Standardized protocol for quantification of nerve bundle density as a biomarker for endometriosis.

Introduction: We propose a standardized protocol for measurement of nerve bundle density in endometriosis as a potential biomarker, including in deep endometriosis (DE), ovarian endometriomas (OMA) and superficial peritoneal endometriosis (SUP). Methods: This was a prospective cohort of surgically excised endometriosis samples from Dec 1st 2013 and Dec 31st 2017 at a tertiary referral center for endometriosis in Vancouver, BC, Canada. Surgical data were available from linked patient registry. Protein gene product 9.5 (PGP9.5) was used to identify nerve bundles on immunohistochemistry. PGP9.5 nerve bundles were counted visually. To calculate nerve bundle density, PGP9.5 nerve bundle count was divided by the tissue surface area (total on the slide). All samples were assessed using NHS Elements software for semi-automated measurement of the tissue surface area. For a subset of samples, high power fields (HPFs) were also counted as manual measurement of the tissue surface area. Intraclass correlation was used to assess intra observer and inter observer reliability. Generalized linear mixed model (GLMM) with random intercepts only was conducted to assess differences in PGP9.5 nerve bundle density by endometriosis type (DE, OMA, SUP). Results: In total, 236 tissue samples out of 121 participants were available for analysis in the current study. Semi-automated surface area measurement could be performed in 94.5% of the samples and showed good correlation with manually counted HPFs (Spearman's rho = 0.781, p < 0.001). To assess intra observer reliability, 11 samples were assessed twice by the same observer; to assess inter observer reliability, 11 random samples were blindly assessed by two observers. Intra observer reliability and inter observer reliability for nerve bundle density were excellent: 0.979 and 0.985, respectively. PGP9.5 nerve bundle density varied among samples and no nerve bundles could be found in 24.6% of the samples. GLMM showed a significant difference in PGP9.5 nerve bundle density between the different endometriosis types (X2 = 87.6, P < 0.001 after adjusting for hormonal therapy, with higher density in DE and SUP in comparison to OMA). Conclusion: A standardized protocol is presented to measure PGP9.5 nerve bundle density in endometriosis, which may serve as a biomarker reflecting local neurogenesis in the endometriosis microenvironment.

Differential effects of renin-angiotensine-aldosteron system inhibition, sympathoinhibition and low sodium diet on blood pressure in women with a history of preeclampsia: A double-blind, placebo-controlled cross-over trial (the PALM study).

Current guidelines lack sufficient evidence to recommend a specific blood pressure lowering strategy to prevent cardiovascular disease after preeclampsia. We conducted a double-blind cross-over trial to identify the most potent antihypertensive strategy: renin-angiotensin-aldosterone system (RAAS) inhibition (losartan), sympathoinhibition (moxonidine), low sodium diet and placebo (n = 10). Due to low inclusion rate our study stopped prematurely. Initiatory analyses showed no significant effect of antihypertensive strategy on office blood pressure and 24-hour blood pressure. However, nocturnal dipping was significantly higher on RAAS inhibition and low sodium diet compared to placebo and sympathoinhibition. Optimal cardiovascular prevention after preeclampsia should be further explored.

Early Onset of Coronary Artery Calcification in Women With Previous Preeclampsia.

BACKGROUND: Preeclampsia, coronary artery calcification (CAC), and atherosclerotic plaque are risk factors for the development of cardiovascular disease. We determined at what age CAC becomes apparent on coronary computed tomography after preeclampsia and to what extent modifiable cardiovascular risk factors were associated. METHODS: We measured cardiovascular risk factors, CAC by coronary computed tomography, and coronary plaque by coronary computed tomography angiography in 258 previously preeclamptic women aged 40-63. Results were compared to 644 age- and ethnicity-equivalent women from the Framingham Heart Study with previous normotensive pregnancies. RESULTS: Any CAC was more prevalent after preeclampsia than after a normotensive pregnancy (20% versus 13%). However, this difference was greatest and statistically significant only in women ages 45 to 50 (23% versus 10%). The degree of CAC advanced 4× faster between the ages of 40 to 45 and ages 45 to 50 in women with a history of preeclampsia (odds ratio, 4.3 [95% CI, 1.5-12.2] versus odds ratio, 1.2 [95% CI, 0.6-2.3]). Women with a preeclampsia history maintained greater advancement of CAC with age into their early 60s, although this difference declined after the perimenopausal years. Women with a previous normotensive pregnancy were 4.9 years (95% CI, 1.8-8.0) older when they had similar CAC scores as previously preeclamptic women. These observations were not explained by the greater prevalence of cardiovascular disease risk factors, and the higher Framingham Risk Scores also observed in women with a history of preeclampsia. CONCLUSIONS: Previously preeclamptic women have more modifiable cardiovascular risk factors and develop CAC ≈5 years earlier from the age of 45 years onwards compared to women with normotensive pregnancies. Therefore, women who experienced preeclampsia might benefit from regular cardiovascular screening and intervention before this age. Registration: URL: https://www.trialregister.nl/trial/5406; Unique identifier: NTR5531.

Longitudinal follow-up of kidney function in patients with a history of preeclampsia: From 11 to 18 years postpartum.

Formerly preeclamptic (fPE) women are reported to have an increased risk to develop end stage kidney disease. To gain more insight in the course of kidney function after preeclampsia we assessed blood pressure, eGFR and urinary protein loss in 75 fPE women at 11 and 18 years postpartum. We found that during follow-up blood pressure did not increase and no cases of CKD were identified. Only a small decrease in eGFR (6-7 mL/min) and a small increase in urinary protein loss were observed, which fall within the expected range of normal aging. In conclusion, our data suggests that progression to kidney disease might not be a major concern in women after preeclampsia within 18 years postpartum.

Circulating Neutrophils Do Not Predict Subclinical Coronary Artery Disease in Women with Former Preeclampsia.

: Introduction: Preeclampsia (PE) represents a hypertensive pregnancy disorder that is associated with increased cardiovascular disease (CVD) risk. This increased risk has been attributed to accelerated atherosclerosis, with inflammation being a major contributor. Neutrophils play an important role in the onset and progression of atherosclerosis and have been associated with vascular damage in the placenta as well as the chronic inflammatory state in women with PE. We therefore investigated whether circulating neutrophil numbers or reactivity were associated with the presence and severity of subclinical atherosclerosis in women with a history of PE. METHODS: Women aged 45-60 years with a 10 to 20 years earlier history of early onset preeclampsia (delivery <34 weeks of gestation) (n = 90), but without symptomatic CVD burden were screened for the presence of subclinical coronary artery disease (CAD) using both contrast-enhanced and non-contrast coronary CT angiography. Subclinical CAD was defined as a coronary artery calcium (CAC) score ≥100 Agatston Units and/or ≥50% coronary luminal stenosis. We assessed whether the numbers and activity of circulating neutrophils were associated with the presence of subclinical CAD and as secondary outcome measurements, with the presence of any calcium (CAC score > 0 AU) or stenosis, categorized as absent (0%), minimal to mild (>0 and <50%), and moderate to severe (≥50%) narrowing of the coronary artery. Blood was drawn just before CT and neutrophil numbers were assessed by flow cytometry. In addition, the presence of the chemokine receptors CXCR2 and CXCR4, which are known to be instrumental in neutrophil recruitment, and neutrophil activity upon stimulation with the bacterial peptide N-Formylmethionyl-leucyl-phenylalanine (fMLF) was assessed by flow cytometry. RESULTS: Of the participating women, with an average age of 49 years, 13% (12 out of 90) presented with subclinical signs of CAD (CAC score ≥100 AU and/or ≥50% luminal stenosis), and 37% (33 out of 90) had a positive CAC score (>0). Total white blood cell count and neutrophil counts were not associated with the presence of subclinical CAD or with a positive CAC score. When assessing the presence of the chemokine receptors CXCR4 and CXCR2, we observed a slight decrease of neutrophil CXCR2 expression in women with CAC (median MFI 22.0 [interquartile range (IQR) 20.2-23.8]) compared to women without CAC (23.8 [IQR 21.6-25.6], p = 0.02). We observed no differences regarding neutrophil CXCR4 expression. In addition, expression of the early activity marker CD35 was slightly lower on neutrophils of women with subclinical CAD (median MFI 1.6 [IQR 1.5-1.9] compared to 1.9 [IQR 1.7-2.1] in women without CAD, p = 0.02). However, for all findings, statistical significance disappeared after adjustment for multiple testing. CONCLUSION: Our findings indicate that neutrophil counts and (re)activity are not directly associated with silent CAD disease burden and as such are not suitable as biomarkers to predict the presence of subclinical CAD in a high-risk population of women with a history of preeclampsia.

Platelet RNA modules point to coronary calcification in asymptomatic women with former preeclampsia.

BACKGROUND AND AIMS: Women who develop preeclampsia during pregnancy are at a higher risk for developing cardiovascular disease. As platelets are affected by preeclampsia, we set out to identify whether platelets carry information in their transcriptome on cardiovascular risk in women with former preeclampsia. METHODS: Platelets were isolated from asymptomatic women with previous preeclampsia, who underwent screening with coronary computed tomography angiography. Platelet RNA was isolated and used to construct gene networks using an unbiased approach. Platelet gene modules assembled from the network were related to risk factors and clinical traits of these women, including coronary artery calcium scores (CACS). RESULTS: We found multiple gene modules which correlated with CACS (correlation coefficients: 0.44 to 0.59, p = 0.05 to 0.007). The genes from two clinically relevant modules were expressed at a higher level in the group with calcifications (p = 3.9 × 10-10 and 0.02) and enriched for platelet-related gene-sets such as platelet activation. The first of these modules was also enriched (ppermutation = 0.0546) for genes mapped to known coronary artery disease susceptibility loci. Additional unbiased network analyses in platelet RNA of patients with overt cardiovascular disease underlined the importance of the identified modules for disease by high preservation. (p = 1.6 × 10-9 to 1.7 × 10-47). CONCLUSIONS: We found platelet RNA modules that correlated with CACS in asymptomatic women with previous preeclampsia. Whether or not platelets directly contribute to this disease trajectory, or reflect the underlying plaque substrate remains to be determined, but enrichment for coronary artery disease susceptibility genes emphasizes the importance for the disease.

Association between parity and persistent weight gain at age 40-60 years: a longitudinal prospective cohort study.

OBJECTIVES: Physiological metabolic adaptations occur in the pregnant woman. These may persist postpartum and thereby contribute to an unfavourable cardiovascular disease (CVD) risk profile in parous women. The aim of the current study is to assess time-dependent changes of cardiometabolic health in parous women compared with nulliparous women. DESIGN AND SETTING: We studied data of 2459 women who participated in the Prevention of Renal and Vascular End-stage Disease study, a population-based prospective longitudinal cohort for assessment of CVD and renal disease in the general population. PARTICIPANTS: We selected women ≥40 years at the first visit, who reported no new pregnancies during the four follow-up visits. All women were categorised in parity groups, and stratified for age. OUTCOME MEASURES: We compared body mass index (BMI), high-density lipoprotein (HDL) cholesterol, blood pressure as continuous measurements and as clinical relevant CVD risk factors among parity groups over the course of 6 years using generalised estimating equation models adjusted for age. RESULTS: The BMI was significantly higher in women para 2 or more in all age categories: per child, the BMI was 0.6 kg/m2 higher. corresponding with 1.5-2.0 kg weight gain per child. HDL cholesterol was significantly lower in women para 2 or more aged 40-49 and 50-59 years: per child, the HDL cholesterol was up to 0.09 mmol/L lower. Blood pressure did not differ among parity groups in any of the age categories. CONCLUSIONS: Higher parity is associated with higher BMI, lower HDL cholesterol and a higher prevalence of cardiovascular risk factors, which is constant over time. These findings warrant for prospective research assessing determinants of cardiometabolic health at earlier age to understand the role of pregnancy in the development of CVD in women.

Trajectory of Cardiovascular Risk Factors After Hypertensive Disorders of Pregnancy.

Women with a history of a hypertensive disorder of pregnancy (HDP) are at increased risk of premature cardiovascular disease. Cardiovascular risk management guidelines emphasize the need for prevention of cardiovascular disease in these women but fail to provide uniform recommendations on when and how to start cardiovascular risk assessment. The aim of this study was to identify a window of opportunity in which to start cardiovascular risk factor assessment by investigating changes in blood pressure, lipids, and fasting glucose levels over time in women with a history of an HDP. We identified women with a history of a normotensive pregnancy (n=1811) or an HDP (n=1005) within a high-risk population-based cohort study. We assessed changes in blood pressure, lipids, glucose, 10-year cardiovascular risk and the occurrence of hypertension, dyslipidemia, and diabetes mellitus longitudinally using 5 measurements at 3-year intervals. Generalized estimating equations were used for statistical analysis, with age as the time variable, adjusting for multiple comparisons using the least significant differences method. In women with an HDP, the overall prevalence of hypertension ( P<0.0001), dyslipidemia ( P=0.003), and diabetes mellitus ( P<0.0001) was significantly higher. They also developed hypertension and diabetes mellitus earlier. At age 35, few women with HDP need to be screened to detect clinically relevant hypertension: 9 need to be screened to detect 1 woman with a treatment indication as opposed to 38 women with history of a normotensive pregnancy. Our data supports cardiovascular follow-up of women with a history of an HDP starting within the fourth decade of life.

Preeclampsia and coronary plaque erosion: Manifestations of endothelial dysfunction resulting in cardiovascular events in women.

Atherosclerosis is the major underlying pathology of cardiovascular disease (CVD). The risk for CVD is increased in women with a history of preeclampsia. Multiple studies have indicated that accelerated atherosclerosis underlies this increased CVD risk. Furthermore, it has been suggested that endothelial dysfunction and inflammation play an important role in the increased CVD risk of women with preeclampsia. Rupture or erosion of atherosclerotic plaques can induce the formation of thrombi that underlie the onset of acute clinical CVD such as myocardial infarction and stroke. In relatively young women, cardiovascular events are mainly due to plaque erosions. Eroded plaques have a distinct morphology compared to ruptured plaques, but have been understudied as a substrate for CVD. The currently available evidence points towards lesions with features of stability such as high collagen content and smooth muscle cells and with distinct mechanisms that further promote the pro-thrombotic environment such as Toll Like Receptor (TLR) signaling and endothelial apoptosis. These suggested mechanisms, that point to endothelial dysfunction and intimal thickening, may also play a role in preeclampsia. Pregnancy is considered a stress test for the cardiovascular system with preeclampsia as an additional pathological substrate for earlier manifestation of vascular disease. This review provides a summary of the possible common mechanisms involved in preeclampsia and accelerated atherosclerosis in young females and highlights plaque erosion as a likely substrate for CVD events in women with a history of preeclampsia.

Cardiovascular RiskprofilE - IMaging and gender-specific disOrders (CREw-IMAGO): rationale and design of a multicenter cohort study.

BACKGROUND: Reproductive disorders, such as polycystic ovary syndrome (PCOS), primary ovarian insufficiency (POI) and hypertensive pregnancy disorders (HPD) like pre-eclampsia (PE), are associated with an increased risk of cardiovascular disease (CVD). Detection of early signs of cardiovascular disease (CVD), as well as identification of risk factors among women of reproductive age which improve cardiovascular risk prediction, is a challenge and current models might underestimate long-term health risks. The aim of this study is to assess cardiovascular disease in patients with a history of a reproductive disorder by low-dose computed tomography (CT). METHODS: Women of 45 - 55 years, who experienced a reproductive disorder (PCOS, POI, HPD), are invited to participate in this multicenter, prospective, cohort study. Women will be recruited after regular cardiovascular screening, including assessment of classical cardiovascular risk factors. CT of the coronary arteries (both coronary artery calcium scoring (CACS), and contrast-enhanced coronary CT angiography (CCTA)) and carotid siphon calcium scoring (CSC) is planned in 300 women with HPD and 300 women with PCOS or POI. In addition, arterial stiffness (non-invasive pulse wave velocity (PWV)) measurement and cell-based biomarkers (inflammatory circulating cells) will be obtained. DISCUSSION: Initial inclusion is focused on women of 45 - 55 years. However, the age range (40 - 45 years and/or ≥ 55 years) and group composition may be adjusted based on the findings of the interim analysis. Participants can potentially benefit from information obtained in this study concerning their current cardiovascular health and expected future risk of cardiovascular events. The results of this study will provide insights in the development of CVD in women with a history of reproductive disorders. Ultimately, this study may lead to improved cardiovascular prediction models and will provide an opportunity for timely adjustment of preventive strategies. Limitations of this study include the possibility of overdiagnosis and the average radiation dose of 3.5 mSv during coronary and carotid siphon CT, although the increased lifetime malignancy risk is negligible. TRIAL REGISTRATION: Netherlands Trial Register, NTR5531 . Date registered: October 21st, 2015.

Determinants of future cardiovascular health in women with a history of preeclampsia.

Women who develop preeclampsia have an increased risk of cardiovascular disease (CVD) later in life. However, current guidelines on cardiovascular risk assessment and prevention are unclear on how and when to screen these women postpartum, and about the role of a positive history of preeclampsia in later-life CVD risk management. The aim of this review is to discuss the present knowledge on commonly used cardiovascular screening modalities available to women with a history of preeclampsia, and to discuss recent developments in early detection of CVD using cardiovascular imaging. Furthermore, we explore how female-specific risk factors may have additional value in cardiovascular screening, in particular in relatively young women, although their implementation in clinical practice is challenged by inconsistent results and lack of long-term outcome data. Non-invasive imaging techniques, e.g., coronary artery intima-media thickness (CIMT), can be helpful to detect subclinical atherosclerotic disease, and coronary artery calcium scoring (CACS) has shown to be effective in early detection of cardiovascular damage. However, while more short-term and long-term follow-up studies are becoming available, few studies have investigated women with a history of preeclampsia in the fourth and fifth decade of life, when early signs of premature CVD are most likely to become apparent. Further studies are needed to inform new and improved clinical practice guidelines, and provide long-term strategies to effectively prevent CVD, specifically targeted at women with a history of preeclampsia. Additionally, evaluation of feasibility, cost-effectiveness, and implementation of CVD screening and prevention initiatives targeted at former preeclampsia patients are needed.