Characterisation of an alternative splice variant of LKB1

The LKB1 protein kinase has previously been implicated in a number of major physiological processes including cell proliferation, polarity and energy metabolism. LKB1 is an upstream kinase for AMP-activated protein kinase (AMPK) and 12 members of the AMPK-related family of kinases. In this study, western blotting of LKB1 from various mouse tissues indicated the existence of two molecular weight bands in testis. This, along with the presence of a potential alternative exon at the 3’ end of the gene, led to the discovery that an alternatively-spliced variant of LKB1 is present in mice. This new splice variant has been termed the ‘short form’ (LKB1S) and the original protein termed the ‘long form’ (LKB1L). The proteins are identical in sequence apart from the C-terminus. The overall aim of this study was to characterise the LKB1S protein; its activity, regulation and possible physiological function, using overexpression studies in mammalian cells and analysis of mice lacking LKB1S. An LKB1S-specific antibody was used to show that LKB1S is primarily expressed in testis. It was demonstrated, using overexpression studies, that both forms can activate AMPK, form an active complex with the regulatory proteins STRADα, STRADβ and MO25 and have a similar sub-cellular localisation. The C-terminus of mouse/human LKB1L can be phosphorylated at serine 431/428 (Sapkota et al., 2001). This residue is absent from LKB1S and so the effect of phosphorylation was investigated to determine if this could lead to differences in activity between the splice forms. However, the results suggest that phosphorylation at this site does not affect the ability of LKB1L to activate AMPK in cells. Male mice lacking expression of LKB1S are infertile. The mice display severe sperm abnormalities and sperm counts are significantly reduced. Histological analysis of testis showed that although early spermatogenesis appears to progress normally, abnormalities become apparent later on, culminating in very few spermatozoa reaching the epididymis. One possible reason that has been investigated is that there is a defect in the release of mature sperm from the seminiferous epithelium (spermiation)

Inflammatory mediators : their roles during pregnancy and parturition

Pregnancy and parturition necessitate profound alterations in the maternal immune response likely effected by paracrine interactions between inflammatory mediators, hormones and various local factors. However, the nature of these interactions and how they effect the recognition and maintenance of pregnancy and initiation of parturition are not well understood. The aim of this thesis was to investigate the role of inflammatory mediators, specifically cytokines, prostaglandin E2 (PGE2), nitric oxide (NO), metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) during pregnancy and parturition.Part 1) This investigated the roles of inflammatory mediators in the initiation and maintenance of pregnancy. The role of seminal plasma, specifically its component PGs (PGE2 and 19- hydroxy PG), in the initial adaptation of the maternal immune response to pregnancy was studied in the non-pregnant cervix, peripheral blood and a monocyte cell line. PGs stimulated release of IL-10, thus favouring development of a T-helper (Th)-2 type, pregnancy favourable immune response. They also stimulated release of the neutrophil chemotactic factor IL-8, which may be involved in mediating post-coital cervical leukocytosis. Furthermore, peripheral blood from pregnant women, specifically the mononuclear cell (CD14+) fraction, released significantly more monocyte chemotactic peptide-1 (MCP-1), which also favours a Th-2 type immune response, than that from non-pregnant women. Release of IL-8 and regulated on activation and normally T-cell expressed and secreted (RANTES) was comparable between groups. The concentrations of MCP-1, IL-8 and RANTES in the fluid compartments within the first trimester uterus were also examined. The chemokines were differentially distributed with MCP-1 and IL- 8 being present in amniotic fluid, maternal serum and extra-embryonic coelom with highest levels of both in the latter compartment. RANTES was only detectable in peripheral serum. These findings support the hypothesis that inflammatory mediators are important in the initiation and maintenance of pregnancy and may play a role in early placental and fetal development.Part 2) This examined the involvement and regulation of inflammatory mediators in cervical ripening and parturition. Cervical ripening involves tissue remodelling mediated by inflammatory mediators, infiltrating cells, MMPs and TIMPs. However their regulation in the cervix are not well understood. In the non-pregnant cervix, IL-8 release was stimulated by PGE2 and NO and inhibited by dexamethasone and progesterone. In addition, the anti-inflammatory factor secretory leukocyte protease inhibitor was stimulated by progesterone and inhibited by PGE2 and PGE2 release was stimulated by NO. In addition, MMP-2 and -9 and TIMPs-1 and -4 were released by the non-pregnant cervix but this secretion was not affected by in vitro administration of PGE2 or NO. Withdrawal of progesterone, which is essential for pregnancy maintenance, also initiates cervical ripening by unknown mechanisms. In vivo administration of the anti-gestogen mifepristone increased protein expression of MMP-1, -8 and -9, CD45 (leukocyte common antigen), neutrophil elastase and CD68 (monocyte marker) but not MMP-2, TIMP-1, -2 and -4 in the first trimester cervix as detected by immunohistochemistry. To summarise the cervical studies, the non-pregnant and pregnant cervix are capable of releasing a wide range of inflammatory mediators, MMPs and TIMPs, co-ordination of which may mediate cervical ripening. Next, the release and regulation of inflammatory mediators at the materno-fetal interface were investigated. MCP-1, IL-8, RANTES and IL-10 were released by third trimester amnion, chorion, decidua and placenta with secretion of MCP-1, IL-8 and IL-10 but not RANTES being stimulated by PGE2 in a perfused placental cotyledon system. Secretory leukocyte protease inhibitor was released predominantly by decidua and was present in increasing concentrations within amniotic fluid during pregnancy and labour. Cytokines may play a role in the inflammatory process of parturition, PGE2 may play an important immunomodulatory role within the placenta at term and secretory leukocyte protease inhibitor might act both to limit the pro¬ inflammatory cascades ongoing during parturition and to protect against microbial invasion. To summarise this section, inflammatory mediators are important in the initiation and regulation of cervical ripening and parturition with complex interactions occurring within the cervix and at the feto-maternal interface

Does body image influence the relationship between body weight and breastfeeding maintenance in new mothers?

Objectives  Obese women have lower breastfeeding initiation and maintenance rates than healthy weight women. Research generally focuses on biomedical explanations for this. Psychosocial factors including body image and well-being after childbirth are less well understood as predictors of breastfeeding. In obese and healthy weight women, we investigated changes in body image between 72 hrs post-delivery and 6–8 weeks post-natal, studying how women's body image related to breastfeeding initiation and maintenance. We also investigated how psychological distress was related to body image.  Design  Longitudinal semi-structured questionnaire survey.  Methods  Body image and psychological distress were assessed within 72 hrs of birth and by postal questionnaire at 6–8 weeks, for 70 obese and 70 healthy weight women initiating exclusive (breastmilk only) breastfeeding or mixed feeding (with formula milk) in hospital. Breastfeeding was re-assessed at 6–8 weeks.  Results  Obese women were less likely to exclusively breastfeed in hospital and maintain breastfeeding to 6–8 weeks. Better body image was related to maintaining breastfeeding and to lower post-natal psychological distress for all women, but education level was the most significant predictor of maintenance in multivariate regression including body image and weight status. Body image mediated, but did not moderate the relationship between weight and breastfeeding maintenance. Body image was lower overall in obese women, but all women had low body image satisfaction around childbirth, reducing further at 6–8 weeks.  Conclusions  Health professionals should consider women's body image when discussing breastfeeding. A focus on breast function over form may support breastfeeding for all women

Qualitative evaluation of a rapid rollout of home blood pressure monitoring in pregnancy during Covid-19

In March 2020, the World Health Organisation named the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2), which causes corona virus disease 2019 (COVID -19), as a pandemic. Pregnant women were considered at increased risk of developing severe COVID-19 after viral infection. In response maternity services reduced face-to-face consultations with high-risk pregnant women by supplying blood pressure monitors for supported self-monitoring. This paper explores the experiences of patients and clinicians of the rapid roll-out of supported self-monitoring programme in Scotland during the first and second wave of the COVID-19 pandemic. We conducted semi-structured telephone interviews with high-risk women and healthcare professionals who were using supported self-monitoring of blood pressure (BP) In four case studies during the COVID-19 pandemic. 20 women, 15 midwives and 4 obstetricians took part in the interviews. Interviews with healthcare professionals showed that while implementation occurred at pace and at scale across the National Health Service (NHS) in Scotland, implementation differed locally, resulting in mixed experiences. Study Participants observed several barriers and facilitators to implementation. Women value the simplicity of use and convenience of the digital communications platforms while health professionals were more interested in their impact on reducing workload for both women and health professionals largely found self-monitoring acceptable, with only a few exceptions. These results show that rapid change can occur in the NHS at a national level when there is a shared motivation. While self-monitoring is acceptable to most women, decisions regarding self-monitoring should be made jointly and on an individual basis

A two-arm parallel double-blind randomised controlled pilot trial of the efficacy of Omega-3 polyunsaturated fatty acids for the treatment of women with endometriosis-associated pain (PurFECT1)

BackgroundEndometriosis is defined by the presence of endometrial-like tissue (lesions) outside the uterus, commonly on the pelvic peritoneum. It affects 6-10% of women and is associated with debilitating pelvic pain. Current management options are often unsatisfactory. Omega-3 polyunsaturated fatty acids (O-PUFA) have the potential to reduce the painful symptoms associated with endometriosis, reduce lesion size, preserve the patient's ability to conceive, and have minimal side effects. We performed a two-arm, parallel double-blinded randomised controlled trial to inform the planning of a future multicentre randomised controlled trial to evaluate the efficacy of O-PUFA for endometriosis-associated pain.ObjectivesThe primary objectives of the trial were to assess recruitment and retention rates. The secondary objectives were to determine the acceptability to women of the proposed methods of recruitment, randomisation, treatments and questionnaires, to estimate the variability in the proposed primary endpoints to inform the sample size calculation and to refine the research methodology for the future definitive trial.MethodsWe recruited women with endometriosis from June 2016 to June 2017 and randomised them to eight weeks of treatment with O-PUFA or olive oil. Pain scores and quality of life questionnaires were collected at baseline and eight weeks. We calculated the proportion of eligible women randomised, and of randomised participants who were followed up to eight weeks. Acceptability questionnaires were used to evaluate women's experiences of the trial.ResultsThe proportion of eligible participants who were randomised was 45.2% (33/73) and 81.8% (27/33) completed the study. The majority of participants described their overall trial experience favourably and there were no adverse events in either group.ConclusionOur pilot trial supports the feasibility of a future larger trial to definitively evaluate the efficacy of O-PUFA for endometriosis-associated pain.Trial registrationThe trial was registered on the ISRCTN registry (registration number ISRCTN44202346)

Recruiting and consenting into a peripartum trial in an emergency setting: a qualitative study of the experiences and views of women and healthcare professionals.

BACKGROUND: Recruiting and consenting women to peripartum trials can be challenging as the women concerned may be anxious, in pain, and exhausted; there may also be limited time for discussion and decision-making to occur. To address these potential difficulties, we undertook a qualitative evaluation of the internal pilot of a trial (Got-it) involving women who had a retained placenta (RP). We explored the experiences and views of women and staff about the information and consent pathway used within the pilot, in order to provide recommendations for use in future peripartum trials involving recruitment in emergency situations. METHODS: In-depth interviews were undertaken with staff (n = 27) and participating women (n = 22). Interviews were analysed thematically. The accounts of women and staff were compared to identify differences and similarities in their views about recruitment and consent procedures. RESULTS: Women and staff regarded recruitment as having been straightforward and facilitated by the use of simplified (verbal and written) summaries of trial information. Both parties, however, conveyed discordant views about whether fully informed consent had been obtained. These differences in perspectives appeared to arise from the different factors and considerations impinging on women and staff at the time of recruitment. While staff placed emphasis on promoting understanding in the emergency situation of RP by imparting information in clear and succinct ways, women highlighted the experiential realities of their pre- and post-birthing situations, and how these had led to quick decisions being made without full engagement with the potential risks of trial participation. To facilitate informed consent, women suggested that trial information should be given during the antenatal period, and, in doing so, articulated a rights-based discourse. Staff, however, voiced opposition to this approach by emphasising a duty of care to all pregnant women, and raising concerns about causing undue distress to the majority of individuals who would not subsequently develop a RP. CONCLUSIONS: By drawing upon the perspectives of women and staff involved in the same trial we have shown that they may operate within different experiential and ethical paradigms. In doing so, we argue for the potential benefits of drawing upon multiple perspectives when developing information and consent pathways used in future (peripartum) trials. TRIAL REGISTRATION: ISCRTN 88609453

Predictors of surgical site skin infection and clinical outcome at caesarean section in the very severely obese : A retrospective cohort study

Introduction The optimal surgical approach for caesarean section is uncertain in women with very severe obesity (body mass index (BMI) >40kg/m2). We aimed to assess maternal and surgical predictors of surgical site skin infection (SSSI) in very severely obese women and to undertake an exploratory evaluation of clinical outcomes in women with a supra-panniculus transverse compared to an infra-panniculus transverse skin incision. Material and methods Using a retrospective cohort design, case-records were reviewed of very severely obese women with a singleton pregnancy delivered by caesarean between August 2011 and December 2015 (n = 453) in two maternity hospitals in Scotland. Logistic regression analysis was used to determine predictors for SSSI. Outcomes were compared between women who had a supra-panniculus transverse compared to infra-panniculus transverse skin incision. Results Lower maternal age was predictive of SSSI, with current smoking status and longer wound open times being marginally significant. Maternal BMI, suture method and material demonstrated univariate associations with SSSI but were not independent predictors. Women with a supra-panniculus transverse skin incision were older (32.9 (4.4), vs. 30.6 (5.7), p = 0.002), had higher BMI (49.2 (7.1), vs. 43.3 (3.3), pPeer reviewe