Effect of vitamin D supplementation on assisted reproduction technology (ART) outcomes and underlying biological mechanisms: protocol of a randomized clinical controlled trial. The “supplementation of vitamin D and reproductive outcome” (SUNDRO) study

Vitamin D plays an important role in human physiology and pathology. The activation of the vitamin D receptor (VDR) may regulate directly and/or indirectly the expression of a very large number of genes (0.5–5% of the total human genome). Accordingly, vitamin D insufficiency has been shown to increase the risk of several diseases including cancers, auto-immune diseases, infections and pregnancy complications [1, 2].

In recent years, a role of vitamin D has been also postulated in reproductive health. VDR is expressed in the ovary, the endometrium and the myometrium [3]. Moreover, vitamin D has been shown to influence female fertility potential in animals and humans [4, 5]. In vitro fertilization (IVF) success was demonstrated to be higher in women with appropriate reserves of vitamin D [6]. In a recent contribution from our group, we detected an adjusted odds ratio (a-OR) (adjusted for age, ethnicity, BMI, parity, duration of infertility, number of retrieved oocytes, number of transferred embryos, and study period) of 2.15 [95% confidence interval (CI): 1.23–3.77] for clinical pregnancy rate in women with vitamin D > 20 ng/ml compared to those with vitamin D < 20 ng/ml. A similar figure was observed when considering the implantation rate. Of note, this beneficial effect was even more evident when using a threshold of 30 ng/ml [7]. The large body of evidence available in animal models and data from in vitro studies in women strongly favour a causal relation, but evidence from RCTs is required to definitely support a role for vitamin D in influencing the chances of pregnancy. To date, only a small RCT has been published and no benefits emerged [8]. However, the study was insufficiently powered to draw definite conclusions (in total 117 women). Moreover, only women scheduled for frozen embryo transfer were included.

Exploring the mechanisms at the basis of the beneficial effects of vitamin D could also be relevant and may open new avenues of research. To date, there are few appealing findings but the scenario is incomplete, especially from interventional studies. Biological evidence from RCTs could conversely provide crucial insights on the role of vitamin D in influencing oocytes quality or endometrial receptivity.

Oocytes cannot be directly tested for molecular analyses. However, the evaluation of the cumulus and granulosa cells of the ovarian follicle could indirectly reveal the quality of the corresponding oocytes. The gene expression profile of cumulus-oocyte-complexes has been suggested to predict which embryos have the best chance of implanting in the uterus. In particular, the expression of COX20, BMP15 and GDF9 has been associated with the establishment of pregnancy or live birth. Other possible potential genes of interest but with less consistency among studies include GREM1, HAS2, VCAN, PTGS [9, 10].

Endometrial samples can conversely be obtained prior to initiate the IVF cycle and can thus be directly studied without interfering with the procedure. Of interest here is that the endometrium and the early pregnancy decidua also express VDR and could be other important targets of vitamin D. In a previous study from our group, vitamin D was shown to modulate the expression of genes important for embryo receptivity and to interact with the local cytokines in human endometrial cells [11].

In the present study the following hypotheses will be tested: 1) 25-hydroxyvitamin D [25(OH)D] deficiency negatively affects pregnancy rates in IVF cycles with an effect mediated through endometrial receptivity and/or oocyte embryo quality; 2) Vitamin D supplementation and restoration of adequate vitamin D levels may actually improve the prognosis of infertile women undergoing IVF procedures. The absolute magnitude of the benefit deemed to be clinically relevant in terms of cumulative clinical pregnancy rate is considered 10% (from 20 to 30%); 3) The molecular profiles of endometrium, follicular fluid and cumulus cells are modified in vitamin D-supplemented women with respect to molecules known to be associated with IVF outcomes and embryo implantation process.

The study has been designed according to the CONSORT methodology.

For the most part, vitamin D is derived from the conversion of pro-vitamin D under the essential effect of solar ultraviolets. This particular mean of actually satisfying our needs represents an important concern. The mutated habits of humans in Western Worlds is not compatible with an adequate exposure to sunlight and thus hamper the provision of sufficient vitamin D [2].

The effect of vitamin D supplementation in women belonging to infertile couples has been insufficiently explored yet, although several groups have highlighted better chances of pregnancy in women replete for serum vitamin D [6]. Therefore, the hypothesis that vitamin D supplementation could improve IVF results deserves further consideration.

The present study can potentially influence the clinical practice of infertility treatment. Vitamin D insufficiency can be simply overcome. Supplementation with vitamin D is economic, simple and effective and would represent an innovative and simple adjuvant treatment of IVF with the potential additional benefit of improving the chances of natural conception. Of further relevance here is that vitamin D supplementation is free of relevant side effects.

Dosage of supplementation was based on previous data showing that a single 600.000 IU dose of cholecalciferol is able to rapidly increase serum 25(OH) D levels and to maintain adequate levels up to 90 days after administration in young subjects, including patients with severe vitamin D depletion. The proposed dosage is indeed considered safe since it gives peak levels well below the widely accepted toxic blood levels of 200 ng/ml [21, 22]. Of note, the single dose administered in-office by the physician guarantees a total adherence to treatment. However, given the lack of causal relationship between vitamin D levels and IVF outcomes and the risk of excessive vitamin D serum levels, patients will be asked to avoid further consumption of vitamin D after randomization. Moreover, at a molecular level, our analysis can provide preliminary data on reproductive cells that can be influenced by circulating vitamin D levels (endometrium, follicle, oocyte cumulus-oocyte complex). Whether the effect of vitamin D in human reproduction is mediated at ovarian, endometrial or both levels represents a matter of controversy. According to our prospective cross-sectional study aiming at investigating IVF outcomes in women with deficient 25(OH) D level, the number of top quality embryos and, as a consequence, the rate of women reaching the stage of blastocyst transfer was higher in women with sufficient levels of serum 25(OH)D. The chances of embryo implantation were also enhanced supporting the idea that the impact of the compound might be exerted in both the reproductive tissues [7]. Disentangling the potential role of vitamin D in the endometrium is particularly relevant given the key role of uterine factors in determining the outcome of IVF treatment, the significant gaps in our knowledge of mechanisms underlying endometrial-embryo asynchrony and most of all, the lack of relevant modifiers of endometrial receptivity status [23]. Also of particular note, there is growing and consistent evidence that vitamin D supplementation may improve birth outcomes and prevent some relevant obstetrics complications. According to a recent systematic review of the randomized studies on vitamin D supplementation, the compound increases mean birth weight of 58.33 g (95%CI 18.88–97.78 g) [24]. Moreover, a lower risk in pre-eclampsia was observed in those women receiving vitamin D supplementation, especially if they received also calcium [25].

Some limitations of our study should be acknowledged. Firstly, it has to be pointed out that the decision for a single dose of 600.000 IU is a pragmatic choice and remarkably simplifies our study design. Moreover, if significant benefits will emerge, this modality could easily enter clinical practice (considering also the low costs) and a high adherence can be expected. On the other hand, a non-significant result will not be a definitive reason to abandon the studies on vitamin D supplementation prior to IVF because one could argue that a more steady and constant administration (such as 2.000 IU daily) could be more beneficial. Secondly, the duration of vitamin D supplementation prior to IVF can be an additional reason of concern, in particular considering that we allowed for a certain variability between Vitamin D administration and the initiation of the cycle (2–12 weeks). In fact, folliculogenesis has been claimed to last 5–6 months and our protocol does not allow us to entirely cover this long period. Our protocol actually consents to properly test the effects of vitamin D on endometrial growth (a menstrual flow will systematically occur after the administration) and on final follicular growth (from dominance to ovulation) but not on the full folliculogenesis process. Again, we decided to privilege pragmatism and simplicity with the aim of facilitating future clinical use but, again, we have to admit that this choice can expose our results to some criticisms if no significant difference will emerge. Thirdly, we will request recruited women to avoid vitamin D consumption but we cannot request them to avoid sun exposure during the whole period of the IVF cycle. Even if one has to expect a similar frequency of this situation in the two study arms, this potential confounder might partly dilute the potential benefits of the tested supplementation of vitamin D. On the other hand, again, we opted for a pragmatic study and this confounder would just reflect real life situations. Fourthly, the decision to focus on women with insufficient vitamin D (< 30 ng/ml) rather than deficient (< 20 ng/ml) or even severely deficient (< 10 ng/ml) could be argued. In fact, the study will be underpowered for a reliable subgroup analysis according to the magnitude of the deficiency. In other words, we will not be able to assess whether the intervention could be of value only in a selected group (in particular those with the lowest levels) rather than for the whole heterogeneous group of women with serum levels below 30 ng/ml. Fifthly, the decision to randomize even women with serum levels below 10 ng/ml could be ethically debatable because this is a condition of possible clinical relevance per se. However, it has to be underlined that there is currently no recommendation for vitamin D testing before IVF and, for this reason, we did not deem unethical to include all women with serum levels below 30 ng/ml.

Overall, despite the above-mentioned limitations of our study design, we believe that this RCT could provide clinically relevant findings, in particular if statistically significant differences in favour of the treatment arm will emerge. In fact, it can influence clinical practice. High doses supplementation with vitamin D is an extremely simple and safe intervention and, if a significant benefit will emerge, one has to expect a rapid and global spread of its use. In addition, given the low costs of the intervention (less than 10 € in Italy), the economical profile can be expected to be highly cost-effective. On the other hand, for the above-mentioned intrinsic limitations consequent to our pragmatic choices, we have to recognize that a negative finding will not definitely close the issue.