Lipopolysaccharide drives alternation of heat shock proteins and induces failure of blastocyst implantation in mouse.

The objective of the present study is to investigate the role of heat shock proteins (Hsps) in preimplantation embryonic development and uterine receptivity during lipopolysaccharide (LPS)-induced pregnancy loss. Mice were treated with PBS or LPS on Day 0.5 of pregnancy, and preimplantation embryos and uterus were collected on Days 1.5-4.42 of pregnancy. The individual preimplantation embryos were assessed for their morphologic appearance and DNA damage during the preimplantation period of pregnancy. The expression of Hsp90, Hsp70, Hsp60, and Hsp25 was determined in preimplantation embryos and uterus by RT-PCR. Comet studies showed that LPS treatment significantly increased the percentage of abnormal embryos and DNA damage in the embryos. The expression of Hsp90, Hsp70, and Hsp60 was significantly lower in preimplantation embryos recovered from LPS-treated mice when compared to their respective controls. The expression of Hsp90, Hsp70, Hsp60, and Hsp25 was altered in uterus of LPS-treated mice when compared to their respective controls. Immunohistochemistry studies showed that at the time of implantation (i.e., Day 4.42), levels of Hsp90 and Hsp60 were decreased in stromal cells of LPS-treated uterus when compared to their respective controls. Hsp25 was highly expressed in the endometrium and stromal cells of LPS-treated uterus. Our results clearly showed that lowering of embryonic expression of Hsps induces DNA damage, which leads to degeneration and degradation of preimplantation embryos, and altered uterine expression of Hsps may not prepare the uterus for implantation. This may ultimately lead to implantation failure in mouse.

Lipopolysaccharide-induced modulation in the expression of progesterone receptor and estradiol receptor leads to early pregnancy loss in mouse.

The objective of the present study was to investigate the effect of Gram-negative bacteria infection on ovarian steroid receptors, i.e. progesterone receptor (PR) and estradiol receptor (ER) during preimplantation days of pregnancy. A well established mouse model of Gram-negative bacteria infection was used to test this objective. Mice were treated with normal saline or lipopolysaccharide (LPS) on day 0.5 of pregnancy and used to collect embryos and uterine horns on day 1.5 to day 4.42 preimplantation day of pregnancy. Total RNA was extracted and reverse-transcription polymerase chain reaction (PCR) was performed to check the expression of PR and ER genes. The mRNA expression of PR and ER was altered in embryos and uterus of LPS-treated animals during preimplantation days of pregnancy studied. These results suggest that PR and ER play an important role in Gram-negative bacteria infection and induced implantation failure in mouse.

Gonadal and nongonadal FSHR and LHR dysfunction during lipopolysaccharide induced failure of blastocyst implantation in mouse.

PURPOSE: The purpose of the present study was to investigate the impact of lipopolysaccharide (LPS) on follicle-stimulating hormone (FSH), luteinizing hormone (LH) and their receptors during preimplantation days of pregnancy. METHOD: The PBS or lipopolysaccharide (LPS) was injected intraperitoneally in the pregnant females on day 0.5 of pregnancy and serum, embryos, ovaries and uterine horns were collected on days 1.5, 2.5, 3.5, 4.0, 4.125, 4.33 and 4.42 of pregnancy. RESULT(S): In the LPS-treated pregnant females, the secretion of FSH and LH is disturbed with respect to normal pregnancy. Furthermore, the expression of FSHR mRNA in embryos and ovaries, LHR mRNA in embryos and uterus get modulated in response to LPS during preimplantation days of pregnancy. CONCLUSION(S): The disturbance in the serum level of FSH and LH in response to LPS leads implantation failure in mouse which suggests that these gonadotropins plays an integral role in the process of the successful implantation. This study also suggests a possible nongonadal role of FSHR and LHR in LPS-induced implantation failure in the mouse.

Development of multilocus putatively neutral DNA markers in the X-chromosome for population genetic studies in humans.

BACKGROUND: It has now been well documented that the type (coding, non-coding) and location (nuclear, mitochondrial etc.) of genetic markers heavily influence evolutionary inferences; realistic assumptions can be drawn if multiple putatively neutral DNA fragments spread across the genome are used. AIM: To infer human population history, Single Nucleotide Polymorphisms (SNPs), located in the non-coding regions of different genes in the X-chromosome have been developed as 'putatively neutral markers'. SUBJECTS AND METHODS: A population sample consisting of 16 male individuals from the western part of India was utilized for sequencing eight DNA fragments located in introns of three genes (Duchenne muscular dystrophy, Factor IX and Pyruvate dehydrogenase E1 sub-unit) on the human X-chromosome. PCR amplification and DNA sequencing confirmed the polymorphic status of all the fragments. RESULTS: Twenty nine SNPs were found to be segregating in the Western Indian population samples. Using these SNPs the nucleotide diversity and demographic parameters of the Western Indian population were estimated. Several tests of neutrality ascertained that all eight fragments evolve putatively neutrally. Further, linkage disequilibrium analyses confirmed this fact. CONCLUSION: All eight DNA fragments seem to bear the characteristics to be considered as 'putatively neutral genetic markers' and thus, could be utilized for inference of human population and demographic histories.

Effect of bacterial endotoxins on superovulated mouse embryos in vivo: is CSF-1 involved in endotoxin-induced pregnancy loss?

Mammalian embryonic development is regulated by several cytokines and growth factors from embryonic or maternal origins. Since CSF-1 plays important role in embryonic development and implantation, we investigated its role in gram-negative bacterial LPS-induced implantation failure. The effect of LPS on normal (nonsuperovulated) and superovulated in vivo-produced embryos was assessed by signs of morphological degeneration. A significantly similar number of morphologically degenerated embryos recovered from both nonsuperovulated and superovulated LPS treated animals on day 2.5 of pregnancy onwards were morphologically and developmentally abnormal as compared to their respective controls (P < .001. Normal CSF-1 expression level and pattern were also altered through the preimplantation period in the mouse embryos and uterine horns after LPS treatment. This deviation from the normal pattern and level of CSF-1 expression in the preimplantation embryos and uterine tissues suggest a role for CSF-1 in LPS-induced implantation failure.

Role of tumor necrosis factor-α in Gram-negative bacterial lipopolysaccharides induced implantation failure.

Background and aims: Gram-negative bacterial lipopolysaccharides (LPS) are known causative agents for pregnancy loss in mothers with genital tract infections. In this study, we attempt to test the role of tumor necrosis factor-α (TNF-α) in the normal physiological processes of preimplantation embryonic development and LPS induced pregnancy loss in mice. Since the preimplantation mouse embryos grow in an unattached state for a considerable period (day 1-4.5) of its development in the maternal environment, it is possible that a critical level of soluble and biologically active TNF-α is maintained in the maternal environment, and that any alteration in this could lead to implantation failure. Here we determine the pattern and level of expression of TNF-α gene in preimplantation stage embryos and uterus collected from control and LPS treated pregnant animals during different stages of preimplantation period of pregnancy by reverse transcriptase-polymerase chain reaction. Methods: The concentrations and biological activity of soluble TNF-α protein present in oviductal fluid (OF) and uterine fluid (UF), in the normal and LPS treated animals, were determined by enzyme-linked immunosorbent assay and 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay on L929 cells, respectively. TNF-α was also given i.p. to study its effect on implantation. Results: An early expression of TNF-α messenger ribonucleic acid in the preimplantation stage embryos collected from LPS treated animals was observed along with a significant rise in the level of biologically active soluble TNF-α in the OF. Similarly, the level of bioactive and soluble TNF-α present in the UF from LPS treated animals was significantly higher as compared to the control on day 4.42 of pregnancy. Conclusions: TNF-α given i.p. exerted similar effects on pregnancy as that of LPS. An incessant exposure of the preimplantation stage embryos to significantly high levels of maternal bioactive free/soluble TNF-α, and an alteration in the normal pattern of its expression in the preimplantation stage embryos may be one of the causes of failure of implantation leading to poor pregnancy outcome in LPS treated mouse. (Reprod Med Biol 2005; 4: 79- 89).

Natural selection mediated association of the Duffy (FY) gene polymorphisms with Plasmodium vivax malaria in India.

The Duffy (Fy) antigens act as receptors for chemokines as well as for Plasmodium vivax to invade human RBCs. A recent study has correlated the occurrence of the FY*A allele of Duffy gene with decreased susceptibility to vivax malaria, but no epidemiological correlation between the distribution of FY*A allele and incidences of vivax malaria has been established so far. Furthermore, if such correlations exist, whether natural selection has mediated the association, is an important question. Since India is highly endemic to P. vivax malaria with variable eco-climatic and varying vivax malaria epidemiology across different regions, such a question could well be answered in Indians. For this, we have genotyped the FY gene at the -33(rd) and the 125(th) nucleotide positions in 250 Indians sampled from six different zonal plus one tribal population covering the whole of India and studied possible correlations with eco-climatic and vivax malaria incidences. No FY*O allele was found, however, both the FY*A and FY*B alleles forming FY*A/FY*A, FY*A/FY*B and FY*B/FY*B genotypes were widely distributed among Indians. Five out of seven population samples significantly deviated from the Hardy-Weinberg equilibrium expectation, and two alleles (FY*A and FY*B) and the homozygote genotype, FY*B/FY*B were clinically distributed over the population coordinates. Furthermore, vivax malaria incidences over the past five years were significantly negatively and positively associated with the frequencies of the FY*A and FY*B alleles, respectively. The Northern Indians were highly differentiated from the other zonal population samples at the FY gene, as evidenced from the reconstructed Neighbor-Joining phylogenetic tree. The results specify the role of natural selection in the distribution of FY gene polymorphism in India. Furthermore, the hypotheses on the part of the FY*A allele in conferring protection to vivax malaria could be validated following population genetic studies in a vivax malaria epidemiological setting, such as India.

Lipopolysaccharide induces alterations in ovaries and serum level of progesterone and 17ß-estradiol in the mouse.

Our objective was to investigate the effect of gram-negative bacterial infection on the ovaries and serum level of P(4) and 17ß-E(2) during the preimplantation days of pregnancy in the mouse. We found that lipopolysaccharide alters the serum level of P(4) and E(2) during the preimplantation days of pregnancy and elevates the E(2)/P(4) ratio, which may keep the uterus nonreceptive during the preimplantation days of pregnancy and also not prepare the developing blastocysts for implantation in the mouse. A large infiltration of macrophages in the corpora lutea and appearance of graafian follicles from day 3.5 of pregnancy because of lipopolysaccharide treatment, which reflect a gram-negative bacterial infection, may be responsible for ovarian dysfunction and altered P(4) and E(2) level in serum.

Effect of polymyxin B on gram-negative bacterial infection during pregnancy.

OBJECTIVE: Polymyxin B (PB) is a naturally occurring cationic cyclic decapeptide which is highly bactericidal to Gram-negative bacteria. The objective of this study was to investigate the effect of PB on the viability of developing embryos during pregnancy and to validate its protective effect on the embryotoxic effect of Gram-negative bacterial lipopolysaccharide (LPS). MATERIAL AND METHODS: Animals were injected intraperitoneally (i.p.) with PB (5-100 µg/animal), (Minimum effective dose) MD of LPS and MD of LPS+PB (5-100 µg/animal) on day 0.5 of pregnancy. The percentage of normal gestational sacs and histopathologic analysis were assessed. RESULTS: PB treatment of pregnant females disturbs the pregnancy in a dose dependent manner and increases the substantial risk of congenital abnormalities in the growing fetuses of the mother. However, PB does not show any adverse effect on implantation of embryos. The embryotoxic effect of LPS can be prevented completely by 25 µg PB/animal; however other lower and higher doses of PB were not able to protect against the effect of LPS on pregnancy. CONCLUSIONS: Our results demonstrate that PB has the ability to protect the LPS-induced pregnancy loss but may not be recommended as a safe drug for the treatment of a mother suffering from Gram-negative bacterial infection during pregnancy.

Bacterial endotoxin (LPS)-induced DNA damage in preimplanting embryonic and uterine cells inhibits implantation.

OBJECTIVE: To investigate lipopolysaccharide (LPS)-induced DNA damage in preimplanting embryonic and uterine cells during preimplantation period of pregnancy that may ultimately inhibit the process of implantation in mouse. DESIGN: Animal study. SETTING: Academic research environment. ANIMAL(S): Sixty four Park strain female mice. INTERVENTION(S): The "minimum dose" (MD) of LPS was injected intraperitoneally in the pregnant females on day 0.5 of pregnancy, and individual embryos and uterine cells were assessed by comet assay on days 1.5, 2.5, 3.5, and 4.375 of the preimplantation period of pregnancy. MAIN OUTCOME MEASURE(S): Percentage of embryos and uterine cells with tail, mean comet tail length, percentage of fragmented DNA in tail. RESULT(S): Significantly higher numbers of embryos with higher mean comet tail length and percentage of fragmented DNA in tail were observed in the LPS-treated compared with control animals as the period of pregnancy approaches the stage of implantation. At the same time, DNA damage was also significantly higher in the uterine cells of LPS-treated compared with control animals. CONCLUSION(S): The MD of LPS can induce DNA damage in the preimplantation-stage embryos and uterine cells, which causes poor embryonic development and improper preparation of uterine horns during the preimplantation period of pregnancy, which may ultimately inhibit the process of implantation in mouse.

Gram-negative bacterial LPS induced poor uterine receptivity and implantation failure in mouse: alterations in IL-1beta expression in the preimplantation embryo and uterine horns.

Genito-urinary tract or systemic infections of the gram-negative bacteria in pregnant women, causes abortions, preterm labor, and several other perinatal complications. LPS is the most potent antigenic component of the gram-negative bacterial cell wall and is known to modulate the expression of various proinflammatory cytokines. Here we investigate the role of the soluble form of IL-1 i.e., IL-1beta in the 'minimum dose' of LPS induced pregnancy loss in mice. Uterine cross-sections on each day of the preimplantation period of pregnancy were examined histopathologically for finding out LPS induced changes in the uterine preparation for embryo implantation. The expression of IL-1beta in the various stages of the preimplantation period of pregnancy was studied by RT-PCR in the embryos and the uterine horns of the LPS treated and normal pregnant mice. We found that LPS significantly alters the proliferation of the glandular epithelium, luminal epithelium and stroma during the preimplantation period. We also found large infiltration of macrophages into the uterine horns of the LPS treated animals. The level and pattern of IL-1beta expression in the preimplantation embryos and uterine horns were also altered in LPS treated animals. These observations indicate that LPS can alter the uterine preparation for blastocyst implantation, which could be due to the change in the IL-1beta expression in the uterine horns. However, a change in the expression pattern of IL-1beta in the preimplantation embryos underlines the significance of this molecule in LPS induced pregnancy loss or implantation failure in mouse.

Gram-negative bacterial endotoxin- induced infertility: a birds eye view.

Alleviation of infertility on the one hand and development of improved methods of contraception on the other are global concerns to woman's health. The molecular signals that regulate implantation are of clinical relevance since understanding the nature of these signals may lead to strategies to correct implantation failure and to develop novel contraceptive approaches. The other pressing concern is the poor pregnancy rate resulting from in vitro fertilization (IVF). The pregnancy rate in IVF programs remains about 20-30% in spite of the high rate of successful fertilization. This has led to the proposition that additional uterine factors, critical for the implantation process, must be limiting. Identification of such parameters could help in determining the appropriate physiological state of the uterus for embryo transfer. Several factors are known to have a direct or indirect impact on the ability of the uterus to develop to a functionally receptive state. This would disrupt the normal coordination between embryonic and uterine development even though all molecular players may seem otherwise normal.

Comprehending the role of LPS in Gram-negative bacterial vaginosis: ogling into the causes of unfulfilled child-wish.

INTRODUCTION: Intrauterine infection is frequently associated with pregnancy loss in pregnant women. DISCUSSION: This article reviews the role of Gram-negative bacterial infection in various complications related to early pregnancy and subsequent pregnancy loss. Here we discuss the pathways of ascending intrauterine infection, microbiology and the pathophysiology of such infections. The clinical impact, therapy, consequences, prevention and implications of Gram-negative bacterial infections in women during their reproductive life span is also discussed. This article also makes an attempt to discuss our studies and findings, related to the effect of the LPS component of the Gram-negative bacterial endotoxin on preimplantation stage embryonic development and implantation. This early phase of pregnancy remains mostly unnoticed by the mother as well as the health care provider, and therefore holds more threat to the life of the fetus and the mother. The molecular mechanisms of LPS-induced pregnancy losses through abnormal embryonic development, implantation failure, and preterm labor and birth with specific references to the role of proinflammatory cytokines like IL-1 and TNF are discussed. CONCLUSION: Once these inflammatory mediators have increased in the feto-maternal tissues, it may be too late or harmful to try and prevent the adverse outcomes of pregnancy.