Aberrant CD8+T cells drive reproductive dysfunction in female mice with elevated IFN-γ levels.

Introduction: Interferon-gamma (IFN-γ) is pivotal in orchestrating immune responses during healthy pregnancy. However, its dysregulation, often due to autoimmunity, infections, or chronic inflammatory conditions, is implicated in adverse reproductive outcomes such as pregnancy failure or infertility. Additionally, the underlying immunological mechanisms remain elusive. Methods: Here, we explore the impact of systemic IFN-γ elevation on cytotoxic T cell responses in female reproduction utilizing a systemic lupus-prone mouse model with impaired IFN-γ degradation. Results: Our findings reveal that heightened IFN-γ levels triggered the infiltration of CD8+T cells in the pituitary gland and female reproductive tract (FRT), resulting in prolactin deficiency and subsequent infertility. Furthermore, we demonstrate that chronic IFN-γ elevation increases effector memory CD8+T cells in the murine ovary and uterus. Discussion: These insights broaden our understanding of the role of elevated IFN-γ in female reproductive dysfunction and suggest CD8+T cells as potential immunotherapeutic targets in female reproductive disorders associated with chronic systemic IFN-γ elevation.

Isolation of single cells from individual mouse ovaries for flow cytometry and functional analysis.

Here, we present a validated workflow to isolate sufficient viable single ovary cells from a single mouse without the need to pool from several mice. We provide steps essential for estrous staging, ovary harvesting and dissociation, ovary cell staining, data collection, and analysis. Our approach allows the use of these single-cell suspensions for flow sorting, flow cytometry analysis, or functional in vitro assays. Importantly, our protocol is designed to maximize the isolation of immune cells, including T cell subsets.

Double Negative T Regulatory Cells: An Emerging Paradigm Shift in Reproductive Immune Tolerance?

Immune regulation of female reproductive function plays a crucial role in fertility, as alterations in the relationship between immune and reproductive processes result in autoimmune subfertility or infertility. The breakdown of immune tolerance leads to ovulation dysfunction, implantation failure, and pregnancy loss. In this regard, immune cells with regulatory activities are essential to restore self-tolerance. Apart from regulatory T cells, double negative T regulatory cells (DNTregs) characterized by TCRαß+/γδ+CD3+CD4-CD8- (and negative for natural killer cell markers) are emerging as effector cells capable of mediating immune tolerance in the female reproductive system. DNTregs are present in the female reproductive tract of humans and murine models. However, their full potential as immune regulators is evolving, and studies so far indicate that DNTregs exhibit features that can also maintain tolerance in the female reproductive microenvironment. This review describes recent progress on the presence, role and mechanisms of DNTregs in the female reproductive system immune regulation and tolerance. In addition, we address how DNTregs can potentially provide a paradigm shift from the known roles of conventional regulatory T cells and immune tolerance by maintaining and restoring balance in the reproductive microenvironment of female fertility.

Thyme (Thymus vulgaris [Lamiaceae]) Leaves Inhibit Contraction of the Nonpregnant Mouse Uterus.

Dysmenorrhea is painful menstrual periods, which affects 25% of women within reproductive age and has a prevalence of 67.2-90.0%. Current treatment has several adverse effects and can be ineffective once the pain is initiated. Thymus vulgaris traditionally used for pain management was investigated in this study for its activity on uterine contraction in the nonpregnant uterus, as a parameter for dysmenorrhea. The dried leaves of T. vulgaris were macerated in water, and the resulting aqueous extract was investigated on the isolated mouse uterus. Parameters investigated included spontaneous contractions, oxytocin-induced contractions, and high potassium chloride (KCl; 80 mM)-induced tonic contractions. Mass spectrometric analysis of the thyme extract was also performed using liquid chromatography-high-resolution Fourier Transform mass spectrometry. Thyme extract inhibited the amplitude and frequency of spontaneous and oxytocin-induced uterine contractions. It also inhibited KCl-induced tonic contractions. The activities observed suggest that T. vulgaris inhibits uterine contractions through blockade of extracellular voltage-gated calcium channels. Secondary metabolites detected included compounds belonging to chlorogenic phytochemical class and flavonoids, which are known to have activities on extracellular calcium blockade. This study has shown that aqueous T. vulgaris extract, also known as thyme, inhibits contractions of the nonpregnant uterus and can be a lead plant in the drug discovery process for the management of dysmenorrhea.

Ascorbic Acid and Alpha-Tocopherol Contribute to the Therapy of Polycystic Ovarian Syndrome in Mouse Models.

Polycystic ovary syndrome (PCOS) affects up to 10% of women within reproductive ages and has been a cause of infertility and poor quality of life. Alteration in the oxidant-antioxidant profile occurs in PCOS. This study, therefore, investigates the contribution of ascorbic acid (AA) and alpha-tocopherol(ATE) on different PCOS parameters. The mifepristone and letrozole models were used, and young mature female mice were randomly assigned to groups of six per group. On PCOS induction with either mifepristone or letrozole, mice were administered AA and ATE at doses ranging from 10-1000mg/kg to 0.1-1000 mg/kg in the respective models. Vaginal cytology, body weights, and temperature, as well as blood glucose, testosterone, and insulin levels, were measured. Total antioxidant capacity and malondialdehyde levels were analyzed. Determination of gene expression of some reactive oxygen species and histomorphological analysis on the ovaries and uteri were performed. At the end of the experiments, AA and ATE restored reproductive cycling, with AA being more effective. AA and ATE increased fasting blood glucose but had no significant effect on serum insulin levels. AA decreased testosterone levels, but ATE caused slight increases. AA and ATE both increased total antioxidant capacity and decreased malondialdehyde levels. AA and ATE also slightly upregulated the mRNA expressions of catalase, superoxide dismutase, and heme oxygenase 1 mainly. AA and ATE also decreased ovarian weight and mostly resolved cysts in the ovaries and congestion in the uterus. This study has shown that AA and ATE are beneficial in the therapy of PCOS.

Justicia flava leaf extract potently relaxes pregnant human myometrial contractility: a lead plant for drug discovery of new tocolytic drugs.

NEW FINDINGS: What is the central question of this study? Can Justicia flava leaf extract (JF) inhibit human myometrial contractility as was previously shown in mouse myometrium? What is the main finding and its importance? JF abolished human myometrial contractions and therefore presents as a lead plant in drug discovery studies involving drugs for preterm birth. ABSTRACT: In the search for new potent therapies for preterm labour, Justicia flava leaf extract (JF) was previously shown to potently inhibit uterine contractility in both pregnant and non-pregnant mouse uterus. This study took the investigation a step further and investigated the activity of JF on pregnant human myometrial contractility. JF potently inhibited human myometrial contractility in a concentration-dependent manner. This pilot study provides evidence that JF should be further investigated as a lead plant in the drug discovery of new uterine relaxants.

The Malaria-High Blood Pressure Hypothesis: Revisited.

Malaria etiologies with pathophysiological similarities to hypertension currently constitute a major subject of research. The malaria-high blood pressure hypothesis is strongly supported by observations of the increasing incidence of hypertension in malaria-endemic, low- and middle-income countries with poor socioeconomic conditions, particularly in sub-Saharan African countries. Malnutrition and low birth weight with persistent symptomatic malaria presentations in pregnancy correlate strongly with the development of preeclampsia, gestational hypertension and subsequent hypertension in adult life. Evidence suggest that the link between malaria infection and high blood pressure involves interactions between malaria parasites and erythrocytes, the inflammatory process, effects of the infection during pregnancy; effects on renal and vascular functions as well as effects in sickle cell disease. Possible mechanisms which provide justification for the malaria-high blood pressure hypothesis include the following: endothelial dysfunction (reduced nitric oxide (NO) levels), impaired release of local neurotransmitters and cytokines, decrease in vascular smooth muscle cell viability and/or alterations in cellular calcium signaling leading to enhanced vascular reactivity, remodeling, and cardiomyopathies, deranged homeostasis through dehydration, elevated intracellular mediators and proinflammatory cytokine responses, possible genetic regulations, activation of the renin-angiotensin-aldosterone system mechanisms and renal derangements, severe anemia and hemolysis, renal failure, and end organ damage. Two key mediators of the malaria-high blood pressure association are: endothelial dysfunction (reduced NO) and increased angiotensin-converting enzyme activity/angiotensin II levels. Sickle cell disease is associated with protection against malaria infection and reduced blood pressure. In this review, we present the state of knowledge about the malaria-blood pressure hypothesis and suggest insights for future studies.

Justicia flava Leaves Exert Mild Estrogenic Activity in Mouse Models of Uterotrophic and Reproductive Cycle Investigations.

This study aimed to assess and determine the estrogenic activity of the leaf extract of Justicia flava (JF) in mice, which may interfere with its therapeutic use in female reproduction. The uterotrophic assay (UTA) utilizing 20 days old female mice and the reproductive cycle assay (RCA) utilizing adult female mice were used in this study. All administrations were performed orally. Reproductive organ and blood samples were collected the day after last administration of JF for histology and hormone analysis. Other parameters such as organ weight, temperature, body weight, and reproductive cycles were analyzed. Our study showed that for UTA, JF increased uterine weights slightly, which were nonsignificant but more pronounced at the highest dose of 1000 mg/kg. JF did not induce vaginal opening, which is a sign of puberty onset. JF also had minimal effect on organ morphology and caused a slight increase in serum estrogen. For RCA, JF did not significantly alter body weight and temperature although an upward trend in temperature was observed. JF did not disrupt cycling significantly (P > .005) compared with estrogen (the positive control drug used). JF also did not significantly alter uterus morphology except at 1000 mg/kg where some increase in the number of glands and cell activity were observed. JF has mild estrogenic activity and will not interfere with reproductive functions at lower doses (10-100 mg/kg) during therapy, but high doses (up to 1000 mg/kg and above) may cause some alterations. Our data, therefore, suggest that JF is a useful candidate in the management of female reproductive health issues at lower doses.

Disruptions in the female reproductive system on consumption of calcium carbide ripened fruit in mouse models.

This study investigated for the first time the outcome of ingestion of calcium carbide-ripened fruit on some female reproductive parameters. A set of unripe mature bananas ripened with calcium carbide (CCRB) and another set ripened via non-artificial means (NARB) were fed orally to prepubertal female mice for three days using the uterotrophic assay procedure. A distilled water group and oestradiol group (10 mg/kg) were also assigned. Food intake, body weights, vaginal openings and cytology were analysed. Samples of blood, uteri, ovaries and cervices were additionally collected and analysed. Increased serum oestrogen level and uterus weight were detected in the CCRB and oestradiol treated groups. Histopathology showed increased numbers of myometrial cells, presence of secondary follicles and regressing corpus lutea as well as thickened cervix epithelia which were evidence of oestrogenic disruptions. This study has shown that consumption of fruits ripened with calcium carbide negatively alters the female reproductive physiology, accelerates puberty onset and increases serum oestrogen levels. Caution must therefore be exercised by fruit sellers in the use of calcium carbide and policies set in place for strict regulation of its use worldwide.

Tocolytic activity assessment of the methanol leaf extract of Justicia flava Vahl (Acanthaceae) on mouse myometrial contractility and preliminary mass spectrometric determination of secondary metabolites.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Justicia flava are traditionally used in the South of Nigeria to prevent preterm births. AIM OF THE STUDY: In this study, the activity of the methanol leaf extract of J. flava (JF) was investigated on uterine contractility in non-pregnant and pregnant isolated mouse tissues. MATERIAL AND METHODS: The effects on spontaneous, oxytocin, and KCl-induced contractions were determined. The effects in calcium-free media were also determined. Possible mechanisms of activity were investigated using receptor and channel modulators. Mass spectrometric analysis was additionally performed on the leaf extract to identify secondary metabolites. RESULTS: JF was observed to inhibit spontaneous, oxytocin and high KCl-induced uterine contractility. JF also inhibited contractions in Ca2+-free media. JF was found to exert its inhibitory effect via interaction with inositol triphosphate and ryanodine receptors and also through modulation of K+- channels. Lignans and alkaloids were identified with the lignans being the most abundant in JF. CONCLUSION: JF has been shown to potently inhibit uterine contractions in non-pregnant and pregnant isolated mouse uterus. The inhibitory activity of JF has been shown to occur via blockade of extracellular and intracellular calcium entry and these effects may be due to the lignans identified in - JF. JF has therefore been shown in this study to be a lead plant in the discovery of new drugs with uterine inhibitory activity.

Evaluation of some neuropharmacological effects of Caladium bicolor aiton (araceae) leaf extracts in mice.

Caladium bicolor Aiton (Araceae) is used in ethnomedicine for the treatment of boils, wound ulcers and convulsion. This study investigated the effects of the leaf extracts on some neuropharmacological parameters. The leaves were collected, dried, powdered and then extracted by maceration in methanol to yield the whole extract (WE). Extraction was also done using n-hexane, ethyl acetate and methanol in a Soxhlet apparatus to obtain n-hexane (HE), ethyl acetate (EA) and methanol (ME) extracts. Preliminary phytochemical screening was done using the whole extract. Some neuropharmacological evaluations were carried out using standard methods. Phytochemical screening revealed the presence of carbohydrates, proteins, alkaloids and flavonoids. WE showed varying levels of protection against strychnine-induced convulsion. Each of HE, EA and ME increased latency (P < 0.01) to pentylenetetrazole-induced convulsion and offered varying levels of protection against maximal electroshock-induced seizure. Each of WE, HE and ME significantly increased the duration of stay on the open arm of the elevated plus maze. Both EA and ME at doses of 100 and 200 mg/kg, and HE at a dose of 400 mg/kg significantly reduced the duration of immobility in forced swim test. It is concluded that the leaf extracts possess anticonvulsant, anxiolytic and antidepressant properties.

Metabolomics-Coupled Functional Pharmacology of Chlorophyll Compounds Isolated From the Leaves of Ficus Exasperata Vahl (Moraceae) Provides Novel Pathways on Myometrial Activity.

New chlorophyll derivatives (pheophytins along with pheophorbide derivatives) were isolated from the leaves of Ficus exasperata and were found to have varying effects on uterine contractility. The current study was therefore aimed at the utilization of mass spectrometry and nuclear magnetic resonance spectroscopy coupled with isolated uterine tissue assay as a platform to assist in the determination of the mechanism of activity of the isolated chlorophyll compounds from the plant F exasperata. The pheophytin and pheophorbide compounds (200 µg/mL) were added to the isolated uterine tissues. Mice uteri, treated with the pheophytin compounds, and the physiological buffer in which the uterine tissues were immersed, were rapidly collected and analyzed using high-resolution Fourier transform mass spectrometry and proton (1H) nuclear magnetic resonance for bioinformatics study. Resulting data were analyzed via pairwise chemometric comparison models, with P < .05 considered statistically significant. Primary signaling pathways found to be correlated with the pheophytins in this study included cyclic adenosine monophosphate, dopamine, extracellular signal-regulated kinases 1/2, and glutamate pathways.

A role of alpha-tocopherol and phylloquinone in the modulation of uterine contractility and reproductive function in mouse models.

BACKGROUND AND AIM: Alpha-tocopherol has been implicated in reproduction processes, and deficiency of phylloquinone has been associated with serious complications in pregnancy. This study was therefore aimed at investigating the effects of phylloquinone and alpha-tocopherol on uterine contractility and female reproductive function using mouse models. MATERIALS AND METHODS: Both in vivo and ex vivo animal models were employed and designed to assess changes on uterine contractility and reproductive functions in the non-pregnant uterus. The effect of alpha-tocopherol and phylloquinone on spontaneous uterine contractions, oxytocin-induced uterine contractions (11.82nM) and high KCl-induced tonic uterine contractions (80mM) were assessed. The effect of subcutaneous administration of alpha-tocopherol (10mg/kg) on reproductive hormone levels and reproductive tissues were also determined. RESULTS: Alpha-tocopherol increased the force of contractions while phylloquinone decreased the force of uterine contractions. Plasma levels of luteinizing hormone (P<0.01), estrogen (P<0.01) and progesterone (P<0.001) were elevated in the presence of alpha-tocopherol after 6 days subcutaneous administration. CONCLUSIONS: Alpha-tocopherol and phylloquinone have been shown to directly modulate uterine contractility and reproductive function and may contribute to the management and treatment of reproductive disorders.

Characterisation of the antiproliferative constituents and activity of Ficus exasperata (Vahl) on ovarian cancer cells -a preliminary investigation.

Ovarian cancer is one of the most common gynaecological cancers today. This study therefore investigates the anticancer effects of Ficus exasperata extracts and fractions on ovarian cancer cells. The antiproliferative activity of the crude extracts (1 mg/mL) was assessed using the MTT assay on A2780 (ovarian cancer) cell line. Bio-activity guided fractionation was performed and preliminary identification was further achieved using high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. All crude extracts tested exhibited antiproliferative activity except for the methanol extract which interestingly showed proliferative effects. Five fatty acids were identified from the active fractions (FB1-10 and FB1-12). FB1-12 exhibited an IC50 value of 15.20 µg/mL. The least potent fraction (FB1-4 + 5) had an IC50 value of 34.51 µg/mL. H1-HEX and H1-MET exhibited 97.2 and 97.9%, respectively, compared to control. This study therefore provides proof-of-principle that fatty acids of Ficus exasperata exhibit significant antiproliferative effects on ovarian cancer cells.

Toward Understanding Myometrial Regulation: Metabolomic Investigation Reveals New Pathways of Oxytocin and Ritodrine Activity on the Myometrium.

In recent times, additional pathways involved in the regulation of the myometrium have been suggested. This also holds true for the effect of drugs such as oxytocin (OT) and ß-adrenergic agonists on the myometrium. Knowledge of these additional pathways will certainly prove useful in designing better therapies for pathologies of the myometrium. This study was therefore aimed at investigating the possibility of other pathways involved in the activities of both OT and ritodrine (RIT; a ß-adrenergic agonist) in the myometrium by utilizing metabolomics and bioinformatics. High-resolution Fourier transform mass spectrometry (HRFTMS) and nuclear magnetic resonance (NMR) spectroscopy coupled with functional uterine assays were used for an innovative assessment. In vitro pharmacological assay of OT (1 nmol/L) and RIT (0.1 nmol/L) on isolated mice uteri mounted in 3 mL organ baths was performed. Mice uteri, treated with OT or RIT, as well as the physiological buffer in which the uterine tissues were immersed, were rapidly collected and analyzed using HRFTMS, proton (1H)-NMR, and bioinformatics. Resulting data were analyzed via pairwise chemometric comparison models, with P ≤ .05 considered statistically significant. In addition to previously known metabolites, nicotinamide adenine dinucleotide, γ-aminobutyric acid, and sphingosine were significantly associated with the activity of OT, whereas the activity of RIT was associated with a downstream involvement of prostaglandin F1 and phosphatidylinositol signaling. These findings add evidence to the reports on additional regulation of myometrial activity by these drugs and suggest newer pathways for therapeutic manipulation.

The leaves of Ficus exasperata Vahl (Moraceae) generates uterine active chemical constituents.

ETHNOPHARMACOLOGICAL RELEVANCE: In the search for new, safe and efficacious uterine active agents, the plant Ficus exasperata was subjected to phytochemical screening and pharmacological analysis. MATERIALS AND METHODS: Ethyl acetate and methanolic leaf extracts of Ficus exasperata were fractionated and purified by a series of chromatographic techniques. The isolation process was guided by in vitro functional uterine assays involving the use of C57Bl/6 female mice. Identification of the active chemical constituents was performed by several spectroscopic techniques which included 1D and 2D nuclear magnetic resonance (NMR) and high resolution mass spectrometry (HRMS). The uterine effects of these compounds were investigated on spontaneous, oxytocin-induced and high KCl-induced contractions using isolated uterine segments of non-pregnant female mice. The activity of different compounds on the amplitude (maximum tension above basal force) and frequency of uterine contractions were simultaneously measured and then statistically analysed. The structure-activity relationships were also examined where possible. RESULTS: These studies led to the identification of some new phytochemical derivatives. Pharmacological assay revealed the presence of both uterine stimulatory and inhibitory constituents. The new pheophytin/pheophorbide derivatives, flavonoids, fatty acids and glycerol derivatives significantly reduced the frequency and amplitude of uterine contraction, while KCl salt, pyrimidine and pheophorbide-b derivatives significantly augmented both spontaneous and agonist-induced contractions. CONCLUSION: This study has demonstrated that Ficus exasperata generates secondary metabolites which have proven effective in the significant inhibition of uterine contractions and thus a potential source of new tocolytic agents. Additionally, uterine stimulatory constituents were also generated some of which may be potential drugs for contraception and/or labour facilitation. Lead compounds generated from this study are the pheophytin/pheophorbide derivatives, pyrimidine derivatives and flavonoid derivatives.

Oxytocin inhibiting effect of the aqueous leaf extract of Ficus exasperata (Moraceae) on the isolated rat uterus.

The leaves of Ficus exasperata Vahl Enum. Pl. vahl (Moraceae) are used by traditional healers in Southern Nigeria and some parts of Africa to avoid preterm births. However, previous reports showed that the plant also exhibited uterine contractions at specific concentrations. This study is therefore aimed at investigating the purported uterine inhibitory aspect of the plant on the isolated rat uterus. The aqueous extract (AET) was tested on rhythmic spontaneous uterine contractions. Concentration-response relationships were obtained for oxytocin (OT), acetylcholine (ACh) and ergometrine (EGM), in the presence or absence of fixed concentrations of AET. Salbutamol (SBL) and verapamil (VER) were used as positive controls. AET, at 1.0 x 10(-2) mg/mL, significantly increased (p < 0.05) the EC50 of oxytocin-induced contractions but had no significant effect on ACh, EGM and spontaneous uterine contractions. However, SBL and VER significantly increased (p < 0.01) the EC50, of OT, ACh and EGM and significantly inhibited (p < 0.01) the frequency and amplitude of spontaneous uterine contractions. The aqueous leaf extract of F. exasperata inhibits oxytocin-induced uterine contractions at the concentration shown in this study. This observation may explain its folkloric use in counteracting preterm contractions and alleviating dysmenorrhoea.

In vitro determination of the mechanism of the uterine stimulatory effect of Newbouldia laevis.

The uterine stimulatory effect of the ethanol leaf extract of Newbouldia laevis (Beauv.) Seemann ex Bureau (Bignoniaceae) was evaluated in the presence of some antagonists in vitro in an attempt to elucidate the mechanism of action of the extract. The extract was tested in the presence and absence of phentolamine (4.09 and 40.91 nM), diphenhydramine (4.45 and 44.47 nM), atropine (1.18 and 11.91 nM), and verapamil (2.03 and 20.35 nM). The effect of the antagonists on the extract and on oxytocin used as a reference drug in this study was evaluated. The EC(50) and E(max) were determined and statistically analyzed using one way ANOVA and Dunnett's post hoc test. There was no significant difference in the EC(50) and E(max) of the extract and oxytocin in the presence of phentolamine. Diphenhydramine and atropine significantly inhibited (p <0.01) the extract but both drugs had no effect on oxytocin. However, significant differences (p <0.01) were observed in the EC(50) and E(max) of the extract and oxytocin in the presence of verapamil. These results suggest that the leaf extract of N. laevis contracts the uterus by opening voltage-operated calcium channels and/or by activation of muscarinic receptors.