Animal and human studies suggest that vitamin D is involved in many processes of the human reproductive system in both genders. Of those, some evidence come from the Assisted Reproduction Technology (ART) [1
]. ART represents a valuable model to draw inferences on vitamin D deficiency in specific aspects of human fertility as it allows the separate evaluation of the various steps of the reproductive process, from sperm function to folliculogenesis to embryo implantation.
Six original articles have investigated the association between serum levels of 25-hydroxy-vitamin D (25(OH)D), the storage form of the vitamin, and pregnancy rates in ART cycles with controversial results. For instance, Rudick et al
] observed that serum 25(OH)D levels were significantly related to implantation, clinical pregnancy, and live birth rates, although opposite trends were found according to patients’ ethnicity being critical in non-Hispanic whites, but not in the Asian ethnicity. In a second study [4
], the same authors examined serum 25(OH)D concentration among recipients of oocyte donation, finding a positive association between vitamin D status and clinical pregnancy rate and suggesting the specific effect of 25(OH)D levels on ART outcomes to be mediated by endometrial receptivity rather than by ovarian stimulation or embryo parameters. To this regard, it should be emphasized that both cyclic and early pregnant endometrium represents an extra-renal site of vitamin D synthesis; moreover, the effect of vitamin D at the uterine level is thought to be exerted via the vitamin D receptor (VDR) through either the regulation of target genes or the hormonal effects on the local immune responses [5
]. In patients who underwent single embryo transfer at blastocyst stage, vitamin D deficiency (<20 ng/mL) emerged as an independent predictor of lower clinical pregnancy rates as compared with non-deficient women [6
]. In contrast, in the largest sample analyzed so far, Franasiak et al
] showed that vitamin D status was unrelated to pregnancy outcomes in women undergoing euploid blastocyst transfer. Two Iranian studies did not confirm any influence of serum 25(OH)D levels in terms of pregnancy rate [8
], but these latter analyses were exposed to a significant risk of type II error given the extremely low proportion of women with sufficient 25(OH)D levels. Using a different approach, Farzadi et al.
reported a correlation between follicular fluid 25(OH)D concentration and assisted reproductive outcomes in an Iranian population [10
Results from a further cross-sectional prospective study supported the role of vitamin D in terms of female fertility [1
]; indeed the odds ratio (OR) for clinical pregnancy in women with vitamin D greater than 20 ng/mL was 2.15 (95% CI 1.23–3.77). Likewise, the group with serum levels >30 ng/mL (sufficient vitamin D) had the highest chances of pregnancy. A similar figure was observed when considering the implantation rate (OR 1.91, 95% CI 1.20–3.05). Finally, women with sufficient 25(OH)D concentration had a significantly higher chance of obtaining top quality embryos and to transfer at the blastocyst stage, thus generally supporting a favorable effect of vitamin D at both ovarian and endometrial level [1
Based on these observations, we comprehensively analyzed vitamin D status of couples attending a single academic infertile center. Such an evaluation would provide an estimation of a cross-sectional epidemiological magnitude of vitamin-D deficiency, while forming the basis for interventions to address the deleterious consequences of vitamin D deficiency-related infertility problems. We aimed to (i) determine the baseline vitamin D profiles of women attending an infertility center; and, (ii) investigate the non-dietary determinants of vitamin D status in the same cohort. We hypothesized that (i) a high proportion of our cohort would be vitamin D deficient; and (ii) vitamin D status in infertile women mainly relies on social habits, degree of sun exposure and health risk factors. This issue is also of particular clinical interest considering the growing and consistent evidence supporting the idea that vitamin D deficiency may influence birth outcomes and may be associated with relevant obstetrics complications [11
The results of this study indicate that in a cohort of female patients attending an infertility center in Northern Italy, levels of serum 25(OH)D follow a seasonal cycle; as a whole, over the entire year, 40.1% of patients showed deficient, and 77.4% insufficient or deficient levels, of 25(OH)D. The median value of serum 25(OH)D concentration reached a sufficient level only for 11% of the entire year period. This means that, excluding a period of 40 days between August and September, during the entire year the majority of women referring to an ART procedure showed insufficient vitamin D status. These findings are of clinical significance considering the recent evidence supporting a critical role of vitamin D in regulating human fertility [26
]. Indeed, in vitro
studies tend to suggest that there might be a relationship between vitamin D deficiency and granulosa cell function [27
]. Vitamin D treatment has been shown to act in these cells down-regulating FSH receptor and antimullerian hormone (AMH) receptor II gene expression and increasing 3β-hydroxysteroid dehydrogenase expression and progesterone production. The favorable effects of vitamin D on the metabolic alterations in PCOS represented by decrease in insulin resistance and androgen levels have been suggested to be translated into a healthier ovarian physiology [2
]. The relationship between 25(OH)D levels and in-vitro
fertilization (IVF) outcomes has been inconsistent in the literature mostly for the multitude of factors involved such as sperm quality and endometrial receptivity and for the retrospective nature of the studies [2
]. Our prospective cross-sectional investigation has demonstrated that vitamin D deficiency negatively affects ART clinical pregnancy rate, potentially opening new therapeutic scenarios for women scheduled for IVF and, further, in general, to all women with infertility [1
]. However, evidence for a causative effect of 25(OH)D levels on IVF outcomes are still poor and studies conducted so far rarely meet all the Hill’s criteria for causation [28
]. An increase in the number of more specific and consistent studies and the demonstration of the existence of a biological gradient of effect for different 25(OH)D levels will be needed in order to confirm previous observations.
Current findings may have major implications both for infertile patients and in terms of national healthcare policy. Since a large proportion of women attending an infertility center show insufficient circulating levels of 25(OH)D, physicians in Reproductive Medicine should consider to monitor serum 25(OH)D levels during the course of any ART cycle. In any case, no evidence of significant decrease in 25(OH)D concentration was found among the various causes of infertility when compared to the male factor group, which was considered a referral cohort that represented the general population since it included healthy women only. Evidence from randomized controlled trials is required to definitively support the benefit of vitamin D supplementation as a simple and inexpensive intervention to positively impact on pregnancy outcomes.
At the national healthcare level it has to be considered that there is a notable discrepancy between mean intakes and dietary recommendations among European countries. In this context, while in Scandinavian countries the consumption of vitamin D with the diet is high, both fortification and supplementation policies have also been implemented. In Germany, the emphasis is on encouraging outdoor sun exposure. Turkey has an infant supplementation program. Conversely, both Spain and Italy do not have formal fortification or supplementation public health policies. This results in lowest mean daily intakes in Spain and Italy compared to Scandinavian countries [29
]. Prevalence of vitamin D deficiency among European population subgroups is difficult to be estimated but it should be considered that in a study population of 58 Italian healthy women (mean age equal to 36.9 years) the mean 25(OH)D serum levels were 15.2 and 30.7 ng/mL in winter and summer, respectively, with prevalence of 25(OH)D concentration lower than 20 ng/mL ranging from 81% in winter to 6.9% in summer [30
]. Whether vitamin D status will prove to be important for fertility outcomes, the adoption of policies aimed at preventing vitamin D deficiency should be considered as a public health priority, and childbearing couples as a vulnerable group in the population.
The effect of seasonality on 25(OH)D levels is highlighted in this study. Median level of serum 25(OH)D was lower in the first trimester of the year, with a consequent increase in the prevalence of deficiency. These findings support previous results and highlight that season is a major factor in determining vitamin D status [32
]. Moreover, we showed that global solar radiation was weakly correlated with 25(OH)D levels. In this context, it should be considered that Italian residents spent more than half of their tourism nights in July and Augusts, as recently reported by Eurostat [34
]. This is supported by the vitamin D peak observed during August. Thus, the weak correlation observed with solar global radiation could be explained by the interference of social factors.
The association between 25(OH)D levels and age has produced controversial results, with some studies supporting an increased prevalence of deficiency with age and others failing to confirm this finding [35
]. Our findings did not support an association between age and 25(OH)D concentration. However, this may be explained by the restricted age range of our cohort of women.
Body weight was negatively associated with vitamin D status and this result is generally consistent with those identified in previous studies [36
]. Our analysis has also evidenced a significant positive correlation between height and 25(OH)D level, which was confirmed at multivariable analyses. This intriguing effect has already been reported for young women in two studies [37
] but still remain unexplained. In fact, vitamin D is key to skeletal development in childhood and its deficiency may result in short stature associated with rickets [39
] but it is difficult to explain how this association could persist in adulthood. A possible explanation is that causes for vitamin D deficiency in childhood might perpetuate over the period of adulthood, but further investigation conducted in adult population are certainly needed.
Interestingly, among the causes of infertility, patients with endometriosis were found to have higher levels of 25(OH)D compared to women with other disorders. These findings are in line with the results of a prospective cohort study from our group demonstrating higher levels of 25(OH)D among reproductive-aged women selected for surgery for gynecologic indications [40
]. On the other hand, they are in contrast with data from Harris et al.
] who calculated a score for predicted 25(OH)D levels in 1385 cases of laparoscopically-confirmed incident endometriosis. Predicted plasma 25(OH)D level was inversely associated with endometriosis. Women in the highest quintile of predicted vitamin D level had a 24% lower risk of endometriosis than women in the lowest quintile [41
]. Thus, the relationship between endometriosis and the vitamin D endocrine system still remains to be clarified. Although vitamin D deficiency may be revealed as a biologically plausible pathway to an increased risk of autoimmune and inflammatory diseases, such as endometriosis, because vitamin D is able to affect several arms of the immune function, genetic and/or epidemiological factors cannot be excluded in the interpretation of these data. Notably, the common phenotype of women with endometriosis might represent a factor predisposing to a higher vitamin D synthesis from the skin [42
]. In fact, it has been demonstrated that the length of time required for endogenous vitamin D3 biosynthesis increases with increasing melanin concentration in the skin [43
]. Women with endometriosis frequently show a typical fair skin phenotype [42
]. Despite different interpretations in this context [45
], this phenotype might probably be the cause of an increased vitamin D synthesis, thus resulting in higher 25(OH)D levels when compared with women with darker skin phenotype from the Italian general population.
Our study is not devoid of limitations. First, the cross-sectional design may limit casual inference on the effects of seasons and infertility causes on vitamin D status. Second, we did not administer a food-recall questionnaire or a questionnaire on vacation habits. However, as already mentioned, the majority of the Italian population spends their vacation during the months of July and August. Third, our results were derived from a single center in Northern Italy; therefore we are uncertain that our results are generalizable to other infertility centers across the country. Finally, the infertility center was located around latitude 45°28′ N thus, it is not certain whether similar results would be obtained in centers in other geographical locations.
One of the strengths of our study is that it is the first to address the prevalence of vitamin D deficiency in a female population attending an infertility center. We had a fairly large cohort to enable us to detect differences between the groups of interest. Indeed, our cohort contained a fair representation of the main indications to infertility treatment, thus enabling us to investigate the effects of various causes on vitamin D status.
Lastly, vitamin D insufficiency has been recently associated with an increased risk of gestational diabetes, pre-eclampsia, and small for gestational age infants [11
]. The high prevalence of vitamin D deficiency in infertile women referring to an ART procedure is likely to be mirrored by a similar prevalence during pregnancy. Furthermore, in general, the demonstration of an advantage of adequate levels of vitamin D in IVF cycles would be strengthened by the potential subsequent advantage also in terms of prevention of obstetrics complications. Even if evidence-based data supporting the benefits of vitamin D supplementation before pregnancy are not yet available, this aspect needs to be carefully considered when approaching our data.