We examined the relation of intakes of total folate and specific sources of folate with reproductive hormones and anovulation in a group of young, healthy women not using folate or other dietary supplements at enrollment. We found that while total folate intake (in dietary equivalents) was not significantly associated with reproductive hormone levels or anovulation, higher intake of dietary synthetic folate was significantly associated with higher luteal progesterone levels and decreased odds of anovulation. The observed association of high dietary synthetic folate intake with increased progesterone levels and decreased risk of anovulation highlights other potential beneficial effects of folic acid fortification that to date may not have been recognized.
To our knowledge this is the first study to look at the association between dietary folate and reproductive hormone levels and anovulation in humans; however, there have been similar studies looking at folic acid supplement use and ovulation in animals. In immature superovulated rats, it was shown that either an excess or deficiency of folates partially inhibited ovulation 
. A later study in rhesus monkeys showed that a folate-restricted diet led to irregular menstrual cycles and progressively decreased pre-ovulatory serum estradiol and mid-luteal progesterone compared to monkeys fed a normal diet 
. Finally, folic acid supplement use in pigs was shown to promote ovulation as documented by increased litter size 
Relevant studies in humans have largely focused on multivitamin supplement use and fertility but for the most part have found consistent results. In humans a significantly higher rate of conceptions was found after preconceptional multivitamin supplement use in comparison with a placebo-like trace element supplement use in a Hungarian randomized, double-blind, controlled trial (n
. Similarly, in a double blind, placebo-controlled study (n
93) looking at the effects of FertilityBlend (a type of multivitamin) for women on fertility, it was found that after 3 months of supplement use, women on FertilityBlend had a trend toward increased mean mid-luteal progesterone, had significantly more days with luteal-phase basal temperatures over 98°F, and had a significantly higher rate of pregnancy than women in the placebo group 
. Finally, a longitudinal cohort study (n
18,555) found an inverse association between frequency of multivitamin use and ovulatory infertility 
. While all of these studies are consistent with our findings of folic acid being beneficial for ovulation, none were able to pinpoint a nutrient that was responsible for this association.
The observed relation between synthetic folate and anovulation is biologically plausible. Specifically, folate deficiency and homocysteine accumulation can lead to reduced cell division, increased inflammatory cytokine production, altered nitric oxide metabolism, increased oxidative stress, elevated apoptosis, and disturbed methylation reactions 
; all of which could subsequently affect oocyte development. Studies of oocyte quality in the in vitro fertilization setting have confirmed this showing that women receiving a folic acid supplement (and who subsequently had lower homocysteine follicular fluid concentrations) had better quality oocytes and a higher degree of mature oocytes compared to women who did not receive folic acid 
. Although currently research is limited, it is possible that ovarian response to endogenous FSH pulses is also decreased in low folate conditions, which could lead to impaired ovulation. In women undergoing controlled hyperstimulation with recombinant FSH, carriers of the T allele in position 677 of the MTHFR gene (which leads to decreased enzyme activity and 5-methyltetrahydrofolate concentrations) have a decreased ovarian responsiveness to this hormone, fewer oocytes to be retrieved 
, and granulosa cells that produce less estradiol, basal and stimulated 
. In regards to the association between higher intake of dietary folate and higher progesterone levels, while evidence in humans is limited, it has been shown that folate deficiency in the rhesus monkeys leads to depleted ovarian granulosa cells 
and decreased levels of luteal progesterone 
. The authors of these articles hypothesized that folate deficiency might led to an "ineffective" process or "abortive" attempt at cell reproduction, the functional consequences of which would be reflected in the reduced biosynthesis of the sex hormones in these germ cells 
The reason for our significant finding with synthetic folate as opposed to natural folate and anovulation could be partly due to the greater absorption rates of synthetic folate 
. While calculating dietary folate equivalents attempted to address this issue, it is known to be an imperfect measure 
. Natural food folate is present primarily in the reduced polyglutamated form while synthetic folic acid is a fully oxidized monoglutamate form of folate. Relative to folic acid, natural food folate has a lower proportion of folate that is absorbed and available for metabolic reactions and/or storage. Several luminal factors also seem to hinder the absorption of natural food folate including its limited release from the food matrix, destruction within the gastrointestinal tract, and incomplete hydrolysis of glutamates in excess of one 
. Since consumption of folic acid fortified foods (such as ready to eat breakfast cereals) is associated with higher usual vitamin B12 intakes this could also lead to a higher bioavailability of synthetic folate 
. Finally, it is possible that the presence of other micronutrients added to fortified foods could explain some of the association between synthetic folate and anovulation; however models, which adjusted for highly correlated dietary nutrients (such as vitamin B12) produced similar results.
The BioCycle Study had a number of strengths including the intensive monitoring of a large number of young, ethnically diverse women throughout 2 menstrual cycles, with multiple measures of reproductive hormones and dietary intake. While self-report of diet is subject to measurement error, the estimation of dietary intake with multiple 24-hour recalls (up to 4 per cycle) decreased the probability of dietary misclassification. Furthermore, a Danish validation study showed that 3 24-hr recalls over 4 weeks had a correlation of 0.49 with red blood cell folate (a biomarker of long-term folate status) 
. The prospective design and exclusion criteria at baseline of the BioCycle Study strengthen the ability to draw inference, having reduced the potential for bias from known risk factors for anovulation. In addition, standardized assessment of a wide variety of participant and dietary characteristics increased the ability to adjust for confounding. Finally, since BioCycle participants were not taking supplements, this allowed us to examine the impact of low levels of folate intake from dietary sources alone.
Nevertheless, the study faced several limitations, including the small number of anovulatory cycles (n=
42), which limited the power of our findings. However, even with this moderate number we observed a significant inverse association between synthetic folate intakes and odds of anovulation. In addition, women were only followed for 2 menstrual cycles. Since anovulation occurs commonly in many women, long-term findings may be quite different. In absence of a daily transvaginal ultrasound or daily first morning urine measurements, the direct detection of ovulation has some degree of misclassification. However, in a sensitivity analysis, the effect of synthetic folate on anovulation was strong and consistent regardless of the definition used for classification. While our study included the use of a fertility monitor to help time visits, bias could have been introduced through mistimed sample collection. However, various indicators of successfully timed visits were found to be unrelated to folate consumption, thus any misclassification is likely to be non-differential 
. As this was an un-supplemented population, we were limited in evaluating the association of dietary folate and reproductive function at modest levels of consumption due to the small number of high consumers (>1000 μ/day). Given that this was an observational study, it is possible that residual confounding by other lifestyle factors that were poorly measured remains; however, adjustment for a variety of demographic and dietary characteristics had little impact on the results. Finally, since we restricted our study sample to healthy, regularly menstruating women, we limited the generalizability of our findings to other populations, specifically women who are known to be chronically anovulatory.
In conclusion, we observed that higher intake of dietary synthetic folate was significantly associated with higher luteal progesterone levels and decreased odds of anovulation. These results highlight the potential beneficial role of folic acid in reproductive outcomes such as sporadic anovulation among healthy women of reproductive age that to date might not have been fully recognized. Future studies are needed to confirm these findings as folic acid supplement use may have important implications in improving fertility outcomes.