported their typical cycle length for the past year. Thus, this study is susceptible to reverse causation, i.e. it is possible that the occurrence of long cycles affects diet, supplement use, or another behavior (such as seeking medical advice) that leads to changes in 25(OH)D status. Third, it is possible that some of the women in our analysis had undiagnosed PCOS. Our finding that vitamin D sufficiency might protect against long cycles, may derive from a subset of women with subclinical PCOS that is characterized by both low vitamin D and long menstrual cycles. We did not have hormonal or ultrasound markers with which to identify women with PCOS. However, our results were robust to the exclusion of women with known PCOS. Fourth, we did not have information regarding current use of oral contraceptives to treat menstrual pain or irregularity, and thus our secondary analysis was confined to data on history of vitamin D deficiency and ever/never use of hormonal contraception for menstrual pain or irregularity. Fifth, 76% of our sample was deficient in vitamin D, while only 13% reported a history of clinically-diagnosed vitamin D deficiency. This indicates that the majority of those with deficient vitamin D concentrations were not previously clinically diagnosed and suggests misclassification of history of vitamin D deficiency, which could bias our observed associations toward the null.
This study has several strengths. The sample size was large and focused on a clinically-relevant population, African-American women, who tend to have lower 25(OH)D levels (49). Several community-based recruitment methods were used to generate a representative sample. Generalizability might be limited somewhat as only motivated women were likely to complete all enrollment steps. Collection of menstrual cycle data was standardized. Vitamin D status was assessed with the accepted biomarker, 25(OH)D (15), which was carefully adjusted for seasonal variation. The study data collection was extensive, allowing for adjustment of potential confounders. In addition, we performed several sensitivity analyses to evaluate the potential for bias and our results were robust to each of them.
Conclusions
These findings suggest that vitamin D status may influence the menstrual cycle and may play a role in ovarian function. The role of vitamin D in reproduction is an emerging field of research (8), and these findings need replication. Further investigation of 25(OH)D and cycle characteristics, including ovarian hormones and symptoms of PCOS, is warranted. Detailed studies of the possible role(s) of vitamin D in follicular recruitment and selection, as well as follicular growth, would be of interest. Prospective trials of vitamin D and cycle length are feasible.
Supplementary Material
Supplemental Table 1. Sensitivity analyses of the association between 25(OH)D and menstrual cycle length and regularity among African-American women in the SELF study (N=1089).
Acknowledgments
Research reported in this publication was supported by NICHD of the National Institutes of Health under award number R00HD079659. This research was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences, by funds allocated for health research by the American Recovery and Reinvestment Act, and by the NIH Office of Dietary Supplements. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. We would like to thank Drs. Anne Steiner and Srishti Shrestha for their comments on an earlier draft of this manuscript.
Footnotes
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Contributor Information
Anne Marie Z. Jukic, Email: annemarie.jukic@yale.edu.
Kristen Upson, Email: kristen.upson@nih.gov.
Quaker E. Harmon, Email: quaker.harmon@nih.gov.
Donna D. Baird, Email: baird@niehs.nih.gov.
References
- 1.Holick MF. Vitamin D deficiency. N Engl J Med. 2007;357:266–81. doi: 10.1056/NEJMra070553. [DOI] [PubMed] [Google Scholar]
- 2.Lerchbaum E, Obermayer-Pietsch B. Vitamin D and fertility: a systematic review. Eur J Endocrinol. 2012;166:765–78. doi: 10.1530/EJE-11-0984. [DOI] [PubMed] [Google Scholar]
- 3.Evans KN, Bulmer JN, Kilby MD, Hewison M. Vitamin D and placental-decidual function. J Soc Gynecol Investig. 2004;11:263–71. doi: 10.1016/j.jsgi.2004.02.002. [DOI] [PubMed] [Google Scholar]
- 4.Luk J, Torrealday S, Neal Perry G, Pal L. Relevance of vitamin D in reproduction. Hum Reprod. 2012;27:3015–27. doi: 10.1093/humrep/des248. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Baird DD, Hill MC, Schectman JM, Hollis BW. Vitamin d and the risk of uterine fibroids. Epidemiology. 2013;24:447–53. doi: 10.1097/EDE.0b013e31828acca0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Dicken CL, Israel DD, Davis JB, Sun Y, Shu J, Hardin J, et al. Peripubertal vitamin D(3) deficiency delays puberty and disrupts the estrous cycle in adult female mice. Biol Reprod. 2012;87:51. doi: 10.1095/biolreprod.111.096511. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Panda DK, Miao D, Tremblay ML, Sirois J, Farookhi R, Hendy GN, et al. Targeted ablation of the 25-hydroxyvitamin D 1alpha -hydroxylase enzyme: evidence for skeletal, reproductive, and immune dysfunction. Proc Natl Acad Sci U S A. 2001;98:7498–503. doi: 10.1073/pnas.131029498. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Irani M, Merhi Z. Role of vitamin D in ovarian physiology and its implication in reproduction: a systematic review. Fertil Steril. 2014;102:460–8 e3. doi: 10.1016/j.fertnstert.2014.04.046. [DOI] [PubMed] [Google Scholar]
- 9.Rashidi B, Haghollahi F, Shariat M, Zayerii F. The effects of calcium-vitamin D and metformin on polycystic ovary syndrome: a pilot study. Taiwanese journal of obstetrics & gynecology. 2009;48:142–7. doi: 10.1016/S1028-4559(09)60275-8. [DOI] [PubMed] [Google Scholar]
- 10.Thys-Jacobs S, Donovan D, Papadopoulos A, Sarrel P, Bilezikian JP. Vitamin D and calcium dysregulation in the polycystic ovarian syndrome. Steroids. 1999;64:430–5. doi: 10.1016/s0039-128x(99)00012-4. [DOI] [PubMed] [Google Scholar]
- 11.Jukic AM, Steiner AZ, Baird DD. Lower plasma 25-hydroxyvitamin D is associated with irregular menstrual cycles in a cross-sectional study. Reprod Biol Endocrinol. 2015;13:20. doi: 10.1186/s12958-015-0012-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Thota C, Farmer T, Garfield RE, Menon R, Al-Hendy A. Vitamin D elicits anti-inflammatory response, inhibits contractile-associated proteins, and modulates Toll-like receptors in human myometrial cells. Reproductive sciences. 2013;20:463–75. doi: 10.1177/1933719112459225. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Lasco A, Catalano A, Benvenga S. Improvement of primary dysmenorrhea caused by a single oral dose of vitamin D: results of a randomized, double-blind, placebo-controlled study. Arch Intern Med. 2012;172:366–7. doi: 10.1001/archinternmed.2011.715. [DOI] [PubMed] [Google Scholar]
- 14.Baird DD, Harmon QE, Upson K, Moore KR, Barker-Cummings C, Baker S, et al. A Prospective, Ultrasound-Based Study to Evaluate Risk Factors for Uterine Fibroid Incidence and Growth: Methods and Results of Recruitment. J Womens Health (Larchmt) 2015 doi: 10.1089/jwh.2015.5277. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Hollis BW. Editorial: The determination of circulating 25-hydroxyvitamin D: no easy task. J Clin Endocrinol Metab. 2004;89:3149–51. doi: 10.1210/jc.2004-0682. [DOI] [PubMed] [Google Scholar]
- 16.Ersfeld DL, Rao DS, Body JJ, Sackrison JL, Jr, Miller AB, Parikh N, et al. Analytical and clinical validation of the 25 OH vitamin D assay for the LIAISON automated analyzer. Clinical biochemistry. 2004;37:867–74. doi: 10.1016/j.clinbiochem.2004.06.006. [DOI] [PubMed] [Google Scholar]
- 17.Wagner D, Hanwell HE, Vieth R. An evaluation of automated methods for measurement of serum 25-hydroxyvitamin D. Clinical biochemistry. 2009;42:1549–56. doi: 10.1016/j.clinbiochem.2009.07.013. [DOI] [PubMed] [Google Scholar]
- 18.Horst RL. Exogenous versus endogenous recovery of 25-hydroxyvitamins D2 and D3 in human samples using high-performance liquid chromatography and the DiaSorin LIAISON Total-D Assay. The Journal of steroid biochemistry and molecular biology. 2010;121:180–2. doi: 10.1016/j.jsbmb.2010.03.010. [DOI] [PubMed] [Google Scholar]
- 19.Sachs MC, Shoben A, Levin GP, Robinson-Cohen C, Hoofnagle AN, Swords-Jenny N, et al. Estimating mean annual 25-hydroxyvitamin D concentrations from single measurements: the Multi-Ethnic Study of Atherosclerosis. The American journal of clinical nutrition. 2013;97:1243–51. doi: 10.3945/ajcn.112.054502. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Kato I, Toniolo P, Koenig KL, Shore RE, Zeleniuch-Jacquotte A, Akhmedkhanov A, et al. Epidemiologic correlates with menstrual cycle length in middle aged women. Eur J Epidemiol. 1999;15:809–14. doi: 10.1023/a:1007669430686. [DOI] [PubMed] [Google Scholar]
- 21.Liu Y, Gold EB, Lasley BL, Johnson WO. Factors affecting menstrual cycle characteristics. Am J Epidemiol. 2004;160:131–40. doi: 10.1093/aje/kwh188. [DOI] [PubMed] [Google Scholar]
- 22.Waller K, Swan SH, Windham GC, Fenster L, Elkin EP, Lasley BL. Use of urine biomarkers to evaluate menstrual function in healthy premenopausal women. Am J Epidemiol. 1998;147:1071–80. doi: 10.1093/oxfordjournals.aje.a009401. [DOI] [PubMed] [Google Scholar]
- 23.Millen AE, Wactawski-Wende J, Pettinger M, Melamed ML, Tylavsky FA, Liu S, et al. Predictors of serum 25-hydroxyvitamin D concentrations among postmenopausal women: the Women's Health Initiative Calcium plus Vitamin D clinical trial. The American journal of clinical nutrition. 2010;91:1324–35. doi: 10.3945/ajcn.2009.28908. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 24.Harlow SD, Campbell B, Lin X, Raz J. Ethnic differences in the length of the menstrual cycle during the postmenarcheal period. Am J Epidemiol. 1997;146:572–80. doi: 10.1093/oxfordjournals.aje.a009316. [DOI] [PubMed] [Google Scholar]
- 25.Symons JP, Sowers MF, Harlow SD. Relationship of body composition measures and menstrual cycle length. Ann Hum Biol. 1997;24:107–16. doi: 10.1080/03014469700004852. [DOI] [PubMed] [Google Scholar]
- 26.Shoben AB, Kestenbaum B, Levin G, Hoofnagle AN, Psaty BM, Siscovick DS, et al. Seasonal variation in 25-hydroxyvitamin D concentrations in the cardiovascular health study. Am J Epidemiol. 2011;174:1363–72. doi: 10.1093/aje/kwr258. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Hornsby PP, Wilcox AJ, Weinberg CR. Cigarette smoking and disturbance of menstrual function. Epidemiology. 1998;9:193–8. [PubMed] [Google Scholar]
- 28.Dietary Reference Intakes for Calcium and Vitamin D. The National Academies Press; 2011. [PubMed] [Google Scholar]
- 29.Brakta S, Diamond JS, Al-Hendy A, Diamond MP, Halder SK. Role of vitamin D in uterine fibroid biology. Fertil Steril. 2015;104:698–706. doi: 10.1016/j.fertnstert.2015.05.031. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 30.Block JP, Subramanian SV, Christakis NA, O'Malley AJ. Population trends and variation in body mass index from 1971 to 2008 in the Framingham Heart Study Offspring Cohort. PloS one. 2013;8:e63217. doi: 10.1371/journal.pone.0063217. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Harlow SD, Park M. A longitudinal study of risk factors for the occurrence, duration and severity of menstrual cramps in a cohort of college women. Br J Obstet Gynaecol. 1996;103:1134–42. doi: 10.1111/j.1471-0528.1996.tb09597.x. [DOI] [PubMed] [Google Scholar]
- 32.Ju H, Jones M, Mishra GD. Smoking and trajectories of dysmenorrhoea among young Australian women. Tob Control. 2014 doi: 10.1136/tobaccocontrol-2014-051920. [DOI] [PubMed] [Google Scholar]
- 33.Sundell G, Milsom I, Andersch B. Factors influencing the prevalence and severity of dysmenorrhoea in young women. Br J Obstet Gynaecol. 1990;97:588–94. doi: 10.1111/j.1471-0528.1990.tb02545.x. [DOI] [PubMed] [Google Scholar]
- 34.Danilenko KV, Samoilova EA. Stimulatory effect of morning bright light on reproductive hormones and ovulation: results of a controlled crossover trial. PLoS Clin Trials. 2007;2:e7. doi: 10.1371/journal.pctr.0020007. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.Hsu MI. Changes in the PCOS phenotype with age. Steroids. 2013;78:761–6. doi: 10.1016/j.steroids.2013.04.005. [DOI] [PubMed] [Google Scholar]
- 36.Sun W, Xie H, Ji J, Zhou X, Goltzman D, Miao D. Defective female reproductive function in 1,25(OH)2D-deficient mice results from indirect effect mediated by extracellular calcium and/or phosphorus. American journal of physiology. 2010;299:E928–35. doi: 10.1152/ajpendo.00378.2010. [DOI] [PubMed] [Google Scholar]
- 37.Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, et al. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nature genetics. 1997;16:391–6. doi: 10.1038/ng0897-391. [DOI] [PubMed] [Google Scholar]
- 38.Rowling MJ, Gliniak C, Welsh J, Fleet JC. High dietary vitamin D prevents hypocalcemia and osteomalacia in CYP27B1 knockout mice. J Nutr. 2007;137:2608–15. doi: 10.1093/jn/137.12.2608. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 39.Barrett ES, Thune I, Lipson SF, Furberg AS, Ellison PT. A factor analysis approach to examining relationships among ovarian steroid concentrations, gonadotrophin concentrations and menstrual cycle length characteristics in healthy, cycling women. Hum Reprod. 2013;28:801–11. doi: 10.1093/humrep/des429. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.Fehring RJ, Schneider M, Raviele K. Variability in the phases of the menstrual cycle. J Obstet Gynecol Neonatal Nurs. 2006;35:376–84. doi: 10.1111/j.1552-6909.2006.00051.x. [DOI] [PubMed] [Google Scholar]
- 41.Ferrell RJ, Rodriguez G, Holman D, O'Connor K, Wood JW, Weinstein M. Hypoestrogenic “inactive phases” at the start of the menstrual cycle: changes with age and reproductive stage, and relationship to follicular depletion. Fertil Steril. 2012;98:1246–53 e1-3. doi: 10.1016/j.fertnstert.2012.07.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.Harlow SD, Baird DD, Weinberg CR, Wilcox AJ. Urinary oestrogen patterns in long follicular phases. Hum Reprod. 2000;15:11–6. doi: 10.1093/humrep/15.1.11. [DOI] [PubMed] [Google Scholar]
- 43.Mumford SL, Steiner AZ, Pollack AZ, Perkins NJ, Filiberto AC, Albert PS, et al. The utility of menstrual cycle length as an indicator of cumulative hormonal exposure. J Clin Endocrinol Metab. 2012;97:E1871–9. doi: 10.1210/jc.2012-1350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.Greb RR, Grieshaber K, Gromoll J, Sonntag B, Nieschlag E, Kiesel L, et al. A common single nucleotide polymorphism in exon 10 of the human follicle stimulating hormone receptor is a major determinant of length and hormonal dynamics of the menstrual cycle. J Clin Endocrinol Metab. 2005;90:4866–72. doi: 10.1210/jc.2004-2268. [DOI] [PubMed] [Google Scholar]
- 45.Zec I, Tislaric-Medenjak D, Megla ZB, Kucak I. Anti-Mullerian hormone: a unique biochemical marker of gonadal development and fertility in humans. Biochem Med (Zagreb) 2011;21:219–30. doi: 10.11613/bm.2011.031. [DOI] [PubMed] [Google Scholar]
- 46.Zhu R, Lee BH, Huang Z, Raja Indran I, Li J, Shen L, et al. Anti-mullerian hormone, antral follicle count and ovarian volume predict menstrual cycle length in healthy women. Clin Endocrinol (Oxf) 2015 doi: 10.1111/cen.12984. [DOI] [PubMed] [Google Scholar]
- 47.Malloy PJ, Peng L, Wang J, Feldman D. Interaction of the vitamin D receptor with a vitamin D response element in the Mullerian-inhibiting substance (MIS) promoter: regulation of MIS expression by calcitriol in prostate cancer cells. Endocrinology. 2009;150:1580–7. doi: 10.1210/en.2008-1555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 48.Merhi Z, Doswell A, Krebs K, Cipolla M. Vitamin D alters genes involved in follicular development and steroidogenesis in human cumulus granulosa cells. J Clin Endocrinol Metab. 2014;99:E1137–45. doi: 10.1210/jc.2013-4161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Looker A, Johnson C, Lacher D, Pfeiffer C, Schleicher R, Sempos C. NCHS data brief, no 59. Hyattsville, MD: National Center for Health Statistics; 2011. Vitamin D status: United States, 2001-2006. [PubMed] [Google Scholar]
Associated Data
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Supplementary Materials
Supplemental Table 1. Sensitivity analyses of the association between 25(OH)D and menstrual cycle length and regularity among African-American women in the SELF study (N=1089).