Nitroxide-HMP-Protects Human Trophoblast HTR-8/SVneo Cells from H2O2-Induced Oxidative Stress by Reducing the HIF1A Signaling Pathway.

Preeclampsia (PE) is a pregnancy-specific syndrome affecting 5-7% of patients. There is no effective treatment available. Early abnormal placental development is associated with oxidative stress (OS) and a release of reactive oxygen species (ROS) in the placenta. This phenomenon leads to downstream signaling, Hypoxia Inducible Factor 1A (HIF1A) stabilization and transcription of the anti-angiogenic factors soluble fms-like tyrosine kinase 1 (sFLT1) and soluble endoglin (sEng), which are known to cause endothelial and trophoblast dysfunction and cardinal features of PE: hypertension, proteinuria and, in severe cases, eclampsia. We tested whether 3-(Hydroxymethyl)-1-oxy-2,2,5,5-tetramethylpyrrolidine (HMP)-a nitroxide-type antioxidant molecule-can reduce placental OS and mitigate PE symptoms in vitro. We induced OS in human trophoblast (HTR-8/SVneo) cells with hydrogen peroxide (H2O2) and assessed whether modulating cell redox function with HMP reduces cell injury, mitochondrial stress and HIF1A and sFLT1 production. Pre-treatment with HMP reduced mitochondrial-derived ROS production, restored LC3B expression and reduced HIF1A and sFLT1 expression in H2O2-exposed HTR-8/SVneo trophoblast cells. HMP improved the mitochondrial electron chain enzyme activity, indicating that a reduction in OS alleviates mitochondrial stress and also reduces anti-angiogenic responses. In reducing placental trophoblast OS, HMP presents a potential novel therapeutic approach for the treatment of PE. Future investigation is warranted regarding the in vivo use of HMP.

Effects of Vitamin D on Fertility, Pregnancy and Polycystic Ovary Syndrome-A Review.

Polycystic ovary syndrome (PCOS) is one of the most common endocrine reproductive disorders in women. Vitamin D deficiency is also quite common in this condition. The degree of vitamin D deficiency correlates with the severity of PCOS. Both male and female vitamin D levels play a role in fertility and affect the outcomes of in vitro fertilization (IVF). Moreover, fertility and IVF indicators are improved by vitamin D not only in healthy women but in those diagnosed with PCOS. Both vitamin D deficiency and PCOS increase pregnancy-related complications. Vitamin D supplementation and optimal vitamin D levels decrease both maternal and fetal risk for complications and adverse events. Furthermore, vitamin D supplementation may ameliorate or even prevent pregnancy-related reversible bone loss in mothers. This review emphasizes the roles of vitamin D deficiency and vitamin D supplementation and their correlation with PCOS regarding reproductive health.

Accurate Quantitative Histomorphometric-Mathematical Image Analysis Methodology of Rodent Testicular Tissue and Its Possible Future Research Perspectives in Andrology and Reproductive Medicine.

Infertility is increasing worldwide; male factors can be identified in nearly half of all infertile couples. Histopathologic evaluation of testicular tissue can provide valuable information about infertility; however, several different evaluation methods and semi-quantitative score systems exist. Our goal was to describe a new, accurate and easy-to-use quantitative computer-based histomorphometric-mathematical image analysis methodology for the analysis of testicular tissue. On digitized, original hematoxylin-eosin (HE)-stained slides (scanned by slide-scanner), quantitatively describable characteristics such as area, perimeter and diameter of testis cross-sections and of individual tubules were measured with the help of continuous magnification. Immunohistochemically (IHC)-stained slides were digitized with a microscope-coupled camera, and IHC-staining intensity measurements on digitized images were also taken. Suggested methods are presented with mathematical equations, step-by-step detailed characterization and representative images are given. Our novel quantitative histomorphometric-mathematical image analysis method can improve the reproducibility, objectivity, quality and comparability of andrological-reproductive medicine research by recognizing even the mild impairments of the testicular structure expressed numerically, which might not be detected with the present semi-quantitative score systems. The technique is apt to be subjected to further automation with machine learning and artificial intelligence and can be named 'Computer-Assisted or -Aided Testis Histology' (CATHI).

Influence of Vitamin D on the Vasoactive Effect of Estradiol in a Rat Model of Polycystic Ovary Syndrome.

We examined the vasoactive effect of estradiol in a rat model of early PCOS and the influence of vitamin D deficiency (VDD). We created a model of chronic hyperandrogenism and VDD in adolescent female Wistar rats (N = 46) with four experimental groups: vitamin D supplemented (T-D+), VDD (T-D-), hyperandrogenic and vitamin D supplemented (T+D+), and hyperandrogenic and VDD (T+D-). T+ groups received an 8-week-long transdermal Androgel treatment, D-animals were on vitamin D-reduced diet and D+ rats were supplemented orally with vitamin D3. Estrogen-induced vasorelaxation of thoracic aorta segments were measured with a wire myograph system with or without the inhibition of endothelial nitric oxide synthase (eNOS) or cyclooxygenase-2 (COX-2). The distribution of estrogen receptor (ER), eNOS and COX-2 in the aortic wall was assessed by immunohistochemistry. VDD aortas showed significantly lower estradiol-induced relaxation independently of androgenic status that was further decreased by COX-2 inhibition. COX-2 inhibition failed to alter vessel function in D+ rats. Inhibition of eNOS abolished the estradiol-induced relaxation in all groups. Changes in vascular function in VDD were accompanied by significantly decreased ER and eNOS staining. Short-term chronic hyperandrogenism failed to, but VDD induced vascular dysfunction, compromised estrogen-dependent vasodilatation and changes in ER and eNOS immunostaining.

Vitamin D Deficiency and Gender Alter Vasoconstrictor and Vasodilator Reactivity in Rat Carotid Artery.

The vitamin-D-sensitivity of the cardiovascular system may show gender differences. The prevalence of vitamin D (VD) deficiency (VDD) is high, and it alters cardiovascular function and increases the risk of stroke. Our aim was to investigate the vascular reactivity and histological changes of isolated carotid artery of female and male rats in response to different VD supplies. A total of 48 male and female Wistar rats were divided into four groups: female VD supplemented, female VDD, male VD supplemented, male VDD. The vascular function of isolated carotid artery segments was examined by wire myography. Both vitamin D deficiency and male gender resulted in increased phenylephrine-induced contraction. Acetylcholine-induced relaxation decreased in male rats independently from VD status. Inhibition of prostanoid signaling by indomethacin reduced contraction in females, but increased relaxation ability in male rats. Functional changes were accompanied by VDD and gender-specific histological alterations. Elastic fiber density was significantly decreased by VDD in female rats, but not in males. Smooth muscle actin and endothelial nitric oxide synthase levels were significantly lowered, but the thromboxane receptor was elevated in VDD males. Decreased nitrative stress was detected in both male groups independently from VD supply. The observed interactions between vitamin D deficiency and sex may play a role in the gender difference of cardiovascular risk.

Vitamin D Deficiency Reduces Vascular Reactivity of Coronary Arterioles in Male Rats.

BACKGROUND: Vitamin D deficiency (VDD) may be considered an independent cardiovascular (CV) risk factor, and it is well known that CV risk is higher in males. Our goal was to investigate the pharmacological reactivity and receptor expression of intramural coronary artery segments of male rats in cases of different vitamin D supply. METHODS: Four-week-old male Wistar rats were divided into a control group (n = 11) with optimal vitamin D supply (300 IU/kgbw/day) and a VDD group (n = 11, <0.5 IU/kgbw/day). After 8 weeks of treatment, intramural coronary artery segments were microprepared, their pharmacological reactivity was examined by in vitro microangiometry, and their receptor expression was investigated by immunohistochemistry. RESULTS: Thromboxane A2 (TXA2)-agonist induced reduced vasoconstriction, testosterone (T) and 17-ß-estradiol (E2) relaxations were significantly decreased, a significant decrease in thromboxane receptor (TP) expression was shown, and the reduction in estrogen receptor-α (ERα) expression was on the border of significance in the VDD group. CONCLUSIONS: VD-deficient male coronary arteries showed deteriorated pharmacological reactivity to TXA2 and sexual steroids (E2, T). Insufficient vasoconstrictor capacity was accompanied by decreased TP receptor expression, and vasodilator impairments were mainly functional. The decrease in vasoconstrictor and vasodilator responses results in narrowed adaptational range of coronaries, causing inadequate coronary perfusion that might contribute to the increased CV risk in VDD.

Network analysis of the left anterior descending coronary arteries in swim-trained rats by an in situ video microscopic technique.

BACKGROUND: We aimed to identify sex differences in the network properties and to recognize the geometric alteration effects of long-term swim training in a rat model of exercise-induced left ventricular (LV) hypertrophy. METHODS: Thirty-eight Wistar rats were divided into four groups: male sedentary, female sedentary, male exercised and female exercised. After training sessions, LV morphology and function were checked by echocardiography. The geometry of the left coronary artery system was analysed on pressure-perfused, microsurgically prepared resistance artery networks using in situ video microscopy. All segments over > 80 µm in diameter were studied using divided 50-µm-long cylindrical ring units of the networks. Oxidative-nitrative (O-N) stress markers, adenosine A2A and estrogen receptor (ER) were investigated by immunohistochemistry. RESULTS: The LV mass index, ejection fraction and fractional shortening significantly increased in exercised animals. We found substantial sex differences in the coronary network in the control groups and in the swim-trained animals. Ring frequency spectra were significantly different between male and female animals in both the sedentary and trained groups. The thickness of the wall was higher in males as a result of training. There were elevations in the populations of 200- and 400-µm vessel units in males; the thinner ones developed farther and the thicker ones closer to the orifice. In females, a new population of 200- to 250-µm vessels appeared unusually close to the orifice. CONCLUSIONS: Physical activity and LV hypertrophy were accompanied by a remodelling of coronary resistance artery network geometry that was different in both sexes.

Vitamin D Deficiency Cause Gender Specific Alterations of Renal Arterial Function in a Rodent Model.

Vitamin D deficiency shows positive correlation to cardiovascular risk, which might be influenced by gender specific features. Our goal was to examine the effect of Vitamin D supplementation and Vitamin D deficiency in male and female rats on an important hypertension target organ, the renal artery. Female and male Wistar rats were fed with Vitamin D reduced chow for eight weeks to induce hypovitaminosis. Another group of animals received normal chow with further supplementation to reach optimal serum vitamin levels. Isolated renal arteries of Vitamin D deficient female rats showed increased phenylephrine-induced contraction. In all experimental groups, both indomethacin and selective cyclooxygenase-2 inhibition (NS398) decreased the phenylephrine-induced contraction. Angiotensin II-induced contraction was pronounced in Vitamin D supplemented males. In both Vitamin D deficient groups, acetylcholine-induced relaxation was impaired. In the female Vitamin D supplemented group NS398, in males the indomethacin caused reduced acetylcholine-induced relaxation. Increased elastic fiber density was observed in Vitamin D deficient females. The intensity of eNOS immunostaining was decreased in Vitamin D deficient females. The density of AT1R staining was the highest in the male Vitamin D deficient group. Although Vitamin D deficiency induced renal vascular dysfunction in both sexes, female rats developed more extensive impairment that was accompanied by enzymatic and structural changes.

Vitamin D Deficiency Induces Elevated Oxidative and Biomechanical Damage in Coronary Arterioles in Male Rats.

BACKGROUND: Several reports prove interconnection between vitamin D (VD) deficiency and increased cardiovascular risk. Our aim was to investigate the effects of VD status on biomechanical and oxidative-nitrative (O-N) stress parameters of coronary arterioles in rats. METHODS: 4-week-old male Wistar rats were divided into a control group (11 animals) with optimal VD supply (300 IU/kgbw/day) and a VD-deficient group (11 animals, <5 IU/kg/day). After 8 weeks, coronary arteriole segments were prepared. Geometrical, elastic, and biomechanical characteristics were measured by in vitro arteriography. O-N stress markers were investigated by immunohistochemistry. RESULTS: Inner radius decreased; wall thickness and wall-thickness/lumen diameter ratio increased; tangential wall stress and elastic modulus were reduced in VD-deficient group. No difference could be found in wall-cross-sectional area, intima-media area %. While the elastic elements of the vessel wall decreased, the α-smooth muscle actin (α-SMA) immunostaining intensity showed no changes. Significant elevation was found in the lipid peroxidation marker of 4-hidroxy-2-nonenal (HNE), while other O-N stress markers staining intensity (poly(ADP)ribose, 3-nitrotyrosine) did not change. CONCLUSIONS: Inward eutrophic remodeling has developed. The potential background of these impairments may involve the initial change in oxidative damage markers (HNE). These mechanisms can contribute to the increased incidence of the cardiovascular diseases in VD deficiency.

Effects of Vitamin D Deficiency on Proliferation and Autophagy of Ovarian and Liver Tissues in a Rat Model of Polycystic Ovary Syndrome.

AIM: We aimed to examine the alterations of the insulin signaling pathway, autophagy, nitrative stress and the effect of vitamin D supplementation in the liver and ovaries of vitamin D deficient hyperandrogenic rats. METHODS: Female Wistar rats received eight weeks of transdermal testosterone treatment and lived on a low vitamin D diet (D-T+). Vitamin D supplementation was achieved by oral administration of vitamin D3 (D+T+). Sham-treated (D+T-) and vitamin D deficient animals (D-T-) served as controls. (N = 10-12 per group). RESULTS: D-T+ animals showed decreased LC3 II levels in the liver and increased p-Akt/Akt and p-eNOS/eNOS ratios with decreased insulin receptor staining in the ovaries. Vitamin D supplementation prevented the increase of Akt phosphorylation in the ovaries. Vitamin D deficiency itself also led to decreased LC3 II levels in the liver and decreased insulin receptor staining in the ovaries. D-T+ group showed no increase in nitrotyrosine staining; however, the ovaries of D-T- rats and the liver of D+T+ animals showed increased staining intensity. CONCLUSION: Vitamin D deficiency itself might lead to disrupted ovarian maturation and autophagy malfunction in the liver. Preventing Akt phosphorylation may contribute to the beneficial effect of vitamin D treatment on ovarian function in hyperandrogenism.

Vitamin D deficiency and androgen excess result eutrophic remodeling and reduced myogenic adaptation in small cerebral arterioles in female rats.

Vitamin D (vitD) insufficiency affects 1 billion people worldwide. Androgen excess (AE) occurs in 8% of fertile females. There are few data about the combined effect of vitD deficiency and AE on the early biomechanical changes of cerebral arterioles in fertile-aged female. Forty-six adolescent female Wistar rats (21-28 day-old, weighing 90-110 g) were grouped randomly in four groups: vitD supplemented groups with and without transdermal testosterone (T) treatment, as well as vitD deficient groups also with and without transdermal T (n = 11 or 12, in all cases). After 8 weeks of treatment, anterior cerebral arterioles (in vivo diameter of 90-130 µm) were obtained and cylindrical segments were examined by pressure arteriography. Myogenic tone, tangential stress and incremental elastic moduli were computed and statistically analyzed. Elastic density was studied on resorcin-fuchsin-stained histological section. VitD deficiency with T treatment resulted in significantly lower inner radii and higher wall thickness values with reduced tangential stress and increased elastic fiber density. VitD deficiency reduced myogenic tone at higher intraluminar pressures (>110 mmHg). Our conclusion is that plasma vitD level is an important factor in the control of myogenic tone in cerebral resistance arteries. AE and vitD deficiency acting parallel induce remodeling of their wall.

Insulin resistance in an animal model of polycystic ovary disease is aggravated by vitamin D deficiency: Vascular consequences.

Hyperandrogenic state in females is accompanied with metabolic syndrome, insulin resistance and vascular pathologies. A total of 67%-85% of hyperandrogenic women suffer also from vitamin D deficiency. We aimed to check a potential interplay between hyperandrogenism and vitamin D deficiency in producing insulin resistance and effects on coronary resistance arteries. Adolescent female rats were divided into four groups, 11-12 animals in each. Transdermal testosterone-treated and vehicle-treated animals were kept either on vitamin D-deficient or on vitamin D-supplemented diet for 8 weeks. Plasma sexual steroid, insulin, leptin and vitamin D plasma levels were measured, and oral glucose tolerance test was performed. In coronary arterioles, insulin receptor and vitamin D receptor expressions were tested by immunohistochemistry, and insulin-induced relaxation was measured in vitro on isolated coronary resistance artery segments. Testosterone impaired glucose tolerance, and it diminished insulin relaxation but did not affect the expression of insulin and vitamin D receptors in vascular tissue. Vitamin D deficiency elevated postprandial insulin levels and homeostatic model assessment insulin resistance. It also diminished insulin-induced coronary arteriole relaxation, while it raised the expression of vitamin D and insulin receptors in the endothelial and medial layers. Our conclusion is that both hyperandrogenism and vitamin D deficiency reduce sensitivity of coronary vascular tissue to insulin, but they do it with different mechanisms.