Clarification and confocal imaging of the nonhuman primate placental micro-anatomy.

Geometry of the placental villous vasculature is a key determinant of maternal-fetal nutrient exchange for optimal fetal growth. Recent advances in tissue clarification techniques allow for deep high-resolution imaging with confocal microscopy; however, the methodology lacks a signal:noise ratio of sufficient magnitude to allow for quantitative analysis. Thus, we sought to develop a reproducible method to investigate the 3D vasculature of the nonhuman primate placenta for subsequent data analysis. Fresh placental tissue was dissected, formalin fixed, clarified using a modified Visikol® protocol and immunolabeled for CD31 (fetal endothelium) and cytokeratin-7 (villous trophoblast) for confocal imaging of the microanatomy. We present a detailed clarification and staining protocol augmented for imaging of nonhuman primate placental tissue. The image stacks generated by this refined staining method and our data acquisition parameters can be analyzed quantitatively to provide insights regarding the villous and vascular micro-anatomy of the placenta.

Early first trimester uteroplacental flow and the progressive disintegration of spiral artery plugs: new insights from contrast-enhanced ultrasound and tissue histopathology.

STUDY QUESTION: Does the use of a vascular contrast agent facilitate earlier detection of maternal flow to the placental intervillous space (IVS) in the first trimester of pregnancy? SUMMARY ANSWER: Microvascular filling of the IVS was demonstrated by contrast-enhanced ultrasound from 6 weeks of gestation onwards, earlier than previously believed. WHAT IS KNOWN ALREADY: During placental establishment and remodeling of maternal spiral arteries, endovascular trophoblast cells invade and accumulate in the lumen of these vessels to form 'trophoblast plugs'. Prior evidence from morphological and Doppler ultrasound studies has been conflicting as to whether the spiral arteries are completely plugged, preventing maternal blood flow to the IVS until late in the first trimester. STUDY DESIGN, SIZE, DURATION: Uteroplacental flow was examined across the first trimester in human subjects given an intravenous infusion of lipid-shelled octofluoropropane microbubbles with ultrasound measurement of destruction and replenishment kinetics. We also performed a comprehensive histopathological correlation using two separately archived uteroplacental tissue collections to evaluate the degree of spiral artery plugging and evaluate remodeling of the upstream myometrial radial and arcurate arteries. PARTICIPANTS/MATERIALS, SETTING, METHODS: Pregnant women (n = 34) were recruited in the first trimester (range: 6+3 to 13+6 weeks gestation) for contrast-enhanced ultrasound studies with destruction-replenishment analysis of signal intensity for assessment of microvascular flux rate. Histological samples from archived in situ (Boyd Collection, n = 11) and fresh first, second, and third trimester decidual and post-hysterectomy uterine specimens (n = 16) were evaluated by immunohistochemistry (using markers of epithelial, endothelial and T-cells, as well as cell adhesion and proliferation) and ultrastructural analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Contrast agent entry into the IVS was visualized as early as 6+3 weeks of gestation with some variability in microvascular flux rate noted in the 6-7+6 week samples. Spiral artery plug canalization was observed from 7 weeks with progressive disintegration thereafter. Of note, microvascular flux rate did not progressively increase until 13 weeks, which suggests that resistance to maternal flow in the early placenta may be mediated more proximally by myometrial radial arteries that begin remodeling at the end of the first trimester. LIMITATIONS REASONS FOR CAUTION: Gestational age was determined by crown-rump length measurements obtained by transvaginal ultrasound on the day of contrast-enhanced imaging studies, which may explain the variability in the earliest gestational age samples due to the margin of error in this type of measurement. WIDER IMPLICATIONS OF THE FINDINGS: Our comprehensive in situ histological analysis, in combination with the use of an in vivo imaging modality that has the sensitivity to permit visualization of microvascular filling, has allowed us to reveal new evidence in support of increasing blood flow to the IVS from 6 weeks of gestation. Histologic review suggested the mechanism may be blood flow through capillary-sized channels that form through the loosely cohesive 'plugs' by 7 weeks gestation. However, spiral artery remodeling on its own did not appear to explain why there is significantly more blood flow at 13 weeks gestation. Histologic studies suggest it may be related to radial artery remodeling, which begins at the end of the first trimester. STUDY FUNDING/COMPETING INTEREST(S): This project was supported by the Oregon Health and Science University Knight Cardiovascular Institute, Center for Developmental Health and the Struble Foundation. There are no competing interests.

Functional imaging of the nonhuman primate Placenta with endogenous blood oxygen level-dependent contrast.

PURPOSE: To characterize spatial patterns of T2* in the placenta of the rhesus macaque (Macaca mulatta), to correlate these patterns with placental perfusion determined using dynamic contrast-enhanced MRI (DCE-MRI), and to evaluate the potential for using the blood oxygen level-dependent effect to quantify placental perfusion without the use of exogenous contrast reagent. METHODS: MRI was performed on three pregnant rhesus macaques at gestational day 110. Multiecho spoiled gradient echo measurements were used to compute maps of T2*. Spatial maxima in these maps were compared with foci of early enhancement determined by DCE-MRI. RESULTS: Local maxima in T2* maps were strongly correlated with spiral arteries identified by DCE-MRI, with mean spatial separations ranging from 2.34 to 6.11 mm in the three animals studied. Spatial patterns of R2* ( = 1/ T2*) within individual placental lobules can be quantitatively analyzed using a simple model to estimate fetal arterial oxyhemoglobin concentration [Hbo,f] and a parameter viPS/Φ, reflecting oxygen transport to the fetus. Estimated mean values of [Hbo,f] ranged from 4.25 mM to 4.46 mM, whereas viPS/Φ ranged from 2.80 × 105 cm-3 to 1.61 × 106 cm-3 . CONCLUSIONS: Maternal spiral arteries show strong spatial correlation with foci of extended T2* observed in the primate placenta. A simple model of oxygen transport accurately describes the spatial dependence of R2* within placental lobules and enables assessment of placental function and oxygenation without requiring administration of an exogenous contrast reagent. Magn Reson Med 76:1551-1562, 2016. © 2015 International Society for Magnetic Resonance in Medicine.

Influence of high fat diet and resveratrol supplementation on placental fatty acid uptake in the Japanese macaque.

INTRODUCTION: Adequate maternal supply and placental delivery of long chain polyunsaturated fatty acids (LCPUFA) is essential for normal fetal development. In humans, maternal obesity alters placental FA uptake, though the impact of diet remains uncertain. The fatty fetal liver observed in offspring of Japanese macaques fed a high fat diet (HFD) was prevented with resveratrol supplementation during pregnancy. We sought to determine the effect of HFD and resveratrol, a supplement with insulin-sensitizing properties, on placental LCPUFA uptake in this model. METHODS: J. macaques were fed control chow (15% fat, n = 5), HFD (35% fat, n = 10) or HFD containing 0.37% resveratrol (n = 5) prior to- and throughout pregnancy. At ∼ 130 d gestation (term = 173 d), placentas were collected by caesarean section. Fatty acid uptake studies using (14)C-labeled oleic acid, arachidonic acid (AA) and docosahexanoic acid (DHA) were performed in placental explants. RESULTS: Resveratrol supplementation increased placental uptake of DHA (P < 0.05), while HFD alone had no measurable effect. Resveratrol increased AMP-activated protein kinase activity and mRNA expression of the fatty acid transporters FATP-4, CD36 and FABPpm (P < 0.05). Placental DHA content was decreased in HFD dams; resveratrol had no effect on tissue fatty acid profiles. DISCUSSION: Maternal HFD did not significantly affect placental LCPUFA uptake. Furthermore, resveratrol stimulated placental DHA uptake capacity, AMPK activation and transporter expression. Placental handling of DHA is particularly sensitive to the dramatic alterations in the maternal metabolic phenotype and placental AMPK activity associated with resveratrol supplementation.

Preoperative serum level of inhibin A is an independent prognostic factor for the survival of postmenopausal women with epithelial ovarian carcinoma.

BACKGROUND: The aim of this study was to determine the prognostic significance of preoperative serum inhibin and activin levels in postmenopausal women with epithelial ovarian carcinoma (EOC) by correlating serum levels with disease parameters, including tumor stage and grade and patient age. METHODS: Serum levels of inhibin A, inhibin B, pro-alpha C, activin A, and activin B were quantitated with sensitive and specific two-site enzyme-linked immunosorbent assays (ELISAs) in samples collected from 44 postmenopausal women diagnosed with EOC. Serum was obtained within 14 days prior to primary tumor reductive surgery and stored at -55 degrees C. All patients underwent definitive surgical staging and cytoreduction at Mayo Clinic and were followed for at least 5 years or until death. Postoperative adjuvant therapy was selected based on stage of disease. Demographics included 5 Stage I, 2 Stage II, 33 Stage III, and 4 Stage IV tumors, and the predominant histology was serous subtype and poorly differentiated grade. RESULTS: Inhibin A was detected in 98% of the serum samples (range, 0-12.18 pg/mL). Univariate analysis was used to demonstrate an association between patients with serum inhibin A levels exceeding the median (1.21 pg/mL) and compromised disease free (P = 0.025) and overall (P = 0.006) survival. While the 5 year disease free survival (DFS) for the entire population was 32%, the corresponding DFS rates for patients with inhibin A levels above and below the median were 10% and 43%, respectively. Similarly, the 5-year overall survival (OS) for the entire population was 35%, compared with 16% for patients above and 47% for patients below the median inhibin A level. Stepwise regression analysis that incorporated age, stage, grade, and inhibin A levels identified serum inhibin A levels above the median to be the most cogent predictor of DFS and OS. CONCLUSIONS: Preoperative serum inhibin A levels provided valuable prognostic information independent of age, stage, and grade in a postmenopausal cohort given standardized treatment for EOC.

Palliative radiotherapy for brain metastases in renal carcinoma.

Between 1968 and 1985, 46 patients with renal cell carcinoma metastatic to the brain parenchyma were treated with radiation. Thirty-nine received whole-brain radiation, mostly 30 Gy in ten fractions. Symptoms improved in 30% of evaluable patients. Partial regression of metastases was documented in two of 11 available sequential computed tomographs (CT) of the brain. Seven patients were treated with surgery and postoperative radiation. In five the excision was complete and associated with clinical improvement. All 46 patients have subsequently died. The median survival time of the entire group was 8 weeks. The ten patients who improved after radiotherapy survived for a median of 17 weeks. Two additional patients were treated in 1986 with fast neutrons; both had a documented maintained complete response. Brain metastasis in renal carcinoma carries a poor prognosis. It is usually unresponsive to conventional photon therapy. In selected cases an alternative treatment with surgery or neutron therapy should be considered.

Multidrug chemotherapy in pulmonary treatment of osteosarcoma.

Forty-three patients with osteosarcoma were treated with amputation and adjuvant chemotherapy utilizing a four-drug combination of cyclophosphamide, vincristine, phenylalanine mustard, and adriamycin (CONPADRI-I regimen). Twenty-four patients (56 per cent) remained free of metastases twelve to sixty-one months after diagnosis. Ten of the twenty-four have been disease-free for more than three years. Another group of thirty patients was treated with amputation and a five-drug adjuvant chemotherapy program which included the administration of massive doses of methotrexate with citrovorum factor (COMPADRI-II regimen). Twenty of the thirty (67 per cent) remained free of metastases from twelve to twenty-six months after amputation (median, sixteen months). Two deaths related to methotrexate toxicity occurred. Late metastases developed in three patients (at sixteen, nineteen, and twenty-six months after operation) in the group treated with the COMPADRI-II regimen.