Uterine fluid cytokine/chemokine levels of women undergoing ART with and without oral Vitamin D supplementation
Authors: Cermisoni Greta Chiara , Reschini Marco , Piccinni Marie-Pierre , Lombardelli Letizia , Logiodice Federica , Sarais Veronica , Giacomini Elisa , Signorelli Simona , Cecchele Anna , Viganò Paola 
Published: April 2022

Abstract

STUDY QUESTION
Is oral Vitamin D supplementation able to modify the intrauterine milieu in terms of cytokine/chemokine pattern?

SUMMARY ANSWER
No significant differences were detected in cytokine and chemokine levels in endometrial secretions between patients undergoing ART with or without Vitamin D supplementation.

WHAT IS KNOWN ALREADY
Cytokines and chemokines secreted into the intrauterine environment are fundamental for the molecular crosstalk between the endometrium and the preimplantation embryo. Whether Vitamin D can regulate these mediators in the endometrial environment is still unclear.

STUDY DESIGN, SIZE, DURATION
This study was an analysis of a secondary outcome from the Supplementation of Vitamin D and Reproductive Outcomes—SUNDRO—clinical trial, a multicenter randomized double-blinded trial designed to explore the effects of Vitamin D replacement in women with Vitamin D levels below 30 ng/ml undergoing autologous ART cycles. Uterine fluid samples were collected from both patients supplemented with Vitamin D (n = 17) and from the placebo group (n = 32).

PARTICIPANTS/MATERIALS, SETTING, METHODS
Based on cutoff points for Vitamin D insufficiency (20–29.9 ng/ml) or deficiency (&lt;20 ng/ml), 67% of patients in the study were insufficient, and 33% deficient, in Vitamin D, although they were considered together for the analysis. Women received a single dose of 600 000 IU 25-hydroxyvitamin D or placebo from 2 to 12 weeks before oocyte retrieval. Inclusion criteria were female age 18–39 years, with a BMI between 18 and 25 kg/m2. Serum 25-hydroxyvitamin D was assessed at the time of hCG administration. Uterine fluid samples were collected during the secretory phase of the menstrual cycle preceding oocyte retrieval. The quantitative determination of 27 cytokines in endometrial secretion samples was performed by using a multiplex immunoassay.

MAIN RESULTS AND THE ROLE OF CHANCE
Uterine fluid samples were collected after a median (range) of 21 (12–41) days after the oral Vitamin D supplementation. Both the supplemented and placebo groups had Vitamin D serum levels below 30 ng/ml at baseline/time of randomization ((median 23.4 ng/ml (interquartile range 19.5–28.4) and 23.4 ng/ml (17.8–25.9), respectively). At the time of hCG administration, serum Vitamin D in supplemented subjects was significantly raised compared to the placebo group ((median 52.9 ng/ml (interquartile range 40.7–64.1) and 24.6 ng/ml (19.3–29.2), respectively, P &lt; 0.001). Our data revealed no significant differences in uterine fluid cytokine/chemokine composition of Vitamin D-supplemented women compared with the placebo group. This finding remained when the concentrations of all mediators studied were normalized to total protein. In a further analysis, no significant differences were found in the content of cytokines/chemokines in uterine fluid from women who conceived (n = 19) compared with the nonpregnant group (n = 30).

LIMITATIONS, REASONS FOR CAUTION
Using a randomized study design (a single dose of 600 000 IU 25-hydroxyvitamin D versus placebo), we found no significant differences between groups. However, we cannot exclude that any benefit of Vitamin D supplementation may be specific for some subgroups of patients, such as those with an imbalance of T-helper 1 and T-helper 2 cell populations. The uterine secretions were collected during the menstrual cycle that preceded oocyte retrieval; therefore, it is possible the uterine fluid collection and analysis in the same cycle of the embryo transfer might have resulted in different conclusions. Moreover, the small sample size could limit the power of the study.

WIDER IMPLICATIONS OF THE FINDINGS
Our analysis of the uterine secretome profiling failed to show any significant difference in endometrial cytokine/chemokine patterns between women with oral Vitamin D supplementation and the placebo group. Vitamin D may act on the uterine environment through a different mechanism.

STUDY FUNDING/COMPETING INTEREST(S)
The study was funded by the Italian Ministry of Health following peer review in the competitive ‘Bando di Ricerca Finalizzata e Giovani Ricercatori 2013’ with reference code RF-2013-02358757. The authors declare that they have no conflict of interest.

TRIAL REGISTRATION NUMBER
EudraCT registration number: 2015-004233-27.