Proposed role for COUP-TFII in regulating fetal Leydig cell steroidogenesis, perturbation of which leads to masculinization disorders in rodents

Reproductive disorders that are common/increasing in prevalence in human males may arise because of deficient androgen production/action during a fetal 'masculinization programming window'. We identify a potentially important role for Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII) in Leydig cell (LC) steroidogenesis that may partly explain this. In rats, fetal LC size and intratesticular testosterone (ITT) increased ~3-fold between e15.5-e21.5 which associated with a progressive decrease in the percentage of LC expressing COUP-TFII. Exposure of fetuses to dibutyl phthalate (DBP), which induces masculinization disorders, dose-dependently prevented the age-related decrease in LC COUP-TFII expression and the normal increases in LC size and ITT. We show that nuclear COUP-TFII expression in fetal rat LC relates inversely to LC expression of steroidogenic factor-1 (SF-1)-dependent genes (StAR, Cyp11a1, Cyp17a1) with overlapping binding sites for SF-1 and COUP-TFII in their promoter regions, but does not affect an SF-1 dependent LC gene (3β-HSD) without overlapping sites. We also show that once COUP-TFII expression in LC has switched off, it is re-induced by DBP exposure, coincident with suppression of ITT. Furthermore, other treatments that reduce fetal ITT in rats (dexamethasone, diethylstilbestrol (DES)) also maintain/induce LC nuclear expression of COUP-TFII. In contrast to rats, in mice DBP neither causes persistence of fetal LC COUP-TFII nor reduces ITT, whereas DES-exposure of mice maintains COUP-TFII expression in fetal LC and decreases ITT, as in rats. These findings suggest that lifting of repression by COUP-TFII may be an important mechanism that promotes increased testosterone production by fetal LC to drive masculinization. As we also show an age-related decline in expression of COUP-TFII in human fetal LC, this mechanism may also be functional in humans, and its susceptibility to disruption by environmental chemicals, stress and pregnancy hormones could explain the origin of some human male reproductive disorders.

Developmentally regulated IL6-type cytokines signal to germ cells in the human fetal ovary

Fetal ovarian development and primordial follicle formation are imperative for adult fertility in the female. Data suggest the interleukin (IL)6-type cytokines, leukaemia inhibitory factor (LIF), IL6, oncostatin M (OSM) and ciliary neurotrophic factor (CNTF), are able to regulate the survival, proliferation and differentiation of fetal murine germ cells (GCs) in vivo and in vitro. We postulated that these factors may play a similar role during early human GC development and primordial follicle formation. To test this hypothesis, we have investigated the expression and regulation of IL6-type cytokines, using quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Expression of transcripts encoding OSM increased significantly across the gestational range examined (8-20 weeks), while expression of IL6 increased specifically between the first (8-11 weeks) and early second (12-16 weeks) trimesters, co-incident with the initiation of meiosis. LIF and CNTF expression remained unchanged. Expression of the genes encoding the LIF and IL6 receptors, and their common signalling subunit gp130, was also found to be developmentally regulated, with expression increasing significantly with increasing gestation. LIF receptor and gp130 proteins localized exclusively to GCs, including oocytes in primordial follicles, indicating this cell type to be the sole target of IL6-type cytokine signalling in the human fetal ovary. These data establish that IL6-type cytokines and their receptors are expressed in the human fetal ovary and may directly influence GC development at multiple stages of maturation.

Prostaglandin E2 as a regulator of germ cells during ovarian development

CONTEXT: The formation of primordial follicles occurs during fetal life yet is critical to the determination of adult female fertility. Prior to this stage, germ cells proliferate, enter meiosis, and associate with somatic cells. Growth and survival factors implicated in these processes include activin A (INHBA), the neurotrophins BDNF and NT4 (NTF5), and MCL1. The prostaglandins have pleiotrophic roles in reproduction, notably in ovulation and implantation, but there are no data regarding roles for prostaglandins in human fetal ovarian development.

OBJECTIVE: The aim of the study was to investigate a possible role for prostaglandin (PG) E(2) in human fetal ovary development.

DESIGN: In vitro analysis of ovarian development between 8 and 20 wk gestation was performed.

MAIN OUTCOME MEASURE(S): The expression patterns of PG synthesis enzymes and the PGE(2) receptors EP2 and EP4 in the ovary were assessed, and downstream effects of PGE(2) on gene expression were analyzed.

RESULTS: Ovarian germ cells express the PG synthetic enzymes COX2 and PTGES as well as the EP2 and EP4 receptors, whereas COX1 is expressed by ovarian somatic cells. Treatment in vitro with PGE(2) increased the expression of BDNF mRNA 1.7 +/- 0.16-fold (P = 0.004); INHBA mRNA, 2.1 +/- 0.51-fold (P = 0.04); and MCL1 mRNA, 1.15 +/- 0.06-fold (P = 0.04), but not that of OCT4, DAZL, VASA, NTF5, or SMAD3.

CONCLUSIONS: These data indicate novel roles for PGE(2) in the regulation of germ cell development in the human ovary and show that these effects may be mediated by the regulation of factors including BDNF, activin A, and MCL1.

A Novel Semi-Automated Method of Tracking Fetal Movements

We have designed software that can â€â€™look’’ at recorded ultrasound sequences. We analyzed fifteen video sequences representing recorded ultrasound scans of nine fetuses. Our method requires a small amount of user labelled pixels for processing the first frame. These initialize GrowCut 1 , a background removal algorithm, which was used for separating the fetus from its surrounding environment (segmentation). For each subsequent frame, user input is no longer necessary as some of the pixels will inherit labels from the previously processed frame. This results in our software’s ability to track movement. Two sonographers rated the results of our computer’s â€vision’ on a scale from 1 (poor fit) to 10 (excellent fit). They assessed tracking accuracy for the entire video as well as segmentation accuracy (the ability to identify fetus from non-fetus) for every 100th processed frame. There was no appreciable deterioration in the software’s ability to track the fetus over time. I

Fetal umbilical artery Doppler pulsatility index and childhood neurocognitive outcome at 12 years

OBJECTIVE: To determine whether an elevated fetal umbilical artery Doppler (UAD) pulsatility index (PI) at 28 weeks' gestation, in the absence of fetal growth restriction (FGR) and prematurity, is associated with adverse neurocognitive outcome in children aged 12 years.

METHODS: Prospective cohort study, comparing children with a normal fetal UAD PI (<90th centile) (n=110) and those with an elevated PI (≥90th centile) (n=40). UAD was performed at 28, 32 and 34 weeks gestation. At 12 years of age, all children were assessed under standardised conditions at Queen's University, Belfast, UK to determine cognitive and behavioural outcomes using the British Ability Score-II and Achenbach Child Behavioural Checklist Parent Rated Version under standardised conditions. Regression analysis was performed, controlling for confounders such as gender, socioeconomic status and age at assessment.

RESULTS: The mean age of follow-up was 12.4 years (±0.5 SD) with 44% of children male (n=63). When UAD was assessed at 28 weeks, the elevated fetal UAD group had lower scores in cognitive assessments of information processing and memory. Parameters included (1) recall of objects immediate verbal (p=0.002), (2) delayed verbal (p=0.008) and (3) recall of objects immediate spatial (p=0.0016). There were no significant differences between the Doppler groups at 32 or 34 weeks' gestation.

CONCLUSIONS: An elevated UAD PI at 28 weeks' gestation in the absence of FGR or prematurity is associated with lower scores of declarative memory in children aged 12 years. A potential explanation for this is an element of placental insufficiency in the presence of the appropriately grown fetus, which affects the development of the fetal hippocampus and information processing and memory long-term. These findings, however, had no impact on overall academic ability, mental processing and reasoning or overall behavioural function.

Campilobacteriose em bovinos de carne no Alentejo – avaliação preliminar

The aim of this study was to assess the prevalence of infection by Campylobacter fetus in beef cattle reared in extensive systems in Alentejo region by direct fluorescent antibody test (DFAT), a technique widely used for determining the prevalence of this disease.

Motor delays in MDMA (ecstasy) exposed infants persist to 2years

Background Recreational use of 3,4 methylenedioxymethamphetamine (ecstasy, MDMA) is increasing worldwide. Its use by pregnant women causes concern due to potentially harmful effects on the developing fetus. MDMA, an indirect monoaminergic agonist and reuptake inhibitor, affects the serotonin and dopamine systems. Preclinical studies of fetal exposure demonstrate effects on learning, motor behavior, and memory. In the first human studies, we found prenatal MDMA exposure related to poorer motor development in the first year of life. In the present study we assessed the effects of prenatal exposure to MDMA on the trajectory of child development through 2 years of age. We hypothesized that exposure would be associated with poorer mental and motor outcomes. Materials and Methods The DAISY (Drugs and Infancy Study, 2003–2008) employed a prospective longitudinal cohort design to assess recreational drug use during pregnancy and child outcomes in the United Kingdom. Examiners masked to drug exposures followed infants from birth to 4, 12, 18, and 24 months of age. MDMA, cocaine, alcohol, tobacco, cannabis, and other drugs were quantified through a standardized clinical interview. The Bayley Scales (III) of Mental (MDI) and Motor (PDI) Development and the Behavior Rating Scales (BRS) were primary outcome measures. Statistical analyses included a repeated measures mixed model approach controlling for multiple confounders. Results Participants were pregnant women volunteers, primarily white, of middle class socioeconomic status, average IQ, with some college education, in stable partner relationships. Of 96 women enrolled, children of 93 had at least one follow-up assessment and 81 (87%) had ≥ two assessments. Heavier MDMA exposure (M = 1.3 ± 1.4 tablets per week) predicted lower PDI (p < .002), and poorer BRS motor quality from 4 to 24 months of age, but did not affect MDI, orientation, or emotional regulation. Children with heavier exposure were twice as likely to demonstrate poorer motor quality as lighter and non-exposed children (O.R. = 2.2, 95%, CI = 1.02–4.70, p < .05). Discussion Infants whose mothers reported heavier MDMA use during pregnancy had motor delays from 4 months to two years of age that were not attributable to other drug or lifestyle factors. Women of child bearing age should be cautioned about the use of MDMA and MDMA-exposed infants should be screened for motor delays and possible intervention.

Desarrollo Embrionario & Fetal

Written in Spanish, this document explains embryonic and fetal development.

Timing and mode of delivery in twins : the ongoing controversy

Tese de doutoramento, Medicina (Ginecologia e Obstetrícia), Universidade de Lisboa, Faculdade de Medicina, 2014

Classificação e previsão de cardiotocogramas usando algoritmos baseados em redes neuronais

Mestrado em Computação e Instrumentação Médica

Consumo de álcool nas mães que amamentam durante o 1º ano de vida da criança, prevalência e fatores associados, em Vila Fanca de Xira, em 2014

RESUMO - Introdução: O consumo de álcool durante o aleitamento materno é um tema que tem vindo a ganhar relevo nos últimos anos. Contrariamente ao consumo de álcool durante a gravidez, em que já se conhecem bem as consequências sobre o feto, sobre os efeitos do consumo de álcool durante a amamentação ainda pouco se sabe. Este estudo visa, assim, caracterizar o consumo de álcool e fatores associados nas mães que amamentam, ou amamentaram, durante o 1º ano de vida da criança no concelho de Vila Franca de Xira em 2014. Metodologia: Foi realizado um estudo transversal, com a aplicação de um questionário de auto-preenchimento, a uma amostra estratificada e com alocação proporcional por Unidade Funcional, de mães que estiveram nas consultas de saúde infantil das unidades de saúde do concelho, entre março e maio de 2014. Resultados: A idade mediana das mães foi 31 anos e das crianças foi 3 meses. O tempo total em aleitamento teve uma mediana de 2 meses e somente 16,3% amamentaram mais de 5 meses. A prevalência do consumo de álcool durante a amamentação foi 7,9% e na gestação foi 8,1%. Os fatores que se associaram, com significância estatística, ao consumo de álcool na amamentação foram a maior idade da mãe, os conhecimentos que esta possuía e o consumo anterior de álcool na gestação. Os hábitos tabágicos durante a amamentação tiveram uma prevalência de 13,4% e o consumo de substâncias ilícitas de 0,6%. Conclusões: A prevalência do consumo de álcool durante a amamentação, a nível local, mantém-se semelhante à da gestação, sendo muito inferior à encontrada em outros estudos de outros países. O tempo total em amamentação é baixo considerando as recomendações nacionais e internacionais.

Cultured human fetal astrocytes can be induced by interferon-gamma to express HLA-DR.

Interferon-gamma (IFN-gamma) modulates the expression of Class II major histocompatibility antigens (MHC), thus providing a potential regulatory mechanism for local immune reactivity in the context of MHC-restricted antigen presentation. Within the central nervous system (CNS), the expression of MHC Class II antigens has been demonstrated on human reactive astrocytes and glioma cells. In order to investigate the modulation of HLA-DR on normal astrocytes, two cell lines were grown from a 20-week-old fetal brain. In situ none of the fetal brain cells expressed HLA-DR as determined by immunohistology on frozen tissue sections. The two cell lines, FB I and FB II, expressed GFAP indicating their astrocytic origin. FB I was HLA-DR negative at the first tissue culture passages, but could be induced to express HLA-DR when treated with 500 U/ml IFN-gamma. FB II was spontaneously HLA-DR positive in the early passages, lost the expression of this antigen after 11 passages and could also be induced to express HLA-DR by IFN-gamma. The induction of HLA-DR expression was demonstrated both by a binding RIA and by immunoprecipitation using a monoclonal antibody (MAB) directed against a monomorphic determinant of HLA-DR. The HLA-DR alloantigens were determined on FB II cells after IFN-gamma treatment, by immunofluorescence and by cytotoxicity assays, and were shown to be DR4, DR6, Drw52, DRw53 and DQwl. These results show that human fetal astrocytes can be induced to express HLA-DR by IFN-gamma in vitro and support the concept that astrocytes may function as antigen-presenting cells.

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.

Demyelination in brain cell aggregate cultures, induced by a monoclonal antibody against the myelin/oligodendrocyte glycoprotein (MOG).

Previous work has shown that aggregate cultures prepared from fetal rat telencephalon and grown in a chemically defined medium offer a useful model to study developmental processes such as myelin synthesis. Since compact myelin is formed in these cultures, we investigated the possibility to use this culture system to study demyelinating mechanisms. In particular, we examined the effect of a monoclonal antibody (8-18C5) directed against the myelin/oligodendrocyte glycoprotein (MOG). We found that addition of anti-MOG antibodies and complement to aggregate cultures led to a highly significant decrease in myelin basic protein (MBP) content and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) specific activity. These results indicate that, in our culture system, anti-MOG antibodies have a strong demyelinating effect.

Screening for foetal malformations: performance of routine ultrasonography in the population of the Swiss Canton of Vaud.

OBJECTIVE: To determine the sensitivity of ultrasonography in screening for foetal malformations in the pregnant women of the Swiss Canton of Vaud. STUDY DESIGN: Retrospective study over a period of five years. METHOD: We focused our study on 512 major or minor clinically relevant malformations detectable by ultrasonography. We analysed the global sensitivity of the screening and compared the performance of the tertiary centre with that of practitioners working in private practice or regional hospitals. RESULTS: Among the 512 malformations, 181 (35%) involved the renal and urinary tract system, 137 (27%) the heart, 71 (14%) the central nervous system, 50 (10%) the digestive system, 42 (8%) the face and 31 (6%) the limbs. Global sensitivity was 54.5%. The lowest detection rate was observed for cardiac anomalies, with only 23% correct diagnoses. The tertiary centre achieved a 75% detection rate in its outpatient clinic and 83% in referred patients. Outside the referral centre, the diagnostic rate attained 47%. CONCLUSIONS: Routine foetal examination by ultrasonography in a low-risk population can detect foetal structural abnormalities. Apart from the diagnosis of cardiac abnormalities, the results in the Canton of Vaud are satisfactory and justify routine screening for malformations in a low-risk population. A prerequisite is continuing improvement in the skills of ultrasonographers through medical education.