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Effects of the Menstrual Cycle on Vascular Function

Permanent Link: http://ufdc.ufl.edu/UFE0022837/00001

Material Information

Title: Effects of the Menstrual Cycle on Vascular Function
Physical Description: 1 online resource (57 p.)
Language: english
Creator: Adkisson, Eric
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: analysis, arterial, augmentation, blood, calf, central, cycle, dilation, effects, endothelial, estrogen, females, flow, forearm, function, guidelines, hormones, index, mediated, menopausal, menstrual, micro, nitric, occlusion, oxide, peripheral, phases, plethysmography, pre, pressue, progesterone, pulse, reactivity, resistance, standardize, stiffness, vascular, velocity, venous, vessels, wave
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Few studies of arterial compliance and endothelial function have been performed in pre-menopausal females. Fluctuations in naturally occurring female sex hormones may alter vascular function. We studied changes in vascular reactivity during the menstrual cycle of 23 healthy pre-menopausal women (age, 19.69 years). Subjects were examined during the four major phases of the menstrual cycle; Early Follicular (EF), Late Follicular (LF), Early Luteal (EL), and Late Luteal (LL). Four non-invasive measures were performed during each of the menstrual cycle phases: augmentation index (AI), central and peripheral pulse wave velocity (PWV), venous occlusion plethysmography (VOP), and brachial artery flow-mediated dilation (FMD). Nitric oxide (NO) levels were also measured during these times. Vascular reactivity was greatest during the LF phase, compared to EF and LL. This is evidenced by increases in brachial artery flow-mediated dilation (EF, 3.35%; LF, 5.12%; LL, 3.71%; P=0.05), as well as, peak forearm (EF, 21.58; LF, 25.86; LL, 21.50 ml/min/100ml tissue; P=0.05) and total calf (EF, 7.26; LF, 8.87; LL, 7.62ml/min/100ml tissue; P=0.05) hyperemic blood flow. Furthermore, there was a reduction in augmentation index (AI) corrected for a heart rate of 75 (EF, 9.17%; LF, 1.35%; LL, 7.04%; P=0.05), an increase in round trip travel time (?tp) of the reflected wave (EF, 104.57; LF, 119.04; LL, 107.74 ms; P=0.05) and a reduction in wasted left ventricular (LV) energy (EF, 10.22; LF, 5.67; LL, 9.91 dynes/cm2/sec; P=0.05). Also, plasma nitrite/nitrate (NOx) increased during the Late Follicular phase (EF, 25.93; LF, 47.97; EL, 26.10; LL, 27.73 micro-mol/L; P=0.05). The temporal pattern of NO was paralleled by plasma levels of estrogen at the 4 measurement phases (EF, 78.85; LF, 158.62; EL, 91.52; LL, 68.32 pg/ml; P=0.05). This is the first study to comprehensively assess systemic arterial function, menstrual cycle hormones and vaso-active mechanisms throughout the menstrual cycle in the same cohort of women. We found that the greatest changes in vascular reactivity occurred during the Late Follicular phase of the menstrual cycle. The mechanism responsible for this phenomenon, in part, is increased NO bioavailability, secondary to an estrogen-mediated response. Due to these variations between phases, laboratory vascular testing in pre-menopausal females must be standardized by menstrual cycle phase to avoid variability.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Eric Adkisson.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Braith, Randy W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022837:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022837/00001

Material Information

Title: Effects of the Menstrual Cycle on Vascular Function
Physical Description: 1 online resource (57 p.)
Language: english
Creator: Adkisson, Eric
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: analysis, arterial, augmentation, blood, calf, central, cycle, dilation, effects, endothelial, estrogen, females, flow, forearm, function, guidelines, hormones, index, mediated, menopausal, menstrual, micro, nitric, occlusion, oxide, peripheral, phases, plethysmography, pre, pressue, progesterone, pulse, reactivity, resistance, standardize, stiffness, vascular, velocity, venous, vessels, wave
Applied Physiology and Kinesiology -- Dissertations, Academic -- UF
Genre: Applied Physiology and Kinesiology thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Few studies of arterial compliance and endothelial function have been performed in pre-menopausal females. Fluctuations in naturally occurring female sex hormones may alter vascular function. We studied changes in vascular reactivity during the menstrual cycle of 23 healthy pre-menopausal women (age, 19.69 years). Subjects were examined during the four major phases of the menstrual cycle; Early Follicular (EF), Late Follicular (LF), Early Luteal (EL), and Late Luteal (LL). Four non-invasive measures were performed during each of the menstrual cycle phases: augmentation index (AI), central and peripheral pulse wave velocity (PWV), venous occlusion plethysmography (VOP), and brachial artery flow-mediated dilation (FMD). Nitric oxide (NO) levels were also measured during these times. Vascular reactivity was greatest during the LF phase, compared to EF and LL. This is evidenced by increases in brachial artery flow-mediated dilation (EF, 3.35%; LF, 5.12%; LL, 3.71%; P=0.05), as well as, peak forearm (EF, 21.58; LF, 25.86; LL, 21.50 ml/min/100ml tissue; P=0.05) and total calf (EF, 7.26; LF, 8.87; LL, 7.62ml/min/100ml tissue; P=0.05) hyperemic blood flow. Furthermore, there was a reduction in augmentation index (AI) corrected for a heart rate of 75 (EF, 9.17%; LF, 1.35%; LL, 7.04%; P=0.05), an increase in round trip travel time (?tp) of the reflected wave (EF, 104.57; LF, 119.04; LL, 107.74 ms; P=0.05) and a reduction in wasted left ventricular (LV) energy (EF, 10.22; LF, 5.67; LL, 9.91 dynes/cm2/sec; P=0.05). Also, plasma nitrite/nitrate (NOx) increased during the Late Follicular phase (EF, 25.93; LF, 47.97; EL, 26.10; LL, 27.73 micro-mol/L; P=0.05). The temporal pattern of NO was paralleled by plasma levels of estrogen at the 4 measurement phases (EF, 78.85; LF, 158.62; EL, 91.52; LL, 68.32 pg/ml; P=0.05). This is the first study to comprehensively assess systemic arterial function, menstrual cycle hormones and vaso-active mechanisms throughout the menstrual cycle in the same cohort of women. We found that the greatest changes in vascular reactivity occurred during the Late Follicular phase of the menstrual cycle. The mechanism responsible for this phenomenon, in part, is increased NO bioavailability, secondary to an estrogen-mediated response. Due to these variations between phases, laboratory vascular testing in pre-menopausal females must be standardized by menstrual cycle phase to avoid variability.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Eric Adkisson.
Thesis: Thesis (M.S.)--University of Florida, 2008.
Local: Adviser: Braith, Randy W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2010-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022837:00001


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1 EFFECTS OF THE MENSTRUAL CYCLE ON VASCULAR FUNCTION By ERIC JOSIAH ADKISSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2008

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2 2008 Eric Josiah Adkisson

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3 To my wife, Lisa; and to my parents, Van and Mary Kay Adkisson, who continually support me and push me to succeed in all of my sc holastic and professional endeavors.

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4 ACKNOWLEDGMENTS I thank Dr. Randy Braith for the opportunity to pursue this degree in his laboratory at the University of Florida. His guidance on this proj ect and all other m atters has been invaluable. I also thank Darren Casey and Darren Beck for thei r friendship and advice. Lastly, I thank the other members of my committee, Dr. Steve Dodd and Dr. Scott Powers, for their suggestions and guidance.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ............................................................................................................... 4LIST OF TABLES ...........................................................................................................................7LIST OF FIGURES .........................................................................................................................8ABSTRACT ...................................................................................................................... ...............9 CHAP TER 1 INTRODUCTION .................................................................................................................. 112 LITERATURE REVIEW .......................................................................................................14Overview of Arterial Compliance ..........................................................................................14Overview of Endothelial F unction and Nitric Oxide ..............................................................16Menstrual Cycle Hormones .................................................................................................... 17Estrogen ...........................................................................................................................17Progesterone .................................................................................................................. ..19Previous Investigations ...........................................................................................................20Studies Reporting Significant Change .............................................................................20Studies Reporting No Significant Change .......................................................................213 METHODS AND MATERIALS ........................................................................................... 23Subjects ...................................................................................................................... .............23Inclusion Criteria .............................................................................................................23Exclusion Criteria ............................................................................................................23Specific Measurements ......................................................................................................... ..23Arterial Stiffness Testing .................................................................................................24Pulse wave analysis (PWA) ..................................................................................... 24Pulse wave velocity (PWV) ..................................................................................... 25Endothelial Function Testing .......................................................................................... 26Brachial artery flow -mediated dilation .................................................................... 26Forearm and calf flow-mediated dilation .................................................................27Blood Collection ..............................................................................................................28Biochemical Analysis ...................................................................................................... 29Menstrual cycle hormones ....................................................................................... 29Vasodilator production ............................................................................................. 294 RESULTS ....................................................................................................................... ........31Subject Characteristics ............................................................................................................31Blood Pressure and Pulse Wave Analysis .............................................................................. 31

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6 Central and Peripheral A rterial Stiffness ................................................................................ 32Forearm and Calf Resistan ce Arterial Blood Flow ................................................................. 32Brachial Artery Endothelial Function .....................................................................................33Menstrual Cycle Hormones and Nitric Oxide ........................................................................ 335 DISCUSSION .................................................................................................................... .....45LIST OF REFERENCES ...............................................................................................................49BIOGRAPHICAL SKETCH .........................................................................................................57

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7 LIST OF TABLES Table page 4-1 Descriptive Characteristics ............................................................................................... .344-2 Blood pressure components and pulse wave analysis ........................................................ 344-3 Pulse Wave Velocity ....................................................................................................... ...344-4 Forearm and calf flow-mediated dilation ........................................................................... 354-5 Brachial artery flow-mediated dilation ..............................................................................354-6 Menstrual cycle hormones and nitric oxide ....................................................................... 35

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8 LIST OF FIGURES Figure page 3-1 Ascending aortic pressure waveform. ................................................................................ 303-2 Determination of the pulse wave transit distances from body surface measurements. ... 304-1 Peripheral systolic blood pressure (PSBP) ........................................................................364-2 Peripheral diastolic blood pressure (PDBP) ......................................................................364-3 Aortic systolic blood pressure (ASBP) ..............................................................................374-4 Aortic diastolic bl ood pressure (ADBP) ............................................................................ 374-5 Mean arterial blood pressure (MAP) .................................................................................384-6 Augmented blood pressure (AgBP) ................................................................................... 384-7 Aortic augmentation index (AI) ......................................................................................... 394-8 Aortic augmentation index normalized for heart rate at 75 bpm (AI@HR=75) ................ 394-9 Roundtrip travel time of reflected wave ( tp). ................................................................... 404-10 Wasted left ventri cular (LV) energy ..................................................................................404-11 Peak forearm blood flow (FBF). ........................................................................................414-12 Total calf blood flow (CBF). .............................................................................................414-13 Absolute dilation ................................................................................................................424-14 Brachial artery flow-m ediated dilation (FMD) .................................................................. 424-15 Normalized brachial artery flow-mediated d ilation (FMD). ..............................................434-16 Estradiol ................................................................................................................ .............434-17 Nitrite/nitrate (NOx) .................................................................................................... ......44

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9 Abstract of Thesis Presen ted to the Graduate School of University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EFFECTS OF THE MENSTRUAL CYCLE ON VASCULAR FUNCTION By Eric Josiah Adkisson December 2008 Chair: Randy Braith Major: Applied Physio logy and Kinesiology Few studies of arterial comp liance and endothelial function have been performed in premenopausal females. Fluctuations in naturally occurring female sex hormones may alter vascular function. We studied changes in vascular reactiv ity during the menstrual cycle of 23 healthy premenopausal women (age, 19.69 years). Subjects were examined dur ing the four major phases of the menstrual cycle; Early Follicular (EF), Late Follicular (LF), Early Luteal (EL), and Late Luteal (LL). Four noninvasive measures were performed during each of the menstrual cycle phases: augmentation index (AI), central and pe ripheral pulse wave ve locity (PWV), venous occlusion plethysmography (VOP), and brachial ar tery flow-mediated dilation (FMD). Nitric oxide (NO) levels were also measured during these times. Vascular reactivity was greatest during the LF phase, compared to EF and LL. This is evidenced by increases in brachial artery flow-mediated dilation (EF, 3.35%; LF, 5.12%; LL, 3.71%; P=0.05), as well as, peak forearm (EF, 21.58; LF, 25.86; LL, 21.50 ml/min/100ml tissue; P=0.05) and total calf (EF, 7.26; LF, 8.87; LL, 7.62ml/min/100ml tissue; P=0.05) hyperemic blood flow. Furthermore, there was a reduction in augmentation index (AI) corrected for a heart rate of 75 (EF, 9.17%; LF, 1.35%; LL, 7.04%; P=0.05), an increase in round trip travel time ( tp) of the reflected wave (EF, 104.57; LF, 119.04; LL, 107.74 ms; P=0.05) and a reduction in wasted

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10 left ventricular (LV) energy (EF, 10.22; LF, 5.67; LL, 9.91 dynes/cm2/sec; P=0.05). Also, plasma nitrite/nitrate (NOx) increased during the Late Follicular phase (EF, 25.93; LF, 47.97; EL, 26.10; LL, 27.73 mol/L; P=0.05). The temporal pattern of NO was paralleled by plasma levels of estrogen at the 4 measuremen t phases (EF, 78.85; LF, 158.62; EL, 91.52; LL, 68.32 pg/ml; P=0.05). This is the first study to comprehensively a ssess systemic arterial function, menstrual cycle hormones and vaso-active mechanisms th roughout the menstrual cycle in the same cohort of women. We found that the gr eatest changes in vascular re activity occurred during the Late Follicular phase of the menstrual cycle. The mechanism responsible for this phenomenon, in part, is increased NO bioavailabil ity, secondary to an estrogen-mediated response. Due to these variations between phases, laboratory vascular testing in pre-menopausal females must be standardized by menstrual cycl e phase to avoid variability.

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11 CHAPTER 1 INTRODUCTION In recent years, due to the increasing risk for cardiovascular disease (CVD) in the years after menopause, research concerning women and menstrual cycle hormones has been primarily focused on post-menopausal women. Arterial comp liance (inverse of arterial stiffness) and endothelial function, measures of cardiovascul ar function, decrease in men and women with age,1-8 but evidence supports postmenopausal women having a steeper age related decline than age-matched men.1-9 In comparison to their age matched male counterparts, women have lower systolic blood pressure (SBP), pulse pressure (PP) and mean arterial pressure (MAP) before menopause, but this sex difference begins to narr ow in the fifth and six decade, disappears by the seventh and is reversed by the eigh th for all 3 measures, respectively.10, 11 CVD and CVD related mortality show a similar trend with re spect to men and women. Pre-menopausal females account for less documented cases of CVD than me n of similar age. However, after menopause this trend is reversed with women no w accounting for the majority of cases.12 This evidence suggests that the menstrual cycle plays an active role on the cardiovascular system, with estrogen protecting pre-menopausal females from CVD. As a measure of prevention against CVD for post-menopausal women, hormone replacement therapy (HRT) has been used exte nsively. However, the effects of HRT are conflicting. Estrogen alone has been shown to at tenuate the age-related decrease in arterial compliance and endothelial function.13-16 In contrast, there are re ports suggesting that estrogen alone has no effect on vascular function.17, 18 Several reports did not identify any changes with combination replacement (e strogen + progesterone),14, 18, 19 which may suggest a possible antagonistic effect of progesteron e on the vasculature. Regardle ss of the effects of HRT on the cardiovascular system, recent evidence sugge sts that HRT does not protect against CVD.20, 21

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12 Few studies of arterial compliance and endot helial function have been performed in premenopausal females. A thorough understanding of variations in vascular function during the menstrual cycle is important. Fluctuations in naturally occurring female sex hormones may alter vascular function. A careful characterization of arterial function duri ng the 4 stages of the female menstrual cycle will be beneficial for 2 reasons. First a systematic analysis of variation in arterial reactivity th roughout the phases of the menstrual cycle may provide information on the underlying sex differences in CVD risk. Secondly, a systematic characterization of cyclical variability in arterial function will help to de velop standardization guid elines for laboratory testing in pre-menopausal women. To our knowle dge there are no standard guidelines for such testing. Accordingly, the purpose of this investigation was to determine the effects of the menstrual cycle on arterial compliance and endothelial function in pre-menopausal females. This is the first study to examine the entire vascul ar tree (central elastic, peripheral muscular and micro-vascular resistance arteries) in the same cohort of wo men throughout their menstrual cycles. Also, this is the first study to assess n itric oxide (NO) production during all phases of the female menstrual cycle. Specific Aim 1: To measure vascular function in centr al elastic, peripheral muscular and microvascular resistance arteries throughout 4 time-points of the normal female menstrual cycle. Hypothesis 1: Cyclic production of estrogen and proge sterone will alter vascular function throughout the menstrual cycle. Specific Aim 2: To determine if laboratory testing of vascular function in females needs to be standardize to the same phase of the menstrual cycle Hypothesis 2: Standardization guidelines are essential to minimi ze laboratory variability when performing vascular tests in pre-menopausal females. Specific Aim 3: To investigate the effect the me nstrual cycle has on NO production.

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13 Hypothesis 3: Production of NO will be altered during the 4 phases of the menstrual cycle.

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14 CHAPTER 2 LITERATURE REVIEW The arterial system is composed of arteries which vary in size and function and work together to maintain blood flow throughout the body. Large elastic arte ries of the central circulation, such as the aorta, serve to cushi on the intermittent output of the left ventricle during systole. In turn, the elas tic recoil during diastole stabilizes the fluctuations in arterial pressure and allows for blood flow in the peripheral circulation. Blood flow pushed into the peripheral circulation is trans ported through muscular conduit arte ries, such as the femoral and brachial arteries. Muscular conduit arteries have the ability to alter smooth muscle tone and therefore, modify the speed of the pressure wave s, commonly referred to as pulse wave velocity. Lastly, the small arterioles, or resistance vessels, transport and c ontrol blood flow into tissues. These vessels are thought to control mean arterial pressure by altering thei r diameter. Overall, the various types and regions of arteries function to convert pulsatile load from the heart into steady flow at the capillary level and maintain a relatively constant perfusion pressure. Overview of Arterial Compliance Arterial compliance is defined as a change in arterial blood volume ( V) for a given change in arterial blood pressure ( P) (i.e. V/ P).22 In general, a compliant artery is able to expand and recoil in response to th e cardiac cycle, stabilizi ng fluctuations in arterial pressure. Reductions in large artery compliance is an important indicator of cardiovascular risk23, 24 and has recently been linked to CVD related mortality.25 There is an age associated decrease in arterial compliance.2-9 ORourke and Nichols26 reported that aortic compliance, measured by PWV, is decreased by approximately 100% betwee n 20 and 80 years of age. Interestingly, it seems that with aging compliance is not affected in peripheral arteries, but is limited to the central elastic arteries.26-28

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15 An explanation for this phenomenon may be found in the morphology of the arteries. When compared to peripheral arteries, central el astic arteries, such as the aorta, have a higher composition of elastic fibers. With age there are 3 morphologic changes in the aorta. First, elastin begins to thin, split, fray and fragment. Secondly, collagen, an extremely stiffer fiber than elastin, doubles in content from age 20 to 70 years.22 Thirdly, arterial ca lcification occurs in some people. The associated decrease in elas tic fibers and increase in stiff fibers with calcification causes profound changes in central co mpliance. This age-related histological transformation is not seen in peripheral muscul ar arteries. Changes in compliance in these muscular arteries are primarily due to changes in smooth muscle tone. A decrease in compliance or increase in stiffne ss in either elastic or muscular vessels can lead to altered blood flow. Additionally, de creased compliance increases the transmission velocity of pressure waves in the arterial syst em. It is important to understand that pressure waves can and do travel forward a nd retrograde in the arterial sy stem. A reflected wave occurs when the primary forward traveling wave encoun ters sudden changes in vessel stiffness or diameter, such as bifurcations or at the inte rface with resistance vesse ls, and a portion of the wave is reflected back toward the heart. In a properly functioning system a summation of reflected waves from many sites arrive back in the ascending aorta post-systole augmenting coronary perfusion pressure. However, with decr eased compliance, the reflected wave returns to the ascending aorta during late systole. The cons equence due to the overlap of the reflected wave into systole is increased arte rial pressures due to higher SB P and lower DBP, thereby causing increased LV afterload and altering coronary perfusion.22 Higher SBP and PP, lower DBP, and LV hypertrophy have all been identified as indepe ndent risk factors of cardiovascular related morbidity and mortality.29-31 Measuring compliance using PWV by applanation tonometry

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16 provides information on large artery stiffness, small artery tone and endothelial function32, 33 and may act as a marker for the development of future CVD. Overview of Endothelial Function and Nitric Oxide Vascular endothelium was once thought of as a quiescent single layer of cells lining the lumen of blood vessels. However, we now know the endothelium to be an extremely important biologically active organ, producing a myriad of mediators that regu late vascular smooth muscle tone and prevent against the development of CVD. One such important mediator is the vasodilator nitric oxide (NO) NO is produced by the reaction of L-Arginine, oxygen, and endothelial nitric oxide synthase (eNOS) with citrulline as a byproduct.34 Upon synthesis, NO diffuses across the endothelial cell membrane a nd into vascular smooth muscle cells. Once in the muscle cell, NO stimulates the movement of intra-cellular calcium out of the cell, thus causing muscle relaxation.35 Proper functioning endothelium continuously produces NO, which is important in maintaining resting vascular tone.36 Increased production above basa l levels is stimulated in 2 ways: shear stress-mediated and agonist-mediated. Mechanical shear stress or pulsatile laminar flow along the endothelial wall causes the synthesi s and release of NO and is an important local mechanism for the maintenance of vascular tone.37 Agonists such as acetylcholine and bradykinin bind to receptors on endothelial cells causing the release of NO.36, 38, 39 Both sheer stress-mediated and agonist-mediated produc tion of NO function by increasing endothelium intra-cellular calcium, which bi nds to an inactive eNOS comp lex releasing the active eNOS molecule. Endothelial dysfunction is often a term used to define a decrease in NO bio-availability. As well as being an important vasodilator, NO al so plays a role in preventing plaque formation by suppressing leukocyte and platelet activity an d inhibiting smooth muscle proliferation.

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17 Decreased levels of NO would promote a vascular environment more prone to plaque formation as well as vasoconstriction. E ndothelial dysfunction is seen in patients with coronary and peripheral vascular disease as well as people with conventional cardi ovascular risk factors40, 41 and is an independent pred ictor of cardiac events.41 Menstrual Cycle Hormones Estrogen Estrogen is a general term used to de fine a group of important steroid hormones synthesized from cholesterol that regulate the fe male menstrual cycle. Multiple estrogens have been identified in plasma of human fe males, the most abundant being beta ( )-estradiol. Estrogens are produced primarily by developing fo llicles in the ovaries and the corpus luteum. During the early follicular (EF) phase follicle-stimulating hormone (FSH) and lutenizing hormone (LH) secreted from the anterior pituita ry initiate and sustain follicular growth. Estrogen concentration rapidly incr eases during the early and late follicular (LF) phases. In the LF phase, just prior to ovulation, there is a surge of LH that tr iggers ovulation and the formation of the corpus luteum. Estrogen levels are at their highest before ovulation and quickly fall in the beginning of the early luteal (EL) phase. Estrogen production begins to slightly increase between the early and late luteal (LL) phases due to a completely functional corpus luteum. Levels then decline at the onset of menstruation. Estrogen plays an essential role in re gulating a womans reproductive process and preparing the uterus for pregnanc y. Along with estrogens important role in the menstrual cycle, estrogen is an active modulator of arterial function. For example, oral administration of estrogen decreases low-density lipoprotein (LDL) choleste rol and increases high -density lipoprotein (HDL) cholesterol in both pre-menopausal and post-menopausal females.20, 42 Also, endogenous sources of estrogen modulate lipid throughout the menstrual cycle. LDL levels decrease and

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18 HDL levels increase when estrogen is high a nd the reverse is seen when estrogen is low.43-45 These changes are associated w ith a decrease risk in CVD,46 but account for only approximately one third of the observed clin ical benefits of estrogen.47 In addition to the affect on lipoproteins, es trogen has direct acti ons on arterial wall physiology. Endothelial cells contain two estr ogen receptors; estrogen receptor alpha (ER ) and estrogen receptor beta (ER ).48, 49 Activation of these receptors by estrogen rapidly elicits nongenomic activation of intracellu lar signaling, as well as, classi cal genomic pathways involving regulation of gene transcription. The rapid vasodilating effect of estrogen in endothelial cells is related to its ability to increase NO by stimulating eNOS activity48, 50 through the activation of a subgroup of ER .51 ER is not associate with rapid non-genomic vasodilation,52 but both ER and ER act as transcription factors increasing eNOS expression,53, 54 thus increasing NO availability in the long-term. In vascular SMCs, estrogen has been shown to produce a rapid dilating response associated with the regulation of intracellular ca lcium. Estrogen receptors have been identified on SMCs55-57 and it is clear that estrogen lowers the concentration of intra-cellular calcium. Even though one study has indicat ed depressed sarcoplasmic retic ulum calcium release due to estrogen,58 other studies suggest attenuating calcium influx59 and/or stimulating calcium efflux.60 Another important means of regulating calcium influx has been identified and works indirectly. Estrogen stimulates the activity of the large-conductance, calcium and voltage-activated potassium (BKCa) channels causing a efflux of potassium that repolarizes membrane potential and closes voltage-dependent calcium channels.61 In aggregate, a decreased concentration of calcium inside the cell would cause a subseque nt acute relaxation of the blood vessels.

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19 Progesterone Progesterone is another ster oid horm one produced during the menstrual cycle. The primary production site of progesterone during the menstrual cycle is the corpus luteum, but is also produced by the adrenal glands, brain, and, during pregnancy, by the placenta. During the follicular phases progesterone production is minimal, but slightly rises just prior to ovulation. After ovulation and the formation of the corpus lu teum, progesterone levels rise sharply and peak approximately one week after ovulation during the luteal phases. Along with estrogen, progesterone is important for preparing the uterus for pregnancy and serve extremely important functions during pregnancy. Progesterones effect on the cardiovascular sy stem remains unclear a nd controversial. It is frequently administered along with estroge n in HRT and is thought to counteract some beneficial features of estrogens As stated previously, suppl ementation of estrogen alone has been shown to attenuate the age-related decrease in arterial comp liance and endothelial function,13-16 but when progesterone is combined w ith estrogen, these benefits are blunted.14, 18, 19 However, not all studies have shown that pr ogesterone blunts the beneficial effects of estrogen.62-64 One primary concern involves progesterones adverse effects on lipoprotein levels. Progesterone opposes estrogen by lowering the level of HDL.65, 66 In contrast to these findings, there are reports of progester one having no effect on estrogen associated lipoprotein changes.63, 64 These differences have been attributed to th e type of progesterone used. Progesterones with lower levels of androgenic act ivity have only small effects66-68 or no effects69 on lipoprotein levels when used in combination with estrogen67 or when used alone.68 Similar to estrogen, progestero ne interacts directly on vasc ular wall physiology, but with possible deleterious effects. Progesterone recep tors (PR) have been localized on vascular SMC,70 endothelial cells71 and two subtypes, PR-A and PR-B, have been identified in human

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20 aorta SMC.72 Both receptors have been previously demonstrated to repress transcriptional activities of estrogen receptors.73 In vascular SMC, progesterone has been shown to up-regulate gene expression of angioten sin II type 1 (AT1) receptors74 that mediate the effects of the reninangiotensin system (RAS ) and induce vasoconstriction.75 In contrast, estrogen down regulates AT1 receptors through a NO dependant mechanism.76 In addition to incr easing gene expression, progesterones effect on AT1 receptors may be part ly due to the repression of estrogen receptor activity, thus regulating NO production and cr eating an environment more prone to vasoconstriction. Previous Investigations The effects of the 4 phases of the menstrual cycle on vascular function are inconclusive. We hypothesize that inconsistencies in the literature exist for two reasons. First, state-of-the-art noninvasive techniques were not utilized. Secondly, measurements were cross-sectional and not carefully standardized in the same cohort of women. Studies Reporting Significant Change W illiams et al.77 measured pre-menopausal females at 4 time points during the menstrual cycle (EF, LF, EL, & LL). They showed that br achial FMD increased from EF to the LF phase, fell in the EL phase, and increased again in the LL phase. Hashimoto et al.78 also saw a increase from EF to LF in brachial FMD, but FMD remained elevated during the luteal phase. In this study, females were only measured once during the luteal phase (5 to 7 days after ovulation), which coincides with the LL phase of the Willia ms study. If another measurement was taken closer to ovulation when estroge n levels were low, Hashimoto et al.78 may have seen an associate fall in brachial FMD as did Williams. In addition, Hashimoto et al.78 measured brachial FMD on age-matched male counterpart s and showed that pre-menopausal females compared to males only during EF.

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21 Williams et al 77 also measured whole body arterial compliance (WBAC), a measurement based on a two-element Windkessel model of the circulation and area method of Liu et al.79 by simultaneous measurement of ascending aortic bl ood flow and carotid blood pressure. WBAC gives a measure of both central and peripheral co mpliance and was shown to increase from the EF phase to the LF phase and fell in both luteal phases. Changes in radial artery compliance were similar Giannattasio et al.80. Measurement of central PWV (carotid-femoral) showed no changes between all phases. Significant changes in WBAC and brachial FMD with no changes in central PWV suggested that peripheral, not central, comp liance changed throughout the menstrual cycle However, Hayashi et al.81 showed no change in periphera l PWV (femoral-dorsalis pedis) during 5 time points of the menstrual cycle (E F, LF, O (Ovulation), EL & LL). Interestingly, central arterial compliance, measured by ultr asound imaging of the common carotid artery, increased from the EF phase and the LF phase a nd into the O phase and decreased in the EL and LL phases making this study the first to suggest that central, not peripheral, compliance changes in response to the menstrual cycle. This dir ectly contradicts the suggestion inferred from the data of Williams Studies Reporting No Significant Change Ounis-Skali et at.82 saw no change in central PWV ( carotid-femoral), peripheral PWV (carotid-radial) or AIa. In this study, females were measur ed on only two occasions, EF and mid luteal. This studys lack of si gnificant change may be due to the small number of measurements and when they were conducted. Early follicular is associated with low estrogen and low progesterone production, whereas in the mid-lu teal phase estrogen a nd progesterone production are increased. Assuming progester one counter acts estrogens e ffects, changes in vascular function may not be seen between EF and mid-luteal. In addition, Willlekes et al.83 tracked

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22 arterial wall properties using ultrasound in th e aorta and femoral arteries in 12 females. Examinations were administered six to eight times during each cycle, which sufficiently encompasses all important time points and hormone fluctuations. Although an adequate amount of examinations were taken, other factors may have contributed to this st udys inability to show significant change. First, a sample size of 12 s ubjects may be too small to see any significant fluctuations. Second, subjects were required to refrain from alcohol, smoking, and caffeine only 3 hours before measurements. Finally, ultrasou nd measurements of the carotid and femoral arteries cannot substitute for a complete ba ttery of vascular tests and therefore cannot definitively suggest that the female menstrua l cycle has no influence on the cardiovascular system.

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23 CHAPTER 3 METHODS AND MATERIALS Our study investigated the eff ects of the menstrual cycle on va scular function. We used a prospective cohort research design to assess the changes in vascular function during four time points during a normal menstrual cycle. Subjects All subjects were recruited using verbal announcements in classes held within the College of Health and Human Performance. All subjects signed a Universi ty of Florida Health Science Center Institutional Review Board (I RB) approved informed consent form prior to participating in the study. We performed a complete batter y of vascular tests on 23 premenopausal females. Inclusion Criteria Clinica lly healthy females Between 18 and 40 years of age Exclusion Criteria Diabetic Receiving any type of contraceptiv es Smoking Resting blood pressure over 135/85 Specific Measurements Subjects were required to make a total of 4 laboratory visits during one menstrual cycle. Each subject was required to track and documen t their menstrual phases for one full menstrual cycle prior to the laboratory visits. This informa tion determined the proper timing for each visit. All testing took place in the Clinical Exercise Physiology (CEP) Laboratory in the Center for Exercise Science, University of Florida, Gainesville, FL.

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24 Arterial Stiffness Testing Pulse wave analysis (PWA) Following a 15 m inute rest period in a supine position, brachial syst olic, diastolic, and pulse blood pressure measurements were perfor med in triplicate in the left arm using an automated noninvasive blood pressure (BP) cuff (V SM MedTech, Ltd.). An average of three BP measurements were used for resting BP values. The assessment of arterial wave reflection characteristics are performed noninvasively us ing the SphygmoCor system (AtCor Medical, Sydney, Australia). High-fidelity radial artery pressure waveforms are recorded by applanation tonometry of the radial pulse using a penci l type micromanometer (Millar Instruments, Houston, Texas). Optimal recording of the pressu re wave is obtained when the hold-down force of the transducer on the artery is such that th e resulting waveform has a stable baseline for at least 10 beats. Using a validated generalized mathematical transfer function the SphygmoCor system synthesizes a central aortic pressure waveform.84, 85 This technique has been shown to be reproducible.86, 87 The reliability of aortic pressure wa veform measurements in our laboratory was established by sequential measurements on 7 young healthy men on 3 separate days. The mean coefficient of variation for these measurements was 6.5%.88 The central aortic pressure wave (Ps-Pd) is composed of a forward traveling wave with amplitude (Pi-Pd), generated by left ventricular ejection and a reflected wave with amplitude (PsPi) that is returning to the ascending ao rta from the periphery (Figure 2-1).89 The contribution or amplitude of the reflected wave to ascending aortic pulse pressure can be estimated by the aortic augmentation index (AIa). AIa is defined as reflected wave amplitude divided by pulse pressure and expressed as a percentage [AIa = (Ps-Pi)/(Ps-Pd) x 100].89, 90 Ps indicates peak systolic pressure, Pi is an inflection point that indicates the beginning upstroke of the reflected pressure wave, and Pd is the minimum diastolic pressure. Th e forward and reflected waves travel in

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25 opposite directions along the arte ry at the same velocity. The round-trip travel time ( tp) of the forward traveling wave from the ascending aorta to the major reflection site and back is measured from the foot of the forw ard traveling pressure wave to Pi. tp is inversely related to arterial pulse wave velocity and arterial stiffness, and directly related to the distance to the reflecting site.89 Pulse wave velocity (PWV) W ith the subject supine, tonometry transit di stances from the supra-sternal notch to the radial (SSN-R), femoral (SSN-F) and carotid (SSN -C) and from the femoral to the dorsalis pedis (F-DP) recording site were measured with a ta pe measure (Figure 2-2). Arterial applanation tonometry (SphygmoCor) were then obtained from the carotid, radial femoral, and dorsalis pedis arteries in that order in rapi d succession. High-fidelity pressu re waveforms were recorded noninvasively using a pencil type micromanometer. Foot-to-foot pulse wave velocity to each peripheral site (dorsalis pedis, radial and femoral) were calculated by determining the delay between the appearance of the pressure waveform foot in the carotid and peripheral sites.91 The distance between recording sites was adjusted for parallel transmission in the aorta and carotid by subtracting SSN-C from SSN-R and SSN-F. The corrected distances will be divided by the respective foot-to-foot transmission delays (Carotid -radial, Carotid-femoral) to give pulse wave velocity. Central pulse wave ve locity (in the mostly elastic aorta) was evaluated using the carotid-femoral (C-F) data and peripheral pulse wave velocity (in the more muscular conduits) using the femoral-dorsalis pedis (F-DP) and caro tid-radial (C-R) data. Pulse wave velocity between the various measuring sites were used as an indirect measur e of regional arterial stiffness. Reliability of pulse wave veloci ty among the different regions was established by sequential measurements on 7 young men on 3 separate days. The mean coefficient of variation for C-R, C-F, and F-DP were 4.5, 2.1, and 5.3%, respectively.88

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26 Endothelial Function Testing Brachial artery flow-mediated dilation Brachial artery reactivity te sting was perform ed using a high-resolution ultrasound (HDI 3000, ATL, Inc). Brachial artery reactivity meas urements were made with the subjects in a supine position following a fast of at least 4 hour s and abstaining from caffeine and alcohol for at least 12 hours prior to the measurements. After lying qui etly for 15 minutes, a 10.5 MHz linear phase array ultrasound transducer was used to imag e the right brachial ar tery longitudinally and was recorded digitally using Pinnacle Studio Plus software (Pinnacle Systems, Inc.). After obtaining resting baseline end di astolic diameters and blood flow velocity, a blood pressure cuff placed on the upper forearm, 1-2 cm below the el bow was inflated to 200 mmHg for 5 minutes. The transducer was held in th e same position for the duration of cuff inflation. Using a cuff position that is distal to the si te of the ultrasound measuremen t has been shown to produce less variability than proximal placement of the cuff92 and has been suggested to serve as a more accurate bio assay of endothelial nitric oxide (NO) availability.93 Immediately following cuff release, brachial artery blood flow velocity was measured for 20 seconds. Brachial artery diameter was then imaged and recorded for an additional 2 minutes. Reactive hyperemia blood flow results in flow-mediated dialation (FMD) of the brachial artery due to shear stress-induced NO release fr om the endothelial wall. About 60 seconds after cuff deflation, peak brachial artery diameter has b een reported to occur an d is a valid measure of endothelial-mediated artery reactivity.94 Brachial FMD is calculated as absolute ( mm) and relative change (%) in brachial ar tery diameter in response to the forearm hyperemic stimulus. Because the main stimulus for FMD is an acute increase in vascular shear stress or blood flow, peak FMD values will be normalized for the magnitude of the hyperemic stimulus.95 Shear rate (velocity/diameter) is used to quantify the hyperemic stimulus.93

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27 Brachial artery diameters are determined during end-diastole by measuring the distance between the near and far wall of the intima us ing image analysis software (Image Pro, Data Translation, Inc.). Five anteri or to posterior point measuremen ts within a 2-3 cm segment are made and the average distances are recorded as the diameter. Forearm and calf flow-mediated dilation Patien ts were tested while in a supine position in a quiet, temperat ure-controlled room, approximately 21-22C and relative humidity of approximately 40-50%. Venous occlusion plethysmography (EC-6, D.E. Hokanson, Inc.) using calibrated mercury strain gauges were used to determine forearm blood flow (FBF) and calf blood flow (CBF) as previously described by others.96, 97 Briefly, the forearm and lower leg was positioned above heart level and a strain gauge was placed around the widest part of th e non-dominant forearm (~5 cm below anticubital fossa) or calf (~10 cm below patella). To m easure FBF, an upper arm cuff (Hokanson SC-10) was placed approximately 5 cm above the antic ubital fossa and inflated to 50 mmHg for 5 seconds every 15 seconds using a rapid cuff infl ator (EC-20, Hokanson, Inc.) to prevent venous outflow.97 To measure CBF, an upper thigh cuff was placed approximately 5 cm above the patella and inflated to 50 mmHg for 5 seconds every 15 seconds. To prevent blood flow to the hand and foot during plethysmography, a cuff (Hokanson, Inc.) was placed around the wrist and ankle, respectively, and inflated to 220mmHg 1 minute before each measurement. The FBF or CBF output signal will transmit to a NIVP3 software program (Hokanson, Inc.) on a PC computer and expressed as milliliters (mL) per minute per 100 mL of tissue (mlmin-1 per 100 mL tissue). Absolute blood flow is determined by the rate of change of limb circumference (e.g. slope) during the 5 second venous occlusion, which co rrelates highly to arteri al blood inflow into the limb.96, 98 The average of 8 plethysmographic measurements over a 2-minute period will be recorded as the resting FBF or CBF.

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28 After baseline FBF is confirmed to be stab le for 2 minutes and recorded, the upper arm cuff was inflated to 200 mmHg for 5 minutes. The cuff was then released and endotheliumdependent FBF was measured during reactive hy peremia blood flow. FBF was measured every 15 seconds for 3 mintues. Peak FBF is recorded as the area under the time x blood flow curve after baseline FBF is subtracted using the trapezium rule.99 Reactive hyperemia induced peak FBF has been shown to correlate highly with acetylcholine-induced FBF in patients with essential hypertension.100 Thus, indicating that reactive hype remia serves as a good noninvasive measurement of endothelium depend ent dilation diameter in resist ance arteries of the forearm. Endothelium-dependent CBF is measured us ing the same techniques and methods as described above for endothelium-dependent FBF. The reliability of peak FBF and CBF using venous occlusion plethysmography is validated in our lab by a pilot study of 9 young healthy males who had peak FBF and CBF during reactive hyperemia performed 3 visits separated by 1 week.101 Mean CV% of resting and peak FBF wa s 17% and 6.6%, respectively, and resting and peak CBF was 15.2% an d 8.4%, respectively. Blood Collection Venous blood sam ples for serum were collected in tubes containing no additive, allowed to clot at room temperature for 15 minutes and immediately centrifuged at 3,000 rpm from 15 minutes at 4C. Venous blood samples for pl asma were collected in tubes containing ethylenediaminetetraacetic acid (EDTA), pl aced on ice for 15 minutes, and centrifuged immediately using the same protocol as the seru m tubes. All serum and plasma samples were separated into 1.5 ml eppendorf tubes and immediatel y stored at -80C until batch analysis at the end of the study.

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29 Biochemical Analysis Menstrual cycle hormones Estrad iol is the major estrogen secreted dur ing the female menstrual cycle. Plasma Estradiol were measured using a commercially available kit (Cayman Chemical, Inc.), which is based on the competition between Estradiol and Estradiol-acetylcholinesterase (Estradiol Tracer) conjugate for Estradiol Antiserum. The amount of Estradiol Tracer that is able to bind to Estradiol Antiserum is determined spectrophotome trically and is inversely proportional to the concentration of estrogen. Levels of progesterone begin to rise during the lut eal phase of the menstrual cycle to help prepare the uterine lining for implantation. Plasma Progesterone were measured using a commercially available kit (Cayman Chemical, Inc.), which is based on the competition between Progesterone and Progesterone -acetylcholinesterase (Proge sterone Tracer) conjugate for Progesterone-specific rabbit antiser um. The amount of Progesterone Tracer that is able to bind Progesterone-specific rabbit antiserum is determ ined spectrophotometrically and is inversely proportional to the concen tration of progesterone. Vasodilator production Because NO is rap idly converted to nitrate a nd nitrite (NOx) in plasma, NOx was used to estimate NO production. Since plasma NOx levels can be influenced by dietary nitrate, subjects were asked to follow the National Institutes of H ealth low nitrate diet guidelines 24 hours prior to each blood draw102. Plasma NOx were measured using a commercially available kit (Cayman Chemical, Inc.), which converts all nitrate to nitr ite using nitrate reductase Spectrophotometric analysis of total nitrite will be performed using Greiss reagent and the absorbance measured at 540 nm.

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30 Figure 3-1. Ascending aortic pressure waveform. Ps = Peak aortic systolic pressure; Pd = minimum aortic diastolic pressure; Pi = inflection point of the reflected wave ; tp = duration of reflected wave from the heart to the periphery and back; tr = systolic duration of the reflected pressure wave 89. Figure 3-2. Determination of the pulse wave tr ansit distances from body surface measurements. The star denotes the supersternal notch. SSN -C = supersternal not ch-carotid distance; SSN-F = supersternal notch-femoral distance; CFTD = carotid femoral transit distance; F-DP = femoraldorsalis pedis distance.

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31 CHAPTER 4 RESULTS A total of twenty-three (n=23) subjects were recruited and tested during the 4 phases of a normal menstrual cycle. The study was approved by the University of Florida Health Science Institutional Review Board and al l subjects signed written informed consent to participate in the study. Subject Characteristics Descriptive characteristics were m easured at all time points (EF, LF, EL and LL) and results are shown in Table 4-1. Ther e were no statistically significant (p 0.05) differences in descriptive characteristics across the 4 measurement time points. Blood Pressure and Pulse Wave Analysis Blood pressure components and pulse wave an alysis results are presented in Table 4-2 and Figures 4-1 4-10. There was no significant change in heart rate, peripheral pulse pressure, and aortic pulse pressure (p 0.05) during the four phases of the me nstrual cycle. LF values were significantly lower than EF values in peripheral systolic blood pressure (105.61 6.53 vs. 110.91 7.76, P 0.05), peripheral diastolic blood pressure (68.17 4.55 vs. 72.87 4.90, P 0.05), aortic diastolic blood pressure (69.22 4.58 vs. 73.78 5.88, P 0.05) and mean arterial blood pressure (80.57 5.10 vs. 85.00 5.82, P 0.05). There were significantly lower LF values than LL values in peripheral systolic blood pressure (105.61 6.53 vs. 110.39 7.51, P 0.05), peripheral diastolic blood pressure (68.17 4.55 vs. 71.69 6.39, P 0.05) and mean arterial blood pressure (80.57 5.10 vs. 84.43 6.39, P 0.05). There was a trend toward decreased aortic systolic blood pressure in LF compared to EF (93.61 5.43 vs. 98.48 6.81, P 0.072).

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32 Pulse wave analysis results demonstrated a significant decrease in aortic augmentation index (AI) (14.04 8.75 vs. 6.96 8.75, P 0.05) and aortic augmentati on index normalized for a heart rate of 75 bpm (AI@75 bpm) (9.17 8.87 vs. 1.35 8.84, P 0.05) between EF and LF. Round trip travel time of the reflected wave ( tp) significantly increased (104.57 15.14 vs. 119.04 19.08, P 0.05) during LF compared to EF. Th ere was a significant decrease in augmented pressure (3.48 2.27 vs. 1.57 2.15, P 0.05) and wasted left ventricular energy (10.22 5.65 vs. 5.67 4.56, P 0.05) from EF to LF. There were significantly lower LF values than LL values in aortic augmentation index (AI) (6.96 8.75 vs.12.52 9.24, P 0.05) and aortic augmentation index normalized for a h eart rate of 75 bpm (AI@75 bpm) (1.35 8.84 vs. 7.04 11.00, P 0.05). Round trip travel time of the reflected wave ( tp) significantly decreased (119.04 19.08 vs. 107.74 10.28, P 0.05) during LL compared to LF There was a significant increase in augmented pressure (1.57 2.15 vs. 3.09 2.94, P 0.05) and wasted left ventricular energy (5.67 4.56 vs. 9.91 6.99, P 0.05) from LF to LL. Central and Peripheral Arterial Stiffness Central and peripheral pulse wave velocity (P WV) results are presented in Table 4-3. No significant change was observed in carotid-radial, carotid-fem oral or femoral-distal PWV throughout all phases. Forearm and Calf Resistance Arterial Blood Flow Forearm and calf resistance arterial blood flow values during reactive hyperemia are presented in Table 4-4 and Figures 4-11 and 4-12. No significant change was observed in resting FBF, total FBF AUC3min resting CBF and peak CBF. There was a significant increase in peak FBF (21.58 4.37 vs. 25.86 5.91, P 0.05) and total CBF AUC3min (7.26 1.30 vs. 8.87 2.55, P 0.05) from EF to LF. There was a tr end towards increased resting CBF (1.74 0.47 vs.

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33 2.04 0.60, P=0.13) and total FBF AUC3min (6.12 1.96 vs. 7.56 2.98, P=0.12) from EF to LF. There were significantly higher LF values than LL values in peak FBF (25.86 5.91 vs. 21.50 5.76, P 0.05) and total CBF AUC3min (8.87 2.55 vs. 7.62 1.72, P 0.05). Brachial Artery Endothelial Function Brachial artery flow-mediated dilation (FMD) results are shown in Table 4-5 and Figures 4-13, 4-14 and 4-15. There were no significant cha nges in baseline diameter (mm), resting SR and peak SR. Significant increases were observed in absolute dilation (mm) (0.16 0.07 vs. 0.23 0.11, P 0.05), brachial FMD (%) (3.35 1.88 vs. 5.12 2.36, P 0.05) and normalized FMD (s-1) (0.44 0.29 vs. 0.64 0.28, P 0.05) during LF compared to EF Significant decreases were observed in absolute dilation (mm) (0.23 0.11 vs. 0.16 0.08, P 0.05), brachial FMD (%) (5.12 2.36 vs. 3.38 1.37, P 0.05) and normalized FMD (s-1) (0.64 0.28 vs. 0.43 0.22, P 0.05) during LF compared to EL. Si gnificant decreases were observe d in absolute dilation (mm) (0.23 0.11 vs. 0.17 0.11, P 0.05), brachial FMD (%) (5.12 2.36 vs. 3.71 2.31, P 0.05) and normalized FMD (s-1) (0.64 0.28 vs. 0.44 0.23, P 0.05) during LF compared to LL. Menstrual Cycle Hormones and Nitric Oxide Menstrual cycle hormones and nitric oxide re sults are presented in table 4-6 and Figures 4-16 and 4-17. There were significant increases in estradiol (78.85.30 vs. 158.62.59, P 0.05) and NOx (25.93.25 vs. 47.97.53, P 0.05) during LF compared to EF. No significant changes were observed in progesterone. LF values were significantly higher than EL and LL values in estradiol (158.62.59 vs. 91.52 0.54 & 68.32 0.35, P 0.05) and NOx (47.97.53 vs. 26.10 21.86 & 27.73 21.05, P 0.05).

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34 Table 4-1. Descriptive Characteristics EF LF EL LL Age (yr) 19.69 1.2919.69 1.2919.69 1.29 19.69 1.29 Height (cm) 165.61 5.78165.61 5.78165.61 5.78 165.61 5.78 Weight (kg) 61.89 12.1061.69 11.8861.69 11.94 62.03 11.96 Body Mass Index (kg/m2) 22.47 3.7022.41 3.6722.39 3.64 22.54 3.67 Values are mean SD; EF=Early Follicular phase; LF=Late Follicular phase; EL=Early Luteal phase; LL=Late Luteal phase Table 4-2. Blood pressure compon ents and pulse wave analysis EF LF EL LL HR (bpm) 64.30 7.1664.30 9.1064.00 7.66 62.96 8.48 PSBP (mmHg) 110.91 7.76105.61 6.53*108.35 5.64 110.39 7.51#PDBP (mmHg) 72.87 4.9068.17 4.55*69.52 5.55 71.69 6.39#PPP (mmHg) 38.09 5.3838.09 6.6238.87 4.71 37.96 5.82 ASBP (mmHg) 98.48 6.8193.61 5.4396.70 6.33 96.52 6.21 ADBP (mmHg) 73.78 5.8869.22 4.58*70.57 5.62 71.39 5.78 APP (mmHg) 25.04 3.5025.04 3.7225.17 3.31 25.13 3.68 MAP (mmHg) 85.00 5.8280.57 5.10*82.57 5.50 84.43 6.39#AgBP (mmHg) 3.48 2.271.57 2.15*2.83 1.99 3.09 2.94#AI (%) 14.04 8.756.96 8.75*11.09 7.56 12.52 9.24#AI @ HR = 75 BPM (%) 9.17 8.871.35 8.84*5.21 7.77 7.04 11.00# t p ( ms ) 104.57 15.14119.04 19.08*116.61 20.81 107.74 10.28#Wasted LV energy 10.22 5.655.67 4.56*8.13 6.11 9.91 6.99#(dynes/cm2/sec) Values are mean SD; *P 0.05 vs. EF. #P 0.05 vs. LF. HR = heart rate; PSBP = peripheral systolic blood pressure; PDBP = peripheral diastolic blood pre ssure; PPP = peripheral pulse pressure; ASBP = aortic systolic blood pressu re; ADBP = aortic diastolic blood pressure; APP = aortic pulse pressure; MAP = mean arterial blood pressure; AgBP = augmented blood pressure; AI = augmentation index; tp = round trip travel time of reflected pressure wave from ascending aorta to peripheral reflecting sites and back; LV = left ventricular. Table 4-3. Pulse Wave Velocity EF LF EL LL CFPWV (m/sec) 6.48 0.586.12 0.586.37 0.49 6.35 0.45 CRPWV (m/sec) 9.52 1.088.87 1.109.17 1.21 9.30 1.21 FDPWV (m/sec) 8.67 1.478.19 1.388.54 1.28 8.59 1.24 Values are mean SD; CFPWV = carotid-femoral pulse wave velocity; CRPWV = carotid-radial pulse wave velocity; FDPWV = femoral-distal pulse wave velocity.

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35 Table 4-4. Forearm and cal f flow-mediated dilation EF LF EL LL Resting FBF 1.64 0.581.84 0.551.67 0.55 1.72 0.63 Peak FBF 21.58 4.3725.86 5.91*22.38 6.91 21.50 5.76#Total FBF AUC3min 6.12 1.967.56 2.986.40 2.07 6.13 1.87 Resting CBF 1.74 0.472.04 0.601.92 0.51 1.75 0.35 Peak CBF 27.32 4.2528.98 5.4226.97 5.63 26.21 4.29 Total CBF AUC3min 7.26 1.308.87 2.55*8.01 1.90 7.62 1.72#Values are mean SD; *P 0.05 vs. EF. #P 0.05 vs. LF. Units are ml/min/100ml tissue; FBF = forearm blood flow; CBF = calf blood flow; AUC = area under flow x time curve. Table 4-5. Brachial artery flow-mediated dilation EF LF EL LL Baseline diameter (mm) 4.46 0.294.64 0.294.51 0.33 4.53 0.37 Absolute dilation (mm) 0.16 0.070.23 0.11*0.16 0.08 0.17 0.11#Brachial FMD (%) 3.35 1.885.12 2.36*3.38 1.37 3.71 2.31#Resting SR 0.76 0.470.63 0.290.68 0.28 0.85 0.73 Peak SR 9.48 2.369.04 1.549.52 2.81 10.15 2.58 Normalized FMD (s-1) 0.44 0.290.64 0.28*0.43 0.22 0.44 0.23#Values are mean SD; *P 0.05 vs. EF. P 0.05 vs. LF. #P 0.05 vs. LF. FMD = flowmediated dilation; SR = shear rate Table 4-6. Menstrual cycl e hormones and nitric oxide EF LF EL LL Estradiol (pg/ml) 78.85 0.30158.62 0.59*91.52 0.54 68.32 0.35# Progesterone (pg/ml) 14.46 9.0117.42 13.4018.93 13.36 11.70 6.41 NOx ( mol/L ) 25.93 21.2547.97 40.53*26.10 21.86 27.73 21.05#Values are mean SD; *P 0.05 vs. EF. P 0.05 vs. LF. #P 0.05 vs. LF. NOx = nitrite/nitrate

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36 EF LF EL LL 50 60 70 80*# PDBP (mmHg) Figure 4-1. Peripheral systolic blood pressure (PSBP) during the 4 phases of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF. EF LF EL LL 50 60 70 80*# PDBP (mmHg) Figure 4-2. Peripheral diastolic blood pressure (PDBP)during the 4 phases of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF.

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37 EF LF EL LL 80 85 90 95 100ASBP (mmHg) Figure 4-3. Aortic systolic bl ood pressure (ASBP) during the 4 phases of the menstrual cycle. EF LF EL LL 65 70 75 80*ADBP (mmHg) Figure 4-4. Aortic diastolic bl ood pressure (ADBP) during the 4 pha ses of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF.

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38 EF LF EL LL 70 75 80 85 90*#MAP (mmHg) Figure 4-5. Mean arterial bl ood pressure (MAP) during the 4 pha ses of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF. EF LF EL LL 0 1 2 3 4 5*# AgBP (mmHg) Figure 4-6. Augmented blood pressure (AgBP) during the 4 phases of th e menstrual cycle. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF.

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39 EF LF EL LL 0 5 10 15 20*#AI (%) Figure 4-7. Aortic augmentation index (AI) during the 4 phases of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF. EF LF EL LL 0 5 10 15*#AI@HR=75 (%) Figure 4-8. Aortic augmentation index norma lized for heart rate at 75 bpm (AI@HR=75) during the 4 phases of the menstrua l cycle. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF.

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40 EF LF EL LL 90 100 110 120 130*# Tp (ms) Figure 4-9. Roundtrip travel time of reflected wave ( tp) during the 4 phases of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF. EF LF EL LL 0 5 10 15*# Wasted LV energy (dynes/cm2/sec) Figure 4-10. Wasted left ventri cular (LV) energy during the 4 pha ses of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF.

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41 EF LF EL LL 10 15 20 25 30*# Peak FBF Figure 4-11. Peak forearm blood flow (FBF) during the 4 phases of the menstrual cycle. Units are ml/min/100ml tissue. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF. EF LF EL LL 0.0 2.5 5.0 7.5 10.0*# Total CBF AUC 3min Figure 4-12. Total calf blood flow (CBF) duri ng the 4 phases of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF. #P 0.05 vs. LF.

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42 EF LF EL LL 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40* #Absolute Dilation (mm) Figure 4-13. Absolute dilation of the brachial artery during the 4 phases of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF. P 0.05 vs. LF. #P 0.05 vs. LF. EF LF EL LL 0.0 2.5 5.0 7.5 10.0*Brachial FMD (%) Figure 4-14. Brachial artery flow-mediate d dilation (FMD) during the 4 phases of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF, EL, & LL.

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43 EF LF EL LL 0.00 0.25 0.50 0.75 1.00* Normalized FMD (S-1) Figure 4-15. Normalized brachial artery flow -mediated dilation (FMD) during the 4 phases of the menstrual cycle. Data are mean SEM. *P 0.05 vs. EF, EL, & LL. EF LF EL LL 0 50 100 150 200*Estradiol (pg/ml) Figure 4-16. Estradiol duri ng the 4 phases of the menstrua l cycle.Data are mean SEM. *P 0.05 vs. EF, EL, & LL.

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44 EF LF EL LL 0 25 50 75*NOx ( mol/L) Figure 4-17. Nitrite/nitrate ( NOx) during the 4 phases of the me nstrual cycle. Data are mean SEM. *P 0.05 vs. EF, EL, & LL.

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45 CHAPTER 5 DISCUSSION The purpose of this study was to assess variab ility in arterial function during the female menstrual cycle. Secondarily, we sought to dete rmine if investigators need to standardize, according to menstrual phase, laboratory vascular function testing in pre-menopausal females. Our data consistently show that the temporal pattern of vascular f unction was significantly different across the 4 phases of the menstrual cycle. Due to this variabil ity, our data argue that vascular testing in pre-menopausal females must be standardized according to menstrual phase. This is the first study to comprehensivel y assess arterial function, menstrual cycle hormones and vaso-active mechanisms throughout the menstrual cycle. The present study demonstrated that the entire vascular system was affected by the 4 temporal phases of the menstrual cycle. Vascular reactivity in central, peripheral and micro-vascul ar arteries increased toward the end of the follicular phase (Phase 2) and fell during the luteal phase (Phase 3-4). Changes in vascular reactivity perfectly mirro red the cyclic production of estrogen during the menstrual cycle. This was the first study to measure NO throughout the 4 phases of the menstrual cycle in the same cohort of women. Our data suggest that NO production may be a primary mechanism driving fluctuat ions in arterial reactivity. As shown in figure 4-17, plasma levels of NO fluctuated in pa rallel with estrogen by increasing by approximately 85% from the early to late follicular (Phase 2) and falling during the luteal phases (Phases 3 & 4). Both endothelial and vascular smooth muscle cells contain estrogen receptors.48, 49, 55-57 Binding of estrogen to these receptors activates pathways th at subsequently increa se NO bio-availability.48, 50, 53, 54 We assume that the increased production of estrogen during the late follicular phase results in a greater opportunity for estrogen + estrogen receptor binding, thereby causing an increase in NO bioavailability. NO is an important modulator of vascular tone36 and vascular

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46 reactivity94, 103 and this estrogen-mediated increase in NO is likely the mechanism responsible for increased vascular reactivity dur ing the late follicular phase. This study demonstrated that during the late follicular ph ase of the menstrual cycle, females experience a dramatic improvement in peripheral endothelial function. This is evidenced by a 53% increase in brachial flow mediated dilation (F MD) between the early follicular (Phase 1) and the late follicular phase (Phase 2) (See Figure 4-14). Brachial FMD returned to baseline values during the luteal phases (Phases 3 & 4). Previous studies measuring brachial FMD have produced conflicting findings. Consistent with results of the present study, Williams et at.77 and Hashimoto et at.78 both reported an increase in brachial FMD from early to late follicular. However both st udies also reported a second ri se in brachial FMD during the luteal phase (Phase 4).77, 78 The explanation for their dispar ate FMD results is unclear. We speculate that their luteal FMD data were collect ed too near the late follicular phase. Indeed, both studies reported estrogen levels during the luteal phase that were not significantly different from values recorded at the late follicular phase.77, 78 Endothelial function in resistance arteries also demonstrated a temporal pattern of improvement during the late follicular phase. Peak forearm blood flow (FBF), measured by venous occlusion plethysmography, increased 20% from early follicular (Phase 1) to late follicular (Phase 2) (See Figure 4-11) as well as total calf blood flow (CBF) (22%). Central hemodynamics also showed significant imp rovement in the late follicular phase. Pulse wave analysis demonstrated an 85% re duction in aortic augmentation index, when corrected for a heart rate of 75 (AI@HR=75), and a 14% increase in round trip travel time of the reflected wave ( tp) from the early follicular (Phase 1) to the late follicular phase(Phase 2) (Figures 4-8, 4-9). Reduction in reflected pressure amplitude le d to a 44% decrease in wasted

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47 left ventricular (LV) energy (F igure 4-10). This reduction is mainly due to a decrease in augmented pressure (55%) (Figure 4-6). To da te, one study has examined central pulse wave characteristics and reported no significan t variations between menstrual phases.82 This study measured subjects once during the early follicul ar phase (Phase 1) a nd once during the early luteal phase (Phase 3). Our findings also show no significant difference between early follicular and early luteal. We speculate that centra l hemodynamics data collected by Ounis-Skali et al.82 was obtained too early in the follicular phase or too late in the luteal phase to see any associated vascular variations. Our study showed no changes in central or peripheral arterial stiffness, when measured by pulse wave velocity (PWV). This is in ag reement with all previ ous studies that have measured PWV throughout the menstrual cycle.77, 81, 82 Our study was the first to repor t significant ch ange in systemic bl ood pressure across the 4 phases of the menstrual cycle. Peripheral syst olic blood pressure (PSBP) peripheral diastolic blood pressure (PDBP), and aortic diastolic blood pressure dropped by approximately 4 mmHg from early follicular (Phase 1) to the late fol licular (Phase 2) (See Figures 4-1, 4-2 & 4-4). Blood pressure began to increase at the onset of the luteal phase (Phase 3) and reached levels equal to early follicular by the end of the luteal phase (Phase 4). Our blood pressure data have clinical relevance and in dicate that clinicians must take in to account menstrual cycle phase in pre-menopausal females when measuring blood pr essure and/or prescribing antihypertensive therapeutic strategies. Cardiovascular disease (CVD) in pre-menopaus al females is conspicuously less than in men of similar age12. Cyclical elevations in circulati on estrogen are cited as one mechanism responsible for this phenomenon. Our data suggests that late follicular (Phase 2) spikes in NO

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48 may confer some degree of cardiova scular protection in females. Besides being an important regulator of vascular tone, NO inhibits leukocyte adhesion, platelet aggregation, expression of adhesion molecules, endothelin-1 (ET-1), vascular growth and inflammation.104 A decrease in NO bio-availability is a direct indication of endothelial dysf unction and is an independent predictor of cardiac events.41 The surge of NO experienced in the late follicular phase would occur approximately every 3 weeks throughout the menstrual years of a female and may provide a lasting protecting affect against CVD. In conclusion, this study shows that central, peripheral and micr o-vascular reactivity increases in the late follicular phase prior to ovulation and falls during the luteal phase. The mechanism responsible, in part, for improved pha se 2 vascular reacti vity is increased NO bioavailability, secondary to an estrogen mediated response. The data also highlights the importance of standardizing vascular testing in pre-menopausal females. Unless laboratory testing is standardized, the tem poral pattern of vascular reacti vity will introduce significant measurement variability in pre-menopausal females. Lastly, our data indicate that the temporal pattern of vascular function in pre-menopausal females should be taken into account when measuring blood pressure in the clinical setting.

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57 BIOGRAPHICAL SKETCH Eric Josiah Adkisson completed his undergra duate degree in exerci se physiology at the University of Florida (UF) in August 2006. Immediately afterward he be gan pursuing a Master of Science degree in applied physiology and kine siology under Dr. Randy Br aith. While at UF, he served as a graduate assistant in the Alan C. Moore Sport and Fitness department, teaching undergraduate conditioning, jogging, we ight training, and backpacking. Eric has been selected for a position at the Marine Corp Officer Candidate School (OCS) in Quantico, VA.