Familial atrial fibrillation mutation M1875T-SCN5A increases early sodium current and dampens the effect of flecainide.

AIMS: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Pathogenic variants in genes encoding ion channels are associated with familial AF. The point mutation M1875T in the SCN5A gene, which encodes the α-subunit of the cardiac sodium channel Nav1.5, has been associated with increased atrial excitability and familial AF in patients. METHODS AND RESULTS: We designed a new murine model carrying the Scn5a-M1875T mutation enabling us to study the effects of the Nav1.5 mutation in detail in vivo and in vitro using patch clamp and microelectrode recording of atrial cardiomyocytes, optical mapping, electrocardiogram, echocardiography, gravimetry, histology, and biochemistry. Atrial cardiomyocytes from newly generated adult Scn5a-M1875T+/- mice showed a selective increase in the early (peak) cardiac sodium current, larger action potential amplitude, and a faster peak upstroke velocity. Conduction slowing caused by the sodium channel blocker flecainide was less pronounced in Scn5a-M1875T+/- compared to wildtype atria. Overt hypertrophy or heart failure in Scn5a-M1875T+/- mice could be excluded. CONCLUSION: The Scn5a-M1875T point mutation causes gain-of-function of the cardiac sodium channel. Our results suggest increased atrial peak sodium current as a potential trigger for increased atrial excitability.

Discordance in Xpert® MTB/RIF assay results among low bacterial load clinical specimens in Bangladesh.

BACKGROUND: The Xpert® MTB/RIF assay detects Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance. RIF-resistant (RIF-R) MTB cases detected using Xpert on sputum specimens at three private-sector TB screening centres in Dhaka, Bangladesh, were subjected to consecutive confirmatory Xpert testing, the results of which were MTB-positive/RIF-susceptible, MTB-positive/RIF-indeterminate or MTB-negative. OBJECTIVE: To assess the possible causes of discordant MTB and RIF-R results. METHODS: Discordant confirmatory Xpert test results were subjected to further investigations using the GenoType® MTBDRplus assay, culture and rpoB gene sequencing. RESULTS: The confirmatory Xpert test was performed on a remnant or a second specimen collected from individuals with an initial RIF-R result (n = 69); 22 (32%) results were discordant, 20 of which had an 'MTB detected-very low' result. Further investigations were mostly concordant with the confirmatory Xpert test. Average variability in paired cycle threshold (Ct) values were higher in 'MTB detected-very low' results vs. specimens with low, medium or high detected MTB results (P < 0.05); discordant results were mostly observed in specimens with 'MTB detected-very low' (20/22). CONCLUSIONS: Repeating the Xpert test and comparing with other available tests should be considered in case of 'MTB detected-very low, RIF resistance detected' results on Xpert.

Evaluation of spermicidal activity of MI-saponin A.

The seed extracts of Madhuca latifolia were reported to have spermicidal activity. The current investigation identified the spermicidal component of the extracts and evaluated its spermicidal potential in vitro. As characterized by infrared, mass, and nuclear magnetic resonance (NMR) spectral analyses, Mi-saponin A (MSA) was found to be the most potent component among a mixture of saponins. The mean effective concentrations of MSA that induced irreversible immobilization were 320 microg/mL for rat and 500 microg/mL for human sperm, as against the respective concentrations of 350 and 550 microg/mL of nonoxynol 9 (N-9). The mode of spermicidal action was evaluated by a battery of tests including (a) double fluoroprobe staining for sperm viability, (b) hypoosmotic swelling test and, assays for 5' nucleotidase and acrosin for physiological integrity of sperm plasma membrane, (c) scanning and transmission electron microscopy for sperm membrane ultrastructure, and (d) plasma membrane lipid peroxidation (LPO). The observations, taken together, were interpreted to mean that the spermicidal effect of MSA involved increased membrane LPO leading to structural and functional disintegration of sperm plasma membrane and acrosomal vesicle. A comparative in vitro cytotoxicity study in human vaginal keratocyte (Vk2/E6E7) and endocervical (End/E6E7) cell lines demonstrated that the 50% cell cytotoxicity (CC(50)) values, and consequently the safety indices, for MSA were >or= 8-fold higher as compared to those of N-9. In conclusion, MSA is a potent spermicidal molecule that may be explored further for its suitability as an effective component of vaginal contraceptive.

A randomized single-blind controlled trial of letrozole as a low-cost IVF protocol in women with poor ovarian response: a preliminary report.

BACKGROUND: Use of letrozole, a selective inhibitor of aromatase, reduces the gonadotrophin dose required to induce follicular maturation. We evaluated whether incorporation of letrozole could be an effective low-cost IVF protocol for poor responders. METHODS: A randomized, controlled, single-blind trial was conducted in the Assisted Reproduction Unit, Institute of Reproductive Medicine, Kolkata, India. Thirty-eight women with a history of poor ovarian response to gonadotrophins were recruited. Thirteen women (Let-FSH group) received letrozole 2.5 mg daily from day 3-7, and recombinant FSH (rFSH) 75 IU/day on days 3 and 8; and 25 women (GnRH-ag-FSH group) underwent long GnRH agonist protocol and stimulated with rFSH (300-450 IU/day). Ovulation was triggered by 10,000 IU of HCG followed by IVF-embryo transfer. The main outcome measures were total dose of rFSH (IU/cycle), terminal estradiol (E2) (pg/ml), numbers of follicles, oocytes retrieved and transferable embryo, endometrial thickness (mm), and pregnancy rate. RESULTS: Compared with the GnRH-ag-FSH group (2865 +/- 228 IU), the Let-FSH group (150 +/- 0 IU) received a significantly (P < 0.001) lower total dose of FSH. Except for terminal E2, which was significantly higher (P < 0.001) in the GnRH-ag-FSH group (380 +/- 46 pg/ml) than the Let-FSH group (227 +/- 45 pg/ml), the treatment outcomes in all other respects, including pregnancy rate, were statistically comparable. CONCLUSIONS: Adjunctive use of letrozole may form an effective means of low-cost IVF protocol in poorly responding women.

Galactose toxicity in the rat as a model for premature ovarian failure: an experimental approach readdressed.

BACKGROUND: The pathophysiological mechanisms underlying premature ovarian failure (POF) are largely unknown. Our objective was to develop a working animal model to explore the pathogenesis of POF. Since galactosaemic women eventually develop POF, we evaluated the potential of experimental galactose toxicity as the proposed model. METHODS: Pregnant rats were fed pellets supplemented with or without 35% galactose from day 3 of conception continuing through weaning of the litters. Female offspring were evaluated for serum levels of galactose and galactose-1-phosphate, growth rate, onset of puberty, reproductive cyclicity, ovarian complement of follicles, hypothalamo-pituitary-ovarian function and follicular response to gonadotrophins. RESULTS: Galactose toxicity delayed the onset of puberty and developed a state of hypergonadotrophic hypoestrogenism. The characteristic low FSH levels at weaning followed by pubertal spurts of gonadotrophins and estradiol (E(2)) secretion of the controls was replaced by a sustained high level of FSH and a low level of E(2) under galactose toxicity. The ovary developed with apparently normal or deficient complement of follicles. Ovarian response to exogenous gonadotrophin stimulation was blunted, but the response improved significantly when the stimulation was preceded by pituitary desensitization. CONCLUSION: Experimental galactose toxicity may serve as a model for exploring some of the basic tenets of POF.

Prenatal exposure to high galactose adversely affects initial gonadal pool of germ cells in rats.

BACKGROUND: In rats, prenatal exposure to high concentrations of galactose may contribute to a condition that is equivalent to the premature ovarian failure (POF) component of human galactosaemia. We investigated if development of POF under experimental galactosaemia-like conditions was attributed to impaired germ cell migration. METHODS: Pregnant rats were fed pellets supplemented with, or without, 35% galactose from day 3 of conception continuing through parturition. Between days 12-15, embryos from one uterine horn were dissected out. Primordial germ cells (PGC) were histochemically localized and counted on the basis of binding of Dolichos biflorus agglutinin, a lectin specific for terminal N-acetylgalactosamine (GalNAc), to the surface glycoconjugate of the germ cells. The embryos from the other uterine horn were maintained until parturition. Liver activity of uridine diphosphate galactose 4-epimerase, the enzyme involved at multiple steps in the process of synthesis of GalNAc, was assayed in 1-2 day old female pups. RESULTS: The numbers of PGC at the day-specific sites on all days of examination were significantly lower (P

Antiovulatory faculty of the flower of Malvaviscus conzattii.

The methanol extract of the flowers of Malvaviscus conzattii was orally administered in cycling unilaterally ovariectomized (ULO) rats at a dose level of 1 g/kg body wt/day for one or two cycles. The effect of the extract on the length of the cycle and ULO-induced compensatory ovulation and hypertrophy of the remaining ovary was assessed on the first oestrus following completion of treatment. Although no adverse influence was observed on either of the parameters, the cycle length was significantly prolonged and both the compensatory phenomena underwent significant inhibition after treatment of the extract for two consecutive cycles. In another experiment, the extract was found to be ineffective in preventing exogenous gonadotropin(s)-induced ovulation in immature and sub-adult rats. It is, therefore, suggested that the extract might have interfered with the synthesis and/or release of gonadotropin(s) from the pituitary while the ovarian utilization of gonadotropin(s) remained unaffected. The LD50 of the extract was found to be 20 g/kg body wt.

Effect of pelvic endometrial implants on overall reproductive functions of female rats.

The effects of pelvic endometrial implants on the overall reproductive potential of female rats were investigated. After homologous transplantation in the peritoneum, the ectopic endometrium developed into highly vascularized nodes that gradually increased in mass until the 9th week postsurgery and then plateaued. In the presence of these implants, overall reproductive function was adversely affected. The effect was of greatest magnitude during 50-70 days posttransplantation. As compared with values in corresponding controls, ovulation was reduced by 43% (6 of 14) (p < 0.05), mating rate was reduced by 44% (12 of 27) (p < 0.025), and premature termination of pregnancy occurred in 34% (5 of 15) of rats. Wastage of pregnancy, which included complete termination or reduction of fetal number, occurred during the postimplantation course of gestation. Furthermore, 100% of the rats with transplants failed to respond to the copulomimetic stimulation for the induction of pseudopregnancy (p < 0.01, compared with corresponding controls). However, on exposure to vasectomized males, 46% (6 of 13) of these rats exhibited development of pseudopregnancy (p < 0.05, compared with corresponding group receiving copulomimetic stimulation). Increased rate of mating failure and differential pseudopregnancy rates after copulomimetic and natural cervical stimulation suggest that the rats with endometrial explants possibly had an absence or a short appearance of behavioral estrus. Hormonal assessment during the preovulatory phase showed a tendency toward lower mean levels of preovulatory estradiol and significantly lower LH (p < 0.01) and progesterone (p < 0.01) concentrations. The adversely affected reproductive functions may be a secondary consequence of these altered endocrine milieus.

The role of nitric oxide in the process of implantation in rats.

Nitric oxide (NO) plays an important role in cell signalling in many physiological systems, including reproduction. During pregnancy, oestrogen modulates uterine NO generation, and NO may play an intermediary role in the oestrogen-mediated effects on the uterus. Since oestrogen is actively involved in inducing endometrial receptivity to support the process of implantation, the role of NO in the process of implantation in rats was investigated. N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase (NOS), was administered in utero with or without sodium nitroprusside (SNP), a generator of NO, on different days during the preimplantation phase of gestation. The status of gestation in respect of implantation failure, endometrial receptivity and embryo development were assessed. L-NAME was administered at various doses (2-5 mg per uterine horn) and on different days of pregnancy (days 2-6 of pregnancy) to optimize the pregnancy terminating dose (absence of implantation site on day 8 of pregnancy) and the effective day of treatment. L-NAME led to failure of implantation when administered at 2.5 mg per uterine horn on day 3 of pregnancy. The characteristic preimplantation permeability changes in the uterus were significantly attenuated and embryo growth was retarded. The L-NAME-mediated effects were significantly reversed when SNP (100 micrograms) was co-administered with L-NAME. These findings suggest a role for NO in the process of implantation. The possible mechanism by which inhibition of the NO-NOS system may interfere with implantation is discussed.

Significance of an abnormal response during pituitary desensitization in an in vitro fertilization and embryo transfer program.

PURPOSE: Our purpose was to evaluate the IVF-ET outcome in patients who did not achieve timely pituitary-ovarian suppression following "long"-protocol GnRH agonist (GnRH-a) administration. METHODS: A retrospective analysis was done on 96 IVF treatment cycles characterized by a delayed response (DR) to long-protocol GnRH-a treatment. The study included those patients who either achieved ovarian suppression (E2 < or = 110 pM) despite an elevated LH level (group DR-A) or had pituitary desensitization (LH < or = 1.5 IU/L) without ovarian suppression (group DR-B) on day 12 of GnRH-a treatment but needed an extended course of GnRH-a treatment to achieve complete suppression. These patients had gonadotropin stimulation either from day 12, despite an elevated level of LH (subgroup DR-A1; n = 13) or elevated E2 levels (subgroup DR-B1; n = 9), or after achieving a complete hypogonadotropic-hypopgonadal state following an extended course of GnRH-a treatment [subgroups DR-A2 (n = 46) and DR-B2 (n = 28)]. The outcome was compared with that of 88 cycles of normal responders (group NR) who had pituitary-ovarian suppression by day 12 day GnRH-a administration. RESULTS: Ovarian response and pregnancy rates in subgroups DR-A1 and DR-A2 were statistically not different and comparable to those in the NR group. In subgroups DR-B1 and DR-B2, E2 response and rates of oocyte retrieval and pregnancy were significantly lower than those in the other groups, but fertilization and cleavage rates were similar. The requirement of gonadotropin for ovarian stimulation was comparatively higher in subgroup DR-A2 and both DR-B subgroups. CONCLUSIONS: There was no treatment cancellation in group NR and both DR-A subgroups, but 22% of the cycles in DR-B1 and 14% of the cycles in DR-B2 were canceled due to poor ovarian response. It therefore appears that during long-protocol pituitary desensitization, the post-GnRH-a level of serum E2, rather than LH, better predicts IVF-ET outcome.

Subclinical hypothyroidism: a determinant of polycystic ovary syndrome.

The present study was an endeavor to explore whether and how hypothyroidism plays a role in the etiology of polycystic ovarian syndrome (PCOS). A composite picture of the hormone profile was assessed in different groups of subjects (control women and hypothyroid women with or without PCOS). Comparative analysis of the results suggests that hypothyroidism is invariably followed by a lowering of sex hormone binding globulin and an increment in the free testosterone level, but further metabolism of testosterone (T) may or may not be directed towards an overproduction of estriol (E3). The factors that dictate the route of T metabolism, and the way by which E3 acts to rescue the ovaries from the development of PCOS under the hypothyroid state are discussed.

Effect of RMI 12,936, an antiprogestational steroid on decidual cell reaction and embryos in rat.

Subcutaneous administration of RMI 12,936 at a dose level of 2 mg/rat on day 5 of unilaterally pregnant rat having trauma-induced decidual cell reaction (DCR) in the contralateral uterine horn, suppresses DCR, induces resorption of implanted embryos and leads to decrease in the plasma level of progesterone. Progesterone replacement (D 5-8) in this situation reverses DCR suppressive effect of RMI 12,936 but fails to prevent resorption of implanted embryos. It is concluded that possibly the drug simultaneously exerts embryotoxic as well as luteolytic effects, but these effects are independent.

Suppressive effect of quinalphos on the activity of accessory sex glands and plasma concentrations of gonadotrophins and testosterone in rats.

Biochemical estimation of prostatic acid phosphatase and fructose content in accessory sex glands, along with radioimmunoassay of plasma gonadotrophins (FSH and LH) and testosterone were performed in Wistar rats following treatment with quinalphos, an organophosphorus insecticide, for 13 and 26 days. Prostatic acid phosphatase activity and fructose content of the accessory sex glands, and plasma levels of testosterone and FSH were significantly lower in all rats treated with quinalphos. However, the degree of inhibition was more extensive in the 26 day-treatment group who, in addition also exhibited a significant reduction in relative weights of the testes and accessory sex organs, and plasma levels of LH. All these adverse effects of quinalphos were prevented when exogenous HCG was administered in concomitant with the insecticide for 26 days. These results suggest that quinalphos may exert a suppressive effect on the functional activity of accessory sex glands by decreasing testicular testosterone production following inhibition of pituitary gonadotrophins release.

Effect of aldrin on accessory sex glands and plasma testosterone levels in rats.

The effects of aldrin, an organochlorine insecticide, on accessory sex glands and plasma testosterone levels in rats were studied. The aldrin was administered i.p. for 13 days and 26 days at a dose of 150 micrograms/kg. Relative weights of prostate, seminal vesicles and coagulating glands were significantly decreased in the treated rats compared to those in controls. In addition, there was a significant fall in acid phosphatase activity in prostate and fructose content in accessory sex glands was also observed in treated animals. Plasma testosterone values showed a decrease with the duration of treatment. HCG supplementation with aldrin treatment prevented all those untoward effects of aldrin in experimental rats.

Flowers of Hibiscus rosa-sinensis, a potential source of contragestative agent. III: Interceptive effect of benzene extract in mouse.

In mouse, oral administration of the benzene extract of Hibiscus rosa-sinensis flowers at a dose level of 1 gm/kg body weight/day from day 5-8 of gestation led to termination of pregnancy in about 92% of the animals. The effect was associated with a significant fall in peripheral level of progesterone and increase in uterine acid phosphatase activity, as measured on day 10. The ovary exhibited signs of luteolysis, and the corpus luteal delta 5-3 beta -hydroxysteroid dehydrogenase activity decreased markedly. The interceptive effect of the extract was prevented completely by exogenous progesterone (1 mg/mouse/day) or chorionic gonadotropin (1 I.U./mouse/day) and partially (62.5%) by exogenous prolactin (500 micrograms/mouse/day). In unilaterally pregnant mouse having trauma-induced deciduomata in the sterile horn, the extract caused resorption of the fetuses, and regression of the deciduomata accompanied by reduction in weight of the ovaries. Luteolysis, may be due to interference with the luteotropic influence, and a consequent fall in plasma level of progesterone have been suggested as the plausible cause of termination of pregnancy.

Flowers of Hibiscus rosa-sinensis, a potential source of contragestative agent: II. Possible mode of action with reference to anti-implantation effect of the benzene extract.

Benzene extractives of Hibiscus rosa-sinensis flowers, administered during day 1-4 of gestation, exerted anti-implantation effect without affecting the tubal transport of zygote. On day 4, normal number of blastocyst was present in the uterus but they did not implant. However, as studied by pontamine blue reaction, it was evident that hyper-permeability of the endometrial capillaries which is the earliest known response of a receptive endometrium to any kind of deciduogenic stimulus was inhibited by the extract. The magnitude of decidualization, as assessed by weight of the traumatized uterine horn and supported by the histological pictures of the uteri was significantly lower in comparison to that of the controls. Ovarian structure exhibited signs of luteolysis. Inadequate progestational development of the endometrium due to interference with the conditioning of the uterus with progesterone during prenidatory phase of pregnancy has been suggested as the plausible cause of the extract-induced implantation failure.

Luteolytic influence of intrauterine dead embryos in the early pregnant rat.

The present investigation was an endeavour to study if annihilation of embryos in the uterus of the rat before the establishment of placental luteotropic functions has any influence on corpus luteal function, and, if there is any, whether it is local or systemic. The responsibility of pregnancy maintenance was imposed on a single ovary by performing unilateral ovariectomy after implantation (on Day 5 postcoitum). The implantation sites in one uterine horn, either ipsilateral or contralateral to the remaining ovary, were selectively destroyed by injecting 0.1 ml of sterile normal saline to that particular horn only, and the peripheral progesterone level and viability of the embryos in the untreated horn, which depended on the functions of the remaining ovary, were examined. Selective killing of embryos in the uterine horn of the ovariectomized side did not exert any influence on the fetal viability in the untreated horn ( nonovariectomized side) and the peripheral progesterone level also remained statistically unaffected. On the contrary, induction of fetal resorptions in the uterine horn of the intact side produced a significant fall in the peripheral level of progesterone and induced resorption of embryos of the ovariectomized side also. The latter could significantly be prevented by simultaneous administration of exogenous progesterone, indicating luteolysis as the major, if not sole, factor responsible for fetal resorption in the untreated horn. The luteolytic effect was attributed neither to saline itself, nor to the distension of the uterine horn caused by saline injection. Luteolytic factors from the dead embryo-bearing horn which act locally on the adjacent ovary only, are discussed.

Flowers of Hibiscus rosa-sinensis, a potential source of contragestative agent: I. effect of benzene extract on implantation of mouse.

In mouse, the benzene extract of Hibiscus rosa-senensis flowers was administered at four different dose levels (250-1000 mg/kg body weight/day) from day 1-4 postcoitus. Anti-implantation response and associated changes in the uterine chemical composition were studied. With an increase in the dosage of the extract, the percentage of implantation failure increased. At the dose level of 1 gm/kg body weight, the extract led to failure of implantation in 93% of the mice. The effect was accompanied by adversely altered uterine weight, its protein content and alkaline and acid phosphatase activity. In another experiment, influence of the extract on uterine uptake of progesterone was studied in bilaterally ovariectomized mice treated with or without estrogen. It exerted neither inhibitory nor stimulatory influence on uterine progesterone uptake in untreated castrated mice but the estrogen-induced increase in the uptake level was significantly inhibited by the extract. Failure of uterine bed preparation due to antiestrogenic potentiality of the extract has been discussed as the plausible cause of implantation failure.

Modulation of luteinizing hormone receptors: effect of an inhibitor of prolactin secretion, p-coumaric acid.

Daily oral administration of p-coumaric acid (PCA) at a dose of 50 mg/kg body wt for 21 days to adult male mice caused a dramatic reduction in serum prolactin concentrations. A significant fall in testicular LH binding was also observed after PCA treatment. Complete recovery of testicular LH binding was obtained by daily administration of prolactin (500 micrograms/mouse) when given simultaneously from day 9 of PCA treatment. A lower daily dose of prolactin (250 micrograms) was found to be ineffective. Scatchard analysis of binding data suggested a decrease in the number of testicular LH binding sites after PCA treatment whereas the affinity constant was unchanged. These results provide direct evidence for an inhibitory effect of PCA on prolactin secretion and also provide additional evidence in favour of a role of prolactin in the modulation of LH receptors.

A probe into the possible mechanism underlying the interceptive action of aristolic acid.

Oral administration of aristolic acid (90 mg/kg body weight) on day 6 pregnant and pseudopregnant mice resulted in the termination of pregnancy with in utero fetal death but the length of pseudopregnancy remained unaffected. Peripheral level of progesterone remained statistically uninfluenced in both conditions and exogenous progesterone failed to prevent aristolic acid-induced pregnancy loss, both of which rule out the involvement of luteolysis as the causal factor for the termination of pregnancy. In another experiment, aristolic acid was found to have no inhibitory effect over the maintenance of decidual cell reaction which indicates no inhibitory influence of the compound on uterine utilization of progesterone but points to the fact that presence of concepts is an essential component in the array of mechanism(s) leading to the termination of pregnancy. Probable toxic effect of aristolic acid on the concepts is discussed.