Dissertation submitted in full compliance with the requirements for the Master's degree in Technology: Radiography, Durban Institute of Technology, Durban, 2004. / The object of this study was to use Transvaginal ultrasound to evaluate the thickness of the endometrium to exclude endometrial abnormality in Black South African women with postmenopausal uterine bleeding. Transvaginal ultrasound is an excellent diagnostic method for assessing endometrial pathology. The study was carried out at the Gynaecological Ultrasound Department, King Edward VIII Hospital. The study included 76 Black women with postmenopausal uterine bleeding. The thickness of the endometrium was measured by Transvaginal ultrasound. The measurement included both endometrial layers (double-layer technique). The Transvaginal ultrasound measurement was compared with the histopathological diagnosis of the biopsy specimens. At the end of the investigation, findings obtained were 3.9% non-representative, 44.8% endometrial adenocarcinomas, 14.5% benign polyp, 3.9% chronic Endometritis, 17.1% benign endometrium, 5.3% endometrial hyperplasia, 9.2% atrophic endometrium, 3.9% myometrial invasion and 1.3% Malignant Mixed Mullerian Tumour. In this study, the thickness of the endometrial echo varied from 5mm to 35mm, with a mean of 18,2mm. When the thickness of the endometrial echo was compared with the histopathological results, the mean value for non-representative was 7.83mm, much lower than the thickness of an active endometrium (13.25mm). In cases with atrophic endometrium, the thickness ranged from 6mm to 30mm with a mean of 15.86mm. The mean value obtained for cases with endometrial adenocarcinoma was 20.32mm (range 11 to 35mm). The sensitivity, specificity and accuracy of Transvaginal ultrasound for detecting endometrial malignancy were 100% if the cutoff limit of 4mm was used In conclusion, this study using Transvaginal ultrasound demonstrated that a thickness limit greater than 8mm was considered in detecting malignancy. No malignant endometrium was thinner than 5mm. Therefore in women with postmenopausal uterine bleeding and an endometrium less than 4mm, it may be justified not to perform further investigations. Transvaginal ultrasound is a simple, well-tolerated safe and reliable method for identifying endometrial thickness in postmenopausal Black South African women. / M

Introduction: The human endometrium undergoes cyclical changes of proliferation, differentiation and shedding. This cyclical process has been described as an inflammatory process. Menstrual abnormalities account for the morbidity of a large population of females in their reproductive age. Aberration in endometrial angiogenesis has been implicated in the mechanism of heavy menstrual bleeding (HMB). Although the precise mechanism for control the endometrial neoangionesis is not fully understood, vascular endothelial growth factor and other cytokines such as cyclooxygenases, prostaglandins, interleukin -8 and leukocytes have been implicated in both endometrial pathologies and angiogenesis dysregulation. In addition, heavy menstrual bleeding results from upregulation of the expression/synthesis of these local markers. Uterine fibroids are the most common benign tumor affecting the female reproductive tract. Heavy menstrual bleeding is the main presenting complaint of women with uterine fibroids. However, the mechanism by which uterine fibroids cause heavy menstrual bleeding has not been elicited yet. Therefore, the mechanism of action of different available treatments for this condition, including uterine artery embolisation is unclear. This thesis is based on the hypothesis that a) uterine fibroid changes the physiology of endometrium and we aimed to find out whether these markers work in a different way in heavy menstrual bleeding in those with uterine fibroids and those without., In addition I we wished to study whether uterine fibroid upregulate these local markers in heavy menstrual bleeding, whereas uterine artery emolisation down-regulates them. Methods: This thesis describes the use of endometrial samples, taken with a Pipelle sampler, collected from women with heavy menstrual bleeding both with uterine fibroids and also with normal uteri, to estimate the difference in the endometrial expression of the factors likely to be involved in the control of menstrual bleeding between the two groups. Results: The study found no differences between the expression of both either proteins or mRNA for the cytokines under investigation By using endometrium, myometrium and different types of fibroid tissue collected from women who had hysterectomies with the complaint of heavy menstrual bleeding, there was higher expression of VEGF, COX-2, PGE2 and IL8 proteins in fibroid than myometrial tissue. However, the level mRNA of expression for VEGF, COX-1, COX-2, IL8 and EP2 showed no differences between myometrial and fibroid tissue. In the same group, endometrial expression of these markers for women who had no hormonal therapy before operation compared with that for women who received gonadotropin releasing hormone agonists (GnRH), higher expression of VEGF mRNA in women who had GnRH agonists than those who had no any hormones. In fibroid tissue, GnRHdownregulated the expression of VEGF protein and other cytokines compared with those not on any hormonal therapy. In addition, the estimated serum levels of these factors, indicating a higher level of IL8 in the GnRH group than in the other group. Conclusion: It seems that theses markers play a role in HMB mechanism in both uterine fibroid and normal uteri group in same manner. In addition, they have a fundamental role in the growth of uterine fibroids as well.

Background: Young women with Polycystic Ovary Syndrome (PCOS) may have increased measures of cardiovascular risk. It is difficult to determine how much of this risk is due to PCOS itself and how much is due to obesity and insulin resistance,which are common in PCOS and are themselves associated with greater cardiovascular risk. Aims and Methods: The study aimed to determine if arterial stiffness, carotid intima-media thickness and diastolic dysfunction were increased in young women with PCOS independently of the effects of obesity. A cross-sectional study of women with PCOS and healthy volunteers aged 16-45 years was undertaken. Subjects had a comprehensive assessment of body composition (including computed tomography assessment of visceral fat), measurements of arterial stiffness (aortic pulse wave velocity; aPWV), common carotid intima-media thickness (ccIMT), diastolic function (longitudinal tissue velocity; e’:a’) and metabolic measures including an oral glucose tolerance test to assess insulin area under the curve (IAUC), a marker of insulin resistance. Results: After adjustment for age and body mass index, PCOS subjects had greater insulin response (IAUC) following glucose challenge (adjusted difference [AD] 35900 pmol min/l, P<0.001), higher testosterone (AD 0.57 nmol/l, P<0.001) and high molecular weight adiponectin (AD 3.01μg/ml, P=0.02) than controls. There were no significant differences in aPWV (AD -0.13m/s, P=0.33), ccIMT (AD - 0.01mm, P=0.13) or e’:a’ (AD -0.01, P=0.86). After adjustment for age, height and central pulse pressure, aPWV and e’:a’ were associated with log visceral fat and IAUC. After adjusting for log visceral fat, the relationships between aPWV or e’:a’ and IAUC were only party attenuated. There was no relationship between cardiovascular measures and adiponectin or testosterone. Conclusions: Insulin resistance and central obesity are associated with subclinical dysfunction in young women, but a diagnosis of PCOS does not appear to confer additional risk at this age.

Breast cancer (BC) is the most commonly diagnosed cancer in the UK and the second leading cause of cancer-related deaths in women worldwide. Limited treatment is available to patients with metastatic BCs, which are resistant to therapy. There is a strong link between metastatic BC and Epithelial Mesenchymal Transition (EMT). There is a need to target EMT, in order to prevent metastasis for which the therapy is not effective. Therefore, there is a need for improved pre-clinical models that will allow studying EMT in real-time. This study aimed to refine current in vivo models of BC, by incorporating inducible bioluminescent reporter(s) that will allow real-time read-out of EMT. The cell-lines used were classified according to their expression of EMT markers and cancer stem cell (CSC) profile. Inducible bioluminescent EMT reporters based on the S100A4 or E-cadherin or N-cadherin promoter driving firefly luciferase were constructed and used to transduce the cell lines. The activity of the S100A4 reporter was validated in hypoxic conditions in MCF-7 and BT549 cells, where it was shown that induction of S100A4-generated bioluminescence was associated with upregulation in S100A4. In vivo it was shown that human mesenchymal stem cells promoted the tumour growth of MCF-7 cells and/or induced EMT-related traits, including downregulation/loss of E-cadherin, upregulation of Vimentin and/or upregulation of S100A4. The activity of the S100A4 reporter was tested in a similar in vivo model, where it was shown that S100A4-generated bioluminescence correlated with protein expression of S100A4. Taken all the data together, we generated an inducible bioluminescent EMT reporter that can detect the baseline levels and changes in the S100A4 expression. The model can be potentially used for giving a real-time read out of EMT/early stages of EMT and subsequently for testing novel drugs targeted at the onset of EMT.

The study investigated the cost-effectiveness of caesarean section (CS) as the major component of Emergency Obstetric Care (EMOC) in a humanitarian context. Research was conducted from December 2007 until June 2008 in Bunia, in the north-east of the Democratic Republic of Congo. Methods A case-control study explored the factors determining whether a woman had a CS or a vaginal delivery. Cases (n=178) were randomly selected from women who had delivered by CS. Controls (n=180) were women who had delivered vaginally within two weeks of a case and were matched by place of residency. Face-to face interviews in the local language used a structured questionnaire about obstetric and socio-economic factors. Obstetric care was assessed during repeat visits to health structures using checklists. Provider cost of CS was calculated for four hospitals, of which one provided free emergency healthcare. Information about cost allocation to CS was collected from hospital managers, maternity staff, and administrators. Costs were verified with local entrepreneurs, international organisations and UN agencies. The social cost of maternal death was discussed in focus groups, which also obtained user cost information additional to the data from the case-control study. Results CS constituted 9.7% of expected deliveries in the Bunia Health Zone. During the study period, the humanitarian hospital performed 75% of all CS. There were no elective CSs in the study sample. The study found no evidence of obstetric surgery for non-medical reasons. Previous CS and prolonged labour during this delivery were the strongest predictive factors for CS. The risk increased with age of the mother and decreased with the number of children alive. Fifteen obstetric deaths were reported to the research team, three among them were women who had a CS. After adjusting the observed number for missed pregnancy-related and late post-partum deaths, the estimated number of maternal deaths avoided by humanitarian EMOC, compared to expected mortality without additional services, ranged from 20 to 228. Compared to recent estimates for the DRC, perinatal deaths avoided ranged from 237 to 453. Cost-effectiveness was expressed as cost per year of healthy life expectancy (HALE) gained. The estimated cost of adding one year of HALE by providing CSs in a humanitarian context ranged from 3.77 USD to 9.17 USD. Comparison of the cost of EMOC and the social cost of maternal death was complicated by the existence of local customs such as “sororate”. The user capacity to pay for health insurance was found to be low. Conclusion Caesarean sections as part of humanitarian assistance were cost-effective. To keep EMOC accessible during and following the transition from emergency relief to development, a change in the national financing policy for health services is advisable.

Recurrent miscarriage (RM) is stressful. One reason for this is because no causes can be found for the pregnancy loss in the majority of cases. Focus has been on the endometrium which undergoes decidualisation in preparation for implantation. Any problems in the finely organised interactions between the endometrium and invading trophoblast cells may contribute towards a miscarriage. Immunological mechanisms are thought to be one of the pathways involved as there is the need of maternal adaptation of her immune response to the semi-allogenic developing embryo. Uterine natural killer (uNK) cells are the most abundant in the endometrium during the window of implantation. They interact with trophoblast cells, and are involved in vascular remodelling, an important step in implantation. Hence, they have a biological plausibility of playing a major role in RM. Both peripheral NK (pNK) and uNK cells tests have been developed as assessments of immunological causes of RM. A systematic review performed showed inadequate evidence for both pNK and uNK cells tests as markers for adverse pregnancy outcomes. There were only twelve studies, with 446 patients reporting pregnancy outcomes. There was no accepted consensus of normality and methodology for analysing NK cells. The conclusion was the need for well-designed studies to assess the role of NK cell tests as a clinically useful marker for screening. This led to the conduct of the pilot phase of a RCT of prednisolone in early pregnancy in women with idiopathic RM and raised uNK cells density. The main aim of this trial was to assess feasibility of recruitment and tolerability of prednisolone. Secondary clinical outcomes included live birth, types of miscarriage, miscarriage karyotype, gestational age at delivery, birthweight and pregnancy complications (eg: pre-eclampsia, gestational diabetes, fetal abnormality, stillbirth, IUGR). 160 women were screened for uNK cells density and 40 were randomised, despite the majority (85%) desiring prednisolone if given a choice. There was a trend towards improved live birth rate with prednisolone treatment but this was not significant. There were equal numbers of biochemical, sac and fetal pregnancy losses in both groups. All completed treatment with main reported side effects in the prednisolone group of insomnia. There were no pregnancy complications. The analysis of uNK cells was found to be very time consuming. To accommodate potentially large numbers who will be screened in the definitive trial, an alternative, quicker and equally accurate method of analysing uNK cells was developed using the colour deconvolution and area measurement plug-ins of a public domain image analysis package, Image J. Women supported this trial. Randomisation was acceptable. The prednisolone was safe. UNK cell density is a valid biomarker of severe outcomes. There was a trend towards improvement in live birth rates. This trial paves the way for the development of an endometrial based test to screen for the subgroup of women with RM that could potentially benefit from individualised treatment.

The extensive assessment of bull’s reproductive potential prior to breeding is highly important and includes examination of general physical soundness, external and internal genitalia and semen quality. Breeding success depends on the efficient use of bulls with high breeding value but simultaneously semen quality imposes restrictions on the use of these bulls in AI. Several techniques have been devised to assess quality of either fresh or frozen-thawed semen. Among a variety of traditional parameters sperm concentration, sperm raw and post-thaw motility and sperm morphology are commonly used for routine semen assessment in the laboratory. In this study, we investigated differences in sperm metabolic activity relative to their motility that may reflect better the fertility of bulls from their non-return rates (NRRs). To investigate the relationship between mid-piece length and fertility of bovine spermatozoa, sperm biometry was performed on ejaculates obtained from 34 bulls representing six breeds: Holstein (yearlings and mature), Friesian, Belgian Blue, Aberdeen Angus, Charolais and Limousin. Significant differences (P<0.01) between ejaculates were found in 9/34 bulls, as well as differences (P<0.001) between individual bulls within the same breed. The average mid-piece length for Aberdeen Angus was 13.35μm, for Belgian Blues and Limousin around 13.8μm, and for Charolais 13.68μm: for dairy breeds (Holstein and Friesian) it was about 13.4μm. The mean value of mid-piece length for breed was compared with their 49 day non-return rate; a negative correlation was found in the dairy breeds, while in bulls from beef breeds this correlation was positive but very low: the small numbers of bulls involved prevented meaningful statistical relationships being established. To differentiate live and dead sperm and non-sperm-specific particles, a flow cytometry method was developed by labelling sperm with JC-1 and propidium iodide (PI) dyes and to determine maximum mitochondrial membrane potential (ΔΨm) at minimum incubation. This method entailed setting regional and logical gates to exclude dead sperm and other non-cellular components from live sperm present within an ejaculate. It was confirmed that spermatozoa of both fresh and frozen-thawed semen exhibited maximum high:low ΔΨm ratio after 40 min incubation. Flow cytometric dot plots of analyses of fresh and frozen-thawed spermatozoa incubated with JC-1 could identify a unified sperm population of membrane-intact cells, each population characterised by both low and high ΔΨm but to varying degrees suggesting that this flow cytometric method simplifies the determination of mitochondrial membrane potential using JC-1. This method serves two purposes: using this method, one could able to evaluate sperm ΔΨm as well as the proportion of live:dead. Changes in mitochondrial structure and integrity appear to be an important component associated with sperm motility and reduced fertility. The ΔΨm was assessed using JC-1 and PI in the presence of glycolytic and respiratory inhibitors. Mean high ΔΨm was significantly greater for control compared to the treatments in fresh and frozen-thawed semen. In samples treated with valinomycin (VAL) and iodoacetamide (IAM) ΔΨm was lowered significantly. The proportion of sperm with a high ratio of high:low ΔΨm was higher in control and 2-deoxy-D-glucose (DOG) treated samples representing more active mitochondria: in samples treated with VAL and IAM the ratio was reduced, representing loss in activity of mitochondria. Cryopreservation significantly decreases high:low ΔΨm ratio in control suggesting that lower mitochondrial activity may be associated with oxidative stress produced by reduced antioxidant levels due to the freeze/thaw cycle. The relationship between ZO2 (µl oxygen consumed /108 spermatozoa/hr) and mitochondrial function was assessed in fresh and frozen-thawed semen. Sperm oxygen consumption was greater in fresh compared to frozen-thawed semen. Insignificant positive correlations existed between ZO2 and ratio of high:low ΔΨm in fresh (r=0.82) and frozen-thawed (r= 0.49) semen suggesting that the ΔΨm measured in this way by flow cytometry can be used as an indicator of ZO2. Finally, the metabolic pathways by which spermatozoa produce energy to support their motility were investigated in fresh and frozen-thawed semen diluted in media containing glycolytic and respiratory inhibitors. Total and progressive motilities were not significantly different in sperm incubated with DOG and VAL but decreased significantly with IAM compared to control. This indicates that sperm can maintain a similar degree of motility when generating their energy exclusively from either glycolysis or mitochondrial activity. IAM significantly lowered sperm motility as well as mitochondrial activity (as described above) and was found to be an inhibitor of both glycolysis and respiration possibly linked with either modification of mitochondrial cysteine and/or glutathione levels. Sperm are considered in a state of hyperactivation/capacitation when their amplitude of lateral head displacement (ALH) increases and path straightness (STR) and linearity (LIN) decrease. In the present study higher ALH and lower STR and LIN were observed when spermatozoa were dependent on mitochondrial energy (DOG), whereas these estimates were reversed when they were on glycolytic energy (VAL) indicating that sperm hyperactivation and capacitation are associated with mitochondrial function. There was a positive correlation of sperm progressive motility, ZO2 and high:low ΔΨm ratio with bull NNRs suggesting that these sperm characteristics may be useful for predicting bull fertility. Furthermore sperm mid-piece length was significantly correlated with sperm average curvilinear velocity and amplitude of lateral head displacement. Since the mitochondria are localized on the sperm mid-piece, it is likely that its energy may contribute in high sperm velocity and also hyperactivation that helps sperm disengagement from oviduct epithelium and positioning at the site of fertilization.

Over 5 million babies have been born as a result of IVF procedures. Worldwide, over 1 million cycles of IVF are performed annually. The IVF procedure involves ovarian stimulation with the purpose of developing multiple follicles and maximising the potential oocyte yield. As a consequence of high oestradiol levels produced during treatment and the use of GnRH agonists or antagonists, a luteal phase deficiency results. This phenomenon is associated with reduced implantation potential and suboptimal conditions for maintenance of early pregnancy. Luteal support in the form of progesterone or HCG has been demonstrated to improve pregnancy rates after IVF. A number of luteal support protocols have been investigated with progesterone the most commonly used drug. The optimum duration of luteal support has yet to be defined. With no agreement in clinical practice evident, the reported use of progesterone ranges from withdrawing luteal support at confirmation of biochemical pregnancy to continuation beyond 12 weeks gestation. Whilst luteal support is considered a very important aspect of IVF treatment, there is very little evidence to support an optimum duration of use. The DOLS trial is a prospective randomised double blind placebo controlled trial investigating the effect of additional luteal support beyond confirmation of pregnancy test after assisted conception. Four hundred and sixty seven patients were randomised after confirmation of biochemical pregnancy to receive a further 8 weeks of vaginal progesterone or 8 weeks of placebo. Summary results were to include a primary outcome defined as viable pregnancy at 12 weeks gestation, whilst secondary outcomes were to report on live birth rates, pregnancy associated complications, neonatal outcomes, effect on first trimester serum screening and effect on uterine artery Doppler velocity. The DOLS trial reported no difference in pregnancy outcome at 12 weeks gestation, with 167/228 (73.3%) women randomised to the extended luteal support treatment arm having a confirmed viable intrauterine pregnancy compared with 167/233 (71.7%) women randomised to the placebo arm of the trial; adjusted risk ratio 0.97 (95%CI 0.87 to 1.09). Similarly live birth rates were not different between the treatment groups; 71.1% versus 70.4% respectively. No effect of extending luteal support beyond positive pregnancy test was observed in reference to complications of pregnancy, neonatal outcome, uterine artery Doppler velocity or antenatal screening outcome. In conclusion, we have confirmed that continuing luteal support using progesterone beyond confirmation of biochemical pregnancy offers no benefit in terms of pregnancy outcomes. However the extended use of progesterone until 12 weeks gestation does not confer harm. We suggest that all clinics worldwide should consider offering luteal support no further than positive pregnancy test, at which point it can be safely withdrawn without compromising live birth rates and reducing treatment burden.

Postpartum haemorrhage (PPH) is the most common cause of maternal mortality leading to an estimated 86, 000 deaths/year. The most common cause of PPH is failure of the uterus to contract properly (uterine atony). Several measures have been introduced to prevent and treat atonic PPH, but in spite of active management of the third stage of labour (AMTSL), maternal deaths from PPH still occur. PPH can kill rapidly within two hours or less. PPH has long been recognised as a dangerous complication for mothers. In order to optimise the prevention and treatment of PPH, different approaches have been introduced and modified over the last century. We reviewed the regimes used in the management of the third stage of labour between 1917 and 2011 as described in the successive editions of the ‘Ten Teachers’ books. Throughout the Ten Teachers series, uterotonic drugs have always been taught as being the best initial measure to manage PPH. However, the importance of bimanual uterine compression (BMC) has increased gradually, moving from third to first treatment option over the editions (Aflaifel and Weeks, 2012a). The components of the AMTSL package for PPH prophylaxis have recently been extensively examined in clinical trials. Its effectiveness in reducing blood loss is now known to be almost all due to the uterotonics (Aflaifel and Weeks, 2012b). However, clinical trials evaluating the efficacy of uterotonics in treating PPH are comparatively rare. Where present they usually compare two uterotonics with an absence of control group, as it is unethical to leave a bleeding woman untreated. A recent innovation is to model the likely outcomes in the absence of uterotonic therapy through histograms. This also allows an assessment of the efficiency of treatment by measuring the number of women who stop bleeding shortly after administering treatments. This model has never previously been applied to databases in which uterotonics were used for prophylaxis. In a secondary analysis of 4 large randomised trials, small secondary histogram peaks (primarily attributed to a treatment effect) were still present even if uterotonic therapy had not been used. Furthermore, the study revealed that women were commonly treated at low levels of blood loss (< 500 mls). It was also seen that, of those diagnosed with PPH (≥ 500 mls), most stopped bleeding at blood losses of around 700 mls even if they did not receive any uterotonic therapy. This should warn against ascribing all the effect to uterotonic therapy. As well as stopping spontaneously, other physical therapies may also have been used concurrently and may have had an effect. The evidence from the histogram study suggested that use of additional uterotonic is not a good surrogate for PPH in the research context. Chapter 4 reports on evaluations of the outcomes that are used by researchers in PPH trials. In the 121 studies evaluated, there was a huge diversity in choosing the outcomes (PPH prevention). The most common was ‘Incidence of PPH ≥ 500 mls’, which was mentioned in 21% (25/121) of trials. The study interestingly showed that use of additional uterotonic was used for sample size calculation in 6% (7/121) of studies as a surrogate for PPH. The above findings emphasise the importance of physical measures in the early treatment of PPH. BMC is thought to help in treating PPH, although there are no clinical trials on its effectiveness. A survey was therefore conducted amongst obstetric care providers in the UK to look at the frequency of BMC use in clinical practice and the attitudes towards its use. The survey found that, although clinicians find BMC effective, it is rarely used as the procedure is considered to be too tiring and too invasive. If, however, BMC could be performed in a less invasive manner, then it could act as an effective low-cost treatment for those PPHs arising from atony. The thesis concludes with an investigation into a new low cost intervention that might contribute to the early physical management of PPH. The ‘PPH Butterfly’ is a new device that is designed to make uterine compression simpler, less tiring and less invasive. It was compared to the standard BMC in a mannequin model. The main objective was to compare the efficacy of the PPH Butterfly to standard BMC in producing sustained uterine compression. The study revealed that the PPH Butterfly is simple to use on a mannequin model, even among obstetric care providers with little experience. It produces an equivalent amount of pressure to BMC, but neither method produced sustained compression over the 5 minutes of use. It also demonstrates the feasibility of using a mannequin model for teaching and performing BMC.

The calpain system is a group of intracellular cysteine proteases. Dysregulated calpain activity, or mutation in calpain isoforms, as well as its endogenous inhibitor calpastatin, has been found to play an important role in tumorigenesis. The main aim of the current study is to explore the differential role of calpain family members in different breast cancer molecular subtypes; validate calpain-1 as a biomarker of trastuzumab response in HER2+ breast cancer patients; and explore the mechanisms by which calpain family regulates trastuzumab response in HER2+ cells. Three representative cell lines for different molecular subtypes were used: MDA-MB-231 (basal-like), MCF-7 (luminal) and SKBR3 (HER2+); and two additional HER2+ cell lines were used in the HER2+ study: acquired trastuzumab-resistant SKBR3 and inherent trastuzumab-resistant JIMT-1. The role of calpain in proliferation, signal transduction and apoptotic response was assessed using growth curves, phosphokinase arrays and Annexin V-FITC apoptosis assays, respectively. The effect of calpastatin knockdown, via shRNA, on cell migration was examined using Haptotaxis assay. The combined effect of calpeptin and trastuzumab on colony formation and cell cycle progression were examined using clonogenic survival and flow cytometry, respectively. Biomarker studies were conducted using standard immunohistochemistry. The results suggested that inhibition of calpain activity showed anti-proliferative effect on breast cancer cells across different subtypes. Knockdown of calpastatin in both MDA-MB-231 and MCF-7 cells did not have significant effects on migratory ability, either with or without calcium ionophore A23187. The study also showed that combining calpeptin and trastuzumab enhanced trastuzumab-induced anti-proliferative effects on SKBR3 and SKBR3/TR cells, but not in JIMT-1 cells. Combined treatment did not further reduce clonogenic survival either in SKBR3 or SKBR3/TR cells, compared with single agent alone. In all three HER2+ cells, combined treatment had no significant effect on trastuzumab-induced G0/G1 cell cycle arrest. Results from the immunohistochemical study suggested that high calpain-1 expression was significantly associated with adverse relapse-free survival in breast cancer patients who received adjuvant trastuzumab. Findings were validated in the expanded Nottingham and independent Newcastle patient cohort. Based on the previous in vitro study, suggesting a role of calpain in regulation of phospho-MSK1/2 expression; and because there is close link between calpain and caspase family. It was decided to explore the correlation between calpain system protein expression with MSK1 and two representative caspases (caspase-3 & -8), as well as their prognostic significance, in a large cohort of invasive breast cancer patients. Results demonstrated significant correlations between calpain-1 vs MSK1, and vs caspase-3; calpastatin vs MSK1, and vs caspase-8, however with low correlation coefficients. High MSK1 expression was significantly associated with improved breast cancer-specific survival. High caspase-3, but not caspase-8, was significantly associated with adverse breast cancer-specific survival. And combinatorial calpain-1 and caspase-3 expression provided additional prognostic values, especially in basal-like subtype. In conclusion, calpain system protein has been found to have roles in different breast cancer molecular subtypes. Calpain-1 is a potential biomarker for trastuzumab response in HER2+ breast cancer patients, and this study suggested MSK1 and caspase-3 could be potential biomarkers in breast cancer.