Obstetrics & Gynaecology - Doctoral Theses
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Item Axis of placental ageing in adverse pregnancy outcomes(University College Cork, 2023) Manna, Samprikta; McCarthy, Fergus; McCarthy, Cathal; European Chiropractors' UnionBackground: Pre-eclampsia (PE), an adverse pregnancy outcome affects 2-5% pregnancies worldwide and significantly adversely impacts both maternal and fetal outcomes. Intrauterine growth restriction (IUGR) is defined as the inability of the fetus to reach normal growth potential within the uterus as a result of various genetic, environmental, or placental factors. Premature ageing of the placenta in pregnancy outcomes such as PE and IUGR is associated with the persistent presence of oxidative stress and placental insufficiency reducing its functional capacity. Placental proteomics has been instrumental in improving our understanding of molecular mechanisms involved in the pathophysiology of placental insufficiency as well as identifying biomarkers to predict and diagnose pregnancy outcomes. In this study, we investigated cellular senescence phenotypes of PE and IUGR pregnancies by simultaneously measuring several biomarkers of senescence, as well as the proteomic signature of the placenta in healthy and adverse pregnancy outcomes PE and IUGR. Method: Maternal plasma and placental samples were collected at term (>37 weeks) and preterm (<37 weeks) gestation from nulliparous women undergoing prelabour elective Caesarean section with PE without intrauterine growth restriction (PE; n=5), PE associated with intrauterine growth restriction (n=8), intrauterine growth restriction (IUGR <10th centile) (n=6) and age-matched controls (n=20) from Cork University Maternity Hospital, Cork, Ireland. To assess cellular senescence absolute telomere length (aTL) and senescence associated genes in the placentas was performed by RTqPCR. Cyclin-dependent kinase inhibitors (p21 and p16) expression were determined by Western blotting. Senescence Associated Secretory Phenotype (SASP) were evaluated in maternal plasma by multiplex ELISA assay. Proteomic analysis of placental samples dissected into 3 sub-anatomical regions (maternal, middle, fetal) taken from 3 nulliparous healthy placentas was performed by mass-spectrometry and pathway analysis was conducted. Based on the differentially expressed proteins (DEPs), a placenta specific disease map using NaviCenta focusing on functional analysis to include the placenta specific context for healthy (n=4) compared to PE affected (n=4) and IUGR affected (n=4) placentas. Results: Placental expression of senescence associated genes CHEK1, PCNA, PTEN, CDKN2A, CCNB-1 was significantly upregulated in PE, while TBX-2, PCNA, ATM and CCNB-1 expression were significantly decreased in IUGR compared to controls. Moreover, placental p16 protein expression was significantly decreased in PE only when compared to controls placentas. We also observed that IL-6 was significantly increased in maternal circulation in PE when compared to controls; while IFN-γ was significantly increased in maternal circulation in women affected with IUGR when compared to controls. Proteomic profiling of healthy placentas divided into three sub-anatomical regions identified 1081, 1086, and 1101 proteins in maternal, middle, and fetal sub-anatomical regions respectively. Depending on sample site location and sub-anatomical regions, 374 differentially expressed proteins (DEP) were identified. When we investigated the proteomic variations between PE and IUGR placentas when compared to controls we observed 314, 391, and 378 proteins in healthy control, PE, and IUGR placenta, respectively. We performed functional analysis by combining ClusterCompare and NaviCenta to analyse a placenta-centric context, and observed regulatory elements predominantly involved in the immune regulation, complement cascade and antioxidant activities in PE and IUGR compared to control placentas. Conclusion: This thesis provides evidence of premature senescence in IUGR, while in PE, evidence of activated cell cycle checkpoint regulators is suggestive of cellular repair and proliferation rather than progression to cellular senescence. The heterogeneity within senescence molecular markers of these phenotypes highlights the complexity and disparity between pathophysiological insults unique to each obstetric complication. Proteomic profiling of sub-anatomical placental regions highlighted the variabilities between regions particularly providing evidence of senescence in these regions. Placental proteomic mapping of healthy placentas compared to adverse pregnancy outcomes PE and IUGR revealed the importance of complement system, inflammatory response, and antioxidant activity in placental function in PE placentas. The identification of novel targets such as transcription factor activity and synergistic miRNAs elements within the core regulatory network, might enlighten future placental research within adverse pregnancy outcomes.Item Placental growth factor; potential for its use in twin pregnancy and evaluation of its benefit in singletons with suspected preterm pre-eclampsia(University College Cork, 2020) Hayes-Ryan, Deirdre; O'Donoghue, Keelin; Kenny, Louise C.Hypertensive Disorders of Pregnancy are common and may result in increased maternal and neonatal morbidity and mortality. Twin pregnancies confer an increased risk of development of a hypertensive disorder of pregnancy. Placental growth factor is an angiogenic protein highly expressed during pregnancy. The pro-angiogenic/anti-angiogenic synergism of PlGF and its receptors is critical for successful placentation in early pregnancy. Circulating maternal levels of placental growth factor correlate well with placental function. Women presenting with suspected pre-eclampsia are currently triaged based on hypertension and dipstick proteinuria. Numerous studies advocate a role for placental growth factor testing as a useful adjunct in the management of women presenting with preterm pre-eclampsia. Several automated immunoassay platforms to quantify placental growth factor are currently available. Comparative studies of these immunoassays are limited. Current reference values and clinical cut-offs for PlGF were constructed from singleton pregnancy cohorts. Given the larger placental volume present in a twin pregnancy, separate reference ranges are likely required. Pregnant women are seldom included in randomised controlled trials and their attitudes and experiences of this are not often investigated. Gathering feedback of their experience is paramount for future trial design to facilitate participation. In this thesis, I reviewed nine years of clinical data in twin pregnancies from a single maternity unit to understand the impact of hypertensive disorders on maternal and neonatal outcomes. I examined cross sectional values from uncomplicated twin pregnancies to assess the potential for using PlGF in this population. I compared the PlGF results obtained from an ELISA to an automated immunoassay, to determine if clinical cut-offs developed for one platform were transferrable to another. I conducted a national multi-site randomised control trial; PARROT Ireland, to evaluate the impact of incorporation of PlGF testing into routine clinical care. Lastly, through one on one interviews with trial participants, I investigated the barriers and facilitators to pregnant women taking part in clinical research. The data from these studies revealed that maternal age >40 years, nulliparity, conception through use of a donor oocyte, and presence of obstetric cholestasis are all important risk factors for the development of a hypertensive disorder in a twin pregnancy. The incidence of iatrogenic late prematurity and neonatal hypoglycaemia are increased when a hypertensive disorder complicates a twin pregnancy. PlGF levels in twin pregnancy differ significantly between those women with a pregnancy that will later be complicated by preeclampsia and those that will not. The difference is present many weeks before clinical signs or symptoms are present, indicating that PlGF has potential to aid diagnosis of pre-eclampsia in twin pregnancies. A dichorionic twin pregnancy specific reference range for PlGF has been developed, which may be utilised for further interventional research on PlGF in twins. The findings also indicate that PlGF biomarker levels vary significantly between different immunoassay platforms, highlighting the importance of developing validated clinical cut-offs for any automated immunoassay before they can be clinically applied. The result of the interim analysis from the PARROT Ireland trial is of no significant reduction in either maternal or neonatal morbidity with the integration of point of care PlGF based testing. These are interim results only however and the final results may differ. Should the final trial results demonstrate a positive impact on maternal morbidity, without a negative impact on neonatal morbidity, it would indicate that PlGF testing should be incorporated into routine clinical investigations for women presenting with suspected pre-eclampsia before 37 weeks’ gestation. The final study of the thesis highlights that pregnant women are interested and willing to participate in research. Identifying the correct timepoint and location to approach women, as well as the manner and language used to communicate with them, are key elements in ensuring their participation. The findings from this thesis, though supportive of the current literature in relation to the potential of PlGF, highlight that there is more research required.Item Predicting successful outcome of singleton and multiple pregnancies after assisted reproductive technologies (ART)(University College Cork, 2020) Geisler, Minna; O'Donoghue, Keelin; Waterstone, JohnPregnancies conceived through ART are increasing in prevalence in maternity units in Ireland. This is due in a large part to the increasing age of women attempting to conceive for the first time but also due to the increasing success of ART. This thesis explores pre-pregnancy characteristics that may aid in predicting pregnancy outcome in women undergoing IVF treatment. The thesis includes three studies. The first is a retrospective study of the pregnancy outcomes of singleton and twin pregnancies conceived from ART. The study found that ART conceived twins had similar perinatal outcomes to spontaneously conceived twins. Overall, singleton pregnancy outcomes were very favourable. Singleton pregnancies, when compared to twin pregnancies, were more likely to result in a livebirth, while twin pregnancies were significantly more likely to result in miscarriage, preterm delivery and/or neonatal unit admission. The second study is a prospective cohort study of 142 nulliparous women at the outset of their IVF cycle. A 3D ultrasound of the uterine volume was performed. Women completed a survey on lifestyle and demographics. None of the interrogated demographics or lifestyle factors demonstrated a significant impact on conception nor on pregnancy loss. Neither uterine length nor volume impacted on pregnancy rates or on preterm delivery rates. The final study details the findings of a survey-based study of 320 women recruited prior to IVF treatment and followed for outcome (negative/positive pregnancy test, first trimester miscarriage). The survey focused on psychological stressors. The findings suggest that stressors do not impact greatly on conception rates from ART. Job-related stress is associated with higher chance of miscarriage. In the absence of any robust predictors of successful, or indeed adverse, pregnancy outcome it is preferable to aim for single embryo transfer and a singleton pregnancy.Item Prenatal stress, the placenta and maternal microbial transmission; implications for health and disease(University College Cork, 2018) Togher, Katie L.; Clarke, Gerard; Khashan, Ali; O'Keeffe, Gerard W.; Kenny, Louise C.; Science Foundation Ireland; University College CorkThere is an extensive amount of epidemiological evidence showing that prenatal maternal distress (PNMD) is a risk factor for a wide range of poor obstetric and neonatal outcomes, as well as an increased risk for the development of metabolic, immune and nervous system disorders in affected children later in life. Whilst many epidemiology studies have supported these associations, the biological mechanisms linking maternal prenatal distress with adverse outcomes remains understudied, particularly in human cohorts. One potential mechanism, known as the glucocorticoid hypothesis proposes that fetal overexposure to stress-induced maternal cortisol during critical windows of development increases the risk of adverse outcomes in the offspring. At the core of this hypothesis is the placenta, which expresses the enzyme 11beta hydroxysteroid dehydrogenase type 2 (HSD11B2), which ultimately controls the amount of cortisol a fetus is exposed to. Prenatal stress has been shown to reduce the placental expression of this enzyme; however the molecular mechanisms through which this occurs have not been well examined. More recently, the transmission of a suboptimal stressed maternal microbiota is emerging as an alternative mechanism that may mediate the impact of prenatal stress on infant development. However this has not yet been examined in a clinical population. We first utilized an in vitro placenta model, JEG-3 cells, to examine the effects of stress on the placental expression of HSD11B2. JEG-3 cells were cultured with exogenous cortisol and interleukin-1 beta (IL-1β), two potential biological mediators of prenatal stress. This study showed both cortisol and IL-1β can reduce HSD11B2 expression, an effect that could be prevented by co-treatment with a histone deacetylase inhibitor. Having established that cortisol can directly affect the expression of HSD11B2, we moved on to our first clinical study to examine this question in a clinical population by examining the impact of prenatal distress on placental gene expression and infant outcomes. A cohort of 121 pregnant women receiving antenatal care at Cork University Maternity Hospital (CUMH) completed the Perceived stressed scale (PSS), State Trait Anxiety Inventory (STAI) and Edinburgh Postnatal Depression Scale (EPDS) in late pregnancy and donated placental biopsies at the time of birth. This study identified a significant reduction in HSD11B2 mRNA along with an increase in the glucocorticoid receptor (NR3C1) in placentae from high distressed pregnancies. Additionally prenatal distress was associated with a number of adverse outcomes including delivering via Caesarean section, reduced Apgar scores and reduced birth temperature, supporting a role for placental glucocorticoid signalling in the relationship between prenatal distress and adverse outcomes. Having reported that stress impacts molecular placental signals and birth outcomes, we moved on to complete the SMArTI (Stressed Microbial Transfer to the Infant) study, a more detailed pregnancy cohort to examine the impact prenatal distress on the maternal and infant microbiome. This study yielded a final cohort off 111 nulliparous pregnant women that were recruited from the IMPROvED consortium at CUMH. Women enrolled in SMArTI completed distress questionnaires and provided saliva and fecal samples in the second and/or third trimester of pregnancy. Vaginal swabs, placenta samples and newborn hair were acquired at birth and infant fecal samples were subsequently collected across the first 5 months of life. We first used this cohort to further examine and validate the relationship between prenatal distress, placental glucocorticoid genes and infant outcomes. We found this relationship to be dependent on the timing of distress, type of distress and infant sex. Most notably we observed second trimester maternal anxiety correlated with reduced birthweight in female infants, a relationship mediated by placental FK506-bind protein 51 (FKBP51) mRNA expression. We finally used the SMArTI cohort to examine, for the first time, the impact of PNMD on the maternal and infant microbiome, using 16S rRNA gene sequencing. Reduced diversity of the maternal gut microbiome in the second trimester was associated with second trimester distress, most substantially with maternal depressive symptoms, an effect that was no longer apparent by the third trimester. The third trimester gut microbiome appeared relatively resistant to change with only modest alterations observed in women who had high second trimester cortisol. Of interest, third trimester distress had no effect on the third trimester gut microbiome, highlighting the experience of distress specifically in the second trimester as an important window of vulnerability. Reduced diversity of the vaginal microbiome, just prior to delivery, was associated with second trimester cortisol, with no alterations linked third trimester distress. When examining the infant gut microbiome we found increased diversity across the first 5 months of life to be associated with second trimester stress with corresponding decreases to the important Bifidobacteriaceae and Lactobacillaceae family. In conclusion, this thesis indicates the experience of PNMD influences key placental genes involved in glucocorticoid signalling in the placentae. The timing of maternal distress and infant sex are important factors in this relationship. Of particular interest we find placental FKBP51 to mediate a relationship between maternal anxiety and infant birthweight, demonstrating a direct role for placental glucocorticoid signalling underlying the relationship between prenatal distress and infant outcomes. The work presented in this thesis is the first of its kind to prospectively examine the influence of PNMD on the maternal gut, vaginal and infant gut microbiome. Stress-induced alterations in the maternal gut microbiome may contribute to adverse obstetric and birth outcomes albeit via a mechanism other than transmission of a suboptimal maternal microbiota during birth. Taken together, our results identify the second trimester as an especially vulnerable period to stress exposures and implicate the placenta and microbiome in mediating these effects. Counteracting the impact of stress during this critical time window may have important obstetric implications. Additionally understanding the consequence of the altered infant gut microbiome as a result of prenatal distress warrants further investigation.Item Exploring the role of mitochondrial dysfunction in the pathophysiology of pre-eclampsia(Elsevier B.V., 2018-06-18) Williamson, Rachel D.; McCarthy, Fergus P.; Khashan, Ali S.; Totorika, Ainhoa; Kenny, Louise C.; McCarthy, Cathal