An algorithm for the management of coagulopathy from postpartum hemorrhage, using fibrinogen concentrate as first-line therapy.

BACKGROUND: We constructed an algorithm for the management of coagulopathy from massive postpartum hemorrhage. Fibrinogen concentrate was administered preferentially, and the dose of both fibrinogen concentrate and fresh frozen plasma given was determined by the plasma fibrinogen concentration and prothrombin time. The efficacy of the algorithm and the amount of fibrinogen concentrate and fresh frozen plasma transfused were determined. METHODS: The study was conducted in a single teaching perinatal center. Nineteen patients were included between April 2011 and March 2014 (patient group). For a historical comparison group, we retrospectively analyzed the records of 19 patients who had been treated for coagulopathy from massive postpartum hemorrhage between April 2006 and March 2011 (control group). RESULTS: Blood loss was significantly lower in the patient group. No adverse events were associated with this management in either group. The dose of fibrinogen concentrate administered was significantly higher and that of fresh frozen plasma administered was significantly lower in the patient group. CONCLUSION: This algorithm appeared to help reduce blood loss and the total amount of fresh frozen plasma transfused when treating coagulopathy from postpartum hemorrhage, and may represent another strategy for achieving hemostasis in this setting.

Identification of a maize neocentromere in an oat-maize addition line.

We report a neocentromere event on maize chromosome 3 that occurred due to chromosome breakage. The neocentromere lies on a fragment of the short arm that lacks the primary centromere DNA elements, CentC and CRM. It is transmitted in the genomic background of oat via a new centromere (and kinetochore), as shown by immunolocalization of the oat CENH3 protein. Despite normal transmission of the maize fragment in most progeny, neocentromeres appear to vary in size within the same tissue, as shown by fluorescent measurements. A secondary truncation in one line lowered mitotic transmission to 3% and precipitously reduced the size of the chromosome. The results support the view that neocentromere formation is generally associated with major genomic disturbances such as wide species crosses or deletion of an existing centromere. The data further suggest that new centromeres may undergo a period of instability that is corrected over a period of several generations.

Maize individualized chromosome and derived radiation hybrid lines and their use in functional genomics.

The duplicated and rearranged nature of plant genomes frequently complicates identification, chromosomal assignment and eventual manipulation of DNA segments. Separating an individual chromosome from its native complement by adding it to an alien genetic background together with the generation of radiation hybrids from such an addition line can enable or simplify structural and functional analyses of complex duplicated genomes. We have established fertile disomic addition lines for each of the individual maize chromosomes, except chromosome 10, with oat as the host species; DNA is available for chromosome 10 in a haploid oat background. We report on instability and transmission in disomic additions of maize chromosomes 1, 5, and 8; the chromosome 2, 3, 4, 6, 7, and 9 additions appear stable. The photoperiodic response of the two recovered maize chromosome 1 addition lines contrasts to the long-day flowering response of the oat parents and the other addition lines. Only when grown under short days did maize chromosome 1 addition lines set seed, and only one line transmitted the maize chromosome 1 to offspring. Low resolution radiation hybrid maps are presented for maize chromosomes 2 and 9 to illustrate the use of radiation hybrids for rapid physical mapping of large numbers of DNA sequences, such as ESTs. The potential of addition and radiation hybrid lines for mapping duplicated sequences or gene families to chromosome segments is presented and also the use of the lines to test interactions between genes located on different maize chromosomes as observed for ectopic expression of cell fate alterations.

Successful laparoscopic treatment of ileo-cecal endometriosis producing bowel obstruction.

Bowel endometriosis manifesting with ileus is difficult to diagnose, often requiring laparotomy for diagnosis and treatment. We report here a case of ileo-cecal endometriosis causing bowel obstruction. A diagnosis of intestinal endometriosis with menstruation-associated bowel symptoms was made, and the patient was successfully treated by laparoscopic ileo-cecal resection.

Successful emergency endometrial ablation for intractable uterine bleeding in a postmenopausal woman complicated with liver cirrhosis and morbid obesity.

Life-threatening intractable uterine bleeding is difficult to treat when concurrent medical complications contraindicate invasive surgery. We present a case of heavy uterine bleeding in a postmenopausal woman that was complicated by liver cirrhosis and morbid obesity. The bleeding was successfully halted through emergency endometrial ablation after failure of uterine artery embolization.

Evidence for the presence of keratinocyte growth factor (KGF) in human ovarian follicles.

The presence of keratinocyte growth factor (KGF) in human follicular fluid (FF) was investigated in a total of 145 FFs obtained during oocyte retrieval for in vitro fertilization (IVF) from 29 patients with no apparent endocrine disorders. The concentrations of KGF, estradiol, progesterone, testosterone and human chorionic gonadotropin (hCG) in FF were measured by enzyme-linked immunosorbent assay. FF samples contained relatively higher amounts of KGF (2194+/-87 pg/ml), whereas its concentrations in serum were below assay limit (<31.2 pg/ml). Concentrations of KGF in FF were positively correlated with both progesterone (r=0.311, p<0.0005) and testosterone (r=0.230, p<0.01) concentrations in FF. However, KGF concentrations were not significantly correlated with estradiol and hCG concentrations. KGF in FF was detected as a broad band (26-29 kD) by immunoblotting, the size being reduced by 7kD after N-glycosidase treatment. In an in vitro experiment, KGF suppressed the basal and hCG-stimulated progesterone production by cultured human luteinized granulosa cells. summary, we demonstrated the presence of KGF in human ovarian follicles, suggesting its possible role as a local factor in regulating human ovarian functions.

Mapping maize sequences to chromosomes using oat-maize chromosome addition materials.

Oat- (Avena sativa) maize (Zea mays) chromosome additions are produced by crossing maize and oat. During early embryo development maize chromosomes are preferentially eliminated, and oat plants are often recovered that retain a single maize chromosome. Each of the 10 maize chromosomes recently has been isolated as a separate oat-maize addition. We describe here the mapping of 400 maize sequences to chromosomes using polymerase chain reaction and DNA from the oat-maize addition material. Fifty of the sequences were from cloned markers that had been previously mapped by linkage analysis, and our results were consistent with those obtained using Southern-blot analysis. Previously unmapped expressed sequence tags and sequence tagged sites (350) were mapped to chromosomes. Maize gene sequences and expression data are rapidly being accumulated. Coupling this information with positional information from high throughput mapping programs provides plant biologists powerful tools for identifying candidate genes of interest.

A complete set of maize individual chromosome additions to the oat genome.

All 10 chromosomes of maize (Zea mays, 2n = 2x = 20) were recovered as single additions to the haploid complement of oat (Avena sativa, 2n = 6x = 42) among F(1) plants generated from crosses involving three different lines of maize to eight different lines of oat. In vitro rescue culture of more than 4,300 immature F(1) embryos resulted in a germination frequency of 11% with recovery of 379 F(1) plantlets (8.7%) of moderately vigorous growth. Some F(1) plants were sectored with distinct chromosome constitutions among tillers of the same plant and also between root and shoot cells. Meiotic restitution facilitated development of un-reduced gametes in the F(1). Self-pollination of these partially fertile F(1) plants resulted in disomic additions (2n = 6x + 2 = 44) for maize chromosomes 1, 2, 3, 4, 6, 7, and 9. Maize chromosome 8 was recovered as a monosomic addition (2n = 6x + 1 = 43). Monosomic additions for maize chromosomes 5 and 10 to a haploid complement of oat (n = 3x + 1 = 22) were recovered several times among the F(1) plants. Although partially fertile, these chromosome 5 and 10 addition plants have not yet transmitted the added maize chromosome to F(2) offspring. We discuss the development and general utility of this set of oat-maize addition lines as a novel tool for maize genomics and genetics.

The maternal transcript for truncated voltage-dependent Ca2+ channels in the ascidian embryo: a potential suppressive role in Ca2+ channel expression.

Ca2+ entry during electrical activity plays several critical roles in development. However, the mechanisms that regulate Ca2+ influx during early embryogenesis remain unknown. In ascidians, a primitive chordate, development is rapid and blastomeres of the muscle and neuronal lineages are easily identified, providing a simple model for studying the expression of voltage-dependent Ca2) channels (VDCCs) in cell differentiation. Here we isolate an ascidian cDNA, TuCa1, a homologue of the alpha(1)-subunit of L-type class Ca2+ channels. We unexpectedly found another form of Ca2+ channel cDNA (3-domain-type) potentially encoding a truncated type which lacked the first domain and a part of the second domain. An analysis of genomic sequence suggested that 3-domain-type RNA and the full-length type have alternative transcriptional start sites. The temporal pattern of the amount of 3-domain-type RNA was the reverse of that of the full-length type; the 3-domain type was provided maternally and persisted during early embryogenesis, whereas the full-length type was expressed zygotically in neuronal and muscular lineage cells. Switching of the two forms occurred at a critical stage when VDCC currents appeared in neuronal or muscular blastomeres. To examine the functional roles of the 3-domain type, it was coexpressed with the full-length type in Xenopus oocyte. The 3-domain type did not produce a functional VDCC current, whereas it had a remarkable inhibitory effect on the functional expression of the full-length form. In addition, overexpression of the 3-domain type under the control of the muscle-specific actin promoter in ascidian muscle blastomeres led to a significant decrease in endogenous VDCC currents. These findings raise the possibility that the 3-domain type has some regulatory role in tuning current amplitudes of VDCCs during early development.

Prolactin, growth hormone, and epidermal growth factor activate Stat5 in different compartments of mammary tissue and exert different and overlapping developmental effects.

Prolactin (Prl)-induced phosphorylation of Stat (signal transducer and activator of transcription) 5 is considered a key event in functional mammary development and differentiation. We now demonstrate that not only Prl, but also growth hormone (GH) and epidermal growth factor (EGF), can activate Stat5 in mammary tissue. We investigated the roles of these hormones in mammary development using mice in which the respective receptors had been inactivated. Although Prl receptor (PrlR)-null mice are infertile, we were able to maintain pregnancies in a few mice by treatment with progesterone. Mammary tissue in these mice was severely underdeveloped and exhibited limited differentiation as assessed by the phosphorylation status of Stat5 and the expression of milk protein genes. PrlR +/- mice showed impaired mammary development and alveolar differentiation during pregnancy, which corresponded with reduced phosphorylation levels of Stat5a and 5b, and impaired expression of milk protein genes. Development of the glands in these mice was arrested at around day 13 of pregnancy. While Prl activated Stat5 only in the epithelium, GH and EGF activated Stat5 preferentially in the stroma. To assess the relevance of the GH receptor (GHR) in the mammary gland, we transplanted GHR-null epithelium into cleared fat pads of wild-type mice. These experiments demonstrated that the GHR in the epithelium is not required for functional mammary development. Similarly, the EGFR in the epithelium is not required for alveolar development. In contrast, epithelial PrlR is required for mammary development and milk protein gene expression during pregnancy. Although GH is not required for alveolar development, we were able to demonstrate its lactogenic function in cultured mammary epithelium from PrlR-null mice. However, ductal development in GHR-null mice was impaired, supporting the notion that GH signals through the stromal compartment. Our findings demonstrate that GH, Prl, and EGF activate Stat5 in separate compartments, which in turn reflects their specific roles in ductal and alveolar development and differentiation.

Is an elevation in basal follicle-stimulating hormone levels in unexplained infertility predictive of fecundity regardless of age?

An elevation in follicle-stimulating hormone (FSH) levels is considered to reflect lowered ovarian function, resulting in poor fecundity in infertile women. However, it remains to be clarified whether or not the significance of FSH levels applies equally to all women irrespective of age. The objective of the present study is to compare basal FSH levels in infertile women who conceived or not after stratification by age. A total of 144 infertile women between ages 25 and 45 who underwent infertility treatment due to unexplained infertility in the University of Tokyo Hospital were included in the retrospective study. Subjects were divided by age into two groups, < 38 (n=98) vs > or = 38 (n=46) years, with ages ranging from 25 to 37, and from 38 to 45, respectively. Blood samples were collected in early follicular phase (day 4-6) for assessment of basal levels of LH, FSH, and PRL. In the older group, pregnant cases had significantly lower FSH levels (6.07 +/- 2.83 mIU/ml) than nonpregnant cases (9.60 +/- 3.67 mIU/ml), whereas no difference in basal FSH levels was observed between pregnant and nonpregnant cases in the younger group. Basal FSH levels of pregnant cases in the older group were significantly lower than those of pregnant cases in the younger group (8.26 +/- 2.95 mIU/ml). Basal LH and PRL levels were not related to fecundity in either group. Thus, an increase in basal FSH levels as a predictor of fecundity should be considered in the context of age.

Stem cell factor (SCF) concentrations in peritoneal fluid of women with or without endometriosis.

PROBLEM: In the quest for possible involvement of stem cell factor (SCF), a cytokine known to have multiple effects, in the pathogenesis of endometriosis, we evaluated concentrations of SCF in peritoneal fluid (PF) of women with or without endometriosis. METHOD OF STUDY: SCF concentrations in PF collected from women undergoing laparoscopy were measured, using a specific enzyme-linked immunosorbent assay (ELISA). Reverse transcription-polymerase chain reaction (RT-PCR) analysis to detect gene expression of c-kit, the receptor for SCF, was performed using the endometriotic tissue and the eutopic endometrium collected during the operation. RESULTS: SCF concentrations in PF of women with endometriosis were significantly higher compared to women without endometriosis. Looking at SCF concentrations in PF of women with endometriosis stratified by disease stage, women with stage I and II exhibited relatively higher SCF levels in PF, whereas SCF levels in PF with stage III and IV were comparable with those without endometriosis. The expression of mRNA for c-kit was detected in both the endometriotic tissue and the eutopic endometrium. CONCLUSION: We demonstrated an elevation in SCF levels in PF associated with endometriosis and the presence of its receptor in endometriotic tissues. Given the known pleiotropic properties of SCF, the present results suggest that SCF might play a role in the pathogenesis of endometriosis.

Increased concentrations of soluble tumour necrosis factor receptor (sTNFR) I and II in peritoneal fluid from women with endometriosis.

Tumour necrosis factor alpha (TNFalpha), a proapoptotic cytokine, is known to be present in peritoneal fluid from women with endometriosis. An emerging view is that soluble TNF receptors (sTNFR) can modulate the effects of TNFalpha by acting as TNFalpha antagonists. To assess the relevance of sTNFRs in the pathophysiology of endometriosis, concentrations of sTNFR I, sTNFR II and TNFalpha in peritoneal fluid from women with endometriosis (n = 53) and without endometriosis (n = 40) were measured. Concentrations of both sTNFR I and sTNFR II in peritoneal fluid from women with endometriosis were significantly higher than in peritoneal fluid from women without endometriosis, both in the follicular and the luteal phases. TNFalpha concentrations did not differ in patients with and without endometriosis in both phases. When stratified by the stage of the disease, women with both stages I/II and stages III/IV exhibited significantly higher concentrations of sTNFR I and sTNFR II in peritoneal fluid, compared with women without endometriosis, whereas no appreciable difference in the concentrations was detected between stages I/II and stages III/IV. A significant correlation was found between the concentrations of sTNFR I and sTNFR II; while the correlations between TNFalpha and sTNFR I or sTNFR II, were either not significant or were very weak. Furthermore, mRNA for the membrane-associated TNF receptor type 1 and TNF receptor type 2, both of which convey the effects of TNFalpha, were shown to be expressed in endometriotic tissues as well as eutopic endometrium. Together, these findings suggest a possible involvement of sTNFRs in the pathophysiology of endometriosis.

Antisense waxy genes with highly active promoters effectively suppress waxy gene expression in transgenic rice.

To regulate Waxy (Wx) gene expression by introducing antisense genes, we connected the 2.3 kb Wx cDNA having 450 bp of the Wx first intron in reverse orientation to rice Wx and maize alcohol dehydrogenase1 (Adh1) promoters and used these constructs to transform rice plants. Of 10 independent transgenic lines analysed, four lines showed various degrees of reduction in amylose and WAXY (WX) protein levels in the endosperm. In two transgenic lines, complete absence of amylose was observed which made the seeds opaque white like glutinous rice (amylose-deficient waxy (wx) mutant). In one of the transgenic lines, A1 line, the presence of the antisense Wx gene cosegregated with reduction of amylose content in the endosperm. In the same line, a reduction in the level of endogenous Wx mRNA was observed in immature endosperm. Interestingly, this reduction was observed only with mature spliced transcripts but not with unspliced transcripts. Reduced amylose synthesis was also observed in pollen grains of four transgenic lines. These results suggest that integrated antisense Wx gene caused a reduction in amylose synthesis in endosperms and pollen grains of transgenic rice carrying the antisense Wx cDNA. These results indicate that manipulation of starch and other carbohydrates in rice grain is possible using antisense genes.

Comparison of the effects of add-back therapy with various natural oestrogens on bone metabolism in rats administered a long-acting gonadotrophin-releasing hormone agonist.

The hypoestrogenic state induced by gonadotrophin-releasing hormone agonist (GnRHa) has been shown to be effective in the treatment of oestrogen-dependent disorders but to induce bone loss. Adding back low doses of oestrogen in GnRHa therapy has been proposed to prevent bone loss. The purpose of this study is to assess the efficacy of add-back therapy with different natural oestrogens such as oestrone (OE(1)), oestradiol (OE(2)) and oestriol (OE(3)). Three-month-old female rats (250 g) were subcutaneously administered microcapsules of leuprorelin acetate in doses of 1 mg/kg of body weight every 4 weeks. GnRHa therapy lasted 16 weeks, and pellets of OE(1), OE(2) or OE(3) (0.5 mg/pellet, 60 day release), as an add-back agent, were implanted at 8 weeks of treatment. At the end of treatment, GnRHa alone decreased bone mineral density of the femur and lumbar vertebrae, and increased serum levels of bone metabolic markers such as alkaline phosphatase and osteocalcin levels. As for cancellous bone histomorphometry, GnRHa decreased bone volume while it increased osteoid volume, osteoid surface, eroded surface, mineral apposition rate and bone formation rate. All the oestrogens tested prevented these changes caused by GnRHa therapy. GnRHa induced a significant increase in body weight and a marked reduction in uterine weight, which was not observed in OE(1) or OE(2) add-back group. Body weight and uterine weight of the OE(3) add-back group were the same as those of the GnRHa group. These findings indicate that GnRHa induces high turnover bone loss which can be prevented by concomitant administration of natural oestrogens such as OE(1), OE(2) and OE(3) to the same extent. In addition, OE(3) is unique in that it is much less effective than OE(1) and OE(2) in blocking body weight gain and in promoting growth of uterine tissues. Because of its tissue-selective actions, OE(3) could be considered as one of the most appropriate oestrogens used for GnRHa add-back therapy.

A core activity associated with the N terminus of the yeast RAD52 protein is revealed by RAD51 overexpression suppression of C-terminal rad52 truncation alleles.

C-terminal rad52 truncation and internal deletion mutants were characterized for their ability to repair MMS-induced double-strand breaks and to produce viable spores during meiosis. The rad52-Delta251 allele, encoding the N-terminal 251 amino acids of the predicted 504-amino-acid polypeptide, supports partial activity for both functions. Furthermore, RAD51 overexpression completely suppresses the MMS sensitivity of a rad52-Delta251 mutant. The absence of the C terminus in the truncated protein makes it likely that suppression occurs by bypassing the C-terminal functions of Rad52p. RAD51 overexpression does not suppress the low level of spore viability that the rad52-Delta251 allele causes and only partially suppresses the defect in rad52 alleles encoding the N-terminal 292 or 327 amino acids. The results of this study also show that intragenic complementation between rad52 alleles is governed by a complex relationship that depends heavily on the two alleles involved and their relative dosage. In heteroallelic rad52 diploids, the rad52-Delta251 allele does not complement rad52 missense mutations altering residues 61 or 64 in the N terminus. However, complementation is achieved with each of these missense alleles when the rad52-Delta251 allele is overexpressed. Complementation also occurs between rad52-Delta327 and an internal deletion allele missing residues 210 through 327. We suggest that the first 251 amino acids of Rad52p constitute a core domain that provides critical RAD52 activities.

Evidence for the presence of hepatocyte growth factor expression in human ovarian follicles.

The presence of hepatocyte growth factor (HGF) in follicular fluid (FF) relative to concentrations of sex steroid hormones and human chorionic gonadotrophin (HCG) was investigated. A total of 69 FF samples were obtained during oocyte retrieval for in-vitro fertilization (IVF) from 11 patients with no apparent endocrine disorders. The concentrations of HGF, oestradiol, progesterone, HCG and testosterone in FF samples were measured by enzyme-linked immunosorbent assay. Transcription of HGF and its receptor, c-met, was detected by reverse transcription-polymerase chain reaction (RT-PCR). Human FF samples contained approximately 90-fold higher amounts of HGF (24.2 +/- 1.2 ng/ml), compared with those of serum (0. 28 +/- 0.04 ng/ml). Concentrations of HGF in FF were positively correlated with those of progesterone (r = 0.649, P < 0.0001) and HCG (r = 0.264, P = 0.026) concentrations in FF. However, HGF concentrations were not significantly correlated with oestradiol and testosterone. HGF in FF was detected by Western blotting, as a single 90 kDa band, corresponding to a single chain form. Additionally, mRNA for both HGF and its receptor were detected in a crude granulosa cell preparation from the pre-ovulatory follicles. These findings suggest that HGF is produced locally in human ovarian follicles and may have a physiological role as an autocrine/paracrine factor.

Hepatocyte growth factor concentrations are elevated in peritoneal fluid of women with endometriosis.

The concentrations of hepatocyte growth factor (HGF) in peritoneal fluid (PF) from women with endometriosis (n = 36) and without endometriosis (n = 40) were measured. All of the PF samples examined contained detectable concentrations of HGF. The HGF concentrations in PF from women with stage III/IV endometriosis (0.906 ng/ml, 0. 561-1.185; median, interquartile range) were significantly higher (P < 0.0001) than those from women without endometriosis (0.315 ng/ml, 0.251-0.472). The HGF concentrations from women with stage I/II endometriosis (0.417 ng/ml, 0.310-1.023) appeared to be intermediate. There were no apparent variations detected among the HGF concentrations in women in the follicular or luteal phases regardless of the presence of endometriosis. Interestingly, HGF concentrations in PF from women on gonadotrophin releasing hormone analogues, independent of the presence of endometriosis, were comparable with those from untreated women. Given the known mitogenic property of HGF in human endometrial cells, these results suggest that HGF might play a role in the progression of endometriosis.

Expression of Ah receptor and dioxin-related genes in human uterine endometrium in women with or without endometriosis.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been suggested as a possible etiologic factor for endometriosis, a condition in which endometrium-like tissues are present outside the uterus. The prevailing view pertaining to the origin of endometriotic cells is that they are from eutopic endometrial cells which regurgitate through fallopian tubes. In order to get insight into the possible involvement of TCDD in the pathogenesis of endometriosis, we suspected that TCDD may act differently on the endometrium with or without endometriosis. To address this, we examined the presence of messenger RNAs of arylhydrocarbon receptor (AhR), AhR nuclear translocator (Arnt) and two dioxin-responsive genes, cytochrome P-450 1B1 (CYP1B1) and downstream of tyrosine kinases (p62(dok)), in the endometrium of women with or without endometriosis using semi-quantitative reverse transcription-polymerase chain reaction. All the genes were expressed throughout the menstrual cycle. The expression level of p62(dok) was higher in the proliferative phase than in the secretory phase. In contrast, the expression levels of AhR, Arnt and CYP1B1 seemed to be constant during the cycle. In terms of the comparison between non-endometriosis and endometriosis group, the mRNA levels of AhR, Arnt, CYP1B1 and p62(dok) were essentially similar. Interestingly, AhR mRNA level was significantly lower in smokers than in non-smokers. Based on the regression analysis, significant linear and positive correlations were observed between AhR and Arnt mRNA levels, and between Arnt and p62(dok) mRNA levels. In summary, expression of AhR and dioxin-related genes in the endometrium did not differ in women with or without endometriosis.