Vitamin D significance in pathogenesis of endometriosis

Abstract

 

Genital endometriosis (GE) is a widespread multifactorial disease thus making it necessary to carry on studying its pathogeny in order to work out target therapy techniques. Studying vitamin D role in GE pathogeny is a new promising research trend.

Patients and technique

25(ОН)D level was determined in peripheral blood (PB) of 440 patients with GE and in peritoneal fluid (PF) – in 49 GE patients; the same test in PB was performed in 30 patients of the control group with the ovulatory menstrual cycle and no gynecologic pathology. 129 patients with GE, as well as 82 women of the control, underwent examination of vitamin D receptor (VDR) polymorphism gene in BsmI, FokI, and TaqI polymorphic locus. We examined vitamin D receptor expression in the eutopic and ectopic endometrium in 32 women with GE and in the endometrium of 20 women from the control group. We also compared the efficacy of combined therapy involving colecalciferol to the standard hormone modulating therapy.

Results

It was established that the prevalent GE forms are characterized by lower 25(ОН)D levels both in PB and in PF. It was also found that G/G genotype of VDR BsmI gene polymorphic variant 1.9 times increases GE occurrence risk. VDR expression was authentically lower in the ectopic endometrium compared to the eutopic endometrium. Patients with GE show no VDR expression cyclic variations in the endometrium which is contrary to the control. Therapy in combination with colecalciferol promotes a more expressed decrease of endometriosis-associated pain syndrome and psycho-emotional stabilization of GE patients compared to the standard hormone modulating therapy.

Conclusion

Vitamin D deficiency plays a significant role in the pathogenesis of GE and Vit D may be applied as its targeted therapy.

Keywords:

• Genital endometriosis
• vitamin D
• colecalciferol
• vitamin D receptor (VDR)

Introduction

Genital endometriosis (GE) is a chronic recidivating progressive hormone-dependent disease. The GE pain syndrome is a serious problem for women and may negatively affect their psycho-emotional state and quality of life [1]. There is no universal treatment that could guarantee complete recovery and exclude disease recurrence [2]. Studies on vitamin D and its receptor in GE pathogeny is a new promising research trend.

The conventional idea of vitamin D has undergone considerable changes due to the discovery of its active hormonal form – calcitriol which is able to produce a number of non-classical effects after binding to vitamin D specific receptors located in various tissues and organs. Some results of studies on experimental models of surgically induced endometriosis in mice and rats show a significant decrease of endometrioid heterotopia area and/or complete damage resorption after treating with colecalciferol and its selective agonist elocalcitol [3–5]. The studies demonstrated non-classical effects of vitamin D such as anti-inflammatory effect based on macrophage quantity and anti-inflammatory cytokine – interleukin-1 (IL-1) in PF level decrease [3]; anti-proliferative effect – fibrosis and apoptosis occurrence in implant stromal components [4] and also anti-angiogenic effect due to the decrease of VEGF (vascular endothelial growth factor) and matrix metalloproteinase levels along with the reduced possibility to increase TIMP-2 (tissue metalloproteinase inhibitor-2) concentration [5].

Our previous study performed on endometriosis experimental model in rats demonstrated a positive dose dependent effect – resorption or regression of endometriosis lesions after oral colecalciferol therapy [6].

Objective

To estimate vitamin D significance in GE pathogenesis, to estimate the efficacy of colecalciferol administration and work out schedules for combined therapy together with its application.

Patients and technique

The screening involved 440 women of reproductive age with GE diagnosis established by operative laparoscopy and confirmed morphologically. Endometriosis-associated pain syndrome was the criteria for inclusion in the study. The control group consisted of 30 women with ovulatory menstrual cycles and without gynecological pathology. The level of 25(ОН)D in PB was detected by enzyme multiplied immunoassay in all patients of both basic and control groups and also in PF – in 49 patients of the basic group.

VDR gene allelic variants were examined in 129 with I-IV degree GE and 82 women from the control group composed by population sampling. The examination was performed by polymerase chain reaction technique – restriction-site polymorphism.

VDR expression level in the ectopic and eutopic endometrium was determined by immune histochemical; analysis in 32 patients with GE and in 20 patients from the control. Visualization was performed applying abcamRabbitSpecific HRP Plus (ABC) Detection IHC Kit (RTU) [ab93697], (Abcam, Great Britain).

Depending on drug treatment variant 440 patients were divided into two groups. The first consisted of 240 patients, 217 of which received colecalciferol in addition to standard hormone modulating therapeutic regimen: 104 in combination with 3.75 mg gonadotropin-releasing hormone agonist (aGnRH) and 113 – in combination with dienogest 2 mg. 23 patients who received colecalciferol as monotherapy either because of contra-indications to standard hormone modulating therapy or patient’s refusal. The second group included 200 patients receiving standard hormone modulating therapy, 103 of which– aGRH 3.75 mg and 97 of which –dienogest 2 mg as monotherapy.

The daily calciferol dose in the first subgroup was personally adjusted considering the initial 25(ОH)D in PB and average values of concentration changes based on daily intake according to the GrassrootsHealth algorithm (Table 1).

Table 1. Median values of 25(ОН)D concentration changes in PB based on daily intake according to GrassrootsHealth algorithm (A Public Health Promotion Organization http://www.grassrootshealth.net).

Expected level (ng/mL)	20	30	40	50	60
Real level (ng/mL)	Colecalciferol recommended daily dose (IU)
10	2000	4000	6000	10000	10000
15	1000	3000	6000	9000	10000
20		2000	5000	8000	10000
25		1000	4000	7000	10000
30			3000	6000	10000
35			1000	5000	9000
40				3000	9000

In drug prescribing preference was given to the highest permissible doses. The recommended 25(OH)D level to achieve the expected effect was 40–60 ng/mL. Before the start of the administration and after 6 months of administration pain syndrome estimation was performed applying the McGill Visual Analogue Scale and also estimation of psycho-emotional state with the application of Hospital Anxiety and Depression Scale (Zigmond A.S. & Snaith R.P).

Statistical analysis

Results statistic processing applied non-parametric statistic technique. The description of the abnormal distribution of quantitative variables was done by median and quartiles 25 and 75 Me (Q1; Q3). The frequency difference authenticity determination was performed applying the exact two-way Fisher test by a standard formula considering Yates correction also applying chi-square (χ²) criterion together with Yates correction and Bonferroni adjustment. The authentic difference between the control and the basic group was treated by odds ratio.

Results

The PB 25(ОН)D level median value in patients with GE (n = 440) was 22.1 (17.1;28.0) ng/mL and was authentically lower (Mann-Whitney test, p < .001) than in the control − 36.0 (19.6;52.8) ng/mL. The PB 25(ОН)D level median value in patients with mild GE (I-II grade) was 23.6 (20.4;28.4) ng/mL while in patients with prevalent GE (III-IV grade) it was 20.6 (16.2;27.6) ng/ml, though the difference was not statistically relevant (Kruskall-Wallis test, p = .267).

The group of patients with GE who underwent (ОН)D level test in PF showed (ОН)D level median value = 27.5 (20.4; 34.9) ng/mL wherein the subgroup of patients with I-II grade GE it was GE − 27.9 (22.3; 34.9) ng/mL while the patients with III-IV grade GE showed 23.1 (13.4; 35.2) ng/mL. The PF (ОН)D level median value was 5.2 (3.6; 9.1) ng/mL where patients with I-II grade GE showed 8.9 (4.9; 11.7) ng/mL, while those with III-IV grade GE − 3.6 (0; 5.2) ng/mL. 24% of patients with GE had 25(ОН)D levels in PF below the technique analytical sensitivity threshold thus we put it as equal to 0 ng/mL. A dependence was detected between 25(ОН)D levels in PB and PF (Wilcoxon rank-sum test, p < .001). The same dependence was preserved after dividing the patients into two groups: first with I-II grade GE and the other with III-IV grade GE (Wilcoxon rank-sum test, p < .001; p = .001). The dependence of 25(ОН)D levels in PB on the disease morbidity rate in patients was not detected (Mann-Whitney test, p = .19). Contrary to that the 25(ОН)D level in PF was significantly dependent on the morbidity rate (Mann-Whitney test, p = .004).

It was established that the frequency of the VDR BsmI gene G allele was authentically higher in the group with GE compared to the population sampling (p = .048). We have also established authentic differences for VDR BsmI gene polymorphic variant G/G genotype in patients with GE compared to the control (p < .05). According to the odds rate GE occurrence risk was 1.9 times higher for this genotype (OR = 1.93 CI = 1.082–3.450). On the contrary, it was also detected that A/A + G/A genotype combination was authentically more frequent in the population (p = .025).

VDR expression was verified in the glad epithelium of eutopic and ectopic endometriosis (Figures 1,2,3) as well as in the endometrium of control group patients. The results of VDR expression estimation are presented in Table 2. It should be mentioned that according to histological examination endometrium hyperplasia was detected in 5 patients and thus the data of these patients were ignored in VDR expression index analysis. In the other, examined cases endometrium structure corresponded to the proliferative (n = 7) and the secretory (n = 20) cycle phases.

Figure 2. VDR expression in glands and stroma of eutopic endometrium – secretory phase. IHC test × 400.

Figure 3. VDR expression in endometrioid heterotopias. IHC test × 200.

Table 2. Estimation of VDR expression in endometrioid heterotopias and the endometrium of patients with GE and in the endometrium in patients from the control.

Groups	VDR expression
	Menstrual cycle phase
	Proliferative (n = 17)	Secretory (n = 30)	Pare-wise comparison*
1 Endometrioid heterotopias (n = 27)	12.88% (10.5;14.49)	10.32% (6.47;15.09)	p = .808
2 Eutopic endometrium (n = 27)	20.12% (18.1;24.24)	21.51% (17.41;24.83)	p = .314
3 Endometrium (control) (n = 20)	14.76% (11.83;23.66)	22.35% (15.82;29.45)	p = .043
Comparison between groups**	p1–3 < .001	p1–3 < .001
Pare-wise comparison***	p1–2 = .01 p1–3 = .309 p2–3 = .413	p1–2 < .001 p1–3 = .917 p2–3 = .007

*Mann-Whitney test.

**Kruskall-Wallis test.

*** DSCF test.

VDR expression in the endometrioid heterotopias was statistically significantly lower compared to the indexes of eutopic endometrium in GE patients: 1.6 times lower in the proliferative phase and 2.1 times lower in the secretory phase of the menstrual cycle (DSCF test p = .01 and p < .001) respectively. The comparison of VDR expression in endometrioid heterotopias to that in the endometrium in patients from the control in the secretory phase showed also a significant decrease of the expression level − 2.2 times in cases of endometriosis (DSCF test, p = .007). No significant difference, however, was detected between the endometrioid heterotopias of the basic group and the control group endometrium in the proliferative phase (DSCF test, p = .413). No statistically significant difference for VDR expression in endometrioid heterotopias could be detected between secretory and proliferative menstrual cycle phases (Mann-Whitney test, p = .808). VDR expression in the endometrium of patients with GE statistically did not vary in different menstrual cycle phases (Mann-Whitney test, p = .314). Along with that the control group in the menstrual cycle secretory phase showed VDR expression 1.5 times increase compared to the same index in the proliferative phase (Mann-Whitney test, p = .043).

The estimation of pain syndrome dynamics showed higher therapeutic efficacy in the group of patients with GE who received combined therapy including colecalciferol. In the group of patients, who received monotherapy with aGnRH 3.75 mg, the absence of pain syndrome was revealed in 77.7% of them. In the group of women, receiving aGnRH 3.75 mg in combination with colecalciferol, the absence of pain syndrome was revealed in 92.3% of them. In 74.2% of patients, receiving dienogest 2 mg as monotherapy, there was no pain syndrome. In the subgroup of patients, receiving 2 mg dienogest combined with colecalciferol, no pain syndrome was revealed in 90.3%. In the subgroup of patients with GE, receiving colecalciferol as monotherapy, the absence of pain syndrome was demonstrated in 69.6% of the women.

The estimation of psycho-emotional status detected anxiety and depression symptoms in 262 women with GE, 52 of them received aGnRH in combination with colecalciferol; 77 of them were given dienogest in combination with colecalciferol and 6 of them were administered colecalciferol as monotherapy. The comparison subgroups consisted of 61 and 66 patients with GE receiving aGnRH 3.75 mg and dienogest 2 mg respectively. The absence of anxiety and depression symptoms was revealed in the majority of patients receiving vitamin D: 67.3% of patients in the group of aGnRH 3.75 mg combined with colecalciferol; 80.5% – in case of combined treatment with colecalciferol and dienogest 2 mg. In the group of patients, who received aGnRH3.75 mg as monotherapy, absence of anxiety and depression symptoms was noted in 42.7% and 72.2% – in the group of dienogest 2 mg as monotherapy. In the subgroup of patients with GE, who received colecalciferol as monotherapy, we did not find symptoms of anxiety and depression in 66.7% of them.

Discussion

Our study demonstrated that vitamin D deficiency (the level of 25(ОН)D is lower than 20 ng/mL) or insufficiency (the level of 25(ОН)D is 20–30 ng/mL) were found in patients with GE. Along with that, the common disease types are characterized by decreased levels both in PB and PF. Hence 25(ОН)D deficiency and/or insufficiency may appear a risk factor for the disease progression.

According to the literature, VDR BsmI gene polymorphic variant allele A is linked to the gene exceeding expression and promotes the serum calcitriol level increase compared to the G variant [7]. Contrary to that G variant is predominant in patients with GE which is probably linked to the gene decreased expression along with calcitriol reduced serum level. This fact may be important for determining the necessary colecalciferol dose for this cohort of patients.

The immune histochemical analysis results allow concluding that VDR is expressed both, in heterotopias and in GE patient endometrium as well as in the endometrium of the control group patients. There are known studies on VDR expression in women’s endometrium [8–10], still, VDR expression in endometrioid heterotopias of GE patients has not been yet investigated. Our study results show that VDR expression in the endometrium of women from the control group in the secretory phase was 1.5 times higher compared to the same index for the proliferative phase, and the value difference was statistically significant (DSCF test, p = .047). VDR expression increase in the secretory phase within the period of the supposed implantation window is probably linked to vitamin D progesterone-like activity which may be important for full-rate secretory transformation and successive embryo implantation.

We did not observe any cyclic variations of VDR expression in the endometrium of patients with GE. This finding may be important for clarifying disease pathogenesis and may indicate endometrium receptivity changes in patients with GE and may also appear one of the reasons for endometriosis-associated infertility.

Existing data on colecalciferol influence on endometriosis-associated pain syndrome are contradictory. Almassinokiani F. et al. did not find any significant difference in dysmenorrhea and chronic pelvic pain decrease after colecalciferol therapy compared to placebo [11]. Contrary to that Lasco A. et al. established that colecalciferol after a single intake of 300,000 IU 5 days before the expected menstruation in patients with endometriosis-associated primary dysmenorrhea leads to the statistically significant decrease of expression of pain syndrome compared to placebo [12]. The pain syndrome intensity decrease may be most probably ascribed to calciferol capability of inhibiting the prostaglandin synthesis in the endometrium and inactivating them by suppressing cyclo-oxigenase-2 [13]. Our study results showed that patients treated with combinations of 3.75 mg aGnRH or 2 mg dienogest with calciferol demonstrated much more expressed pain syndrome decrease and Psycho-emotional stabilization compared to the standard hormone modulating therapy.

Conclusion

Our data demonstrate that 25(ОН)D decreased levels both in PB and PF may appear a risk factor for disease progressing. G/G genotype of VDR BsmI gene polymorphic variant 1.9 times increases endometriosis occurrence risk. Patients with GE show no VDR expression cyclic variations which may be one of the risk factors of endometriosis-associated infertility. Colecalciferol administration in patients with GE is an effective treatment both as monotherapy and as an addition to standard treatment schedules. Taking into consideration its good tolerance together with efficacy and comparably low cost, the possibility of long-term administration and of prescription for anti-relapse treatment including the pregnancy planning stage, colecalciferol may be applied as a promising pathogenically approved targeted therapy of endometriosis.

Disclosure statement

The authors report that they have no conflict of interest.

Figure 1. VDR expression in glands and stroma of eutopic endometrium – proliferative phase. IHC test × 400.

Additional information

Funding

This research was supported in the frame of Fundamental Scientific research АААА-А19-119030490009-6 by Russian Ministry of Science and Higher Education.