Principal findings
In this study, the total follicle 25(OH)D3 and follicular free 25(OH)D3 levels were higher in the TQE group than in the non-TQE group. The results of this study were consistent with those reported by Alawad et al. Intrafollicular vitamin D levels are positively correlated with oocyte maturity, fertilization rate, and top-quality embryos.[14] Arnanz et al found that total follicle 25(OH)D3 levels and follicular free 25(OH)D3 levels were predictors of euploid embryos in the vitamin D-deficient group.[13] However, a study conducted by Ciepiela et al. showed different results; the TQE group had lower follicular 25(OH)D3 levels than the non-TQE group [4]. Rudick et al. revealed that race had an effect on the relationship between vitamin D status and post-IVF pregnancy rates.[15] The results of this study were in line with those of Zainab M. Alawad and Arnanz et al. due to the same sample (Asian racial group), while in the study of Ciepiela et al. the study subjects were the Hispanic race group.
A study conducted by Cai et al. showed that serum total 25(OH)D3 levels correlate with serum-free 25(OH)D3 levels.[16] In this study, total follicle 25(OH)D3 levels correlate with follicular free 25(OH)D3 levels. However, in this study, total follicular 25(OH)D3 levels were not correlated with total serum 25-(OH)-vitamin D3 levels, in contrast to the study by Liu et al., in which the levels of vitamin D in follicular fluid correlated with serum levels of vitamin D.[15] This is thought to be related to the pleiotropic effect of vitamin D, which is related to the vitamin D response element (VDRE) gene, which gives a different phenotype in each cell.
The median follicular calcium level in the TQE group was higher than in the non-TQE group (6.30 (3.74–18.49) mg/dL vs 6.23 (3.60–17.82) mg/dL respectively), but not significantly different statistically (p = 0.805). Follicular calcium levels were positively correlated with serum 25(OH)D3 levels in the TQE group (r = 0.218, p = 0.046) and all study samples (r = 0.216, p = 0.004). However, calcium levels did not correlate with serum 25(OH)D3 levels in the non-TQE group (r = 0.051, p = 0.216). Follicular calcium levels were positively correlated with serum calcium levels in the TQE group (r = 0.222, p = 0.040), the non-TQE (r = 0.215, p = 0.046), and all study samples (r = 0.224, p = 0.003). The results of this study are in line with those of Ozdamar et al., in which follicular calcium levels in the IVF group in pregnant vs. non-pregnant patients were not significantly different.[17] It was hypothesized that during follicular fluid sampling, a specific calcium oscillation process did not occur, so there was no activation of intracellular calcium ions.
Patients with total follicular 25(OH)D3 levels of > 12.1 ng/mL had a 2.38 times tendency to obtain high-quality embryos (TQE). Ciepiela et al. reported that the cut-off value of serum 25(OH)D3 as a predictor of top-quality embryos was > 12.7 ng/mL [4]. In a study by Liu et al., a cut-off value of 25(OH)D3 serum of > 14.1 ng/mL can predict a normal fertilization rate, pregnancy rate, and live birth rate.[15] The cut off value of follicular free 25(OH)D3, which can be used as a predictor of top quality embryo (TQE), is > 0.00175 ng/mL (95% CI 0.605 (0.528–0.678), p = 0.0146, sensitivity 34.88%, specificity 85.06%). The relationship between the cut off value of follicle-free 25(OH)D3 levels > 0.00175 ng/mL between the TQE and non-TQE groups differed statistically significantly (p = 0.002, OR (95% CI) 3.05(1.46–6) ,38)). Patients with follicle-free 25(OH)D3 levels > 0.00175 ng/mL showed a 3.05 times tendency to obtain top-quality embryos (TQE).
The mechanism that could explain the results of this study is that follicular free 25(OH)D3 affects AMH signaling and steroidogenesis in granulosa cells; thus, good follicle development can occur. Modulation of steroidogenesis in granulosa cells through activation of the AMP-activated protein kinase (AMPK) pathway increases follicle maturation. Vitamin D also regulates oxidative stress at the cellular level, which plays an important role in follicular development. Mature oocytes can fertilize and form high-quality embryos.