Uterine fluid cytokine/chemokine levels of women undergoing ART with and without oral Vitamin D supplementation.

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 (<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 < 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 INTERESTS: 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.

Folate Levels and Pregnancy Rate in Women Undergoing Assisted Reproductive Techniques: a Systematic Review and Meta-analysis.

Maternal nutrition is believed to be closely related to reproductive success and the importance of folate in the reproductive process and its involvement in fundamental biological systems are well known. The present systematic review and meta-analysis will focus on two main aspects: level of folate in women undergoing infertility treatments and association between folate status and success rate in assisted reproductive techniques. Although the importance of folate in the preconceptional phase is known, available data regarding the levels of folate in women who undergo infertility treatments are scarce. Referring to the threshold values generally used for the general population or for supplement users, the concentration of folate in the serum and erythrocytes of infertile women is adequate in the majority of the population with differences related to the geographic origin of the study populations. However, using the red blood cells folate threshold specifically indicated to prevent neural tube defects, the majority of available studies do not offer sufficient data to conclude on the real folate situation. As for the probability of success of ART treatments based on folate levels, our review did not reveal a significant effect.

Single oral dose of vitamin D3 supplementation prior to in vitro fertilization and embryo transfer in normal weight women: the SUNDRO randomized controlled trial.

BACKGROUND: Improving in vitro fertilization success is an unmet need. Observational studies have suggested that women with deficient or insufficient vitamin D have lower chances of in vitro fertilization success, but whether supplementation improves clinical pregnancy rate remains unclear. OBJECTIVE: This study aimed to determine whether oral vitamin D3 supplementation improves clinical pregnancy in women undergoing an in vitro fertilization cycle. STUDY DESIGN: The "supplementation of vitamin D and reproductive outcome" trial is a 2-center randomized superiority double-blind placebo-controlled trial. Subjects were recruited between October 2016 and January 2019. Participants were women aged 18 to 39 years with low vitamin D (peripheral 25-hydroxyvitamin D of <30 ng/mL), serum calcium of ≥10.6 mg/dL, body mass index of 18 to 25 kg/m2, and antimüllerian hormone levels of >0.5 ng/mL and starting their first, second, or third treatment cycle of conventional in vitro fertilization or intracytoplasmic sperm injection. The primary outcome was the cumulative clinical pregnancy rate per cycle. Pregnancies obtained with both fresh or frozen embryo transfers were included. Clinical pregnancy was defined as an intrauterine gestational sac with a viable fetus. The primary analysis was performed according to the intention-to-treat principle and could also include natural conceptions. Secondary outcomes included total dose of gonadotropins used, embryologic variables (number of oocytes retrieved, number of suitable oocytes retrieved, fertilization rate, and rate of top-quality embryos), and clinical outcomes (miscarriage rate and live birth rate). RESULTS: Overall, 630 women were randomized 2 to 12 weeks before the initiation of the in vitro fertilization cycle to receive either a single dose of 600,000 IU of vitamin D3 (n=308) or placebo (n=322). Interestingly, 113 (37%) and 130 (40%) women achieved a clinical pregnancy in the treatment and placebo groups, respectively (P=.37). The risk ratio of clinical pregnancy in women receiving vitamin D3 was 0.91 (95% confidence interval, 0.75-1.11). Compared with the placebo, vitamin D3 supplementation did not improve the rate of clinical pregnancy. Exploratory subgroup analyses for body mass index, age, indication to in vitro fertilization, ovarian reserve, interval between drug administration and initiation of the cycle, and basal levels of 25-hydroxyvitamin D failed to highlight any clinical situation that could benefit from the supplementation. CONCLUSION: In women with normal weight with preserved ovarian reserve and low vitamin D levels undergoing in vitro fertilization cycles, a single oral dose of 600,000 IU of vitamin D3 did not improve the rate of clinical pregnancy. Although the findings do not support the use of vitamin D3 supplementation to improve in vitro fertilization success rates, further studies are required to rule out milder but potentially interesting benefits and explore the effectiveness of alternative modalities of supplementation.

Oral Vitamin D supplementation impacts gene expression in granulosa cells in women undergoing IVF.

STUDY QUESTION: Does oral Vitamin D supplementation alter the hormonal milieu of follicular fluid (FF) and the transcriptomic profile of luteinised granulosa cells (GCs) in women with Vitamin D deficiency undergoing IVF? SUMMARY ANSWER: A transcriptomic signature relevant to oral Vitamin D supplementation in luteinised GCs was demonstrated, although Vitamin D supplementation did not alter hormone levels in FF. WHAT IS KNOWN ALREADY: Vitamin D deficiency is linked to lower live birth rates among women undergoing IVF. It is unclear whether Vitamin D elicits a targeted action in reproductive physiology or is a surrogate marker of overall well-being. Several in-vitro studies, but none in vivo, have examined the impact of Vitamin D on the periovulatory follicle, focusing on GCs as a proxy marker of oocyte competence. STUDY DESIGN, SIZE, DURATION: We present a report of secondary outcomes from the SUNDRO clinical trial, which was launched in 2016 to determine whether Vitamin D supplementation can improve the IVF outcomes of women who are deficient in Vitamin D (<30 ng/ml). FF samples of 145 women who were randomised to receive Vitamin D or placebo from March 2017 to January 2019 were collected. All follicles that were aspirated in our study measured ≥11 mm on the day of hCG trigger. The first cohort of samples was collected from the dominant follicle of each participant and utilised for hormone profiling (n = 50 Vitamin D, n = 45 Placebo). For the second cohort, the follicle aspirates of each participant were pooled to create a single FF sample, which was used for the isolation of GCs for gene expression studies (n = 20 Vitamin D, n = 30 placebo). Six of the samples from the second cohort were used for RNA-sequencing analysis (n = 3 Vitamin D, n = 3 placebo). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two academic infertility units were involved in the recruitment of the participants, who received a single dose of oral 25-hydroxyvitamin D (600 000 IU) or placebo, 2-12 weeks before oocyte retrieval. Women in both groups were deficient in Vitamin D, aged 18-39 years with a normal BMI (18-25 kg/m2) and <3 previous IVF cycles. The FF was aspirated at the time of oocyte retrieval and stored. Liquid chromatography tandem mass spectrometry was used to measure FF abundance of 25-hydroxyvitamin D, aldosterone, androstenedione, cortisol, cortisone, corticosterone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, dihydrotestosterone, oestradiol (E2), 17-OH-hydroxyprogesterone, progesterone (P4) and testosterone. GCs were isolated from pooled FFs and the transcriptome was evaluated by RNA-sequencing and RT-PCR. Ingenuity pathway analysis (IPA) was used to assess the top canonical pathways and upstream regulators mediating the action of Vitamin D. MAIN RESULTS AND THE ROLE OF CHANCE: At oocyte retrieval, FF concentration of 25-hydroxyvitamin D was 2.8-fold higher (P < 0.001) in the Vitamin D group (39.5 ng/ml; n = 50) compared to placebo (13.8 ng/ml; n = 45) but no other hormonal differences were detected. In the placebo group, but not the Vitamin D group, weak correlations of 25-hydroxyvitamin D concentration with P4 (r = 0.31, P = 0.03) and E2 (r = 0.45, P = 0.002) were observed. RNA-sequencing identified 44 differentially expressed genes in the GCs of patients who received Vitamin D (n = 3) compared to placebo (n = 3). RT-PCR demonstrated upregulation of VDR (vitamin D receptor), GSTA3 (glutathione S-transferase A3) and IL21R (interleukin 21 receptor), and downregulation of P T GS2 (prostaglandin-endoperoxide synthase 2), KLF4 (kruppel-like factor 4), T RP C4 (transient receptor potential cation channel subfamily C member 4), VEGF (vascular endothelial growth factor), RXRB (retinoid X receptor beta) and AGER (advanced glycosylation end-product specific receptor) genes in the Vitamin D (n = 17) versus placebo (n = 27) group. IPA suggested roles of Vitamin D in antioxidant defence. LIMITATIONS, REASONS FOR CAUTION: Interpretation of the data is influenced by our intervention strategy (2-12 weeks prior to retrieval). As folliculogenesis may last 5-6 months, our protocol can only examine with confidence the impact of Vitamin D on the final stages of follicular growth. Furthermore, we examined the hormonal profile of the dominant follicle only, while the GC data reflect the transcriptome of all (pooled) follicles large enough to be used for IVF. Luteinised GCs from controlled ovarian stimulation were used in this study, which may be functionally distinct from the GCs of developing follicles. Moreover, the sample size for RNA-sequencing analysis was low (n = 3 per group), regardless of validation by RT-PCR that was performed on a larger cohort, introducing complexity to the IPA analysis, which required an input of data with P-adjusted <0.08 instead of <0.05 to be informative. WIDER IMPLICATIONS OF THE FINDINGS: This is the first in-vivo study to show that Vitamin D supplementation alters gene expression in luteinised GCs. In contrast to some in-vitro evidence, no effect of the intervention on expression of genes encoding steroidogenic enzymes was observed. Unlike other studies, our results suggest that supplementation with Vitamin D is unlikely to directly influence hormone availability in FF. Our findings instead reinforce the hypothesis that Vitamin D could be considered one of the gatekeepers in protecting against an exaggerated response to ovarian stimulation. STUDY FUNDING/COMPETING INTEREST(S): The study has been funded by the Italian Ministry of Health (RF-2013-02358757) following peer review in the competitive 'Bando di Ricerca Finalizzata e Giovani Ricercatori 2013' for the clinical trial SUNDRO (EudraCT registration number 2015-004233-27). There are no competing interests. TRIAL REGISTRATION NUMBER: EudraCT registration number 2015-004233-27.

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.

BACKGROUND: Vitamin D plays an important role in human physiology and pathology. The receptor for vitamin D regulates 0.5-5% of the human genome. Accordingly, vitamin D insufficiency has been shown to increase the risk of several diseases. In recent years, based on growing evidence, on a role of vitamin D has been also postulated in reproductive health both in animals and humans, especially in female fertility female fertility. In vitro fertilization success was shown to be higher in women with appropriate reserves of vitamin D. However a causal relation has not been demonstrated and randomized controlled trials testing the effectiveness of vitamin D supplementation in IVF are warranted. METHODS: This is a multicenter randomized double blinded placebo controlled study aimed at determining the benefits of vitamin D [25(OH)D] supplementation in improving clinical pregnancy rate in women undergoing IVF. Eligible women with a serum level of 25-hydroxyvitamin D [25(OH)D] < 30 ng/ml will be randomized. Recruited women will be given the drug (either 600,000 IU of 25(OH) D or placebo in a single oral administration) at the time of randomization. Two centres will participate and the sample size (700 women) is foreseen to be equally distributed between the two. Patients will be treated according to standard IVF protocols. DISCUSSION: The primary aim of the study is the cumulative clinical pregnancy rate per oocyte retrieval. Clinical pregnancy is defined as the presence of at least one intrauterine gestational sac with viable foetus at first ultrasound assessment (3 weeks after a positive human chorionic gonadotropin [hCG] assessment). Secondary outcomes include: 1) clinical and embryological variables; 2) oocyte and endometrium quality at a molecular level. To investigate this latter aspect, samples of cumulus cells, follicular and endometrial fluids will be obtained from a subgroup of 50 age-matched good-prognosis cases and controls. TRIAL REGISTRATION: The protocol was included in EudraCT on 22nd September 2015 with the registration number assigned ' 2015-004233-27 '; it was submitted through the database of the Italian "Osservatorio Nazionale della Sperimentazione Clinica (OsSC)" - (National Monitoring Centre of Clinical Trials) to the National Competent Authority on 8th March 2016 and approved on 23rd June 2016.