An investigation of the effect of oestrogen on longitudinal growth

In the absence of readily available physiological models of human growth, the effects of oestradiol on the human C28/I2 chondrocyte cell line were studied. The classical oestrogen receptors, ERα and ERβ, were shown to be expressed in both murine and human chondrocyte cell lines. Oestradiol and related chemicals, which alter the function of the oestrogen receptors (ER), were exploited to tease out the different functions of each ER in the growth plate. In the absence of foetal bovine serum, oestradiol had no effect on proliferation, differentiation or apoptosis of chondrocyte cells in monolayer culture or on the growth of the foetal metatarsal culture system. In addition, oestradiol did not convey a protective effect on chondrocytes exposed to the pro-inflammatory cytokines, tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in monolayer culture. However, endogenous oestrogen appears to play an important role in maintaining chondrocyte proliferation in monolayer culture and mineralisation in metatarsal culture as reflected by the inhibitory effects of Faslodex, the non-specific ER antagonist, on chondrocytes and metatarsals in culture. In the presence of methyl-piperidino-pyrazole (MPP), a selective ERα antagonist, and raloxifene, a selective oestrogen receptor modulator with higher ERβ binding affinity, a reduction in chondrocyte proliferation and increase in apoptosis was observed in murine and human chondrocytes. Similarly, a marked reduction in linear growth occurred when foetal murine metatarsals were exposed to MPP and raloxifene in combination. A less marked reduction in growth was observed in MPP-treated metatarsals. These findings suggest that the oestrogen receptors may have opposing actions in the growth plate with ERβ acting like a brake on chondrocyte growth and ERα promoting growth. ERβ may regulate cell proliferation through control of cell cycle modulators affecting G1/S phase transition as MPP and raloxifene in combination reduced cyclin E and p53 levels on Western blot analysis. The aim of the second part of my thesis was to investigate the effect of oral oestrogen on linear growth in girls with primary ovarian insufficiency (POI). A retrospective review of girls with POI treated at a tertiary endocrinology clinic over an 11 year period was performed. As expected the majority of girls with POI had Turner syndrome (TS; 83.7%). Non-TS associated POI was rare and the leading cause was iatrogenic secondary to the effects of total body irradiation for bone marrow transplantation (12.8%). A significant proportion of these girls developed POI after full pubertal development so few cases were available to investigate the effect of oestrogen on growth. The oral oestrogen regime followed in individual patients with TS was highly variable so it was not possible to assess the effects of dose on height velocity or bone maturation in this retrospective audit. However, the second clinical study examined in detail the effect of oestrogen on growth in TS girls who received a standardised course of oral ethinylestradiol for pubertal induction and a standard dose of growth hormone (10 mg/m2/week). These girls participated in a prospective randomised double-blind placebo-controlled multi-centre study of growth promoting treatment in TS. The girls were initially randomised to oxandrolone or placebo at 9 years of age and further randomised to oral ethinylestradiol at 12 or 14 years of age. The results of this study are embargoed until published. The laboratory effects of oestradiol found in this thesis suggest that ERα may stimulate or maintain growth, and ERβ may inhibit growth. The obvious question is how these observations might be involved in the complex relationship between puberty, oestrogen and height velocity in humans. As affinity studies show that the half maximal effective concentration (EC50) of ERα is achieved at slightly lower concentrations of oestradiol than ERβ it is conceivable that the ERα effect could predominate at lower systemic oestradiol concentrations and that ERβ could become more important at higher concentrations for example in later puberty. Alternatively, it is possible that the expression of ERα reduces or ERβ increases in the growth plate after reaching peak height velocity

The effect of environmental, physical, and nutritional factors on in vitro fertilisation

Folliculogenesis, fertilisation and implantation of a human embryo requires unity of many different pathways. The literature review discusses folliculogensis, implantation and the potential affect that endocrine disrupting agents (EDAs) and Vitamin D can have on infertility and Polycystic Ovary Syndrome (PCOS). Can physical and immune-modulating treatments such as Endometrial scratching (ES) and intralipid aid in the treatment of recurrent implantation failure (RIF)?EDAs were detected in the PCOS and controls, only the polyfluoroalkyl-agent (PFAA) perfluorooctane sulfonate (PFOS) had a significantly higher concentration in the PCOS group, (4.11±1.62 ng/ml vs. 3.11±1.05ng/ml, p=0.03). Whole group analysis showed PFAAs demonstrated significant positive correlations with testosterone. PFAAs, Polychlorinated Biphenyls and Dichlorodiphenyldichloroethylene (p,p-DDE) demonstrated significant positive correlations with cleavage rates (p=0.01 to 0.04), thus these chemicals may disrupt cell division in early embryo development. There was no correlation between EDAs and pregnancy in either group.A pilot study was designed to see what effects Vitamin D levels had on IVF outcomes in PCOS and control subjects. A linear trend was observed between Vitamin D levels and fertilisation rates in the PCOS group suggesting a possible relationship between Vitamin D and oocyte maturation in this distinct population of women.An observational study was designed to assess the effect of ES on women undergoing IVF. No increase in clinical pregnancy rates (p=0.54) was demonstrated in women with RIF, however clinical pregnancy rates were significantly reduced (p=0.04) in women undergoing their second cycle of IVF. These findings suggest that this treatment is not effective in the treatment of RIF.The effect of intralipid to aid implantation in women with RIF has been postulated but not confirmed. A pilot study was designed to observe the effect of intralipid on NK-cell populations of women with RIF and controls undergoing IVF. The findings demonstrated no effect of intralipid on NK-cell populations in women with RIF

POLYCYSTIC OVARY SYNDROME COAGULATION AND METABOLIC STUDIES

The polycystic ovary syndrome (PCOS) is a heterogeneous disorder in women characterised by chronic ovulatory failure, hyperandrogenaemia, and insulin resistance. Some women are completely asymptomatic and others present with extreme menstrual disturbance, severe hirsutism, infertility and recurrent miscarriage. The pathophysiology of PCOS is not completely understood, but it is thought that insulin resistance plays a central role. In normal subjects, non-diabetic obese patients and patients with non-insulin dependent diabetes, insulin resistance is associated with elevated plasminogen activator inhibitor-1 (PAI-1) levels. PAI-1 is a glycoprotein, which inhibits the formation of plasmin (a proteolytic enzyme). Plasmin aids fibrinolysis and extracellular proteolysis. High PAI-1 and low plasmin levels increase the risk of thrombosis and impair extracellular proteolysis required in ovarian follicle growth, ovulation and embryo implantation. This study was designed to determine whether elevated plasminogen activator inhibitor-1 (PAI-1) was associated with the insulin resistance present in PCOS, investigate its possible role in the causation of anovulation and recurrent pregnancy loss in these women and ascertain whether it was an additional thrombotic risk factor so that clinicians and patients could take appropriate measures to reduce this risk In a pilot study, systemic PAI-1 activity was significantly elevated in oligomenorrhoiec women with PCOS. A larger study supported these findings, but demonstrated that obesity was a significant confounding factor, as the increase in PAI-1activity disappeared when standardised for weight. Activated Protein-C (APC) resistance was subsequently tested in these women because of the unexpected finding of an increased prevalence of a positive family history of thrombosis in women with PCOS compared with controls, but there was no increase in the prevalence of APC-resistance in PCOS. In another project, the cellular distribution of PAI-1 protein in human ovaries was described for the first time using immunohistochemistry. It was localised to the granulosa and theca cell compartments in both polycystic and normal ovaries, however there was no significant difference in the intensity of PAI-l staining between both groups on image analysis. PAI-1 messenger RNA expression was also evaluated in these biopsies by in-situ hybridisation, but no signal was detected suggesting that there was either a low overall RNA preservation in the tissues, or an insufficient sensitivity of the cocktail of oligonucleotide probes used. This study did not support the hypothesis that elevated PAI-1 was a feature of PCOS, however the in-situ location of PAI-1 protein was demonstrated for the first time in the human ovary and consistent with a previously suspected role in ovulation. The results did not support a role for PAI-1 in anovulation, recurrent miscarriage or increased thrombosis in PCOS

Regulation of sex hormone binding globulin and insulin-like growth factor binding protein-1

Women with polycystic ovary syndrome (PCOS) present most commonly with hirsutism and/or anovulation. The prevalence of hirsutism and/or menstrual irregularity is increased in obese women with PCOS compared with lean women with PCOS. It is, therefore, possible that factors associated with obesity exacerbate the symptoms and the underlying hormonal abnormality of this condition. Sex hormone binding globulin (SHBG) and insulin-like growth factor binding protein-1 (IGFBP1) concentrations are reduced in PCOS and decline during puberty in boys and girls. The regulation of sex hormone binding globulin has been thought to be primarily by the sex steroid hormones and that of IGFBP1 by insulin. The hypothesis of this thesis is that dietary factors are more important than the sex steroids in the regulation of hepatic SHBG production and that this is of relevance in understanding the relationship between obesity and hirsutism in women with polcystic ovary syndrome. In addition insulin also regulates hepatic IGFBP1 production implying that the two binding proteins may be co-regulated. The role of sex steroids in the regulation of IGFBP1 is not known. The regulation of hepatic production of SHBG and IGFBP1 by insulin, IGF-1 and the sex steroids was studied in-vitro by cell culture experiments on Hepatocarcinoma G2 cells. The relationship of dietary factors on androgen metabolism and circulating SHBG and IGFBP1concentrations was investigated in three separate studies. In the first two studies women with PCOS were studied before and after calorie restriction and during an oral glucose tolerance test (OGTT). A third study conducted over a 24 hour period investigated the diurnal variation in SHBG and IGFBP1 related to that of insulin and IGF-1. The cell culture experiments demonstrated a role of insulin and IGF-1 as inhibitors of hepatic SHBG production but only insulin inhibited the secretion of IGFBP1. The effect of the sex steroids was less clear since testosterone increased SHBG production while oestradiol had no effect. The sex steroids had no effect on IGFBP1 production. Weight loss following a four week very low calorie diet was associated with a significant increase in SHBG and IGFBP1 concentrations mirrored by a decrease in insulin and IGF-1. This resulted in a decrease in free testosterone although the total testosterone concentration did not change. The inverse relationship of SHBG and IGFBP1 with insulin was further confirmed in the OGTT and 24 hour study although the role of IGF-1 was less clear. The time course of the changes was significantly longer for SHBG than IGFBP1. These studies demonstrate that insulin is a primary regulator of SHBG and IGFBP1 synthesis by the liver. The two binding proteins may be co-regulated by insulin but other factors including the sex steroids may alter their half-lives in the circulation and therefore their serum concentration under various physiological and pathological conditions

Role of Sex Hormones in Human Body

Gonadal Steroids hormones play an important role in the reproductive and non-reproductive systems. Estrogen has important rule in cardiovascular system as it has vasodilator effect and reduces or prevents platelet activation. In addition, it improves the profile of circulating lipoproteins. All of which may explain why women at premenopausal age are less likely to have heart disease than menopause women or men. E2 play a grate effect on the skeletal system as it is one of the strongest regulators of osteoblast and osteoclast function, and it is responsible for the reduction of adipose tissue and regulation of the body weight, and also has dermatological effect,hence it stimulates the proliferation of keratinocytes and prevents their apoptosis, in addition to the progesterone which increases collagen synthesis. Estrogen is necessary for the functioning and integrity of the tissues of the urinary system specially of the lower urinary tract. Sex steroid are crucial for nervous system, as progesterone is important for production of neurosteroid, and estrogen is currently used in Parkinson’s and Alzheimer’s disease because of its effects on mental health. The androgens also have a crucial biological effects on neural, muscle, bone, adipose tissue,prostate, cardiovascular, haemopoietic, and the reproductive systems. The gonadal steroid hormones play an important role in immune system and regulating the immune response against different viral or bacterial infections

Clinical applications of the progesterone receptor antagonist mifepristone during early pregnancy and its application in therapeutic abortion

Mifepristone (RU 38,486) is a new synthetic steroid which is antagonistic to progesterone at the receptor level. Mifepristone has been demonstrated to cause spontaneous abortion of early pregnancies when taken orally. When used in combination with a prostaglandin analogue the termination success rate is increased. The studies performed in this thesis were designed to further investigate the clinical effects of mifepristone in the first and second trimesters of pregnancy, and to study the mechanism of action of the drug. These studies examined: 1) . The efficacy and side-effects of mifepristone (600mg) in combination with a prostaglandin E1 lmg (Gemeprost) pessary for early termination of pregnancy. 2) . The effect of mifepristone on prostaglandin metabolite levels in the first and second trimesters of pregnancy. 3). The effect of mifepristone on progesterone and oestrogen receptor concentrations in the decidua and placenta in early pregnancy. 4). The physiological and clinical effects of progesterone inhibition with mifepristone in the second trimester. 5). The placental transfer of mifepristone during the second trimester and its influence upon maternal and fetal steroid concentrations