Laurence Loubiere

Expression of thyroid hormone transporters in the human placenta and changes associated with intrauterine growth restriction

Thyroid hormones (TH) are important for the development of the human fetus and placenta from very early gestation. The transplacental passage of TH from mother to fetus and the supply of TH into trophoblasts require the expression of placental TH plasma membrane transporters. We describe the ontogeny of the TH transporters MCT8, MCT10, LAT1, LAT2, OATP1A2 and OATP4A1 in a large series (n = 110) of normal human placentae across gestation and describe their expression changes with intrauterine fetal growth restriction (IUGR n = 22). Quantitative RT-PCR revealed that all the mRNAs encoding TH transporters are expressed in human placenta from 6 weeks gestation and throughout pregnancy. MCT8, MCT10, OATP1A2 and LAT1 mRNA expression increased with gestation. OATP4A1 and CD98 (LATs obligatory associated protein) mRNA expression reached a nadir in mid-gestation before increasing towards term. LAT2 mRNA expression did not alter throughout gestation. Immunohistochemistry localised MCT10 and OATP1A2 to villous cytotrophoblasts and syncytiotrophoblasts, and extravillous trophoblasts while OATP4A1 was preferentially expressed in the villous syncytiotrophoblasts. Whilst MCT8 protein expression was increased, MCT10 mRNA expression was decreased in placentae from IUGR pregnancies delivered in the early 3rd trimester compared to age matched appropriately grown for gestational age controls. No significant change was found in the mRNA expression of the other transporters with IUGR. In conclusion, several TH transporters are present in the human placenta from early 1st trimester with varying patterns of expression throughout gestation. Their coordinated effects may regulate both transplacental TH passage and TH supply to trophoblasts, which are critical for the normal development of the fetus and placenta. Increased MCT8 and decreased MCT10 expression within placentae of pregnancies complicated by IUGR may contribute to aberrant development of the fetoplacental unit.

Monocarboxylate Transporter 8 Modulates the Viability and Invasive Capacity of Human Placental Cells and Fetoplacental Growth in Mice

Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo.

The expression of thyroid hormone transporters in the human fetal cerebral cortex during early development and in N-Tera-2 neurodifferentiation

Non‐technical summary  Thyroid hormones are important in brain development and they enter cells through thyroid hormone transporters at the cell membrane. Thyroid hormone transporters are thought to play an important role since gene defects in one of these transporters, MCT8, have been associated with severe mental retardation. This paper describes the expression of a range of thyroid hormone transporters in the human fetal brain during early pregnancy, and suggests that these transporters could regulate the supply of thyroid hormones into brain cells from very early in development. Surprisingly, the reduction of thyroid hormones and MCT8 expression do not affect the differentiation of an unspecialised cell to a specialised human nerve cell in the brain. Thyroid hormones and MCT8 are thus likely to affect other processes during human brain development. To find out how thyroid hormones influence human fetal brain development requires further research.

Pituitary Tumor Transforming Gene Binding Factor: A New Gene in Breast Cancer

Pituitary tumor transforming gene (PTTG) binding factor (PBF; PTTG1IP) is a relatively uncharacterized oncoprotein whose function remains obscure. Because of the presence of putative estrogen response elements (ERE) in its promoter, we assessed PBF regulation by estrogen. PBF mRNA and protein expression were induced by both diethylstilbestrol and 17beta-estradiol in estrogen receptor alpha (ERalpha)-positive MCF-7 cells. Detailed analysis of the PBF promoter showed that the region -399 to -291 relative to the translational start site contains variable repeats of an 18-bp sequence housing a putative ERE half-site (gcccctcGGTCAcgcctc). Sequencing the PBF promoter from 122 normal subjects revealed that subjects may be homozygous or heterozygous for between 1 and 6 repeats of the ERE. Chromatin immunoprecipitation and oligonucleotide pull-down assays revealed ERalpha binding to the PBF promoter. PBF expression was low or absent in normal breast tissue but was highly expressed in breast cancers. Subjects with greater numbers of ERE repeats showed higher PBF mRNA expression, and PBF protein expression positively correlated with ERalpha status. Cell invasion assays revealed that PBF induces invasion through Matrigel, an action that could be abrogated both by siRNA treatment and specific mutation. Furthermore, PBF is a secreted protein, and loss of secretion prevents PBF inducing cell invasion. Given that PBF is a potent transforming gene, we propose that estrogen treatment in postmenopausal women may upregulate PBF expression, leading to PBF secretion and increased cell invasion. Furthermore, the number of ERE half-sites in the PBF promoter may significantly alter the response to estrogen treatment in individual subjects.

MCT8 expression in human fetal cerebral cortex is reduced in severe intrauterine growth restriction

The importance of the thyroid hormone (TH) transporter, monocarboxylate transporter 8 (MCT8), to human neurodevelopment is highlighted by findings of severe global neurological impairment in subjects with MCT8 (SLC16A2) mutations. Intrauterine growth restriction (IUGR), usually due to uteroplacental failure, is associated with milder neurodevelopmental deficits, which have been partly attributed to dysregulated TH action in utero secondary to reduced circulating fetal TH concentrations and decreased cerebral thyroid hormone receptor expression. We postulate that altered MCT8 expression is implicated in this pathophysiology; therefore, in this study, we sought to quantify changes in cortical MCT8 expression with IUGR. First, MCT8 immunohistochemistry was performed on occipital and parietal cerebral cortex sections obtained from appropriately grown for gestational age (AGA) human fetuses between 19 weeks of gestation and term. Secondly, MCT8 immunostaining in the occipital cortex of stillborn IUGR human fetuses at 24–28 weeks of gestation was objectively compared with that in the occipital cortex of gestationally matched AGA fetuses. Fetuses demonstrated widespread MCT8 expression in neurons within the cortical plate and subplate, in the ventricular and subventricular zones, in the epithelium of the choroid plexus and ependyma, and in microvessel wall. When complicated by IUGR, fetuses showed a significant fivefold reduction in the percentage area of cortical plate immunostained for MCT8 compared with AGA fetuses (P<0.05), but there was no significant difference in the proportion of subplate microvessels immunostained. Cortical MCT8 expression was negatively correlated with the severity of IUGR indicated by the brain:liver weight ratios (r2=0.28; P<0.05) at post-mortem. Our results support the hypothesis that a reduction in MCT8 expression in the IUGR fetal brain could further compromise TH-dependent brain development.

Triiodothyronine regulates angiogenic growth factor and cytokine secretion by isolated human decidual cells in a cell-type specific and gestational age-dependent manner

STUDY QUESTION Does triiodothyronine (T3) regulate the secretion of angiogenic growth factors and cytokines by human decidual cells isolated from early pregnancy? SUMMARY ANSWER T3 modulates the secretion of specific angiogenic growth factors and cytokines, with different regulatory patterns observed amongst various isolated subpopulations of human decidual cells and with a distinct change between the first and second trimesters of pregnancy. WHAT IS KNOWN ALREADY Maternal thyroid dysfunction during early pregnancy is associated with complications of malplacentation including miscarriage and pre-eclampsia. T3 regulates the proliferation and apoptosis of fetal-derived trophoblasts, as well as promotes the invasive capability of extravillous trophoblasts (EVT). We hypothesize that T3 may also have a direct impact on human maternal-derived decidual cells, which are known to exert paracrine regulation upon trophoblast behaviour and vascular development at the uteroplacental interface. STUDY DESIGN, SIZE, DURATION This laboratory-based study used human decidua from first (8–11 weeks; n = 18) and second (12–16 weeks; n = 12) trimester surgical terminations of apparently uncomplicated pregnancies. PARTICIPANTS/MATERIALS, SETTING, METHODS Primary cultures of total decidual cells, and immunomagnetic bead-isolated populations of stromal-enriched (CD10+) and stromal-depleted (CD10−) cells, uterine natural killer cells (uNK cells; CD56+) and macrophages (CD14+) were assessed for thyroid hormone receptors and transporters by immunocytochemistry. Each cell population was treated with T3 (0, 1, 10, 100 nM) and assessments were made of cell viability (MTT assay) and angiogenic growth factor and cytokine secretion (immunomediated assay). The effect of decidual cell-conditioned media on EVT invasion through Matrigel® was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE Immunocytochemistry showed the expression of thyroid hormone transporters (MCT8, MCT10) and receptors (TRα1, TRβ1) required for thyroid hormone-responsiveness in uNK cells and macrophages from the first trimester. The viability of total decidual cells and the different cell isolates were unaffected by T3 so changes in cell numbers could not account for any observed effects. In the first trimester, T3 decreased VEGF-A secretion by total decidual cells (P < 0.05) and increased angiopoietin-2 secretion by stromal-depleted cells (P < 0.05) but in the second trimester total decidual cells showed only increased angiogenin secretion (P < 0.05). In the first trimester, T3 reduced IL-10 secretion by total decidual cells (P < 0.05), and reduced granulocyte macrophage colony stimulating factor (P < 0.01), IL-8 (P < 0.05), IL-10 (P < 0.01), IL-1β (P < 0.05) and monocyte chemotactic protein -1 (P < 0.001) secretion by macrophages, but increased tumour necrosis factor-α secretion by stromal-depleted cells (P < 0.05) and increased IL-6 by uNK cells (P < 0.05). In contrast, in the second trimester T3 increased IL-10 secretion by total decidual cells (P < 0.01) but did not affect cytokine secretion by uNK cells and macrophages. Conditioned media from first trimester T3-treated total decidual cells and macrophages did not alter EVT invasion compared with untreated controls. Thus, treatment of decidual cells with T3 resulted in changes in both angiogenic growth factor and cytokine secretion in a cell type-specific and gestational age-dependent manner, with first trimester decidual macrophages being the most responsive to T3 treatment, but these changes in decidual cell secretome did not affect EVT invasion in vitro. LIMITATIONS, REASONS FOR CAUTION Our results are based on in vitro findings and we cannot be certain if a similar response occurs in human pregnancy in vivo. WIDER IMPLICATIONS OF THE FINDINGS Optimal maternal thyroid hormone concentrations could play a critical role in maintaining a balanced inflammatory response in early pregnancy to prevent fetal immune rejection and promote normal placental development through the regulation of the secretion of critical cytokines and angiogenic growth factors by human decidual cells. Our data suggest that there is an ontogenically determined regulatory ‘switch’ in T3 responsiveness between the first and second trimesters, and support the notion that the timely and early correction of maternal thyroid dysfunction is critical in influencing pregnancy outcomes. STUDY FUNDING/COMPETING INTEREST(S) This study is funded by Wellbeing of Women (RG/1082/09 to S.Y.C., M.D.K., J.A.F., L.S.L., G.E.L.) and Action Medical Research – Henry Smith Charity (SP4335 to M.D.K., S.Y.C., L.S.L., J.A.F.). The authors have no conflicts of interest to disclose.

Differential Triiodothyronine Responsiveness and Transport by Human Cytotrophoblasts from Normal and Growth-Restricted Pregnancies

Context: Abnormal placentation in human pregnancy is associated with intrauterine fetal growth restriction (IUGR). Our group has previously reported the association between severe IUGR, lower fetal circulating concentrations of thyroid hormones (THs), and altered expression of TH receptors and TH transporters within human placental villi. We postulate that altered TH bioavailability to trophoblasts may contribute to the pathogenesis of IUGR. Design and Objective: Cytotrophoblasts were isolated from normal and IUGR human placentae to compare their responsiveness to T3 and their capability for T3 transport. Results: Compared with normal cytotrophoblasts, the viability of IUGR cytotrophoblasts (assessed by methyltetrazoleum assay) was significantly reduced (P < 0.001), whereas apoptosis (assessed using caspase 3/7 activity and M30 immunoreactivity) was significantly increased after T3 treatment for 48 h (P < 0.001 and P < 0.01, respectively). The secretion of human chorionic gonadotropin was significantly increased by IUGR cytotrophoblasts compared with normal cytotrophoblasts (P < 0.001), independently of T3 treatment. Net transport of [125I]T3 was 20% higher by IUGR cytotrophoblasts compared with normal cytotrophoblasts (P < 0.001), and this was accompanied by a 2-fold increase in the protein expression of the TH transporter, monocarboxylate transporter 8, as assessed by Western immunoblotting (P < 0.01). Conclusions: IUGR cytotrophoblasts demonstrate altered responsiveness to T3 with significant effects on cell survival and apoptosis compared with normal cytotrophoblasts. Increased monocarboxylate transporter 8 expression and intracellular T3 accumulation may contribute to the altered T3 responsiveness of IUGR cytotrophoblasts.

Radiofrequency-induced Thermo-chemotherapy Effect Versus a Second Course of Bacillus Calmette-Guérin or Institutional Standard in Patients with Recurrence of Non–muscle-invasive Bladder Cancer Following Induction or Maintenance Bacillus Calmette-Guérin Therapy (HYMN): A Phase III, Open-label, Randomised Controlled Trial

BACKGROUND There is no effective intravesical second-line therapy for non-muscle-invasive bladder cancer (NMIBC) when bacillus Calmette-Guérin (BCG) fails. OBJECTIVE To compare disease-free survival time (DFS) between radiofrequency-induced thermo-chemotherapy effect (RITE) and institutional standard second-line therapy (control) in NMIBC patients with recurrence following induction/maintenance BCG. DESIGN, SETTINGS, AND PARTICIPANTS Open-label, phase III randomised controlled trial accrued across 14 centres between May 2010 and July 2013 (HYMN [ClinicalTrials.gov: NCT01094964]). INTERVENTION Patients were randomly assigned (1:1) to RITE (60min, 40mg mitomycin-C, 42±2°C) or control following stratification for carcinoma in situ (CIS) status (present/absent), therapy history (failure of previous induction/maintenance BCG), and treatment centre. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Primary outcome measures were DFS and complete response (CR) at 3 mo for the CIS at randomisation subgroup. Analysis was based on intention-to-treat. RESULTS AND LIMITATIONS A total of 104 patients were randomised (48 RITE: 56 control). Median follow-up for the 31 patients without a DFS event was 36 mo. There was no significant difference in DFS between treatment arms (hazard ratio [HR] 1.33, 95% confidence interval [CI] 0.84-2.10, p=0.23) or in 3-mo CR rate in CIS patients (n=71; RITE: 30% vs control: 47%, p=0.15). There was no significant difference in DFS between treatment arms in non-CIS patients (n=33; RITE: 53% vs control: 24% at 24 mo, HR 0.50, 95% CI 0.22-1.17, p=0.11). DFS was significantly lower in RITE than in control in CIS with/without papillary patients (n=71; HR 2.06, 95% CI 1.17-3.62, p=0.01; treatment-subgroup interaction p=0.007). Disease progression was observed in four patients in each treatment arm. Adverse events and health-related quality of life between treatment arms were comparable. CONCLUSIONS DFS was similar between RITE and control. RITE may be a second-line therapy for non-CIS recurrence following BCG failure; however, confirmatory trials are needed. RITE patients with CIS with/without papillary had lower DFS than control. HYMN highlights the importance of the control arm when evaluating novel therapies. PATIENT SUMMARY This study did not show a difference in bladder cancer outcomes between microwave-heated chemotherapy and standard of care treatment. Papillary bladder lesions may benefit from microwave-heated chemotherapy treatment; however, more research is needed. Both treatments are similarly well tolerated.