Vitamin D Supplementation Improves Uterine Receptivity in a Rat Model of Vitamin D Deficiency: A Possible Role of HOXA-10/FKBP52 Axis

Abstract

Synchronized uterine receptivity with the time of implantation is crucial for pregnancy continuity. Vitamin D (VD) deficiency has been linked to the failure of implantation. Therefore, we tested the link between the Homeobox transcription factor-10/immunophilin FK506-binding protein 52 (HOXA-10/FKBP52) axis and the uterine receptivity in VD-deficient rats. The effect of VD supplementation at different doses was also investigated. Forty-eight pregnant rats were divided into six groups (eight/group); normal control rats fed with standard chow (control), control rats supplemented with VD (equivalent dose of 400 IU/day) (control-D400). VD-deficient group (DEF) and the three VD deficiency groups with VD supplementation were equivalent to 400, 4,000, and 10,000 IU/day (DEF-D400, DEF-D4000, and DEF-D10000, respectively). The expression levels of HOXA-10/FKBP52, progesterone level, and histological evaluation of decidualization using osteopontin (OSN) and progesterone receptor (PGR) were estimated. An assessment of the uterine contractility was conducted for all rats. This study showed the downregulation of HOXA-10/FKBP52 together with increased amplitude and frequency of the uterine contractility in the DEF group compared to control. VD dose-dependent supplementation restored progesterone/receptor competency, upregulated the expressional response of HOXA-10 and its downstream FKBP52, and improved uterine receptivity and endometrial decidualization at the time of implantation that was documented by increased area% of OSN and the number of implantation beads.

Keywords: HOXA-10/FKBP52; contraction; decidualization; progesterone receptor; vitamin D.

FIGURE 1

A timeline represents the included groups with the scheduled experimental interventions. D: refers to vitamin D (VD) supplementation in different doses equivalent to 400, 4,000, and 10,000 IU. DEF: refers to VD deficiency.

FIGURE 2

(A) Representation of the baseline level of VD, (B) the changes in the serum VD levels at the study end, (C) the serum progesterone, and (D) the number of uterine implantation sites. D: refers to VD supplementation in different doses equivalent to 400, 4,000, and 10,000 IU. DEF: refers to VD deficiency, *: A statistically significant sign compared to the control group, #: compared to control-400, $: compared to the deficiency group, @: compared to DEF-400, and &: significant compared to the DEF-4000 group (p < 0.05).

FIGURE 3

Representation of maintained serum (A) calcium and (B) phosphate within the normal range in all the groups. D: refers to VD supplementation in different doses equivalent to 400, 4,000, and 10,000 IU. DEF: refers to VD deficiency. No statistical significance between the groups.

FIGURE 4

Represents the levels of (A) H₂O₂, (B) Glutathione peroxidase activity (GSH-Px) in uterine tissues, and the expression levels of (C) Homeobox transcription factor (HOXA-10), as well as (D) Immunophilin FK506-binding protein 52 (FKBP52). D: refers to vitamin D supplementation in different doses equivalent to 400, 4,000, and 10,000 IU. DEF: refers to vitamin D deficiency. *: Statistically significant sign compared to the control group, ^#: compared to control-400, $: compared to deficiency group, and @: compared to DEF-400 (P < 0.05).

FIGURE 5

The figure represents (A) the curves of the uterine contraction traced during 10 min after equilibration for 60 min, the scale of the Y axis is 15 g and the axis is 10 min. (B) The amplitude (mg) and (C) frequency (n/10 min) of the wave were calculated. D: refers to vitamin D supplementation in different doses equivalent to 400, 4,000, and 10,000 IU. DEF: means vitamin D deficiency. *: Statistically significant sign compared to the control group, ^#: compared to control-400, $: compared to the deficiency group, and @: compared to DEF-400 (P < 0.05).

FIGURE 6

Photomicrograph of an antimesometrial region of the rat uteri at the 8th day of pregnancy (H&E ×400): (A) Control and (B) Control-D400 revealing different sized rounded decidual cells illustrating eosinophilic cytoplasm, vesicular nuclei, and numerous prominent nucleoli. (C) DEF displaying an impaired differentiation of stromal cells into decidual cells. (D) DEF-D400 illustrating alternating decidual cells and impaired differentiated stromal cells (arrows) into decidual cells. (E) DEF-D4000 and (F) DEF-D10000 revealing an obvious differentiation of stromal cells into different sized rounded decidual cells. Scale bar: 20 μm.

FIGURE 7

Photomicrograph of the progesterone receptor (PGR) immunostained sections in the mesometrial region of the rat uteri at 8th day of pregnancy (×400): (A) Control and (B) Control-D400 revealing strong nuclear immunostaing of PGR in the stromal cells (arrows) and vascular endothelium (curved arrows). (C) DEF displaying weak nuclear immunostaining of PGR in few stromal cells. (D) DEF-D400 illustrating moderate nuclear immunostaining of PGR in some stromal cells. (E) DEF-D4000 and (F) DEF-D10000 demonstrating obvious strong nuclear immunostaing of PGR in the stromal cells (arrows) and vascular endothelium (curved arrows). Scale bar 20 μm. (G) Quantification of the mean area % of PGR positive immunostaining in the mesometrial region of the rat uteri at 8th day of pregnancy. D: refers to vitamin D supplementation in different doses equivalent to 400, 4,000 and 10,000 IU. DEF: refers to vitamin D deficiency *: Statistically significant sign compared to the control group, ^#: compared to control-400, $: compared to the deficiency group, and @: compared to DEF-400 (P < 0.05).

FIGURE 8

Photomicrograph of osteopontin (OSN) immunostained sections of the antimesometrial region of the rat uteri at 8th day of pregnancy (×400): (A) Control and (B) Control-D400 revealing strong cytoplasmic immunostaing of OSN in many decidual cells (arrows). (C) DEF displaying weak cytoplasmic immunostaining of OSN in few cells. (D) DEF-D400 illustrating moderate cytoplasmic immunostaining of OSN in some cells. (E) DEF-D4000 and (F) DEF-D10000 demonstrating strong cytoplasmic immunostaing of OSN in many decidual cells (arrows). Scale bar 20 μm. (G) Quantification of the mean area % of OSN positive immunostaining in the antimesometrial region of the rat uteri at 8th day of pregnancy. D: refers to vitamin D supplementation in different doses equivalent to 400, 4,000 and 10,000 IU. DEF: refers to vitamin D deficiency *: Statistically significant sign compared to the control group, ^#: compared to control-400, $: compared to the deficiency group, and @: compared to DEF-400 (P < 0.05).

FIGURE 9

The correlation represented the direct relationship between VD levels and (A) the serum progesterone level and (B) the uterine progesterone receptor expression. VD revealed a direct correlation to the (C) uterine immunophilin FK506-binding protein 52 (FKBP52), which was correlated significantly to (D) the progesterone receptor expression in the uterine tissues.

FIGURE 10

Representation of strong positive correlations between the VD levels and (A) HOXA-10 and (B) GSH-Px. Interestingly, the uterine contractility represented by (C) the contraction wave amplitude and (D) the frequency of contractions was negatively correlated to VD status.

FIGURE 11

A schematic representation demonstrating the role of VD level in early pregnancy and implantation. VD-deficient rat uteri revealed a small number of uterine implantation sites (beads). By VD supplementation and in a dose-dependent effect, enhanced progesterone secretion, uterine progesterone receptor, and its co-chaperone FKBP52 expression associated with HOXA-10 upregulation were obtained. This causes improving the oxidative stress markers; GSH-px and the H₂O₂ levels and favors decasualization and controlled uterine contractions, thus improved uterine receptivity and promoted the implantation process. The role of VD on increased receptivity was mediated through the modulation of the immunophilin FKBP52, HOXA-10.