Organised Medicine Lectures PDF

Iron Fe2+ Calcium Ca2+ Copper Cu2+ Phosphorus PO4 Bicarbonate HCO3- CCK cholecystokinin WEEK 1 MALNUTRITION Nutrition – the provision of nourishment to cells, tissues, organs, systems and the body as a whole. It is how food influences our body as a whole. It includes public health, agricultur...

Iron Fe2+ Calcium Ca2+ Copper Cu2+ Phosphorus PO4 Bicarbonate HCO3- CCK cholecystokinin WEEK 1 MALNUTRITION Nutrition – the provision of nourishment to cells, tissues, organs, systems and the body as a whole. It is how food influences our body as a whole. It includes public health, agriculture, animal production, biochemistry and physiology. It is clinically important for feeding patients and dietetics (using diet as therapy/prevention of disease). The core concepts of nutrition: - Nutrient balance is the buffering effect of body stores. - Nutrient turnover is when metabolic substrates are continually being utilised and replaced. It allows for rapid adjustment to changes in metabolic state and the potential dysfunction if rates of utilisation and synthesis are mismatched. - Nutrient flux – a measure of the activity of the metabolic pathway. Not necessarily related to the size of the metabolic pool. - Metabolic pool: o Functional pool – direct involvement in body functions o Storage pool – provides buffering effect (can be made available to the functional pool if required) o Precursor pool – provides the substrate for nutrient/metabolite synthesis. - Adaptation to altered nutrient supply – minimises the consequences of such alterations. The greater the capacity to respond to adverse nutritional states the greater the capacity to survive those states. Provides all the nutrients which satisfy all the needs of our body. Macronutrients provide energy, they include fat, protein, carbohydrates and alcohol. Micronutrients are vitamins and minerals. The body also needs water as part of a well-balanced diet. Dietary carbohydrates are categorised based on polymerisation: - Monosaccharides and disaccharides – glucose (honey, grapes), fructose (fruits, veg, honey), galactose, lactose (milk) and sucrose. - Oligosaccharides – inulin (fructose polymer), between 3 and 9 monomers. Mostly not digested. - Polysaccharides – starches (potatoes, cereal grains, absorbed in small intestine) and non- starch polysaccharides (NSPs – dietary fibre, not digested and end up in colon). Carbohydrates provide energy and fuel for central nervous system (CNS). It allows for the control of blood glucose and insulin metabolism. Carbohydrates affect satiety/gastric emptying (affecting absorption), cholesterol and triglyceride metabolism, fermentation (production of short-chain fatty acids) and control of colonic epithelial cell function and effects bowel laxation/motor activity. Glycaemic index – allows quantitative comparison of blood glucose responses to ingestion of equivalent amounts of CHO from different foods relative to pure glucose (GI=100). Not all foods containing free sugars have high GI (complex and simple carbohydrates are irrelevant for this). Low GI foods (70) include white bread, they cause a greater response. Diets with high glycaemic index foods are detrimental to diabetes (lead to increased risk of type 2). Glycaemic load is important for measuring total carbohydrate content of the diet. It determines whether the diet is high or low GI. The type of carbohydrate is more important than the amount when controlling diabetes. Recommended intake of: energy requirements - Carbohydrates – 50% of dietary energy. 5% of calorie intake should be from free sugars (cereals, chocolate, sweets). - Dietary fibre – 30g a day for people over 16. Dietary fibre, by definition should have a health benefit and end up in the colon. They are classified as soluble (e.g. pectin) and insoluble (cellulose). They have a bulking effect (increases bulk of the stool so reduces constipation), speed up colonic transit (reduces among of time that carcinogenic compounds are present int the body) and lower cholesterol. Another benefit is fermentation provides short-chain fatty acids (e.g. butyrate, acetate and propionate), these are the main energy source of the colonocytes. - Fat – 35%. Saturated fat should be less than 11% of energy intake (ideally under 7%). Dietary cholesterol should be below 300mg/day, especially in diabetics. Substitute saturated fats with unsaturated fats, particularly PUFAs and whole grain sources. Substituting saturated fats with refined sugars doesn’t reduce CV risk. - Protein requirements – 15%. Needed for growth and repair. Highest in 0-5 year olds. In adults, it is RN I= 0.8g of protein/kg/day. Amino acids should be essential and non-essential and from animals and plants due to differences in content, balance and digestibility. Nitrogen balance is used to determine the protein requirements. 1g N2 = 6.25g of protein. - Energy requirements – to estimate resting metabolic rate, use Henry equations then multiply by physical activity level. Resting energy requirements increase in illness, trauma and infection. 95% of what we eat is digestible and can be used as metabolizable energy. - Dietary recommendations to reduce risk of cardiovascular disease: less than 6g of salt intake per day, Mediterranean style diet (tomatoes – flavonoids, 2 portions of fish per week, one being oily – mackerel, herring, salmon, sardines, trout), increase fibre to 30g, 5 portions of fruit and vegetables a day, maintain healthy weight, do 30 minutes of moderate physical activity 5 times per week so it makes you warmer and breathe harder. Physical exercise reduces mortality due to cardiovascular disease, improves vascular function, lowers cholesterol and triglyceride levels and reduces bp. 15% of salts are naturally present, 70% is from processed food and 15% is added ourselves. Factors affecting energy requirements – resting metabolism (depends on size, age, growth, food) and physical activity (job and non-occupational). Overall physical activity level (PAL) is the ratio of total energy expenditure to resting metabolic rate (RMR). It is 1.4 for sedentary individuals, 1.7 for moderately active individuals etc. Vitamins – organic substances required in small amounts for normal metabolism but cannot be synthesized by the body in sufficient quantities. They are divided on solubility. Water soluble compounds are B and C. Fat soluble vitamins are A, D, E and K, they have multiple actions. Water soluble vitamins are needed for:  - Intermediary energy metabolism – thiamine, nicotinic acid etc. Coenzymes involved in glycolysis etc. - Anaemia preventing – B12 and folate. They are involved in thymidylate synthesis (important for rapid cell division and DNA synthesis). B12 is present in animal products (but also required an intrinsic factor from the gut to be absorbed). If this intrinsic factor is not present it causes a function deficiency, not a dietary deficiency. Deficiency can cause megaloblastic anaemia and neuropathy (impaired synthesis of myelin). Folate in found in green vegetables. Deficiency can cause anaemia and increased risk of neural tube defects (so is supplemented in pregnant women). Methionine synthetase (a B12 dependent enzyme which catalyses a reaction that yields free folate in tissues via methylation of homocysteine to methionine) provides the link between the physiological functions of B12 and folate. - Antioxidants – vitamin C and E Vitamin D is a hormone as well as a vitamin because it can by synthesised in our skin. D3 (cholecalciferol) is present in fish oils, egg yolk and butter. 7-dehydrochoelsterol is found in animal fats, plant sterols (skin-UV light). UV light converts 7-dehydrocholesterol to pre-vitamin D3. Heat then converts this into vitamin D3. It then enters the circulation. It hydroxylated in the liver to be stored/transported as calcidiol. It then hydroxylated by the kidneys to become calcitriol (active form). It targets intestine, kidney, bone and parathyroid gland. Actions of vitamin D: - Intestinal absorption of Ca2+ and PO4. It increased the synthesis of the calcium binding protein (calbindin). - Renal reabsorption of Ca2+ and PO4 for normal bone formation. Low levels of plasma Ca2+ stimulates STH and vitamin D synthesis. Activated vitamin D increases intestinal Ca2+ absorption and regulated Ca2+ excretion from the kidney and bone. When plasma Ca2+ is high, calcitonin is secreted from the thyroid gland and promotes Ca2+ excretion in kidney and prevents bone from releasing Ca2+. - Neuromuscular and immune functions, apoptosis and inflammation (VDRs form dimers with RXR and regulate gene expression). - Vitamin d deficiency causes rickets (in children, weight of body is too much for poorly mineralised bones) and osteomalacia (in adults). - Vitamin d toxicity – (>250μg/day) causes weakness, nausea, loss of appetite, headache, abdominal pains, cramp and diarrhoea. Also hypercalcaemia and calcification of soft tissue, vessels etc which will impair function. Minerals – inorganic elements that have a physiological function. Must be supplied by diet. If they are required in g/day, they are macrominerals. If in mg or μg they are trace elements. Fe2+ is needed for haemoglobin, electron transport and cytochrome P450. The body stores it in reticulo-endothelial system (spleen and liver) and bone marrow. Plasma ferritin is the main storage protein. Transferrin transfers Fe2+ to bone marrow. Requirements change with age, gender, illness etc. Dietary sources are haem Fe2+ (from meat) or non-haem Fe2+ (from vegetables). There are different absorption mechanisms into the mucosal cells. Absorption is approximately 10% of intake and can be up or down regulated. Absorption is enhanced by reducing agents (e.g. ascorbic acid) as Fe2+. Other enhancers of absorption are vitamin C, fructose, alcohol, meat (dietary) and Fe2+ deficiency and anaemia and pregnancy (physiological). Inhibitors are tannins, phosphates, bran, lignin, some minerals (e.g. Ca2+), Fe2+ overload and Cu2+ deficiency. Positive energy balance – development of obesity, growth, pregnancy, recovery from depletion. Negative energy balance – easting disease, anorexia nervosa, starvation, voluntary weight loss. Units of energy – SI unit (joules) or non-SI unit (calorie – kcal=103 cal). 1kcal=3.184kJ Components of energy expenditure: - Basal metabolic rate or resting metabolic rate (BMR or RMR) – 60-70% of total energy expenditure. Heart and respiratory functions account for 10%, protein turnover is 25%, there is also fat turnover and carbohydrate turnover and the maintenance of ion gradients across membranes. - Energy expenditure of physical activity (EEA) – 25-30% - Diet induced thermogenesis (DIT) – increase in metabolic rate after a meal, about 10%. Obligatory thermogenesis includes protein (20-25%), CHO (5-7%) and fat (2-4%). Adaptive thermogenesis or facultative thermogenesis occurs due to an increase in sympathetic nervous system activity in response to carbohydrate feeding. Thermic effect of exercise and illness: the thermic effect of exercise in sedentary individuals is 10% and about 15-30% in athletes. The thermic effect of illness is varied, e.g. the thermic effect of post- operation is +10%, for severe infection it is +30-60% and for 3rd degree burns it is +50-100%. Dietary reference values (DVR): - Estimated average requirement (EAR) – average, if we all have the same, half will be underfed and half will be overfed. - Reference nutrient intake (RNI) – meets or exceeds needs of 97.5% of a group. It is the normal distribution using 2 standard deviations. CHD, Lifestyle and Comorbidities Risk factors for cardiovascular disease – obesity, diabetes, diet, smoking, lack of physical exercise, biological (genetics, gender). Generally modifiable. Hypertension diet – DASH diet (dietary approaches to stop hypertension): - High in fruit and veg and non-starch polysaccharides (NSPs), low fat dairy and reduced red meat. - Increase intake of K+ (reduces bp), calcium and magnesium - Reduce Na+ in the diet to reduce bp. Combining the DASH diet with low-Na+ diet reduces bp more than either of them by themselves. Increasing consumption of vegetables increases consumption of flavonoids which are antioxidants. Hypercholesterolaemia leads to increased risk of atherosclerosis, coronary artery disease, angina and heart attack. There is no relationship between dietary cholesterol and coronary heart disease. Increasing dietary cholesterol leads to small increases in LDL-C levels but effects vary between individuals. Saturated fats and trans fats increase LDL-C levels and are associated with increased cardiovascular risk. Soluble non-starch polysaccharides decrease LDL-C, bind bile salts and prevent their reuptake. Higher intake of fibre is associated with lower rates of cardiovascular disease. They also have favourable effects on weight and insulin sensitivity. Phytosterols (stanols) also reduce cholesterol uptake from the GIT (as well as bile), they have a similar structure to cholesterol but are found in plants. They are not absorbed and inhibit the absorption of cholesterol. Platelet aggregation – n-3 PUFAs can be plant derived or marine derived. People who were taking these fatty acids supplements were less likely to die in the days following a heart attack. The mechanism is unknown but it may be due to decreased thrombosis (inhibition of platelet aggregation), improvement of endothelial function (releases compounds that release vasodilatation and inhibit platelet aggregation). Normally via the COX pathway, arachidonic acid is metabolised to thromboxane (TXA2), causing platelet aggregation. The formation of PGI2 causes vasodilatation but also reduced platelet activation. EPA (e.g. of a fish oil) is metabolised the same way but produces TXA3 which is worse at activating platelets. PGI3 is also produced, which is equally effective as PGI2 (prostacyclin). Undernutrition and Dietetics Dietician – state registered health care professional Dietetics – use of diet in the prevention/treatment of disease. To use diet to treat disease, there are some basic requirements: - Know normal nutritional requirements - Identify the nature of the disorder - Assess patient’s nutritional status and intake – this involves: o Anthropometry – height, weight, girth using BMI, growth charts and reference values. o Body composition – skin folds, MUAC, body water (BIA, DEXA, MRI) o Biochemistry and haematology – plasma protein/electrolytes, vitamins/lipids, full blood count o Function – muscle strength, physical signs e.g. wound healing o Dietary assessment – dairy, FFQs, 24 hour recall - Know the composition of foods, supplements and artificial foods - Devise a meal plan/regimen - Monitor compliance and progression - Reassess and adjust as appropriate. Malnutrition Universal Screening Tool (MUST) – involves questions regarding unintentional weight loss, changes in eating habits, normal weight and height. Undernutrition – can be general (deficiency of calories, negative energy balance) or specific (deficiency of an essential nutrient). It can also be primary (related to the diet) or secondary (related to an illness or condition). Undernutrition can be caused by: - Reduced delivery of nutrients to the gastrointestinal system – decreased food availability, mechanical or functional (neurological) - Increased demand for nutrients – physiological, pathological - Inability for gastrointestinal system to absorb nutrients – intrinsic problem, post-surgery In response to calorie undernutrition: - Fatty acids are used as energy source, 3 weeks for full adaptation. Persists while fat stores last. Reduced insulin suppression of hydrolysis. - Ketone bodies supply brain requirements.  - Lack of carbohydrate/protein leads to reduced insulin - Reduced insulin suppression of skeletal muscle proteolysis – amino acids released - Finally tissue protein breakdown and death Protein-energy malnutrition (PEM) – huge problem worldwide. The form depends on protein- carbohydrate balance: - Dry PEM – Marasmus (no oedema, general). Severe calorie and protein deficiency - Wet PEM – kwashiorkor (oedema, specific). Severe protein deficiency – linked to low plasma albumin concentrations (hypoalbuminaemia). Effects of undernutrition (general): - Immune system – impaired ability to fight infections and repair wounds - Skeletal muscle – reduced muscle size and strength, impairing the ability to exercise or work - Digestive system – decreased production of stomach acid. Shrinking of stomach. Frequent, often fatal, diarrhoea. - Cardiovascular system – reduced heart size, low bp. Ultimately heart failure. - Respiratory system – slow breathing and reduced lung capacity. Ultimately respiratory failure. - Reproductive system – reduced size of ovaries and testes. Cessation of menstrual periods. - Nervous system – apathy and irritability. Mental dysfunction - Metabolism – low body temperature (hypothermia). Fluid accumulation in the arms, legs and abdomen. Disappearance of fat - Blood – anaemia - Skin and hair – thin, dry, inelastic skin. Dry, sparse hair that falls out easily. Weight loss is considered high/significant if over 5%, clinically 10% is used more. Refeeding syndrome – carbohydrate intake increases, causing increase in circulating insulin and greater demand for intracellular electrolytes. Causes hypophosphataemia (30% women>35%. - High % abdominal fat. - % tissue fat NAFLD >5% - Waist circumference men>102cm women>88cm. more useful. Waist circumference can be a good indicator of intra-abdominal fat. It is related to waist:hip ratio. Apples have a greater risk of complications over pears. It is more related to visceral fat than BMI. Abdominal obesity is more significant of a risk factor for heart attack than raised BMI. - Waist:hip ratio men>0.9 women>0.85. - Measuring skin folds assumes a constant relationship between subcutaneous and % body fat. It can be measured all over the body, commonly on biceps and triceps Fat can be measured using MRI. The prevalence of obesity is rising in many developed countries and also in eastern countries. In the Asian population there are different levels of BMI risks due to greater amount of abdominal obesity, increased CV risk. Obesity is more common among people from more deprived areas, older age groups, some black and minority ethnic groups and people with disabilities. Dietary risk factors for obesity: - Fat – unbalanced proportions of saturated fats and reduced n-3 fatty acids, ‘fast food’ etc, passive overconsumption of energy, reduced ability to oxidise fat causes higher risk of gaining weight (genetic/higher predisposition). - Sugar – rapid increase in blood glucose, overconsumption of energy, free sugars - Alcohol – decreases fat oxidation and increases fat storage. 7kcal/g. Causes of Obesity Genetic aspects of obesity – due to rare genetic disorders such as Prader-Willi syndrome, hypothalamic disorder (excessive appetite). There is higher correlation of obesity measures among monozygotic pairs than among dizygotic pairs. Genetics appear to account for 50-70% of the differences in BMI in later life in twins brought up apart. There is no relationship between adoptive parents and adoptee in relation to body weight class. Leptin deficiency – leptin reduces appetite, however, very few people have been found to be leptin deficient (rare genetic disorder). People lacking in leptin are massively obese. Obese patients normally have raised leptin levels. Genetic susceptibility – there is a strong relationship between biological parents and adoptee for a whole range of body fatness. There is no relationship between adoptive parents and adoptee in relation to body class. This suggests body weight is caused by genetics not the environment (i.e. some people may be more susceptible). The recent increase in obesity cannot be due to genetic change of population but due to lifestyle changes. There are populations of Pima Indians in Arizona and in Mexico, both with similar genetics. However, there are high levels of obesity in Arizona due to sedentary lifestyle and high fat diet, and low levels of obesity in Mexico where people are more physically active. Control of food intake – rats fed ad libitum (as desired) on laboratory chow maintain weight (hunger and satiety). Rats given palatable foods gain weight (appetite more powerful than hunger). The hypothalamus controls food intake. The hypothalamus (arcuate nucleus – ARC) has a role in integration of appetite signals. It is a region of the brain not protected by BBB, which allows gut hormones to pass into the brain. Energy dense foods mean you still have a positive energy balance, even without eating a large volume of food. Fat is energy dense and increases palatability of food. Conversion of excess fat to body fat is very efficient. An increase in carbohydrate intake will lead to increase in conversion of fat in the diet to body fat. There is reduced requirement for physical activity due to occupations, cars, telephones and computers. Inactivity leads to weight gain and weight gain leads to inactivity. Evolutionary pressure – drive to eat as much as possible when food is available. Encase it becomes no longer available. This is driven by the arcuate nucleus (ARC) in the hypothalamus which has a role in integration of appetite signals. There are 2 sets of hormones in the arcuate nucleus, neurones which release NPY or AgRP (release neuropeptides which stimulate orexigenic neurons which stimulate food intake and reward circuits) and neurones which release POMC (proopiomelanocortin) and CART (cocaine and amphetamine regulated transcript) (which stimulate anorexigenic neurones which inhibit food intake and reward circuits). Food reinforcement – eating is a pleasurable activity as it stimulates reward centres. Food deprivation may increase reinforcing value of food, it increases motivation to eat. Food environment and drivers of obesity – marketing, information labelling, price, access, out of home food offer. The sugar tax is an attempted prevention strategy. Foods such as pure fruit juice and drinks with high milk content are exempt. Thin outside fat inside (TOFI) – visceral fat leads to cardiovascular disease. You can be obese but still metabolically healthy (if fat accumulates only under the skin). This is rare. Location of fat storage is important. Energy balance is mainly regulated through control of food intake affected by: hunger (desire/need for food), appetite (psychological desire to eat certain food), satiation (feeling of fullness that terminates meal) and satiety (feeling of repletion that inhibits further meals). POMC undergoes post translational modification to generate melanocortins (act at melanocortin receptors). AgRP is an endogenous antagonist of the melanocortin-4 receptor. Gut peptides involved in regulation of food intake: - Gastric distension leads to release of a number of hormones from the enteroendocrine cells: cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1), oxyntomodulin, peptide YY (PYY), apolipoprotein A-IV, enterostatin. These all inhibit food intake. Leptin is relased from adipose tissue and inhibits food intake. Leptin is a peptide hormone secreted from adipose tissue. Decreased food intake leads to decreased leptin levels. This is reversed by refeeding or insulin. Production of leptin correlates with amount of adipose tissue (energy stores). Leptin resistance can occur so it works less. Mutations causing absence of leptin leads to severe obesity. You can give human recombinant leptin which helps control appetite and lose weight. Leptin deficiency is a rare genetic mutation and does not explain the increase in obesity. Leptin is transported into the brain where it: - Inhibits NPY AgRP release - Activates the POMC/CART neurons  - Switch from appetite stimulation to appetite suppression. Insulin – plasma increase at times of positive energy balance and decrease at times of negative energy balance. Crosses the BBB through receptor-mediated process. Decreases intake and body weight due to actions on ARC. Ghrelin – produced from GIT, secreted on anticipation of food, acts centrally at ARC or brain stem to stimulate food intake through NPY and AgRP. Hunger at certain times of day is due to ghrelin. Levels peak before a meal as it stimulates hunger. If you are obese and trying to lose weight, ghrelin levels increase. Ghrelin may be involved in reward, which causes increase in risk taking. Ghrelin antagonists are in development for treatment of obesity. It causes weight loss. PYY – PYY is released in repsonse to certain foods entering the GI tract, it can act directly on the ARC to inhibit appetite or via the panncreas to stimulate the relase of insulin (acts within ARC to inhbit appetite). Secreted from distal GIT dependent (e.g. protein > fat or carbohydrate). Also stimulated by CCK, gastric acid and bile. PYY1-36 cleaved to PYY3-36- acts at NPY Y2 receptor. It inhibits food intake, possibly through vagal inputs. PYY levels stay elevated for 12 hours post meal. It decreases food intake in lean and obese people (no resistance). Release of NPY is switched off. CCK (cholecystokinin) – released from duodenum and jejunum in response to fatty acids/fatty foods. Acts on receptors on the vagal nerve and possible crosses the BBB. This inhibits food intake. Can act directly at the ARC or indirectly via the vagus to inhibit food intake. Reward centres are involved in food intake. Appetite is linked to reward processes in the brain, e.g. opioid receptors (endorphins), cannabinoid receptors and dopamine. If you eat food that tastes nice, this reward will be greater. There is possible reinforcement of motivation to find and consume foods of high incentive/energy content. Dopamine and reward – feeding is associated with dopamine release in the dorsal striatum. The degree of pleasure correlates with the amount of dopamine released. Bupropion is a dopamine reuptake inhibitor which is involved in weight loss. The inhibition of this reuptake leads to an increase in the amount of dopamine in the synapse, leading to a prolonged activation of the dopamine receptor. Nicotine suppresses appetite, so people who have given up smoking may notice weight gain because they are eating more. Cannabinoid receptors – food reward depends on CB1 receptor activation. It may regulate dopaminergic system. Rimonabant is a CB1 receptor antagonist. Management of Obesity Ladder of intervention (tackling obesity) – eliminate/restrict choice, guide choice through disincentives/incentives, change default, provide information, do nothing. Becomes less intrusive. Overview of the whole systems approach – set-up, building the local picture, mapping the local system, action, managing the system network, reflect and refresh. To produce clinically significant weight loss, you must restrict calories and do aerobic exercise. Lifestyle modification is most important, including dietary energy restriction and physical activity. There are limited drug therapies which can assist and maintain weight loss. Bariatric surgery induces large weight loss and leads to remission of type 2 diabetes but is restricted to the severely obese with significant co-morbidities. Treatment is affected by willingness to give up enjoyment, ability to alter long-term feeding habits, physiological responses to weight loss. It is therefore important to set realistic goals and to be committed. NICE obesity guidelines – total energy intake4) linkage. After about eight to ten glucosyl residues, there is a branch containing an α(1->6) linkage. Uridine diphosphate (UDP)-glucose, the building block of glycogen, is synthesised from glucose 1-phosphate and UTP by UDP-glucose pyrophosphorylase. Glucose from UDP-glucose is transferred to the nonreducing ends of glycogen chains by primer- requiring glycogen synthase, which makes α(1->4) linkages. The primer is made by glycogenin. Branches are formed by amlyo-α(1->4)->alpha(1->6)- transglucosidase (common name, glucosyl 4:6 transferase), which transfers a set of six to eight glucosyl residues from the nonreducing end of the glycogen chain (breaking an α(1->4) linkage, and attaches it with an α(1->6) linkage to another residues in the chain. Pyridoxal phosphate-requiring glycogen phosphorylase cleaves the α(1->4) bonds between glucosyl residues at the nonreducing ends of the glycogen chains, producing glucose 1-phosphate. This sequential degradation continues until four glucosyl units remain before a branch point. The resulting structure is called a limit dextrin that is degraded by the bifunctional debranching enzyme. Glucosyl 4:4 transferase removes the outer three of the four glucosyl residues at a branch and transfers them to the non-reducing end of another chain, where they can be converted to glucose 1-phosphate by glycogen phosphorylase. The remaining single glucose residue attached in an alpha(1->6) linkage is removed hydrolytically by the amylo-(1->6) glucosidase activity of debranching enzyme, releasing free glucose. Glucose 1-phosphate is converted to glucose 6-phosphate by phosphoglucomutase. In the muscle, glucose 6-phosphate enters glycolysis. In the liver, the phosphate is removed by glucose 6-phosphatase, releasing free glucose that can be used to maintain blood glucose levels at the beginning of a fast. A deficiency of the phosphatase causes glycogen storage disease Type 1a (Von Gierke disease). This disease results in an inability of the liver to provide free glucose to the body during a fast. It affects both glycogen degradation and gluconeogenesis. Glycogen synthesis and degradation are reciprocally regulated to meet whole-body needs by the same hormonal signals (namely, an elevated insulin level results in overall increased glycogenesis and decreased glycogenolysis, whereas an elevated glucagon, or adrenaline, level causes increased glycogenesis and decreased glycogenesis). Key enzymes are phosphorylated by a family of protein kinases, some of which are cyclic adenosine monophosphate dependent (a compound increased by glucagon and adrenaline). Phosphate groups are removed by protein phosphatase-1 (active when its inhibitor is inactive in response to elevated insulin levels). Glycogen synthase, phosphorylase kinase, and phosphorylase are also allosterically regulated to meet tissues needs. In the well fed state, glycogen synthase is activated by glucose 6-phosphate, but glycogen phosphorylase is inhibited by glucose 6-phosphate as well as by ATP. In the liver, glucose also serves as an allosteric inhibitor of glycogen phosphorylase. The Ca2+ released from the endoplasmic reticulum in muscle during exercise and in liver in response to adrenaline activates phosphorylase kinase by binding to the enzyme’s calmodulin subunit. This allows the enzyme to activate glycogen phosphorylase, thereby causing glycogen degradation. AMP activates glycogen phosphorylase in muscle. Triacylglycerol metabolism – Generally, a linear hydrocarbon chain with a terminal carboxyl group, a fatty acid can be saturated or unsaturated. Two fatty acids are dietary essentials: linoleic and alpha- linolenic acids. Fatty acids are synthesised in the cytosol of liver following a meal containing excess carbohydrate and protein. Carbons used to synthesise fatty acids are provided by acetyl coenzyme A (CoA), energy by ATP, and reducing equivalents by NADPH provided by the pentose phosphate pathway and malic enzyme. Citrate carries two carbon acetyl units from the mitochondrial matrix to the cytosol. The regulated step in fatty acid synthesis is catalysed by biotin-requiring acetyl CoA carboxylase (ACC). Citrate allosterically activates ACC and long-chain fatty acyl CoA’s inhibit it. ACC can also be activated by insulin and inactivated by adenosine monophosphate-activated protein kinase (AMPK) in response to adrenaline, glucagon, or a rise in AMP. The remaining steps in fatty acid synthesis are catalysed by the multifunctional enzyme, fatty acid synthase, which produces palimotyl CoA by adding two-carbon units from malonyl CoA to a series of acyl receptors. Fatty acids can be elongated and desaturated in the endoplasmic reticulum (ER). When fatty acids are required for energy, adipocyte hormone-sensitive lipase (activated by adrenaline and inhibited by insulin), along with other lipases, degrades stored triacylglycerol (TAG). The fatty acid products are carried by serum albumin to the liver and peripheral tissues, where oxidation of the fatty acids provides energy. The glycerol backbone of the degraded TAG is carried by the blood to the liver, where it serves as an important gluconeogenic precursor. Fatty acid degradation occurs in the mitochondria. The carnitine shuttle is required to transport long-chain fatty acids from the cytosol to the mitochondrial matrix. A translocase and the enzymes carnitine palmitoyltransferases (CPT) I and II are required. CPT-I is inhibited by Malonyl CoA, thereby preventing simultaneous synthesis and degradation of fatty acids. In the mitochondria, fatty acids are oxidised, producing acetyl CoA, NADH, and FADH2. the first step in the B-oxidation pathway is catalysed by one of four acyl CoA dehydrogenases, each with chain-length specificity. Medium-chain fatty acyl CoA dehydrogenase (MCAD) deficiency causes a decrease in fatty acid oxidation (process stops once a medium chain fatty acid is produced), resulting in hypoketonemia and severe hypoglycaemia. Oxidation of fatty acids with an odd number of carbons proceeds two carbons at a time (producing acetyl CoA) until three-carbon propionyl CoA remains. This compound is carboxylated to methylmalonyl CoA (by biotin-requiring propionyl CoA carboxylase), which is then converted to succinyl CoA (a gluconeogenic precursor) by vitamin B12-requiring propionyl CoA mutase. Beta oxidation of very-long chain fatty acids occurs in peroxisomes. Integrated metabolism – The integration energy metabolism is controlled primarily by insulin and the opposing actions of glucagon and the catecholamines, particularly adrenaline. Changes in the circulating levels of these hormones allow the body to store energy when food is abundant or to make stored energy available in times of physiologic stress. Insulin is a peptide hormone produced by the beta cells of the islets of Langerhans of the pancreas. It consists of disulphide-linked A and B chains. A rise in blood glucose is the most important signal for insulin secretion. The catecholamines, secreted in response to stress, trauma, or extreme exercise, inhibit insulin secretion. Insulin increases glucose uptake (by muscle and adipose) and the synthesis of glycogen, protein, and triacylglycerol: it is an anabolic hormone. These actions are mediated by binding to its tyrosine kinase receptor. Binding initiates a cascade of cell-signalling responses, including phosphorylation of a family of proteins called insulin receptor substrate proteins. Glucagon is a monomeric peptide hormone produced by the α- cells of the pancreatic islets (both insulin and glucagon synthesis involves peptide hormone produced by the α-cells of the pancreatic islets (both insulin and glucagon synthesis involves formation of inactive precursors that are cleaved to form the active hormones). Glucagon, along with adrenaline, NA, cortisol, and growth hormone (the “counterregulatory” hormones”, opposes many of the actions of insulin. Glucagon secretion is stimulated by low blood glucose, amino acids, and the catecholamines. Its secretion is inhibited by elevated blood glucose and by insulin. Glucagon binds to high-affinity receptors of hepatocytes. Binding results in the activation of adenylyl cyclase, which produces the second messenger cyclic adenosine monophosphate (cAMP). Subsequent activation of cAMP-dependent protein kinase A results in the phosphorylation-mediated activation or inhibition of key regulatory enzymes involved in carbohydrate and lipid metabolism. Both insulin and glucagon affect gene transcription. Hypoglycemia is characterised by low blood glucose accompanied by adrenergic and neuroglycopenic symptoms that are rapidly resolved by the administration of glucose. Insulin-induced, postprandial, and fasting hypoglycaemia result in release of glucagon and adrenaline. The feed-fast cycle – The flow of intermediates through metabolic pathways is controlled by four mechanisms: 1) the availability of substrates, 2) allosteric activation and inhibition of enzymes, 3) covalent modification of enzymes, and 4) induction-repression of enzyme synthesis. In the absorptive state, the 2-4 hour period after ingestion of a meal, these regulatory mechanisms ensure that available nutrients are captured as glycogen, triacylglycerol (TAG), and protein. During this interval, transient increases in plasma glucose, amino acids, and TAG occur, the last primarily as components of chylomicrons synthesised by the intestinal mucosal cells. The pancreas responds to the elevated levels of glucose with an increased secretion of insulin and a decreased secretion of glucagon by the pancreas. The elevated insulin-to-glucagon ratio and the ready availability of circulating substrates make the absorptive state an anabolic period during which virtually all tissues use glucose as a fuel. In addition, the liver replenishes its glycogen stores, replaces any needed hepatic proteins, and increases TAG synthesis. The latter are packaged in very-low density lipoproteins, which are exported to the peripheral tissues. Adipose tissue increases TAG synthesis and storage, whereas muscle increases protein synthesis to replace protein degraded since the previous meal. In the fed state, the brain uses glucose exclusively as a fuel. In the absence of food, plasma levels of glucose, amino acids, and TAG fall, triggering a decline in insulin secretion and an increase in glucagon and epinephrine release. The decreased insulin/counterregulatory hormone ratio and the decreased availability of circulating substrates make the fasting state a catabolic period. This sets into motion an exchange of substrates among liver, adipose tissue, skeletal muscle, and brain that is guided by two priorities: 1) the need to maintain adequate plasma levels of glucose to sustain energy metabolism of the brain and other glucose-requiring tissues and 2) the need to mobilise fatty acids (FAs) from adipose tissue and release ketone bodies from liver to supply energy to other tissues. To accomplish these goals, the liver degrades and reducing equivalents needed for gluconeogenesis and to supply the acetyl coenzyme A building blocks for ketogenesis. The adipose tissue degrades stored TAG, thus providing FAs and glycerol to the liver. The muscle can also use fatty acids as fuel as well as ketone bodies supplied by the liver. Muscle protein is degraded to supply amino acids for the liver to use in gluconeogenesis, but decreases as ketone bodies increase. The brain can use both glucose and ketone bodies as fuels. From late fasting into starvation, the kidneys play important roles by synthesising glucose and excreting the proteins from ketone dissociation as ammonium (NH4+). WEEK 10, 11, 12 The Male Reproduction System Male puberty – increase in steroid hormones from gonads and adrenal glands. Testicular androgens control development of genitalia and body hair as well as enlargement of larynx and laryngeal muscles (voice breaking). Male genital system: the urethra goes through the prostate gland, a membranous part (through pelvic floor muscles) and the penile part (through corpus spongiosum). Functions: - Penis – intromission of spermatozoa suspension - Testes – production and temporary storage of spermatozoa, synthesis and secretion of testosterone, oestrogen, activin, inhibin and oxytocin - - Secretory glands – seminal fluids and nutrients to support and nourish spermatozoa. - Ductal system – carriage of spermatozoa to the exterior, maturation of spermatozoa. Testes – the testes descend from the posterior abdominal wall to the scrotum outside the body at 7th month gestation. It keeps gametes to 2-3oC below core body temperature. Highly vascular. There is skin, dartos muscle and tunica vaginalis. Fibrous septa from tunica albuginea divides the testes into 250-350 lobules. Each lobule contains 1-4 coiled seminiferous tubules. Each 80cm long and 150 micron diameter. Androgens are produced from in utero onwards. There are different stages of maturation of spermatozoa for different ductal compartments. In the testes there are 2 distinct compartments, the tubular (germinal compartment) and extra-tubular (endocrine compartment): - Tubular compartment – has tunica propria (basement membrane), germinal cells (contain spermatogonia – stem cells, primary spermatocytes, secondary spermatocytes, spermatids and spermatozoa). The spermatogonia are close to the basement membrane, the spermatozoa are towards the middle) and Sertoli cells (tall columnar, oval nucleus at right angle to basement membrane) and myeloid cells - Extra-tubular compartment – Leydig cells (LC) lie in the interstitial spaces between seminiferous tubules. There are LH receptors on Leydig cells. LH stimulation leads to synthesis of testosterone and oxytocin which enters the tubules and neighbouring blood vessels. Sertoli cells create basal, adlumenal and lumenal compartments in the tubular compartment. Tight junctions between adjacent Sertoli cells, at the baso-lateral location creates the blood-testis barrier. Tight junctions form a fence between the basal and adlumenal spaces. - Basal compartment – spermatogonia reside here. The basal compartment is accessible to proteins, charged sugars from the extra-tubular fluid and immune cells from neighbouring blood and lymphatic system. - Adlumenal compartment – the site of spermatogenesis, upon release of spermatogonium. - Lumenal compartment – spermatids are found on the luminal surface. Fully formed spermatozoa are found in the lumen of seminiferous tubules. Importance of the blood-testis barrier – tight junctions cause complete apposition of adjacent plasma membranes forbidding movement of solutes from the basal to the adlumenal compartment and vice versa. It isolates the adlumenal compartment from the basal compartment. Interstitial fluid cannot get in. This protected niche is ideal for vulnerable spermatocytes. Only unidirectional movement of germ cells are allowed through the tight junctions. Tight junctions open transiently and allow spermatogonia to enter the adlumenal compartment. The developing spermatocyte or spermatozoa cannot leak back out to the basal compartment. This means the body cannot elicit an immune reaction and create antibodies against sperm antigens revealed during spermatogenesis. It also protects the developing spermatocytes from pathogens and mutagens. Sertoli cells also actively secrete molecules and nutrients into the adlumenal compartments for the developing sperm. Spermatogenesis – development of sperm in the testes: - Spermatocytogenesis: o Mitotic proliferation – results in secondary spermatocytes from spermatogonium (undifferentiated male germ cells/stem cells). Produces large numbers of cells. Spermatogonia undergo mitosis to form 1 reserve stem cell (type A dark) and 1 cell for differentiation (type A pale, this differentiates into type B). Type B undergoes maturation to become primary spermatocytes. These replicate their DNA shortly  after formation to become 4n. Important for genetic recombination. Prolonged period in prophase (c22 days). o Meiotic division – They then undergo meiosis to form secondary spermatocytes (2n). These remain attached by cytoplasmic bridges. - Spermatidogenesis – They rapidly undergo a second meiotic division to produce haploid spermatids (n) present by now near lumen. Generates genetic diversity and halves the chromosome number. - Spermiogenesis – packaging of chromosomes for effective delivery to the oocyte. Formation of head, neck and tail. Cytodifferentiation packaging of chromosomes for effective delivery. Spermiogenesis occurs in small hollows in the luminal surface of Sertoli cells to create spermatozoa. Formation of acrosome close to nucleus (n). Has enzymes needed for fertilisation. Also creates polarity of cell. Nuclear elongation. Anterior pole has acrosome, posterior pole has developing flagella/microtubule to make tail. Cytoplasm migrates to posterior part of cell. Excess cytoplasm pinched off. So head contains nucleus only. Prominent mitochondria appears in the neck region, will help with motility when needed. The head is the only bit which goes into the egg, this is why all mitochondria comes from mum, as there is none in the sperms head. Production rate – 300-600 spermatozoa per gram of testis per second. Continuous post-puberty. Sperm count goes down as you get old, but does not cease. Sperm production is continuous, not cyclical or pulsatile. It takes 64 days to develop a mature spermatozoan. In humans, spermatogonia divide in sequence every 16 days. At any one time, there are a number of cells dividing (spermatogenic cycle). Before they have completed maturation, a new initiation starts (staggered entry). Spermatogenesis is initiated at different times in different regions of the testis thus giving a smooth non-pulsatile flow of spermatozoa. Sertoli cells co-ordinate the temporal and spatial organisation of spermatogenesis. The ejaculate contains the spermatozoa produced over a period of a few hours. Endocrine support of spermatogenesis – high levels of testosterone are needed for spermatogenesis (this starts at puberty). LH stimulates Leydig cells to produce testosterone which binds to receptors on Sertoli cells. Testosterone induces receptors for FSH on Sertoli cells. FSH from pituitary now stimulates Sertoli cells to produce androgen binding protein (ABP) which binds and carries testosterone in testicular fluid to the entire ductal system. Sertoli cells also produce inhibin which is part of a negative feedback loop. It inhibits FSH production by pituitary gland. In the absence of FSH and LH spermatogenesis do not occur so sperm count will be low. High levels of testosterone can cause negative feedback to production of gonadotropins, which cause a decrease in LH and FSH. This highlights the danger of overuse of synthetic androgens. Structure of exit ducts and accessory glands: - Rete testis – network of channels which fluid and spermatozoa (from seminiferous tubules) empty into. They are lined with simple cuboidal or low columnar epithelial cells. - Efferent ductules leading to the epididymis – tall ciliated epithelia propel the immobile spermatozoa. Non-ciliated cells have microvilli and absorb testicular transport fluid (spermatozoa still immobile and immature, will not be able to fertilise). - Epididymis – contains convoluted 5m long epididymal duct. Tall columnar epithelium with asymmetric modified microvilli (stereocilia). Important for absorption. Rhythmic contraction of underlying smooth muscle cells moves maturing sperm forward to vas deferens. Their function is to absorb fluid (concentrates sperm hundredfold) and phagocytosis. Secretion of  glycoproteins, sialic acid, defensin, glycerophosphocholine (for maturation of spermatozoa) under influence of testosterone. - Spermatic duct – important site for sperm storage. The vas deferens (within which sperm reside) runs through the spermatic cord. The testicular artery runs along with it. The veins in this system form an unusual plexus (pampiniform) around the testicular artery. The spermatic cord is encased in cremaster muscle. It is drained by the pampiniform veins (multiple tendrils) forms a complex interconnecting plexus around the testicular artery. Excellent heat exchange, protects contents of vas deferens from overheating. The veins are difficult to distinguish from the arteries as they also have muscular walls. - Structure of vas deferens – 3 muscle layers (outer, central, inner). A fibroelastic lamina propria and inner epithelium. Longitudinal folds create stellate lumen. Tall columnar ciliate epithelium. Peristalsis. Spermatozoa storage (2-3days). A vasectomy is ligation of the vas deferens. It doesn’t cause fluid accumulation as spermatozoa build up is removed by phagocytosis. - Seminal vesicles (accessory gland) – mucosal folds create vast surface area. Secretes seminal fluid: fructose (energy), prostaglandins (contraction of smooth muscle in male and female tract), proteins, amino acids, alkaline secretions to neutralise acid conditions of vagina), fibrinogen (or clotting). Contraction of seminal vesicle propels secretion into ejaculatory ducts which open into the prostatic urethra. - Prostate gland – collection of concentric secreting glands which open into urethra. It secretes citric acid, proteolytic enzymes, clotting enzymes, prostate-specific antigen. The prostate gland progressively enlarges from age 45. Benign prostatic hyperplasia is compression of the urethra causing retention of urine. Older men have stored secretions in the lumen that can form corpora amylacea (calcified, condensed glycoprotein). The glands are lined with stratified columnar to cuboidal epithelia. The epithelium is arranged in folds or papillary ingrowths. - Bulbourethral glands (an accessory gland) – opens into the membranous urethra. Produces watery fluid rich in galactose and sialic acid. Acts as lubricant and neutralises acidic urine in urethra and vaginal fluids. Precedes semen during emission. The penis – erectile tissue contains 2 dorsal cylinders (corpora cavernosa) which are highly vascular and 1 ventral cylinder (corpus spongiosum which contains the penile urethra). Erectile tissue has interconnecting vascular spaces. The cavernous tissue is a mesh of interconnected cavernous spaces lined by vascular endothelium. The cavernous spaces are separated by trabeculae of smooth muscle fibres with an extracellular matrix (collagens, elastic fibres, numerous unmyelinated and preterminal autonomic nerves). Erectile physiology – the corpora cavernosa (spongey tissue containing deep arteries) contain helicine vessels arising from deep arteries which supply the erectile tissue. Smooth muscle cells are important here in regulation of contraction/dilation. Smooth muscle fibres in trabeculae allow relaxation and dilation of sinusoidal spaces. Vascular cavernous spaces or sinusoidal spaces are lined with simple squamous endothelium. The corpora cavernoas is supplied with blood from the internal pudendal artery, which gives rise to the bulbourethral artery, cavernous artery and dorsal artery supplying the glans penis. The cavernous (central) artery leads to helicine arteries that opens into the cavernous spaces/vascular sinusoids. Blood returns through the venous system to the deep dorsal vein. There is anastomosis between the central artery and the deep dorsal vein (Arterio-venous anastomosis). Venous drainage occurs from the post cavernous venules, sub-albugineal venous plexus, emissary veins, circumflex veins and deep dorsal vein.  - Flaccid state – In flaccid state, the vessels are coiled, restricting blood flow. Sinusoidal smooth muscles remain contracted, so spaces are empty, and blood flows from the internal pudendal arteries via central deep artery and helicine arteries to vascular cavernous spaces and out through the open emissary veins. Low volume, low pressure. AV anastomosis between central artery and deep dorsal vein helps shunt blood from artery to vein. - Erection – During erection, the helicine vessels straighten and dilate. The sinusoidal spaces fill with blood (tumescence) during erection. The AV shunts are closed, so more blood flows into Helicine arteries. Smooth muscles of sinusoids relax, so blood flows into the lacunar spaces. The resultant pressure compresses the emissary veins, reducing venous outflow. Large volume, high pressure (veno-occlusive function). Erection is a complex neurovascular physiological process. It involves interplay among neural, vascular, hormonal and psychological factors, as well as the integrity of the vascular bed of the penis. Nerve supply gives sensation and regulates vascular supply. CNS control allows for psychological and tactile stimuli (internal pudendal nerves). Psychogenic stimuli (visual cues, erotic imagery) using limbic system. Parasympathetic non-adrenergic and non-cholinergic nerves. Neurotransmitters are dopamine, acetylcholine, VIP and nitrous oxide. Secretion of nitrous oxide causes relaxation of smooth muscle, dilation and relaxation of central cavernosal and helicine arteries so blood flow into the cavernous spaces and there is veno-occlusion. Erectile Dysfunction Occurs in 50% of 50-70 year olds. It is not associated with mortality. Causes of erectile dysfunction include: cardiovascular disease (damage to blood vessels in penis), smoking, alcohol, long-term diabetes (nervous and microvascular damage, impaired release of nitrous oxide), prostate surgery or drug-induced (thiazides have a role in hypertension, prazosin, antidepressants, anti-psychotics). Prolonged erections are dangerous as can cause ischaemia. Erectile dysfunction may be due to tears in the fibrous capsule of cavernosa, obstructed vessels or damage to the fibro-elastic architecture. It was found that nitrous oxide was released by nerves in the penis and is important in erections. Under parasympathetic control, when you become sexually aroused there are parasympathetic nerves that release nitrous oxide. There is also local release of nitrous oxied due to local stimulation. It causes arterial vasodilatation but doesn’t affect venous flow, leading to engorgement with blood and erection. The corpus cavenosum becomes full with blood. Nitrous oxide is the endothelium-derived relaxant factor, it causes vasodilatation and regulates bp. It activates adenylyl cyclase increasing cGMP. This is broken down by phosphodiesterase V to inactivate it. A drug was developed to inhibit this enzyme, so there is a build up of cGMP. Manipulating nitrous oxide is used in GTN spray (nitrous oxide given off). Phosphodiesterase V inhibitors are generally well tolerated. However, there is a significant interaction with nitrates (e.g. GTN) due to increased activity. Can cause severe hypotension. The absence or presence of random erections is important in diagnosing whether it is psychogenic or disease related. Using nitrous oxide was ineffective for treating angina, but patients complained of erections. The drug was then adapted to make Viagra. There are alternatives to Viagra including an injection, a pessary and pumps with a ring placed on the penis to prevent venous flow. Viagra is effective however side effects include headaches and visual disturbances. Viagra can be bought over the counter but is cheaper on prescription. Role of testosterone in maintenance of smooth muscle intracavernosal injection of synthetic prostaglandins will lead to tumescence. Sildenafil – tmax is about an hour, so it will take between 30-60 minutes before sexual activity. It also has a short half-life and will last 3-4 hours. Dose is 25-100mg. Tadalafil has a half-life of 17 hours, so duration is much longer. The drug enhances the effects of sexual stimulation, so the erection wouldn’t last for 17 hours. Prolonged erection (priapism) can endanger oxygenated blood supply. The Female Reproduction System Functions of female reproductive system – produce haploid female and receive haploid male gametes prior to fertilisation. Provide a suitable environment for fertilisation and implantation. Accommodate and nourish the embryo and fetus during pregnancy. Expel the mature fetus at the end of pregnancy, protect against pathogens and produce steroid hormones. The clitoris, labium minora and labium majora make up the external genitalia. The hymen is a thin, fibrous membrane with an irregular frill, it is in the vestible. It can be quite strong or weak. At the enterance of the vestible are the paired greater vestibular gland (Bartholin gland), it important for lubrication during intercourse. The vagina itself doesn’t have glands, but the cervix does. The histology of the clitoris is similar to the penis, there are vascular sinusoids (so can gorge with blood during arousal). There are 2 corpora cavernosa (erectile vascular tissue) and nerve endings (pacinian touch receptors). Female genital mutilation (FGM) can involve removing any of the external genitalia. The vagina is at about 90 degrees to the anteverted uterus. The vagina is a fibromuscular tube (7- 9cm) which is capable of marked distension and elongation. The vagina hugs the urterus at the inner end, it forms a cuff, creating anterior, posterior and lateral fornices. Don’t go into the fornices when doing a cervical swab. Layers of the vaginal tube: - Epithelial mucosa, non-keratinised statified squamous epithelia. Glycogen rich superficial layers. It is under influence of oestrogen, before puberty and after menopause this layer is thin. Glycogen is involved in the maintenance of pH. At ovulation there is increased glycogen production by the vaginal epithelium. The breakdown of glycogen by commesnal lactobacilli leads to production of lactic acid and an acid pH of 5.7-3 (fluctuation depends on menstrual cycle). This restricts vaginal flora to acid loving bacteria and deters pathogens such as candida albicans (thrush). - Lamina propria - Sub mucosa, highly vascularised, has elastic fibres which allow distension - Adventitia elastic fibres and irregular smooth muscle. Cervix (neck or lower part of uterus) – c ylinderical tube 3-4cm long and 2.5cm wide, partly protruding into the vagina. The cervix can distend to 10cm diameter during childbirth. Composed of: - Ectocervix (the projecting bit into the vagina). The opening of the ectocervix into the vagina is called the external Os. - Endocervix is the passageway betewen the external Os and uterine cavity. It terminates at the internal OS, the opening of the cervix to the uterine cavity. There are differences in histology between endo and ectocervix due to differences in environment. On histology, there appears to be glands in the endocervix, however they are not glands they are invaginations. The endocervix is made of a single layer of tall columnar mucus secreting epithelial cells, a basal layer of reserve cells and stroma composed of a matrix of fibromuscular tissues, elastin and collagent fibres, glysocaminoglycans. Rich in lympahtic vessels and nerves. The stroma is important for softening the cervix as it can cause hydration of the matrix and alterations in the collagen and elastin fibres. Softening is important for childbirth. There is rapid reversal to normal dimensions after childbirth. There are deep invaginations of columnar epithelium into the cervical stroma. This increases surface area. The cells secrete mucin (increases during ovulation). They are not true glands. The pH of cervical mucus is about 7, sperm can rest in these crypts. Function of mucin: - Lubrication during sex - Protection against bacterial ascent into uterus - Allows ascent of sperm into the uterus at the correct time Sperm-mucin interaction – the amount and properties change during the menstrual cycle. In the proliferative phase the mucin is thin, watery and abundant with a more alkaline pH (allows sperm viability). Just prior to ovulation there is 10x more in volume, less viscous than at the outset of the menstrual cycle and it facilitates movement of spermatozoa. In the secretory phase after ovulation the mucin is more viscid, more acidic and deters the penetration by any more spermatozoa. It seals the uterus in preparation of possible embryo implantation. Sperm slows down in the endocervix. The ectocervix is very similar to the vagina. There is a transformation zone (squamo-columnar junction). This transformation process is called squamous metaplasia. It is a normal physiological replacement of the everted columnar epithelium by a newly formed squamous epithelium. There is irritation of exposed columnar epithelium by the acidic vaginal environment which results in the proliferation of reserve cells to two layers. These cells further proliferate and differentiate (metaplasia) to form an immature squamous metaplastic multi-layered epithelium. It is at the transformation zone that cervical smears are taken. The actual extend and location can vary with age, menstrual cycle and hormonal secretion. Consequences of squamous metaplasia: - Development of Nabothian cysts – the newly formed stratified squamous epithelium can grow over the simple columnar epithelium at the transformation zone. Some invaginations of the columnar epithelium become covered and lose their connection to the surface. Continuing mucin secretion of these blocked invaginations results in the formation of small Nabothian cysts. - Development of abnormal epithelium – lose regular stratified pattern, high nucleus to cytoplasm ratio, increased mitotic activity (intraepithelial neoplasia) - Progression to cancer. These cells can breach the basement membrane and invade cervical stoma (invasive carcinoma). Cervical smears are taken to ensure early diagnosis of cancer. Human papillomavirus – cervical cancer is the 2nd most prevalent cancer in women. Infection with HPV is a major causative agent for cervical cancer. Over 40 of the 100 types are transmitted sexually. Low risk HPV types can cause genital warts but not cervical cancer (e.g. type 6). Type 16 and 18 are high risk and can cause cervical cancer. In men, HPV can cause genital warts, cancer of anus, penis or throat. Vaccination of women has indirectly protected men. Men who have sex with men up to and including the age of 45 are eligible for free HPV vaccination on the NHS. Girls are immunised at age 12-13, before most people are sexually active. Uterus – the uterine wall is composed of an external serosa, covered with the peritoneum of the pelvic cavity, the perimetrium. There is a middle muscular layer called the myometrium. It is hormone sensitive so can undergo hyperplasia and hypertrophy during pregnancy. There is also an internal mucosal layer called the endometrium which lines the entire uterus and is under influence of the menstrual cycle. The fertilised egg will implant in the endometrium of the uterus. The endometrium is continuous with the lining of the cervix. The endometrium has a functional layer rich in true glands and blood vessels and a basal layer that is capable of producing the functional layer. Under the influence of progesterone, the glands become saw-toothed and rich in secretions during the secretory phase of the menstrual cycle. Fallopian tubes: about 10-12cm long. Open at the infundibulum, which is surrounded by fimbrae, finger-like projections into the peritoneal cavity. The released ovum, from the ovary is wafted into and lodges at the ampulla region of the tube. Sperm tends to stay at the isthmus (first part of the tube coming from the uterus) until ovulation draws nearer (for up to 2 days), they then travel to the ampulla. Fertilisation occurs at the ampulla. There are 2 layers of smooth muscle in the wall of the tube: the inner being a tight spiral and the outer being a loose spiral which makes them appear circular and longitudinal respectively. The epithelial lining of the tube shows marked invagination and is now called the papillary mucosa. These invaginations are ideal for storing the egg and sperm. Sperm binding protein is present on the surface of the egg. Contraction occurs to push the fertilised egg into the uterus. Capacitation of sperm occurs at the isthmus (sperm rests), leading to removal of its glycoprotein coat and seminal proteins. Sperm movement alters here once ovulation draws near and sperm can swim to the ampulla. There is gradual release of sperm, which may avoid polyspermy. The isthmus us also more muscular, so can aid the swim. The epithelial mucosa of the entire fallopian tube has ciliated and non-ciliated cells. Ciliated cells are more numerous near the ovarian end of the tube. Secretory, non-ciliated cells predominate close to the uterus. Cilia beat towards the uterus, creating a flow in that direction. Ciliary height is cycle dependent, they are highest at the time of ovulation then decrease in length due to progesterone. Non-ciliated cells secrete mucus to aid motion of cilia. The tubal fluid contains K+ and Cl- ions, immunoglobulins and serum proteins to provide nutrients to the egg during its migration. Disorders of the uterine tube: - Tubal ectopic pregnancy – implantation of fertilised ovum - Acute and chronic salpingitis – bacterial infection can lead to acute inflammation, pus formation, abscess, scarring and blocked tube. Will require IVF. - Stone pregnancy Structure of Ovary Blood supply to the ovary – the ovarian artery arises from the abdominal aorta at the level of the renal arteries. It passes laterally, anterior to the ureter, and inferiorly into the pelvis. It supplies the ovary and then continues towards to uterus. On the right side, the ovarian vein (ROV) drains into the inferior vena cava. On the left side, the left ovarian vein drains into the left renal vein. Anastomosis of vascular elements – the ovarian artery gives branches to the fallopian tube. It anastomoses with the uterine artery (branch of internal iliac) to supply the uterus. The uterine artery also anastomoses with the vaginal artery (another branch of internal iliac). This vascular system arrangement allows rapid transfer of hormones from ovary to uterus and vagina, which enables co- ordination of physiological and histological changes in the menstrual cycle. Histology of the ovary: - Cortex – epithelial lining, stromal cells, gametes (oogonia/follicles). Gamete formation and follicle maturation occurs in the cortex. Stromal cells are spindle shaped and display a whirling pattern. - Medulla (middle) – stromal cells, mesenchymal cells, vestigial remnants of embryonic Wolffian duct. - Hilum – where blood vessels and lymphatic vessels enter and leave etc. - Blood vessels, lymphatics, hilus cells (similar to Leydig cells in males, they produce testosterone). Development of follicles in the ovary: oogonia – primary oocytes (in utero) and primordial follicle – primary follicles (at puberty) – secondary follicles – tertiary/Graafian follicle – preovulatory follicle – oocyte and corpus luteum – atretic follicles (died ones). Gamete development in cortex: - Embryonic period – first trimester/12 weeks of pregnancy. At 4-5 weeks gestation there is migration of the stromal cells, mesenchymal/granulosa cells and primordial germ cells (PGCs) from the yolk sac to the ovary. Primordial germ cells give rise to oogonia (spermatogonia in males). From 5 weeks onwards, the migrated primordial germ cells in the ovary undergo mitotic divisions to form numerous oogonia. The stromal cells proliferate and cluster around oogonia. Mesenchymal cells increase in number. - Fetal period – second trimester of pregnancy. Mitotic divisions of oogonia cease, oogonia increase in size and then enter first meiotic division. They then stop and remain in prophase (for many years until puberty). They are now called primary oocytes (several millions found in this trimester). Degradation and loss of primary oocytes throughout gestation – at birth, there are about a million oocytes. However, this number gets smaller due to degeneration or germ cell loss or atresia occurs in utero. This loss continues after birth. At puberty, a quarter of a million primary oocytes are left. Only around 500 will ovulate during reproductive period. Formation of primordial follicles – during fetal and neonatal period, the surviving primary oocytes acquire a single layer of granulosa cells and are now called primordial follicles. Granulosa cells are mesenchymal cells, they form this first wrapping around the oocyte, the stromal cells will do the second wrap later. Puberty, Menstrual Cycle and Contraception and Further Follicular Development Changes occurring in the menstrual cycle involve the hypothalamus, anterior pituitary and the ovary 5 major hormones: - Gonadotropin releasing hormone (GnRH) – found in hypothalamus - Follicle stimulating hormone (FSH) – anterior pituitary. A protein. It is responsible for follicle development/maturation and proliferation of granulosa cells and oestradiol production. - Luteinizing hormone (LH) – anterior pituitary. A protein. It is responsible for ovulation, follicular luteinization and progesterone production. - Oestradiol – ovary (oestrogens = oestradiol and oestrone). A steroid hormone - Progesterone – ovary. A steroid hormone. Proliferative phase/follicular phase – day 0-14. - Bleeding occurs at day 0-7 and new follicles start to develop. If pregnancy doesn’t occur, cessation of progesterone and oestrogen results in involution/shrivelling of functional layer of endometrium, rise in endothelin and thromboxane, vasoconstriction of spiral arteries, cessation of blood flow and ischaemia of functional endometrium, rupture of arteries and shedding of blood into uterus. Apototic/necrotic tissue from functional layer is shed with blood. The basal layer of endometrium remains. Histology of stratum functionale – oestrogen stimulates mitotic activity in glands and proliferation of stromal cells. It increases thickness of endometrium (inner layer of the uterus) and increases length of spiral arteries. The functional layer is rich in uterine glands and blood vessels. The basal layer is capable of producing function layer when it is sloughed off in menstruation. The functional layer is lost during menstruation. - The endometrium starts to regrow after bleeding. There is thinning of cervical mucus to allow passage of sperm and increased fat deposition in mammary glands. There are episodic pulses of GnRH within the hypothalamus which vary across the menstrual cycle. There are more pulses in this phase. They cause the production and secretion of FSH and LH from the pituitary. They cause the ovary to produce oestrogen (oestradiol). There is a negative feedback mechanism which tells the pituitary and hypothalamus to regulate the amount of oestrogen (oestradiol). Progesterone levels remain quite low. - Development of primary follicle at puberty – FSH stimulates development of primordial follicles. Primordial follicles mature to primary follicles by: enlargement of the oocyte, proliferation and increase in granulosa cells (which are more cuboidal in shape), multilayering of granulosa cells and formation of zona pellucida between oocyte and the granulosa cells. Zona pellucida proteins are important for protection of the oocyte and avoidance of polyspermy. Gap junctions between oocyte and granulosa cells allow transfer of metabolites and regulatory substances between the 2 cell types so the oocyte can be provided with nutrients. cAMP from granulosa cells maintains meiotic arrest of oocyte. The preovulatory follicle has a much larger diameter than the primary follicle. - Development of secondary follicles – granulosa cells continue to proliferate. Fluid-filled cavities appear, they are called antral cavities which are rich in hyaluronic acid (important for fertilisation and implantation). Stromal cells condense around the follicle to form a theca interna and a theca externa. Thecal cells produce androgens, granulosa and thecal cells together produce oestrogen, so levels rise. - Development of tertiary/Graafian follicle – fluid filled antral cavities join to form a large antrum. The oocyte is pushed to one side (eccentrically located). Oocyte is separated from the cavity by a coat of granulosa cells, the cumulus oophorus. There is an increase in oestrogen secretion and proliferation of granulosa cells. Granulosa cells now start to express LH receptors. The antral state is the longest period. Secretory phase/luteal phase (day 14-28) – - At day 14 (ovulation), we reach a point where the negative feedback switches and becomes positive. Ovulation normally results in only 1 follicle per month, the others undergo atresia. After ovulation there is plugging of the cervix by thickening mucus, preventing more sperm from entering. The high oestrogen (oestradiol) levels in the late follicular phase cause a surge in release of LH and FSH from the pituitary. This induces oocyte to come out of arrest and complete meiosis. Cell division produces a haploid gamete (secondary oocyte) and a small polar body (first polar body). Meiosis arrests again. Oestrogen and FSH cause a change in the action of LH. The granulosal cells begin to express receptors for LH, and LH binding causes cholesterol to produce progesterone (becomes the dominant hormone). Granulosal cells no longer bind oestrogen or FSH. - Development of pre-ovulatory follicle (shortest stage) – due to high LH, the oocyte is released from the follicle (proteolytic activity to breakdown collagen). It is surrounded by granulomas cells (now called corona radiata). Rupture of the follicle can be accompanied with bleeding. And there can be mid-cycle pain. Oocytes travel into the fallopian tube. There is recruitment of 5-9 new follicles. One of these will go on to form the graafian follicle in the next cycle. - Glands in the endometrium become saw-toothed and secretory (under progesterone control). Spiral arteries coil more. Surface epithelial cells have short microvilli and lose surface negative charge. Endometrial glands are full of secreted material which can be used as nutrients by conceptus. Optimal vascular supply. Endometrium is in a receptive phase now, allowing implantation. The implantation window is short (5-7 days after ovulation). - Formation of the corpus luteum – forms from the Graafian follicle after expulsion of oocyte. The remaining follicle is called the corpus luteum. This has a central blood clot and is surrounded with lipid rich cells. LH is still being produced and induces enlargement of granulosa cells which fill up with yellow lipid (luteinization). Granulosa cells start producing lots of progesterone. The high progesterone causes a negative feedback, supressing GnRH, FSH, LH and oestrogen production/secretion, so no new follicles can develop (puts all eggs in one basket). o If pregnancy occurs, there is a second LH surge which maintains the corpus luteum and progesterone maintains the endometrium. hCG (human chorionic gonadotrophin) from the placenta prevents degeneration of corpus luteum. hCG signals to pituitary to secrete LH. The corpus luteum continues to secrete progesterone until end of fourth month of pregnancy. The placental progesterone is then enough now to take over. o If no fertilisation occurs corpus luteum becomes an acellular corpus albicans (10-12 days), progesterone levels drop and the corpus luteum gets smaller and smaller and degenerates and the cycle starts again. GnRH secretion increases so there is more LH and FSH. This allows new follicles to mature. There is onset of menstruation. There is a rise in uterine prostaglandins, which lead to period cramps. Effect of FSH and LH – there are 2 cell layers to the developing follicle (granulosal cells and thecal cells). FSH acts on granulosal cells to increase synthesis of oestrogen. LH acts in the thecal cells to produce androgens. Cholesterol is the precursor for these hormones. Progestagens/progesterones are made and then this can be changed to form glucocorticoids, mineralocorticoids, and androgens. Oestradiol can be formed from either testosterone or oestrone (which form from androstenedione). These molecules are structurally very similar but have very different physiological effects. A receptor to LH is expressed on the thecal cells, so LH can bind, this causes cholesterol to form androstenedione (very hydrophobic, so diffuses into granulosal cells as the thecal cells don’t have the correct enzymes). When FSH binds onto the granulosal cells, androstenedione forms oestradiol within the cell, it then exits the cell. Oestradiol increases proliferation of granulosal cells so lots of oestradiol is made even though levels of LH and FSH are relatively low. Hormonal contraception – suppress ovulation by negative feedback of progesterone on the pituitary and hypothalamus. This causes a decrease in GnRH secretion resulting in low FSH and LH levels so there are no new follicles so you can’t get pregnant: - Combined oral contraceptive pill (COC) – synthetic oestrogen and progesterone in varying amounts. Oestrogen helps progesterone work more effectively. It is monocyclic. 21 days on (output of GnRH, FSH and LH suppressed), 7 days off (endometrium breaks simulating menstruation). - Progesterone only pill (POP) – may be taken if family history of breast cancer e.g. Can be in the form of a pill or an implant or in an intra-uterine device. The pill must be taken continuously, compliance is essential. Cessation of menstrual cycle: - In pregnancy, no menstrual cycle occurs as high levels of progesterone are present. It inhibits secretion of pituitary gonadotrophins. - Menopause – for women over 50, menstruation becomes less regular. The ovaries lose the ability to respond to FSH and LH so there are low oestrogen (oestradiol) levels so a follicle doesn’t develop. 51 is the average age. In established menopause there is high LH and FSH. Onset of menstruation – puberty – activation of GnRH pulses to anterior pituitary begin 2-4 years before your first period. Possibly due to maturation within CNS and weight. Increase in LH and FSH cause an increase in oestradiol and androgen synthesis. Ovarian oestrogens regulate growth of breast and female genitalia. Androgens from the ovary and adrenals control growth of pubic and axillary hair. Menstrual irregularities: - Amenorrhoea – absence of menstruation. Primary is an endocrine abnormality, secondary is life-style linked (anorexia, excessive exercise). - Menorrhagia – excessive bleeding endometrial/myometrial disorder. - Dysmenorrhoea – excessive pain, endometrial/myometrial disorder - Post-menopausal bleeding – important symptom of malignant disease. Fertilization and Implantation Fertilisation is a sequence of co-ordinated events that begins with contact between a sperm and an oocyte and ends with the intermingling of maternal and paternal chromosomes. An ovulated/secondary oocyte still has its first polar body, but this will degenerate. The ovulated/secondary oocyte (haploid nucleus) continues to obtain nutrients from the cytoplasm. The granulosa cells suspended in hyaluronic acid rich matrix produces progesterone and chemo- attractants (aromatic aldehydes). The zona pellucida remains as a protective shell. Spermatozoa – the acrosome contains the enzyme acrosin needed for the acrosomal reaction. The plasma membrane of the sperm head has odorant receptors (similar to olfactory receptors in the nose) that can react to the chemo-attractants from the oocyte. The head also has 3 surface binding molecules (ADAM family – fertilin α and β and cyritestin). Spermatozoa also secrete hyaluronidase to get to the egg. ADAM is a disintegrin and metalloproteinase domain containing protein. Spermatozool movement from the vagina to the oviduct can take 2-7 hours. About 99% of sperm don’t enter the cervix and are lost. In the absence of progesterone domination mucus permits sperm penetration. Successful sperms can survive for many hours in cervical crypts. Sperm travel to the oviduct by regular wave-like swimming. Capacitation – needed for spermatozoa to attain full fertilisation capacity. Occurs in female genital tract, when it is oestrogen-primed (pH around 5.7 which normally occurs just prior to ovulation and 2- 3 days after). Full capacity is reached by the time the spermatozoa travels through the isthmus to the ampulla region of the oviduct. In the isthmus, sperm linger in the mucosal foldings and become immotile (24hours). Only at ovulation they acquire motility and swim to the ampulla: - Changes in spermatozooal surface by stripping of glycoproteins. Exposure to atmospheric O2 at ejaculation leads to production of hydrogen peroxide, a potent capacitation agent. This along with the low pH leads to changes in surface charge, macromolecular organisation and loss of cholesterol. It also leads to reduced stability of plasma membrane and enhances fusibility. It also increases permeability to Ca2+ and causes actin polymerisation (F-actin) between surface membrane of acrosome and plasma membrane to prevent a premature acrosome reaction (a wall is formed). The capacitated spermatozoon is now metastable. Unless it finds an oocyte, it will die. Thus, capacitation doesn’t occur in the male. - Changes in movement characteristics. Regular wave-like changes to wide amplitude whiplash beats (hyperactivated motility pattern) needed to swim upstream from isthmus to ampulla. Sperm has to swim against the flow created by the cilia in the fallopian tubes. Fertilisation: - Penetration of the corona radiata (granulosa cells) – secretion of hyaluronidase, digestion of extra cellular matrix, active movement to reach zona pellucida. - Penetration of zona pellucida – the zona pellucida is made of 4 sulphated glycoproteins (ZP4 in humans). The structure is different in different species, so there can’t be cross species fertilisation. - Attachment to zona pellucida – the receptors for ZP proteins (e.g. ZP2R like what mice have) are present on different membrane components of the sperm. The receptor for ZP3 is on the surface of the sperm head, the receptor for ZP2 is on the inner acrosomal membrane. - The acrosome reaction – in mice, the binding of ZP3 to its receptor on sperm-head plasma membrane leads to a Ca2+ influx that causes depolymerisation of the F-actin present between the acrosome and the sperm-head plasma membrane. The wall is now not there so the acrosome can expand. The acrosome swells and fuses with overlying surface plasma membrane. This gives a vesiculated appearance. Acrosin and contents are released by exocytosis. The inner acrosomal membrane now faces the exterior. This leads to availability of the ZP2 receptor for binding. The revealed ZP2 receptor binds to ZP of the zona pellucida. There is mechanical propulsion along ZP2 filaments until the oocyte plasma membrane surface is reached. The release of acrosin aids digestion of a pathway through the zona pellucida. Gamete fusion: after penetration of zona pellucida, spermatozoon lies tangential to the oocyte surface. The oocyte microvilli envelop sperm head. There is sperm-oocyte binding. The process involves adhesion molecules such as disintegrins (alpha and beta fertilin) and izumo 1 (on the sperm) and integrins and cd9 (on the egg). Only specific areas on the egg surface are rich in integrins to allow binding in correct areas. Spermatozoon sinks into the oocyte to form a zygote. Post fusion events: after fusion there is a rise in Ca2+ in the oocyte, which leads to its exit from metaphase II. One set of chromosomes is dispatched as the second polar body. The other half set of 23 unpaired chromosomes remain behind in the female pronucleus and can unite with the 23 paternal chromosomes of the penetrating sperm. Gynogenetic triploidy (don’t want both sets of chromosomes to come from mum) is avoided by dispatching the second polar body. If a spermatozoa tries to get in at the point of the exiting polar body, they will bang into each other and cause mutual interference and all 3 haploid sets of chromosomes could remain inside. This would result in fetal death. To avoid this, sperm do not bind to the oocyte membrane immediately overlying the second metaphase spindle. This region is devoid of binding proteins like integrins, CD9 and of microvilli. This ensure avoidance of encounter between the exiting second polar body and the entering sperm. Prevention of polyspermy (more than one sperm entering the cell) – avoidance of androgenetic triploidy/polyploidy. Changes in the electrical activity or membrane potential of the zygote leads to a wave of Ca2+ from the site of sperm entry, followed by a Ca2+ release from internal stores (lasts 2-3 minutes). This causes a 1-2 minute Ca2+ spike every 15 minutes. This leads to the cortical reaction which involves the release of cortical granules (enzymes) by oocyte into perivitelline space. These enzymes cleaves ZP2 and hydrolyses binding of ZP3. The ZPR are no longer available for further sperm binding. Tyrosine residues on adjacent ZPs start to cross link and the zona becomes indissoluble and impenetrable to sperm. If the oocyte is fertilised by 2 spermatozoa there is a partial molar pregnancy. There is abnormal placentation and no viable fetus. There is establishment of diploid genetic constitution. For most genes, we inherit two working copies however, errors can occur. Aneuploidy is an abnormality in number of chromosomes by loss duplication. The loss of a chromosome is lethal, an extra chromosome causes trisomy. 20% of first trimester miscarriages show autosomal trisomy. Cytoplasmic inheritance – fertilizing spermatozoa contributes the centriole (essential for karyokinesis and cytokinesis). Without this, cell division during early development is compromised. Most cytoplasmic inheritance comes from the female, e.g. cell membrane, cell organelles and mitochondria. In humans, activation of embryonic genome occurs from 4-8 cell stage. Maternal RNA dominates prior to that stage, so any protein synthesis in the zygotes is maternally regulated in these early stages. All adult mitochondria are maternally derived. Paternal mitochondria, if any, undergo autophagy. Genetically based defects of mitochondrial function will be transmitted to offspring. Transport of conceptus – zygote to blastocyst. Cell division occurs as it travels to the uterus. There is rising ration of progesterone to oestrogen. The cilia of tubal epithelium push the zygote towards the uterus for correct implantation. Implantation has 3 phases – attachment (apposition), adherence (stable adhesion) and invasion (burrows into the wall). Compaction in the morula leads to polarity and asymmetric division, resulting in 2 groups of cells: trophoectoderm (will form the placenta, outermost layer of cells) and inner cell mass (will form embryo). The zona pellucida prevents blastomeres falling apart during early cleavage, it prevents dizygotic twins sticking together and making a chimeric conceptus. Conceptus-uterus communications – after attachment, for successful implantation and continued pregnancy: - Conceptus must signal its presence to the mother to avoid commencement of menstruation  - Must continue to signal its presence to the mother throughout pregnancy (maternal recognition of pregnancy must be maintained) - This is achieved by long range communication which alters the pituitary-ovarian axis. Long range communications – maternal recognition of pregnancy is required to stop cyclical changes. The trophoblast secretes hCG, which is carried to the ovary and binds to LH receptors on luteal cells of the corpus luteum. The luteotrophic signal supresses luteolysis and prolongs life of corpus luteum. The corpus luteum of pregnancy continues to produce progesterone (up to 6 weeks of pregnancy) but then placental production is high enough to take over. The progesterone dominated phase of endometrium is prolonged throughout gestation (also ensures further ovulation ceases). Trophoblast cells of blastocyst secrete human chorionic gonadotrophin (hCG), which is an analogue of LH. This is carried to the ovary where it binds to the LH receptors on granulosa cells of the corpus luteum (continues to produce progesterone and suppresses luteolysis). This prolongs the life of the corpus luteum which continues to produce progesterone up to 4 months of pregnancy, thereafter the placenta takes over production. The endometrium remains in the progesterone dominated secretory phase throughout gestation. Short range communication – attachment involves dissolution of zona pellucida and adhesion between trophoblast and epithelial cells of the endometrium. There is increased stromal reaction in the endometrium underlying the attaching blastocyst which spreads. There is also increased angiogenesis, vascular permeability and oedema in the endometrium. This process of endometrial change is called decidualisation. The high levels of progesterone and hCG initiates the decidualisation of the endometrium. LIF promotes receptivity (come here message) and there is downregulation of cell surface mucin (go away message). Zipper theory is where HSPG of both trophoblast and endometrium bind together. The conceptus must establish physical and nutritional contact with the mother. Adhesion between trophoblast and epithelial cells of the endometrium starts changes in the endometrium stroma. The changes are deep and spread to the areas surrounding the blastocyst and all along the uterus as the trophoblast invades into the stroma. This process of endometrial change is called secondary decidualisation. The high levels of progesterone and hCG is behind the secondary decidualisation. Attachment initiates a stromal reaction at the site of attachment, which spreads to create 3 decidual layers: - Decidua basalis – endometrium underlying the conceptus. This shows the highest changes as this is where the conceptus needs to burrow into. This is also called the basal plate of the placenta. - Decidua capsularis – superficial portion overlying the conceptus. - Decidua parietalis/vera – remaining uterine mucosa. Molecular language of attachment: - Endometrium – endometrial glands produce LIF (leukaemia inhibitory factor) which acts as a ‘come hither’ message for a short period of time. Endometrium down regulates anti-adhesive cell surface mucin (MUC1) which normally acts as a ‘go away’ molecular message. Clinically, high levels of MUC1 may be behind miscarriages. Epithelial cells adjacent to blastocyst produces heparin binding EGF-like growth factors and heparan sulphate proteoglycans (HSPG). - Trophoblast cells – these cells also upregulate heparan sulphate proteoglycans. HSPG from both cell types bind together (homophilic binding). Integrin binding occurs between the 2 cell  types. Endometrial EGF binds to trophoblast EGF receptors. Blastocysts deficient in EGF (epidermal growth factor) receptors die. Molecular and cellular language of invasion: - After attachment, the single layer of trophoblast in the trophoectoderm proliferates to form numerous cytotrophoblast cells and creates the chorionic plate of the placenta. - The uppermost cytotrophoblast cells also fuse with each other to become a single multinucleated tissue layer without cell boundaries, called the syncytiotrophoblast. This syncytiotrophoblast forms numerous finger-like projections into the endometrium. It releases proteases (including matric metallo proteases which digests endometrial tissue and allows the blastocyst to burrow into the endometrium). There is loosening of the epithelial cells of the endometrium to allow invasion. Pro-inflammatory reaction results in increase in histamine, prostaglandins, TGFβ etc. This is good for the blastocyst as it can utilise them to continue burrowing. All secreted by stromal cells, macrophages, decidual natural killer cells. - The burrowing and expanding syncytial fingers further degrade endometrial tissue. They tap uterine glands and can use the uterine milk for histiotrophic nutrition of the developing placenta and embryo (for first 10 weeks). The conceptus burrows into the wall completely. There is plug formation and repair of endometrium at the entrance site. This type of implantation is called interstitial implantation. This is unique to humans. The baby doesn’t grow in the uterine cavity, it continues to grow in the wall of the uterus and slowly obliterates it as it grows. Parental imprinting – for the majority of autosomal nuclear genes, both the mother and the father pass on functionally active copies to their offspring. Expression is bi-allelic. Parental imprinting compels us to reproduce sexually. For some genes, one parent actively silences their own gene meaning that there is a parent-of-origin specific monoallelic expression of the gene in the offspring. The packaging of chromosomes in oocyte or spermatozoa can influence the organisation of genes and their ability to become transcriptionally active or silenced (epigenetics). This epigenetic imprinting is important. Genes affected in spermatogenic lineage may differ from oogenetic lineage. To be fully functional a majority of genes require both parental imprints. The importance of parental imprinting may be behind the birth of sexual reproduction. In 1% of genes we inherit only 1 working copy of a gene, the other is silent. These genes are called imprinted genes. Depending on the gene, either copy (maternal or paternal) can be silenced. Silencing usually happens through the addition of methyl groups. The epigenetic tags on imprinted genes usually remain for the life of the organism. Such silencing leads to the direct phenotype expression of the allele that is not being silenced. E.g. IGF II/insulin-like growth factor 2 is paternally expressed, it binds to its receptor to promote growth and nutrient transfer in the placenta and fetus. The dad is ensuring growth and good nutrient transfer to the baby. There is a second receptor for IGF II that can bind to it (weaker affinity), which is maternally expressed so can mop up some IGF II if there is too much nutrient transfer. Suckling behaviour and smiling in babies are paternally expressed genes. Epigenetics – mechanism of resulting gene expression, maintained across cell divisions without altering the DNA sequence itself. The epigenome is the machinery that regulates gene expression and thus may alter phenotype. Methylation inhibits/silences the gene (via the CpG). Histone modification (tightness of wrapping) that also can silence a gene. Epigenetic modifications can occur when: - Epigenetic reprogramming during gametogenesis – behind uniparental imprinting of alleles. Genes affected by epigenetic changes in the spermatogenic lineage differ from those imprinted in the oogenetic lineage. One of the parents is silenced.  - Epigenetics in early development – important epigenetic processes can occur during development in the periconceptual period. - Epigenetics after fertilisation – upon fertilisation there is rapid demethylation of the entire parental genome in all genes. However, some genes remain imprinted. - Throughout life – epigenetic mechanisms can be continually modulated. Differential environmental exposure between monozygotic twins results in differing phenotypes, regardless of an identical genome. Dizygotic twins are also vulnerable to epigenetic modifications. Implantation Incorrect implantation can occur at the cervix, internal os (placental praevia) etc. Attachment and adherence of the blastocyst to the endometrium only occurs in the luteal phase when endometrium is receptive and when it can provide nutrition. At this phase there is loss of glycocalyx from epithelial surface of cells of the endometrium, there is also loss of anionic charges on the epithelium flattening of epithelial microvilli and thinning of the mucin coat. This is a very small window only 2-3 days long. These changes reverse in the regression phase. The endometrium in the luteal phase has saw toothed secretory glands which produce lipids, carbohydrates, nucleic acids and proteins (uterine milk). There is diffusion of these nutrients to the blastocysts via tapping (this is called histiotrophic nutrition). After the first 10 weeks of histotrophic nutrition – as syncytiotrophoblast digest endometrial tissue, small spaces appear which are called lacunae. Cellular debris from eroded endometrial uterine glands and maternal blood from ruptured endometrial capillaries fill these lacunae. The lacunae become interlinked and creates large spaces filled wit