Prognostic value of follicular fluid 25-OH vitamin D and glucose levels in the IVF outcome

Animal and in vitro studies have shown that 25-OH vitamin D may play a significant role in glucose tolerance through its effects on insulin secretion and insulin sensitivity [1]. Few data exist about the role of 25-OH vitamin D in reproduction. Insufficient levels during pregnancy are potentially associated with increased risk of preeclampsia, insulin resistance and gestational diabetes mellitus [2–4], while experimental data indicate that 25-OH vitamin D insufficiency is critical for fetal development, and especially for fetal brain development and immunological functions [5]. Evidence has shown that 25-OH vitamin D levels did not vary during the phases of the menstrual cycle [6] while endometriosis has been associated with higher serum levels of 25-OH vitamin D [7]. The most advantageous serum 25-OH vitamin D levels for its proper action appeared to be 30 ng/ml approximately [8], but the effects of higher concentrations need to be addressed. However, the association of 25-OH vitamin D levels with embryo quality and IVF outcome in IVF-ET treatments has not yet been thoroughly investigated.

Regarding the glucose level in follicular fluid (ff), a substantial utilization of glucose by the oocyte/cumulus cells complex has been reported [9], suggesting a possible influence of ff glucose in the fertilization process. Glucose is a pivotal metabolite for the cumulus oocyte complex (COC) which provides substrates to the oocyte for energy production [10]. It has been reported that the oocyte is exposed to glucose levels at a percentage between 31-82% of ff concentrations when the latter concentrations are at physiological levels [11]. The environment to which the COCs is exposed during maturation affects oocyte development and therefore subsequent embryonic development [12]. Impact of altering levels of glucose metabolism on oocytes and COCs function are likely to be a major cause for diminished oocyte competence and reduced fertility.

Considering the interactions of 25-OH vitamin D with glucose we aimed to measure serum and ff 25-OH vitamin D and glucose levels in women undergoing IVF-ET treatment in order to investigate whether 25-OH vitamin D in combination with glucose concentrations have any impact on the IVF outcome.

From January 2009 to August 2009 a total of 101 consecutive women underwent 101 IVF-ICSI cycles. All women studied, after ovarian stimulation, reached the ovum pick up stage, but only 86 managed to reach embryo transfer. All 101 women were allocated to one of the three groups according to their ff 25-OH vitamin D levels. Group A (n = 31), group B (n = 49), group C (n = 21) with less than 20 ng/ml, 20.1-30 ng/ml and more than 30 ng/ml ff vitamin levels, respectively. The biological rationale for this categorization lays in the fact that physiological serum 25-OH vitamin D levels range between 20 and 30 ng/ml [8]. Upper or lower concentrations have been characterized as hypervitaminosis D and hypovitaminosis D, respectively.

Stimulation characteristics in terms of number of ampoules used, number of stimulation days and number of oocytes retrieved, were similar between the three groups (Table 1). In 86 from the total of 101 women studied, embryos were transferred on day 3 post-OPU. The remaining 15 women (group A: 6, group B: 7, group c C: 2) did not have an embryo transfer due to fertilization or cleavage failure. Characteristics, such as age, and BMI, did not differ between the three groups studied (Table 1). Serum vitamin levels of all women studied were significantly correlated with ff vitamin levels (r = 0.79, p < 0.001) (Figure 1), Moreover, ff vitamin levels negatively correlated with embryo quality as depicted in Figure 2 (r = -0.27, p < 0.05). This correlation is reliably reflected in the significant differences in embryo quality between the three groups, with group C demonstrating the lower embryo quality (Table 1). Clinical pregnancy rates of groups A and B were significantly higher as compared to group C (Table 1). The ff vitamin levels of women who achieved pregnancy were significantly lower than those of women that did not become pregnant (22.1 ± 5.5 vs 29.8 ± 8.7 ng/ml, p < 0.05). Furthermore, there were no significant differences between age and BMI values between pregnant and not pregnant women.

	Patient Groups (ff vitamin D, ng/ml)			P value
	A (< 20)	B (20.1 - 30)	C (>30)
No of women	31	49	21	NS
Age (years)	35.76 + 5.8	36.35 + 4.5	36.58 + 5.8	NS
BMI (kg/m2)	23.8 + 2.6	24.2 + 3.1	24.5 + 3.5	NS
Serum vitamin D (ng/ml)	18.7 + 5.74	24.39 + 8.9	30.3 + 11.9	< 0.05
Serum glucose (mg/dl)	85.46 + 17.3	86.04 + 18.3	82.3 + 16.2	NS
ff glucose (mg/dl)	70.44 + 12.7	66.14 + 12.3	52.82 + 16.7	< 0.05
No of stimulation days	10.4 + 2.1	11.6 + 2.6	12.1 + 2.9	NS
No of FSH ampoules (75 IU each)	54.5 + 4.8	56.8 + 5.5	57.7 + 5.9	NS
No of oocytes	3.9 + 3.2	5.91 + 4.3	6.14 + 5	NS
CES	16.29 + 11.2	15.67 + 11.1	15 + 8.1	NS
MSEQ	7.96 + 2.6	7.02 + 2.5	5.6 + 3.6	< 0.05
CPR/woman	32.3% (10/31)	32.7% (16/49)	14.3% (3/21)	< 0.05
CPR/ET	40% (10/25)	38.1% (16/42)	15.8% (3/19)	< 0.05

NS: not significant, BMI: body mass index, ff: follicular fluid, CES: cumulative embryo score, MSEQ: mean score of embryo quality, CPR: clinical pregnancy rate, ET: embryo transfer

A significant negative correlation between ff vitamin levels and ff glucose levels was found (r = -0.25, p < 0.05). Moreover, ff glucose levels were significantly different (p < 0.05) between the three groups with group C demonstrating the lower glucose levels (Table 1).

Our results have shown that the vitamin D levels in the ff were negatively correlated to the quality of embryos and the higher values of vitamin D were associated with lower possibility to achieve pregnancy.

Over the past decade the physiological role of 25-OH vitamin D has been investigated extensively [13], but data regarding its role in human reproduction are scarce. More recently, Ozkan and colleagues [14] reported that replete ff vitamin levels predicts the IVF outcome, proposing therefore the beneficial role of excess vitamin concentrations on endometrial receptivity, since 25-OH vitamin D receptors have been identified in endometrium [15]. The above suggestion seems to contrast with the finding of decreased expression of vitamin D binding protein (VDBP) in the ff of IVF success group reported by Estes and colleagues [16]. This discrepancy is difficult to explain, and more data are needed to elucidate it. However, it is important to note that in the report by Ozkan a significantly higher number of embryos were transferred in the IVF success group compared to the IVF failure group. Although a multivariate regression analysis was performed to show that vitamin D levels in ff independently determined the IVF outcome, the total number of cases was small to draw solid conclusions. Furthermore, in that study [14], the quality of embryos transferred in relation to vitamin D levels was not investigated.

According to the present data, upon increase of ff vitamin levels a small non-significant increase of collected oocytes was observed (Table 1). Although the CES was similar between the three groups, the MSEQ of group C was significantly lower than that of groups A and B (p < 0.05), suggesting that embryos in group C were of poor quality. Taking into account the observed significant negative association between vitamin levels and the quality of embryos (Figure 2) we may hypothize that ff vitamin levels possibly reflect the quality of embryos. It appears that ff vitamin levels, out of the normal range, have a negative impact on the developmental capability of embryos. This effect seems to be even more critical on IVF outcome. Increased ff vitamin levels via the poor quality of produced embryos may result in a poor IVF outcome. The mechanism that vitamin levels exert such an action remains to be investigated, but it appears that glucose levels have a pivotal role in this pathway.

The physiological role of glucose metabolism in regard to reproduction has been determined by several researchers [12, 17], but there is no data on the impact of vitamin levels on glucose metabolism. The ff glucose levels were different between the three groups (p < 0.05), suggesting that higher levels of vitamin D corresponded to lower levels of ff glucose. High serum vitamin concentrations probably increase the utilization of insulin, resulting in the proper metabolism of glucose. In contrast to the comparable serum glucose concentrations, significant differences in ff glucose concentrations between the three groups of women were observed. The explanation for this contrast lays in the fact that the lower ff glucose concentrations are influenced not only by the higher vitamin levels (thus increased glucose transportation into granulosa-cumulus cell complex) but also by the presence of other factors which are responsible for follicle/oocyte maturation. It seems that the interaction between extra-ovarian and intra-ovarian factors determine the fate of the follicle and the quality of the oocyte. Nevertheless, glucose concentrations too low or too high are detrimental for oocyte maturation and growth of granulosa and cumulus cells affecting directly the oocyte competence [12, 17]. According to our results it seems that high ff vitamin concentrations (>30 ng/ml), probably affecting insulin action resulted in modulation of ff glucose metabolism. This situation seems to be undesirable, since it appears to have directly negative impact on oocyte and embryo quality and subsequently on IVF outcome. Women with vitamin D deficiency (group A) appear to react as women with sufficient vitamin D levels (group B). Women with hypervitaminosis D (group C) during assisted reproduction cycles are associated with poor IVF result, something which in line with the significant higher (p < 0.05) vitamin D levels of IVF failure women as compared to the respective levels of IVF success women. Data about the role of vitamin D in follicle function are lacking in women, however, a study in vitamin D receptor null mutant mice suggested that vitamin D is essential for full gonadal function in both sexes [18].

The measurement of 25-OH vitamin D levels in combination with glucose levels are reported for the first time in women undergoing IVF-ET treatments. It seems that increased ff 25-OH vitamin D levels in combination with the decreased ff glucose levels have a negative impact on embryo quality and therefore on IVF outcome.