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Women who experience early natural menopause, or surgical menopause, may be prone to any morbidity and mortality risk related to oestrogen deficiency. The thesis aimed to explore the long-term sequelae of the menopause by investigating current knowledge about the relationship between early age at natural menopause, any surgical menopause, and early age at surgical menopause, and future mortality, incident cancer, and incident circulatory disease. The thesis also investigated the long-term sequelae of menopause using data from a nested cohort of UK women from the Royal College of General Practitioners’ Oral Contraception Study [RCGP OCS]. Methods A portfolio of systematic reviews was conducted. To assess whether a young age at natural menopause, any surgical menopause, and a young age at surgical menopause were associated with mortality and ill-health, data from the RCGP OCS were analysed using Cox regression and competing risks. Results Systematic reviews: 153 papers were included, of varying quality. The heterogeneity of included studies’ exposure and reference groups meant that meta-analysis was not possible. Younger age at natural menopause was strongly associated with an increased risk of all-cause mortality, but not cancer-related death. A suggestion of an increased risk was found for cardiovascular death. Young natural menopause protected strongly against incident endometrial and weakly against ovarian cancers. Few studies examined cervical cancer and age at natural menopause was not a risk factor. Colorectal cancer was not consistently associated with age at natural menopause. Young age at natural menopause was associated with an increased risk of coronary heart disease [CHD], but not cerebrovascular disease. Little evidence was found that surgical, compared with natural menopause was associated with any mortality outcomes. Surgical menopause tended to protect against ovarian cancer but no effect was found for cervical cancer. The relationship between colorectal cancer, or cerebrovascular disease, and surgical menopause is unclear. More evidence showed that surgical menopause may increase the risk of CHD. Limited evidence for young age at combined natural and surgical menopause found it to increase the risk of all-cause mortality and perhaps cardiovascular death. There was no association with risk of cancer death. No clear associations were found for early age at combined natural and surgical menopause and any of the specific cancers examined, or cerebrovascular disease. There may be an association with CHD, but its evidence base was weak. Nested cohort of the RCGP OCS: Premature menopause was associated with decreased risk of: all-cause, cancer-, and circulatory-related mortality, all incident cancer, other cancers, and CHD. Premature menopause was not associated with: incident breast, main gynaecological, or colorectal cancer; all circulatory disease, cerebrovascular disease, or other circulatory disease. For early natural menopause, a significantly decreased risk was observed for: death from all causes, cancer, and non-cancer, non-circulatory causes; incident all cancer, colorectal cancer (borderline), other cancers; incident all circulatory disease and CHD. No significant risk was observed for circulatory mortality; incident breast or main gynaecological cancer; incident cerebrovascular disease or other circulatory disease. Compared with natural menopause, surgical menopause increased circulatory-related mortality only. The risk of incident main gynaecological cancers was decreased. The other incident cancer outcomes were not associated with surgical menopause. Risk of any, and each type of incident circulatory disease was not significantly altered with surgical menopause.

Introduction: Sporadic CJD (sCJD) has a varied clinical and pathological phenotype. This variability is felt to be influenced by patients' genotype at codon 129 on the prion protein gene (PRNP); and has associations with PrPres protein type. There is a polymorphism at codon 129 of PRNP, which results in three genotypes (MM, MV, VV). Additionally, the protease resistant protein (PrPres) seen in patients with CJD can also be classified according to certain biochemical characteristics. The protein types are determined by their mobility on western blot electrophoresis and their degree of glycosylation (type 1, 2a, 2b). In 1996, Parchi and Gambetti categorised cases by sub-types relating to these findings (broadly speaking: MM1, MM2, MV1, MV2, VV1 and VV2) suggesting this as an explanation for the clinical and pathological diversity of the condition (Parchi 1996). Relatively recently a significant minority of patients have been found to have both PrPres protein types present in the brain. Two recent papers have proposed a revised classification system, taking into account the co-occurrence phenomenon, suggesting there are further distinct groups of patients with sCJD (Cali 2009, Parchi 2009). Methods: 108 patients referred to the UK CJD surveillance system, confirmed to have had sporadic CJD at post-mortem, were included in the study. All cases had PRNP genetic analysis to exclude genetic CJD and establish their codon 129 status. Biochemical analysis of disease-associated prion protein via western blot was undertaken in the temporal, parietal and occipital lobes and the thalamus in all patients. A structured assessment of the clinical features seen both at presentation and during the patients' illness was undertaken. A summary of the pathological findings for the patients was also recorded. The clinical and pathological features were then analysed for the relative influence of the codon 129 genotype, protein typing and the combination of the two as the revised sub-type, according to the 2009 Parchi classification system. Results: 26 out of 108 patients had mixed PrP isotypes on western blot analysis. On comparing the most common of these mixed groups [MM 1(+2)] with those patients with the same genotype and predominant protein type [MM1], there were subtle differences in the clinical phenotype and pattern of investigation results. However, the major clinical findings were the same. In general, there was a trend towards increased disease duration with increasing relative ratios of type 2 proteins. On reviewing the pathological findings there were no findings unique to any genotype-isotype sub-group. Conclusions: While there were differing trends, the lack of distinct clinical or pathological findings by genotype-isotype sub-type suggest that these groups do not represent unique strains of prions, but rather groupings over a spectrum of disease. Therefore, more detailed biochemical analysis leading to a revised classification may be more accurate, but currently adds limited useful extra clinical information.

(1) Human trypsin was purified from human pancreatic tissue and used as an immunogen in the production of monoclonal antibodies. It was also used as a standard preparation of human trypsin in the screening of monoclonal antibodies and in the development of an immunoradiometric assay (IRMA). (2) Eight monoclonal antibodies were raised to human trypsin, two of which were subsequently employed in the development of an IRMA to measure immunoreactive trypsin (IRT) in blood and blood spots. (3) An antiserum to human trypsin was raised in sheep and employed in a sandwich ELISA. (4) Monoclonal antibodies were tested by a number of different screening systems including ELISA, immunoblotting and immunoadsorption assays with 125I-labelled human trypsin. (5) Six selected monoclonal antibodies were tested for use (a) as the solid phase antibody and (b) as the radioactive tracer in an IRMA, and against each other for compatability in an IRMA. (6) One combination of antibodies was chosen and experiments performed to determine (a) lack of competition of binding to antigen between the two and (b) positive binding of the combination to serum IRT. (7) An IRMA to measure IRT in blood and blood spots was developed using two monoclonal antibodies, 56/C5/33 (IgG2a) and 125I-labelled 55/A4/31 (IgM). (8) The detection limit of the assay with purified cationic human trypsin as the analyte was determined to be 0.8 ng trypsin. (9) The IRMA was performed with reconstituted blood containing standards of human trypsin (either blood or dried blood spots on filter paper). (10) There is potential for this assay to be further developed and employed as a routine screening assay to detect elevated concentrations of IRT in dried blood spots from newborn infants with cystic fibrosis.