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The effect of menstrual cycle phase on diffusing capacity of the lung Bacon, Catherine 1997

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THE EFFECT OF MENSTRUAL CYCLE PHASE ON DIFFUSING CAPACITY OF THE LUNG by CATHERINE BACON B.Sc, The University of Otago, 1991 B.Phed. (Hons.)/ The University of Otago, 1993  A THESIS  SUBMITTED IN PARTIAL FULFILLMENT OF THE  REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES SCHOOL OF HUMAN KINETICS  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA September 1997 © Catherine Jane Bacon  In presenting this thesis in partial fulfilment  of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library, shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department, or  by his or  her  representatives.  It  is understood that  copying or  publication of; this thesis for financial gain shall not be allowed without my written permission.  Department of  HoMAW  ICtfJCTiCS  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  ^  ScSpreMfe^  i^f  ABSTRACT Pulmonary diffusing capacity (DL) has been observed to decrease d u r i n g menses. Nonetheless, a descriptive study of alterations i n this parameter w i t h menstrual cycle phase has not been completed and the mechanism of change is not clear. Changes i n resting single-breath diffusing capacity of carbon monoxide  (DLQO),  and i n its two components: pulmonary capillary blood volume ( V c ), and membrane diffusing capacity ( D M ) were measured i n 13 normally menstruating w o m e n at points within the menstrual cycle chosen to best discriminate between the effects of oestradiol, progesterone and prostaglandins. In addition, haemoglobin concentration ([Hb]), packed cell volume ( P C V ) , and percent of carboxyhaemoglobin (COHb) were measured. Measurements of  DLQO, V C , D M , and  [Hb] were undertaken  at five testing points throughout three menstrual cycles, whilst C O H b and P C V were assessed at four points within one cycle.  The phase of the menstrual cycle was  determined by quantitative analysis of basal body temperature recorded daily by subjects. N o changes i n resting Dlco, P C V or for [Hb] corrected  DLCO  divided by alveolar volume ( V A ) , C O H b ,  DLCO, D L C O / V A , D M  or V c were found using one-way  repeated measures analyses of variance ( A N O V A s ) of the most representative ovulatory menstrual cycle for each subject. Two-way repeated measures A N O V A s of  DLCO  and  DLCO/VA;  and [Hb] corrected  DLCO  and  DLCO/VA,  which separated the  effects of the five testing points and the ovulatory or anovulatory status of a menstrual cycle were also performed and no significant changes were observed. W h e n the effect of the large hormonal differences between an ovulatory and an anovulatory cycle were removed, a trend towards an increase i n D L independent of the effects of [Hb] at mid-cycle and during the luteal phase compared to the early follicular phase were observed.  ii  Notwithstanding the extreme variability of hormonal changes within the human menstrual cycle, without the benefit of hormonal analysis we have not found consistent alterations in  DL O C  with menstrual phase in normally  menstruating women. This is despite careful effort to time diffusion test with points in the cycle that should best discriminate between the hormonal effects of oestradiol, progesterone and prostaglandins.  iii  TABLE OF CONTENTS Abstract  ii  Table of Contents  iv  List of Tables  vii  List of Figures  viii  Acknowledgment  ix  CHAPTER ONE:  INTRODUCTION  1  Cyclic Alterations in Diffusing Capacity  1  Respiratory Changes Within the Menstrual Cycle  3  Circulatory Changes Within the Menstrual Cycle  3  Further Explanations of D L C O Changes During Menses  5  Limitations of Previous Work  6  Research Questions  6  CHAPTER TWO: METHODOLOGY  9  Ethical Approval  9  Subject Recruitment and Selection  9  Documentation of Menstrual Data  10  Timing of Testing  11  Documentation of Exercise  15  Testing Protocol  15  Measurement of Height, Weight and Anthropometric Variables  16  Measurement of Resting Forced Vital Capacity and Forced Expired Volume in One Second  18  Measurement of Resting Diffusing Capacity  18  Calculation of Diffusing Capacity  20  Correction for Haemoglobin Concentration  21  Quantification of D M and Vc  21  The Reliability of Diffusing Capacity and its Components  22  Collection and Batching of Blood Samples  22  iv  Measurement of Packed Cell Volume and Haemoglobin Concentration  23  Data Analysis  23  Criteria for the Selection of the "Best" Cycle for Analysis  24  Statistical Analysis  25  C H A P T E R THREE: RESULTS  27  Recruitment of Subjects  27  General Subject Characteristics  27  Subject A c t i v i t y  28  L u n g Function Characteristics  28  Analysis of Cycles  30  Analysis of Test Points  30  Blood Sample Analysis Testing Dates  32  Best Ovulatory Cycle Analysis  34  Ovulatory Versus Anovulatory Cycles  38  Changes i n Carboxyhaemoglobin w i t h Menstrual Cycle Phase  38  Changes i n Packed Cell Volume W i t h Menstrual Cycle Phase  42  Reliability of the Packed Cell Volume Measurements  42  Reliability and Validity of the Haemoglobin Concentration Measurements  C H A P T E R FOUR:  42  DISCUSSION  43  Recruitment and Cycle Characteristics of Subjects  43  Diffusion Changes W i t h i n the "Best" Ovulatory Menstrual Cycle  44  Haemoglobin Concentration Changes W i t h i n the "Best" Ovulatory Menstrual Cycle  45  Diffusion Changes i n Ovulatory Versus Anovulatory Cycles  46  Further Mechanisms of Diffusion Change  48  Reliability and Validity of Packed Cell Volume and Haemoglobin Concentration Measurements  C H A P T E R FIVE: C O N C L U S I O N  49  51  v  References  52  Abbreviations  56  APPENDICES  Appendix I: Pulmonary Diffusion: Its Measurement, Components and Determinants  57  Appendix II: The Human Menstrual Cycle  67  Appendix III: Calculation of Intraclass Correlation Coefficient  70  Appendix IV: Ethical Approval  72  Appendix V: Poster Advertisement for Subjects  76  Appendix VI: Menstrual Cycle Diary  77  Appendix VII: Daily Exercise Record  79  Appendix VIII: Initial Questionnaire  81  Appendix IX: Data Sheets  88  Appendix X: Final Questionnaire  91  Appendix XI: Timetables of Testing Dates  104  Appendix XII: Raw Data Summary  115  vi  LIST OF TABLES Table 1. L u n g Function Characteristics of Subjects.  29  Table 2. Summary of Menstrual Cycles.  31  Table 3. A n Analysis of Test Points.  33  vii  LIST OF FIGURES  Figure 1. The T i m i n g of Testing at Points i n the Menstrual Cycle.  13  Figure 2. Diffusing Capacity Measured at Five Test Points W i t h i n Ovulatory Menstrual Cycles of 13 Women.  35  Figure 3. Diffusing Capacity/Alveolar Ventilation Ratio Measured at Five Test Points W i t h i n Ovulatory Menstrual Cycles of 13 W o m e n .  35  Figure 4. Haemoglobin Corrected Diffusing Capacity Measured at Five Test Points W i t h i n Ovulatory Menstrual Cycles of 13 W o m e n .  36  Figure 5. Haemoglobin Corrected Diffusing C a p a c i t y / A l v e o l a r Ventilation Ratio Measured at Five Test Points W i t h i n Ovulatory Menstrual Cycles of 13 W o m e n .  36  Figure 6. Haemoglobin Changes Measured at Five Test Points W i t h i n Ovulatory Menstrual Cycles of 13 Women.  37  Figure 7. Diffusing Capacity Measured at Five Test Points W i t h i n Ovulatory and Anovulatory Menstrual Cycles of 6 W o m e n .  39  Figure 8. Haemoglobin Corrected Diffusing Capacity Measured at Five Test Points W i t h i n Ovulatory and Anovulatory Menstrual Cycles of 6 Women. 39 Figure 9. Diffusing Capacity/Alveolar Ventilation Ratio Measured at Five Test Points W i t h i n Ovulatory and Anovulatory Menstrual Cycles of 6 Women.  40  Figure 10. Haemoglobin Corrected Diffusing C a p a c i t y / A l v e o l a r Ventilation Ratio Measured at Five Test Points Throughout Ovulatory and A n o v u l a t o r y Menstrual Cycles of 6 Women.  40  Figure 11. Percent Carboxyhaemoglobin Measured at Five Test Points W i t h i n the Menstrual Cycles of 7 Ovulating and 3 N o n - O v u l a t i n g Women. Figure 12. The H u m a n Menstrual Cycle.  41 68  viii  ACKNOWLEDGMENT I w o u l d like to thank Drs. Jerilynn Prior, D o n McKenzie, Raja A b b o u d and A l a n M a r t i n for their advice i n the various stages of putting this thesis together. I w o u l d also like to acknowledge the assistance of Bill Sheel and Drs Jim Potts and Angelo Belcastro. Finally, my deepest gratitude goes to the subjects involved i n this study: those who provided insight with their questions, those w h o maintained enthusiasm for the project even when mine was waning, and those w h o waited expectantly for the appropriate stages of their menstrual cycles, left messages of their progress for me on answer machine and voice mail, and turned up to the laboratory at all hours of the day for testing.  ix  CHAPTER ONE: INTRODUCTION  The movement of metabolic and other gases across the alveolar membrane of the l u n g is a vital physiological process which takes place solely via passive diffusion. The physical properties of the alveolar membrane, the respiratorycardiovascular interface, i n particular its extreme thinness (around 1pm) and vast surface area (approximately 70m ) allow this exchange to take place freely i n a 2  healthy lung. It is doubtful i n fact that lung diffusion limits the transfer of oxygen (O2) from the atmosphere to metabolising tissues i n healthy, untrained individuals, either i n rest or d u r i n g maximal exercise. Despite this, under certain circumstances lung diffusion may limit oxygen delivery, for example i n diseased lungs (Crapo and Forster, 1989), at high altitude (Hastala and Berger, 1996), or i n elite athletes during maximal exercise (Dempsey, 1986). The capacity of the lungs to transfer O 2 from the l u n g alveoli to the red blood cell haemoglobin (Hb) i n the pulmonary capillaries is called pulmonary diffusing capacity ( D L ) . D L is usually measured clinically by determining the rate of disappearance of a k n o w n concentration of carbon monoxide from a single breath of a m i x e d gas: hence the name for the measurement: "diffusing capacity of the lungs for carbon monoxide" ( D L C O ) " - For a substantial explanation of the basis of l u n g diffusion measurement and calculation refer to A p p e n d i x I: L u n g Diffusion: Its Measurement, Components and Determinants (p48).  CYCLIC ALTERATIONS IN DIFFUSING CAPACITY  Because  DLCO  is an important clinical measurement, often measured serially  in one patient to assess changes i n pulmonary function occurring as a result of pulmonary disease, it is important to be aware of other factors that may alter lung diffusion on a day to day basis as well as any cyclic variations i n  1  DLCO  DLCO  has  traditionally been thought to undergo circadian variation, because it has been shown to decrease in individuals of both sexes by 1 to 2 percent per hour from morning to night (Cinkotai and Thompson, 1966). Nonetheless, a cyclic circadian pattern of D L c o variation has not been demonstrated. Moreover, a more recent study (Frey et al., 1987) found no change in D L c o at different times of the day after correcting it for COHb backpressure (an artifact of repeated diffusion testing), and for small circadian changes in haemoglobin concentration ( [Hb] ). There are two ovarian phases of the human menstrual cycle The first, the follicular phase, begins on the first day of menses and lasts until ovulation. The subsequent luteal phase lasts from ovulation until the beginning of the next cycle and is characterised by vastly increased production of ovarian steroid hormones by the corpus luteum, a remnant of the ruptured ovarian follicle. For a complete review of the human menstrual cycle refer to Appendix II: The Human Menstrual Cycle. A change in D L c o with menstrual cycle phase might be expected from the recent results of Sansores et al. (1995). These researchers noted a 9.5% decrease on the third day of menses relative to a premenstrual baseline measurement. In this study, 14 healthy women (including one smoker and eight who were taking oral contraceptives), underwent six single-breath carbon-monoxide lung diffusion ( D L c o ) measurements. A baseline measurement was obtained premenstrually (1 to 7 days before menses), then repeated measurements made on each of the first 4 days of menses, and on one occasion following menses (5 to 10 days after the onset of bleeding). In ten of the 14 women the two components of DLco, pulmonary capillary blood volume (Vc ), and membrane diffusing capacity (DM), were determined via duplicate D L c o measurements at two different O 2 fractions. The authors observed no significant changes in Vc, D M , or [Hb]. The mechanisms of this diffusion reduction are difficult to explain. D L c o is a function of both pulmonary and circulatory parameters and will alter with changes  2  in the diffusing properties of the alveolar membrane (DM), the pulmonary capillary blood volume (Vc), or the reaction kinetics of carbon monoxide (CO) and Hb (expressed as theta or 6). These might alter as a consequence of respiratory alterations in the luteal phase or as a direct hormonal effect.  RESPIRATORY CHANGES WITHIN THE MENSTRUAL C Y C L E The luteal phase of the menstrual cycle is associated with an increased resting and exercise minute ventilation (VE), (Schoene et al., 1981 and Jurkowski et al., 1981), raised resting hypercapnic and hypoxic drives (Schoene et al., 1981; Dombovy et al., 1987) and greater inspiratory muscle endurance (Chen and Tang, 1989) compared to the follicular phase. A potential mechanism for DLco changes is not apparent from established alterations in any of these respiratory parameters. It is possible that mild respiratory alkalosis occurring as a result of increased V E might have a direct effect on 0. Deep breathing also has the potential to elevate DLco by raising the compliance of previously under inflated alveoli and consequently increasing the exchange surface area. Whilst large inspirations prior to a single breath might therefore raise DLco measurement, it seems unlikely that the small increase in tidal volume occurring in the luteal phase of the menstrual cycle would have a physiologically significant or even detectable effect on lung diffusion.  CIRCULATORY CHANGES WITHIN THE MENSTRUAL CYCLE A substantial (17%) elevation of Vc (Seaton, 1972), and small (2.6%) increases in [Hb] (Jurkowski et al., 1981) have been observed in the luteal compared to the follicular phase of the menstrual cycle. The cause of these alterations is unclear but the timing of changes implicates a hormonal mechanism.  3  Progesterone mediated increases i n V c may have occurred during the premenstrual measurement i n the Sansores et al., (1995) study. These researchers regarded hormonal changes as a possible explanation for the  DLCO  reduction they  noted d u r i n g menses. Although alterations i n V c i n the same study were non significant, the sample size for the assessment of V c changes was small and a corresponding alteration i n V c might not have been detected. Progesterone levels increase around 10 to 40 fold i n the luteal phase of a menstrual cycle i n w h i c h normal ovulation occurs. A progesterone mediated change i n  DLCO  would  therefore be expected to occur i n the luteal phase of a normal ovulatory cycle, but not i n an anovulatory cycle. The difference i n diffusion alterations between menstrual cycles of differing ovulatory status has not been investigated. H i g h levels of oestrogen which occur immediately prior to ovulation i n a normal menstrual cycle and following the administration of synthetic oestrogen, prevent the plasma volume drop associated with bed-rest (Fortney et al., 1988). A l t h o u g h unsubstantiated, a potential mechanism for this oestrogenic effect on plasma volume may involve the cardiovascular vasodilator nitric oxide (NO). Expired N O levels were shown i n one study to increase almost threefold from menses until days 13 to 19 of the menstrual cycle and rapidly drop again later i n the cycle (Kharitonov et al., 1994). Although ovulation was not documented i n this study and the timing of reproductive hormone changes therefore u n k n o w n , the profile of N O fluctuations suggest that oestradiol, luteinising hormone or follicle stimulating hormone are the most likely hormones to initiate the response. Mid-cycle increases i n plasma volume w o u l d result i n similar increases i n V c and therefore  DLCO  by definition. Moreover, depending on the ability of the blood  to restore [Hb] to normal levels, plasma volume alterations might affect the measured  DLCO  through changes i n [Hb]. Plasma volume, if it were raised too high,  might also have the potential to induce pulmonary oedema v i a increased pressure and stress failure of pulmonary capillaries thus increasing the thickness of the  4  diffusion membrane and lowering D M (and DLCO)- If raised oestradiol is responsible for menstrual alterations i n DLco, a large increase i n D L c o w o u l d be expected i n the late follicular phase prior to ovulation compared to moderate elevations d u r i n g the luteal phase.  FURTHER EXPLANATIONS OF D L  c o  CHANGES DURING MENSES  Alterations i n the endogenous production of C O w i t h menstrual phase have been reported i n the past but not well studied (Coburn, 1970). A change i n production w o u l d affect the assessment of D L C O via changes i n the diffusion gradient for carbon monoxide. Nonetheless, the expected magnitude of endogenous C O increase during menses as a result of red blood cell breakdown was calculated by Sansores et al. (1995), to result i n only negligible declines i n Dlco, w e l l short of the observed reduction. Direct effects of steroid hormones on 6, the reaction rate of C O and haemoglobin might be considered as a potential mechanism. Reductions i n D L c o occurring around menstruation might also be mediated by vasoconstrictive agents (prostaglandins for example), which might alter the vasomotor tone of pulmonary capillaries thus affecting pulmonary capillary blood volume and hence D L c o Prostaglandin A2 has a k n o w n vasoconstrictor action on pulmonary b l o o d vessels (Patton et al., 1989) and may be involved i n uterine vasoconstriction occurring just before and around the time of flow. Although its exact role i n the h u m a n endometrium i n vivo has not been established, Prostaglandin F2a has also been implicated i n the process of luteolysis or break-down of the corpus luteum occurring at the end of the luteal phase (Adashi et al., 1996).  5  LIMITATIONS OF PREVIOUS W O R K In addition to the small number of subjects who were able to undergo the diffusion partitioning procedure, the Sansores et al. (1995) study is limited i n its ability to either characterise changes in  DLCO  over the menstrual cycle or determine  a likely mechanism for the observed change because it considered only the effect of a single menstrual cycle phase (menses itself) on  DLCO  and because only one cycle for  each subject was investigated. Furthermore, 8 of the 14 subjects were taking oral contraceptives and the post-menses measurement (obtained between D a y 5 and Day 10 of the menstrual cycle) may have corresponded w i t h a point i n time when some of these 8 w o m e n were still taking placebo tablets, while others had resumed hormone tablets for the new cycle.  R E S E A R C H QUESTIONS The main purpose of this study is to describe changes i n D L c o and the two components of  DLCO (VC  and DM), within an ovulatory menstrual cycle of healthy  regularly menstruating women.  DLCO  expressed as a ratio to alveolar ventilation  (VA) is also analysed i n an attempt to reduce error due to varying inspired volumes i n the  DLCO  procedure. Haemoglobin concentration [Hb], may also vary w i t h  menstrual cycle phase and values of  DLCO  , DLco / V A , V c and  DM  are reported  corrected for haemoglobin. Measurement of packed cell volume ( P C V or haematocrit), w h i c h reflects alterations i n plasma volume, and C O H b percent provide further explanatory power for significant alterations i n diffusion. following research questions provide a specific basis for the investigation.  6  The  1. Is there a change in DLco within an ovulatory menstrual cycle in regularly menstruating women? 2. Is there a change in D L C O / V A within an ovulatory menstrual cycle in regularly menstruating women? 3. Is there a change in [Hb] corrected  DLCO  within an ovulatory menstrual cycle in  regularly menstruating women? 4. Is there a change in [Hb] corrected D L C O / V A within an ovulatory menstrual cycle in regularly menstruating women? 5. Is there a change in [Hb] corrected Vc within an ovulatory menstrual cycle in regularly menstruating women? 6. Is there a change in [Hb] corrected D M within an ovulatory menstrual cycle in regularly menstruating women?  If a significant alteration in DLco is found, the following research questions will be investigated as explanatory variables of the above.  7. Is there a change in percent COHb within an ovulatory menstrual cycle in regularly menstruating women?  8. Is there a change in PCV within an ovulatory menstrual cycle in regularly menstruating women?  The characteristics of diffusion changes between ovulatory and anovulatory menstrual cycles of individuals who display both within the study period will be compared.  7  9 -12. Is the change i n D L C O /  DL O/VA, C  [Hb] corrected  DLCO/  [Hb] corrected  DL O/VA C  w i t h menstrual cycle phase different for ovulatory versus anovulatory cycles of the same regularly menstruating woman.  Finally, the reliabilities of the P C V measurement and [Hb] measurement procedures used i n the study were assessed. A further comparison of the validity of the portable [Hb] analyser compared to a hospital spectometry unit was undertaken.  8  CHAPTER TWO: METHODOLOGY  ETHICAL APPROVAL Ethical approval for the study was obtained from the University of British Columbia C l i n i c a l Screening Committee for Research and other Studies Involving H u m a n Subjects. The ethical approval certificate and approved subject consent form is included (Appendix IV).  SUBJECT R E C R U I T M E N T A N D S E L E C T I O N Volunteers were obtained through advertising on bulletin boards around the University of British Columbia (Appendix V) and through w o r d of mouth. Initial contact w i t h people indicating their interest i n participating i n the study was normally made by telephone. Subjects included i n the study had not been taking oral contraceptives for the previous 3 months, had no history of respiratory medical conditions and had not smoked regularly i n the last 2 years since smoking has been shown to be associated w i t h a decline i n  DLCO  (Frans et al., 1975). A subject who is an ex-smoker but has not  smoked regularly for 2 years or more was thought to be less likely to resume smoking d u r i n g the study. A l l subjects had also menstruated regularly for at least 5 years and a normal average cycle length of 21-36 days over the previous 12 months (Barr and Prior, 1994). Subjects selected for the study also reported the recognition of cyclic alterations of physiological or psychological parameters d u r i n g the menstrual cycle from w h i c h they believed they were able to estimate the time of ovulation and predict the occurrence of menstruation. Examples of commonly reported m o l i m i n a l changes are increased viscosity of cervical mucous just prior to  9  ovulation and cramps just prior to menses. Subjects who had no experience with completing The Menstrual Diary (© Prior, 1996; Appendix VI) and who were not totally familiar with the timing of key events in their menstrual cycle, kept a record using this instrument for at least one month before testing. An appointment was then made with subjects who met all selection criteria for the study. At this appointment, subjects read and signed the consent form. The timing of the testing points in the cycle was carefully explained and tentative bookings were made for the next four or five tests.  D O C U M E N T A T I O N OF MENSTRUAL D A T A The Menstrual Cycle Diary (© Prior, 1996) that allows daily recording of menstrual flow, molimina, mood fluctuations, and sub-lingual temperature was used by subjects in the study. The Diary was modified to include a measurement of supine resting heart-rate and hours of sleep (Appendix VI). Subjects were instructed to measure sub-lingual basal body temperature at approximately the same time each morning before rising using a Becton Dickenson digital thermometer. Daily basal body temperature measurements were used to provide an index of ovulation and luteal phase onset using the method of least mean squares (Prior et al., 1990b). Luteal phase onset was defined as the number of days from the first day of the quantitatively determined mean temperature rise to the day before the onset of bleeding inclusive. Daily reports of menstrual cycle experiences (molimina), were used to increase subject interest in the project and their adherence to the study, identify the best time of testing and in the analysis to help confirm possible cycle-related changes in diffusion. Results from the menstrual charts were explained to subjects to help clarify the testing points and an attempt was made to answer any questions they had relating to their own menstrual cycle.  10  TIMING OF TESTING Testing of subjects occurred at five points during the menstrual cycle (figure 1) for three cycles. Day 1 of the cycle was defined as beginning at midnight preceding the day that menstrual bleeding began irrespective of the exact time at which bleeding started. Subjects were instructed to report to the lab at the following five test points outlined below.  Test Point a. Early Menstrual This took place on the first or second day of flow or the last day of the previous cycle. It was preferable that this Test Point occurred while menstrual cramps were were present. This time point was chosen as a time in the cycle where levels of oestrogen and progesterone are low but prostaglandins (as indicated by cramping) are present.  Test Point b. Late Menstrual This testing point took place a few days later between Day 3 to Day 4 of the cycle (inclusive), when menstrual cramps if they had been present had subsided. At this time point levels of oestrogen, progesterone and prostaglandins are minimal.  Test Point c. Early Follicular This took place a few days later again on Day 5 to Day 8 inclusive. At this time point follicular oestrogen levels would be expected to be moderate whilst levels of progesterone and prostaglandins are very low.  Test Point d. Midcycle This testing point took place as close as possible to subject observed and reported thickening of cervical mucous in The Daily Menstrual Diary©. This time point would be expected to coincide with peak levels of oestrogen and low levels of  1 1  progesterone and prostaglandins that occur just prior to ovulation. Daily monitoring of cervical mucous was crucial in determining the correct time for this test point as the timing of ovulation within the cycle is quite variable (Landgren et al., 1980).  Test Point e. Mid Luteal This took place between 3 to 7 days after a subject observed an increase in basal body temperature of around 0.35°C (Prior et al., 1990b) or between Day 17 to Day 22 of the cycle if a basal body temperature increase was not clear. A n increase in temperature signifies the luteal phase onset and was quantitatively determined from basal body temperature measurements using the least mean squares analysis of Prior et al. (1990b). Analysis of the ovulatory pattern of the subject's past cycles was also used to help identify when ovulation was likely to occur and estimate the best time for mid-luteal testing for that individual. A mid-luteal test occurring within an ovulatory cycle would be expected to coincide with moderate levels of oestrogen, high levels of progesterone and low levels of prostaglandins.  12  TEST POINTS  a 8)  .C. LU  z  § LU r—  LU  i  22 24 26 23 DAYS OF MENSTRUAL CYCLE Figure 1: The T i m i n g of Testing at Points i n the Menstrual Cycle  13  Subjects began their first test at any of the five points depending upon the stage of their cycle they were at when they were first available for testing. For the purposes of this study, all test points were given a number and letter code. The number corresponds to the cycle number while the letter corresponds to the testing point (eg: "2a" refers to Cycle Number 2, Test Point a). Apart from a fingerprick blood sample, which was used for [Hb] measurement, and haematocrit measurement if there was enough blood, no blood or urine samples were collected at Test Point a (Early Menstrual). At the completion of the study, the timing of visits was checked retrospectively against the subjects Menstrual Diary to ensure that the timing corresponded with the criteria. While advance warning of Test Point d (Midcycle) was provided by alterations in cervical mucous or other menstrual cycle experiences that the individual associated with ovulation (eg: mid-cycle cramps), it was possible to retrospectively compare the actual laboratory testing day with the quantitatively determined day of luteal phase onset. Ovulation and peak oestrogen would be expected to preceed the basal temperature rise by 2 to 3 days (Prior et al., 1990b) and peak progesterone levels might be expected around midway between the basal body temperature rise and the onset of the following cycle. The criteria for these two test points was clarified based on basal temperature analysis and was as follows.  Test Point d. Mid Cycle This test point should have occurred between 5 days before basal temperature rise and the day before basal temperature rise to correspond with expected peak oestrogen.  14  Test Point e. M i d Luteal This test point should have occurred between the second day of increased body temperature and three days before the onset of menses for the next cycle to correspond w i t h expected peak progesterone.  D O C U M E N T A T I O N O F EXERCISE Subjects used the Daily Exercise Record (Appendix VII) to record the amount of time spent engaging i n either vigorous or strenuous exercise (sufficient to elevate heart-rate to greater than 150 beats per minute) or m i l d or moderate exercise (sufficient to elevate heart-rate to between 90 and 150 beats per minute). The primary mode of exercise each day was also recorded. Daily exercise was measured so that a reterospective assessment of exercise patterns could be used to explain aberrant D L c o measurements and to help explain changes that occurred i n menstrual hormonal status, particularly those changes sufficient to alter D L c o or its components.  TESTING PROTOCOL O n an initial visit to the laboratory subjects completed a questionnaire detailing descriptive data including age, occupation, a brief menstrual, exercise and health history as well as a 24 hour recall of the consumption of foods h i g h i n calcium (Appendix VIII). Basic anthropometric variables (height, body mass and sum of six skinfolds) were measured and the subject's date of birth was recorded. The timing of subsequent visits to the lab was explained to subjects and tentative dates were recorded. Where possible, each subject came into the lab at the same time for each of the 15 laboratory tests i n order to minimise the effect of diurnal variation i n lung diffusion (Cinkotai and Thompson, 1966). Subjects were  15  asked to be as consistent as possible with the timing of meals and caffeinated drinks prior to each session. Because pulmonary diffusing capacity of C O has been found to decline from 1 hour to 24 hours following maximal exercise (Sheel, 1995), subjects were also instructed not to exercise intensively 24 hours prior to their laboratory visit. The time of last vigorous exercise, last meal and last coffee or caffeinated drink was recorded. Upon arrival in the laboratory, the subject's menstrual diary was reviewed, the date (or tentative date) and time of the next testing point were confirmed and any questions they had relating to their menstrual cycle were addressed. At Test Points b, c, d, and e for the first two cycles only blood was drawn and overnight urine collected from 11 subjects willing to undergo this procedure. At each Test Point body weight, forced vital capacity (FVC) and the forced expired volume in 1 second (FEV1) were measured prior to assessment of diffusing capacity of the lung (DLco) and its two components D M and Vc. At Test Point "a." when no venous blood was drawn, [Hb] was measured from a finger capillary sample. At the final testing session, skinfolds were again measured, items of the questionnaire which might have changed over the study period were readministered and selected items from the Canadian Multicentre Osteoporosis Study (CaMOS) were administered via interview (Appendix X). Sheets for raw data entry are included in Appendix IX.  MEASUREMENT OF HEIGHT, WEIGHT A N D ANTHROPOMETRIC VARIABLES  Height The subject stood erect without shoes against a Holtain stadiometer. Subjects were instructed look straight ahead and visual inspection was used to determine if  16  their head was held in the Frankfort Plane: the position where an imaginary line joining the orbitale (most inferior point on the margin of the eye socket) to the tragion (notch superior to the flap of the ear at the superior aspect of the zygomatic bone) is horizontal. Upon a full inspiration, the measurement was taken as the maximum distance from the floor to the most superior point on the skull. Height was measured both on the initial and final testing session and, if the measurement differed, an average was recorded.  Weight Body weight was determined with subjects wearing light clothing, using a Horns beam scale and measured to the nearest 0.05 kg. A n average of the measurement at the initial and final testing session was recorded in the descriptive data. The subjects weight was also measured at all other test sessions to ascertain that no large fluctuations over the duration of the study occurred.  Skinfolds Skinfold measurements were obtained following the procedure described by Ross and Marfell-Jones (1982), with the exception that all measurements, including the abdominal skinfold, were made on the right side of the body. The Triceps, Subscapular, Iliac Crest, Supraspinal, Abdominal, Anterior Thigh and Medial Calf skinfolds were each measured twice. If the two measurements varied more than 10% a third measurement was taken. The average between the two measurements varying less than 10% was recorded. If no measurement differed from the next closest by more than 10% an average of all three was used. Six skinfolds (all the above excluding Iliac Crest) were summed and recorded as the sum of six skinfolds. As skinfold assessment also took place at at the initial and final testing session, the average of the two sum of six skinfolds was reported.  17  MEASUREMENT OF RESTING FORCED VITAL CAPACITY (FVC) A N D FORCED EXPIRED V O L U M E IN ONE SECOND (FEV1) Forced Vital Capacity (FVC) and Forced Expired Volume in Is (FEV1) were measured at each testing session using the spirometry functions of a Collins/DS pulmonary function analyser. The subject was instructed to inspire deeply and then expire forceably: "as hard and fast as possible" until all air was "squeezed out" of their lungs. A flow versus volume loop for a whole breath cycle was completed when the subject reinspired to vital capacity. The largest FVC and FEV1 from at least two maximal tests not varying by more than 10% for either variable was recorded. The forced expired volume from 25% to 75% of the expiration (FEV25-75) and the peak expired flow rate (PEFR) were recorded from the test with the greatest summed FVC and FEV1.  MEASUREMENT OF RESTING DIFFUSING CAPACITY  (DL  C O  )  Resting pulmonary diffusing capacity was determined via the single-breath method first developed by Krogh (1915) and modified by Ogilvie et al. (1957) using the Collins analyser. Single breath methods as opposed to steady state measurements of diffusion are thought to better reflect the alveolar membrane and pulmonary capillary characteristics of the ventilated parts of the lungs (Forster et al., 1986). Moreover, they do not require the measurement of arterial pC02 for their most accurate determination. According to Forster et al. (1986) however, singlebreath techniques may be less sensitive to unevenness of gas distribution and probably to non-uniformity of diffusion throughout the lung. It was thought that in healthy subjects unevenness and non-uniformity are likely to be minimal hence the use of the single-breath measurement in this investigation. The single-breath method used to measure  DLCO  in our laboratory consists of  a rapid inspiration, a 10s breath-hold and a rapid expiration of the test gas containing  18  about 21% oxygen, 10% helium, 0.3% carbon monoxide and the balance nitrogen. A sample of expired gas is collected in a collection bag attached to the Collins analyser. Breath-hold during the test is timed from the beginning of inspiration to the beginning of sample collection as outlined by Ogilvie et al. (1957). While the latest American Thoracic Society (A.T.S.) recomendations (A.T.S., 1995) suggest the use of the Jones and Meade (1961) protocol which adjusts the calculation of breath hold to better reflect the CO concentration profile in the alveolar space, the results of Graham et al., (1981), suggest that in healthy subjects measures of diffusing capacity calculated via the Ogilvie method are likely to be similar to those measured via the Jones and Meade method. Both carbon dioxide ( C O 2 ) and water ( H 2 O ) were removed from the expired gas sample prior to analysis as the gas sample passed through a divided canister containing calcium sulphate and barium hydroxide. Concentrations of CO were measured using an infrared analyser. The concentration of expired O 2 is assumed for the purposes of calculation. In the measurement of D L C O , subjects were encouraged to relax against a closed glottis and remain calm during the breath-hold to avoid performing either a Valsalva or Muller manoeuver that could under- or overestimate DLco respectively. Each diffusion measurement was also examined to ensure that the inspired volume was at least 85% of the FVC, the total time of inspiration was less than two seconds and the breath-hold time was between nine and eleven seconds as the accuracy of DLco measurements are increased in this range (Graham et a l , 1981). The American Thoracic Society (A.T.S., 1995) recommends that 90% FVC be attained for each inspiration. However, subjects in this study were unable to attain this standard consistently and so the criterion was lowered to 85%. On a few occasions for 9 subjects, only one of the two tests reached 85%. If the two tests were above 80% and acceptably close to each other (below), then an average of these two tests was recorded. In line with A.T.S. recommendations (1995), DLco was determined in duplicate and repeated a third time if the initial two measurements varied from each other by more than 10% of their average. The  19  average of tests differing from the mean by 10% or less was reported. A n interval of at least 4 minutes was allowed between tests to ensure elimination of the test gas from the lungs.  C A L C U L A T I O N O F DIFFUSING C A P A C I T Y DLCO  was calculated automatically by the Collins system using equation 1  (below). The rationale for its use is outlined i n A p p e n d i x I (p57).  Equation 1: Calculation of Diffusing Capacity 60 x L n [(FEHe/FECO) x (FiCO/FiHe)] x STPD correction 713 x t  DLCO = V A X  where  DL O C  VA  = diffusing capacity for C O ( m l C O ( S T P D ) / m i n / m m H g ) = alveolar volume ( A T P S i n ml) = V i x FiHe x 1.05 FEHe  F i H e = inspired H e fraction FEHe = expired H e fraction F i C O = inspired C O fraction F E C O = expired C O fraction V i = volume inspired t = breath-hold time (s) ATPS  = ambient body temperature and pressure  STPD  = standard temperature and pressure dry  1.05 = correction factor for 5% carbon dioxide i n expired air removed prior to analysis 713 =  PB  of 760mmHg -  P w a t e r vapour at 37°  20  c of 4 7 m m H g  CORRECTION FOR HAEMOGLOBIN CONCENTRATION The diffusion value was adjusted for haemoglobin concentration using the formula below recommended by the American Thoracic Society in its 1995 recommendations (equation 2). Both adjusted and unadjusted diffusion measurements are reported and analysed.  Equation 2:  where  DLCO  [Hb]adjusted =  DLCO  [Hb]adjusted = diffusing capacity for CO corrected for [Hb] (g/dl)  DL O C  measured (9.38 + [Hb]) / 1.7 [Hb]  (mlCO(STPD)/min/mmHg) DLCO  measured = unadjusted diffusion measurement  QUANTIFICATION OF D M A N D Vc The two components of pulmonary diffusing capacity ( D M and Vc) were measured using the single-breath method of Roughton and Forster (1957) as modified by Ogilvie et al. (1957). Two measurements of resting diffusing capacity were made using two different inspired fractions of 02 (21% and 90%). Subjects breathed for 5 minutes through a low resistance valve (Hans Rudolph, #2700B) attached to a Douglas bag filled with a gas mixture of 90% ± 5% O 2 , and the balance  N 2 . The D L C O 90% 02 test was performed in the same manner as the 21% 02. The reciprocal of  D L C O (1/DLCO)  or total resistance to diffusion, is the sum of two  component resistances ( 1 / D M and l / V c ) . Mean pulmonary capillary oxygen partial pressure required for a calculation of 1/6 was estimated using the alveolar gas equation. The calculation of D M and Vc and the formula and underlying  21  assumptions for the use of the alveolar gas equation are outlined in Appendix I (p57).  THE RELIABILITY OF DIFFUSING CAPACITY A N D ITS COMPONENTS The reliability of the single breath procedure in measuring DLco and its components has been determined in our laboratory in a study of 9 individuals measured twice on separate days (Sheel et al., 1996). Pearson's product-moment correlation coefficients between the two measurements were r = 0.98, r = 0.84 and r = 0.92 for  DLCO/ D M  and Vc respectively.  COLLECTION A N D BATCHING OF BLOOD A N D URINE SAMPLES On the first cycle a total of approximately 16ml of blood was drawn from those subjects willing to undergo this procedure into 2, 6ml SST® vacutubes containing clot activator for later serum production, and into a 5ml vacutube (3 to 4 ml only) containing 15% K3 EDTA for haemoglobin (Hb) and packed cell volume (PCV) analysis. On the second cycle, approximately 20ml of blood was drawn as above with an additional 3 to 4ml drawn into an airtight vacutube containing EDTA for later carboxyhaemoglobin concentration [COHb] analysis. In addition, eight subjects who were willing collected a 45ml urine sample from their first morning excretion of the day that they were scheduled to come into the laboratory. Upon arrival at the lab, the urine sample was transferred to a standard household freezer in the laboratory. At the end of the week urine samples were transferred to -70°C.  22  M E A S U R E M E N T OF P A C K E D CELL V O L U M E A N D H A E M O G L O B I N CONCENTRATION Haemoglobin was measured at each testing session. For sessions when venous blood was not drawn, a pin-prick capillary blood sample was used. W h e n venous samples were already taken, the whole, unclotted blood was slowly d r a w n into a 3 m l syringe from the gently rotated vacutube and a drop from the syringe was allowed to saturate the measuring cuvette. Total [Hb] was then analysed using a H e m o C u e A13 portable (J-hemoglobin photometer i n the laboratory. In addition, [COHb] and total [Hb] were measured for one cycle (four time-points) i n each subject at Vancouver General Hospital using an O S M 3 hemoximeter. Samples were taken from the refrigerated storage in the laboratory and transported on ice-packs for this analysis. Both same day and backdated samples were checked to ascertain that a delay d i d not affect percent C O H b determination. A sample from the remaining whole unclotted blood was injected into two microcapillary tubes and spun i n an International Electric C o m p a n y (IEC) microcapillary centrifuge model M 8 centrifuge for 3 minutes. P C V was determined from the spun samples using a Sherwood Micro-Hematocrit Tube Reader and the average from the two microcapillary tubes recorded.  DATA ANALYSIS In order to answer the research questions, the data analysis was divided into three parts. Firstly to investigate changes i n lung diffusion and diffusion-related variables, a "best" ovulatory cycle was selected for each subject and a difference over the five test points investigated. Secondly, the different profiles of diffusion change between ovulatory and anovulatory cycles in the same subject were investigated by selecting the best ovulatory and best anovulatory cycle.  23  Finally, analyses of reliability of the [Hb] and P C V measurements were made in two ways. Firstly, intraclass correlation coefficients for four repeated measurements (consecutive where possible), from the Test Points at w h i c h blood was d r a w n were determined. Secondly, at a random Test Point for each subject, duplicate measurements were analysed. Thus the stability (or reliability of the measurements over time), controlled for changes i n [Hb] and P C V w i t h menstrual cycle phase, were compared to a same day reliability of the measurement procedures. The validity of the [Hb] measurements made at our laboratory was determined from cross-measurements of venous blood total [Hb] on our laboratory portable H e m o C u e [Hb] analyser and the total [Hb] measurement made on the O S M hemoximeter located at Vancouver General Hospital. One Test i n w h i c h both measurements were made w i l l be selected at random for each subject. A l t h o u g h any spectrometry measurement uses only a very small sample of whole blood (cf. blood chemistry analysis), this O S M hemoximeter was deemed as a suitable criterion for the determination of validity of our equipment. The manufacturer reports the total standard error for measurements of total [Hb] and [HbCO] made on the equipment to be 0.4 g / d l and 0.6% respectively for a 13.8 g / d l sample of oxygenated blood. N o comprehensive study of Hemo-Cue reliability is reported i n the manufacturer's manual although the stated accuracy of the apparatus is ±0.3 g / d l .  C R I T E R I A F O R T H E S E L E C T I O N O F T H E "BEST" C Y C L E F O R A N A L Y S I S One cycle was chosen for each subject for investigation of diffusion alterations. This procedure prevented the overrepresentation of one subject i n the investigation whilst providing a solution to effectively deal w i t h missing test points and anovulatory menstrual cycles. The criteria for selecting the best cycle were as follows. 1. Diffusion tests met the criteria outlined previously (pl8-20).  24  2. Cycle data was complete. N o more than 33% of temperature readings (or any 3 at mid-cycle) were missing or affected by reported illness or taking at the w r o n g time (Prior et al., 1990b). 3. The cycle was ovulatory as confirmed by basal body temperature methods. 4. The menstrual period was between 3 and 6 days i n length and the cycle (defined by the luteal phase cycle point (point "e") was of normal length: between 21 and 36 days (Prior, 1996). The cycle chosen normally began at the same cycle point that the subject originally started participating i n the laboratory sessions, unless it was impossible to select five sequential testing points meeting the criteria above starting at this test point. O v u l a t i o n was assessed using The Menstrual Diary© which begins each cycle w i t h the onset of menstruation. Whether a cycle for analysis was to be classed as ovulatory or not depended on the basal body temperature analysis of Test Points d and e. ( M i d Cycle and Mid-Luteal Test Points) irrespective of where the cycle began. If the cycle began at Test Point e, both the preceding and proceeding cycle needed to be ovulatory.  STATISTICAL ANALYSIS Descriptive statistics of subject characteristics including age, height, weight and l u n g function parameters were calculated using Microsoft™ Excel 5.0. The first analysis of changes i n diffusion parameters d u r i n g a normal ovulatory cycle was made via a One-Way Repeated Measures Analysis of Variance ( R M - A N O V A ) over the five Test Points. Significant trends across the Test Points were also investigated using trend analysis. A l l pair-wise Tukey's post-hoc tests were chosen as the statistic used to investigate the nature of any significant changes i n the overall F statistics that were observed. The second analysis of ovulatory and anovulatory cycles was investigated using a Two-Way (Ovulatory Status (2 levels) x  25  Test Point (5 levels)) R M - A N O V A and testing for a significant interaction between the groups. The significance of the regression equation determined was also tested. Finally for the reliability and validity studies, Intraclass Correlation Coefficients were calculated for the repeated measurements using Statistical Package for the Social Sciences, Version x (SPSSx) for Windows. The Intraclass Correlation Coefficient is a more appropriate measurement of reliability than Pearson's (interclass) Correlation Coefficient (Vincent, 1995, pl78). Refer to A p p e n d i x III for the formula and an explanation. For all analyses a was set at 0.05.  26  CHAPTER THREE: RESULTS  RECRUITMENT OF SUBJECTS Subjects were largely self-selected because the criteria for entry were outlined on the advertisement, however three individuals who were ineligible to participate contacted by telephone. A total of 19 subjects agreed to enter the study. Four withdrew from the study before the first laboratory visit due to other time commitments. Two subjects withdrew after their first laboratory visit, one because of other commitments and one for feelings of discomfort with the laboratory procedure. One subject agreed to participate but was not willing to have blood drawn and no blood was drawn from another subject after medical staff experienced undue difficulty in drawing blood from her. One subject (subject 21) was only able to participate for two rather than three menstrual cycles and. Thus, a total of 13 subjects (11 from whom blood was drawn) are included in the analysis.  GENERAL SUBJECT CHARACTERISTICS All the subjects were associated with the university either as staff (n=3), students (n=9) or in one case, a recently graduated student. They were 168.8±4.2cm in height and weighed 67.1±21.2kg. In accordance with the selection criteria, no subjects smoked regularly, none had a respiratory or endocrine medical condition and none had taken oral contraceptives in the last year. All subjects had a history of regular menstruation and all reported that they were normally able to predict the onset of menses. They reported a cycle length of 29.6±2.0 days and a 4.9±1.3 day duration of menses. Their average age at menarche was 12.5±1.3 years. None of the subjects had been pregnant in the past. Summary data of subject characteristics is included in Appendix XII (p59).  27  SUBJECT ACTIVITY A l l subjects were healthy and active. Three were competitive athletes at the time of the study. Analysis of the open question for subjects to list the vigorous activity they performed regularly revealed that they performed 6.0±3.1 hours per week of vigorous activity ranging from 0 to 10.1 hours per week at the time of the study. A l l had spent more than one hour per week vigorously active i n the past for an average of 9.3+4.2 years. The question from the Canada Multicentre Study of Osteoporosis (CaMOS), w h i c h divides activity into strenuous sport, vigorous w o r k and moderate activity elicited values of 6.5±4.9,1.5±1.3, and 6.1+3.9 hours per week respectively. Hours of sitting per week ranged from 1.5 to 13.5 hours per week (7.2±3.3 hours/week).  L U N G F U N C T I O N CHARACTERISTICS Since all subjects were active and had no respiratory conditions, all l u n g function variables were within the normal range (Table 1). Blood haemoglobin concentration (recorded as an average between the initial and final laboratory reading was 13.1+1.0 m g / d l . The packed cell volume was 39.0±2.9%. Ten of the thirteen subjects experienced difficulty i n consistently attaining an inspired volume of 90% of forced vital capacity (FVC) i n a l l four diffusion tests during each test session. Ten subjects were able to attain 85% i n at least one of the two tests at both O 2 fractions at every session. For the three subjects w h o could not, care was taken i n the selection of the "best" cycle for analysis to include test sessions that met this criterion.  28  TABLE ONE: Lung Function Characteristics of Subjects showing forced vital capacity (FVC), forced expired volume in Is (FEV1), forced expired volume between 25% and 75% of expiratory time (FEV25-75), and peak expiratory flow rate (PEFR)  SUBJECT  FVC (1)  FEV1 (1/s)  FEV 25-75 (1)  PEFR (1/s)  00  4.39  3.67  3.58  9.29  01  4.14  3.40  3.11  7.69  03  3.76  3.43  4.17  7.04  04  5.77  4.91  5.18  9.59  08  3.95  3.25  3.22  6.58  12  4.47  3.92  4.35  9.20  13  3.76  3.46  4.59  7.02  16  4.08  3.74  4.47  6.99  18  3.35  2.72  2.55  5.30  19  3.89  3.50  4.40  9.52  20  3.96  3.50  4.07  8.36  21  4.46  3.85  4.48  9.93  22  4.56  3.82  3.76  7.72  MEAN  4.19  3.63  3.99  8.02  S.D.  0.59  0.49  0.72  1.42  29  ANALYSIS OF CYCLES The 13 subjects completed testing over a total of 38 full menstrual cycles (two for Subject 21 and three for all the rest). The cycle characteristics for each subject are shown i n Table 2. Because subjects began attending the lab at different times throughout their cycle, but kept their Menstrual Diary for the period of time leading up to and following their initial and final laboratory visit, there are more than 3 cycles listed for some subjects. In addition one subject, (Subject 12), was not able to attend laboratory sessions for one month, but kept her Menstrual Diary d u r i n g the intervening time. One subject (Subject 19), missed a menstrual period. This is represented i n the table below as a particularly long cycle. Basal body temperature analysis confirmed that 6 (16%) of the 37 menstrual cycles w i t h sufficient data recorded were anovulatory.  A N A L Y S I S O F T E S T I N G POINTS A timetable which shows the dates of diffusion testing for each subject, and corresponding cycle day for the diffusion measurements is included (Appendix XI). The timetable also shows the cycle length (CL) for the cycle beginning at Point "a" and the day of luteal phase onset as determined by the basal body temperature rise. The Test points chosen for the "best" ovulatory cycle and the anovulatory cycle are also shown i n A p p e n d i x XI on a separate timetable. W h e n the best ovulatory cycles were chosen for each subject, data from four Test Points (in three subjects) were missing because the subject was unable to attend at that time. For the anovulatory cycles recorded by six subjects, there were two missing Test Points. Missing values were replaced i n the statistical analysis w i t h the grand cell mean corrected for the subject mean and for the test point mean. In two cycles chosen as the best ovulatory cycle, the length of menses was more than 6 days (7 days for Subject 13 and 11 days for Subject 19). Subject 04, whose best ovulatory  30  TABLE TWO:  Summary of Menstrual Cycles  showing cycle number i n study, cycle length (days), length of menses (days), and length of the luteal phase* . 1  SUBJECT  CYCLE NO.  CYCLE LENGTH  MENSES LENGTH  L U T E A L L E N G T H (t value)  1 2 3  26 23 30  5 6 5  11 0 9  (2.07)  01  1 2 3  27 26 28  6 5 5  14 11 13  (5.75) (6.57) (11.67)  03  1 2 3  38 27 41 31  6 6 7 6  9 9 0 8 •„  (3.86) (5.26)  1 2 3 4  26 27 28 26  5 5 7 7  6 11 I.D.* ID.  (2.49) (2.29)  08  1 2 3  28 28 34  4 3 5  6 10 9  (2.53) (5.99) (3.78)  12  1 2 3 4 5  29 26 27 33 ID.  4 I.D. I.D. I.D. I.D.  15 0 I.D. I.D. I.D.  (2.14)  13  1 2 3  27 29 25  7 5 7  10 7 0  (3.49) (4.76)  16  1 2 3 4  29 27 28 27  7 5 5 5  14 11 14 12  (2.07) (4.96) (3.44)  18  1 2 3 4  32 32 34 31  7 6 8 5  13 14 0 17  (8.06) (9.30)  19  1 2 3  29 59 I.D.  11 10 I.D.  6 0 I.D.  (3.75)  20  1 2 3 4  38 31 35 ID.  6 6 5 I.D.  14 15 13 I.D.  (7.92) (8.27) (6.31)  21  1 2 3  30 34 33  I.D. 5 4  10 9 I.D.  (6.16) (4.56)  22  1 2 3 4  28 29 27 26  I.D. I.D. 4 4  I.D. 9 14 13  (6.45) (12.25)  00  04  (7.94)  (4.64)  2  *1 Luteal length was measured from the day of onset of basal body temperature rise (inclusive) to the day of onset of menses (exclusive). A luteal length of 0 means that the menstrual cycle was anovulatory. The t value is for the difference in basal temperatures between the follicular and luteal phase (greater than 2.0 is considered acceptable). *2 Insufficient data to determine this value  3 1  cycle was 26 days i n length, had recorded only 15 basal temperatures for that cycle and therefore fell just short of the criteria for sufficient data. Despite there being inadequate recordings, the mean difference between temperatures i n the follicular and luteal phase for that subject was allowable (t = 2.49) and was included as an ovulatory cycle. The anovulatory cycles appeared to be relatively more disturbed i n terms of cycle length and length of bleeding. T w o of the six anovulatory cycles were longer than 36 days and four exhibited menstrual periods greater than 6 days. One anovulatory cycle w i t h only 16 (out of 26) recorded temperatures was also used. The proportion of cycle Testing Days that occurred outside the strict timing criteria previously outlined (pll-13) is shown below (Table 3). The details of this information may be examined by comparing Diffusion Testing Timetable (Appendix XI) w i t h Table 2. A total of 10 tests for ovulatory cycles d i d not occur at the correct time. N i n e were only one day outside the criteria and one (Subject 20, Test Point d) occurred two days late. Three tests originally scheduled as Test Point e met the criteria for Test Points d (2) and a (1) and were thus analysed as such. A l l tests occurred w i t h i n the correct time frame for the anovulatory cycles. Test Points "d" and "e" took place between Day 15 and 19, and between Day 19 and 23 of the menstrual cycle respectively.  B L O O D S A M P L E A N A L Y S I S TESTING DATES T w o timetables showing the date of testing, and corresponding cycle day for collection of blood samples for H b and C O H b assessment are included (Appendix XI). In addition to the smaller number of samples, they differ from the diffusion time-table i n minor details when blood testing took place on the previous or subsequent day.  32  TABLE THREE: An Analysis of Test Points  BEST O V U L A T O R Y CYCLE  a  b  c  d  e  1  1  0  0  2  Timing of Tests Outside Criteria 2  2  0  5  1  13  13  13  13  13  a  b  c  Number of Missing Test Points  Total Test Points  A N O V U L A T O R Y CYCLE  Number of Missing Test Points  d  e  1  0  0  0  1  Timing of Tests Outside Criteria 0  0  0  0  0  Total Test Points  6  6  6  6  6  33  BEST C Y C L E A N A L Y S I S N o significant change across the five test points was noted i n  DLCO  or  DLCO/VA  (Figure 2 and 3). Adjustment for [Fib] alterations made no difference to the  results for  DLQO,  and  DLCO/VA  (Figures 4 and 5), although a significant difference i n  [Hb] between Test Points of an ovulatory cycle was observed (Figure 6). Tukey's post-hoc test for all pairwise comparisons at the a=0.05 significance level showed that only the largest difference from Test Point a to Test Point e was significant. The change i n [Hb] was characterised by an average 5% increase from the early follicular to the m i d luteal phase. N o changes were found i n [Hb] adjusted or unadjusted V c or D M over 5 Test Points. For these parameters however, 16 missing values (25%), were replaced w i t h mean corrected values.  34  Figure 2: Diffusing Capacity Measured at Five Test Points Within Ovulatory Menstrual Cycles of 13 Women. Bars represent standard deviations from the mean. No significant alterations were observed.  5.00 T  4.50  1) X 4.00 E  +  +  i  E E  3.50  +  3.00  +  2.50  +  2.00 <  TEST POINTS  Figure 3: Diffusing Capacity/Alveolar Ventilation Ratio Measured at Five Test Points Within Ovulatory Menstrual Cycles of 13 Women. Bars represent standard deviations from the mean. No significant alterations were observed.  35  Figure 4: Haemoglobin Corrected Diffusing Capacity Measured at Five Test Points Within Ovulatory Menstrual Cycles of 13 Women. Bars represent standard deviations from the mean. No significant alterations were observed.  Figure 5: Haemoglobin Corrected Diffusing Capacity/Alveolar Ventilation Ratio Measured at Five Test Points Within Ovulatory Menstrual Cycles of 13 Women. Bars represent standard deviations from the mean. No significant alterations were observed.  36  15.0  14.0 +  £  12.0  1  11.0 < -  10.0  I  I  a  b  I  I  c  d  1 e  T E S T POINTS  Figure 6: Haemoglobin Changes Measured at Five Test Points Within Ovulatory Menstrual Cycles of 13 Women. Bars represent standard deviations from the mean. Test Point e is significantly different from Test Point a.  37  O V U L A T O R Y VERSUS A N O V U L A T O R Y CYCLES W h e n diffusion measurements of the 6 subjects w h o showed a variation i n menstrual cycle status over the 3 months were analysed, [Hb] adjusted D L c o changes over the 5 test points approached significance (p=0.066). There was also a slight trend towards a difference i n  DLCO/VA  and  DLCO/VA  adjusted for [Hb]. A l l three  measurements tended to fall from the late menstrual measurement to mid-cycle. [Hb] adjusted D L c o tended to undergo steady decline from Test Point a to d and then a small rise at Test Point e. Figures 7 to 10 show trends in the changes of these variables i n ovulatory and anovulatory cycles of subjects who recorded both during the study. There was no change i n non [Hb] adjusted D L c o , nor i n adjusted or unadjusted D M or V c over the 5 test points. N o changes were observed i n any of the diffusion variables measured from the ovulatory to the anovulatory cycle. A d d i t i o n a l l y , there were no significant interactions between menstrual status and Test Point for any of the diffusion variables.  C H A N G E S IN CARBOXYHAEMOGLOBIN WITH MENSTRUAL CYCLE PHASE There was no change i n C O H b percentage measured at 4 Test Points for all 11 subjects. Seven of the subjects exhibited ovulatory cycles for this analysis. Separating out the potential differences i n C O H b changes i n ovulatory subjects from the three subjects w h o d i d not ovulate during that cycle by using a M i x e d Method Repeated Measures A N O V A , revealed no change i n C O H b over the test points and no interaction between menstrual status and Test Point (Figure 11).  38  Figure 7: Diffusing Capacity Measured at Five Test Points Within Ovulatory and Anovulatory Menstrual Cycles of 6 Women. Bars represent standard deviations from the mean. No significant alterations were observed.  Figure 8: Haemoglobin Corrected Diffusing Capacity Measured at Five Test Points Within Ovulatory and Anovulatory Menstrual Cycles of 6 Women. Bars represent standard deviations from the mean. No significant alterations were observed.  39  Figure 9: Diffusing Capacity/Alveolar Ventilation Ratio Measured at Five Test Points Within Ovulatory and Anovulatory Menstrual Cycles of 6 Women. Bars represent standard deviations from the mean. No significant alterations were observed. 5.00 x  |  4.50  i  4.00  f -OVULATORY - - ANOVULATORY  < ^  3.50 +  3.00 +  I  2.50 +  2.00 TEST POINT  Figure 10: Haemoglobin Corrected Diffusing Capacity/Alveolar Ventilation Ratio Measured at Five Test Points Within Ovulatory and Anovulatory Menstrual Cycles of 6 Women. Bars represent standard deviations from the mean. No significant alterations were observed.  40  Figure 11: Percent Carboxyhaemoglobin Measured at Five Test Points Within the Menstrual Cycles of 7 Ovulating and 3 Non-Ovulating Women. Bars represent standard deviations from the mean. No significant changes between test points, differences between the two groups or interactions found.  41  C H A N G E S IN PCV WITH MENSTRUAL PHASE N o alteration i n P C V over 4 Test Points was observed w h e n measurements from all 11 subjects were analysed. Data from nine subjects w h o exhibited an ovulatory cycle during the phase of P C V measurement were also analysed and no changes over the menstrual cycle were found.  RELIABILITY OF THE P A C K E D C E L L V O L U M E M E A S U R E M E N T S The test-retest reliability of two measurements of P C V , d r a w n from the same whole blood sample and spun at the same time i n our laboratory was very high and significant (r intraclass = 0.99; r = 0.98; p<0.01). Over four tests measured at different 2  phases i n the menstrual cycle and after the separation of any consistent change across the Test Points, the reliability of P C V measurement i n one subject was m u c h lower and non-significant (r intraclass = 0.25; r = 0.06). 2  RELIABILITY A N D VALIDITY OF THE H A E M O G L O B I N  CONCENTRATION  MEASUREMENTS Test retest reliability for the measurement of [Hb] i n our laboratory was also very high (r = 0.98; r = 0.96; p<0.01). The reliability of this measurement procedure 2  undertaken at four points i n the menstrual cycle after removing consistent changes w i t h menstrual phase was still high (r = 0.96; r = 0.92; p<0.01). 2  The Intraclass Correlation Coefficient for the relationship between our measurement procedure and hospital based spectroscopy was (r = 0.89; r = 0.85; 2  p<0.01). There was a significant trial effect (p<0.01) and our laboratory procedure overestimated [Hb] compared to the O S M unit by an average of 0.63 g / 1 0 0 m l or 5%.  42  CHAPTER FOUR: DISCUSSION  RECRUITMENT A N D CYCLE CHARACTERISTICS OF SUBJECTS Of the subjects who were initially recruited for the study, only 68% completed the entire project. As only two of subjects who later withdrew attended the first laboratory visit, there is little information available to assess a difference in characteristics between those who completed the study and those who did not. The subjects who withdrew were of a similar educational and occupational background to the subjects who remained in the study and no obvious selection bias is apparent. Moreover, since the initial sample was non-random, the consequences of a selection bias as a result of subject withdrawal is likely to be relatively unimportant. Only 72% (81 out of 113) carefully selected women met the further screening criterion of two consecutive ovulatory cycles in the study of Prior et al. (1990a). Moreover, of the 81 women who remained in their 12 month study, only 66 (81%) completed it and only 13 (20%) of these women had consistently ovulatory menstrual cycles throughout the year. Screening for the current study was not as rigid as for the Prior et al. study. In the current study, subjects were not required to demonstrate two consecutive ovulatory cycles before entering the study. Subjects in the current study did however report a good knowledge of the timing and nature of events within their cycle and it was assumed that this was likely to be related to hormonal consistency. It was anticipated that at least 35% of subjects recruited for the study would either withdraw or not exhibit consistent ovulatory cycles for the three cycles. In fact, 32% of recruited subjects withdrew from the study and of the 13 who remained only six ovulated consistently for the time they were in the study. Two subjects recorded insufficient data to determine if they ovulated consistently or not.  43  DIFFUSION C H A N G E S WITHIN THE "BEST" OVULATORY MENSTRUAL CYCLE Neither  DLCO  nor its components was found to alter during ovulatory  menstrual cycles in this group of women. While a change in [Hb] was observed, correction of the diffusion measurements for [Hb] did not affect the result. Differences in V A from test to test or over the three month period could not explain the lack of significance in D L c o either because there was no change in D L c o expressed as a ratio of V A . In addition, V A was very consistent. The mean intrasubject standard deviation of V A was 0.23 litres, less than 4% of the mean. For a One Way Repeated Measures A N O V A , power is correctly determined using the noncentrality parameter lambda (k) (Winer et al., 1991). X is essentially an effect size measure, analogous to Cohen's "d", whose variance term in the denominator is equivalent to the mean squared error table.  In turn,  MSerror  (MSerror)  from the A N O V A  can be estimated from the mean intratrial variance and the  mean correlation coefficient of all pairwise trials as shown in Equation 3.  Equation 3:  X= n X  (Lti - It)  C e 2  where  2  (1 - p)  A, (lambda)  = the noncentrality coefficient  n  = number of subjects  X(Lti-Ll)2  = the sum of the squared differences between each trial mean and the overall mean = the common within cell variance (estimated from the average variance among scores of the dependent variable within a group)  p (rho)  = the average of the correlation coefficients between all pairs of trials  44  In the current study, the power to detect a 10% difference in  DLCO  from the  highest to the lowest Test Point, assuming an even spread of DLco at other Test Points, was greater than 99.9%. The power to detect the same magnitude difference was even greater for [Hb] adjusted  DLCO  and [Hb] adjusted  DLCO/VA,  but was lower  (75.5%) for [Hb] adjusted Vc. The power to detect a 5% change in DLQO, [Hb] adjusted DLCO  and [Hb] adjusted  DLCO/VA  was still high: 59%, 74% and 70% respectively.  More than the required number of subjects to detect with confidence the 9.5% difference in  DLCO  noted by Sansores et al (1995) were recruited for this study in  order to ensure enough consistently ovulating women.  H A E M O G L O B I N CONCENTRATION CHANGES WITHIN THE "BEST" OVULATORY MENSTRUAL CYCLE The luteal phase increase in [Hb] is in agreement with the findings of Jurkowski et al. (1981). While these researchers noted a 3% increase in the luteal phase of healthy subjects in whom ovulation was hormonally confirmed, in the current study a 5% alteration was observed. Past studies of [Hb] changes throughout the menstrual cycle have produced spurious results. While Vellar (1994) and Dombovy et al. (1987) noted luteal phase decreases in [Hb] neither of these studies confirmed the timing or existence of a luteal phase using basal body temperature or hormonal analysis. Lebrun et al. (1993), in a study quantitatively confirming ovulation, observed no change in [Hb] from the follicular to luteal phase. In the past, cyclic changes in [Hb] have been thought to occur as a result of increases in plasma volume and lowered PCV.  Both progesterone and oestradiol may mediate  fluid retention probably through stimulation of the renin-angiotensin system and increased production or activity of anti-diuretic hormone. Exogenous oestrogen also prevents plasma volume reduction with bed rest and the positive influence of endogenous oestradiol on plasma volume appears to be greater around the time of  45  ovulation when progesterone levels are low (Fortney et al., 1988). Luteal increases in plasma volume and concomitant reduction in PCV would result in a lowered [Hb] and cannot explain the luteal increases in [Hb] noted in this study. Further evidence that [Hb] changes occur independently of hormonally mediated haemostatic alterations in this study are the lack of change noted in PCV and the lack of difference in [Hb] changes over the Test Points between ovulatory and anovulatory cycles. The [Hb] rise from menses to luteal phase in this study may simply reflect the gradual restoration of red blood cells lost during menstruation. A 16% increase in caloric consumption in the luteal phase compared to the follicular phase has been previously noted (Barr et al., 1995). Although this study did not analyse differences in micronutrient intake, it is reasonable to suggest that iron intake is also higher in this phase and may accelerate this restoration process. Menstrual phase alterations in iron intake would be likely to have an important effect in the [Hb] of individuals with a suboptimal iron status.  DIFFUSION CHANGES IN OVULATORY VERSUS A N O V U L A T O R Y CYCLES While no alterations in any of the diffusion measurements were noted with this smaller group of subjects, a non-significant tendency for [Hb] corrected D L C O to change existed, as did weak trends in both corrected and uncorrected D L C O / V A . At Test Point c in the ovulatory cycle, one subject (00), recorded a value much higher than normal. Removal of this subject from the analysis and correction of this data point to an average value, resulted in a significant change in [Hb] corrected DLco with menstrual cycle phase. However, no reason for this discrepancy could be determined from the diffusion test results. The subject had not exercised intensely in the previous day and had not consumed a large meal or recently drunk coffee. The test was completed at the same time of day as usual. The elevated record could  46  be due in part to the unusually high V A but correction for V A fluctuations in all the subjects did not produce significant results. The pattern of these trends in D L fluctuation was towards a small (5%) increase from early in menses until later in menses followed by a drop later in the follicular and luteal phases. Seaton (1972) noted a substantial decline (14.8%) in Vc from 7 to 10 days following menstruation compared to 2 to 4 days prior to menstruation. Although there was on average a corresponding 4.4% drop in  DLCO/  this difference was not statistically significant. The author suggests that progesterone mediated premenstrual distension of the pulmonary capillary bed, possibly secondary to respiratory arteriole or venous distension is the most likely explanation of his findings. The results of this study which carefully documented rises in basal body temperature, a secondary effect of progesterone, and found no corresponding change in  DLCO  do not support this rationalisation. A n alternative  interpretation of Seaton's results would be that peaking oestradiol (around day 7 to 10 of the menstrual cycle) caused the alteration in Vc. This would be in agreement with the trend of a midcycle rise in  DLCO  observed in the current study, although  subjects in this study tended to ovulate much later than Day 7 to 10. Sansores et al. (1995), in contrast, noted a decline in diffusion during menses. These researchers investigated  DLCO  change during menses itself and recorded daily  changes during this time of the cycle thus investigating more closely a specific phase of the menstrual cycle, menses itself. Mean intra-individual coefficients of variation for the parameter  DLCO  in the Sansores et al. (1995) study was lower than  in the current study: 4.8% as opposed to 8.8%. This is not surprising given that in the Sansores et al. (1995) study, the trials were conducted over a few days compared to the current study which analysed trials conducted over a monthly cycle. The current study has carefully controlled for the effects of hormonal changes by selecting testing points to coincide with peak oestradiol and progesterone levels and assessing menstrual cycles for the occurrence of ovulation. Accordingly, the  47  lack of significant change in diffusion would appear to make the possibility of hormonally induced diffusion changes unlikely.  FURTHER MECHANISMS OF DIFFUSION C H A N G E Percentage COHb changes with menstrual cycle phase might be at least partially responsible for the DLco changes during menstruation observed by Sansores et al. (1995). These researchers reject COHb changes as an explanation of their observed DLco decline as the magnitude of COHb increase required to lower DLco by 10% was too great to be accounted for by red cell break-down. Coburn et al. (1970) report a doubling of C O production in the luteal phase of the menstrual cycle. Excess endogenous CO production may be a result of the breakdown of haemoglobin from senescent blood cells but may also reflect increased degradation of other haem compounds particularly of hemoproteins in the liver and other organs (Coburn, 1970). This excess production is unlikely to be reflected to any great extent in the blood as it is cleared from the body via the lungs and is masked by uptake from the environment. No change in whole blood COHb percent with menstrual cycle phase was noted in the current study. Repeated measurements of DLco will raise blood [COHb] and thus lower successive determination of DLco. We have found that percentage COHb increases around 1.5% following a partitioned diffusion test in our laboratory. Following the test or repeated tests it declines at a rate of approximately 1% per hour (Stewart et al., 1997). Complete clearance of raised [COHb] may therefore take some hours and depends upon alveolar ventilation, pulmonary capillary P O 2 and upon DLco itself. It seems very unlikely that any of the decrease in DLco noted by Sansores et al. (1995) was explained by [COHb] accumulation with repeated testing, given that tests were completed 24 hours apart.  48  Prior to this investigation we felt that levels of prostaglandins associated w i t h menstrual cycle cramps might have been responsible for the observed changes i n DLCO/  perhaps acting via pulmonary arteriole vasoconstriction or alveolar  membrane fluid retention. The incidence of menstrual cramps although it was assessed i n The Menstrual Diary© was very unpredictable i n both its occurrence for a particular cycle and i n its timing. The few testing points that coincided w i t h subjects reported cramping seemed to elicit  DLCO  measurements w i t h i n the subject's  normal range. Moreover, neither changes i n V c nor D M w i t h menstrual cycle phase (including the early and late menses testing points) were significant and this does not provide support for the prostaglandin initiated mechanisms described. Hormone independent alterations i n N O are a possible alternative explanation for  DLCO  changes during menses. If this were the case however, it is  hard to explain w h y hormone dependent changes i n N O production (Kharitonov et al., 1994) were not observed.  RELIABILITY A N D VALIDITY OF P A C K E D C E L L V O L U M E A N D H A E M O G L O B I N CONCENTRATION  MEASUREMENTS  Both P C V and [Hb] measurements obtained i n our laboratory were highly reproducible. Approximately 98% and 96% of the variation i n one measurement of P C V and [Hb] respectively could be explained by the variation i n a measurement just preceding it. In addition, 92% of the variation i n [Hb] was consistent over time after the effect of systematic alterations over the menstrual cycle were removed. Over time P C V determination was much less reliable reflecting changes i n P C V over time that were not consistent between subjects and were not related to menstrual cycle changes since this was controlled. O n l y 6% of the variation i n a P C V measurement was retained i n repeated test over a month. It seems therefore  49  that PCV undergoes fluctuation with time that is not systematic between individuals. The portable Hemocue analyser used in our lab for [Hb] determination appears to overestimate [Hb] compared to hospital based spectometry. The overestimation for a subject with a [Hb] of approximately 12 g/dl would be in the order of about 0.6 g/dl. This is outside the stated accuracy of both pieces of equipment (0.4 and 0.3 g/dl for the OSM and Hemocue respectively). In assessing the validity of the Hemocue, there is obviously a question of faith regarding the authenticity of manufacturer accuracy reports. The OSM spectometer in Vancouver Hospital outlined comprehensive laboratory trials in the determination of its reliability and accuracy compared to blood chemistry analysis. It therefore seems to be a more trustworthy standard and it could be assumed that the Hemocue in our laboratory did indeed consistently overestimate [Hb] by at least 0.2 g/dl.  50  CHAPTER FOUR:  CONCLUSION  This study has resulted in the following findings.  1. There is no change in D L C O or D L C O / V A either corrected or uncorrected for [Hb] with menstrual cycle phase of an ovulatory cycle in regularly menstruating healthy women. 2. There is no change in [Hb] corrected Vc or D M ; or in [COHb] or PCV with menstrual cycle phase of an ovulatory cycle in regularly menstruating healthy women. 3. There is a small (5%) increase in [Hb] from the early follicular to mid luteal phase of an ovulatory menstrual cycle in regularly menstruating healthy women. 4. There is no difference in menstrual cycle related  DLCO  or D L C O / V A changes  between ovulatory and anovulatory cycles in the same subject. 5. There is no difference in menstrual cycle related [COHb] changes between ovulatory and anovulatory cycles in different subjects.  Given that the timing of diffusion testing was designed to maximise reproductive hormone changes and that no difference in diffusing capacity exists between ovulatory and anovulatory cycles, a hormonally mediated alteration in D L over the menstrual cycle is unlikely. The avoidance of female subjects in studies related to pulmonary diffusion because of supposed phase related changes appears to be unfounded.  5 1  References Adashi, E.Y., Rock, J A . and Rosenwaks, Z. Reproductive Endocrinology, and Technology. Raven Press Ltd, New York, 1996.  Surgery,  American Thoracic Society. Single breath carbon monoxide diffusing capacity (transfer factor). Recommendations for a standard technique - 1995 update. American Journal or Respiration and Critical Care Medicine 152: 2185-2198, 1995. Barr, S.I., Janelle, K.C. and Prior, J.C. Energy intakes are higher during the luteal phase of ovulatory menstrual cycles. American Journal of Clinical Nutrition 61(1): 39-43,1995. 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Dombovy, M.L., Bonekat, H.W., Williams, T.J. and Staats, B.A. Exercise performance and ventilatory response in the menstrual cycle. Medicine and Science in Sports and Exercise 19(2); 111-117, 1987. Forster, R.E., Dubois, A.B., Briscoe, W.A., and Fisher, A.B. In: The Lung: Physiological Basis of Pulmonary Function Tests. Year Book Medical: Chicago, 3rd edition, 1986. Fortney, S.M., Beckett, W.S., Carpenter, A.J., Davis, J., Drew, H . , LaFrance, N.D., Rock, J.A., Tandersley, C.G. and Vromen, N.B. Changes in plasma volume during bed rest: effects of menstrual cycle and estrogen administration. Journal of Applied Physiology 65(2): 525-533, 1988.  52  Frans, A., Stanescu, D.C., Veriter, C. Smoking and pulmonary diffusing capacity. Scandinavian Journal of Respiratory Disease 56; 165, 1975. Frey, T.M., Crapo, R.O., Jensen, R.L. and Elliott, C.G. Diurnal variation of the diffusing capacity of the lung: Is it real?American Reviews in Respiratory Diseases 136; 1381,1987. Graham, B.L., Mink, J.T. and Cotton, D.J. Improved accuracy and precision of singlebreath C O diffusing capacity measurements. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology 51(5); 1306-1313, 1981. Guyton, A . C Textbook of Medical Physiology 9th Edition. J.E. (eds). W.B. Saunders Company, 1996  Guyton, A . C . and Hall,  Hastala, M.P. and Berger, A.J. Physiology of Respiration. 1996.  Oxford University Press,  Jones, F.S. and Meade, F.A. A theoretical and experimental analysis of anomalies in the estimation of pulmonary diffusing capacity by the single breath method. Quarterly Journal of Experimental Physiology 46; 131-43, 1961. Jurkowski, J.E.H., Jones, N.L., Toews, C.J., and Sutton, J.R. Effects of menstrual cycle on blood lactate, 02 delivery and performance during exercise. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology 51(6); 1493-1499, 1981. Kharitonov, S.A., Logan-Sinclair, R.B., Busset, C M . and Shinebourne, E.A. Peak expiratory nitric oxide differences in men and women: relation to the menstrual cycle. British Heart Journal 72; 243-245, 1994. Krogh, M . The diffusion of gases through the lungs of man. Journal of Physiology 49(4); 271-300,1915. Landgren, B.M., Unden, A.L. and Diczfalusy, E. Hormonal profile of the cycle of 68 normally menstruating women. Acta Endocrinologica 94: 89-98, 1980. Lebrun, C M . , McKenzie, D . C , Prior, J.C and Taunton, J.E. Effects of menstrual cycle phase on athletic performance. Medicine and Science in Sports and Exercise 27(3): 437-444,1995. Manier, G., Moinard, J. and Stoicheff, H . Pulmonary diffusing capacity after maximal exercise. Journal of Applied Physiology 75(6); 2580-2585, 1991. Miles, D.S., Christopher, C.E., Doerr, E., Schonfeld, S.A., Sinks, D.E. and Gotshall, R.W. Changes in pulmonary diffusing capacity and closing volume after running a marathon. Respiratory Physiology 52; 349-359, 1983.  53  Ogilvie, C M . , Forster, R.E., Blake, W.S. and Morton, J. A standardized breath holding technique for the clinical measurement of the diffusing capacity of the lung for carbon monoxide. Journal of Clinical Investigation 36; 1-17, 1957. Prior, J.C, Vigna, Y.M., Schechter, M.T. and Burgess, A.E. Spinal bone loss and ovulatory disturbances. New England Journal of Medicine 323; 1221-1227, 1990a. Prior, J.C, Vigna, Y.M., Schulzer, M., Hall, J.E. and Bonen, A. Determination of luteal phase length by quantitative basal temperature methods: validation against the midcycle L H peak. Clinical and Investigative Medicine 13(3); 123-131, 1990b. Ross, W.D. and Marfell-Jones, M.J. Kinanthropometry In McDougall, J.D., Wenger, H.A. and Green, H.J. (eds). Physiological Testing of the High Performance Athlete 2nd Edition.. Human Kinetics Books, 1982. Roughton, F.J.W. and Forster, R.E. Relative importance of diffusion and chemical reaction rates in determining rate of exchange of gases in the human lung, with special reference to true diffusing capacity of pulmonary membrane and volume of blood in the lung capillaries. Journal of Applied Physiology 11; 290, 1957. Sansores, R.H., Abboud, R.T., Kennell, C. and Haynes, N . The effect of menstruation on the pulmonary carbon monoxide diffusing capacity. American Journal of Respiratory and Critical Care Medicine 152; 381-384. Schoene, R.B., Robertson, T., Pierson, D.J. and Peterson, A.P. Respiratory drives and exercise in menstrual cycles of athletic and nonathletic women. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology 50: 13001305,1981. Scoggin, C.H., Doekel, R.D., Kryger, M.H., Zwillich, C.W. and Weil, J.V. Familial aspects of decreased hypoxic drive in endurance athletes. Journal of Applied Physiology: Respiratory, Environmental and Exercise Physiology 44; 464-468, 1978. Seaton, A. Pulmonary capillary blood volume in women: normal values and the effect of oral contraceptives. Thorax 27, 75-79, 1972. Sheel, A.W. The time-course of pulmonary diffusion capacity changes following maximal exercise. Unpublished Master's Thesis, University of British Columbia, 1995. Sheel, A.W., Potts, J., Lama, I., Coutts, K. and McKenzie, D . C Reliability of measurement of diffusion capacity of the pulmonary membrane and pulmonary capillary blood volume. The Physiologist 39(5): A-47, abstract 21:3, 1996 Stewart, I.B., Bacon, C.J., McKenzie, D.C. Carboxyhaemoglobin accumulation and clearance following repeated pulmonary diffusion testing. Unpublished data.  54  Turcotte, R.A., Perrault, H . , Marcotte, J.E. and Beland, M . A test for the measurement of pulmonary diffusion capacity during high-intensity exercise. Journal of Sport Sciences 10; 229-235, 1991. Vellar, O.D. Changes in hemoglobin concentration and hematocrit during the menstrual cycle. Acta Obstetrica Gynecologica Scandivnavica 53: 243-246, 1974. Vincent, W.J. Statistics in Kinesiology. Human Kinetics, 1995 Winer, B.J., Brown, D.R. and Michels, K.M. Statistical Principles in Experimental Design. 3rd Edition. McGraw-Hill Inc, 1991.  55  Abbreviations CO  Carbon Monoxide  C O H b Carboxyhaemoglobin [COHb] Carboxyhaemoglobin Concentration DLco Diffusing Capacity of Carbon Monoxide Units: m l C O (STPD) per m m H g Partial Pressure Change DM  Diffusing Capacity of the Alveolar Membrane  Hb  Haemoglobin  [Hb]  Haemoglobin  NO  N i t r i c Oxide  O2  Molecular Oxygen  PACO  Partial Pressure of Alveolar Carbon Monoxide  Concentration  P c C O Partial Pressure of Pulmonary Capillary Carbon Monoxide p C 0 2 Partial Pressure of Carbon Dioxide PCV p02  Packed C e l l V o l u m e (haematocrit) Partial Pressure of Molecular Oxygen  VA  Alveolar Volume Units: m l (BTPS)  Vc  Pulmonary Capillary Blood V o l u m e  VCO  Rate of Carbon Monoxide Production  V C 0 2 Rate of Carbon Dioxide Production V02  Rate of Oxygen Production  56  Appendix I: Lung Diffusion: Its Measurement, Components and Determinants.  DIFFUSING CAPACITY OF THE L U N G Diffusing capacity of the lung entails the transfer of O 2 from the atmospheric side of the alveolar epithelium to its binding with erythrocytic haemoglobin. While both steady-state and a number of variations of single-breath techniques are available for the measurement of DLco, lung diffusion is most commonly assessed clinically using a single-breath method originally developed by Krogh (1915). This method quantifies the rate of disappearance of C O from the alveolar space within the lung following the inspiration and breath hold (usually 10s) of a known concentration of CO and assumes that the diffusion gradient for C O across the exchange surface does not limit its transport. This assumption is valid because carbon monoxide has a very high affinity to haemoglobin and at low concentrations is effectively removed from the blood plasma upon its diffusion across the membrane. This ensures that a large C O concentration driving force is maintained between the epithelial layer of the alveolar membrane (on the atmospheric side) and the pulmonary capillary blood plasma even when pulmonary capillary blood perfusion is relatively low. In general,  DLCO  is the volume of carbon monoxide diffusing across the  alveolar membrane per mmHg partial pressure change from mean alveolar air to mixed pulmonary capillary blood (equation 1).  57  Equation 1: Basic Equation for Diffusing Capacity DLco =  VCO PACO-PCCO  where  and  DLco  =  diffusing capacity of CO  VCO  =  volume (ml) of CO transferred per minute  PACO  - PcCO =  difference between mean alveolar and capillary CO partial pressure  In the single breath procedure, the lungs may be considered as a closed bag (of volume V A : the alveolar volume) from which CO is removed at an exponential rate proportional to its concentration gradient. Under normal circumstances, the pulmonary capillary CO partial pressure (PcCO) is negligible and ignored.  DLco  at  breath-hold time (t) is a function of the initial and final alveolar fraction of CO and the alveolar volume ( V A ) (equation 2).  58  Equation 2: The K r o g h Equation for Single Breath Diffusing Capacity 60 x L n 713 x t  DLCO = V A X  where  DL O C  [FACOo/FACOt]  x STPD correction  ( m l C O / m i n / m m H g ) STPD  V A = alveolar volume (ATPS i n ml) t = breath-hold time (60 i n the numerator converts seconds to minutes) 713 constant reflecting C O transfer L n = natural logarithm FACOO  = initial alveolar C O concentration  FACOC  = alveolar C O concentration at the end of breath-hold  STPD = standard temperature and pressure dry 1.05 = correction factor for 5% carbon dioxide i n expired air removed prior to analysis 713 = the correction factor for conversion from concentration gradient to partial pressure difference = P B Of  760mmHg -  P w a t e r vapour at 37°  c of 4 7 m m H g  In the single breath procedure, an inert gas that w i l l not diffuse across the alveolar membrane (usually helium (He)) is present i n the inspired mixture and has two purposes: to assess the initial alveolar C O fraction from the inspired fraction; and to determine the V A via its dilution i n the total l u n g volume. Because helium does not diffuse to any great extent, the ratio of inspired H e fraction to alveolar H e fraction (assumed to be equivalent to the expired H e ~ fraction), w i l l equal the ratio of inspired C O fraction to alveolar C O fraction after inspiration but before any diffusion has occurred. Thus, the initial fraction of C O  59  (FACOO)  may be determined from the change in helium fraction from inspired to  alveolar air multiplied by the inspired C O fraction (equation 3).  Equation 3: Calculation of the Initial Alveolar CO Fraction. FACOO  where  = FiCO x FEHe FlHe  FiCO = inspired CO fraction FEHe = expired helium fraction FlHe = inspired helium fraction  If both sides of the equation are divided by the expired CO fraction (assumed equivalent to the alveolar CO concentration at the end of breath-hold time FACOO,  (FECO =  the following equation is obtained (equation 4).  Equation 4: Calculation of the Initial to Final Alveolar CO Fraction Ratio. FACOO  = FiCO x FEHe FlHe  FACOt  FECO  =  where  FECO  FEHe x F I C O F E C O x FlHe  = expired CO fraction  As long as the change in the FCO:FHe ratio from inspired to expired gas remains proportional to changes in either CO or He fraction, the relationship is independent of the actual quantites of inspired CO or He. It is convenient to assume  60  that FiCO is equal to FiHe cancelling these variables out of the equation. The ratio [FACOO/FACOI]  may thus be regarded as equal to [(FEHe/FECO) x (FiCO/FiHe)] and is  used in the calculation of  DLCO  in this study (Equation 2, p20).  V A may also be determined via He dilution in the total lung volume (which includes the inspired volume and the residual volume) with a correction made for the dead-space of the diffusion instrument and for anatomical dead-space (equation 5).  Equation 5: Calculation of the Alveolar Volume. V A = (Vi-VD) x FiHe x 1.05 FEHe  where  V A = alveolar volume Vi = inspired volume V D = dead space (anatomical and instrument) FiHe = inspired He fraction FEHe = expired CO fraction 1.05 = dilution factor for the C02 fraction which is normally chemically removed from the sample  DLCO  a s  a  ratio of V A may be reported to correct for variations in inspired  ventilation and lung size (equation 6).  Equation 6: Calculation of Diffusing Capacity to Alveolar Volume Ratio  DL O/VA = DL O/VA C  where  C  (1 BTPS)  BTPS = Body Temperature and Pressure Saturated 6 1  COMPONENTS OF LUNG DIFFUSION  Because diffusing capacity of the lung may be considered as a conductance of the lung for CO, its inverse, the total resistance of the lung (1/DLco)/ can be partitioned into two component resistances as shown (equation 7).  Equation 7: Partitioning of Diffusing Capacity into its Membrane Component and Pulmonary Capillary Volume Component  + DLco  where  and  DM  6X VC  DLco = diffusing capacity of the lung for carbon monoxide DM  = diffusion across the alveolar membrane  0  = the reaction rate of carbon monoxide with haemoglobin  Vc  = volume of the pulmonary capillaries  In essence, l/0Vc may be considered as the component of resistance that varies with changes in PA02 and 1 / D M as all other resistance (Crapo and Forster, 1989). These authors describe a useful anatomical model for the partitioning which though not strictly correct is helpful in understanding the factors affecting diffusion. Using this model, the first resistance (1/DM), can be thought of as the resistance due to the movement of CO across the alveolar membrane to the surface of a red blood cell. This movement requires the crossing of 3 cell layers (the alveolar epithelium, the alveolar basement membrane and the capillary endothelium), the interstitial fluid within the alveolar membrane and a layer of blood plasma. Aveolar membrane diffusion ( D M ) therefore depends on the thickness and composition of 62  the involved pathways, which may be altered i n disease (as for example i n pulmonary fibrosis); and the surface areas of air-tissue and tissue-blood interfaces, w h i c h may in turn be affected by changes in the pulmonary capillary blood supply or alveolar ventillation. D M may also be decreased by changes i n the back pressure of C O caused by C O bound haemoglobin already circulating i n pulmonary arterial blood. The second resistance (1 / 9 V c ) , is a product of two resistances: one due to the reaction of C O w i t h the red blood cell haemoglobin (1/0), and the other to the pulmonary capillary blood volume ( 1 / V c ) . The combined resistance, depends upon the surface area of the erythrocytes, diffusion across the erythrocyte membrane and w i t h i n the blood cell as well as the chemical reaction of C O w i t h haemoglobin. The chemical reaction term (0) refers to a predetermined rate that 1ml of normal [Hb] can pick up C O per I m m H g concentration gradient. Consequently, changes i n the concentration of erythrocytic haemoglobin and factors which affect the saturation kinetics of the reaction (namely the partial pressures of 02 and C O 2 (p02 and pC02), p H , and temperature) all affect the combined resistance. Because 0 varies i n a standard way w i t h alterations i n p02, both 1 / V c and 1 / D M may be calculated from the slope and y-intercept respectively of a linear line of 1 / D L c o plotted against 1/0. Specifically, the slope of the line estimates 1 / V c , while the Y-intercept represents 1 / D M . Each value of 1/0 is normally calculated as described by Forster et al. (1986). It is the mean capillary O 2 tension ( P c 0 2 ) which determines 0 and this is very difficult to quantify. While the alveolar partial pressure of oxygen ( P A O 2 ) is typically the same as the end capillary partial pressure of oxygen (PecC02), Pc02 is assumed to be 1 5 m m H g lower. Note that 0 is also a function of [Hb], as its uptake depends on the number of red cells present (equation 8).  63  Equation 8: Determination of Theta. 0.34 + f0.006 x P0O2I iHbJ 15  where  9 (theta) = the reaction rate of C O and red blood cell haemoglobin Pc02 = mean capillary partial pressure of oxygen = PA02-15  [Hb] = haemoglobin concentration  P A 0 2 can be estimated by using the alveolar gas equation (equation 9), assuming a respiratory exchange ratio (RER) of 0.8 and that the arterial pressure of carbon dioxide (PaC02) is equal to an alveolar P A C O 2 of 40 mmHg.  Equation 9. Alveolar Gas Equation  P A 0 2 = [Fi02 x (PB - 47)] - P A C 0 2 [FI02 + (1 - FiQ2)]  RER  where  PA02 = partial pressure of alveolar oxygen F1O2 = fraction of inspired oxygen PB = barometric pressure P A C 0 2 = partial pressure of alveolar carbon monoxide F1O2 = fraction of inspired oxygen RER = respiratory exchange ratio  64  FACTORS AFFECTING L U N G DIFFUSION An individuals pulmonary diffusing capacity is largely determined by anthropometric factors. Larger D L C O is correlated with greater body dimensions including weight, height, surface area and lung volume (Crapo and Forster, 1989). Reduction in D L C O results from both obstructive and fibrotic lung disorders, marijuana and cigarette smoking, pulmonary oedema, and anaemia. Increases may occur in association with asthma, pulmonary haemorrhage, and left to right circulatory shunts (Crapo and Forster, 1989). In healthy individuals, D L C O also depends on body position, increasing 15% from the upright to supine or prone position (Chang et al., 1992). In addition, D L C O increases during exercise. Turcotte et al. (1992), noted 42% and 65% increases in diffusing capacity during moderate and intense exercise respectively. Paradoxically, a number of researchers have noted reductions in D L C O following maximal exercise (reviewed by Sheel, 1995). The magnitude of the postexercise decrease reported in the literature varies from 2% to 19% depending on intensity and duration of the exercise bout and the time following exercise that diffusion capacity is measured. Miles et al. (1983) observed 2% reductions in DLQO, while Manier et al. (1991), found 10% reductions in DLCO/ 24 hours and 28 minutes respectively after the completion of a marathon. Sheel (1995), in a study designed to follow the timecourse of post-exercise alterations in DLCO, observed a peak (13%), reduction in pulmonary diffusion six hours following maximal exercise which was still 6% below baseline 24 hours after the exercise bout. Although exercise training induced changes in D L C O have not been reported in the literature, physically active people generally have a higher D L C O value (Crapo and Forster, 1989). A n increase in D L C O is likely to occur in trained athletes as a result of increased total blood volume and hence pulmonary capillary blood volume. Sheel (1995), found small (7.5%) but statistically non-significant differences  65  between the  DLCO  of a group of highly trained and a group of moderately trained  males.  66  Appendix II: The Human Menstrual Cycle  THE HUMAN MENSTRUAL CYCLE The menstrual cycle of humans and old world primates is characterised by circa-lunar fluctuations in the levels of hormones of the hypothalamic-pituitaryovarian (HPO) axis, early-cycle bleeding (menstruation), and mid-cycle release of a mature ovum (ovulation) (figure 2). The phases of the menstrual cycle may be categorised by ovarian hormone changes or changes in the uterine lining. During the early part of the ovarian "follicular" phase, reproductive hormone levels are low and menstruation occurs. Increasing levels of oestrogen cause the cessation of menstruation and the onset of development of an encapsulated ovum (the follicle). At this time, the uterine lining (endometrium) undergoes proliferation: it begins to thicken and develop a new blood supply. A late follicular shift from the negative feedback regulatory action of oestrogens on the hypothalamus to a positive feedback mechanism results in a rapid but short-lived increase in oestrogen levels and a slightly delayed gonadtrophin level rise. This results in the rupture of the mature follicle and exocytosis of the ovum from the ovary marking ovulation. The remainder of the follicle becomes a steroid hormone producing body known as the corpus luteum.  During the ovarian "luteal" phase, the corpus luteum produces  large amounts of both progesterone and oestradiol which act to maintain the uterine lining. In the latter part of the luteal phase corpus luteal function declines. Lowered steroid hormonal levels, mediated by the action of locally produced prostaglandins, initiate transient vasoconstriction of arteries leading to the endometrium. Necrotic erosion of the superficial layer of the ishaemic endometrium by proteolytic enzymes released following leukocyte and macrophage invasion, results in the shedding of outer cell layers of the endometrium and rupture of blood vessels causing the onset of menstruation. 67  0  2  4  6  8  10 12  14  16 13 20 22 24 26 28  DAYS OF MENSTRUAL CYCLE Figure 12: The H u m a n Menstrual Cycle (Adapted from Guyton, 1996)  68  C H A N G E S IN RESPIRATORY FUNCTION OVER THE H U M A N MENSTRUAL CYCLE Menstrual cycle phase is already known to influence respiratory function. Both resting (Shoene et al., 1981) and maximal exercise (Jurkowski et al., 1981) minute ventilation (VE) increases in the luteal phase of the menstrual cycle compared to the follicular phase. Resting hypercapnic and hypoxic drives are also higher in the luteal phase (Schoene et al., 1981; Dombovy et al., 1987). Menstrual phase dependent alterations in ventilatory drive are thought to be a result of progesterone mediated stimulation of central respiratory centres. Firstly, the synthetic progestin medroxyprogesterone acetate (MPA), which increases ventilatory drive, has been previously identified in the cerebrospinal fluid (Scoggin et al., 1978). Secondly, the changes in oral occlusion pressure found by Schoene and coworkers (1981) to coincide with changes in ventilatory drive are generally thought to reflect total neural drive. Peripheral factors may also be involved in ventilatory changes over the menstrual cycle; Chen and Tang (1989) have noted a higher inspiratory muscle endurance in the mid to late luteal phase compared to the mid follicular phase.  69  Appendix III: Calculation of Intraclass Correlation Coefficient The Intraclass Correlation Coefficient may be calculated from a Repeated Measures A N O V A using the following formula (Equation 1, from Vincent, 1995, p g 179).  Equation 1: Intraclass Correlation Coefficient  Tintraclass = MSsubject - MStrial+error MSsubject  where  r traclass = Intraclass Correlation Coefficient m  MStrial+error = Mean Square of Trial Effect and Error MSsubject = Between Subjects M e a n Square  Because both P C V and [Hb] repeated measurements were made on separate days at different points of the menstrual cycle, day to day variation a n d / o r menstrual cycle effects w i l l cause changes i n the means of the separate trials. In order to assess only the measurement error (or reliability of the measurement procedure), the trial effect should be ignored. Vincent (1995) recommends the use of the following modified formula i n this situation (Equation 2).  70  Equation 2: Modified Intraclass Correlation Coefficient  ^ i n t r a c l a s s = MSsubject - M S e r r o r MSsubject  where  r2i traclass = Modified Intraclass Correlation Coefficient n  M S e r r o r = Error Mean Square MSsubject = Between Subjects M e a n Square  71  Appendix IV: Ethical Approval Certificate and Consent Form  72  Appendix VI: Menstrual Cycle Diary  Menstrual Cycle Diary Name:  Year:  M o n t h :  1  Cycle Day  2  3 . 4 5  6  7  8  9  10 11 12 13 14 15 16 17  18  19 20 21 22 23 24 25 26 27 28 29 30 31  Date Tampons/pads/day Record 0 = none,  1 = minimal, 2 = moderate, 3 = moderately intense, 4 = very intense  Amount Flow Cramps Breast Sore: Front Breast Sore: Side Fluid Retention Mucous Secretion Constipation Headache Sleep Problems Feeling Frustrated Feeling Depressed Feeling Anxious Record M = much less, L = a little less, U = usual, Y = a little increased, Z = much increased Appetite Breast Size Interest in sex Feeling of energy Feeling of self-worth Outside stresses Basal Temperature  SUPIN£ n£ARr«AT£ Comments (temperature taken late, feeling sick, poor sleep, etc)  JC Prior  78  Copyright 1990  Appendix VII: Daily Exercise Record  79  DAILY EXERCISE RECORD Year:  Month:  Name:  Date Men Vigorous/ Day Strenuous Exercise (HR 10s >25) (hrs:mins)  Mild/ Moderate Exercise (HR 10s 15-25) (hrs:mins)  Comments  Mode  (eg: running, walking, swimming, resistance training)  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31  80  Appendix VIII: Initial Questionnaire  8  1  INITIAL  VERSION  GENERAL AND HEALTH QUESTIONNAIRE Code Number  ~~~  Date/Time  Birthday 1. H o w would y o u  describe your  work?  2- What's your partnership status?  3 . W h o is at h o m e with y o u ? n  °-°  n  family /  e  D  a  r  t  n  e  r  partner  roammate(s}  4, H o w m a n y y e a r s of schooling d o you h a v e ? l e s s than S  8  ~12  5  w  "  A  m  y  D  U  o  n  a  « ___™illl*?L 1 f t  m m  more than 16  ^ ^ r m o n e s (including the pill) or other m e d i c a t i o n s ? Y e s  No  ^Jfy^ptea$eJistihem  Sx D o you h a v e a s t h m a , other lung problems or significant Hlness? Y e s  No  7. Is there a n y o n e related to you who has/had a broken bone (without a fall or accident) or who is getting/became shorter and developing/developed a h u n c h e d b a c k ? 8, D o y o u currently $moke?  Yes  No  & A r e you a past s m o k e r ?  Yes  No  If y e s , how long since you stopped? mnnthi'V^aro  82  10, D o y o u currently drink alcohol?  Yes  No  if yes, how many glasses of wine or beer do you drink per week? 1or2  3-7  8 - 12  more than 12  If yes, how many glasses of spirits d o you drink per week? lor 2  3-7  8-12  more than 12  11. Do you currently drink caffeine containing drinks (non-herbal tea, coffee, or coke)? If yes, how m a n y glasses do you drink per week? 1or2  3*7  8-12  12. A r e you on a diet for a health concern or to lose weight?  more than 12  Y  es  No  If yes, p l e a s e explain?  13, H a s your weight c h a n g e d in the past 5 years? If y e s , has it?  Increased  Yes  decreased  How much h a s It changed/fluctuated?  No fluctuated  ^  &  ^  s  14, How much do you feel you should weigh?  gpp  lbs  15, What w a s your highest non-pregnant weight?  kgs  tos  16- What w a s your lowest non-pregnant weight?  kgs  lbs  83  NUTRITION 17. A r e you taking any multiple vitamins?  Yes  No  18, A r e you taking any calcium supplements?  Yes  No  If yes:  H o w many m g are there in each pill? H o w many pills d o you take each day? H o w long h a v e you been taking them?  1 0 , H a v e you h a d any calcium containing foods (milk, yoghurt, c h e e s e , c r e a m e d c h e e s e , c a n n e d s a l m o n , etc) over the last 24 hours? Pood  Amount  : Breakfast  Lunch  Dinner  84  EXERCISE 20.  Do you d o a n hour or more of vigorous exercise per w e e k ?  Yes  No  If y e s , please indicate the type of activity, the average frequency a n d duration. T y p e  o  f A c t i v i t y  F r e q u e n c y  ( n o  s e s s i o n s  p e r  w e e k )  D u r a t i o n  ( t i m e  p e r  21.  H o w many months over the last year have you exercised a similar amount?  22.  H o w m a n y years (over t h e last 5) have you exercised a similar amount?  23.  H a v e you d o n e more than 1 hour of aerobic exercise per w e e k in the past?  Yes  No  If y e s , how many years did you do this for?  MENSTRUAL  s e s s i o n )  years  CYCLE  24.  A r e you having regular periods?  Yes  25.  H o w long is your cycle length?  days  £6,  H o w m a n y days Jong is your flow?  days  27. C a n you usually tell, by the way y o u feel, that your period is c o m i n g ?  85  Yes  . No  No  2 $ , D o you usually experience the following symptoms? If yes, p l e a s e indicate the time in your cycle that you notice them (respond "intermittently" if this s e e m s the most appropriate answer). Breast T e n d e r n e s s  Yes  No  Appetite C h a n g e s  Yes  No  Mood Changes  Yes  No  Fluid Retention  Yes  No  Stretchy V a g i n a l Mucous  Yes  No  T i m e  i n  c y c l e  29. Are there any c h a n g e s in your symptoms after your flow starts?  Illlllllllllllllllillllllllllllillllllll  lllllllll  If yes, p l e a s e explain?  30. H o w m a n y times did you menstruate in the last year? none  1-2  3-6  7-9  10-17  3 1 . H a v e you menstruated in the last 6 months?  m o r e than 17  Yes  3 2 . H o w m a n y periods have you m i s s e d in the last 5 years? (normal = I0>i4/year) 3 3 . H a v e you been pregnant in the last 5 years (full « 10 months)? Y e s  If y e s : H o w many months after delivery until your period started?  86  No  periods No  periods  34. H a v e you recently h a d P M S ?  Yes  No  If yes, p l e a s e describe your symptoms and their timing in relation to flow?  36, A r e you currently taking oral contraceptives?  yes  No  36. H a v e you taken them in the past?  Yes  No  If y e s : H o w long did you u s e them for? months/years  H o w long a g o s i n c e you u s e d them last?  37* Did you have regular periods when you stopped the pills? y e s  No  If no: H o w long w e r e they irregular for? months/years  87  Appendix IX: Data Sheets INITIAL S U B J E C T D A T A S H E E T Date  /  /  Code  DOB  Height (m)  /__/  (  )  day month year  Triceps  Subscap  SupSpi  Abdom  AntThi  MedCalf  S of Six  Suplliac (IliacCrest)  Any Respiratory or Endocrine Medical Conditions Y/N Current Smoking Y / N Past Smoking Y / N  Length ago  No per day  Oral Contraceptives Y / N Past Oral Contraceptives Y/N  Menstruate Regularly Y / N  Type  Length ago  Able to predict onset of bleeding Y / N  No of times menstruated in the last year  None  1-3  4-7  8-10  10-17  Training/Sport  Hrs/Week  (average training or intensive exercise)  88  Months/Year  >17  SUBJECT DATA SHEET Date day  P  B  /  /  month  Time  Time at:  Weight (kg)  year  (mmHg)  Test Time  Code  T  1  2  3  R  O  O  4  Last V i g Ex  F V C (ml)  5  Last Meal  Hb (mg/lOOdl)  (%)  Day in menstrual cycle  FEV1 (ml)  %  PCV  Oes  Humidity  M (°C)_  Last Coffee  FEV25-75  PEFR  (haematocrit)  Prog  COHb  Fi02 (between)  21% Tests I and II Fi02  21%  VinBTPS  D L C O (ave)  He  CO  V A (ave)  Hold  D/VA  (ave)  90% Tests III and IV Fi02  90%  VinBTPS  D L C O (ave)  DM  He  V A (ave)  VC  89  CO  Hold  D/VA  (ave)  FINAL SUBJECT DATA SHEET  Date  /__/  Code  Height (m)  DOB  __/__/  day month  (  )  year  Triceps Subscap SupSpi Abdom AntThi  MedCalf  Suplliac (IliacCrest)  .  ._  S of Six  Any Respiratory or Endocrine Medical Conditions Y/N Current Smoking Y / N Oral Contraceptives Y / N Menstruation During Study (circle) Did not Menstruate  Irregular Cycles  Regular Cycles  Training/Sport  Hrs/Week  (average training or intensive exercise)  90  Months/Year  Appendix X: Final Questionnaire  91  FINAL  VERSION  GENERAL AND HEALTH QUESTIONNAIRE Code Number  Date/Time  1. A r e you o n a n y hormones (including the pill) or other m e d i c a t i o n s ? Y e s  No  Jf y e s , p l e a s e list them  2. D o you have asthma, other lung problems or significant illness? Y e s  3. D o you currently s m o k e ?  Yes  4, D o you currently drink alcohol?  No  No  Yes  No  If y e s , how m a n y g l a s s e s of wine or beer do you drink per w e e k ? more than 12 If yes, h o w m a n y glasses of spirits do you drink per w e e k ? more than 12  5. D o you currently drink caffeine containing drinks {non-herbal t e a , coffee, or coke)? Jf y e s , how m a n y g l a s s e s do you drink per w e e k ? 1 or 2  3*7  8-12  92  more than 12  NUTRITION 6, Are you taking a n y multiple vitamins? 7.  Are you taking any calcium supplements?  If yes:  Yes  No  Yes  No  H o w m a n y m g are there in each pill? H o w m a n y pills Oo you take each day? H o w long h a v e you been taking them?  S, H a v e y o u h a d any calcium containing foods (milk, yoghurt, c h e e s e , c r e a m e d c h e e s e , c a n n e d s a l m o n , etc) over the last 24 homsl Food  Amount  Breakfast  Lunch  Dinner  93  9. Are you on a diet for a health concern or to lose weight?  Y  es  No  If y e s , p l e a s e explain?  EXERCISE 10.  Do you do a n hour or more of vigorous exercise per week?  Yes  No  If yes, p l e a s e indicate the type of activity, the average frequency a n d duration. T y p e  o  f A c t i v i t y  F r e q u e n c y  ( n o  s e s s i o n s  p e r  w e e k )  D u r a t i o n  ( t i m e  11.  H o w many months over the last year have you exercised a similar amount?  12.  H o w many years (over t h e l a s t 5) have you exercised a similar amount?  p e r  s e s s i o n )  If y e s , how many years did you do this for?  13.  years  H a v e you d o n e more than 1 hour of aerobic exercise per week in the past?  Yes  ^^^^II^^^^BIIIIIIIiHliili^^HiHB^Bi^HBiBI  94  No  .111111  MENSTRUAL CYCLE 14. A r e you having regular periods?  Yes  No  15. H o w long is your cycle length?  days  16. H o w m a n y days long is your flow?  days  17. C a n you usually tell, by the way you feel, that your period Is c o m i n g ?  Yes  No  18. D o you usually experience the following symptoms? If y e s , please indicate the time in your cycle that you notice them (respond "intermittently" if this s e e m s the most appropriate answer). T i m e  Breast T e n d e r n e s s  Yes  No  Appetite C h a n g e s  Yes  No  Mood Changes  Yes  No  Fluid Retention  Yes  No  Stretchy V a g i n a l Mucous  Yes  No  I n  c y c l e  19. A r e there a n y c h a n g e s in your symptoms after your flow starts?  If y e s , please explain?  20. Over the study have you? not menstruated at all  menstruated abnormally  2 1 . O v e r the study have you taken oral contraceptives?  95  menstruated normally y  e  s  No  Respondent I.D. #  In this section I would like to ask you questions that will help us understand how women's hormones relate to bone structure. We ask everyone these questions.  5*  R E P R O D U C T I V E H I S T O R Y (FEMAU3S)  5.1 *  Before menopause, have you ever gone 3 months or more without a menstrual period? (not including pregnancy or during breastfe ding) •  5.2*  Yes  • No '—* Go to 5.2  What was the longest single period of time without a menstrual flow?  months  If you count all the periods you have missed throughout your menstruating years, how many months would that be? (this question asks for the cumulative time)  months  Have your menstrual periods stopped for more than one year? (No period one year or more after last menstruation) •  Yes  •  No  — l » At what age?  .--  years  5.12  How old were you when you had your first menstrual period?  5.3  Have you had your uterus removed (hystereaomyf! • Yes  • No  — I » At what age? 5.4*  years  years  Have you ever had one or both ovaries removed? • Yes, one ovary removed • Yes, both ovaries removed (if ovaries  were  removed  on separate  • Yes, do not know how many • No  at what age? at what age? occasions,  write  the age al which  at what age?  See notes in manual  96  the second  ovary  was  removed)  Respondent I.D. #  I'm going to ask you a few questions on your eating habits.  8.7  a) I am going to read two sentences for you. Please answer True (T) or False (F) for each statement as it pertains to you. I enjoy eating too much to spoil it by counting calories of watching my weight.  T •  F  •  I consciously hold back at meals in order not to gain weight.  T •.  F  •  b) Which of these best describes you? On a scale of 0 to 5, where 0 means no restraint in eating (eating whatever you want, whenever you want it) and 5 means total restraint (constantly limiting food intake and never "giving in"), what number would you give yourself? 0 1 2 3 4 5  Eat whatever you want, whenever you want it Usually eat whatever you want, whenever you want it Often eat whatever you want, whenever you want it Often limit food intake, but often "give in" Usually limit food intake, rarely "give in" Constantly limiting food intake, never "giving in"  Now the questions I will ask will relate to the use of tobacco.  9,  TOBACCO  9.1  Have you ever used any of the following tobacco products daily for at least 6 months? Cigarettes Pipes Cigars Chewing tobacco  • • • •  Yes Yes Yes Yes  • No • No . • No • No  97  «-»  cUu 3  O >>  <o >  JS  CU  o  Q  'i?  u  CTJ  ^3  <£ o == o 1  c  o 125 ml 250 ml  • •  o a o.  9 §  M  (0.5 cup) (1.0 cup)  (1 tbsp) (2 tbsp) (4 tbsp)  (.25 cup) (0.5 cup) (1.0 cup)  125 ml 250 ml 375 ml 125 ml 160 ml 250 ml  • • • • • •  Ice-cream, ice milk or frozen yogurt  Cream soups made with milk  (0.5 cup) (.67 cup) (1.0 cup)  (0.5 cup) (1.0 cup) (1.5 cup)  125 ml (0.5 cup) 175 ml (single) 250 ml (1 cup)  • • •  Yogurt  Hard cheese (to eat, in sandwich or mixed dish)  Milk desserts (tapioca, rice pudding)  0J  • 15 g (0.5 oz) • 30 g (1 oz) • 60 g (2oz)  15 ml 30 ml 60 ml  •a  Milk to drink incl. choc, milk & hot cocoa w/milk  (0.5 cup) (1.0 cup) (1.5 cup)  Serving Size  • • •  c u  Milk/cream in tea/coffee  J? 60 ml 125 ml 250 ml  u  • • •  I 125 ml 250 ml 375 ml  week  o  month  c  • • •  33 sei •vings p  no  Never  S  Fold  c u  During the last 12 months?  In your 30's  Never Less  Same  More  (If subject 40 years or over)  Never  Less  Same  In your teens?  More  Never  Less  Same  As a child?  More  Respondent I.D. #  •  8  Respondent I.D. # o  2 "O  B CQ C/3  a 13 O  >  CD  z o  S  a  " et) Vi  2.  5-1  cu  Z  g  01  g  8 SP.S := .o a.  g  _  .a 1/3  - em .3 00 00  S  60  88  a  a .s 1  is ^ a, ~ S S  j  " <=L  a  •no  • ••  ODD  o £  §• g - ^ • o o g.  •all  •all  •all  11  *  s  •  ,  I  1 cu JS M  C  CD  z o  o o ft.  2  5g  §  a & 3$  1.9  ja° a^  cd O ii  5 U  •a U  o •-  ae  =9  cd  S  3  a  a, p. 3  J3  cS o H  .Is ed t_  U  -  -5  © a  cu  a C  o  c S  o  cu  5  o  "5  c/3  M  ©^ .2  60  •S a  £ >»  S ©  cu  ©"  8  a ©  .?<>  ^ cu  * 2  k  5 ©  1| © ©  cu  —  ° -a  £ ©  £s  oo s ;  8  2  inn  it  CU  cu  i  3  <u  Alcoholic beverages  C  £^ C3 -Ct  caffeinated  B  -——  a  ."2 •3 a  rnlaq  3 3  1——:—-—-  4 c/3  decaffeinated  o  -s;  O o o >» ©  caffeinated  •s <u £ Less  Same  More  toe too  Tea  cu O  3  decaffeinated  None  None  8 -  caffeinated  43  Serving /day  cu cu c-s c-i  Serving /week  I  Serving /month  CU  None  In your 30's  Less  Same  When in your teens?  CO  (If subject is 40 years or over)  c  During the past 12 months?  More  Respondent I.D. #  K)  1  )^  Respondent I.D. #  In this section I will ask you about your physical activities and exercise.  11.  PHYSICAL ACTIVITY  11.1  During a typical week in the past 6 months, how much time did you usually spend walking to work or school or while doing errands? •  11.2  11.3  None  •  Between 6-10 hours  •  Less than 1 hour  •  Between 11-20 hours  •  Between 1-5 hour  •  More than 20 hours  Which of the following describes the paid work you usually do or what you consider your job? Or if retired or unemployed, which best describes your (past or longest) job? •  I am usually sitting during the day and do not walk around very much  •  I stand or walk quite a lot during the day but I do not have to lift or carry heavy things  •  I usually lift or carry light loads or I often have to climb stairs or hills  •  I do heavy work or have to carry loads  Do you currently participate in any regular activity or programme (either on your own or in a formal class)! •  Yes  •  No  —• How many times a week? —* How long per session ?  L  101  minutes  Respondent I.D. #  11.4* On the average, during the last year, how many hours in a week did you spend in the following activities? Never,  1/2-1  2-3  4-6  7-10  11-20  21-30  hr  hrs  hrs  hrs  hrs  hrs  31  hrs +  STRENUOUS SPORTS  (such as jogging, bicycling on hills, tennis, racquetball, swimming laps, aerobics) VIGOROUS WORK  (such as moving heavy furniture, loading or unloading trucks, shovelling, weight lifting, or equivalent manual labour)  -  MODERATE ACTIVITY  (such as housework, brisk walking, golfing, bowling, bicycling on level ground, gardening) c  U. of Hawaii Cancer Research Center  11.5 * On the average, during the last year, how many hours in a day did you spend in the following sitting activities? Never  Less than 1 hr  1  to 2 hrs  3  to 4 hrs  5  to 6 hrs  7  to 10 hrs  11 hrs or more  Sitting in car or bus Sitting at work Watching T V Sitting at meals Other sitting activities (such as reading, playing cards, sewing) •U. of Hawaii Cancer Research Center  11.6  On the average, during the last year, how many hours in a day did you sleep (include naps)! • •  5 hours or less 6 hours  • 7 hours • 8 hours  • 9 hours • 10 hours or more  See notes in manual  102  Respondent I.D. #  11.7 * Rate your overall level of physical activity compared to your peers during certain times in your past life.  When you were about 50  When you were about 30  if subject 60 y. and over  if subject 40 y. and over  Teenager  A lot less active Somewhat less active About the same Somewhat more active A lot more active  Now I want to ask you questions about being in the sunlight  12.1 * Did you ever expose a considerable part of your body to direct sunlight? A.  During the past 12 months?  • • • •  never seldom regularly often  • • • •  never seldom regularly often  • • • •  never seldom regularly often  • • • •  never seldom regularly often  If 60 years old or more. B.  When you were about 50 years old?  If 40 years old or more. C.  When you were about 30 years old?  For all. D.  When you were a child or teenager?  See notes in manual  103  Child  Appendix XI: Timetables of Testing Dates  104  co Q  o O  O Q  o  z  in  tco LU t—  z  o  to  Q  O co  a  CO  S Q  o  o O CO  O O  O  co  CM  3  CO  Si o  o  CM  o  105  3  CD CO  co in  Oo  co CM  CM  i  o w  to  CM  a> co in  CM  TO CO C O CD II  ,3 •a  1  o -O  CM  p  10 CO  TJ  :  CM  in  <3 | co  e.  iffl  co CO CM CO  CM Q  CM  s  re in o  co  CM CM >> CO  O  co CM|  m  o  o  o CO  m o m o  > v CO  co  in  g  co "J  o  m  Xi  CM  CM Xi '  m o  CM  in  S5 II  ~TO"  CM  TOin g  Q  m  CM >.  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ASU RED  c o CO  \—  35.7  z  CO  CO  29.5  C O  1—  «_  T—  CO  CO  2  rv  CM  — i o T t OJ rv' CO CO  Tt  §  CO  gj  2.64  I  <&  CM o CO CO CO CM  38.1  z < z  OJ rv LO CO CO CM co  Tt  Tt  CO CM CO CO 00 OJ o T— CM CM CM CO CO CO CO CO CO CO  o o o CO o o CO C OC OC O C O  O o o o o o o o o o O O o  122  2 CO  111 If  5 CO  CO  CO  CO  CM CO  cd  eo  CO  CO CD  CO  TJ co  s CO  12 3  LO  LU  _l'  o >o  CO  >cc  o I3  > O  z  <  CM  z  < Q  z  <  > cc  o 3 >  o Z < Z  X  z O  CO  0_ r-  co LU ILU >  < Q UJ CC 3 CO < UJ  JEj CO  CO  LU  CD Z  < X  o  cj  <  CO  cc rZ LU  o z o o z  CO  CO  CO  o -J  CD O 2 UJ  < X  124  IUJ  >  <  o  UJ  cc  LU  o tt  cc Iz  LU  o z o o TJ  O  CO ZD  <  JO  ICBMS00 1 CBMS01 CBMS03 CBMS04 ICBMS08 iCBMS12 ICBMS16 miss ICBMS18 1.05 ICBMS19 0.50 ICBMS20 1.00 ICBMS21 0.50 MEAN 0.79 STDEV 0.18  LU  CD  0.65 0.70 0.55 miss 0.90 1.20 1.00 miss 1.10 0.85 1.00 0.90 1.00 1.50 0.90 0.85 0.80 0.75 0.40 0.75 0.50 0.90 1.15 0.90 0.55 0.25 0.50 0.90 0.95 0.65 0.80 miss 1.00 0.79 0.81 0.88 0.22 0.19 0.36  5 MEA status  0.66 AN 0.97 0.98I 0.88 1.08 0.79 0.55 1.00 0.45 AN 0.88 0.77 0.82 AO AO  CO g  AO AO  CL  0.75 0.80 0.85 0.75 0.90 0.75  z  I  |  |  I I  > o  I  o  AO AO NV/AO AO!  >cc  ! 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