Continuous dosing of a novel contraceptive vaginal ring releasing Nestorone® and estradiol: pharmacokinetics from a dose-finding study.

BACKGROUND: As part of a program to develop a novel estradiol-releasing contraceptive vaginal ring (CVR), we evaluated the pharmacokinetic (PK) profile of CVRs releasing segesterone acetate (Nestorone® (NES)) combined with one of three different estradiol (E2) doses. STUDY DESIGN: A prospective, double-blind, randomized, multi-centered study to evaluate a 90-day CVR releasing NES [200mcg/day] plus E2, either 10mcg/day, 20mcg/day, or 40mcg/day in healthy reproductive-age women with regular cycles. Participants provided blood samples twice weekly for NES and E2 levels during the first 60 days (ring 1) and the last 30 days (ring 2) of use. A subset underwent formal PK assessments at ring initiation, ring exchange (limited PK), and study completion. RESULTS: The main study enrolled 197 women; 22 participated in the PK substudy. Baseline characteristics between the main and PK participants were comparable, with an average BMI of 25.8 kg/m2 (SD 4.3). In the PK substudy, all three rings showed similar NES PK: mean area under the curve (AUC(0-72)) 34,181 pg*day/mL; concentration maximum (Cmax) 918 pg/mL; time to maximum concentration (Tmax) 3.5 h. For E2, the Cmax occurred at 2 h, and was significantly higher with the 20 mcg/day ring (mean 390 pg/mL); 10 mcg/day, 189 pg/mL, p=.003; 40 mcg/day, 189 pg/mL, p<.001), and declined rapidly to≤50 pg/mL for all doses by 24 h. For all subjects, the median E2 levels remained under 35 pg/mL during treatment. CONCLUSION: PK parameters of NES were not affected when paired with different doses of E2, but E2 levels from all three doses were lower than anticipated and no dose response was observed. IMPLICATIONS: While these novel estradiol-releasing combination contraceptive vaginal rings provided sustained release of contraceptive levels of Nestorone over 90 days, the E2 levels achieved were not consistent with bone protection, and a dose-response was not observed.

Therapeutic targeting of membrane-associated GRP78 in leukemia and lymphoma: preclinical efficacy in vitro and formal toxicity study of BMTP-78 in rodents and primates.

Translation of drug candidates into clinical settings requires demonstration of preclinical efficacy and formal toxicology analysis for filling an Investigational New Drug (IND) application with the US Food and Drug Administration (FDA). Here, we investigate the membrane-associated glucose response protein 78 (GRP78) as a therapeutic target in leukemia and lymphoma. We evaluated the efficacy of the GRP78-targeted proapoptotic drug bone metastasis targeting peptidomimetic 78 (BMTP-78), a member of the D(KLAKLAK)2-containing class of agents. BMTP-78 was validated in cells from patients with acute myeloid leukemia and in a panel of human leukemia and lymphoma cell lines, where it induced dose-dependent cytotoxicity in all samples tested. Based on the in vitro efficacy of BMTP-78, we performed formal good laboratory practice toxicology studies in both rodents (mice and rats) and nonhuman primates (cynomolgus and rhesus monkeys). These analyses represent required steps towards an IND application of BMTP-78 for theranostic first-in-human clinical trials.

A snapshot of biologic drug development: Challenges and opportunities.

Since the approval of insulin as the first recombinant therapeutic protein, the prominence of biologic therapies in drug development has grown significantly. Many modalities beyond traditional biologics are now being developed or explored for various indications with significant unmet medical needs. From early traditional replacement proteins to more recent, highly engineered antibodies, oligonucleotides, fusion proteins, and gene constructs, biologic agents have delivered life-changing therapies, despite often having scientifically and technically challenging development programs. This brief review outlines some of the major biotherapeutic classes and identifies the advantages and challenges with the development of these products.

Cost-effectiveness of salpingotomy and salpingectomy in women with tubal pregnancy (a randomized controlled trial).

STUDY QUESTION: Is salpingotomy cost effective compared with salpingectomy in women with tubal pregnancy and a healthy contralateral tube? SUMMARY ANSWER: Salpingotomy is not cost effective over salpingectomy as a surgical procedure for tubal pregnancy, as its costs are higher without a better ongoing pregnancy rate while risks of persistent trophoblast are higher. WHAT IS KNOWN ALREADY: Women with a tubal pregnancy treated by salpingotomy or salpingectomy in the presence of a healthy contralateral tube have comparable ongoing pregnancy rates by natural conception. Salpingotomy bears the risk of persistent trophoblast necessitating additional medical or surgical treatment. Repeat ectopic pregnancy occurs slightly more often after salpingotomy compared with salpingectomy. Both consequences imply potentially higher costs after salpingotomy. STUDY DESIGN, SIZE, DURATION: We performed an economic evaluation of salpingotomy compared with salpingectomy in an international multicentre randomized controlled trial in women with a tubal pregnancy and a healthy contralateral tube. Between 24 September 2004 and 29 November 2011, women were allocated to salpingotomy (n = 215) or salpingectomy (n = 231). Fertility follow-up was done up to 36 months post-operatively. PARTICIPANTS/MATERIALS, SETTINGS, METHODS: We performed a cost-effectiveness analysis from a hospital perspective. We compared the direct medical costs of salpingotomy and salpingectomy until an ongoing pregnancy occurred by natural conception within a time horizon of 36 months. Direct medical costs included the surgical treatment of the initial tubal pregnancy, readmissions including reinterventions, treatment for persistent trophoblast and interventions for repeat ectopic pregnancy. The analysis was performed according to the intention-to-treat principle. MAIN RESULTS AND THE ROLE OF CHANCE: Mean direct medical costs per woman in the salpingotomy group and in the salpingectomy group were €3319 versus €2958, respectively, with a mean difference of €361 (95% confidence interval €217 to €515). Salpingotomy resulted in a marginally higher ongoing pregnancy rate by natural conception compared with salpingectomy leading to an incremental cost-effectiveness ratio €40 982 (95% confidence interval -€130 319 to €145 491) per ongoing pregnancy. Since salpingotomy resulted in more additional treatments for persistent trophoblast and interventions for repeat ectopic pregnancy, the incremental cost-effectiveness ratio was not informative. LIMITATIONS, REASONS FOR CAUTION: Costs of any subsequent IVF cycles were not included in this analysis. The analysis was limited to the perspective of the hospital. WIDER IMPLICATIONS OF THE FINDINGS: However, a small treatment benefit of salpingotomy might be enough to cover the costs of subsequent IVF. This uncertainty should be incorporated in shared decision-making. Whether salpingotomy should be offered depends on society's willingness to pay for an additional child. STUDY FUNDING/COMPETING INTERESTS: Netherlands Organisation for Health Research and Development, Region Västra Götaland Health & Medical Care Committee. TRIAL REGISTRATION NUMBER: ISRCTN37002267.

Ectopic pregnancy prediction in women with a pregnancy of unknown location: data beyond 48 h are necessary.

STUDY QUESTION: Are there improvements in the accuracy of prediction of ectopic pregnancy (EP) in women with early symptomatic pregnancy using human chorionic gonadotrophin (hCG) curves when clinicians consider visits beyond the first 48 h after initial presentation? SUMMARY ANSWER: Two hCG values, measured 48 h (2 days) apart, are often not sufficient to accurately predict the outcome of a woman with a pregnancy of unknown location (PUL), but adding a third visit on Day 4 or 7 significantly improved the prediction for 1 in 15 women. WHAT IS KNOWN ALREADY: The use of serial hCG values is commonly used to aid in the prediction of the final diagnosis in women with a PUL. Initial outcome predictions based on two hCG values may often be incorrect. STUDY DESIGN, SIZE, DURATION: This retrospective multicenter cohort study included 646 women with a PUL, recruited over 2 years. Of these women, 146 were ultimately diagnosed with EP. PARTICIPANTS/MATERIALS, SETTING, METHODS: Women presenting to the emergency room with first trimester pain or bleeding, with a PUL, at least 2 hCG values and a definitive final diagnosis from the University of Pennsylvania, University of Miami and University of Southern California, were recruited from 2007 to 2009. MAIN RESULTS AND THE ROLE OF CHANCE: Using currently recommended prediction rules, adding a third hCG evaluation on Day 4 after initial presentation significantly improved the accuracy of initial prediction from the first two values (48 h apart, or Day 2) by 9.3% (P = 0.015). Adding a third value on Day 7 improved prediction significantly by 6.7% (P = 0.031), compared with prediction based on first two values. The improvement in prediction by assessing four hCG values (Days 0, 2, 4 and 7) compared with three values (Days 0, 2 and 4) was 1.3% and not statistically significant. LIMITATIONS, REASONS FOR CAUTION: Missing data imputation likely biased results toward the null; predicted outcomes may not match those made by clinicians; and the study does not predict intrauterine pregnancy and spontaneous miscarriage separately. WIDER IMPLICATIONS OF THE FINDINGS: This study provides useful information for the prediction of outcomes for women with a symptomatic first trimester pregnancy of unknown location, but may not be generalizable to all pregnant women. STUDY FUNDING/COMPETING INTEREST(S): Supported by NIH grant numbers R01-HD036455 to Dr Barnhart and Dr Sammel, K24HD060687 to Dr Barnhart, and 5T32MH065218 to Ms. Zee. The authors have no conflicts of interest to declare.

Prognostic profiles and the effectiveness of assisted conception: secondary analyses of individual patient data.

BACKGROUND: At present, it is unclear which treatment strategy is best for couples with unexplained or mild male subfertility. We hypothesized that the prognostic profile influences the effectiveness of assisted conception. We addressed this issue by analysing individual patient data (IPD) from randomized controlled trials (RCTs). METHODS: We performed an IPD analysis of published RCTs on treatment strategies for subfertile couples. Eligible studies were identified from Cochrane systematic reviews and we also searched Medline and EMBASE. The authors of RCTs that compared expectant management (EM), intracervical insemination (ICI), intrauterine insemination (IUI), all three with or without controlled ovarian stimulation (COS) and IVF in couples with unexplained or male subfertility, and had reported live birth or ongoing pregnancy as an outcome measure, were invited to share their data. For each individual patient the chance of natural conception was calculated with a validated prognostic model. We constructed prognosis-by-treatment curves and tested whether there was a significant interaction between treatment and prognosis. RESULTS: We acquired data from 8 RCTs, including 2550 couples. In three studies (n = 954) the more invasive treatment strategies tended to be less effective in couples with a high chance of natural conception but this difference did not reach statistical significance (P-value for interaction between prognosis and treatment outcome were 0.71, 0.31 and 0.19). In one study (n = 932 couples) the strategies with COS (ICI and IUI) led to higher pregnancy rates than unstimulated strategies (ICI 8% versus 15%, IUI 13% versus 22%), regardless of prognosis (P-value for interaction in all comparisons >0.5), but at the expense of a high twin rate in the COS strategies (ICI 6% versus 23% and IUI 3% versus 30%, respectively). In two studies (n = 373 couples), the more invasive treatment strategies tended to be more effective in couples with a good prognosis but this difference did not reach statistical significance (P-value for interaction: 0.38 and 0.68). In one study (n = 253 couples) the differential effect of prognosis on treatment effect was limited (P-value for interaction 0.52), perhaps because prognosis was incorporated in the inclusion criteria. The only study that compared EM with IVF included 38 couples, too small for a precise estimate. CONCLUSIONS: In this IPD analysis of couples with unexplained or male subfertility, we did not find a large differential effect of prognosis on the effectiveness of fertility treatment with IUI, COS or IVF.

Spontaneous high-grade glial intramedullary tumor of the spine in a rhesus macaque (Macaca mulatta).

BACKGROUND: A 4-year-old rhesus macaque presented with acute, progressive paresis of the extremities. METHODS: A complete blood count, serum biochemical analysis, neurologic exam and necropsy were performed. RESULTS: The clinical, histopathological, and immunohistochemical findings confirmed a high-grade intramedullary glial tumor of the spinal cord that was most consistent with an ependymoma. CONCLUSIONS: We describe a case of a naturally occurring spontaneous spinal cord neoplasia in a non-human primate.

Diagnostic value of serum hCG on the outcome of pregnancy of unknown location: a systematic review and meta-analysis.

BACKGROUND The term 'pregnancy of unknown location' (PUL) refers to cases where a pregnancy test is positive but the pregnancy cannot be visualized by transvaginal sonography (TVS). Various strategies integrating TVS and serum hCG measures are used to follow-up until the location and/or viability of the pregnancy becomes clear; however, the optimal strategy to predict the outcome of pregnancy in women with PUL is unknown. Therefore, we performed a systematic review and meta-analysis to determine the diagnostic accuracy of the various serum hCG strategies in women with PUL. METHODS We searched Medline and EMBASE for articles which were published (in any language) from 1980 to January 2012 on strategies using serum hCG in women with PUL and reporting on the final outcome of pregnancy. RESULTS From 980 selected titles, 23 articles, all cohort studies, were included. There were 10 studies on a single serum hCG cut-off level, 4 on serum hCG ratio (hCG 48 h/hCG 0 h) and 6 on logistic regression modelling. Three other strategies were reported using serum hCG, serum progesterone and/or uterine curettage findings; each of these strategies comprised a single study. Comparative diagnostic studies have not been performed on the diagnostic value of serum hCG in women with PUL. Included studies showed substantial clinical heterogeneity in the definition of the outcome, and only data for the pregnancy outcome ectopic pregnancy (EP) were suitable for meta-analysis. The receiver operating characteristic curves showed that the serum hCG ratios and logistic regression models had a better performance as compared with an absolute single serum hCG level (as the curve was considerably closer to the diagonal, indicating no diagnostic value). CONCLUSIONS Overall the study was limited by the high clinical heterogeneity of the data but in women with PUL diagnostic strategies using serum hCG ratios, either alone or in logistic regression models, have the best diagnostic performance in the case of EP. Well defined prospective comparative studies using standardized diagnostics and clinical application plus agreed definitions of outcome are required to identify the best strategy to diagnose pregnancy outcome in women with PUL.

The prognostic profile of subfertile couples and treatment outcome after expectant management, intrauterine insemination and in vitro fertilisation: a study protocol for the meta-analysis of individual patient data.

OBJECTIVE: The current evidence concerning the best treatment option for couples with unexplained and male subfertility is inconclusive. Most studies that have evaluated the effectiveness of treatment options, such as expectant management (EM), intrauterine insemination (IUI), with or without controlled ovarian stimulation (COS), and in vitro fertilisation (IVF), have not taken the couples' prognosis into account. It is very likely that the individual prognosis of the couple influences the effect of treatment. Individual patient data analyses allow us to take these prognostic factors into account, and to evaluate their effect on treatment outcome. This study aims to use anonymised data from relevant published trials to perform an individual patient data meta-analysis, evaluating the effect of couples' prognosis on the effectiveness of EM, IUI, with or without COS, and IVF. METHODS: Based on earlier systematic reviews and an updated search, randomised controlled trials will be considered for inclusion. Untreated subfertile couples with unexplained or male subfertility included in trials comparing EM, IUI, with or without COS, and IVF are included. Authors of the included studies will be invited to share their original anonymised data. The data will be assessed on validity, quality and completeness. The prognosis of the individual couple will be calculated with existing prognostic models. The effect of the prognosis on treatment outcome will be analysed with marker-by-treatment predictiveness curves, illustrating the effect of prognosis on treatment outcome. This study is registered in PROSPERO (registration number CRD42011001832). CONCLUSION: Ultimately, this study may help to select the appropriate fertility treatment, tailored to the needs of an individual couple.

Does a prediction model for pregnancy of unknown location developed in the UK validate on a US population?

BACKGROUND: A logistic regression model (M4) was developed in the UK to predict the outcome for women with a pregnancy of unknown location (PUL) based on the initial two human chorionic gonadotrophin (hCG) values, 48 h apart. The purpose of this paper was to assess the utility of this model to predict the outcome for a woman (PUL) in a US population. METHODS: Diagnostic variables included log-transformed serum hCG average of two measurements, and linear and quadratic hCG ratios. Outcomes modeled were failing PUL, intrauterine pregnancy (IUP) and ectopic pregnancy (EP). This model was applied to a US cohort of 604 women presenting with symptomatic first-trimester pregnancies, who were followed until a definitive diagnosis was made. The model was applied before and after correcting for differences in terminology and diagnostic criteria. RESULTS: When retrospectively applied to the adjusted US population, the M4 model demonstrated lower areas under the curve compared with the UK population, 0.898 versus 0.988 for failing PUL/spontaneous miscarriage, 0.915 versus 0.981 for IUP and 0.831 versus 0.904 for EP. Whereas the model had 80% sensitivity for EP using UK data, this decreased to 49% for the US data, with similar specificities. Performance only improved slightly (55% sensitivity) when the US population was adjusted to better match the UK diagnostic criteria. CONCLUSIONS: A logistic regression model based on two hCG values performed with modest decreases in predictive ability in a US cohort for women at risk for EP compared with the original UK population. However, the sensitivity for EP was too low for the model to be used in clinical practice in its present form. Our data illustrate the difficulties of applying algorithms from one center to another, where the definitions of pathology may differ.

Transglutaminase 1-deficient recessive lamellar ichthyosis associated with a LINE-1 insertion in Jack Russell terrier dogs.

BACKGROUND: Congenital, nonepidermolytic cornification disorders phenotypically resembling human autosomal recessive ichthyosis have been described in purebred dog breeds, including Jack Russell terrier (JRT) dogs. One cause of gene mutation important to humans and dogs is transposon insertions. OBJECTIVES: To describe an autosomal recessive, severe nonepidermolytic ichthyosis resembling lamellar ichthyosis (LI) in JRT dogs due to insertion of a long interspersed nucleotide element (LINE-1) in the transglutaminase 1 (TGM1) gene. METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Phenotypic information and genotyping with a canine microsatellite marker suggested TGM1 to be a candidate gene. Genomic DNA samples and cDNA generated from epidermal RNA were examined. Consequences of the mutation were evaluated by Western blotting, quantitative reverse transcription-polymerase chain reaction (RT-PCR) and enzyme activity from cultured keratinocytes. RESULTS: Affected dogs had generalized severe hyperkeratosis. Histological examination defined laminated to compact hyperkeratosis without epidermolysis; ultrastructurally, cornified envelopes were thin. Affected dogs were homozygous for a 1980-bp insertion within intron 9 of TGM1. The sequence of the insertion was that of a canine LINE-1 element. Quantitative RT-PCR indicated a significant decrease in TGM1 mRNA in affected dogs compared with wild-type. TGM1 protein was markedly decreased on immunoblotting, and membrane-associated enzyme activity was diminished in affected dogs. CONCLUSIONS: Based on morphological and molecular features, this disease is homologous with TGM1-deficient LI in humans, clinically models LI better than the genetically modified mouse and represents its first spontaneous animal model. This is the first reported form of LI due to transposon insertion.

Clinical indicators for success of misoprostol treatment after early pregnancy failure.

OBJECTIVE: To identify clinical indicators for success of misoprostol treatment after early pregnancy failure. METHODS: A total of 473 women with early pregnancy failure received 800 microg of vaginal misoprostol on treatment day 1. At the follow-up visit on day 3, a second dose was given if expulsion was incomplete. On day 8, vacuum aspiration was offered if expulsion had not occurred. Ultrasonography was used as gold standard for success. A Classification and Regression Tree analysis was undertaken to derive two decision trees for the success of misoprostol treatment on study days 3 and 8. RESULTS: Heavy bleeding after the first dose and an open cervical os were identified as clinical indicators of treatment success on day 3. Treatment success occurred in 84% of women with either or both indicators. Reporting passage of tissue after a second misoprostol dose and old blood in the vagina were potential indicators of treatment success or failure on day 8. A woman with either of these indicators has a 65% chance of treatment success after the second dose. Conversely, a woman with neither indicator on day 8 has a 94% chance of treatment failure. CONCLUSION: Standard clinical findings may be useful as indicators for success or failure of medical management of early pregnancy failure in settings with limited or no access to ultrasonography. More research to identify even better indicators is warranted.

An atypical case of Trypanosoma cruzi infection in a young English Mastiff.

A case of Trypanosoma cruzi infection in a young English Mastiff from Texas is presented. Clinical signs and laboratory findings included subcutaneous edema, lymphadenopathy, weight loss, and hypoalbuminemia. Cytology of a lymph node revealed numerous amastigotes. No trypomastigotes were observed in buffy coat preparation of peripheral blood, and on histologic evaluation, most organs contained numerous interstitial pseudocysts. Initial serology was positive for both T. cruzi and Leishmania, and immunohistochemistry supported a diagnosis of Leishmania. However, additional serology supported a T. cruzi infection, and cultivation of organisms isolated from a lymph node revealed morphology consistent with T. cruzi. In addition, PCR analysis resulted in a 504 bp fragment with 99% homology to a flagellar protein of T. cruzi. Although uncommon, autochthonous cases of both T. cruzi and Leishmania have been reported in the United States. Clinical signs observed with both diseases can show many similarities, cytology may be indistinguishable, as in this case, and serological cross-reactivity is common. This case demonstrates an unusual presentation of T. cruzi and the use of multiple testing strategies to support its diagnosis.

Comparative sequence analysis and radiation hybrid mapping of the canine keratin 10 gene.

The type I keratin, K10, is expressed in epidermal keratinocytes undergoing terminal differentiation to form the stratum corneum, a barrier essential for life. In order to facilitate the study of keratinization disorders in the dog, the sequence and mapping of KRT10 is reported. The coding region of KRT10 is 1707 bp and is comprised of eight exons. Although the length of KRT10 has been reported to be polymorphic in humans, this was not observed in the eight domestic dog breeds studied, although one wild canid displayed a size difference. The structure and sequence of this gene is highly conserved across mammalian species. Canine K10 had an 86% amino acid identity with the human gene. KRT10 was localized to the on-going canine radiation hybrid map to chromosome 9 in the type I keratin gene cluster.

Mild recessive epidermolytic hyperkeratosis associated with a novel keratin 10 donor splice-site mutation in a family of Norfolk terrier dogs.

BACKGROUND: Epidermolytic hyperkeratosis in humans is caused by dominant-negative mutations in suprabasal epidermal keratins 1 and 10. However, spontaneous keratin mutations have not been confirmed in a species other than human. OBJECTIVES: To describe an autosomal recessive, mild, nonpalmar/plantar epidermolytic ichthyosis segregating in an extended pedigree of Norfolk terrier dogs due to a splice-site mutation in the gene encoding keratin 10 (KRT10). METHODS: Dogs were evaluated clinically, and skin samples were examined by light and electron microscopy. Genomic DNA samples and cDNA from skin RNA were sequenced and defined a mutation in KRT10. Consequences of the mutation were evaluated by assessing protein expression with immunohistochemistry and Western blotting and gene expression with real-time RT-PCR (reverse transcriptase-polymerase chain reaction). RESULTS: Adult dogs with the disease had generalized, pigmented hyperkeratosis with epidermal fragility. Light microscopic examination defined epidermolysis with hyperkeratosis; ultrastructural changes included a decrease in tonofilaments and abnormal filament aggregation in upper spinous and granular layer keratinocytes. Affected dogs were homozygous for a single base GT-->TT change in the consensus donor splice site of intron 5 in KRT10. Keratin 10 protein was not detected with immunoblotting in affected dogs. Heterozygous dogs were normal based on clinical and histological appearance and keratin 10 protein expression. The mutation caused activation of at least three cryptic or alternative splice sites. Use of the cryptic sites resulted in transcripts containing premature termination codons. One transcript could result in shortening of the proximal portion of the 2B domain before the stutter region. Quantitative real-time PCR indicated a significant decrease in KRT10 mRNA levels in affected dogs compared with wild-type dogs. CONCLUSIONS: This disease is the first confirmed spontaneous keratin mutation in a nonhuman species and is the first reported recessive form of epidermolytic hyperkeratosis.

Comparative sequence analysis and radiation hybrid mapping of two epidermal type II keratin genes in the dog: keratin 1 and keratin 2e.

In order to extend knowledge of the process of cornification across species and to be better able to recognize inborn errors in keratin synthesis in the dog, we describe the organization and chromosome mapping of canine KRT1 and KRT2E and compare these results to human and murine sequence data. The coding regions of KRT1 and KRT2E are 1,860 bp and 1,902 bp respectively, distributed over nine exons. Both genes are localized on the canine radiation hybrid map to chromosome 27 in the type II keratin gene cluster close to polymorphic markers. These genes are highly conserved across species and based on both genomic and amino acid sequences, canine KRT1 and KRT2E share greater homology with humans than with mice.

A serum proteomics approach to the diagnosis of ectopic pregnancy.

An ectopic pregnancy (EP) occurs when implantation of the embryo occurs outside of the uterus. If left untreated, the developing fetus will continue to grow, leading to life-threatening consequences for the mother. A major difficulty with the diagnosis of ectopic pregnancy is that methods of detection are limited, and some, such as ultrasound, are not very reliable in the earliest days of gestation. Currently, no effective serum test exists to distinguish an ectopic pregnancy from a normal intrauterine pregnancy. The incidence of ectopic pregnancy is increasing and has doubled in the last 20 years. It is now the second most common cause of maternal death in the first trimester of pregnancy. To address this issue, we initiated a project to identify serum markers of ectopic pregnancy. The subjects for these studies presented at the Hospital of the University of Pennsylvania. We obtained over 140 serum samples from women with suspected ectopic pregnancy: women presenting with pain and/or bleeding in the first trimester of pregnancy. The approximate racial breakdown of the subjects is as follows: African American, 36%; Caucasian, 3%; Asian, 2%; Hispanic, 1%; unknown, 58%. Serum samples from 139 women (62 with ectopic pregnancy and 77 with a normal intrauterine pregnancy) were applied to WCX2 (weak ion exchange) protein chip surfaces and analyzed for serum markers using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS). Several proteins in the 7500-18,000 Da mass range were identified that may discriminate an ectopic pregnancy from an intrauterine pregnancy. The most promising markers were analyzed using classification and regression tree analysis (CART) with and without clinical variables (serum hCG value, length of amenorrhea). Two different algorithms were developed that classify the patients on the basis of sensitivity (number of EPs who screen positive/# of EPs) or specificity (# of healthy patients who screen negative/# of healthy). Our current approach is to refine these two "rule sets" to segregate patients into three groups: those who need immediate intervention for a probable ectopic pregnancy, those who appear to have a normal pregnancy, and those who need further monitoring for diagnosis.

A heritable keratinization defect of the superficial epidermis in norfolk terriers.

Although well-characterized in man, abnormal cornification secondary to heritable superficial keratin defects is rarely reported in animals. This report describes a mild cornification defect in seven related Norfolk terrier dogs. Lesions were present at birth and pedigree analysis suggested an autosomal recessive mode of inheritance. The affected dogs had hyperpigmented skin with scaling following mild trauma. The lesions were generalized but most prominent in the glabrous skin of the axillary and inguinal regions-areas where the epidermis is not protected by hair and is subject to frequent trauma. The most striking histological change was vacuolation in the upper epidermis, which often resulted in epidermolysis and blister formation. All of the affected dogs showed similar gross and histological changes. Ultrastructural changes included abnormal keratin filament clumping, prominent clear spaces in the cytoplasm of suprabasal keratinocytes, and abnormal keratohyaline granules. Immunohistochemical labelling for keratin 10 demonstrated a lack of expression in the superficial epidermis of affected dogs. All of the morphological changes noted in the Norfolk terriers were consistent with a mild form of a heritable defect in superficial keratin synthesis.

Comparison of two culture systems for the in-vitro growth and maturation of mouse preantral follicles.

The objective of our study was to compare to ability of collagen-treated membranes and bovine collagen gels to maintain murine preantral follicle growth and development in-vitro. To fulfill that objective, murine follicle and oocyte growth rates were followed for ten days in culture. Meiotic competence and the capacity to reach the two-cell stage after in-vitro maturation and fertilization, respectively, were then assessed. We used preantral follicles from 12 day-old CF-1 female mice that were isolated by enzymatic digestion from ovaries. Follicles were placed either on collagen-treated membranes or embedded in a bovine collagen matrix. The follicles were grown, changing the media and obtaining measurements every other day for ten days. Following culture, the granulosa-oocyte complexes were matured; the resultant metaphase II arrested oocytes were inseminated and cultured to the two-cell stage. The data was analyzed with significance considered for probability values of p < 0.05. We performed individual measurements on 650 follicles in seven separate experiments. Forty-eight hours after initial seeding and throughout the entire length of culture, both the follicles and oocytes grown in the collagen matrix were larger than follicles cultured on collagen-treated membranes (p < .0001). However, oocyte recovery rates were higher among follicles cultured on collagen-treated membranes (p < .01). Similar percentages of meiotically competent oocytes, fertilization and cleavage rates were observed in both groups. Our results show that mouse preantral follicles display a greater growth rate when grown embedded in a collagen gel matrix. This may be due to the maintenance of a normal three-dimensional organization of the follicle within the collagen matrix. However, this system does not enhance meiotic competency or fertilization rates in the mouse when compared to culture on collagen-treated membranes.

Immunohistochemical staining patterns of canine meningiomas and correlation with published immunophenotypes.

This study examined immunohistochemical staining patterns for several meningioma variants involving either the brain or spinal cord of dogs. Formalin-fixed, paraffin-embedded tissue from 15 tumors was obtained. The selected tumor group included seven meningothelial, three transitional, two malignant (anaplastic), one myxoid, one papillary, and one osteomatous meningiomas. Tumors were evaluated for reactivity to the following six immunohistochemical markers: vimentin, pancytokeratin, glial fibrillary acidic protein (GFAP), S100, neuron-specific enolase (NSE), and synaptophysin. Vimentin expression was detected in all meningiomas, and 14 of 15 tumors demonstrated intense vimentin staining in more than 50% of the neoplastic cells. Pancytokeratin expression was present in 11 of 15 neoplasms; however, positive staining frequently was focal and often involved a small percentage of the neoplastic cells. GFAP expression was detected in a single, anaplastic meningioma. Although expression of NSE and S100 was detected in 12 of 25 meningiomas, the intensity of the staining and the percentage of positive neoplastic cells was highly variable. Synaptophysin was uniformly negative. These results will help to establish immunohistochemical profiles for meningiomas that will improve our ability to correctly differentiate these neoplasms of meningeal origin from central nervous system tumors originating from other sites.