Дисертації з теми "Oestradiol/progesterone"

Endometriosis and breast cancer are sex hormone dependent diseases characterised by the local production of high levels of 17β-oestradiol. The relationship between prostaglandins and sex steroid hormones is one of the focal questions in endometriosis, breast cancer and other sex steroid hormone related disorders. Therefore, the main hypothesis was that the aldo-keto reductase (AKR) 1C isoenzymes are responsible for controlling the availability of 17β-oestradiol, progesterone and prostaglandins in the microenvironment of the endometrium, and surrounding adipose tissues of endometriotic lesions and breast tumours. This was investigated using quantitative real-time polymerase chain reaction (PCR) for measuring the gene expression of AKR1C1-3 enzymes, and prostaglandin E (1-4) and F receptors in the endometrium, and surrounding adipose tissues of endometriotic lesions and breast tumours. This was then followed by investigating the role of one of the AKR1C enzymes - AKR1C3 - by inhibiting its catalysis using bimatoprost, followed by using PGE2 as one of the main candidates acting as a transcription factor for upregulating the expression of AKR1C3 which in turn upregulates the production of the local 17β-oestradiol. The gene expression of AKR1C1 was significantly higher in endometriotic lesions compared to eutopic endometrium of endometriosis patients. However, there was no significant difference in the gene expression of AKR1C (1-3) enzymes in the surrounding adipose tissues of endometriotic lesions between patients with or without endometriosis. Also, there was no significant difference in the gene expression of AKR1C (1-3) enzymes in the breast adipose tissues of patients with breast tumours, regardless of the oestrogen or progesterone receptor status. The gene expression of prostaglandin E (EP) receptor subtype 3 was significantly higher in the endometriotic lesions compared to eutopic endometrium of endometriosis patients. In the omental adipose tissue, there was no significant difference in the gene expression of EP1-4 and FP receptors between endometriosis and non-endometriosis patients. In the breast adipose tissue, there was also no significant difference in the gene expression of EP1-4 and FP receptors in patients with breast cancer regardless of the oestrogen or progesterone receptor status. The inhibitory constant (Ki) of bimatoprost was determined using oestrone as a substrate: Ki = 2.9µM and αKi = 0.7µM. Bimatoprost also significantly inhibited the production of 17β-oestradiol and inhibited the production of 9α,11β PGF2 in a dose dependent manner in the human endometrial cells. The effect of PGE2 on the expression of AKR1C1 and AKR1C3 was assessed in the human endometrial cells. The EP4 receptor agonist, L-902688, increased the gene expression of AKR1C1 and AKR1C3. Despite gene expression elevation, L-902688 did not increase the production of 17β-oestradiol. In conclusion, the results were contradictory and highlighted the need for further investigation into the relationship between prostaglandins and sex steroid hormones in the microenvironment of the endometrium, and surrounding adipose tissues of endometriotic lesions and breast tumours.

Dans l'etiologie de l'osteoporose, les oestrogenes et la progesterone semblent jouer un role important. C'est pourquoi nous avons etudie le role de ces steroides sexuels sur l'activite des cellules osseuses au cours du vieillissement chez la ratte. Des rattes lou de 30 mois ainsi que des femelles wistar agees de 6, 12 et 30 mois ont ete ovariectomisees (ovx), supplementees ou non en 17-oestradiol (e : 10g/kg pv/48 h), progesterone (p : 140g/kg pv/48 h), et 17-oestradiol + progesterone (ep : memes doses), ou pseudo-operees (sh). La duree de la carence oestrogenique et des differents traitements est respectivement de 30 et 60 jours selon que les animaux sont de souche lou ou wistar. Chez nos rattes wistar agees de 30 mois, l'excretion urinaire de ca et pi est deux fois plus elevee que celle mesuree chez les animaux de 6 et 12 mois. Simultanement les coefficients de retention et d'utilisation digestive apparente du ca et du pi sont tres diminues. Chez les lou ovx, e et ep, ainsi que chez les wistar adultes et senescents, nous observons une augmentation parallele des taux d'arnm calcitonine et de la calcitoninemie. En ce qui concerne l'hormone parathyroidienne, l'augmentation de l'hormone au niveau systemique est parallele a une baisse de la calcemie dans les groupes e, p et ep mais uniquement chez les lou. L'age s'accompagne d'une accentuation des concentrations plasmatiques de pth. Enfin, l'ovx augmente alors que les traitements e et ep reduisent la somatomedinemie des rattes wistar agees de 6, 12 et 30 mois. Les marqueurs du turnover osseux, mesures chez nos animaux de 6 et 12 mois, sont eleves apres ovx et retournent a des valeurs proches de celles des temoins apres l'administration d'oestrogene seul ou combine a la progesterone. Quant aux animaux de 30 mois, et contrairement aux lou, apres deux mois de carence oestrogenique, e et ep corrigent totalement aussi bien l'osteocalcinemie que l'excretion urinaire des agents de pontage du collagene. La senescence ainsi que l'ovx s'accompagnent d'une reduction significative de la bmc (bone mineral content) et de la bmd (bone mineral density) distales. L'administration d'e ameliore la bmc et la bmd distales ainsi que l'aire, le perimetre et l'epaisseur des trabecules osseuses des rattes wistar quel que soit leur age. Ep corrige aussi la bmc et la bmd distales des wistar et la bmd distale des rattes lou. L'administration d'ep est le seul traitement qui ameliore significativement la resistance osseuse des tibias de rattes wistar agees de 12 et 30 mois ainsi que ceux de souche lou. La force necessaire a la rupture des os de rattes lou augmente egalement apres supplementation oestrogenique. En conclusion, chez les rattes ovx agees de 30 mois, l'administration d'ep previent l'osteopenie induite par castration.

It is the first time in Lithuania that practical application of semen filtration by Sephadex G-15 for the needs of artificial insemination industry was assessed. The efficacy of semen filtration was assessed applying a battery of sperm quality assays to study bovine spermatozoa quality before and after filtration, as well as after cryopreservation. The estimation of effect of different concentrations of progesterone, oestradiol, heparin separately or in combination, on different functional parameters of bull spermatozoa after thawing was performed. These are the first published studies applying integrated approach to study the effects of sperm challenge with several biologically active substances.

Orientador: Cezinande de Meira
Resumo: Diferentes tratamentos hormonais com a utilização de estrógenos e progestágenos são comumente utilizados para aumentar a oferta de receptoras nos programas de TE. Porém, pouco se sabe sobre a ação destes hormônios na expressão gênica e proteica dos receptores endometriais de estrógeno e progesterona em éguas acíclicas. O presente estudo teve como objetivo avaliar os efeitos de três tratamentos hormonais utilizados durante a preparação de éguas acíclicas sobre o edema, tônus uterino e expressão gênica e proteica de receptores de estrógeno e progesterona endometriais. Éguas em anestro foram divididas em três grupos: Dose Total 10 mg BE+P4, (n=7), Dose Total 5 mg BE+P4 (n=7), Priming Hormonal (n=7) e comparadas com o grupo de éguas cíclicas (n=7). Foram avaliados: a expressão proteica e gênica relativa dos transcritos para os receptores de estrógeno e progesterona presentes no endométrio por meio das técnicas de imunohistoquímica e RT-qPCR; e as características morfológicas do útero por palpação retal e ultrassonografia em modo B. Os tratamentos hormonais utilizados no presente estudo foram eficazes em promover edema e tônus uterinos semelhante ao que ocorre em éguas cíclicas. Adicionalmente, o grupo Priming Hormonal demonstrou induzir características uterinas similares as observadas nos grupos 5 mg BE+P4 e no grupo controle, após 14 dias de intervalo. O tratamento hormonal com dose total de 10 mg de BE+P4 LA utilizando para o preparo de éguas acíclicas, demonstrou ser similar ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Different hormonal treatments with the use of estrogen and progestogen are commonly used to increase the supply of receiving the TE programs. However, little is known about the effect of these hormones on gene and protein expression of endometrial receptors for estrogen and progesterone in non-cyclic mares. This study aimed to evaluate the effects of three hormonal treatments used for the preparation of non-cyclic mares on the edema and uterine tone and gene and protein expression of estrogen and endometrial progesterone receptors. Mares anestrus were divided into three groups: total dose 10 mg EB + P4 (n = 7) total dose 5 mg EB + P4 (n = 7) Hormonal Priming (n = 7) and compared with the group of cyclic mares (n = 7). Were evaluated: protein expression and gene transcripts related to the estrogen and progesterone receptors present in the endometrium by the techniques of immunohistochemistry and RT-qPCR; and the morphological characteristics of the uterus by rectal palpation and ultrasound in B mode. Hormonal treatments used in this study were effective in promoting edema and uterine tone similar to what occurs in cyclic mares. Additionally, the Hormonal Priming group demonstrated induce uterine similar characteristics observed in the groups 5 mg EB + P4 and control group, after 14 days apart. Hormonal treatment with a total dose of 10 mg EB + P4 LA using for the preparation of non-cyclic mares, was shown to be similar in tone and uterine edema, protein expression and relative... (Complete abstract click electronic access below)
Mestre

O objetivo deste trabalho foi verificar a presença e a localização de receptores para hormônios esteróides e gonadotróficos, através da técnica de imunohistoquímica, pelo método de peroxidase-antiperoxidase (PAP), nos diferentes tecidos que compõe o trato genital da égua e a variação de reatividade destes receptores durante o ciclo estral e no anestro fisiológico. Também se objetivou verificar se há diferença de reatividade em éguas com e sem endometrose. Foram coletadas amostras de útero, cérvice e oviduto, de 41 éguas sem raça definida e com histórico reprodutivo desconhecido, em um abatedouro. Quinze éguas se encontravam em estro, dezoito em diestro e oito éguas em anestro. Concluiu-se que a intensidade e a distribuição da coloração para os receptores de estrógeno (RE), progesterona (RP) e hormônio luteinizante (RLH) variaram de acordo com o tipo de célula e o estágio do ciclo estral. Nas amostras de endométrio observou-se imunorreatividade alta no epitélio luminal para RE e RP tanto no estro quanto no diestro; o epitélio glandular, estroma e miométrio mostraram reatividade moderada para os dois receptores durante as duas fases. Durante o anestro os resultados foram semelhantes aos encontrados durante a fase cíclica. Na avaliação da reatividade para RLH, durante o estro e diestro, o epitélio luminal mostrou reatividade de fraca a moderada, mas no diestro houve maior reatividade média. O epitélio glandular apresentou menor reatividade do que o luminal. No miométrio a coloração foi fraca durante todo o ciclo. Durante o anestro a reatividade foi fraca no epitélio luminal, ausente em quase todas as amostras no epitélio glandular e de fraca a ausente no miométrio. Neste experimento, não foi observada diferença significativa de reatividade entre os endométrios com e sem endometrose, mas as áreas afetadas mostraram coloração assíncrona para RE, RP e RLH. Na cérvice, foi observada imunorreatividade moderada a alta para RE e RP no epitélio luminal, no estroma e no músculo. A intensidade de coloração das células epiteliais e musculares variou pouco entre o estro e o diestro, mas durante o anestro houve maior reatividade no tecido muscular e no estroma. Foi observada reatividade para RLH no epitélio e camada muscular, sem variação significativa nas fases do ciclo. A intensidade de coloração foi de fraca a moderada no epitélio e fraca na camada muscular. No oviduto, observou-se imunorreatividade para RE e RP nos três tecidos, durante a fase cíclica e o anestro. No epitélio, os valores encontrados foram de moderados a altos, sem variação significativa nas três fases. A coloração das células epiteliais do oviduto foi nitidamente irregular, com o núcleo muito corado no que parecem ser células secretoras e pouco corado ou sem coloração nas células ciliadas, refletindo provavelmente as diferentes funções das células epiteliais neste órgão. No estroma a reatividade foi moderada durante a fase luteal, mostrando reatividade mais alta no estro e no anestro. A camada muscular apresentou reatividade máxima para RE no estro e no diestro. A reatividade para RLH no epitélio luminal foi de fraca a moderada durante todo o ciclo. No músculo também foi observada reatividade, porém bem mais fraca do que no epitélio. Durante o anestro somente três das oito amostras apresentaram reatividade no tecido muscular. No diestro foi observada maior reatividade do que no estro. Os resultados do presente estudo evidenciam, pela primeira vez, a presença de receptores para LH nos diferentes tecidos do trato reprodutor extragonadal da égua. Embora existam relatos da expressão e localização de RE e RP no endométrio equino, esta é a primeira vez que se utiliza a técnica de imunohistoquímica para localizar estes receptores na cérvice e no oviduto desta espécie. Foi observada variação individual bastante acentuada entre as amostras, em uma mesma fase cíclica. Provavelmente estes resultados sejam o reflexo da variação entre o dia do ciclo em que os animais se encontravam, bem como da complexidade dos mecanismos envolvidos na presença desses receptores. Os achados deste estudo indicam que tanto os hormônios gonadais quanto o LH atuam por meio de seus receptores nos diferentes tecidos do trato reprodutivo da égua, podendo servir para a elaboração de novas estratégias para melhorar a eficiência reprodutiva nesta espécie.
The aim of this study was to demonstrate the presence and localization of gonadotropic and steroid hormone receptors, in different tissues of the mare genital tract and the different reactivity to these receptors during the endometrial cycle and physiologic anestrus. Another objective was to compare the reactivity to theses receptors in mares with and without endometrosis. Immunohistochemistry was performed using the peroxidase anti-peroxidase technique (PAP). Uterus, cervix and oviduct of 41 criollo mares were collected in an abattoir. There was variation in the intensity of the staining and distribution for estrogen receptors (PR), progesterone receptors (PR) and luteinizing hormone receptors (LHR) with the endometrial cycle and different tissues. The endometrial surface epithelial cells were stained strongly for ER and PR in the estrous and dioestrus; glandular epithelial cells, stromal cells and smooth muscle cells of the myometrium had moderate staining for ER and PR during these two phases and in anestrus too. The immunoreactive score for LHR in the surface epithelial cells during endometrial cycle was weak to moderate but, in general, strong staining was observed in dioestrus. More weak staining intensity was observed in the glandular epithelial cells than luminal epithelial cells. Smooth muscle cells of the myometrium showed weak staining for LHR throughout the endometrial cycle. During the anestrus, the immunoreactivity score was weak in the surface epithelial cells. In general, the glandular epithelium was not stained. Myometrium cells were weak to not staining for LHR, in this phase. In this study there was no significant difference in immunoreactive score for ER, PR and LHR in endometrium with or without endometrosis but fibrotic glands showed different expression patterns of ER, PR and LHR, could evidence for functionally glandular maldifferentiation in endometrosis. The cervical epithelial surface, stromal cells and smooth muscle cells were moderate to strongly staining for ER and PR, with little variation throughout the endometrial cycle but the immunorectivity was strongest during the anestrus in muscular and stromal cells. Surface epithelial cells of cervix were weak to moderate stained for LHR; smooth muscle cells showed weak staining for these receptors. There was no variation during cycle. In the oviduct, epithelial, stromal and muscle cells showed reactivity for RE and RP, during cycle and anestrus. Epithelial cells were moderate to strongly staining for these receptors, with evident irregularity in different types of cells. Apparently ciliated epithelial cells were stained but the intensity was much less than that observed in nonciliated epithelial cells, probably reflecting different functions of these cells. Stromal cells showed moderate staining during dioestrus and strongest reactivity in estrous and anestrus; muscle cells showed strong reactivity for ER throughout the cycle. The reactivity for LHR was weak to moderate throughout the cycle in the epithelial cells and weak in the muscle cells. During anestrus only three strains of muscle cells showed reactivity for LHR. In dioestrus the intensity was strongest. These findings evidence for the firs time the presence for LHR in extra-gonadal reproductive organs of mare. Though there were reports of ER and PR expression in equine endometrium, this is the first report of localization of these receptors in cervix and oviduct of mare using immunohistochemistry. It was found marked individual variation among the strains. These results probably were caused by the variation among the day of cycle and the complexity of mechanisms involved in the presence of these receptors. The findings of the present study allow us to infer that the ovarian steroid hormones nad LH function through their receptors in different tissues of mare reproductive tract, can help us to elaborate new strategies to improve the reproductive efficiency in this specie.

RESUMO:A determinação da fracção exalada de óxido nítrico (FENO) é amplamente utilizada como um biomarcador da inflamação eosinofílica das vias aéreas. Alguns estudos sugerem que a produção de óxido nítrico (NO) é influenciada pelas variações cíclicas hormonais na mulher,porém os dados não são consensuais. Deste modo, o objectivo do nosso estudo foi avaliar como varia a FENO ao longo do ciclo menstrual. Com esta finalidade, avaliamos um grupo de 20 voluntárias, em idade fértil, com ciclo menstrual regular, não fumadoras, que não utilizavam contraceptivos hormonais, nem suplementos alimentares e/ou medicamentosos e que não se encontravam grávidas, nem a amamentar. Todas referiram não ter conhecimento de qualquer patologia que afecte a FENO. A existência de atopia foi controlada através de testes cutâneos por prick, tendo-se excluído as participantes que apresentaram testes positivos. Realizamos quatro visitas de estudo, com base na periodicidade do ciclo de cada participante, nas quais, efectuamos a determinação da FENO, a quantificação dos níveis plasmáticos de óxido nítrico e nitratos (NO/NO3 -) e o doseamento hormonal de 17 -estradiol e progesterona. As avaliações realizaram-se no período da manhã, em jejum absoluto, tendo respeitado uma dieta pobre em nitratos no dia anterior e abstido da prática de exercício vigoroso uma hora antes da avaliação. Com este trabalho, verificamos um aumento significativo da FENO na fase secretora (17.97 ppb ± 5.8) comparativamente com a fase menstrual e proliferativa (16.48 ppb ± 3.6 e 15.95 ppb ±2.8, respectivamente). Não observamos variações significativas dos níveis plasmáticos de NO/NO3 - ao longo do ciclo. Constatamos uma correlação positiva entre a FENO e os níveis plasmáticos de NO/NO3 - durante a ovulação e verificamos que, para a nossa amostra, os níveis hormonais de estradiol e progesterona não são preditores do valor da FENO, nem dos níveis plasmáticos de NO/NO3-. Os resultados deste trabalho mostram uma variação da FENO ao longo do ciclo, ainda assim, mantendo-se os seus valores dentro do intervalo de referência, reforçando a fiabilidade deste biomarcador.--ABSTRACT:The determination of fractional exhaled nitric oxide (FENO) is widely used as a biomarker of eosinophilic airway inflammation. Some studies suggest that nitric oxide (NO) is influenced by cyclical hormonal changes in women, but those are not consensual. The aim of our study was to assess how FENO varies throughout the menstrual cycle. With this purpose, we studied a group of 20 volunteers within childbearing age, with regular menstrual cycle, non-smokers, who were not taking any medications including hormonal contraception and food supplements and who were not pregnant or breast-feeding. All participants reported not being aware of any condition that could affect the FENO. The presence of atopy was controlled by a skin prick test, having been excluded participants with positive test. We conducted four study visits, based on the periodicity of the cycle of each participant. In each visit, we made the determination of the FENO, the quantification of plasmatic levels of nitric oxide and nitrates (NO/NO3 -) and the blood levels of hormone estradiol-17 and progesterone. The evaluations occurred at morning, after overnight fasting. The participants were request to follow a low-nitrate diet in the previous day and refrained from vigorous exercise, for at least one hour before the visit We found a significant increase of FENO on secretory phase (17.97 ppb ± 5.8) compared with the menstrual and proliferative phase (16.48 ppb ± 3.6 and 15.95 ppb ± 2.8, espectively). No significant variations were found throughout the menstrual cycle in plasmatic levels of NO/NO3 -. We found a positive correlation between FENO and plasmatic levels of NO/NO3 - during ovulation. Finally, in our sample, the levels of oestradiol and progesterone are not predictors of FENO value nor of plasmatic levels of NO/NO3-. This study shows a variation of FENO over the menstrual cycle, nevertheless, the values remain within the reference range, reinforcing the reliability of this biomarker.

Pour évaluer l'influence des stéroïdes sur la synthèse de LH et FSH l'auteur mesure chez le rat le taux de RNAm en utilisant la traduction en milieu acellulaire et l'hybridation directe. Tous les stéroïdes testés in vivo exercent sur l'accumulation des RNAm un effet inhibiteur. Leur action s'exerce directement sur les cellules hypophysaires, selon un processus qui est fonction de la dose et du temps. Ceci pourrait expliquer les variations rythmiques des RNAm des gonadotrophines observées au cours du cycle oestrien chez la femelle. Ces résultats indiquent que chez le rat la synthèse des gonadotrophines est, comme leur sécrétion, soumise à une régulation par les stéroïdes gonadiques. Leur action s'exerce probablement au niveau transcriptionnel.

Dosage chez 226 hommes et 179 femmes ages de 16 a 60 ans, des gonadotrophines et des hormones steroides sexuelles. L'etude des resultats en fonction des donnees concernant des populations de divers continents, fait apparaitre de petite differences mais surtout une grande stabilite ausi bien dans les profils que dans les valeurs des hormones etudiees par l'ensemble des populations

This study, investigating 263 women undergoing trans-vaginal oocyte retrieval for in vitro fertilisation (IVF) found that microorganisms colonising follicular fluid contributed to adverse IVF (pre-implantation) and pregnancy (post-implantation) outcomes including poor quality embryos, failed pregnancy and early pregnancy loss (< 37 weeks gestation). Some microorganisms also showed in vitro growth patterns in liquid media that appeared to be enhanced by the hormonal stimulation protocol used for oocyte retrieval. Elaborated cytokines within follicular fluid were also associated with adverse IVF outcomes. This study is imperative because infertility affects 16% of the human population and the numbers of couples needing assistance continues to increase. Despite significant improvements in the technical aspects of assisted reproductive technologies (ART), the live birth rate has not increased proportionally. Overt genital tract infection has been associated with both infertility and adverse pregnancy outcomes (including miscarriage and preterm birth) as a direct result of the infection or the host response to it. Importantly, once inflammation had become established, medical treatment often failed to prevent these significant adverse outcomes. Current evaluations of fertility focus on the ovary as a site of steroid hormone production and ovulation. However, infertility as a result of subclinical colonisation of the ovary has not been reported. Furthermore, identification of the microorganisms present in follicular fluid and the local cytokine profile may provide clinicians with an early indication of the prognosis for IVF treatment in infertile couples, thus allowing antimicrobial treatment and/or counselling about possible IVF failure. During an IVF cycle, multiple oocytes undergo maturation in vivo in response to hormonal hyperstimulation. Oocytes for in vitro insemination are collected trans-vaginally. The follicular fluid that bathes the maturing oocyte in vivo, usually is discarded as part of the IVF procedure, but provides a unique opportunity to investigate microbial causes of adverse IVF outcomes. Some previous studies have identified follicular fluid markers that predict IVF pregnancy outcomes. However, there have not been any detailed microbiological studies of follicular fluid. For this current study, paired follicular fluid and vaginal secretion samples were collected from women undergoing IVF cycles to determine whether microorganisms in follicular fluid were associated with adverse IVF outcomes. Microorganisms in follicular fluid were regarded as either "colonisers" or "contaminants"; colonisers, if they were unique to the follicular fluid sample, and contaminants if the same microorganisms were detected in the vaginal and follicular fluid samples indicating that the follicular fluid was merely contaminated during the oocyte retrieval process. Quite unexpectedly, by these criteria, we found that follicular fluid from approximately 30% of all subjects was colonised with bacteria. Fertile and infertile women with colonised follicular fluid had decreased embryo transfer rates and decreased pregnancy rates compared to women with contaminated follicular fluids. The observation that follicular fluid was not always sterile, but contained a diverse range of microorganisms, is novel. Many of the microorganisms we detected in follicular fluid are known opportunistic pathogens that have been detected in upper genital tract infections and are associated with adverse pregnancy outcomes. Bacteria were able to survive for at least 28 weeks in vitro, in cultures of follicular fluid. Within 10 days of establishing these in vitro cultures, several species (Lactobacillus spp., Bifidobacterium spp., Propionibacterium spp., Streptococcus spp. and Salmonella entericus) had formed biofilms. Biofilms play a major role in microbial pathogenicity and persistence. The propensity of microbial species to form biofilms in follicular fluid suggests that successful treatment of these infections with antimicrobials may be difficult. Bifidobacterium spp. grew, in liquid media, only if concentrations of oestradiol and progesterone were similar to those achieved in vivo during an IVF cycle. In contrast, the growth of Streptococcus agalactiae and Escherichia coli was inhibited or abolished by the addition of these hormones to culture medium. These data suggest that the likelihood of microorganisms colonising follicular fluid and the species of bacteria involved is influenced by the stage of the menstrual cycle and, in the case of IVF, the nature and dose of steroid hormones administered for the maturation of multiple oocytes in vivo. Our findings indicate that the elevated levels of steroid hormones during an IVF cycle may influence the microbial growth within follicular fluid, suggesting that the treatment itself will impact on the microflora present in the female upper genital tract during pre-conception and early post-conception phases of the cycle. The effect of the host immune response on colonising bacteria and on the outcomes of IVF also was investigated. White blood cells reportedly compose between 5% and 15% of the cell population in follicular fluid. The follicular membrane is semi-permeable and cells are actively recruited as part of the normal menstrual cycle and in response to microorganisms. A previous study investigated follicular fluid cytokines from infertile women and fertile oocyte donors undergoing IVF, and concluded that there were no significant differences in the cytokine concentrations between the two groups. However, other studies have reported differences in the follicular fluid cytokine levels associated with infertile women with endometriosis or polycystic ovary syndrome. In this study, elevated levels of interleukin (IL)-1 á, IL-1 â and vascular endothelial growth factor (VEGF) in vaginal fluid were associated with successful fertilisation, which may be useful marker for successful fertilisation outcomes for women trying to conceive naturally or prior to oocyte retrieval for IVF. Elevated levels of IL-6, IL-12p40, granulocyte colony stimulating factor (GCSF) and interferon-gamma (IFN ã) in follicular fluid were associated with successful embryo transfer. Elevated levels of pro-inflammatory IL-18 and decreased levels of anti-inflammatory IL-10 were identified in follicular fluid from women with idiopathic infertility. Successful fertilisation and implantation is dependent on a controlled pro-inflammatory environment, involving active recruitment of pro-inflammatory mediators to the genital tract as part of the menstrual cycle and early pregnancy. However, ongoing pregnancy requires an enhanced anti-inflammatory environment to ensure that the maternal immune system does not reject the semi-allergenic foetus. The pro-inflammatory skew in the follicular fluid of women with idiopathic infertility, correlates with normal rates of fertilisation, embryo discard and embryo transfer, observed for this cohort, which were similar to the outcomes observed for fertile women. However, their pregnancy rate was reduced compared to fertile women. An altered local immune response in follicular fluid may provide a means of explaining infertility in this cohort, previously defined as 'idiopathic'. This study has found that microorganisms colonising follicular fluid may have contributed to adverse IVF and pregnancy outcomes. Follicular fluid bathes the cumulus oocyte complex during the in vivo maturation process, and microorganisms in the fluid, their metabolic products or the local immune response to these microorganisms may result in damage to the oocytes, degradation of the cumulus or contamination of the IVF culture system. Previous studies that have discounted bacterial contamination of follicular fluid as a cause of adverse IVF outcomes failed to distinguish between bacteria that were introduced into the follicular fluid at the time of trans-vaginal oocyte retrieval and those that colonised the follicular fluid. Those bacteria that had colonised the fluid may have had time to form biofilms and to elicit a local immune response. Failure to draw this distinction has previously prevented consideration of bacterial colonisation of follicular fluid as a cause of adverse IVF outcomes. Several observations arising from this study are of significance to IVF programs. Follicular fluid is not always sterile and colonisation of follicular fluid is a cause of adverse IVF and pregnancy outcomes. Hormonal stimulation associated with IVF may influence whether follicular fluid is colonised and enhance the growth of specific species of bacteria within follicular fluid. Bacteria in follicular fluid may form biofilms and literature has reported that this may influence their susceptibility to antibiotics. Monitoring the levels of selected cytokines within vaginal secretions may inform fertilisation outcomes. This study has identified novel factors contributing to adverse IVF outcomes and that are most likely to affect also natural conception outcomes. Early intervention, possibly using antimicrobial or immunological therapies may reduce the need for ART and improve reproductive health outcomes for all women.

Hepatocellular carcinoma (HCC), or primary liver cancer, is the fifth most common cancer worldwide and the third most common cause of cancer mortality (El-Serag 2012). The development of HCC is thought to be a multi-staged process that involves several risk factors including the chronic hepatitis B and C infection, carcinogen exposure, metabolic disease, excessive alcohol consumption and male gender. Accumulation of genetic and epigenetic alterations with DNA-damaged hepatocytes can also contribute to the molecular pathogenesis of HCC. A better understanding of molecular mechanisms associated with HCC could ultimately improve our current strategies for screening and targeted therapy of this disease. Array comparative genomic hybridisation studies in murine diethylnitrosamine (DEN)-induced HCC have identified ccne1 (cyclin E) as a candidate gene associated in accelerated liver carcinogenesis (Teoh et al. 2008). In order to investigate the role of cyclin E and its impact on hepatocyte cell cycle regulation in early HCC development, we employed a well-known and highly reproducible rodent model of DEN-induced hepatocarcinogenesis. C57BL/6J male mice were injected with DEN (10 mg/kg i.p.), age day 12-15. In this model, male animals develop hepatocyte dysplasia at 6 mths and HCC in >90%. Transcript and protein expression of cyclin E was evident as early as 6 mths in dysplastic nodules (DNs), and significantly increased in HCCs. In contrast, there was little or undetectable cyclin E in normal liver and liver surrounding HCCs at all timepoints. Glutathione S transferase-pi form and cyclin E expression co-localised in DNs from liver in mice at 6 and 9 mths. Cyclin E/cdk2 kinase activity was also significantly upregulated in DNs, while increased proliferative activity by cyclin D1 and proliferating cell nuclear antigen (PCNA) in HCCs was observed at 9 mths, a timepoint where there was maximal p53 and p21 tumour suppressor expression. Aberrant cyclin E protein expression, including low molecular weight (LMW) isoforms were detected in HCCs and liver surrounding HCCs. Interestingly, sequencing analyses of p53 revealed a 1093-1361 nucleotide deletion in up to 90% of DNs, causing dysfunctional p53 nuclear localisation and export signalling. Because p53 directly signals to p21, co-immunoprecipitation studies were performed and revealed preferential binding of p21 to cyclin D1, rather than cyclin E, thereby allowing "escape" from the G1/S checkpoint. To directly test whether cyclin E regulates p53 expression in HCCs derived from DEN-treated male mice at 9 mths, we conducted cyclin E knockdown in primary HCC cells. This strategy resulted in increased p53 and p21 expression, as well as significant diminution of Bcl-xL, the p53-induced anti-apoptotic marker. Cell viability tetrazolium/formazan assay was significantly impaired in cyclin E RNAi targeted primary HCC cells. Conversely, chemical inhibition of p53 by pfithrin-α, augmented cyclin E, PCNA and Bcl-xL protein expression whilst cell viability was restored following co-treatment with MG-262, a 26S proteasome inhibitor. In contrast, overexpressing cyclin E in naïve primary hepatocytes enhanced PCNA expression, increased hepatocyte viability, downregulated p53 and its downstream signalling intermediate, p21. We next determined whether miRNA-34, a co-regulator of cyclin E and p53, was instrumental in the reciprocity between cyclin E and p53 as key "drivers"of hepatocarcinogenesis. Dysplastic liver and HCCs obtained from DEN-injected male mice were assayed for miR-34a,b,c. miR-34a and c were significantly upregulated in HCCs and dysplastic liver compared with normal liver. Similar trends were noted for miR-34a,b,c in human hepatitis C-related HCCs when compared with normal human liver. Importantly, this was associated with significantly enhanced cyclin E and p53 mRNA expression in human HCCs compared to normal and cirrhotic liver. In this murine model, there was disproportional and upregulation of functionally active cyclin E, miR-34a,c in DNs and early HCCs with congruent loss of p53 function associated with cell cycle checkpoint failure, diminished apoptosis and increased proliferative drive. In human HCV-related HCCs, miR-34a, p53 and cyclin E transcript levels were universally upregulated. When we performed in vitro experiments in murine primary hepatocytes and primary HCC cells, knocking down or overexpressing cyclin E did not affect miR-34 expression. However, stabilising p53 with MG-262 enhanced miR-34a,c expression (though not significant), whilst inhibiting p53 using pfithrin-α significantly reduced miR-34a,c. miR-34 may provide a plausible link to increased cyclin E expression, activity and increased proliferative drive in dysplastic and neoplastic liver in mice and humans. Gender disparity in human HCC is well described, with a strong male predominance. However, the role of sex hormones in hepatocarcinogenesis remains poorly defined. In order to determine if there are gender differences in the expression of cyclin E, effects on cell cycle regulators and tumour suppressors, dysplastic liver and HCCs were studied in DEN-treated C57BL/6J female mice. These mice displayed a significant reduction in dysplastic hepatocytes compared with intact DEN-treated males at 3, 6 mths, while HCC incidence, number and size of tumours were significantly diminished in females at up to 15 mths. In carcinogen-treated female mice, cyclin E (native and LMW isoforms) protein expression and kinase activity were reduced compared to males at 6-12 mths, with concomitant reduction in hepatocyte proliferation by PCNA and cyclin D1 expression. Unlike male mice, G1/S checkpoint is evident by robust p53-mediated apoptosis. To ascertain if these differences are attributable to the effects of oestradiol/progesterone (E/P) and/or testosterone, we conducted hormonal manipulation studies using the same carcinogen-model by performing ovariectomy in female animals and orchidectomy in male mice, in which some animals received E/P or testosterone supplementation. Castration of DEN-injected male mice resulted in a loss of cyclin E LMW isoforms compared to intact males, diminution of cyclin E kinase activity and phospho-retinoblastoma expression. There was also induction of p53-mediated apoptosis in dysplastic hepatocytes, leading to a reduction in number of DNs by 6 mths. These anti-proliferative and pro-apoptotic effects were magnified by E/P replacement in castrated DEN-treated males. In contrast, testosterone-replacement in ovariectomised-female mice exhibited accelerated hepatocarcinogenesis compared to intact female DEN-treated animals and displayed LMW cyclin E isoforms similar to those detected in DEN-injected intact males. In further analyses, there was increased oestrogen receptor-α (ERα) transcript and protein expression in HCCs derived from DEN-injected intact male mice, and in dysplastic liver from castrated male mice replaced with E/P. E/P replacement and testosterone withdrawal were associated with ERα expression, the loss of cyclin E LMW isoforms, intact cdk2 expression, functional G1/S checkpoint control and induction of p53-mediated apoptotic cell death in preneoplastic hepatocytes. Further, oestrogen (E2) stimulation had varying effects on cell cycle regulation and viability in primary hepatocytes and HCC cells. As there was little to no significant correlation between ERα and its downstream target, c-myc transcript levels, we propose that E2/ERα signalling may be operative via other pathways to subsequently activate p53. These findings open up tantalising avenues to further explore the inter-regulatory signalling pathways between E2/ERα, cell cycle regulators and the tumour suppressor, p53.