Journal Pre-proof

Impact of micronised progesterone and medroxyprogesterone

acetate in combination with transdermal oestradiol on cardiovascular
markers in women diagnosed with premature ovarian insufficiency or
an early menopause: a randomised pilot trial

Monica Mittal , Carmel McEniery , Prasanna Raj Supramaniam ,

Linda Cardozo , Mike Savvas , Nick Panay , Haitham Hamoda

PII: S0378-5122(22)00025-1
DOI: https://doi.org/10.1016/j.maturitas.2022.01.012
Reference: MAT 7602

To appear in: Maturitas

Received date: 16 September 2021

Revised date: 15 January 2022
Accepted date: 19 January 2022

Please cite this article as: Monica Mittal , Carmel McEniery , Prasanna Raj Supramaniam ,
Linda Cardozo , Mike Savvas , Nick Panay , Haitham Hamoda , Impact of micronised progesterone
and medroxyprogesterone acetate in combination with transdermal oestradiol on cardiovascular mark-
ers in women diagnosed with premature ovarian insufficiency or an early menopause: a randomised
pilot trial, Maturitas (2022), doi: https://doi.org/10.1016/j.maturitas.2022.01.012

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Highlights

 Risk stratification of arterial disease can be performed with carotid femoral pulse

wave velocity.

 Menopause augments the age-dependent increase in arterial stiffness.

 Hormone replacement therapy is the mainstay of management of an early

menopause and premature ovarian insufficiency.

 Micronised progesterone/medroxyprogesterone acetate can both be used in this

cohort.

 Positive changes in traditional markers with micronised progesterone were not

reflected in carotid femoral pulse wave velocity.

TITLE: Impact of micronised progesterone and medroxyprogesterone acetate in

combination with transdermal oestradiol on cardiovascular markers in women diagnosed

with premature ovarian insufficiency or an early menopause: a randomised pilot trial

RUNNING TITLE: Impact of HRT on cardiovascular risk markers

AUTHORS: *Monica Mittal1, Carmel McEniery2, Prasanna Raj Supramaniam3, Linda

Cardozo4, Mike Savvas4, Nick Panay5, Haitham Hamoda4

*Corresponding author
1
Imperial College Healthcare NHS Trust, St Mary’s and Hammersmith Hospitals, Department

of Obstetrics and Gynaecology, Praed Street, London W2 1NY

2
University of Cambridge, Division of Cardiovascular Medicine, Addenbrooke's Hospital,

Cambridge, Hills Road, Cambridge CB2 0QQ

3
Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Department of

Obstetrics and Gynaecology, Headley Way, Headington, Oxford OX3 9DU

4
King’s College Hospital NHS Foundation Trust, Department of Obstetrics and Gynaecology,

Denmark Hill, Brixton, London SE5 9RS

5
Imperial College Healthcare NHS Trust and Chelsea and Westminster NHS Foundation

Trust, Queen Charlotte's and Chelsea Hospital, Department of Obstetrics and Gynaecology,

Du Cane Rd, White City, London W12 0HS

CORRESPONDING AUTHOR:
Monica Mittal BSc, MBBS, MRCOG, MD Imperial College Healthcare NHS Trust, St Mary’s

and Hammersmith Hospitals, Praed Street, Paddington, London W2 1NY, UK

Tel no.: 07958086986

monica.mittal@nhs.net

https://orcid.org/0000-0002-6684-0964

INSTITUTION WHERE THE WORK WAS UNDERTAKEN:

King’s College Hospital NHS Foundation Trust, Department of Obstetrics and Gynaecology,

Denmark Hill, Brixton, London SE5 9RS

ABSTRACT

Objective: To compare the difference between micronised progesterone (MP) and

medroxyprogesterone acetate (MPA) in combination with transdermal oestradiol (t-E2) on

cardiovascular disease (CVD) risk markers in women diagnosed with an early menopause

and premature ovarian insufficiency (EMPOI).

Background: The European Society for Cardiology has identified carotid femoral pulse wave

velocity (cfPWV) as the gold standard cardiogenic biomarker for risk stratification of arterial

disease. Menopause has been shown to augment the age-dependent increase in arterial

stiffness, with hormone replacement therapy (HRT) being the mainstay of management of

women diagnosed with EMPOI.

Study design: A pilot randomised prospective open-label trial. Women were randomised to

either cyclical MP (Utrogestan 200mg) or MPA (Provera 10mg) in conjunction with t-E2
(Evorel Patches 50mcg/day) for 12 months. Seventy-one subjects were screened, and

baseline data are available for 57 subjects.

Main outcome measure: Carotid-femoral pulse wave velocity (cfPWV).

Results: PWV did not significantly change from baseline in either treatment arm. MP + t-E2

demonstrated a positive effect on traditional CVD markers, with a significant improvement

seen in cardiac output (CO) (0.71±1.01mL/min, 95% CI 0.20 to 1.21) and reduction in

diastolic blood pressure (DBP) (-3.43±6.31mmHg, 95% Cl -6.57 to -0.29) and total peripheral

resistance (TPR) (-0.15±0.19mmHg⋅min⋅mL-1, 95% CI -0.24 to -0.05) after 12 months. MPA +

t-E2, in contrast, did not demonstrate significant changes from baseline in traditional

haemodynamic parameters.

Conclusion: The positive changes in traditional markers were not reflected in the

cardiogenic biomarker, cfPWV, which has demonstrated a higher positive predictive value

for cardiovascular events than traditional measurements.

Keywords

Medroxyprogesterone acetate, micronised progesterone, transdermal oestrogen,

progesterone, hormone replacement therapy (HRT), carotid femoral pulse wave velocity

(cfPWV).
INTRODUCTION

Vascular aging can now be predicted through an assessment of carotid-femoral Pulse Wave

Velocity (cfPWV), a non-invasive cardiogenic biomarker of aortic stiffness [1]. PWV is

determined by the time taken for the arterial pulse pressure, generated by the systolic

contraction of the heart, to propagate along the arterial tree [1-2]. It provides a reflection of

the properties of arterial vascular health, including arterial wall elasticity and its dimensions

[1-2], with higher values correlated to a relative fall in arterial compliance [2-3]. Aortic

stiffness is increased in conditions such as hypertension and is a strong independent

indicator for subsequent cardiovascular events [1, 4-5]. The association between increased

arterial stiffness and altered coronary perfusion is postulated to be through its effects on

systolic blood pressure (SBP), subsequently increasing left ventricular afterload.

The Framingham Risk Score [6] has shown brachial pulse pressure (PP) to be a strong

independent determinant of recurrent cardiac events and all-cause mortality in the general

population. The risk of a major clinical event is said to increase by 10-40% for every

10mmHg increase in PP [7]. However, PP measurements and augmentation index (AI) are

indirect surrogate measures of arterial stiffness which are influenced by factors related to

cardiac function, such as heart rate (HR), limiting their interpretation [7-8]. In contrast,

aortic PWV is influenced to a lesser degree by other cardiac function parameters [7] and

may be superior in the prediction of cardiovascular events over the Framingham Risk Score

[9], brachial artery stiffness (carotid radial PWV), AI, central PP and PP amplification [10].

PWV, however, is affected by the age and body mass index (BMI) of the study population.
The structural composition of the arterial walls changes with advancing age, contributing to

the increased PWV [3].

The menopause has been shown to augment the age-dependent increase in arterial

stiffness. Oestrogen deficiency is associated with increased concentrations of

proinflammatory cytokines, which through a sequence of pathways, have been implicated in

the inhibition of endothelium dependent vasodilation and nitric oxide synthesis [11].

Studies have shown that women diagnosed with premature ovarian insufficiency (POI) have

a higher incidence of total cardiovascular disease (CVD) (hazard ratio [HR] 1.61, 95%

confidence interval [CI] 1.22 to 2.12, p=0.0007) [12] and mortality (80%) [13] secondary to

this [14-15], irrespective of the cause. The Framingham Study documented an increased risk

of coronary heart disease (CHD) in postmenopausal women, particularly in the age category

40-44 years [16]. However, this risk has been shown to be two-fold higher in women

diagnosed with POI (HR 2.2, 95% CI 1.0 to 4.9) compared to an early menopause (EM) (HR

1.2, 95% CI 0.7 to 2.0) [17]. A sub-analysis of the Multi-Ethnic Study of Atherosclerosis

reported a 4% lower risk of heart failure for every year of increased menopausal age (HR

0.96, 95% CI 0.94-0.99) [18].

Oestrogen deficiency is also associated with atherogenic changes in the lipid profile [19]

contributing to cardiovascular risk in this population. Knauff et al., (2008) [20] compared the

lipid profile of 90 POI patients with 198 control subjects and demonstrated a significantly

higher triglyceride level (mean difference 0.17 log mmol/L, 95% CI 0.06-0.29) and borderline

lower high-density lipoprotein (HDL) cholesterol level in women with POI [20].
The mainstay of management of women diagnosed with POI or an EM (EMPOI) is hormone

replacement therapy (HRT). Most international guidance documents recommend treatment

until at least the natural age of the menopause [21-26]. The number of trials assessing the

impact of HRT on cardiovascular events in women diagnosed with EMPOI are, however,

limited. Kalantaridou et al., (2004) [27] assessed endothelial function in women with POI

before and after 6 months of HRT (oral 0.625mg conjugated equine estrogen [CEE] with

cyclical medroxyprogesterone acetate [MPA] 5mg). Women receiving HRT demonstrated a

more than two-fold increase in flow-mediated dilation, comparable to the control group,

but the findings are limited by the small sample size (n=18 versus controls n=20) and did not

reach statistical significance [27].

Langrish et al., (2009) [26] compared the impact of HRT (transdermal estradiol [t-E2] 100-

150mcg/day with cyclical vaginal progesterone 400mg/day for two weeks every month)

with the combined oral contraceptive pill (COCP) (ethinylestradiol 30µg with 1·5mg

norethisterone) on cardiovascular risk markers in women with POI. They concluded that HRT

significantly lowered mean 24-hour systolic and diastolic blood pressure and had a greater

benefit on renal function than the COCP. The number who completed the study, however, is

also small (n=18) [26].

The Postmenopausal Estrogen/Progestin Interventions (PEPI) Trial analysed the impact of

oestrogen (CEE 0.625mg daily) alone or in combination with one of three progestogen

regimens (MPA 2.5mg/day; MPA 10mg on days 1-12; or micronised progesterone [MP]

200mg on days 1-12) on heart disease risk factors (HDL cholesterol levels; SBP; serum insulin
levels; and fibrinogen levels) in postmenopausal women aged between 45-64 years. They

demonstrated that unopposed oestrogen produced the greatest beneficial effects, but the

high rate of endometrial hyperplasia restricted its use to women without a uterus. For

women with a uterus, the cyclic use of MP produced the most favourable cardiovascular

effects [28]. MP in combination with oestrogen has previously been shown to have no

adverse effect on blood pressure [29] and insulin levels, both surrogate markers of CVD risk,

with the most favourable impact seen on HDL levels [26].

This pilot study aimed to assess the impact of MP and MPA in combination with t-E2 on the

cardiovascular risk profile of women with EMPOI, through an analysis of both traditional

surrogate markers and cfPWV, identified as the gold standard cardiogenic biomarker for risk

stratification of arterial disease by the European Society for Cardiology [2, 4].

METHODOLOGY

Study objective

To compare the CVD risk of MP versus MPA in combination with t-E2 in the management of

women diagnosed with EMPOI.

Primary endpoint

The main outcome measure was cfPWV.

Secondary outcome measures

The secondary endpoints were HR, SBP, diastolic blood pressure (DBP), PP, AI, cardiac

output (CO), stroke volume (SV), total peripheral resistance (TPR), total cholesterol, HDL,

low density lipoprotein levels (LDL), triglyceride (TG) levels and cholesterol ratio (total

cholesterol/HDL ratio).

Study design

Women under the age of 45-years diagnosed with EMPOI, with an intact uterus, were

prospectively invited and recruited from two tertiary referral Reproductive Endocrine and

Menopause Clinics between April 2013 and August 2015. Subjects fulfilling the inclusion and

exclusion criteria (Table 1) were randomised into one of two treatment arms using a web-

based computer randomisation software, Graph Pad. Both groups were prescribed

50mcg/day of t-E2 in the form of Evorel Patches in conjunction with either cyclical MP

(Utrogestan) 200mg orally on days 15-26 of a 28-day cycle or, MPA (Provera) 10mg

orally on days 16-26 of a 28-day cycle. The medication was prescribed and administered in

accordance with the Summary of Product Characteristics of each of the medications. The

total duration of the study period was 12-months. The study was open labelled where

neither the participant nor researcher were blinded to the randomisation secondary to

limited funds.

Table 1. Inclusion and exclusion criteria.

Prior to randomisation, participants previously prescribed hormonal treatment were asked

to stop it for a minimum of four weeks, designated a washout period, to enable a baseline

assessment to be undertaken.
Measurements were undertaken at baseline and repeated at intervals of 3-, 6-, and 12-

months.

Carotid-Femoral Pulse Wave Velocity

Carotid-femoral PWV was measured using an oscillometric technique to acquire the pulse

waveform (Vicorder device, Skidmore Medical). Patients were placed into the supine

position at approximately 450, in room temperature, and allowed to rest and re-climatise for

5-10 mins prior to assessment. A 10cm-wide cuff was placed over the upper thigh, in the

region of the femoral pulse. Simultaneously, a 3cm-wide cuff was placed around the neck

for the carotid pulse. The distance between the suprasternal notch and mid upper thigh cuff

was measured and utilised in the assessment, as per the manufacturer’s instructions. The

cuffs were simultaneously inflated, and the pulse waveforms recorded. Measurements were

made in duplicate/triplicate, and the mean values used for analysis.

Sample collection

Venous blood was collected for total cholesterol, HDL, LDL, and triglyceride levels. Blood

was collected into a serum separating vacutainer with clot activator (BD Diagnostics,

Plymouth, UK). All laboratory measurements were carried out at King’s College Hospital,

Haematology and Clinical Biochemistry Laboratories.

Statistical analysis and sample size

Univariate analysis was performed to compare age, BMI, and ethnicity between the two

groups. Continuous variables were compared using paired t-test between visits and baseline
for each group. Multiple logistic regression was undertaken to compare continuous

variables adjusting for age, BMI, and ethnicity. A p-value of <0.05 was considered

statistically significant. All analyses were performed using IBM SPSS Statistics version 22.0

(Statistical Package for Social Sciences, Chicago, USA).

We aimed to recruit 90 women in total to allow for a 10% drop out and loss to follow up.

The sample size was calculated using the Altman nomogram [30] and based on changes in

the AI in response to exposure to the two different progestogens assessed in the study.

Baseline data were obtained from a reference population, Pulse Wave Analysis (PWA) and

AI data as reported by McEniery et al., (2005) [31]. Based on the reported findings, we

considered a change of 8% with Standard Deviation of 12 to be a clinically significant

difference to detect. Using the Altman nomogram, this would give a standardised difference

of 0.80. A sample of 80 women in two groups would detect a standardised difference of

0.80 with 80% power at the 5% level of significance.

RESULTS

Patient characteristics

Seventy-one participants consented to the study. Five were excluded as they did not meet

the study inclusion criteria (Table 1) (diabetes [n=1]; hereditary thrombophilia [n=3];

spontaneous pregnancy [n=1]). Figure 1 outlines the study algorithm.

Figure 1. Study algorithm.

The baseline demographic characteristics and haemodynamic parameters for the two study

populations did not differ (Table 2). The age of the participants ranged between 19 years to

44 years of age. Of these, 43 of the participants were 40 years of age (POI), and 23 were

>40 years of age (EM).

Table 2. Baseline demographic characteristics and haemodynamic parameters for the two

study populations.

PWV did not demonstrate significant changes from baseline for the duration of the study, in

either treatment arm (Table 3).

Table 3: Impact of MP and MPA in combination with t-E2 on the haemodynamic variables

after 3-, 6- and 12-months, presented as mean difference (±SD), with 95% confidence

intervals.

MP + t-E2 demonstrated a positive effect on traditional CVD markers, with a significant

improvement seen in CO (0.71±1.01mL/min, 95% CI 0.20 to 1.21, p=0.01) and reduction in

DBP (-3.43±6.31mmHg, 95% Cl -6.57 to -0.29, p=0.03) and TPR (-0.15±0.19mmHg⋅min⋅mL-1,

95% CI -0.24 to -0.05, p=0.01) after 12-months duration compared to baseline. A significant

increase in cholesterol ratio was seen after 12-months duration (0.18±0.30mmol/l, 95% CI

0.02 to 0.33, p=0.03) during the same study period.

MPA + t-E2 in contrast, did not demonstrate significant changes from baseline in HR, SBP,

PP, AI, CO or TPR. Total cholesterol levels, however, were initially lowered at 3-months

duration (-0.28±0.55mmol/l, 95% Cl -0.52 to -0.04, p=0.02). HDL levels demonstrated a

significant decline after 3-months (-0.13±0.24mmol/l, 95% CI -0.23 to -0.02, p=0.02) and 6-

months (-0.16±0.27mmol/l, 95% CI -0.29 to -0.02, p=0.03) duration from baseline. This

difference was not maintained by 12-months duration (-0.11±0.21mmol/l, 95% CI -0.22 to

0.01, p=0.07). After 12-months duration, significant reductions were seen in the TG levels (-

0.21±0.37mmol/l, 95% CI -0.42 to -0.01, p=0.04).

DISCUSSION

Central arterial stiffness has been shown to be an independent predictor of cardiovascular

risk and all-cause mortality [32-34]. Invasive catheterisation of the ascending aorta remains

the gold standard technique for central haemodynamic assessment, but the invasive nature

of the technique has limited its clinical applicability. The analysis of peripheral waveforms

with dedicated devices has provided the opportunity to assess central haemodynamics non-

invasively and has shown good correlation with direct central blood pressure measurements

obtained during invasive catheterisation of the ascending aorta [32]. Salvi et al., (2019) [35]

demonstrated a strong correlation between non-invasive devices measuring cfPWV

(Complior Analyse, PulsePen ET, PulsePen ETT, and SphygmoCor) and invasive aortic PWV

measurements (r>0.83) [35]. The Vicorder system (Skidmore Medical Limited) used in this

study has been calibrated to non-invasively record mean aortic and diastolic pressures and

has also been validated against other devices used in this context [32].
The difference in obtained measurements between invasive aortic PWV and non-invasive

cfPWV readings can be attributed to three main causes [35]. Firstly, cfPWV assessments

do not incorporate the ascending aorta in the path of travel. Secondly, cfPWV

measurements include arterial segments in which the path of travel of the pulse can be in

an opposite direction (brachiocephalic trunk and common carotid artery) to the thoracic

aorta. Thirdly, cfPWV measurements include segments of the femoral artery in their

evaluation. The muscular component of the femoral artery is greater than that of the aorta.

This difference between muscular and elastic arteries can increase the PWV assessment in

younger individuals. This difference, however, is reversed with advancing age, thus, cfPWV

measurements may be overestimated in younger adults and underestimated in older

individuals. Furthermore, PWV exponentially increases in aortic arteries with increasing age,

but only weakly and linearly increases with age within the muscular arteries of the lower

limbs [36].

Overall, cfPWV is deemed to be a highly reproducible, non-invasive emerging cardiogenic

biomarker of arterial stiffness and thus, cardiovascular risk stratification [2, 37]. It is

considered to be the gold standard measure for arterial stiffness [8, 37], however, its clinical

application is limited by population-based differences seen within the vascular behaviour in

varying physiological and pathological conditions [38], including the different methodologies

available to measure the path length and algorithms used to calculate PWV [37].

MP + t-E2 resulted in a significant improvement in CO and reduction in DBP and TPR after

12-months of treatment. PWV, however, did not significantly change from baseline in either
treatment arm over the course of the study, consistent with the neutral changes observed

in the AI and PP readings over the same period.

No significant changes were seen in the lipoprotein profile with MP + t-E2 treatment in

keeping with MP having a more selective effect on progesterone receptors [39-40].

Furthermore, no significant changes were demonstrated in the LDL levels, the serum levels

of which have been closely correlated to the development of coronary artery disease,

supporting the notion of a neutral impact of MP on CVD risk [41-42].

MPA + t-E2, in contrast, resulted in changes in the lipoprotein profile that included a

reduction in total cholesterol levels at 3-months of treatment, HDL levels at 6-months and

TG levels after 12-months of treatment. The TG lowering effect seen with MPA + t-E2 may

have occurred secondary to its antagonistic effect on oestrogen stimulated hepatic TG

synthesis or due to increased activity of the lipoprotein lipase enzyme [41]. MPA + t-E2,

however, did not demonstrate significant changes in any of the other measured traditional

haemodynamic parameters, including CO and TPR.

The findings overall, showed that MP and MPA given in combination with t-E2 did not

adversely impact CVD risk markers as assessed by the gold standard cardiogenic biomarker,

cfPWV when used in the management of women with EMPOI. Furthermore, MP + t-E2

resulted in a significant improvement in CO and reduction in DBP and TPR.

The European Society of Hypertension have stated that a PWV of 12m/s can signify vascular

damage and/or cardiovascular risk. The baseline PWV for both treatment arms and the
change from baseline throughout the study duration was approximately 50% lower than the

single cut-off value used by the European Society of Hypertension. This single value,

however, cannot be extrapolated to all populations, as it can underestimate the level of risk

in young subjects and overestimate the level of risk in older individuals [38]. More directed

ranges accounting for age and ethnicity are therefore needed for clinical application of this

cardiogenic biomarker in everyday practice [43].

A meta-analysis by Laugesen E et al., (2013) [44] concluded that a 1m/s increase in PWV is

associated with a 14% (95% CI 9 to 20%), 15% (95% CI 9 to 21%) and 15% (95% CI 9 to 21%)

increased risk of total cardiovascular events, cardiovascular mortality, and all-cause

mortality, respectively when controlled for risk factors [44]. In this study, the PWV for both

treatment arms changed by <1m/s from baseline, consistent with no significant changes

observed in this parameter throughout the study.

Vlachopoulos C et al., (2010) [45] conducted a meta-analysis of 11 longitudinal studies

including 5,648 subjects with a mean follow up duration of 45-months. The studies included

both men and women of broad age categories, the youngest of which were 42.611.2 years.

They found that the relative risk of cardiovascular events increased by 8.8% (n=3285; 95% CI

1.04 to 1.14) with a 10mmHg increase in central systolic pressure, 13.7% (n=4778; 95% CI

1.06 to 1.22) with a 10mmHg increase in central PP and 31.8% (n=1326; 95% CI 1.09 to 1.59)

with a 10% absolute increase in central AI. A 10% increase in central AI was also correlated

to a 38.4% (n=569; 95% CI 1.19 to 1.61) increased risk of all-cause mortality [45]. Adkisson EJ

et al., (2010) [46] reported a 5-9% reduction in cardiac morbidity and 4% reduction in all-

cause mortality when the SBP is lowered by 3mmHg [46]. This study did not demonstrate
significant changes in PP, AI or SBP, in either treatment arm from baseline, with absolute

levels differing by <10mmHg and <10% change in AI at 12-months, consistent with the

neutral changes observed in the PWV readings over the same study period.

Hypertension, overall, is an important risk predictor for CVD. Current practice estimates

greater prediction of all cardiovascular events with raised SBP, but both systolic and

diastolic hypertension being independent predictors of adverse cardiovascular outcomes

[47]. Systolic hypertension occurs because of increased SV and/or arterial stiffness [48].

DBP, has been proposed by researchers, including the Framingham investigators, to drive

coronary risk in younger subjects [49-52], peaking in the fifth decade of life [45], with SBP

becoming of greater importance in older people [49-52]. This study did not identify changes

in the SBP over the course of the research, consistent with no changes observed in SV and

AI. The DBP, however, did demonstrate a significant reduction after 12-months duration (-

3.43±6.31mmHg, 95% Cl -6.57 to -0.29, p=0.03) in the MP + t-E2 treatment arm. This could

reflect the younger cohort included in this study and support the more favourable impact of

MP in combination with t-E2 when traditional surrogate markers of CVD risk are assessed.

The HR increased by 4-5 beats per minute after 12-months duration from baseline in both

treatment arms, with a significant increase recorded in the MP + t-E2 (7.69±12.80bpm, 95%

CI 1.10 to 14.27, p=0.03) treatment arm at 6-months duration. Cardiac function, such as HR,

has been shown to influence the parameters PP and AI [7-8], with this difference potentially

providing an explanation for the fluctuations seen in the PP and AI readings over the course

of the study.
Epidemiological studies, such as the Framingham cohort studies in the USA, have identified

several CVD risk factors including the lipid profile. Risk stratification models to estimate a

patient's 10-year risk of developing CVD incorporate lipid parameters in their equation. Both

the Framingham-based equations [53] and the European Systematic COronary Risk

Evaluation (SCORE) algorithm [54] consider total cholesterol, HDL cholesterol and the ratio

of total cholesterol to HDL to be the strongest predictors. The Framingham Offspring study

followed a cohort of individuals for 20-years and identified that any combination of low

levels of HDL cholesterol, high levels of LDL cholesterol and high levels of triglyceride were

associated with an increased risk of CVD [55]. The National Clinical Guideline Centre for

Cardiovascular Risk Assessment for primary prevention of CVD, however, recommend using

the QRISK2 risk assessment tool to assess cardiovascular risk for the primary prevention of

CVD in people aged 84 years which incorporates the cholesterol ratio, total

cholesterol/HDL in its calculation [56].

LDL is the predominant cholesterol-carrying lipoprotein and main atherogenic lipoprotein.

Epidemiological data has suggested that isolated low HDL levels is a strong and independent

risk factor for CVD [57-59]. HDL particles may act as a protective factor against

atherosclerosis through several biological mechanisms including effluxing cellular

cholesterol, diminishing cellular death, decreasing vascular constriction, reducing

inflammatory response, protection against pathological oxidation, combating bacterial

infection, lessening platelet activation, regulating gene expression by virtue of micro-RNAs,

and improving glucose metabolism [59].

A subsidiary analysis of a clinical trial in Australia, New Zealand, and Finland, looking at the

impact of Long-Term Intervention with Pravastatin in Ischaemic Disease (LIPID study),

(n=9014 aged between 31-75 years with recorded acute coronary syndrome), acknowledged

that all lipid parameters were linked with future coronary events. They concluded that the

total cholesterol/HDL ratio, LDL/HDL ratio or apolipoprotein B/apolipoprotein A1 ratio were

more superior in predicting the time to coronary event than a single lipid measure [60].

The ESCARVAL Study Group found that HDL levels, total cholesterol/HDL ratios and

triglyceride/HDL ratios are better predictors for mortality and CVD than other lipid

parameters commonly used in clinical practice [61]. The athero-protective function of HDL is

to prevent endothelial activation, inflammation, and oxidative stress, as well as enhance

nitric oxide production and promote cholesterol efflux to reduce lesion formation and

maintain barrier integrity [62]. They demonstrated a positive association with the

triglyceride/HDL ratio and an inverse association with total cholesterol, HDL, and LDL on

age-adjusted mortality rates (deaths/10,000 person-years) [63]. The subfractions of HDL

were not measured as they have not shown an added benefit in the identification of

persons at risk above the measurement of standard HDL.

The main strength of the study is in its’ aim to bridge the gap in knowledge regarding the

impact of HRT on surrogate markers of cardiovascular disease through an analysis of both

traditional markers and cfPWV in this cohort of women. The study, despite highlighting

several findings is not without its limitations.

The main caution that needs to be considered when interpreting non-invasively measured

cfPWV readings is the calculation of path length between the carotid and femoral sites

which can significantly influence PWV, with differences of up to 30% being cited. The

distance is commonly measured by one of four different methodologies: (i) the direct

distance between the carotid and femoral sites; (ii) the distance between the sternal notch

and femoral sites; (iii) the subtracted distance between the carotid and sternal notch from

the total distance; or (iv) the subtracted distance between the carotid and sternal notch

from the sternal notch and femoral site [64]. The expert consensus document on the

measurement of aortic stiffness recommended using 80% of the direct distance between

the common carotid artery and common femoral artery [10]. This study, however, utilised

the distance between the sternal notch and femoral site to calculate the distance between

the two points as per the device’s manufacturer’s instructions, but is largely dependent on

body habitus, potentially introducing an error in the PWV estimation.

Furthermore, the PWV measurements were undertaken at different points within the

cyclical hormonal medication phases of oestrogen only or oestrogen and progesterone

combined. Each visit occurred after a set period plus or minus two weeks to allow flexibility

for the trial subjects. Hormonal influences can occur directly on the arterial wall physiology,

affecting blood pressure and vascular reactivity through oestrogen driven activation of

endothelial nitric oxide synthase activity. Adkisson et al., (2010) [46] postulated cyclic

variations within the haemodynamic parameters, with lower central and systemic blood

pressure (approximately 4mmHg) readings in the late follicular and early luteal phases of

the menstrual cycle mirrored by a proportionate increase in oestrogen levels at this time,
and a subsequent increase in the bioavailability of nitric oxide [46]. The progestogen

component may antagonise the oestrogen mediated responses.

The main limitation is the small sample size which did not achieve the intended power

calculation. The number of recruits who voluntarily decided to leave the study or were lost

to follow up, however, appears consistent across the two treatment arms; this, however,

does not detract from needing larger studies to demonstrate significance. Furthermore, the

loss of subjects is unlikely to be solely attributable to the widely reported progestogen

related adverse effects as loss is seen across both treatment arms. The largest fall in

numbers occurred after the initial 3-month period. This needs to be considered when

interpreting the findings.

The open-label design means that neither the participants nor the investigators were

blinded to the study arms. This was due to the limitation in funding that restricted the

option of packaging the medication identically.

CONCLUSION

MP combined with t-E2 was shown to have a more favourable effect on traditional surrogate

markers of CVD risk in women with EMPOI, with changes observed in CO, DBP and TPR,

when compared to MPA + t-E2. No significant differences, however, were noted when the

cardiogenic biomarker, cfPWV, was assessed. Further studies are needed to establish

reference values for cfPWV to help guide assessment of CVD risk and evaluate the response

to hormone replacement in women with EMPOI.

Contributors

Monica Mittal conducted the study and analysed the data.

Carmel McEniery checked the manuscript for intellectual content.

Prasanna Raj Supramaniam analysed the data and checked the manuscript for intellectual

content.

Linda Cardozo checked the manuscript for intellectual content.

Mike Savvas checked the manuscript for intellectual content.

Nick Panay checked the manuscript for intellectual content.

Haitham Hamoda formulated the hypothesis, oversaw the manuscript, and checked the

manuscript for intellectual content.

All authors approved the final submission.

Funding

This research did not receive any specific grant from funding agencies in the public,

commercial, or not-for-profit sectors.

Ethical approval

Study approval was obtained by the Research and Development Department at King’s

College Hospital and ethical approval was granted by the London and GTAC Ethics

Committee (REC Number: 12/LO/1957; EudraCT Number: 2012-004511-30

https://www.clinicaltrialsregister.eu/ctr-search/search?query=2012-004511-30) on the 16th

January 2013. All patients gave informed written consent prior to data collection.

Provenance and peer review

This article was not commissioned and was externally peer reviewed.
Research data (data sharing and collaboration)

There are no linked research data sets for this paper. Data will be made available on

request.

Declaration of competing interests

The authors declare that they have no competing interests.

Acknowledgements

I would like to extend my gratitude to all respondents, without whose cooperation I would

not have been able to conduct this study.

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Figure 1. Study algorithm.
Table 1. Inclusion and exclusion criteria.

Inclusion Criteria Exclusion Criteria

Females aged between 18 and up to Age <18 or >45-years of age
45-years of age
Confirmed diagnosis of POI or EM Pregnant or lactating females
Willingness to participate Contraindication to the use of hormonal
preparations
No concomitant co-morbidities that Factors present in the medical history:
would contraindicate the use of 1. that would contribute to an increased
hormonal preparations risk of CVD
2. known thrombophilia
3. known porphyria
4. known liver disease
5. known past or suspected breast cancer
6. undiagnosed vaginal bleeding
7. genital tract carcinoma
Smokers
Body mass index >35Kg/m2
The use of concomitant medications that could
influence the results, such as anti-hypertensives
Known hypersensitivity to any of the active
substances or excipients contained within
Utrogestan, Provera or Evorel patches;
known allergy to peanuts or soya
Key: POI – premature ovarian insufficiency; EM – early menopause; CVD – cardiovascular

disease; MPA – medroxyprogesterone acetate.

Table 2. Baseline demographic characteristics and haemodynamic parameters for the two
study populations.

Category PWV cohort Lipoprotein profile cohort

MP + t-E2 MPA + t-E2 P- MP + t-E2 MPA + t-E2 P-value
value
Age (years) 35.75±6.52 37.97±6.02 0.53 35.75±6.52 37.97±6.02 0.17
BMI (Kg/m2) 25.23±5.17 24.90±4.21 0.79 25.23±5.17 24.90±4.21 0.79
Ethnicity 0.30 0.83
Asian 2 7 2 7
Black 11 7 12 7
White 13 13 14 13
Other 2 2 4 2
HR (bpm) 68.38±10.04 69.89±10.34 0.58
SBP (mmHg) 122.67±12.32 124.05±12.61 0.68
DBP (mmHg) 69.02±8.32 69.58±7.18 0.79
PP (mmHg) 53.69±7.33 54.96±8.57 0.55
AI (%) 20.73±7.35 19.87±5.62 0.63
CO (mL/min) 5.87±1.03 6.02±1.13 0.61
SV (ml) 89.03±12.48 89.62±16.98 0.88
TPR (mmHg⋅min⋅mL-1) 0.98±0.18 0.96±0.15 0.63
PWV (m/s) 6.39±1.72 6.39±1.17 1.00
Total cholesterol 4.65±0.67 4.77±0.79 0.09
(mmol/l)
HDL (mmol/l) 1.74±0.42 1.54±0.40 0.40
LDL (mmol/l) 2.47±0.58 2.66±0.63 0.20
TG (mmol/l) 0.98±0.45 1.26±0.78 0.35
Cholesterol ratio 2.79±0.67 3.31±1.05 0.55
(mmol/l)

Key: Data expressed as mean±standard deviation [SD]; t-E2 – transdermal oestradiol; MP –

MP; MPA – medroxyprogesterone acetate; BMI – body mass index; HR – heart rate; SBP –
systolic blood pressure; DBP – diastolic blood pressure; PP – Pulse pressure (difference
between the systolic and diastolic blood pressure); AI – Augmentation index (quantifies the
extent of augmented pressure relative to the central pulse pressure, and is the difference
between the second and first systolic peaks expressed as a percentage of the pulse pressure);
CO – cardiac output; SV – stroke volume; TPR – total peripheral resistance; PWV – pulse
wave velocity (speed of travel of the pulse along a specified arterial segment); HDL – high
density lipoprotein; LDL – low density lipoprotein; TG – triglyceride; cholesterol ratio – total
cholesterol/HDL.
Table 3: Impact of MP and MPA in combination with t-E2 on the haemodynamic variables

after 3-, 6- and 12-months, presented as mean difference (±SD), with 95% confidence

intervals.

Variables MP + t-E2 MPA + t-E2

Mean difference Mean difference Mean difference Mean difference Mean difference Mean difference
after 3-months after 6-months after 12-months after 3-months after 6-months after 12-months
(95% CI) (95% CI) (95% CI) (95% CI) (95% CI) (95% CI)
HR 1.16±12.88 (-4.56 7.69±12.80 (1.10 4.71±13.97 (-2.24 2.89±12.67 (-2.72 1.99±11.49 (-3.55 4.03±18.07 (-5.98
to 6.87) to 14.27)* to 11.65) to 8.51) to 7.53) to 14.03)
SBP -0.65±10.20 (- -3.61±7.39 (-7.41 -1.69±7.78 (-5.56 -2.14±11.64 (- -2.42±9.78 (-7.13 1.18±8.95 (-3.77
5.18 to 3.87) to 0.19) to 2.18) 7.30 to 3.02) to 2.30) to 6.14)
DBP -1.75±6.57 (-4.66 -2.94±5.60 (-5.81 -3.43±6.31 (-6.57 -2.08±7.18 (-5.26 -2.12±6.86 (-5.42 0.97±8.40 (-3.69
to 1.17) to -0.06) to -0.29)* to 1.10) to 1.19) to 5.62)
PP 1.32±9.85 (-3.05 -1.29±6.50 (-4.63 1.83±4.69 (-0.50 -0.32±8.63 (-4.15 -0.42±7.98 (-4.27 -0.80±6.43 (-4.36
to 5.68) to 2.05) to 4.17) to 3.51) to 3.43) to 2.76)
AI -1.16±10.48 (- -1.16±10.48 (- 2.14±6.52 (-1.21 1.19±5.83 (-1.39 0.73±6.12 (-2.21 2.49±5.60 (-0.61
5.81 to 3.48) 5.81 to 3.48) to 5.49) to 3.78) to 3.68) to 5.59)
CO 0.46±1.37 (-0.15 0.34±0.95 (-0.14 0.71± 1.01 (0.20 -0.07±1.37 (-0.68 0.03±1.18 (-0.54 -0.06±1.29 (-0.77
to 1.06) to 0.83) to 1.21)* to 0.54) to 0.60) to 0.66)
SV 3.31±18.76 (-5.01 -3.51±14.63 (- 3.54±12.07 (-2.47 -3.10±16.07 (- -0.68±16.69 (- 0.20±11.38 (-6.10
to 11.62) 11.03 to 4.02) to 9.54) 10.22 to 4.03) 8.73 to 7.36) to 6.50)
TPR -0.08±0.21 (-0.18 -0.07±0.16 (-0.15 -0.15±0.19 (-0.24 -0.02±0.18 (-0.10 0.00±0.22 (-0.11 0.04±0.24 (-0.09
to 0.02) to 0.01) to -0.05)* to 0.06) to 0.11) to 0.18)
PWV 0.40±3.32 (-1.07 0.22±1.38 (-0.49 0.28±0.87 (-0.15 -0.02±1.04 (-0.48 -0.07±1.05 (-0.58 0.64±1.62 (-0.26
to 1.87) to 0.93) to 0.71) to 0.44) to 0.43) to 1.54)
Total -0.16±0.45 (-0.37 -0.13±0.50 (-0.37 0.02±0.47 (-0.23 -0.28±0.55 (-0.52 -0.21±0.51 (-0.46 -0.23±0.51 (-0.51
cholesterol
HDL to 0.06) to 0.11) to 0.26) to -0.04)* to 0.04) to 0.06)
LDL
TG -0.07±0.17 (-0.15 -0.03±0.21 (-0.13 -0.09±0.20 (-0.20 -0.13±0.24 (-0.23 -0.16±0.27 (-0.29 -0.11±0.21 (-0.22
Cholesterol to 0.01) to 0.07) to 0.01) to -0.02)* to -0.02)* to 0.01)
ratio -0.04±0.35 (-0.20 -0.01±0.35 (-0.17 0.09±0.31 (-0.07 -0.12±0.46 (-0.32 -0.11±0.45 (-0.34 -0.02±0.45 (-0.27
to 0.13) to 0.16) to 0.26) to 0.08) to 0.13) to 0.23)
-0.12±0.49 (-0.34 -0.16±0.59 (-0.45 0.06±0.48 (-0.18 -0.13±0.54 (-0.37 0.17±0.73 (-0.19 -0.21±0.37 (-0.42
to 0.11) to 0.12) to 0.31) to 0.10) to 0.53) to -0.01)*
0.03±0.30 (-0.11 0.03±0.37 (-0.16 0.18±0.30 (0.02 0.07±0.46 (-0.14 0.25±0.67 (-0.10 0.05±0.38 (-0.17
to 0.18) to 0.21) to 0.33)* to 0.27) to 0.59) to 0.27)
Key:
*p<0.05.
Data expressed as mean±standard deviation [SD]; t-E2 – transdermal oestradiol; MP – MP;
MPA – medroxyprogesterone acetate; HR – heart rate (bpm); SBP – systolic blood pressure
(mmHg); DBP – diastolic blood pressure (mmHg); PP – Pulse pressure (mmHg); AI –
Augmentation index (%); CO – cardiac output (mL/min); SV – stroke volume (ml); TPR – total
peripheral resistance (mmHg⋅min⋅mL-1); PWV – pulse wave velocity (m/s); HDL – high
density lipoprotein (mmol/l); LDL – low density lipoprotein (mmol/l); TG – triglyceride
(mmol/l); cholesterol ratio – total cholesterol/HDL.