W.C. Duncan

Immunolocalization of bcl-2 in the human corpus luteum

The mechanisms of luteal regression and rescue in women are unknown but forms of programmed cell death may be involved. The proto-oncogene bcl-2 is an important inhibitor of apoptosis but has not previously been described in the human corpus luteum. Immunohistochemical localization of bcl-2 protein was investigated in human corpora lutea obtained from women undergoing surgery during endocrine monitored menstrual cycles as well as from women who had been treated with human chorionic gonadotrophin (HCG) to prolong the luteal phase. Bcl-2 was found to be localized in granulosa-lutein, theca-lutein (as identified by co-localization of P450(17)alpha-hydroxylase) and the endothelial cells around some blood vessels. Immunoblotting demonstrated the presence of a single band of approximately MW 26 kDa. There was no apparent change in either the intensity of immunostaining or the histological localization during the normal luteal phase or following treatment with human chorionic gonadotrophin. The product of the proto-oncogene bcl-2 is present in the human corpus luteum. It is unlikely that bcl-2 expression alone is responsible for prolongation of the lifespan of the corpus luteum in early pregnancy although it is possible that the action of the bcl-2 gene present is modified by changes in other members of the bcl-2 family.

Economic evaluation of diagnosing and excluding ectopic pregnancy

BACKGROUND The diagnosis of ectopic pregnancy in women presenting in early pregnancy is often protracted, relying on costly investigations that are psychologically burdensome to the patient. The aim of this study was to evaluate the financial costs to the health services in Scotland of the current methods used to diagnose and exclude ectopic pregnancy, and compare these with that of a theoretical single diagnostic serum biomarker. METHODS We conducted a retrospective cost-description analysis (with and without costs of diagnostic laparoscopy) of the health-care costs incurred by all patients presenting to a large Scottish teaching hospital between June and September 2006 with pain and bleeding in early pregnancy, where ectopic pregnancy was not excluded. Additionally, a cost minimization analysis was performed for the costs of current ectopic pregnancy investigations versus those of a theoretical single diagnostic serum biomarker. This included sensitivity analyses where the biomarker was priced at increasing values and assumed to have less than 100% diagnostic sensitivity and specificity. RESULTS About 175 patients were eligible to be included in the analysis. Forty-seven per cent of patients required more than three visits to diagnose or exclude ectopic pregnancy. The total yearly cost for diagnosing and excluding ectopic pregnancy was 197K pound sterling for the hospital stated, and was estimated to be 1364K pound sterling for Scotland overall. Using a theoretical diagnostic serum biomarker we calculated that we could save health services up to 976K pound sterling (lowest saving 251K pound sterling after subanalysis) every year in Scotland. CONCLUSIONS Ectopic pregnancy is expensive to diagnose and exclude, and the investigation process is often long and might involve significant psychological morbidity. The development of a single diagnostic serum biomarker would minimize this morbidity and lead to significant savings of up to 1 million pounds per year in Scotland.

Molecular mechanisms of ovarian hyperstimulation syndrome: paracrine reduction of endothelial claudin 5 by hCG in vitro is associated with increased endothelial permeability

BACKGROUND Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening complication of ovarian stimulation associated with severe vascular hyperpermeability. Primary co-cultures of human luteinized granulosa cells (LGCs) and human umbilical vein endothelial cells (HUVECs) were used as a model of steroidgenic/endothelial cell interaction in OHSS. METHODS hCG and the vascular endothelial growth factor (VEGF) inhibitor, Flt-1Fc, were added to co-cultures of LGCs and HUVECs separated by a micropore membrane. Endothelial permeability to labeled bovine serum albumin was measured and the expression of the endothelial cell-specific adhesion protein claudin 5 was investigated using immunocytochemistry and western blotting. RESULTS The addition of hCG increased HUVEC permeability in the presence of LGCs (P < 0.05). hCG increased VEGF concentrations in both chambers of the co-culture system (P < 0.05). The increased permeability in the presence of LGCs and hCG was inhibited when VEGF was blocked by Flt-1Fc (P < 0.05). Endothelial membrane claudin 5 protein was reduced in the presence of hCG and LGCs, as measured by immunocytochemistry (P < 0.05) and western blotting (P < 0.05) and this reduction was inhibited by Flt-1Fc. hCG had no direct effects on endothelial cell claudin 5. CONCLUSIONS For OHSS, this novel paradigm suggests that hCG can increase endothelial permeability by up-regulating VEGF in LGCs which causes reduction in endothelial claudin 5 expression.

Bone Morphogenetic Proteins Are Mediators of Luteolysis in the Human Corpus Luteum

Bone morphogenetic proteins (BMPs), members of the transforming growth factor β (TGFβ) superfamily, play important roles in folliculogenesis in various species; however, little is known about their role in luteal function. In this study, we investigated the expression, regulation, and effects of BMP2, BMP4, and BMP6 in carefully dated human corpora lutea and cultured human luteinized granulosa cells. The mRNA abundance of BMPs was increased in the regressing corpus luteum in vivo (P<.01-.001). Human chorionic gonadotropin (hCG) down-regulated BMP2, BMP4, and BMP6 transcripts both in vivo (P=.05-.001) and in vitro (P<.001), and decreased the mRNA abundance of BMP receptors (BMPR1A, BMPR1B, BMPR2; P<.05-.01) in vitro. Three BMPs were regulated by differential signaling pathways. H89, a protein kinase A inhibitor, increased the expression of both BMP2 (P<.05) and BMP4 (P<.05) while decreasing BMP6 (P<.01). PMA, a protein kinase C activator, decreased both BMP4 and BMP6 expression (P<.0001) while enhancing the mRNA abundance of BMP2 (P<.01). BMPs significantly down-regulated transcripts for LH/choriogonadotropin receptor (LHCGR; P<.001) and steroidogenic acute regulatory protein (STAR; P<.001), whereas up-regulating those of follicular stimulating hormone receptor (FSHR; P<.01) and aromatase (CYP19A1; P<.05-.01) in vitro, possessing an effect opposite to hCG but similar to Activin A. Like Activin A, BMP4 and BMP6 stimulated the expression of Inhibin/Activin subunits with a marked effect on INHBB expression (P<.05-.01). These data confirm that BMPs are increased during luteal regression and negatively regulated by hCG via differential mechanisms, suggesting that BMPs are one of the mediators of luteolysis in women.

Using a decline in serum hCG between days 0–4 to predict ectopic pregnancy treatment success after single-dose methotrexate: a retrospective cohort study

BackgroundThe 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.MethodsWe 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.ResultsIn 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.ConclusionsWe 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.

Cardiac imaging with high frame rate contrast enhanced ultrasound: In-vivo demonstration

This work presents the first in-vivo High-frame rate Contrast Enhanced Ultrasound (HFR CEUS) for cardiac application. The in-vivo acquisition has been made on a sheep. A coherent compounding of diverging waves combined with Pulse Inversion (PI) transmission allow a frame rate of 250 frame per seconds which is 8 times faster than standard CEUS acquisition in cardiac application. The proposed method improves the image contrast compared to the CEUS and allows a better tracking of fast movement of the heart.

Cardiac flow mapping using high frame rate diverging wave contrast enhanced ultrasound and image tracking

Contrast echocardiography (CE) ultrasound with microbubble contrast agents have significantly advanced our capability in assessing cardiac function. However in conventional CE techniques with line by line scanning, the frame rate is limited to tens of frames per second, making it difficult to track the fast flow within cardiac chamber. Recent research in high frame-rate (HFR) ultrasound have shown significant improvement of the frame rate in non-contrast cardiac imaging. In this work we show the feasibility of microbubbles flow tracking in HFR CE acquisition in vivo with a high temporal resolution and low MI as well as the detection of vortex near the valves during filling phases agreeing with previous study.

Effects of motion on high frame rate contrast enhanced echocardiography and its correction

Contrast echocardiography (CE) ultrasound with microbubble contrast agents have significantly advanced our capability in assessing cardiac function, including myocardium perfusion imaging and quantification. However in conventional CE techniques with line by line scanning, the frame rate is limited to tens of frames per second and image quality is low. Recent research works in high frame-rate (HFR) ultrasound have shown significant improvement of the frame rate in non-contrast cardiac imaging. But with a higher frame rate, the coherent compounding of HFR CE images shows some artifacts due to the motion of the microbubbles. In this work we demonstrate the impact of this motion on compounded HFR CE in simulation and then apply a motion correction algorithm on in-vivo data acquired from the left ventricle (LV) chamber of a sheep. It shows that even if with the fast flow found inside the LV, the contrast is improved at least 100%.