Expression of vitamin D and vitamin D receptor in chorionic villous in missed abortion.

The contemporary world despite its enough developed medicine and generally highly enlightened population faces a great problem of vitamin, micro-element and nutrient deficiency turning to become the XXI century pandemic. Along with that significant growth of interest can be seen towards vitamin D importance for reproductive physiology. The fact is that vitamin D receptors (VDR) have been detected in women's ovarium tissue, fallopian tubes, decidua and placenta. Some recent years studies have proven that vitamin D may act as immune regulator during implantation. During early pregnancy the trophoblast release vitamin D, which produces anti-inflammatory reaction and also induce decidual tissue growth for successive pregnancy. It was a comparison between the expression of Vitamin D and VDR in chorionic villous in cases of normal pregnancy and missed abortion groups. 64 samples of chorionic villous were taken: 32 from missed abortion and 32 from the induced abortion group. Abortive material was taken from two groups of women residing in North-West region of Russia: missed abortion and pregnancy terminated at woman's wish (induced abortion); 6-12 weeks of gestation, singleton pregnancy. Immune histochemical examination showed homogenous distribution of vitamin D and VDR expression in syncytiotrophoblasts, cytotrophoblasts and chorion villus stroma.Vitamin D expression relative area was 10,3% which is statistically different from the induced abortion group - 15,4% (p<0,01). VDR expression analysis showed its homogenous distribution in chorionic villus structures in both groups. High VDR expression was detected in chorion villus stromal components. In missed abortion group, the morphometry results showed distinctly lower relative area of vitamin D expression against the comparison group (35,9 ± 1,8; 56,1 ± 2,4 p < 0,01). Also in missed abortion group, positively significant correlation has been determined between the level of vitamin D in blood and VDR relative area expression (r = 0,412). In missed abortion group, definite vitamin D and VDR expression decrease was detected compared to the induced abortion group. The results witness vitamin D importance for pregnancy progress.


Introduction
The human implantation process takes a very short time, which is called implantation window or receptivity period (approximately 6th-8th day after LH peak). Normally the formation is ensured by steroid sex hormone cyclic impact on the endometrium, causing its transformation on tissue, cellular, subcellular, and molecular organization levels [1]. Moreover, an active trophoblast invasive process is seen on 5th-6th pregnancy week, causing considerable spiral artery lumen extension and significant growth of placental perfusion. After 6-8 weeks, chorion starts producing the basic amount of progesterone and this is also the very period of placenta formation.
The majority of spontaneous pregnancy terminations happen in the first trimester. In high miscarriage risk groups, threatened miscarriage is common and permanent not only during the whole gestation period but used to be common for each next pregnancy. High miscarriage risk groups are such as repeated miscarriages, ART failure, sterility, endocrine pathology, endometrium receptor apparatus disturbances, chronic endometritis with a persistent opportunistic pathogen and/or virus, genital abnormality, intrauterine synechia, antiphospholipid syndrome, autoimmune disorders and immunologic factors, chromosome rearrangement bearing marriage partners. Repeated failure risk is also high and if no rehabilitation or treatment measures are taken on pre-conceptional and on early gestation stages, the result is 50% pregnancy failures [2].
It is known that low progesterone level in the 1st pregnancy trimester is a prognostic marker for threatened miscarriage development. It is common to take into consideration and accentuate only the progesterone insufficiency and assign maintaining monotherapy based on "pregnancy corpus luteum" function supplementing, which is often not enough (especially for normal fetal karyotype). It has been proven that miscarriages may be caused not only by progesterone deficiency in the pregnancy but also by chorion insufficiency including its own hormone and active biological molecule release.
The modern world despite its highly developed medicine and enlightened population faces a great problem of vitamin, microelement and nutrient deficiency turning to become the XXI CONTACT Olesya Bespalova shiggerra@mail.ru The Research Institute of Obstetrics, Gynecology and Reproductology Named after D.O. Ott, St. Petersburg, Russia century pandemic. References it has been shown that vitamin deficiency may lead to hypovitaminosis or even of vitamin deficiency. Thus, 50-80% of pregnant women have vitamin deficiency [3] which is a disadvantageous context for gestation and delivery. May cause placenta formation disorders and increase pathologic condition or disease risk in newborns and infants [4]. While folic acid importance is well accepted and vitamin d9 addition on pregravidal and gravidal stages is recommended in all world guideline [5] as well as that of magnesium for successful conception and pregnancy prolongation, the evidence base for the necessity of other biologically active substance addition important for correct and harmonious pregnancy support and development is still discussed.
The recent years have witnessed a rising interest in vitamin D importance for reproduction [6]. Vitamin D metabolism within the duration of pregnancy is characterized by a physiologic increase of vitamin D active form [calcitriol 1,25 (OH)2D] and its subgroup calcidiol [25(jy)D] in the maternal organism aimed at reaching its optimal level in the fetus [7,8]. It is been shown that calcidiol freely penetrates the placental barrier and appears to be the basic pool of vitamin D in the fetus.
It is known that the maternal adaptive immune system is physiologically suppressed during pregnancy, while the inborn immunity receives certain stimulation. These changes in motherplacenta-fetus system are beneficial for immune tolerance and exclude fetus rejection possibility. Fact is that vitamin D may act as an immune regulator in time of implantation. In early pregnancy, trophoblast works out vitamin D and in the meantime responds to the impact of exogenous vitamin D, which produces a local anti-inflammatory reaction in tandem with inducing decidual tissue growth for successive pregnancy. T-and B-activated lymphocytes have vitamin D receptors, due to which 1,25(OH)2D appears to be an effective immune system modulator being capable of inhibiting 1 type T-helper proliferation and limiting the production of cytokines: IFN-c, IL-2, and TNF-a. On the contrary, vitamin D is capable of inducing 2 type T-helper cytokines by having a protective effect on pregnancy [9,10].
Vitamin D receptors (VDR) are expressed in 36 tissue types [11,12]:stomach, amphiblestrodes, somatic, cardiac and smooth muscles, prostate, skin, hair follicles, thyroid and parathyroid gland, salivary gland, fatty tissue, pituitary, testicles lungs, adrenal glands, bony and cartilaginous tissue, osteoblasts, bone marrow, cerebral and cancerous cells, poroophoron, small intestine, kidney, pancreatic cells, thymus, and fetal liver. In women, VDR is expressed in breast tissue, ovary, endometrium, fallopian tubes, decidual tissue, placenta, and ovarian membrane. Human placenta expresses all the vitamin D signaling components (VDR, associate to VDR retinoid X-receptor (RXR)nuclear to vitamin A receptor. The VDR-RXR complex connects itself to the respective genome region in the presence of calcitriol and launches gene transcription mechanism followed by the translation of corresponding protein molecules, mitochondrial enzyme CYP27B1, and cytochrome P-450 isoenzyme CYP24A1). In 1979, Weisman et al. [13] discovered that during in vitro studies decidual and placenta tissue cell culture is capable of synthesizing biologically highly active calcitriol metabolite [1,25(OH)2D and 24,25(OH)2D] able to suppress cytokine expression, such as granulocyte-macrophage colony-stimulating factor 2 (GMCSF-2), tumor necrosis factor a (TNF-a), anti-inflammatory interleukin 6 (IL-6), and also to stimulate cathelicidin antimicrobial peptide (CAMP) expression in cytotrophoblasts and primarily cultivated decidual cells [14,15]. Thus the Escherichia coli toxin effect on tertiary chorion villi in the experimental model along with simultaneous vitamin D administration was less traumatic than a similar effect on a cell culture without vitamin D [3] which could be due to a CAMP elevated level. Consequently, vitamin D addition in case of 25(OH)D deficiency in serum may reduce infection process frequency within the gestation period.
No less than 50% of the world population has differently expressed vitamin D deficiency [3]. Nowadays WHO experts estimate vitamin D deficiency as new XXI century pandemic [16]. Vitamin D deficiency has been registered in more than half of gravidas and newborns [17,18]. Vitamin D deficiency increases risks of such obstetric pathologies as secondary hypertension and gestosis, gestational diabetes [19][20][21], Cesarean, spontaneous premature delivery, bacterial vaginosis starting from early gestation stages. Still the number of proper quality investigations does not allow setting up an evidence base to determine cause-and-effect-relationship for vitamin D and pregnancy sequela etiopathogenesis. According to 2016 Cochrane review, vitamin D administration at pre-gravidal and gravidal levels does not improve pregnancy start and outcome [21]. Such results were obtained due to the absence of polycentric randomized studies as well as distinct criteria for terms, treatment duration, dosage, initial and reached vitamin D blood-level, they analyzed 2833 women in 15 trials, they showed a decrease in risk of preeclampsia and some studies showed a decreased risk of preterm birth. It is known that placenta forms a physical and functional barrier between maternal and fetal perfusions. 1,25(OH)2D may have autocrine, paracrine, and endocrine importance in mother-placenta-fetus system for immune defense regulation, trophoblast invasion, nutrient materials and gases, hemopoiesis, hormone release, fetal growth, and development. It is well known since the 1970s, that trophoblast and decidual cells transform calcidol into vitamin D active formcalcitriol. Later studies established a definite connection between this transforming process and CYP27B1 expression [22]. In 1983, VDR expression in placenta tissue was demonstrated by binding radioactive calcitriol marked CYP27A1 with VDR in rat trophoblasts, then further investigations showed that trophoblast, vitelline sac, and decidual tissue in sheep, mice, rats as well as human gravida express VDR. Apart from that CYP24A1is also expressed in trophoblast, vitelline sac and decidual tissue, where it facilitates transforming 25(OH)D and 1,25(OH)2D into inactive forms.
There are no studies dealing with vitamin D expression from early pregnancy in cases of complicated or and physiological pregnancy.
The present study objective was vitamin D and VDR expression assay in chorionic villous both in cases of the normal (medical abortion) and the missed abortion group.

Study design
sixty-four samples of chorionic villous were obtained: 32 of missed abortion and 32 of induced abortion. Criteria of inclusion were: abortive material from two groups of women residing in North-West region of Russia either with missed abortion or pregnancy terminated at woman's desire (Induced abortion); 6-12 weeks gestational age, singlet pregnancy, normal fetal karyotype. Criteria of exclusion were: abortive material in case of proved fetal genetic pathology, gestational age over 12 weeks, multiple pregnancies, diabetes mellitus. Both group patients did not differ in age, body mass index, gestation number and age for the moment of termination. First group patients had twice less abrasio cases in an anamnesis which was authentically different from the second group (p < .05). Homocysteine and vitamin D blood-levels in women were determined only for the first group of missed abortion cases. It was noted that women of this group had vitamin D deficiency with vitamin D blood concentration level -26.3 ± 7.04 mmol/L.
The morphology study applied histological analysis (using hematoxylin and eosin for survey staining), immune histochemical examination of vitamin D and VDR expression in villous chorion (this technique implied quantity and quality estimation of vitamin D and VDR expression in villous chorion by applying antibodies), confocal laser scanning microscopy, and 3D reconstruction (Table 1).

Histological technique
The histological study was carried out according to the standard histological design. Hematoxilin and eosin were used for survey staining. Embryohistologic gestation terms, chorial syncytium, villus stroma, and vessel component state were estimated by light microscopy along with detecting chorion villi pathologic changes. Immune histochemical examination of vitamin D and VDR expression in villous chorion for the first trimester non-developing pregnancy was carried out by applying DakoCytomation

Digital microscopy and morphometry
The quantity analysis of immune histochemical reaction results was based on photomicrography obtained through microscopic image fixation system, consisting of Olympus BX46 microscope and «CellSens 47 Entry» NikonEclipse E400 software. The studied marker expression occupied area was calculated by Video-Test-Morphology 5.0 software (VIDEOTEST, Russia). Following indexes were estimated for each section in five visual fields: (1) expression optic density automatically measured according to the Bouguer-Lambert-Beer law (the so-called "optic density expression" index basic for Video-Test-Morphology 5.0 software is considered applicable for microphotography optic parameter analysis because measures are taken similarly to spectrophotometric analysis); (2) expression relative area calculated as proportion of immune positive cells area to specimen general area: After that, the studied index average was figured.
To figure and compare the digital data average as well as to estimate the obtained results, the authenticity share difference valuation method was applied along with average tendency analysis (Student's t-criterion) and correlation analysis. The average rate difference was considered veracious with p < .05. The statistical analysis supposed comparing two excerpts by one character (present or absent). The correlation analysis was based on the Pearson correlation coefficient with following correlation force indexes: r ¼ 0-0.2 (very weak), r ¼ 0.2-0.4 (notable), r ¼ 0.4-0.6 (significant), r ¼ 0.6-0.8 (high), and r ¼ 0.8-1.0 (very high).

Histological study
For each case of the presented material 64 samples, embryohistologic gestational age was identified with their peculiarities strictly dependent on the age (Tables 2 and 3).

Missed abortion group
In abortive material sample of the missed abortion group (n ¼ 32), the chorion villi correspond the 6-8 week embryohistological age in 6-12 week obstetric gestational age. Chorion villi with differently expressed spread dystrophic changes were spotted in 60% (19) of studies.

Immunohistochemical study
The immunohistochemical study showed the homogenous distribution of vitamin D and VDR expression in syncytiotrophoblasts, cytotrophoblasts, and chorion villus stroma both in the missed abortion group and the induced abortion group. Vitamin D expression is presented in Figure 2. Vitamin D expression relative area in the missed abortion group was 10.3%, while in the comparison group, 15.9 %. The studied marker comparative assay showed veracious vitamin D expression decrease in the missed abortion group. The expression optic density had no statistically significant variation. It was 0.12 ± 0.01 in the basic group and 0.13 ± 0.1 in the comparison group. VRD expression analysis showed its homogenous distribution in chorion villus structures in both groups. High VDR expression was detected in syncytiotrophoblasts, cytotrophoblasts, and chorion villus stromal componentsdata expressed in Figure 2.
The morphometric results showed veraciously smaller vitamin D average area expression in chorion villi in the missed abortion group than in the comparison group (p < .01). The VRD expression optic density index decrease was noted in the missed abortion group compared to the comparison group: 0.17 ± 0.01 and 0.20 ± 0.01 respectively.
Notable connections have been discovered thanks to the undertaken correlation analysis (Table 3). Thus, significantly positive relation has been established between vitamin D bloodlevel and VDR expression relative area (r ¼ 0.412) in pregnancy termination cases which was not observed for the expression of the vitamin itself (r¼ À0.084). An evident trend can be pointed out: the absence of any correlation between vitamin D and VDR expression both in the missed abortion group and normal fetal development cases (r ¼ 0.15, r¼ À0.088). Thus, there is a notable positive correlation between vitamin D expression and pregnancy termination stage in the missed abortion group (r ¼ 0.29), which is not observed in the normal fetal development group (r ¼ 0.0001). A curious fact is that VDR expression level dependence on pregnancy termination time is completely contrary, i.e. the earlier was pregnancy termination the higher was VDR expression (r¼ À0.32). The immunofluorescent study applying confocal laser scan microscopy allowed detecting verified vitamin D expression in syncytiotrophoblasts, cytotrophoblasts, and chorion villus stromal components. Vitamin D expression area was comparable to histochemical study results. Figure 3 presents vitamin D expression in chorion villi structures in both study groups. The immunofluorescent study showed VDR expression ultimately homogeneous distribution in syncytiotrophoblasts. In chorion, villi stroma VDR expression minimum value and nonuniform distribution were determined. Perhaps VDR expression decrease is bound to dystrophic changes in chorion villi ( Figure 3).
Taking into consideration that this was the first vitamin D and VDR morphology study, we have estimated the studied markers co-expression in decidual tissue fragments of the investigation groups. Vitamin D expression (green fluorescence) was homogeneously distributed and had no more than 53% of the general expression area. VDR expression was also homogeneously distributed having high intensity and about 80% expression area. VDR expression homogeneous distribution was also observed in placenta bad zones. Vitamin D and VDR expression in extravillous trophoblasts are also homogeneously distributed.
r ¼ À0.088 4. Correlation between vitamin D expression and pregnancy termination stages in Group 1 r ¼ 0.0001 5. Correlation between VDR expression and pregnancy termination stages in Group 1 r ¼ 0.003 6. Correlation between vitamin D expression and reproductive loss number Correlation between VDR expression and reproductive loss number

Conclusion
It is well known that women experience increasing vitamin and microelement demand during pregnancy. According to WHO, child's future health and mental development ensuring factors are: young parental age (20-30 years), mother's healthy lifestyle before and during pregnancy, good high-vitamin nutrition, rich of microelements and essential polyunsaturated aliphatic acids. Fact is that in case of normal fetal karyotype chorion (and then placenta as well) is an independent and self-sufficient organ producing a constellation of hormones and biologically active molecules. In the case of steroid disbalance in pregnant women, the chorion function is damaged and finally suffers insufficiency. Chorion sustainable work may be influenced not only by endocrine profile but also by the immune system and vitamin D, the receptors of which are present in villi. Vitamin D may appear to be an immune regulator during implantation. In early gestation  ages, trophoblasts simultaneously produce vitamin D and respond to its impact, having a local anti-inflammatory effect and in the meantime inducing decidual tissue growth for a successful pregnancy. Thus, vitamin D sufficient administration is very important for successive pregnancy development as well as for fetal and newborn health. Every second termination threatened pregnant woman has vitamin D deficiency, which directly affects positive implantation results, chorion development, and pregnancy prolongation. It is also suggested that immune mechanisms underlie the recurrent pregnancy loss (RPL). Vitamin D is capable of producing a powerful immune modulating effect, strong enough to influence the pregnancy outcome.
The results of our immunohistochemical and immunofluorescent study applying confocal laser scanning microscopy show vitamin D and VDR expression to be ultimately verified in syncytiotrophoblasts, cytotrophoblasts, and extravillous trophoblasts, in chorion villus stroma and decidual tissue. In case of the missed abortion group, veracious not only vitamin D and VDR expression decrease is noted in chorion villi structures comparing to the physiological pregnancy group but also a decrease of vitamin D transporting formcalcidiol [25(OH)D] blood-level in pregnant women. In a half of cases, it turns into deficiency or notable insufficiency.
Similar data have been obtained by a group of Chinese scholars [23]. Their investigation objective was to study 25-hydroxyvitamin D [25(OH)D] concentration and VDR expression in decidual tissue in patients with recurrent pregnancy loss (RPL). Western-blotting technique allowed to obtain data witnessing not only 25(OH)D blood-level considerable reduction in gravida from RPL group compared to the control but also notable VDR expression decrease in decidual tissue. The data of an additional 25(OH)D concentration examining in decidual tissue were comparable to vitamin D blood-level (in the RPL group the indexes were lower comparing to the control). The Chinese scholars suggested that modified maternal vitamin d localized state may affect the pregnancy outcome. The study also showed 5(OH)D concentration decrease in decidual tissue (as well as in serum) in women with RPL compared to women with physiological pregnancy.
Finally, reasoning from our investigation, it can be stated that vitamin D protectively affects pregnancy prolongation and successful pregnancy development in the first trimester. Vitamin D deficiency may be associated with pregnancy loss and may also be a reason for a spontaneous early abortion. The study has also seemed to indicate that vitamin D administering in pregnant women with vitamin D deficiency/insufficiency may be a protective therapy.

Disclosure statement
No potential conflict of interest was reported by the authors.