Diurnal differences in milk composition and its influence on in vitro growth of Staphylococcus aureus and Escherichia coli in bovine quarter milk.

In experimental intramammary inoculation studies, it has been observed that mastitis susceptibility is influenced, among others, by cow factors. To identify milk characteristics leading to these differences, quarter milk samples of morning and evening milk were collected and analyzed for their composition (protein, fat, lactose, urea, lactoferrin, lactoperoxidase, and ß-lactoglobulin concentrations), somatic cell count, and antibodies against Staphylococcus aureus. Furthermore, in vitro growth of S. aureus and Escherichia coli in fresh quarter milk samples was determined. All measured parameters differed significantly between quarters and also between morning and evening milk with the exception of lactose levels. In addition, quantitative growth of S. aureus and E. coli was significantly different in morning milk compared with evening milk. Mixed model analysis revealed that replication of S. aureus was negatively associated with the presence of fat, S. aureus-specific IgG1 antibodies, contamination of the milk sample and morning milk. Replication of E. coli was negatively associated with fat concentrations, and positively associated with morning milk. The significant difference between morning and evening milk supports the theory that changes in milk composition influence bacterial growth. Although all determined milk components differed significantly between quarters and in time no significant association with bacterial growth could be identified with the exception of fat for both studied species and IgG1 titers for S. aureus. The negative association of fat with bacterial growth was assumed to occur due to activation of lipolysis by milk handling and can most likely be neglected for in vivo relevance. The fact that S. aureus-specific IgG1 titers were negatively associated with S. aureus growth in vitro encourages the ongoing effort to develop a vaccine against S. aureus-induced mastitis.

Transmission of Actinobacillus pleuropneumoniae among weaned piglets on endemically infected farms.

Clinical outbreaks due to Actinobacillus pleuropneumoniae occur recurrently, despite the wide-scale use of antimicrobials or vaccination. Therefore, new approaches for the prevention and control of these outbreaks are necessary. For the development of alternative measures, more insight into the transmission of the bacterium on farms is necessary. The aim of this cohort study was to quantify transmission of A. pleuropneumoniae amongst weaned piglets on farms. We investigated three possible transmission routes: (i) indirect transmission by infected piglets within the same compartment, (ii) transmission by infected pigs in adjacent pens and (iii) transmission by direct contact within pens. Additionally, we evaluated the effect of independent litter characteristics on the probability of infection. Two farms participated in our study. Serum and tonsil brush samples were collected from sows pre-farrowing. Serum was analysed for antibodies against Apx toxins and Omp. Subsequently, tonsil brush samples were collected from all piglets from these dams (N=542) in three cohorts, 3 days before weaning and 6 weeks later. Tonsil samples were analysed by qPCR for the presence of the apxIVA gene of A. pleuropneumoniae. Before weaning, 25% of the piglets tested positive; 6 weeks later 47% tested positive. Regression and stochastic transmission models were used to assess the contribution of each of the three transmission routes and to estimate transmission rates. Transmission between piglets in adjacent pens did not differ significantly from that between non-adjacent pens. The transmission rate across pens was estimated to be 0.0058 day(-1) (95% CI: 0.0030-0.010), whereas the transmission rate within pens was ten times higher 0.059 day(-1) (95% CI: 0.048-0.072). Subsequently, the effects of parity and serological response of the dam and litter age at weaning on the probability of infection of pigs were evaluated by including these into the regression model. A higher dam ApxII antibody level was associated with a lower probability of infection of the pig after weaning; age at weaning was associated with a higher probability of infection of the pig after weaning. Finally, transmission rate estimates were used in a scenario study in which the litters within a compartment were mixed across pens at weaning instead of raising litter mates together in a pen. The results showed that the proportion of infected piglets increased to 69% if litters were mixed at weaning, indicating that farm management measures may affect spread of A. pleuropneumoniae.

A cohort study on Actinobacillus pleuropneumoniae colonisation in suckling piglets.

Actinobacillus pleuropneumoniae causes respiratory disease in pigs and despite the use of preventive measures such as vaccination and antimicrobials clinical outbreaks still occur. At weaning often many piglets are not colonised. If differences in prevalence between litters are large and if factors were known that could explain these differences, this may provide an opportunity to raise groups of A. pleuropneumoniae free piglets. To this end, a cohort study was performed on two endemically infected farrow-to-finish farms. Seventy-six of 133 sows were selected using stratified random selection by parity. Farmers complied with a strict hygiene and animal management protocol to prevent transmission between litters. Tonsil brush and serum samples taken three weeks before parturition were tested for antigen with an apxIVA qPCR and antibodies with Apx and Omp ELISAs, respectively. Three days before weaning tonsil brush samples from all piglets (n=871) were collected and tested for antigen. Whereas all sows tested positive both in serology tests as well as qPCR, 0.41 of the litters tested fully negative and 0.73 of all piglets tested negative. The proportion of positively tested piglets in positive litters ranged from 0.08-1.0 (median=0.36). A grouped logistic regression model with a beta binomial distribution of the probability for piglets to become infected was fitted to the data and associations with explanatory variables were explored. To test the possibility that alternatively the clustering was caused by onwards transmission among the piglets, a transmission model was fitted to the data incorporating sow-piglet and piglet-piglet transmission, but this model did not fit better. The results of this study showed that the number of colonised suckling piglets was highly clustered and mainly attributable to the variability of infectiousness of the dam, but no dam related risk factor for colonisation status of litter or piglets within litters could be identified.

Changes in Cesarean section scar dimensions during pregnancy: a prospective longitudinal study.

OBJECTIVES: To describe changes in Cesarean section (CS) scars longitudinally throughout pregnancy, and to relate initial scar measurements, demographic variables and obstetric variables to subsequent changes in scar features and to final pregnancy outcome. METHODS: In this prospective observational study we used transvaginal sonography (TVS) to examine the CS scar of 320 consecutive pregnant women at 11-13, 19-21 and 32-34 weeks' gestation. For scars visible on TVS, the hypoechoic part was measured in three dimensions and the residual myometrial thickness (RMT) was also measured. Analyses were carried out using one-way repeated measures ANOVA and mixed modeling. The incidence of subsequent scar rupture was recorded. RESULTS: The CS scar was visible in 284/320 cases (89%). Concerning length and depth of the hypoechoic part of the scar and RMT, the larger the initial scar measurement, the larger the decrease observed during pregnancy. For the hypoechoic part of the scar, the width increased on average by 1.8 mm per trimester, while the depth and length decreased by 1.8 and 1.9 mm, respectively (false discovery rate P < 0.0001). Mean RMT in the first trimester was 5.2 mm and on average decreased by 1.1 mm per trimester. Two cases (0.62%) of uterine scar rupture were confirmed following a trial of vaginal delivery; these had a mean RMT of 0.5 mm at second scan and an average decrease of 2.6 mm over the course of pregnancy. CONCLUSION: This study establishes reference data and confirms that the dimensions of CS scars change throughout pregnancy. Scar rupture was associated with a smaller RMT and greater decrease in RMT during pregnancy. There is the potential to test absolute values and observed changes in CS scar measurements as predictors of uterine scar rupture and outcome in trials of vaginal birth after Cesarean section.

Impact of ovulation and implantation timing on first-trimester crown-rump length and gestational age.

OBJECTIVE: To determine the impact of ovulation and implantation timing on first-trimester crown-rump length (CRL) and the derived gestational age (GA). METHOD: One hundred and forty-three women who were trying to conceive were recruited prospectively. The timing of ovulation and implantation and the ovulation to implantation (O-I) interval were established in 101 pregnancies using home urinary tests for luteinizing hormone and human chorionic gonadotropin. In 71 ongoing pregnancies, GA determined by measurement of fetal CRL at 10-14 weeks' gestation was compared with GA based on ovulation and implantation day. First-trimester growth was determined by serial ultrasound scans at 6-7, 8-9 and 10-14 weeks. RESULTS: The median ovulation and implantation days were 16 and 27, respectively, with an O-I interval of 11 days. GA estimated from CRL at 10-14 weeks was on average 1.3 days greater than that derived from ovulation timing. CRL Z-score was inversely related to O-I interval (ρ= -0.431, P=0.0009). There was no significant relationship between CRL growth rate and the difference between observed CRL and expected CRL based on GA from last menstrual period (ρ=0.224, P=0.08). CONCLUSIONS: Early implantation leads to a larger CRL and late implantation to a smaller CRL at 10-14 weeks, independent of CRL growth rate. Implantation timing is a major determinant of fetal size at 10-14 weeks and largely explains the variation in estimates of GA in the first trimester derived from embryonic or fetal CRL.

Do pocket-sized ultrasound machines have the potential to be used as a tool to triage patients in obstetrics and gynecology?

OBJECTIVES: To evaluate the performance and potential impact on patient management of a pocket-sized ultrasound machine (PUM) in comparison to high-specification ultrasound machines (HSUM). METHODS: This was an observational cohort study with 204 unselected patients in three categories: 1) women with pain and bleeding in early pregnancy (101 patients); 2) women presenting for routine obstetric ultrasound assessment (53 patients); 3) women with possible gynecological pathology (50 patients). Scans were carried out transabdominally using a PUM. A second operator repeated the examination transvaginally and/or transabdominally, depending on the clinical indication, using an HSUM. The operators were blind to each other's findings. RESULTS: In the early pregnancy group, there was good to very good agreement between the PUM and HSUM for identifying the presence or absence of an embryo, gestational sac, fetal heart motion, pregnancy location and final diagnostic outcome (kappa coefficients, 0.844, 0.843, 0.729, 0.785 and 0.812, respectively; P < 0.0001). In the obstetric ultrasound group there was good to very good agreement for fetal presentation, placental location and placental position (kappa coefficients, 0.924, 0.924 and 0.647, respectively; P < 0.0001). In the gynecological pathology group, there was very good agreement for final diagnosis and type of ovarian mass (low risk or complex) (kappa coefficients, 0.846 and 0.930, respectively; P < 0.0001). For the measured continuous variables, there was close agreement for crown-rump length, mean sac diameter, femur length and mean diameter of an ovarian mass, but not for endometrial thickness. Neither patient demographics (age, body mass index, ethnicity) nor operator experience and familiarity with a PUM had an impact on agreement between the two machines. If a PUM had been the only equipment available for an initial assessment, only two cases would have led to a suboptimal patient management plan. CONCLUSION: The findings and final diagnosis in the three study groups were similar for both a PUM used transabdominally and an HSUM used transvaginally and/or transabdominally.

Detection of Actinobacillus pleuropneumoniae in pigs by real-time quantitative PCR for the apxIVA gene.

A real-time quantitative PCR (qPCR) for detection of the apxIVA gene of Actinobacillus pleuropneumoniae was validated using pure cultures of A. pleuropneumoniae and tonsillar and nasal swabs from experimentally inoculated Caesarean-derived/colostrum-deprived piglets and naturally infected conventional pigs. The analytical sensitivity was 5colony forming units/reaction. In comparison with selective bacterial examination using tonsillar samples from inoculated animals, the diagnostic sensitivity of the qPCR was 0.98 and the diagnostic specificity was 1.0. The qPCR showed consistent results in repeatedly sampled conventional pigs. Tonsillar brush samples and apxIVA qPCR analysis may be useful for further epidemiological studies and monitoring for A. pleuropneumoniae.

Visibility and measurement of cesarean section scars in pregnancy: a reproducibility study.

OBJECTIVES: To evaluate the visibility of cesarean section (CS) scars by transvaginal sonography (TVS) in pregnant women, to apply a standardized approach for measuring CS scars and to test its reproducibility throughout the course of pregnancy. METHODS: In this observational cohort study, 320 consecutive pregnant women with a previous cesarean delivery were examined to assess scar visibility by two independent examiners. TVS was carried out at 11-13, 19-21 and 34-36 weeks' gestation. A scar was defined as visible when an area of hypoechogenicity representing myometrial discontinuity at the anterior wall of the lower uterine segment was identified. In a subset of patients (n = 111), visible scars were measured by two independent examiners in three dimensions: scar width, depth and length as well as the residual myometrial thickness (RMT). Descriptive analysis was used to assess scar visibility, and the intraclass correlation coefficient (ICC) was calculated to show the strength of absolute agreement between two examiners for scar measurements. For RMT, a cut-off of 2.4 mm was used and measurement agreement was assessed using Cohen's kappa coefficient. RESULTS: The scar was visible in 284/320 cases (88.8%). Visible scars were significantly associated with anteverted uteri (P < 0.0001). Both examiners had 100% agreement on scar visibility at 12 and 20 weeks' gestation, while agreement was 96% at 34 weeks. The intra- and interobserver agreements for scar measurements were generally good (ICC 0.86 and 0.89, respectively). The kappa coefficient for the RMT was 0.27 in the first trimester, compared with 0.51 and 0.72 in the second and third trimesters, respectively. CONCLUSION: CS scars remain visible in the majority of women throughout pregnancy. They can be reproducibly measured in three dimensions when assessed by TVS in all trimesters of pregnancy. The agreement between two observers for CS scar measurement can be considered good for the first trimester, compared with relatively moderate agreement for the second and third trimesters.

Does the presence of a cesarean section scar influence the site of placental implantation and subsequent migration in future pregnancies: a prospective case-control study.

OBJECTIVES: To describe placental location in the first trimester of pregnancy and subsequent placental migration in women with and without a history of previous cesarean delivery. METHODS: In this prospective case-control study, placental location was defined according to five anatomical sites in relation to the endometrial cavity. Placental localization was carried out by transabdominal ultrasound between 11 and 14 weeks' gestation. We recruited 738 women who had undergone one or more previous cesarean sections (CS) and 1856 patients without previous cesarean delivery. Comparative analysis was performed of placental location between the two groups, and to assess placental migration of those classified as being low lying at 20 and 32 weeks' gestation. RESULTS: There were significant differences in placental location between the two groups. In the CS group there were significantly more posterior and fewer fundal placentae than in the control group (47.2 vs 31.5% and 4.7 vs 15.5%, respectively). The number of previous cesarean deliveries did not have a significant effect on placental location. There was no significant difference in the incidence of anterior low-lying placenta between groups (1.5 vs 0.9%). Placental migration of the low-lying subtypes was similar in both groups (62 vs 64%). CONCLUSION: The presence of CS scars in the uterus are associated with an increase in the number of posterior placentae and a reduced number that implant in the fundus of the cavity. Migration of a low-lying placenta is independent of the presence of a CS scar in the uterus.

Homologous whole bacterin vaccination is not able to reduce Streptococcus suis serotype 9 strain 7997 transmission among pigs or colonization.

Streptococcus suis (S. suis) is an important porcine pathogen worldwide, and antibiotics are often applied to treat or prevent clinical signs. Vaccination could be an alternative measure to reduce the abundant use of antimicrobials. The aim of this study was to determine the effect of vaccination with homologues whole bacterin vaccine containing S. suis serotype 9 strain 7997 on transmission of this serotype among pigs and on mucosal colonization. Caesarean derived, colostrum deprived pigs (N=50) were housed pair wise. Thirteen pairs were vaccinated intramuscularly with 2-3×10(9) colony forming units (CFU) inactivated S. suis serotype 9 per dose and α-tocopherolactetaat as adjuvant at 3 and 5 weeks of age; twelve pairs served as non-vaccinated controls. At 7 weeks of age, one pig of each pair was intranasally inoculated with 1-2×10(9)CFU of the homologues strain, whereas the other pig of each pair was contact-exposed. Tonsil brushings and saliva swabs were collected for 4 weeks, and tested for the presence of S. suis by bacteriological culture. No differences in number of S. suis in the tonsils or saliva samples or in clinical signs were observed between vaccinated and control pigs. In all pairs, transmission between inoculated and contact exposed pigs occurred, and no difference was observed in rate at which this occurred. The estimated transmission rate parameter ß between vaccinated pigs was ß(v)=5.27/day, and for non-vaccinated pigs ß(nv)=2.77/day (P=0.18). It was concluded that vaccination against S. suis serotype 9 did not reduce transmission, nor colonization and that there were no indications that protection against clinical signs was induced.

Limitations of current definitions of miscarriage using mean gestational sac diameter and crown-rump length measurements: a multicenter observational study.

OBJECTIVES: There is significant variation in cut-off values for mean gestational sac diameter (MSD) and embryo crown-rump length (CRL) used to define miscarriage, values suggested in the literature ranging from 13 to 25 mm for MSD and from 3 to 8 mm for CRL. We aimed to define the false-positive rate (FPR) for the diagnosis of miscarriage associated with different CRL and MSD measurements with or without a yolk sac in a large study population of patients attending early pregnancy clinics. We also aimed to define cut-off values for CRL and MSD that, on the basis of a single measurement, can definitively diagnose a miscarriage and so exclude possible inadvertent termination of pregnancy. METHODS: This was an observational cross-sectional study. Data were collected prospectively according to a predefined protocol. Intrauterine pregnancy of uncertain viability (IPUV) was defined as an empty gestational sac or sac with a yolk sac but no embryo seen with MSD < 20 or < 30 mm or an embryo with an absent heartbeat and CRL < 6 mm or < 8 mm. We recruited to the study 1060 consecutive women with IPUV. The endpoint was presence or absence of a viable pregnancy at the time of first-trimester screening ultrasonography between 11 and 14 weeks. The sensitivity, specificity, positive and negative predictive values were calculated for potential cut-off values to define miscarriage from MSD 8 to 30 mm with or without a yolk sac and from CRL 3 to 8 mm. RESULTS: Of the 1060 women with a diagnosis of IPUV, 473 remained viable and 587 were non-viable by the time of the 11-14-week scan. In the absence of both embryo and yolk sac, the FPR for miscarriage was 4.4% when an MSD cut-off of 16 mm was used and 0.5% for a cut-off of 20 mm. There were no false-positive test results for miscarriage when a cut-off of MSD ≥ 21 mm was used. If a yolk sac was present but an embryo was not, the FPR for miscarriage was 2.6% for an MSD cut-off of 16 mm and 0.4% for a cut-off of 20 mm, with no false-positive results when a cut-off of MSD ≥ 21 mm was used. When an embryo was visible with an absent heartbeat, using a CRL cut-off of 4 mm the FPR for miscarriage was 8.3%, and for a CRL cut-off of 5 mm it was also 8.3%. There were no false-positive results using a CRL cut-off of ≥ 5.3 mm. CONCLUSIONS: These data show that some current definitions used to diagnose miscarriage are potentially unsafe. Current national guidelines should be reviewed to avoid inadvertent termination of wanted pregnancies. An MSD cut-off of > 25 mm and a CRL cut-off of > 7 mm could be introduced to minimize the risk of a false-positive diagnosis of miscarriage.

Gestational sac and embryonic growth are not useful as criteria to define miscarriage: a multicenter observational study.

OBJECTIVES: We studied changes in mean gestational sac diameter (MSD) and embryonic crown-rump length (CRL) in intrauterine pregnancies of uncertain viability (IPUVs). We aimed to establish cut-off values for MSD and CRL growth that could be definitively associated with either viability or miscarriage, and to establish the relationship between growth in MSD and appearance of embryonic structures in the gestational sac. METHODS: One thousand and sixty consecutive IPUVs were recruited prospectively from four London University hospitals: 462 with no yolk sac or embryo, 419 with a yolk sac but no embryo, and 179 with an embryo but no heartbeat visible. IPUV was defined as an empty gestational sac with or without a yolk sac but no embryo seen with MSD < 20 or < 30 mm (depending on center) or an embryo with no heartbeat and CRL < 6 mm or < 8 mm (depending on center). Scans were repeated 7-14 days later. The endpoint was viability at first-trimester screening ultrasonography between 11 and 14 weeks. Change in MSD and CRL between the first and second scans of each pregnancy was compared with respect to viability and appearance of embryonic structures using the two-sample t-test. RESULTS: The study included 359 pregnancies in which a gestational sac with or without embryo was identified at the follow-up scan 7-14 days later. Of these, 192 were viable and 167 non-viable at the 11-14-week scan. MSD growth was significantly higher in viable than non-viable pregnancies (mean 1.003 vs. 0.503 mm/day; P < 0.001, 95% CI of difference 0.403-0.596). A difference in CRL growth was found between the two groups (mean 0.673 vs. 0.148 mm/day; P < 0.001, 95% CI of difference 0.345-0.703). MSD growth of 0.6 mm/day was associated with a specificity for diagnosing miscarriage of 90.1%, a sensitivity of 61.7% and 19 false-positive test results. A cut-off of CRL growth rate of 0.2 mm/day gave a sensitivity of 76.3% and there were no false-positive test results for miscarriage. On repeat scan the failure of either a yolk sac or embryo to be visualized was always associated with miscarriage. CONCLUSION: There is an overlap in MSD growth rates between viable and non-viable IPUV. No cut-off exists for MSD growth below which a viable pregnancy could be safely excluded. A cut-off value for CRL growth of 0.2 mm/day was always associated with miscarriage. These data suggest that criteria to diagnose miscarriage based on growth in MSD and CRL are potentially unsafe. However, finding an empty gestational sac on two scans more than 7 days apart is highly likely to indicate miscarriage, irrespective of growth.

HE4 and CA125 as a diagnostic test in ovarian cancer: prospective validation of the Risk of Ovarian Malignancy Algorithm.

BACKGROUND: Recently, a Risk of Ovarian Malignancy Algorithm (ROMA) utilising human epididymis secretory protein 4 (HE4) and CA125 successfully classified patients as presenting a high or low risk for epithelial ovarian cancer (EOC). We validated this algorithm in an independent prospective study. METHODS: Women with a pelvic mass, who were scheduled to have surgery, were enrolled in a prospective study. Preoperative serum levels of HE4 and CA125 were measured in 389 patients. The performance of each of the markers, as well as that of ROMA, was analysed. RESULTS: When all malignant tumours were included, ROMA (receiver operator characteristic (ROC)-area under curve (AUC)=0.898) and HE4 (ROC-AUC)=0.857) did not perform significantly better than CA125 alone (ROC-AUC=0.877). Using a cutoff for ROMA of 12.5% for pre-menopausal patients, the test had a sensitivity of 67.5% and a specificity of 87.9%. With a cutoff of 14.4% for post-menopausal patients, the test had a sensitivity of 90.8% and a specificity of 66.3%. For EOC vs benign disease, the ROC-AUC of ROMA increased to 0.913 and for invasive EOC vs benign disease to 0.957. CONCLUSION: This independent validation study demonstrated similar performance indices to those recently published. However, in this study, HE4 and ROMA did not increase the detection of malignant disease compared with CA125 alone. Although the initial reports were promising, measurement of HE4 serum levels does not contribute to the diagnosis of ovarian cancer.

Umbilical artery pulsatility index and fetal abdominal circumference in isolated gastroschisis.

OBJECTIVES: To investigate changes in abdominal circumference (AC) and umbilical artery pulsatility index (UA-PI) with gestation in fetuses with isolated gastroschisis, and to determine whether a relationship exists between UA-PI and fetal AC. METHODS: Data from 58 pregnancies with isolated gastroschisis diagnosed at between 24 and 36 weeks' gestation were included in the study. Z-scores were calculated with respect to expected UA-PI values in normal pregnancies after log-transformation. AC-Z-scores were calculated with respect to expected size in normal pregnancies according to a standard chart. Functional linear discriminant analysis (FLDA) was applied to generate 50(th), 5(th) and 95(th) percentile curves for changes in both AC and UA-PI with gestational age in fetuses with gastroschisis. These curves were compared with the standard curves, as were the means. UA-PI was also plotted against AC. For this relationship, a robust Spearman correlation coefficient was obtained with FLDA. RESULTS: In fetuses with gastroschisis, there was a highly significant negative correlation between UA-PI and AC, normalized for gestation using Z-scores (median correlation coefficient, - 0.289; median P = 0.000023). Moreover, compared with standard curves AC was lower and UA-PI higher in the gestational-age range studied. Both the AC and UA-PI curves showed a significantly different rate of change with gestation compared with the normal ranges. The mean values for fetuses with gastroschisis compared with the standard AC and UA-PI range curves were significantly different for AC throughout gestation, and for UA-PI from 32 weeks' gestation. CONCLUSIONS: In fetal gastroschisis, it is well known that AC tends to be smaller, though UA-PI has not been reported to be abnormal in any consistent way. There is a clear relationship between the fetus's AC for gestation and UA-PI, which is not the case for normally grown fetuses. The data suggest that the growth restriction seen in gastroschisis may be explained by hypoxia, and not simply by the classical explanation of extra-abdominal displacement of the abdominal viscera.

Lidocaine does not reduce pain perception during gel instillation sonography or subsequent office hysteroscopy: results of a randomized trial.

BACKGROUND: To evaluate if the addition of lidocaine to the gel used for gel infusion sonohysterography (GIS) reduces pain experienced during GIS or subsequent hysteroscopy. METHODS: A total of 142 consecutive patients were randomized using computer-generated random integers. In 79 patients, GIS was performed with a gel containing lidocaine (Instillagel®) and in 63 patients the gel did not contain lidocaine (Endosgel®). Immediately after GIS, 132 patients (94%) underwent office hysteroscopy. The women were asked to fill in a questionnaire including a 100-mm visual analogue scale (VAS) score after each examination. RESULTS: The mean age (SD) was 50.8 (12.1) years; 58.5% were premenopausal and 15.6% were nulliparous. The median (interquartile range (IR)) VAS score during GIS for all women was 6 (19.5): 8 (21) for the lidocaine group versus 5 (18.2) for those who received gel without lidocaine. The median (IR) VAS scores during hysteroscopy in the total group, the Instillagel group and the Endosgel group were 15.5 (43.2), 24 (35) and 9 (52), respectively. None of the differences were statistically significant. CONCLUSION: The addition of lidocaine to the gel used either for GIS or prior to office hysteroscopy does not reduce the procedure-related pain.

Prediction of lymph node involvement in breast cancer from primary tumor tissue using gene expression profiling and miRNAs.

The aim of this study was to investigate whether lymph node involvement in breast cancer is influenced by gene or miRNA expression of the primary tumor. For this purpose, we selected a very homogeneous patient population to minimize heterogeneity in other tumor and patient characteristics. First, we compared gene expression profiles of primary tumor tissue from a group of 96 breast cancer patients balanced for lymph node involvement using Affymetrix Human U133 Plus 2.0 microarray chip. A model was built by weighted Least-Squares Support Vector Machines and validated on an internal and external dataset. Next, miRNA profiling was performed on a subset of 82 tumors using Human MiRNA-microarray chips (Illumina). Finally, for each miRNA the number of significant inverse correlated targets was determined and compared with 1000 sets of randomly chosen targets. A model based on 241 genes was built (AUC 0.66). The AUC for the internal dataset was 0.646 and 0. 651 for the external datasets. The model includes multiple kinases, apoptosis-related, and zinc ion-binding genes. Integration of the microarray and miRNA data reveals ten miRNAs suppressing lymph node invasion and one miRNA promoting lymph node invasion. Our results provide evidence that measurable differences in gene and miRNA expression exist between node negative and node positive patients and thus that lymph node involvement is not a genetically random process. Moreover, our data suggest a general deregulation of the miRNA machinery that is potentially responsible for lymph node invasion.

Improving the preoperative classification of adnexal masses as benign or malignant by second-stage tests.

OBJECTIVE: The aim of this study was to establish when a second-stage diagnostic test may be of value in cases where a primary diagnostic test has given an uncertain diagnosis of the benign or malignant nature of an adnexal mass. METHODS: The diagnostic performance with regard to discrimination between benign and malignant adnexal masses for mathematical models including ultrasound variables and for subjective evaluation of ultrasound findings by an experienced ultrasound examiner was expressed as area under the receiver-operating characteristics curve (AUC), sensitivity and specificity. These were calculated for the total study population of 1938 patients with an adnexal mass as well as for subpopulations defined by the certainty with which the diagnosis of benignity or malignancy was made. The effect of applying a second-stage test to the tumors where risk estimation was uncertain was determined. RESULTS: The best mathematical model (LR1) had an AUC of 0.95, sensitivity of 92% and specificity of 84% when applied to all tumors. When model LR1 was applied to the 10% of tumors in which the calculated risk fell closest to the risk cut-off of the model, the AUC was 0.59, sensitivity 90% and specificity 21%. A strategy where subjective evaluation was used to classify these 10% of tumors for which LR1 performed poorly and where LR1 was used in the other 90% of tumors resulted in a sensitivity of 91% and specificity of 90%. Applying subjective evaluation to all tumors yielded an AUC of 0.95, sensitivity of 90% and specificity of 93%. Sensitivity was 81% and specificity 47% for those patients where the ultrasound examiner was uncertain about the diagnosis (n = 115; 5.9%). No mathematical model performed better than did subjective evaluation among the 115 tumors where the ultrasound examiner was uncertain. CONCLUSION: When model LR1 is used as a primary test for discriminating between benign and malignant adnexal masses, the use of subjective evaluation of ultrasound findings by an experienced examiner as a second-stage test in the 10% of cases for which the model yields a risk of malignancy closest to its risk cut-off will improve specificity without substantially decreasing sensitivity. However, none of the models tested proved suitable as a second-stage test in tumors where subjective evaluation yielded an uncertain result.

Effect of cancer prevalence on the use of risk-assessment cut-off levels and the performance of mathematical models to distinguish malignant from benign adnexal masses.

OBJECTIVE: Two logistic regression models have been developed for the characterization of adnexal masses. The goal of this prospective analysis was to see whether these models perform differently according to the prevalence of malignancy and whether the cut-off levels of risk assessment for malignancy by the models require modification in different centers. METHODS: Centers were categorized into those with a prevalence of malignancy below 15%, between 15 and 30% and above 30%. The areas under the receiver-operating characteristics curves (AUC) were compared using bootstrapping. The optimal cut-off level of risk assessment for malignancy was chosen per center, corresponding to the highest sensitivity level possible while still keeping a good specificity. RESULTS: Both models performed better in centers with a lower prevalence of malignant cases. The AUCs of the two models for centers with fewer than 15% malignant cases were 0.97 and 0.95, those of centers with 15-30% malignancy were 0.95 and 0.93 and those of centers with more than 30% malignant cases were 0.94 and 0.92. This decrease in performance was due mainly to the decrease in specificity from over 90 to around 76%. In the centers with a higher percentage of malignant cases, a sensitivity of at least 90% with a good specificity could not be obtained by choosing a different cut-off level. CONCLUSIONS: Overall the models performed well in all centers. The performance of the logistic regression models worsened with increasing prevalence of malignancy, due to a case mix with more borderline and complex benign masses seen in those centers. Because the cut-off of 0.10 is optimal for all three types of center, it seems reasonable to use this cut-off for both models in all centers.

Effect of Eimeria acervulina infection history on the immune response and transmission in broilers.

Heterogeneity in exposure to Eimeria spp. of chickens in a flock will result in differences between individual birds in oocyst output and acquired immunity, which subsequently affects transmission of the parasite in the population. The aim of this study was to quantify effects of previous infection of broilers with Eimeria acervulina on immune responses, oocyst output and transmission. A transmission experiment was carried out with pair-wise housed broilers, that differed in infection history. This "infection history" was achieved by establishment of a primary infection by inoculation of birds with 50,000 sporulated E. acervulina oocysts at day 6 of age ("primed"); the other birds did not receive a primary infection ("naïve"). The actual transmission experiment started at day 24 of age: one bird (I) was inoculated with 50,000 sporulated oocysts and was housed together with a non-inoculated contact bird (C). Oocyst excretion and parameters describing transmission, i.e. the number of infected C birds and time passed before start of excretion of C birds, were determined from day 28 to day 50 for six pairs of four different combinations of I and C birds (I-C): naïve-naïve, naïve-primed, primed-naïve and primed-primed. Immune parameters, CD4(+), CD8(+), αßTCR(+) and γδTCR(+) T cells and macrophages in duodenum, were determined in an additional 25 non-primed, non-inoculated control birds, and in the naïve-naïve and naïve-primed groups, each group consisting of 25 pairs. Although the numbers of CD4(+) T cells and γδTCR(+) T cells increased after primary infection, none of the immunological cell types provided an indication of differences in infectivity, susceptibility or transmission between birds. Oocyst output was significantly reduced in primed I and C birds. Transmission was reduced most in the primed-primed group, but nonetheless transmission occurred in all groups. This study also showed that acquired immunity significantly reduced oocyst output after inoculation and contact-infection, but not sufficiently to prevent transmission to contact-exposed birds.