High mobility group box 1 (HMGB1) protein in human uterine fluid and its relevance in implantation.

STUDY QUESTION: Does a differential abundance of high mobility group box 1 (HMGB1) protein in uterine fluid (UF) have a functional significance? SUMMARY ANSWER: In rats, an excess of HMGB1 in UF during the receptive phase is detrimental to pregnancy. WHAT IS KNOWN ALREADY: The identification of constituents of the human uterine secretome has been a subject of renewed interest, due to the advent of high throughput proteomic technologies. Proteomic-based investigations of human UF have revealed the presence of several proteins such as mucins, host defense proteins S100, heat shock protein 27 and haptoglobin, etc. The present study reports on the presence of HMGB1, a nuclear protein, in human UF. Activated macrophages/monocytes, natural killer cells, mature dendritic cells, pituicytes and erythroleukemic cells are also known to secrete HMGB1. Existing data suggest that extracellular HMGB1 plays a role in inflammation. STUDY DESIGN, SIZE, DURATION: The human part of this study was cross-sectional in design. UF and endometrial tissues were collected from regularly cycling women in the early secretory (i.e. pre-receptive phase, Day 2 post-ovulation, n = 7) or secretory phase (i.e. receptive phase, Day 6 post-ovulation, n = 7) of their menstrual cycles. Samples were also collected from cycling rats in the proestrous (n = 8) or metestrous (n = 8) phase of their estrous cycles. Uteri were also collected from HMGB1-treated pregnant (n = 7) and untreated pseudo-pregnant (n = 7) rats and from pregnant rats at Day 3-5 post-coitum (p.c.) (n = 18, 3 each for six-time points). PARTICIPANTS/MATERIALS, SETTING, METHODS: In each group of human samples, four samples were used for isobaric tag for relative and absolute quantification (iTRAQ) analysis and three samples were used for immunoblotting experiments to determine the abundance of HMGB1 in pre-receptive and receptive phase UF samples. HMGB1 levels in rat UF and endometrial tissue samples were estimated by ELISA and immunohistochemical studies, respectively. The expression of inflammation-associated molecules, such as nuclear factor kappa B (NFκB), receptor for advanced glycation end products (RAGEs), tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), was analyzed by immunohistochemistry in HMGB1-treated and pseudo-pregnant rats. MAIN RESULTS AND THE ROLE OF CHANCE: HMGB1 was identified as one of the differentially abundant proteins in the list generated by 8-plex iTRAQ analysis of receptive and pre-receptive phase UF samples. In both humans and rats, secreted and cellular levels of HMGB1 showed a similar pattern, i.e. significantly (P < 0.05) lower abundance in the receptive phase compared with that in the pre-receptive phase. A significant (P < 0.05) decline was also observed in the endometrial expression of HMGB1 on the day of implantation in pregnant rats. Exogenous administration of recombinant HMGB1, on Day 3 p.c., led to pregnancy failure, whereas administration of recombinant leukemia inhibitory factor or saline had no effect on pregnant rats. Further investigations revealed morphological changes in the endometrium, an increase in the expression of luminal epithelial NFκB and significantly (P < 0.05) higher expression levels of endometrial RAGE, TNF-α and IL-6 in HMGB1-treated rats, compared with untreated pseudo-pregnant rats. LIMITATIONS, REASONS FOR CAUTION: The mechanisms, contributing to a decline in the cellular and extracellular levels of HMGB1 during the receptive phase, remain to be ascertained. WIDER IMPLICATIONS OF THE FINDINGS: An excess of HMGB1 in the UF may be associated with infertility in women.

Uterine secretome and its modulation in rat (Rattus norvegicus).

The present study identifies uterine fluid (UF) proteins that display differential abundance during the embryo-permissive phase in nonconception and conception cycles in rats. UF samples were collected from nonpregnant rats in the proestrous (n=17) and metestrous (n=18) phases and also from pregnant (n=17) and pseudopregnant (n=17) rats on day 4 post coitus. UF protein profile in the metestrous phase was compared with that in the proestrous phase. Similarly, UF protein profile of the pregnant rats was compared with that of the pseudopregnant rats. Two-dimensional PAGE, followed by densitometric analysis of the paired protein spots, revealed differential abundance of 44 proteins in the metestrous phase, compared with that in the proestrous phase. Of these, 29 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight or liquid chromatography-tandem mass spectrometry. Functional groups such as proteases, protease inhibitors, and oxidoreductases were enriched in differentially abundant proteins. Total protease activity in UF was found to be significantly (P<0.05; t-test) higher in the proestrous phase, compared with that in the metestrous phase. Furthermore, 41 UF proteins were found to be differentially abundant in pregnant rats, compared with pseudopregnant rats. Of these, 11 proteins could be identified. Immunoblotting analysis confirmed significantly higher (P<0.05; t-test) abundance of ß-actin, Rho-specific guanine nucleotide dissociation inhibitor alpha (Rho-GDIα), and peroxiredoxin-2 and -6 in the metestrous phase, compared with that in the proestrous phase. Compared with pseudopregnant rats, pregnant rats had significantly higher (P<0.05; t-test) levels of UF ß-actin and Rho-GDIα. Furthermore, these proteins could be detected in the culture supernatants of endometrial epithelial cell lines, thereby providing an evidence of their secretion from endometrial epithelial cells. Data obtained from the study expand our knowledge on the uterine milieu that favours embryo implantation.

Endometrial polyps associated with endometrial hyperplasia in an obese bonnet monkey (Macaca radiata): a case report.

BACKGROUND: A 10-year-old, female bonnet monkey (Macaca radiata) showed abnormal menstrual cycle length with heavy menstrual bleeding for 6-8 days. METHODS: Uterine ultrasound and histological examinations of endometrium by endometrial biopsy. RESULTS: An ultrasound examination of the uterine cavity showed presence of an enlarged polypoid mass. Further endometrial histology confirmed the presence of simple endometrial hyperplasia. CONCLUSIONS: We report for the first time that endometrial polyp is associated with endometrial hyperplasia in obese bonnet monkey.

Discrepancies in identification of Vibrio cholerae strains as members of Aeromonadaceae and Enterobacteriaceae by automated microbial identification system.

AIMS: Incidental observation of a discrepancy in identification of Vibrio cholerae prompted a study to understand the ability of an automated microbial identification system to identify this important pathogen. METHODS AND RESULTS: Twenty clinical isolates of V. cholerae showing difference in genetic profiles by random amplified polymorphic DNA (RAPD) fingerprinting, serologically confirmed as O1, and showing presence of ctxA and tcpA genes in PCR were subjected to analysis by Vitek 2 Compact automated identification system for identification. Vitek 2 Compact detected 10 of 20 isolates correctly, whereas the remaining 10 were identified as various members of Aeromonadaceae and Enterobacteriaceae. CONCLUSIONS: Our results indicate that Vitek 2 Compact automated microbial system does not always identify V. cholerae strains correctly. SIGNIFICANCE AND IMPACT OF STUDY: These observations should create awareness among end users about possible misidentifications by automated systems and encourage simultaneous use of serology and/or PCR for correct identification at least for V. cholerae, which is one of the most important enteric pathogens.

Hospitalisation due to infectious and parasitic diseases in District Civil Hospital, Belgaum, Karnataka.

To assess the burden of infectious and parasitic diseases on hospital services at District Civil Hospital (DCH) Belgaum, a retrospective study was carried out using discharge records concerning 8506 inpatients due to infectious and parasitic diseases among 95,655 patients admitted for all causes during the reference period 2000-2003. Out of the 21 causes of infectious and parasitic diseases, only 5 contributed maximally towards hospital admission. The most frequent cause was intestinal infections (44.0%) followed by tuberculosis (35.4%). 57.5% of these admissions were from the productive age group of 20-54 years. Tuberculosis is the most important disease in terms of hospital bed days (59.7%). Tuberculosis and intestinal infectious diseases represent more than three-fourth of the overall burden in terms of hospital bed days.

Estrus behavior and fecal steroid profiles in the Asiatic lion (Panthera leo persica) during natural and gonadotropin-induced estrus.

In this paper the behavior of the Asiatic lion was studied during natural and gonadotropin-induced estrus in relation to fecal estradiol and progesterone concentration. The average length of estrus was 5.4 days and no significant difference was observed between natural and gonadotropin-induced estrus. Vocalization and rolling were the major estrus behavioral activities of Asiatic lions and the frequency of these activities were similar in both natural and gonadotropin-induced estrus and treatment with exogenous gonadotropin did not alter estrus behavioral activities. A significant positive correlation was observed between fecal estradiol and frequency of estrus behavior during natural and gonadotropin-induced estrus. Following gonadotropin treatment estrus could be induced in 69% of animals and these induced animals ovulated following hCG treatment. This study reports for the first time the successful use of the non-invasive fecal steroid assay for monitoring the induction of estrus and ovulation in the Asiatic lion.

Herbal care for reproductive health: ethno medicobotany from Uttara Kannada district in Karnataka, India.

Traditional herbal medicine is predominantly practiced by the rural people of India, especially remote areas such as the Uttara Kannada District in Western Ghats of Karnataka. Local traditional healers play an important role in the management of reproductive health problems of the native population due to socio-economical and geographical factors. In the present study, 92 traditional medicine practitioners/healers from various regions of Uttara Kannada district were interviewed to collect information on the use of herbal treatments for a range of female and male reproductive disorders. Information was also collected on the method of preparation, dose and duration along with the botanical names, family and local names of the medicinal plants. The plants were then collected and identified. A total of 18 formulations from 25 plant species belonging to 17 families were identified, which are commonly used to treat 12 different reproductive ailments. This study identifies herbal remedies not previously documented, that are used by indigenous people in the treatment of reproductive disorders. Additionally, the paper highlights the need to retain and explore the rich biodiversity associated with Indian rain forests that may result in the discovery of new medical treatments. Finally, this paper notes the continuing reliance on herbal medicines and healing traditions by local people in remote areas. Understanding and working with local healers and tribes provides a unique opportunity to learn about the use of potentially new herbal and plant medications.

Spasmolytic effect of traditional herbal formulation on guinea pig ileum.

BACKGROUND: The herbal formulation consisting of Andrographis paniculata Nees., Cassia fistula L., Foeniculum vulgare Mill. and Cuminum cyminum L. is widely used by the local traditional practitioners in rural Northern Karnataka for spasmodic abdominal pain. OBJECTIVE: The present study was undertaken to evaluate safety and spasmolytic effect of poly-herbal formulation. MATERIALS AND METHODS: Acute toxicity studies were carried out in Swiss mice, as per the Organization for Economic Co-operation and Development (OECD) guidelines. The spasmolytic activity of the formulation was studied in isolated guinea pig ileum model using histamine and acetylcholine as agonists. The data were analyzed by one-way ANOVA, followed by Dunnetts post-hoc test and P ≤ 0.05 was considered as significant. RESULTS: The formulation did not show any adverse toxic effects and found to be safe. It also showed significant (P < 0.05) relaxation in different agonist like histamine and acetylcholine-induced contractions in guinea pig ileum. CONCLUSION: Antispasmodic activity of the herbal formulation can be attributed to its atropine-like activity. The present findings, therefore, support its utility in spasmodic abdominal pain.

Shewanella algae in acute gastroenteritis.

Shewanella algae is an emerging bacteria rarely implicated as a human pathogen. Previously reported cases of S. algae have mainly been associated with direct contact with seawater. Here we report the isolation of S. algae as the sole etiological agent from a patient suffering from acute gastroenteritis with bloody diarrhoea. The bacterium was identified by automated identification system and 16S rRNA gene sequence analysis. Our report highlights the importance of looking for the relatively rare aetiological agents in clinical samples that does not yield common pathogens. It also underscores the usefulness of automated systems in identification of rare pathogens.

Isolation of NDM-1-producing multidrug-resistant Pseudomonas putida from a paediatric case of acute gastroenteritis, India.

Pseudomonas putida is an uncommon opportunistic pathogen, usually susceptible to antimicrobial agents. Data concerning resistance to antimicrobial agents in clinical P. putida isolates are limited. To the best of our knowledge we report for the first time the isolation of NDM-1-producing multidrug-resistant P. putida from a case of acute gastroenteritis. The isolate showed resistance to a wide range of antimicrobials, including fluoroquinolones, third-generation cephalosporins and carbapenems. The isolate also exhibited multiple mutations in the quinolone resistance determining region and showed the presence of qepA, bla TEM , bla OXA1 and bla OXA7 genes. The present study highlights the importance of looking for the relatively rare aetiological agents in clinical samples that do not yield common pathogens.

Termination of pregnancy in common marmosets (Callithrix jacchus) following administration of antiserum to chicken riboflavin carrier protein.

The presence of protein(s) which is immunologically and functionally similar to the well characterised avian riboflavin carrier protein has been demonstrated in female common marmosets during pregnancy. The protein(s) that interacts with [14C]riboflavin has an immunological homology to chicken riboflavin carrier protein. The termination of pregnancy in four out of six marmosets following administration of antiserum to chicken riboflavin carrier protein supports the hypothesis that vitamin carrier protein ensures an uninterrupted supply of the vitamin to the growing embryo and that immunoneutralization of maternal riboflavin carrier protein leads to fetal death and termination of pregnancy.

Endometrial contraception: modulation of molecular determinants of uterine receptivity.

Modulation of endometrial receptivity is a promising approach for fertility regulation since it allows a contraceptive to act specifically at the endometrium. This was corroborated by our previous observations that treatment with low doses of a pure progesterone antagonist (PA, antiprogestin), onapristone (ZK 98299), in bonnet monkeys inhibited fertility by selectively retarding endometrial development, without affecting the hypophyseal-hypothalamic function. In the present study, further investigations, undertaken to analyze the molecular repertoire of a nonreceptive primate endometrium, determined expression of: steroid hormone receptors, i.e. progesterone receptor (PR) and estrogen receptor (ER); cytokines, i.e. leukemia inhibitory factor (LIF): transforming growth factor beta (TGFbeta) and its receptor (TGFbetaR); and cell adhesion molecules, i.e. integrins (alpha(v)beta(3), alpha(1)beta(1)). These studies were conducted during the different phases of the normal menstrual cycle and following treatment with different doses of onapristone (2.5 mg, 5 mg, or 10 mg every third day for one cycle) in bonnet monkeys. The molecules were analysed collectively to explore the possibility of a correlation between expression of these markers and endometrial receptivity and to investigate whether there exists a regulatory link between expression of these molecules under in vivo conditions. Three types of expression patterns of endometrial factors were observed during the peri-implantation period following onapristone treatment: 1) LIF, alpha(v)beta(3), and alpha(1)beta(1) showed significant (P < 0.02) down regulation in glandular epithelium of endometria in animals treated with all three doses of onapristone as compared to the control group. This was indicative of their critical role in the progesterone-driven cascade leading to implantation. 2) PR, TGFbeta, and TGFbetaR remained unaffected in the endometria from 2.5 mg treated animals and showed down regulation in animals treated with 5 and 10 mg onapristone as compared to the control group, thereby suggesting that the expression of these markers may not truely reflect endometrial receptivity per se. However, their facilitatory role in preparing the endometrium for implantation can not be ruled out since continued perturbation in the expression of these molecules may affect endometrial growth, remodelling, and differentiation, which in turn may render the endometrium nonreceptive; 3) ER remained unaltered in endometria of animals rendered infertile with 2.5, 5, and 10 mg onapristone. This observation indirectly suggests that onapristone-induced endometrial changes are mediated via some specific mechanisms. The present study clearly demonstrates that endometrial non-receptivity induced at low doses of onapristone is associated with changes in the expression pattern of specific molecular markers. However, no direct correlation was observed between in vivo expression of TGFbeta, LIF, and integrins, thereby lending support to the concept that there exists redundancy or multiple pathways which regulate implantation events.

Reproductive toxicity of Aroclor-1254: effects on oocyte, spermatozoa, in vitro fertilization, and embryo development in the mouse.

Polychlorinated biphenyls (PCBs) have been reported to adversely affect reproduction in laboratory and wild animals. The present study was undertaken to determine the toxic potential of Aroclor-1254 (A-1254) on in vitro fertilizing ability of oocytes and epididymal sperm and on preimplantation embryo development in the mouse. A-1254 was added to the IVF medium at concentrations of 0.01, 0.1, 1.0, and 10.0 micrograms/mL. Cumulus masses containing the oocytes were obtained from superovulated B6D2F1 mice and were placed in the culture medium containing A-1254 to which epididymal sperm, capacitated in a medium without A-1254, were added. The IVF rate was assessed 20 to 24 h after insemination. A-1254 significantly reduced the mean percent ova fertilized even at 0.1 microgram/mL. Incubation of the cumulus masses in various concentrations of A-1254 for 6 h, followed by insemination with sperm capacitated in the presence of A-1254, also significantly reduced the IVF rate. Capacitation of sperm in A-1254-containing medium, followed by coculture with untreated oocytes, failed to affect the IVF rate. No significant effect on sperm motility was observed following exposure to 1 and 10 micrograms/mL of A-1254. Estradiol-17 beta also reduced the IVF rate, however, the effect of A-1254 was more severe compared to the estradiol treatment. Furthermore, addition of A-1254 to the embryo culture medium was associated with a significant decrease in embryo growth at 48 h and 96 h. These results demonstrate adverse effects of A-1254 on oocytes, IVF, and embryonic development in the mouse.

Effects of polychlorinated biphenyls (PCBs) on in vitro fertilization in the mouse.

Polychlorinated biphenyls (PCBs) are industrial chemicals that are long-lasting global environmental contaminants. PCBs have been reported to adversely affect reproduction in laboratory and wild animals by reducing the incidence of breeding and the survival rate of young. The present study was undertaken to determine the toxic potential of PCBs on in vitro fertilization (IVF) in the mouse. Aroclor 1221, 1254, and 1268, and 3, 3', 4, 4'-tetrachlorobiphenyl (TCB), a PCB congener, were added to IVF medium at various concentrations (0.01, 0.1, 1, and 10 micrograms/mL). Cumulus masses containing oocytes were obtained from superovulated B6D2F1 mice and cultured in medium containing PCB to which capacitated sperm were added. Oocytes were assessed for fertilization 20 to 24 h after insemination. A-1221, A-1268, and TCB reduced the fertilization rate at the 1 microgram/mL and 10 micrograms/mL doses, while inhibition of fertilization by A-1254 reached significance at 0.1 microgram/ml. Furthermore, all of these chemicals caused an increased incidence of degenerative ova and abnormal 2-cell embryos at the higher dose levels (1 microgram/mL and 10 micrograms/mL). The results suggest that higher dosages of PCB and TCB adversely affect fertilization and cause an increased incidence of degeneration of oocytes and abnormality in the early mouse embryos.

Interference with ovulation and luteal function by human ovarian follicular fluid peptide in bonnet monkeys, Macaca radiata.

Partially purified human ovarian follicular fluid peptide (hGF2) was administered during follicular phase in 5 bonnet monkeys. In control as well as hGF2-injected animals, ovulation was synchronized by treatment with FSH, Pergonal and hCG. All the 5 control bonnet monkeys showed plasma estradiol peak followed by increased progesterone levels. In 4 out of 5 hGF2-injected animals, plasma progesterone levels were drastically reduced. Plasma estradiol levels were decreased only in 3 animals in which amenorrhea was observed. These data reveal that hGF2 injection disrupted ovarian function in bonnet monkeys.

Luteal deficiency caused by human ovarian follicular fluid peptide in post-partum marmosets, Callithrix jacchus.

A peptide purified from human ovarian follicular fluid (hOFFP) was administered during the follicular phase of post-partum marmosets. Four of the five animals treated with hOFFP, ovulated as evidenced by the presence of ovarian stigmata at the time of laparotomy. However, only 2 of these animals became pregnant, one had a normal delivery while the other aborted. A shortening of the luteal phase was observed in the other three animals and all animals conceived in subsequent cycles. The results indicated that treatment with the ovarian follicular fluid peptide resulted in impairment of fertility as a result of luteal insufficiency.

Shortening of luteal phase in common marmosets by sheep ovarian follicular fluid peptide.

A low molecular weight peptide has been partially purified from sheep follicular fluid. It inhibited FSH binding to granulosa cells from ovarian follicles of common marmosets (Callithrix jacchus). When injected into cycling marmosets during the follicular phase, it reduced the area under the curve (AUC) of circulating progesterone. The peptide also shortened the luteal phase in all marmosets during the treatment cycle compared to the pretreatment control cycle. These results indicate that the ovarian follicular fluid peptide inhibited FSH binding to granulosa cells thereby probably resulting in decreased progesterone secretion (AUC) from these cells and subsequently inducing luteal insufficiency.

Release of norethisterone from a bioabsorbable implant in female bonnet monkeys (Macaca radiata).

Bioabsorbable implants prepared by fusion of 85% norethisterone (NET) and 15% cholesterol were inserted subdermally in four cycling bonnet monkeys (Macaca radiata). No skin reaction or inflammation was observed at the site of implantation. Plasma concentration of norethisterone (NET) measured by radioimmunoassay were monitored for 14 to 16 months at monthly intervals. In the first month weekly samples were analysed. NET was released into circulation within 24 hours after insertion of the implant. In all the monkeys, except one, a sharp rise in NET (1.9 to 20 ng/ml) occurred immediately after insertion. Levels then remained between 1.7 and 0.6 ng/ml for about 4 months. Thereafter they remained steady up to about 9 months in two monkeys and gradually declined to about 0.4 ng/ml in the remaining two. In all except one, there was a sudden burst of NET release between the 10th and 11th month. The hormone almost cleared out of circulation by the 14th to 16th month. Initial menstrual cycles after pellet insertion were disturbed, leading to spotting and irregular bleeding. Regular cycles appeared from 5 to 8 months after the insertion of the implant. These cycles were ovulatory, as determined by the progesterone levels.

Effect of Hibiscus rosa sinensis Linn. on oestrous cycle & reproductive organs in rats.

PIP: The effect of Hibiscus rosa sinensis Linn. on the estrous cycle and reproductive organs was studied in female albino rats. Depending on the dose and duration of treatment, the benzene extract of the flowers disrupted the estrous cycle. Treatment for 30 days resulted in a significant (p less than .05) reduction in the weight of the ovaries, uterus, and pituitary gland. Ovarian follicular atresia and uterine atrophy were observed. Treatment resulted in degranulated gonadotrophs in the pituitary, with the extent of damage being dose-dependent.^ieng

Localization of estrogen and progesterone receptors in the endometrium of common marmosets Callithrix jacchus.

In the present study, changes in the immunohistochemical localization of endometrial estrogen receptor (ER) and progesterone receptor (PR) during various stages of the ovarian cyclicity in common marmoset, have been reported. Ovarian cyclicity was monitored by estimating plasma estradiol and progesterone. During the early follicular phase, weak ER immunolocalization was observed in the endometrial stroma. During the late follicular phase under the influence of rising estradiol levels, stromal ER localization was intense. During the luteal phase, ER localization was absent in the stroma indicating that high concentrations of progesterone suppressed ER. PR localization was not observed in the stroma during the early follicular phase, while weak staining was seen in the stroma during the late follicular phase. PR localization was maximum during the mid luteal phase. However in marmoset, endometrial ER and PR localization was restricted only to the stroma. This unique feature may be due to the characteristic reproductive profile of this nonmenstruating species and needs to be studied further. Thus it can be hypothesized that in the marmoset endometrium, steroid hormone mediated effects possibly occur directly in the stroma and are then transmitted to the epithelium by autocrine/paracrine action of growth factors and cytokines.