TRP channel expression correlates with the epithelial-mesenchymal transition and high-risk endometrial carcinoma.

Transient receptor potential (TRP) channels excel in cellular sensing as they allow rapid ion influx across the plasma membrane in response to a variety of extracellular cues. Recently, a distinct TRP mRNA expression signature was observed in stromal cells (ESC) and epithelial cells (EEC) of the endometrium, a tissue in which cell phenotypic plasticity is essential for normal functioning. However, it is unknown whether TRP channel mRNA expression is subject to the phenotypic switching that occurs during epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET), and whether TRP channel mRNA expression is associated with aggressive phenotypes in endometrial cancer (EC). Here, we induced EMT and MET in vitro using in primary EEC and ESC, respectively, and analyzed expression and functionality of TRP channels using RT-qPCR and intracellular Ca2+ imaging. The outcome of these experiments showed a strong association between TRPV2 and TRPC1 mRNA expression and the mesenchymal phenotype, whereas TRPM4 mRNA expression correlated with the epithelial phenotype. In line herewith, increased TRPV2 and TRPC1 mRNA expression levels were observed in both primary and metastatic EC biopsies and in primary EC cells with a high EMT status, indicating an association with an aggressive tumor phenotype. Remarkably, TRPV2 mRNA expression in primary EC biopsies was associated with tumor invasiveness and cancer stage. In contrast, increased TRPM4 mRNA expression was observed in EC biopsies with a low EMT status and less aggressive tumor phenotypes. Taken together, this dataset proved for the first time that TRP channel mRNA expression is strongly linked to cellular phenotypes of the endometrium, and that phenotypic transitions caused by either experimental manipulation or malignancy could alter this expression in a predictable manner. These results implicate that TRP channels are viable biomarkers to identify high-risk EC, and potential targets for EC treatment.

Mapping the expression of transient receptor potential channels across murine placental development.

Transient receptor potential (TRP) channels play prominent roles in ion homeostasis by their ability to control cation influx. Mouse placentation is governed by the processes of trophoblast proliferation, invasion, differentiation, and fusion, all of which require calcium signaling. Although certain TRP channels have been shown to contribute to maternal-fetal transport of magnesium and calcium, a role for TRP channels in specific trophoblast functions has been disregarded. Using qRT-PCR and in situ hybridisation, the spatio-temporal expression pattern of TRP channels in the mouse placenta across gestation (E10.5-E18.5) was assessed. Prominent expression was observed for Trpv2, Trpm6, and Trpm7. Calcium microfluorimetry in primary trophoblast cells isolated at E14.5 of gestation further revealed the functional activity of TRPV2 and TRPM7. Finally, comparing TRP channels expression in mouse trophoblast stem cells (mTSCs) and mouse embryonic stem cells (mESC) confirmed the specific expression of TRPV2 during placental development. Moreover, TRP channel expression was similar in mTSCs compared to primary trophoblasts and validate mTSC as a model to study TRP channels in placental development. Collectivity, our results identify a specific spatio-temporal TRP channel expression pattern in trophoblasts, suggesting a possible involvement in regulating the process of placentation.

The functional expression of transient receptor potential channels in the mouse endometrium.

STUDY QUESTION: Does mouse endometrial epithelial cells and stromal cells have a similar transient receptor potential (TRP)-channel expression profile and to that found in the human endometrium? SUMMARY ANSWER: Mouse endometrial epithelial and stromal cells have a distinct TRP channel expression profile analogous to what has been found in human endometrium, and hence suggests the mouse a good model to investigate the role of TRP channels in reproduction. WHAT IS KNOWN ALREADY: An optimal intercellular communication between epithelial and stromal endometrial cells is crucial for successful reproduction. Members of the TRP family were recently described in the human endometrial stroma; however their functional expression in murine endometrium remains unspecified. Furthermore, epithelial and stromal cells have distinct functions in the reproductive process, implying the possibility for a different expression profile. However, knowledge about the functional expression pattern of TRP channels in either epithelial or stromal cells is not available. STUDY DESIGN, SIZE, DURATION: In this study, the expression pattern of TRP channels in the murine (C57BL/6 J strain) endometrium was investigated and compared to the human expression pattern. Therefore, expression was examined in uterine tissue isolated during the natural estrous cycle (n = 16) or during an induced menstrual cycle using the menstruating mouse model (n = 28). Next, the functional expression of TRP channels was assessed separately in endometrial epithelial and stromal cell populations. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative RT-PCR was used to evaluate the relative mRNA expression of TRP channels in murine uterine tissue and cells. To further assess the functional expression in epithelial or stromal cells, primary endometrial cell cultures and Fura2-based calcium-microfluorimetry experiments were performed. MAIN RESULTS AND THE ROLE OF CHANCE: The expression pattern of TRP channels during the natural estrous cycle or the induced menstrual cycle is analog to what has been shown in human samples. Furthermore, a very distinct expression pattern was observed in epithelial cells compared to stromal cells. Expression of TRPV4, TRPV6 and TRPM6 was significantly higher in epithelial cells whereas TRPV2, TRPC1/4 and TRPC6 were almost exclusively expressed in stromal cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Although relevant mRNA levels are detected for TRPV6 and TRPM6, and TRPM4, lack of selective, available pharmacology restricted functional analysis of these ion channels. WIDER IMPLICATIONS OF THE FINDINGS: Successful reproduction, and more specifically embryo implantation, is a dynamic developmental process that integrates many signaling molecules into a precisely orchestrated program. Here, we describe the expression pattern of TRP channels in mouse endometrium that is similar to human tissue and their restricted functionality in either stromal cells or epithelial cells, suggesting a role in the epithelial-stromal crosstalk. These results will be very helpful to identify key players involved in the signaling cascades required for successful embryo implantation. In addition, these results illustrate that mouse endometrium is a valid representative for human endometrium to investigate TRP channels in the field of reproduction. STUDY FUNDING/COMPETING INTEREST(S): The Research Foundation-Flanders (G.0856.13 N to J.V.); the Research Council of the Katholieke Universiteit Leuven (OT/13/113 to J.V. and PF-TRPLe to T.V.); the Planckaert-De Waele fund (to J.V.);  Fonds Wetenschappelijk Onderzoek Belgium (to K.D.C. and A.H.). None of the authors have a conflict of interest.

In Vitro Assessment of the Expression and T Cell Immunogenicity of the Tumor-Associated Antigens BORIS, MUC1, hTERT, MAGE-A3 and Sp17 in Uterine Cancer.

BACKGROUND: While immunotherapy moved to the forefront of treatment of various cancers, it remains underexplored for uterine cancer. This might be due to the small patient population with advanced endometrial carcinoma and uterine sarcoma. Data about immunotherapeutic targets are scarce in endometrial carcinoma and lacking in uterine sarcoma. METHODS: Expression of five tumor-associated antigens (TAA) (BORIS, MUC1, hTERT, MAGE-A3 and Sp17) was validated in uterine tumor samples by immunohistochemistry (IHC) and/or quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). TAA immunogenicity was analyzed by determining spontaneous T cell responses towards overlapping peptide pools covering the whole TAA in patient blood. RESULTS: At mRNA level, MAGE-A3 and Sp17 were overexpressed in a minority of patients and BORIS was moderately overexpressed (26% in endometrial carcinoma and 62% in uterine sarcoma). hTERT was overexpressed in the vast majority of tumors. On protein level, MUC1 was upregulated in primary, recurrent and metastatic EMCAR and in metastatic US tumors. hTERT protein was highly expressed in both normal and malignant tissue. Spontaneous TAA-specific T cell responses were detected in a minority of patients, except for hTERT to which T cell responses occurred more frequently. CONCLUSIONS: These data point to MUC1 and hTERT as most suitable targets based on expression levels and T cell immunogenicity for use in immunotherapeutic regimens.

In Vitro Validation of Survivin as Target Tumor-associated Antigen for Immunotherapy in Uterine Cancer.

Survivin is an antiapoptotic protein, not expressed in terminally differentiated adult tissues, yet overexpressed in several tumors. Therefore, it is an interesting target for immunotherapeutic strategies. In addition to specific overexpression in tumors, tumor survival is mediated by survivin and hence, tumor survival can be tackled by targeting survivin. Survivin expression in uterine cancer was validated by quantitative real-time polymerase chain reaction and immunohistochemistry. In addition, we evaluated survivin immunogenicity by analyzing spontaneous B-cell and T-cell responses in patients. Survivin as a protein was expressed in only a minority of normal tissues, whereas it was being expressed in all of the currently analyzed uterine cancers, both endometrial carcinoma (n = 52) and uterine sarcoma (n = 52). Survivin RNA transcripts were overexpressed in more aggressive tumors and survivin protein was overexpressed in recurrent endometrial tumors compared with primary tumors. Spontaneous T-cell responses were seen in 10/39 endometrial cancer patients and 3/16 uterine sarcoma patients. In normal controls, T-cell responses were found only in 1 donor (n = 21). Although increased antibody titers were found in more aggressive and far-advanced tumors, no differences in B-cell responses were seen. Overall, when compared with normal controls, a B-cell response was only measured in 1/41 uterine sarcoma patients. In conclusion, we currently validated the presence of survivin in uterine cancer. In addition, spontaneous T-cell responses were found in 23.6% of the total patient population. These data indicate that a survivin-specific immune response may be induced spontaneously in patients, further fortifying the eligibility of survivin as an immunotherapeutic target.

Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle.

STUDY QUESTION: Are members of the transient receptor potential (TRP) channel superfamily functionally expressed in the human endometrial stroma? SUMMARY ANSWER: The Ca(2+)-permeable ion channels TRPV2, TRPV4, TRPC6 and TRPM7 are functionally expressed in primary endometrial stromal cells. WHAT IS KNOWN ALREADY: Intercellular communication between epithelial and stromal endometrial cells is required to initiate decidualization, a prerequisite for successful implantation. TRP channels are possible candidates as signal transducers involved in cell-cell communication, but no fingerprint is available of the functional distribution of TRP channels in the human endometrium during the luteal phase of the menstrual cycle. STUDY DESIGN, SIZE, DURATION: Endometrial biopsy samples (previously frozen) from patients of reproductive age with regular menstrual cycles, who were undergoing diagnostic laparoscopic surgery for pain and/or infertility, were analysed. Samples were obtained from the menstrual (Days 1-5, n = 3), follicular (Days 6-14, n = 6), early luteal (Days 15-20, n = 5) and late luteal (Days 21-28, n = 5) phases. In addition, a total of 13 patient samples taken during the luteal phase were used to set up primary cell cultures for further experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Quantitative real-time PCR (qRT-PCR), immunocytochemistry, Fura2-based Ca(2+)-microfluorimetry and whole-cell patch clamp experiments were performed to study the functional expression pattern of TRP channels. Specific pharmacological agents, such as Δ(9)-tetrahydrocannabinol, GSK1016790A and 1-oleoyl-2-acetyl-glycerol, were used to functionally assess the expression of TRPV2, TRPV4 and TRPC6, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Expression of TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4 and TRPM7 was detected at the mRNA level in endometrial biopsies (n = 19) and in primary endometrial stromal cell cultures obtained from patients during the luteal phase (n = 5) of the menstrual cycle. Messenger RNA levels of TRPV2, TRPC4 and TRPC6 were significantly increased (P < 0.01) in the late luteal phase compared with the early luteal phase. Immunocytochemistry experiments showed a positive staining for TRPV2, TRPV4, TRPC6 and TRPM7 in the plasma membrane and in the cytoplasm of primary endometrial stromal cells. Ca(2+)-microfluorimetry revealed significant increases (P < 0.001) in intracellular Ca(2+) levels when stromal cells were incubated with specific activators of TRPV2, TRPV4 and TRPC6. Further functional characterization was performed using whole-cell patch clamp experiments. Taken together, these data provide evidence for the functional activity of TRPV2, TRPV4, TRPC6 and TRPM7 channels in primary stromal cell cultures. LIMITATIONS, REASONS FOR CAUTION: Although mRNA levels are detected for TRPV6, TRPC1, TRPC4 and TRPM4, the limited supply of specific antibodies and lack of selective pharmacological agents restricted any additional analysis of these ion channels. WIDER IMPLICATIONS OF THE FINDINGS: Embryo implantation is a dynamic developmental process that integrates many signalling molecules into a precisely orchestrated programme. Our findings identified certain members of the TRP superfamily as candidate sensors in the epithelial-stromal crosstalk. These results are very helpful to unravel the signalling cascade required for successful embryo implantation. In addition, this knowledge could lead to new strategies to correct implantation failure and facilitate the development of novel non-hormonal contraceptives. STUDY FUNDING/ COMPETING INTERESTS: This work was supported by grants from the Research Foundation-Flanders (G.0856.13N to J.V.), the Research Council of the KU Leuven (OT/13/113 to J.V. and T.D. and PF-TRPLe to T.V.) and by the Planckaert-De Waele fund (to J.V.). K.D.C. and K.H. are funded by the FWO Belgium. None of the authors have a conflict of interest.

Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release.

Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation.

Lack of Evidence That Male Fetal Microchimerism is Present in Endometriosis.

INTRODUCTION: Fetal microchimerism has been implicated in the etiology of autoimmune diseases. This study was done to test the hypothesis that male fetal microchimerism is present in eutopic and ectopic endometrium (EM) obtained from women with endometriosis but not in eutopic EM from women without endometriosis. METHODS: A total of 31 patients were selected, including women with endometriosis (paired eutopic and ectopic EM; n = 19) and women without endometriosis (eutopic EM; n = 12). Tricolor interphase fluorescence in situ hybridization analysis was performed by cohybridization of CEP Y SpectrumAqua and CEP X SpectrumGreen (SG)/CEP Y SpectrumOrange probes. RESULTS: Ectopic EM from women with endometriosis had 75% XX chromosomes (double SG signals) and 25% X chromosomes (single SG signal). Y chromosomes were not observed in any of the eutopic/ectopic endometrial tissues from cases or controls. CONCLUSIONS: We were unable to confirm our hypothesis that male fetal microchimerism is present in eutopic and/or ectopic EM obtained from women with endometriosis.

Investigating the influence of maternal cortisol and emotional state during pregnancy on the DNA methylation status of the glucocorticoid receptor gene (NR3C1) promoter region in cord blood.

BACKGROUND: The methylation status of the human glucocorticoid receptor gene NR3C1 in newborns has been reported to be sensitive to prenatal maternal mood. This study investigates both the association between maternal cortisol and emotional state during pregnancy and the methylation state of the promoter region of NR3C1 gene. METHODS: We examined 83 pregnant women. Psychological data and diurnal cortisol data were assessed to evaluate maternal stress once each trimester. DNA methylation at different loci of the NR3C1 gene, including exon 1B, 1D and 1F, was analyzed in genomic DNA from cord blood mononuclear cells. RESULTS: Univariable analyses indicated pregnancy related anxiety to be the strongest psychological parameter throughout pregnancy. Most significant findings concerned 1F. Particularly the methylation state of CpG9 was significantly associated with maternal emotional wellbeing. In a multivariable model the proportion of variance in methylation state of F9 explained (PVE) by pregnancy related anxiety was 7.8% (p = 0.023) during T1. Furthermore different CpG-units located at the nerve growth factor inducible protein A (NGFI-A) binding sites of 1F were associated with maternal anxiety [(F20.21: PC PRAQ and fear of integrity in T1: respectively PVE:8.9% and PVE:9.0%; Fear of delivery T2: PVE:8.0%, Fear of integrity T2: PVE:6.0% and STAI T2: PVE:5.9%) - (F12.13: PC PRAQ T1: PVE:6.9%, fear of integrity T2: PVE:6.0% and fear of delivery T2: PVE:8.0%)] and cortisol (F38.39: PVE:8.9%) in T2. CONCLUSION: These data indicate that prenatal maternal emotional state, particularly pregnancy related anxiety, are associated with the methylation state of the NR3C1 gene in the child.

The influence of maternal cortisol and emotional state during pregnancy on fetal intrauterine growth.

BACKGROUND: This exploratory study investigates the influence of maternal cortisol and emotional state during pregnancy on fetal intrauterine growth (IUG). We expected higher basal cortisol levels, or more depressive and anxious complaints during pregnancy, to be associated with slower IUG and lower birth weight. METHODS: A total of 91 pregnant women were recruited from the antenatal clinic and were seen once each trimester. In addition to psychological assessments, a diurnal cortisol profile was derived from saliva samples. IUG was evaluated using ultrasound. RESULTS: In mid-pregnancy (trimester (T)2), basal cortisol levels significantly predicted the variance of weight (proportion of variance in growth variable explained (PVE) = 11.6%) and body mass index (BMI) at birth (PVE = 6.8%). In late pregnancy (T3) emotional state, particularly depressive symptoms (BMI at birth: PVE = 6.9%; ponderal index (PI) at birth: PVE = 8.2%; head circumference at T3: PVE = 10.3%; head circumference at birth PVE = 9.1%) and attachment (BMI at birth: PVE = 6.9%; PI at birth: PVE = 7.2%) had an influence on growth. Analysis of growth between T2 and T3 showed that attachment and cortisol in T3 had an influence on the variation in increase in estimated fetal weight (PVE = 12.5-8.6%). CONCLUSION: These data indicate basal cortisol levels were more important in T2 whereas emotional state was more important in T3.

Substantial variation in transplacental transfer of chemotherapeutic agents in a mouse model.

OBJECTIVE: Data on the transplacental transfer of chemotherapeutic agents are lacking. We aimed to measure the maternofetal transfer of cytotoxic drugs in a mouse model. STUDY DESIGN: The transplacental transfer of doxorubicin (9 mg/kg), epirubicin (11 mg/kg), vinblastine (6 mg/kg), carboplatin (50 mg/kg), paclitaxel (10 mg/kg), and cytarabine (100 mg/kg) was tested in a C57/Bl6J mouse model. Ninety minutes after intravenous (IV) drug injection on gestational day 18.5, maternal and fetal blood were collected simultaneously. Plasma drug levels were determined using high performance liquid chromatography or atomic absorption spectrometry. RESULTS: Fetal plasma concentrations of doxorubicin, epirubicin, vinblastine, and cytarabine were 5.1% ± 0.6% (n = 8), 4.8% ± 3.8% (n = 8), 13.8% ± 5.8% (n = 6), and 56.7% ± 22.6% (n = 6) of the maternal concentrations, respectively. Total platinum passed the mouse placenta easily (117.0% ± 38.9%, n = 6). Paclitaxel could not be detected in fetal plasma samples (n = 6). CONCLUSIONS: Substantial variations in transplacental transfer were noted among the tested drugs. Current findings contribute to the understanding of reported pregnancy outcomes in humans.

Pharmacokinetics of chemotherapeutic agents in pregnancy: a preclinical and clinical study.

OBJECTIVE: To determine the impact of physiologic changes of pregnancy on pharmacokinetics of chemotherapeutic agents. DESIGN: A preclinical and a clinical case-control trial. SETTING: Institute of Primate Research Nairobi and collaborating hospitals in Belgium, the Netherlands and Czech Republic. POPULATION: Pregnant and nonpregnant women and baboons receiving chemotherapy. METHODS: Chemotherapy pharmacokinetics was compared between the pregnant and nonpregnant state. Standard-dosed chemotherapy regimens were administered in pregnant and nonpregnant baboons/women, followed by serial blood samplings. Drug plasma levels were determined using high performance liquid chromatography and atomic absorption spectrometry. MAIN OUTCOME MEASURES: Area under the curve (AUC), maximal plasma concentration, terminal elimination half-life, clearance and distribution volume of each drug in pregnant and nonpregnant state. RESULTS: Intraindividual comparative pharmacokinetic data were obtained for doxorubicin and paclitaxel/platinum in three and two baboons, respectively. In the clinical trial, two patients were exposed to doxorubicin and one patient was exposed to paclitaxel/platinum during and after pregnancy. Furthermore, a pooled analysis was performed based on 16 cycles of pregnant and 11 cycles of nonpregnant women. Numbers of pregnant/nonpregnant patients were 5/2, 7/5, 4/4 and 2/2 for paclitaxel, doxorubicin, epirubicin and platinum, respectively. For all drugs tested in the preclinical and clinical study, a decreased AUC and maximal plasma concentration and an increased distribution volume and clearance were observed in pregnancy. CONCLUSIONS: Although numbers were too small for statistical significance, pregnancy-associated physiologic alterations appear to lead to a decrease in plasma exposure of chemotherapeutic drugs. The importance of long-term follow-up of women treated with chemotherapy during pregnancy is underscored.

Transplacental transfer of paclitaxel, docetaxel, carboplatin, and trastuzumab in a baboon model.

BACKGROUND: The paucity of data on fetal effects of prenatal exposure to chemotherapy prompted us to study the transplacental transport of commonly used anticancer agents in a pregnant baboon model. METHODS: Single or combination chemotherapy with paclitaxel, docetaxel, carboplatin, and trastuzumab was administered to 9 baboons at a mean (SD) gestational age of 117 (26) days (paclitaxel, 100 mg/m2 [n = 2]; docetaxel, 100 mg/m2 [n = 2]; paclitaxel, 175 mg/m2 with carboplatin, area under the curve of 6 at standard dosage [n = 2] and 50% dosage [n = 1]; docetaxel, 75 mg/m2 with carboplatin, area under the curve 6 [n = 1]; and docetaxel, 75 mg/m2 with trastuzumab, 8 mg/kg [n = 1]). Serial fetal and maternal blood samples, amniotic fluid, maternal urine, and fetal and maternal tissue samples were collected for the first 76 hours after drug infusion. Levels of carboplatin were determined by atomic absorption spectrometry, docetaxel and paclitaxel by high-performance liquid chromatography, and trastuzumab by enzyme-linked immunosorbent assay. RESULTS: Fetal plasma concentrations of carboplatin averaged 57.5% (14.2%) of maternal concentrations (n = 7). Fetal plasma concentrations were 1.5% (0.8%) of maternal concentrations (n = 7). Immediately after ending the infusion, paclitaxel was not detectable in fetal tissues, whereas, after 3 hours, fetal tissues contained 15% of maternal tissue concentrations.Docetaxel could not be detected in fetal blood samples (n = 9). In the first 3 hours after docetaxel infusion, fetal tissues contained 5.0% to 50.0% of maternal tissue concentrations, whereas equal fetal and maternal tissue concentrations were found after 26 and 76 hours.The transplacental passages of trastuzumab were 85.0% and 3.0%, 2 and 26 hours after trastuzumab infusion, respectively. After 26 hours, amniotic fluid contained 36.4% of the fetal plasma concentration. Fetal tissue concentrations varied between 5.0% and 14.0% of the maternal concentration. CONCLUSION: Variable plasma and/or tissue concentrations of taxanes, carboplatin, and trastuzumab were encountered in the fetal compartment. These data are important when cancer treatment is considered during pregnancy and underline the need for long-term follow-up of children after prenatal exposure to these cytotoxic agents.

Vinblastine and doxorubicin administration to pregnant mice affects brain development and behaviour in the offspring.

BACKGROUND: Administration of chemotherapy during the fetal phase of pregnancy may put late-developing organs like the central nervous system at risk. METHODS: Transplacental transfer of doxorubicin and vinblastine was measured in C57/BJ mice by high-performance liquid chromatographic detection of the drugs in maternal and fetal plasma, 90 min after intravenous injection. Further, doxorubicin, vinblastine or saline were administered to pregnant C57/6J mouse dams on gestational day 17.5. Effects on brain morphology of the offspring were examined at 24h p.i. (immediate phase) and at 4-5 months p.i. (residual phase), using light- and electron microscopy. At the age of 3 months, offspring performed a behavioural test battery addressing neuromotor performance, exploration and anxiety, and learning and memory. RESULTS: Fetal plasma levels of doxorubicin and vinblastine reached respectively 5.0+/-0.2% and 13.9+/-2.4% of the maternal plasma levels. In the immediate phase, pathological examination revealed endothelial and perivascular parenchymal damage to the neocortical subventricular zone and a less constant thickening of the leptomeninx, in some cases also cortical lamination defects were noted. Brain histology was within normal limits in the mice of the residual phase group. Behavioural testing revealed subtle differences between drug-exposed and control mice. Grip strength was reduced in drug-exposed mice, but other tests for motor performance were normal. Several exploratory measures were altered, and there were some indications of increased anxiety in the drug-exposed mice. In the passive avoidance task, step-through latency was shorter in the drug-exposed mice, but their normal performance in the Morris water maze indicated that this was probably not due to impaired memory. CONCLUSION: The current preclinical data reveal subtle changes in behaviour and transiently also in brain morphology in the mice that were prenatally exposed to vinblastine or doxorubicin.

Prevention by maternal pancreatic islet transplantation of hypothalamic malformation in offspring of diabetic mother rats is already detectable at weaning.

Exposure to gestational diabetes (GD) in rats leads to dysplasia of the ventromedial hypothalamic nucleus (VMN), decisively involved into the regulation of body weight and metabolism. Recently, we have shown here that VMN malformation is absent in adult offspring of GD mothers treated by pancreatic islet transplantation during gestation. We therefore now investigated whether VMN malformation and its prevention are already present at the early postnatal end of the critical hypothalamic differentiation period. Already at weaning, the total number of VMN neurons, the volume of the VMN relative to total brain volume, and the numerical density of neurons in the anterior subnucleus of the VMN were reduced in offspring of sham-transplanted mothers (all P<0.05), but did not differ between offspring of islet-transplanted mothers and controls. No morphometric alterations occurred in the paraventricular hypothalamic nucleus. In conclusion, prevention of VMN malformation in offspring of islet-transplanted diabetic mothers is a direct consequence of normalized maternal metabolism during critical perinatal development.