OBJECTIVE: To determine the safety, tolerability, and efficacy of combination gefitinib and methotrexate to treat ectopic pregnancy.METHODS: We performed a phase I, single-arm (non-randomized), open-label study. Twelve women with ectopic pregnancies were administered methotrexate (50 mg/m(2), intramuscular) and 250 mg oral gefitinib in a dose-escalation protocol: one dose (day 1) n=3; three doses (days 1-3) n=3; seven doses (days 1-7) n=6. Efficacy was examined by comparing human chorionic gonadotrophin (hCG) decline and time to resolution with historic controls administered methotrexate only.RESULTS: Common side effects were transient acneiform rash in 67% (8/12) and diarrhea in 42% (5/12) of participants. There was no clinical or biochemical evidence of serious pulmonary, renal, hepatic, or hematologic toxicity. Of six participants with a pretreatment serum hCG level between 1,000 and 3,000 international units/L, hCG levels declined significantly faster than in the control group. Median serum hCG levels by day 7 after treatment were less than one fifth of levels observed among 71 historic controls treated with methotrexate alone (median [interquartile range] hCG in participants 261 [55-1,445] international units/L compared with controls 1,426 [940-2,573]; P=.008). Median time for the ectopic pregnancies to resolve with combination therapy was 34% shorter compared with methotrexate alone (21 days compared with 32 days; P=.018).CONCLUSION: Combination gefitinib and methotrexate has potential as a treatment for ectopic pregnancy but is commonly associated with minor side effects such as transient rash and diarrhea. The treatment requires validation of safety and efficacy in a larger trial.

Objective: Fetal growth restriction (FGR) is a key cause of adverse pregnancy outcome where maternal and fetal factors are identified as contributing to this condition. Idiopathic FGR is associated with altered vascular endothelial cell functions. Decorin (DCN) has important roles in the regulation of endothelial cell functions in vascular environments. DCN expression is reduced in FGR. The objectives were to determine the functional consequences of reduced DCN in a human microvascular endothelial cell line model (HMVEC), and to determine downstream targets of DCN and their expression in primary placental microvascular endothelial cells (PLECs) from control and FGR-affected placentae.Approach: Short-interference RNA was used to reduce DCN expression in HMVECs and the effect on proliferation, angiogenesis and thrombin generation was determined. A Growth Factor PCR Array was used to identify downstream targets of DCN. The expression of target genes in control and FGR PLECs was performed.Results: DCN reduction decreased proliferation and angiogenesis but increased thrombin generation with no effect on apoptosis. The array identified three targets of DCN: FGF17, IL18 and MSTN. Validation of target genes confirmed decreased expression of VEGFA, MMP9, EGFR1, IGFR1 and PLGF in HMVECs and PLECs from control and FGR pregnancies.Conclusions: Reduction of DCN in vascular endothelial cells leads to disrupted cell functions. The targets of DCN include genes that play important roles in angiogenesis and cellular growth. Therefore, differential expression of these may contribute to the pathogenesis of FGR and disease states in other microvascular circulations.

Uterine fibroids are a prevalent gynaecological condition in reproductive-aged women and are the commonest reason for hysterectomy. The cellular composition of clonal fibroids are heterogeneous, with phenotypically dissimilar cells that include smooth muscle cells (SMC), vascular SMC (VSMC) and fibroblasts. The aim of our study was to investigate genes that are commonly differentially expressed between fibroid and myometrial whole tissues in phenotypically different sub-populations of cells isolated from fibroid and myometrium. Genes to be investigated by fluorescence-activated cell sorting, quantitative real-time PCR and immunocytochemistry include transforming growth factor beta (TGFB) and retinoic acid (RA) signalling families and steroid hormone receptors. We hypothesised that each cell population isolated from fibroid and myometrium would differ in the expression of fibroid-associated genes. We demonstrated that phenotypically different cellular constituents of uterine fibroids differentially express cellular RA-binding protein 2 (CRABP2), progesterone receptor B (PRB) and TGFB receptor 2 mRNA in fibroid-derived cells of VSMC and SMC phenotype. CRABP2 mRNA was also differentially expressed in fibroblasts and VSMC sub-populations from within clonal fibroid tumours. We conclude that differential regulation of RA, TGFB and PR pathway transcription occurs in fibroid-associated SMC and -fibroblasts and that investigation of paracrine interactions between different cell types within the fibroid microenvironment provides an important new paradigm for understanding the pathophysiology of this common disease.

Methods. HPV genoprevalence in women aged 18-24 years attending family planning clinics in the prevaccine period (2005-2007) was compared with prevalence among women of the same age group in the postvaccine period (2010-2011). The same recruitment and testing strategies were utilized for both sets of samples, and comparisons were adjusted for potentially confounding variables.Results. The prevalence of vaccine HPV genotypes (6, 11, 16, and 18) was significantly lower in the postvaccine sample than in the prevaccine sample (6.7% vs 28.7%; P < .001), with lower prevalence observed in both vaccinated and unvaccinated women compared with the prevaccine population (5.0% [adjusted odds ratio, 0.11; 95% confidence interval, 0.06-0.21] and 15.8% [adjusted odds ratio, 0.42; 95% confidence interval, 0.19-0.93], respectively). A slightly lower prevalence of nonvaccine oncogenic HPV genotypes was also found in vaccinated women (30.8% vs 37.6%; adjusted odds ratio, 0.68; 95% confidence interval, 0.46-0.99).Conclusions. Four years after the commencement of the Australian HPV vaccination program, a substantial decrease in vaccine-targeted genotypes is evident and should, in time, translate into reductions in HPV-related lesions.

Uterine fibroids are the most common benign tumour afflicting women of reproductive age. Despite the large healthcare burden caused by fibroids, there is only limited understanding of the molecular mechanisms that drive fibroid pathophysiology. Although a large number of genes are differentially expressed in fibroids compared with myometrium, it is likely that most of these differences are a consequence of the fibroid presence and are not causal. The aim of this study was to investigate the expression and regulation of NR2F2 and CTNNB1 based on their potential causal role in uterine fibroid pathophysiology. We used real-time quantitative RT-PCR, western blotting and immunohistochemistry to describe the expression of NR2F2 and CTNNB1 in matched human uterine fibroid and myometrial tissues. Primary myometrial and fibroid smooth muscle cell cultures were treated with progesterone and/or retinoic acid (RA) and sonic hedgehog (SHH) conditioned media to investigate regulatory pathways for these proteins. We showed that NR2F2 and CTNNB1 are aberrantly expressed in fibroid tissue compared with matched myometrium, with strong blood vessel-specific localisation. Although the SHH pathway was shown to be active in myometrial and fibroid primary cultures, it did not regulate NR2F2 or CTNNB1 mRNA expression. However, progesterone and RA combined regulated NR2F2 mRNA, but not CTNNB1, in myometrial but not fibroid primary cultures. In conclusion, we demonstrate aberrant expression and regulation of NR2F2 and CTNNB1 in uterine fibroids compared with normal myometrium, consistent with the hypothesis that these factors may play a causal role uterine fibroid development.

Background: Ectopic pregnancy (EP) occurs in 1-2% of pregnancies, but is over-represented as a leading cause of maternal death in early pregnancy. It remains a challenge to diagnose early and accurately. Women often present in early pregnancy with a `pregnancy of unknown location' (PUL) and the diagnosis and exclusion of EP is difficult due to a lack of reliable biomarkers. A serum biomarker able to clearly distinguish between EP and other pregnancy outcomes would greatly assist clinicians in diagnosing and safely managing PULs. This study evaluates the ability of maternal serum macrophage inhibitory cytokine-1 (MIC-1) levels to differentiate between EP and other pregnancy outcomes in women with a PUL.Methods: Sera were collected from 120 women with a PUL at first clinical presentation and assayed for MIC-1 by ELISA. Results were classified according to ultimate pregnancy outcome and the discriminatory ability of MIC-1 to diagnose EP was assessed.Results: Serum MIC-1 levels were lower in women with histologically confirmed (definite) EP (dEP) (median 552 ng/mL; interquartile range (IQR) 414-693 ng/mL) compared to women with definite viable intra-uterine pregnancies (dVIUPs) (722 ng/mL; IQR 412-1122 ng/mL), and higher when compared to women with definite non-viable intra-uterine pregnancies (dNVIUPs) (465 ng/mL; IQR 341-675 ng/mL). MIC-1 levels were significantly higher in women with dEP compared to women whose PULs resolved without medical intervention (srPUL) (401 ng/mL; IQR 315-475 ng/mL) (p<0.003). There were no women with an ectopic pregnancy where serum MIC-1>1000 ng/mL.Conclusion: Serum MIC-1 levels in PUL were not able to categorically diagnose EP, however, MIC-1 could distinguish women with an EP that required medical intervention and those women whose PULs spontaneously resolved. A single serum MIC-1 measurement also excluded EP at levels above 1000 ng/mL. MIC-1 may play a role in the development of a combined assay of biomarkers for the diagnosis of EP.

Background: The current measure of treatment efficacy of single-dose methotrexate for ectopic pregnancy, is a fall in serum hCG of >= 15% between days 4-7 of treatment, which has a positive predictive value of 93% for treatment success. Two small studies have proposed a fall in serum hCG between days 0-4 after treatment confers similar, earlier prognostic information, with positive predictive values of 100% and 88% for treatment success. We sought to validate this in a large, independent cohort because of the potentially significant clinical implications.Methods: We conducted a retrospective study of women (n=206) treated with single-dose methotrexate for ectopic pregnancy (pre-treatment serum hCG levels <= 3000 IU/L) at Scottish hospitals between 2006-2011. Women were divided into two cohorts based on whether their serum hCG levels rose or fell between days 0-4 after methotrexate. Treatment outcomes of women in each cohort were compared, and the test performance characteristics calculated. This methodology was repeated for the current measure (>= 15% fall in serum hCG between days 4-7 of treatment) and an alternate early measure (>20% fall in serum hCG between days 0-4 of treatment), and all three measures were compared for their ability to predict medical treatment success.Results: In our cohort, the positive predictive value of the current clinical measure was 89% (95% CI 84-94%) (121/136). A falling serum hCG between days 0-4 predicted treatment success in 85% (95% CI 79-92%) of cases (94/110) and a >20% fall in serum hCG between days 0-4 predicted treatment success in 94% (95% CI 88-100%) of cases (59/63). There was no significant difference in the ability of these tests to predict medical treatment success.Conclusions: We have verified that a decline in serum hCG between days 0-4 after methotrexate treatment for ectopic pregnancies, with pre-treatment serum hCG levels <= 3000 IU/L, provides an early indication of likelihood of treatment success, and performs just as well as the existing measure, which only provides prognostic information on day 7.

The transcription factor Kruppel-like factor 5 (KLF5) has been shown to associate with nuclear factor kappa B (NFκB) to regulate genes involved in inflammation. However, there are no studies on the expression and regulation of KLF5 in the processes of human labour and delivery. Thus, the aims of this study were to determine the effect of i) human labour on KLF5 expression in both foetal membranes and myometrium; ii) the pro-inflammatory cytokine interleukin 1 beta (IL1β), bacterial product flagellin and the viral dsRNA analogue poly(I:C) on KLF5 expression and iii) KLF5 knockdown by siRNA in human myometrial primary cells on pro-inflammatory and pro-labour mediators. In foetal membranes, there was no effect of term or preterm labour on KLF5 expression. In myometrium, the term labour was associated with an increase in nuclear KLF5 protein expression. Moreover, KLF5 expression was also increased in myometrial cells treated with IL1β, flagellin or poly(IC), likely factors contributing to preterm birth. KLF5 silencing in myometrial cells significantly decreased IL1β-induced cytokine expression (IL6 and IL8 mRNA expression and release), COX2 mRNA expression, and subsequent release of prostaglandins PGE2 and PGF2 α. KLF5 silencing also significantly reduced flagellin- and poly(I:C)-induced IL6 and IL8 mRNA expression. Lastly, IL1β-, flagellin- and poly(I:C)-stimulated NFκB transcriptional activity was significantly suppressed in KLF5-knockout myometrial cells. In conclusion, this study describes novel data in which KLF5 is increased in labouring myometrium, and KLF5 silencing decreased inflammation- and infection-induced pro-labour mediators.

Erratic, "unscheduled", vaginal bleeding continues to be the greatest disadvantage associated with widespread use of long-acting, progestogen-only methods of contraception. As a consequence, it is also the main reason for premature discontinuation of use of these methods in most cultures. From other perspectives, these methods have high acceptability, very high contraceptive efficacy and a range of valuable, added, non-contraceptive health benefits. There has been widespread awareness of the variability of these vaginal bleeding patterns associated with long-acting methods for several decades and much research has been invested into studying their patterns and implications. Considerable research has also been directed towards trying to understand the underlying mechanisms responsible for the unpredictable bleeding. Much has been clarified about the multiple mechanisms contributing to the appearance of superficial, thin-walled fragile vessels within the endometrium of many of those women with troublesome bleeding, but there is still little understanding of why some women develop these vessels and others have no fragile vessels (and may therefore develop amenorrhea). We now have several medical approaches to reliably stopping a prolonged episode of troublesome bleeding, but no good therapy to produce long-lasting relief from recurrence of erratic bleeding in predisposed women. Future understanding of the variability in individual endometrial responses in different women may be a key to solving this frustrating symptom.