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Blood flow variations in internal carotid and middle cerebral arteries induced by postmenopausal hormone replacement therapy
Penotti et al.
primarily a result of marked hypersecretion of estradiol. Second, a literature search failed to reveal any report of polycystic ovarian disease that resulted in such a massive secretion of estradiol as to mimic an estrogenproducing tumor. The occurrence of this case also suggests that polycystic ovarian disease should be included in the differential diagnosis of estrogen-producing neoplasms, in spite of its being a very rare cause of such excessive estradiol production.
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We thank Laura Molho, MD, for the pathology reports and for preparing the photomicrographs. REFERENCES 1. Chang RJ, Mandel FP, LuJK, Judd HL. Enhanced disparity of gonadotropin secretion by estrone in women with polycystic ovarian disease. J Clin Endocrinol Metab 1982;54: 490-4. 2. Hoffman D, Lov K, Lobo RA. The prevalence and significance of elevated DHEA-S levels in anovulatory women. Fertil Steril 1983;39:404-5.
Blood flow variations in internal carotid and middle cerebral arteries induced by postmenopausal hormone replacement therapy Mauro Penotti, MD,· Torquato Nencioni, MD,· Livio Gabrielli, MD,b Massimiliano Farina, MD,b Elisabetta Castiglioni, MD,· and Filippo Polvani, MD· Milan, Italy OBJECTIVE: Our purpose was to clarify the mechanisms by which postmenopausal estrogen replacement therapy exerts its protective effect on cardiovascular risk. STUDY DESIGN: By means of a bidirectional Doppler ultrasonographic system we measured pulsatility index variations the internal carotid artery and middle cerebral artery in 25 early postmenopausal women during a 6-month period of hormone replacement therapy. Transdermal estradiol (50 IJog/day) was continuously administered. A 12-day course of medroxyprogesterone acetate (10 mg/day) was added every second month. RESULTS: The pulsatility index showed a significant (p = 0.0001) reduction in both arteries after 6 weeks. At 22 weeks a 25% reduction was measured. No variation of the estrogen-induced pulsatility index reduction was observed at the end of every cyclic progestogen supplementation. CONCLUSIONS: In early postmenopausal women hormone replacement therapy causes a rapid reduction of pulsatility index in brain arteries. Cyclical progestational supplementation does not modify this positive effect on reactivity of the blood vessels. (AM J OesTET GVNECOL 1993;169:1226-32.)
Key words: Hormone replacement therapy and cardiovascular protection, hormone replacement therapy and blood flow, pulsatility index in brain arteries
Hormone replacement therapy is known to reduce the risk of ischemic heart disease in postmenopausal women.':" although the mechanism by which it exerts this effect is not completely understood. Favorable plasma lipid variations induced by hormone replacement therapy (high-density lipoprotein' increase and From the Second Obstetrical and Gynecological Department' and the Institute of Vascular Surgery,' University of Milan. Received for publication February 28, 1993; revised April l ), 1993; accepted May 26, 1993. Reprint requests: M. Penotti, MD, Second Obstetrical and Gynecological Department, University of Milan, Via Commenda 12, 20122 Milano, Italy. Copyright © 1993 by Mosby-Year Book, Inc. 0002-9378 $1.00 + .20 6/1/48938
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low-density lipoprotein decreasej'" seem to account for about half the effect on cardiovascular mortality risk.' In fact, experimental studies performed on monkeys with food-induced hypercholesterolemia, in which a surgical menopause was obtained, showed that hormone replacement therapy highly reduces both atherosclerotic plaque size? and coronary artery low-density lipoprotein uptake," but that these effects seem to be only partially related to plasma lipoprotein variation. Besides these favorable actions on both lipid profile and atherosclerotic process, evidence exists about the presence and functionality of estrogen receptors on female baboon myocardium and aorta" so that a direct, receptor-mediated effect of estrogens on the cardiovascular
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system can be postulated. As for humans, Bourne et al. 10 initially reported on uterine artery pulsatility index variations during postmenopausal hormone replacement therapy. Recently a positive effect of hormone replacement therapy on cardiac function in postmenopausal women has been documented suggesting a combined action on both inotropism and peripheral vasodilatation. II To study the effect of hormone replacement therapy on peripheral blood vessels, in May 1991 we began a study of how it affects blood flow characteristics III internal carotid and middle cerebral arteries. Material and methods
Twenty-five normotensive, postmenopausal Italian women entered the study. They were all within 6 months to 2 years from the last menses. The mean age of patients at enrollment in the study was 51.68 years (± 3.30), mean body mass index was 23.50 (± 3.26). All women were complaining of menopausal symptoms and had gonadotropin and estradiol serum values within the postmenopausal range for our laboratory. All women underwent a pretreatment endometrial evaluation by Vabra curettage. Seven of the 25 patients who participated in the study were smokers. Of these, two smoked 10 to 20 cigarettes per day and five smoked < 10 cigarettes per day. No subject had received any exogenous sex steroid since the time of menopause. Patients were informed of the aims of the study and gave consent. The design of the study was approved by the Ethical Committees of our institutes. Women were given a continuous estradiol transdermal supplementation at a dosage of 50 ILg/day (Estraderm ITS 50, Ciba-Geigy). Patients used patches in the way suggested by the manufacturer, attaching them on the buttocks or on the abdominal wall and replacing them twice weekly. In the first 12 days of every second month we added a 10 rug/day course of medroxyprogesterone acetate (Farlutal, Carlo Erba). Visits were scheduled as follows: two control visits before the beginning of therapy, with a 6-week interval between the first and second visits; 6 weeks after the beginning of therapy (at the end of the estrogen-only period); 8 weeks from the beginning of therapy (at the end of the progestogen supplementation); 22 weeks from the start of therapy (at the end of the estrogen-only period); and at the end of the twenty-fourth week, during the progestogen supplementation. Therapy started at the second control visit. According to our schedule the first estrogen-only control was performed at least 40 days after the beginning of therapy and the second at least 40 days after the end of the previous progestogen supplementation. During therapy patients were studied on the second day of the patch administration. A 24-hour urine collection
Penotti et al. 1227
on the day before each visit was obtained from all patients. We asked the patients to keep the used patches and bring them at the following control visit. Compliance to the therapy was assessed at each control visit by asking the patients, evaluating the bleeding pattern, counting used patches, and assessing estrone3-glucuronide levels on the 24-hour urine collection. Routine blood laboratory examinations and complete urinalysis were performed on each patient before the beginning of the study. No subject had history of significant illness (e.g., diabetes mellitus; hypertension; cardiovascular, pulmonary, peripheral vascular, or renal disease). Estrone-3-glucuronide was measured on a sample from a 24-hour urine collection obtained at each control visit and stored at - 20° C until the assay. All samples were assayed together in duplicate with a radioimmunoassay method using dextran-charcoal for the separation of antibody-bound and free hormone fractions. Antibody was provided by Dr. P. Samarajeewa, Department of Biochemistry, University College, London. Sensitivity was 0.33 pmol/ml. Doppler studies. This study was carried out with a color flow imaging (Philips QAD - PV) system with a 7.5 MHz phased linear-array transducer and a bidirectional Doppler ultrasonographic system (Oxford Sonicaid Vasoflo 4) supplied with a spectrum analyzer and two probes (2 MHz pulsed wave and 4 MHz continuous wave for intracranial and extracranial evaluation, respectively). Both devices were used in the first control, whereas in the following examinations only the Doppler ultrasonography system was used. During the first control in each patient both common carotid arteries, carotid bulbs, and internal and external carotid arteries were scanned with color flow imaging, to identify plaques or stenoses. The presence of parietal alterations excluded subjects from study. Women who passed this preliminary examination were later subjected to the bidirectional Doppler study. The internal carotid artery on both sides within the first centimeter from bifurcation and the middle cerebral artery on both sides to a depth between 40 and 55 mm from the temporal surface were explored. For each evaluation, the pulsatility index on internal carotid and middle cerebral arteries was considered. Carotid blood flow is influenced by noise, ambient temperature, and food intake. 12, 13 Patients fasted for at least 2 hours were studied in a noiseless laboratory with constant temperature and light by the same examinator (M.F.) with earphones connected to the Doppler's audio output at the same time of day. Before examination they rested in a supine position for 15 minutes, until pulse rate varied by < 5 beats/min, and systolic-diastolic blood pressure varied by < 5 mm Hg over two consecutive measurements.
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Penotti et al.
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OL-----,----,------.-------r----r-----.--~ 22w 24w 6w 8w -6w o Time (weeks) Fig. 1. Plots of urinary concentration of estrone-3-glucuronide. Boxes contain 50% of observed values. Dots, Single case values; +, mean; - - -, median; whiskers, upper and lower quartiles of distribution of cases. Outliers (values that exceed 1.5 times the correspondent whisker) are not included.
In our laboratory the coefficient of variation of the pulsatility index on internal carotid and cerebral middle arteries was 1.5% and 2.2%, respectively. It was calculated over twelve Doppler evaluations in each of two premenopausal women taking a combined oral contraceptive (Ginoden, Schering), to exclude the influence of endogenous plasmatic estradiol on the impedance in explored arteries. Doppler evaluations were performed in 2 days, six measurements a day for each of the two subjects, with a 5-minute interval between measurements. Patients who entered the study underwent the bidirectional Doppler evaluation of internal carotid and middle cerebral arteries during each control visit from the first control visit to the end of the twenty-fourth week of the study. Statistical analysis. The statistical analysis of time changes of urinary estrone-3-g1ucuronide and pulsatility index measured in internal carotid and middle cerebral arteries were performed by analysis of variance (PROC GLM, SAS/PC version 6.04) for repeated measurements. A set of pairwise nonorthogonal contrasts between the two measurements before treatment and between each treatment time and baseline was performed. Measurements at each time were also compared with values at each other time. The level of significance of all pairwise contrasts was adjusted according to the Bonferroni procedure" to cope with the inference problem caused by multiple nonorthogonal contrasts.
Results
Twenty-one patients completed the 24-week study period, were compliant, and are still carrying on the therapy scheme. Four patients were lost at follow-up: two at the eighth week and two at the twenty-second week. Of these four patients, three did not want to continue because of personal difficulties in adhering to the close follow-up rules. The fourth patient was switched to different clinical care after the sixth week because of the pathologic result of basal time endometrial sampling. During the period of the study no significant changes took place either in blood pressure or in pulse rate. The results of estrone-3-g1ucuronide assessment in each patient are shown in Fig. 1. Mean values of estrone-3-g1ucuronide (SD) changed as follows: first control visit, 20.66 nmol per 24 hours (16.83); second control visit, 17.26 nmol per 24 hours (10.45); 6 weeks after starting therapy, 40.77 nmol per 24 hours (22.73), p < 0.05 versus basal time values; 8 weeks after starting therapy, 41.11 nmol per 24 hours (21.62), P < 0.05 versus basal time values; 22 weeks after starting therapy, 32.95 nmol per 24 hours (16.96); at the end of 24 weeks, 29.2 nmol per 24 hours (15.11). A preliminary statistical evaluation showed, at each control time, no difference in the results of Doppler evaluations between the right- and left-side arteries. Thus the mean value of the two sides was used in the analyses, both for the internal carotid and the middle cerebral artery. Single patient values of the pulsatility
Penotti et al. 1229
Volum e 169, Number 5 Am J Ob stet Gynecol
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0.5 '----.----r-----.---------r----=--r~---_.__----J 24 w 6w 8w 22w -6w o Tim e (weeks) Fig. 3. Plots of pu lsatility ind ex values in midd le cerebra l artery. Boxes contain 50% of observed values. Dots, Single case values; + , mean ; - - -, median ; whiskers, upper and lower quartiles of distribution of cases.
index in the internal carotid and middle cerebral arteries a re depicted in Figs . 2 and 3. Values of pulsatility index at each assess me nt (me an, SD, m inimum and maxim um value) in the internal caro tid and middle cerebral artery are shown in Tables
I and II , together with the number of measurements (left plus right side) perfor med at each time . No variatio n in pulsatility index was observed between th e two basal assess me nts in both arteries. In the in ternal carotid artery a highly significant
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Table 1. Internal carotid artery pulsatility index: Descriptive statistics Time (wk)
Scans Mean SD Minimum Maximum
- 6
o
6
8
22
24
44 0.833 0.\08 0.590 1.040
38 0.843 0.\09 0.570 1.070
44 0.795* 0.110 0.620 1.190
46 0.735 t 0.096 0.520 0.900
40 0.66 3! 0.076 0.530 0.830
38 0.647 0.067 0.530 0.780
*P = 0.001 versus basal time. t p < 0.01 versus previous time. !P = 0.0001 versus previous time.
-------I
., -
Table II. Middle cerebral artery pulsatility index: Descriptive statistics
Scans Mean SD Minimum Maximum
-6 44 0.873 0.120 0.640 1.130
T_im_e_(W_k_)
-r-r-
_
o
6
8
22
24
38 0.883 0.102 0.660 1.100
44 0.794* 0.12\ 0.600 1.080
46 0.777 0.104 0.620 1.030
40 0.658t 0.097 0.500 0.850
38 0.651 0.084 0.530 0.830
*P = 0.0001 versus basal time. tp = 0.0001 versus previous time.
(jJ = 0.001 ) reduction of pulsatility index was observed at 6 weeks. At 8 weeks, at the end of the progestogen supplementation, pulsatility index value showed a highly significant (jJ < 0.0 I ) reduction versus the previous time assessment. At week 22 a further highly significant (jJ = 0.0001) reduction was observed, when the pulsatility index showed a 25 .3% reduction versus basal time . No variation in pulsatility index was observed at week 24, at the end of the cyclic progestogen supplementation, compared with its value at th e previous time . In middle cerebral artery a highly significant (jJ = 0.000 I) reduction of pulsatility index was ob served at the sixth week . No variation was measured at 8 weeks, at the end of the first cyclic progestogen supplementation, in comparison with the previous value. At week 22 the pulsatility index showed a highly significant (jJ = 0.0001) reduction with respect to its value at the previous time, up to a maximum of 24.5% compared with basal time . At week 24 the pulsatility index value did not show any significant variation compared with the previous time . Co mment
Doppler examination of venous and arterial circulation was introduced in 1970 for a better knowledge of circulatory physiology. Since then new techniques have imp roved analysis of ultrasonic signal and have widened the field of investigation to abdominal an d intra-
cranial vasal diagnostics. Man y parameters automatically calculated by Doppler de vices are used to recognize and quantify hemodynamic changes of blood flow. Among these parameters pulsatility index has been used extensively and has been considered an expression of resistance to blood flow downstream of the point where measurements are performed. Its values vary in relation to age an d exploration site.":" We measured the pulsatility index on internal carotid and middle cerebral arteries to determine the correlations, if any, between hormonal status and blood flow. Our data clearly show a huge effect of estradiol administration in the cerebral blood flow of postmenopausal women. Our results showed no significant variation in pul satility index values between the two pretreatment measurements. These data illustrate the basal situation during postmenopausal estrogen deprivation. After 6 weeks of estradiol supplementation the pulsatility index already showed a significant reduction in the internal carotid arery. This reduction means a blood flow increase in cerebral arteries. This datum is confirmed by the ob servation of a significant reduction of pulsatility index in the middle cerebral artery at 6 weeks . This artery, which has no significant resistance district downstream, is subjected to a local autoregulation related to systemic blood pressure and metabolite change (Pco j, PO z, pH, hormones, tissue lactic acid). " ?" Therefore a middle cerebral artery pulsatility index reduction in po stmeno-
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pausal subj ects during hormon e replacement therap y, without blood pressure and metabolite change, confirm s an action of estradiol on middle cerebral artery. We did no t ob serve an y vari ation in the pulsatility index of the middle cerebral artery as mea sured at 8 weeks versus previous time evalu ation . On th e other hand, a further significant reduction in pulsatility index was mea sured in th e int ernal carotid artery at the same time. These facts see m to point out th at no impairmen t of estradiol's dilating effect should be ascrib ed to progestoge n sup pleme n tation. At th e twenty-second week a further significan t de crease in the pulsatility ind ex of both arteries was observed , when the pulsatility index was reduced by > 20% of basal time values . In both arteries no significant variation was measured at th e twenty-fourth week, at the end of the cyclic progestogen sup plementation . This datum again suggests the absence of a worsening effect of progestogen on estradio l-me diated vascular reactivity. Most epide miologic studies report that hormone re placem ent therap y can reduce the card iovascular mortality risk in postmenopausal wome n . A recent report from Stampfer et al." showed a 49% redu ction in the risk of major coronary artery disease among current estr ogen users. In this lar ge coho rt prospe ctive study the autho rs could demonstrate a significant reduction of the risk for this group of patients only. However, the mechanism by which these result s can be obtained remain s cont roversial. Un til now alm ost all epidemiolog ic and clinical studies have de alt with data referring to patients on estrogen repl acem ent therapy onl y, and the favorable effect of estrogen s on seru m lipid profile is the best known mechanism by which the protection ag ainst coronary artery disease is obtained: Furthermore, stud ies on coronary ang iogr aphy showed a redu ction of atherosclerosis in patients on estrogen replacement therapy." : 22 Whether progestogen supplementati on could be a factor th at reduces estrogeninduced protective effect is still open to debate. The reduction in pul satility index cause d by ho rmone replacement therapy that we observed in some arteries of the brain can be con sider ed an expression of a general (perha ps locally differ entiated ) reactivity of arteri es to estrad iol. In fact, in postmenopausal women Bourne et al." reported a 50% reduction in uterine artery p ulsatility index after tran sdermal estradiol admin istrati on. This action on vascular reactivity increases blood peripheral supply and can be considered one of the mechanisms by which estro gens exert their protective effect from cardiovas cular disease. In this regard it sho uld be very in teresting to kn ow whether the pul satility index in internal caro tid and middle cerebral art er ies changes with menop au se (i.e., from lower values to higher ones). Unfortunately, no research has yet
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Table III. Internal carotid artery and middle cer ebral artery pul satil ity ind ex in normal fertile and postmenopau sal women
Internal caro tid artery Middle cere bral artery No. of cases Age (yr)
Normal fertile women
Postmenopausal women
0.71 (0.09)
0.83 (0. 10)*
0.74 (0.1 I )
0.88 (0.10)*
15 28. 10 (4.07)
25 5 1.68 (3.30)
Values are mean ± SD. *P < 0.001 versus normal fertil e wom en .
been produced on this . However, the onl y published re sults (as far as we know) on premenopau sal middle cere bra l ar tery pul satil ity ind ex values see m to support th is hypothesis." Our own unpublished results on the pul satility index of these arteries of you ng fertile women are in agreement; mean values seem to be lower in th is group of patients compar ed with postmenopausal basal values (Table III ). Gangar et aJ.2 4 first showed a d irec t effect of estradiol su p pleme ntation on th e reactivity of the internal carotid art ery in postmenopau sal women and demonstra ted a correlation between men opausal age and pulsatility index. These authors studied a gr oup of women with a greater menopau sal age than in our study and used a different therapeutic sche m e in estrogen administr ati on . They did not report any variation in pulsatility index after 6 weeks of therapy. Our re sults show instead th at estrogen administration in early postmenopausal women is able to induce a rapid reduction of blood flow resistan ce in cerebral circulation . It has recently been shown that th e onset of menopause causes a worsening of th e lipid profile." It can be speculated th at the preventi on of thi s worsening exerte d by hormone repl acem ent therap y, together with the protective effect on the progression of atherosclerot ic pro cess, as proved in anima ls," s could be the lasting factor in cardiovascular protective action. A function al, and perhaps tran sient, act ion on vascular reactivity could be the facto r th at makes such a protective effect no longer so evident in past users.' Furthermore, because it seems th at women at lower risk for cardiovascular disease enj oy th e same relative benefit aga inst coronary disease as women in general,' it would ap pear th at such a fun ctional effect of estrogens on blood vessel reactivity could actually playa pivotal role in re alizing this protective action. H owever, a much lon ger follow-up on such gro ups of pat ients is needed to ascertain whether this effect on vascular reactivity is
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Penotti et al.
maintained throughout the p eriod of es trogen su p p lementation .
A limit o f o ur study is the lack of information as to the effect of unopposed estrogen supplementation on va scular rea ctivity. In fact , it is possible that the effect we evaluated co u ld be m o re in ten se and rap id without the cyclic proge st o gen su p p le men tatio n. H o wever, it is re co gnized wo r ld wid e that h ormone r epla cement ther apy in wo men who still h ave a n intact uterus sh o u ld b e r ealized with a cycl ic pro ge st ogen su p p le menta t io n to prevent e n dom e tr ia l hyperpla sia ; a ll wo men wh o to ok p art in o ur stu dy h ad n ot und ergone a ny previous gynecolo g ic su r gery. On th e o th er hand, it seem s to u s ve ry interesting that in o ur stu d y no r eduction in the favo r able, estrogen-induced pulsatility in dex variation was ever o b se rved at the e n d o f the cyclic progest o ge n su p p le me n tati ons. Thus in the light of o ur data a n d those of Gangar et al." it seems that progest o gen su p p le me n tation is n ot a b le to wo rse n the estro gen-induced favorable vascu lar m odifications. H o weve r, fu rther stu d ies a re n eeded to con cl u d e that medro xypr o gesterone acetate doc s n ot d e creas e th e beneficial effec t of es trogen on the art erial wall. For instance, a double-blind, crosso ver study with medroxyprogest erone ace ta te al one , es tr o ge n al one, then estrogen-medroxyprogest erone a ce ta te co u ld be carried o u t. We thank E. Kowarich for his wo r k in e strone- 3glucuronide urinary assays a n d Dr. M. Sanarico (N o us tat) for sta tistical evaluatio n o f d ata a n d p r oduction o f grap h ics . REFERENCES
I. Stampfer M], Willett WC, Colditz .lA, et al. A prospective study of postmenopausal estr ogen th erap y and coronary heart disease. N Eng l J Med 1985;313:1044-9. 2. Hen derson BE, Pagan ini-H ill A, Ross RK. Estrogen replacem ent th erapy and prot ection fro m acu te myocardi al infarc tion. Av ] OBSTET GYNECOL 1988;159:31 2-7. 3. Stampfer Mj , Co ld itz GA, Willett WC, et al. Postmen op ausal estrogen therapy an d cardiovasc ular disease. N Eng l J Med 1991; 325:756-62. 4. Bush Tl., Barrett- Connor E,Cowan LD, et al. Cardiovascular mo rta lity and non- con traceptive use of estrogen in wome n: res ults from the Lipid Research Clinics Pro gram follow-up study. Circulation 1987;75:1102-9. 5. Paganini-Hill A, Ross RK, Henderson BE. Postmen opausal estrogen treatmen t and stroke: a pr ospective study. BMJ 1988;295:519-22. 6. Fah raeu s L. The effects of estra dio l on blood lipid s and lipoprotein s in postm en op au sal wome n . Obs te t Gynecol 1988;72:18-22S. 7. Ada ms MR, Kapl an jR, Manuck SB, et al. Inh ibition of
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coro nary artery athero scler osis by 1713-estrad iol in ovariectomized monk eys. Arteriosclerosis 1990;10:105 1-7. 8. Wagner JD, Clark son TB, St. Clair RW, Schwenke DC, Adams MR. Estrogen replace ment th er ap y and coronary artery (CA) atherogen esis in surgically postmenopausal cynomolgus monkeys. Circulation 1989;80 (sppl 2):3 31, A-1320. 9 . Lin AL, Gonzales R, Carey KD, Shain SA. Estradiol-1713 affects estro gen recep tor d istribu tion and elevates pro ge stero ne receptor content in baboon aorta. Arte riosclerosis 1986;6:495-50 4. 10. Bourne T , Hillard TC, Whiteh ead MI, Croo k D, Campbe ll S. Oe strogens, ar te rial sta tu s, and postmenopausal women . Lan cet 1990;335:1470-1. I I. Pines A, Fisman EZ, Levo Y, et al. T he effects of hormon e repl acem ent therap y in normal postm en opau sal wome n: measur em ents of Dop pler- derived parameter s of aortic flow. AM J OBSTET GYN ECOJ. 1991;164 :80 6-12. 12. Meire HB, Kossoff G, Lod ge T. Recent advances in ultrasound. In : Steiner RE, cd. Recent advances in radiology and med ical imag ing. Ed inburgh: Churchill Livingst on e, 1983:63. 13. Grant EG. In: Grant EG, White EM, eds. Dupl ex sonography of th e cerebrovascular system. New York: Springer 1988:37. 14. Kirk RE. Experimental design : procedu res for the beh aviora l sciences . Monter ey, Californ ia: Bro oks Cole, 1982: 105-6. 15. Vr iens EM, Kraaier V, Mussbach M, Wieneke GH, Van Huffel en AC. T ran scranial pul sed Dop pler measur em ents of blood velocity in th e middl e cerebral artery. Ultrasound Med BioI 1989; 15:40 5-8. 16. Otis SM, Rossman ME, Schneid er PA, Rush MP, Ringelstein EB. Relationship of cerebr al blood flow regulation to acute mountain sickness . J Ultrasoun d Med 1989;8: 143-8. 17. Matti e H, Gro limun d P, Hu bert P, Stu rzen egger M, Zurbrugg HR . Transcra nia l Doppler sonogra p hy findings in midd le cerebral artery disease . Arch Neurol 1988;45:28995. 18. Reivich M. Arterial Pco, and cer ebra l haemod ynami cs. Am J Physiol 1964 ;25:206. 19. Bishop CCR, Powell S, Rutt D, Browse NL. Transcra nial Doppler mea surem ent of middle cerebral artery blood flow velocity: a validation study. Stroke 1986;17 :913- 5. 20. Ringelstein EB, Sievers C, Ecker S, Schn eid er PA, Otis SM. Noninvasive assessm ent of CO 2-indu ced cerebral vasom otor respon se in normal indi viduals an d patients with internal carotid artery occlusio ns. Stroke 1988 ;19:963 -9 . 21. Sullivan JM , Zwagg RV, Lemp GF, et al. Postmenop au sal estrogen use and coronary at heros clerosis. Ann Int ern Med 1988;108:358-63. 22. Gro uchow HW, And er son AJ , Barboriak j], Soboci nski KA. Postm en op au sal use of estrogen and occlusio n of coronary art eries. Am Heart ] 1988;115:954-63. 23 . Sha mma FN, Fayad P, Brass L, Sarrel P. Middl e cere bra l artery blood veloci ty during co nt rolled ovarian hyperstimulation . Fertil Steri l 1992;57:1 022 -5. 24. Gangar KF, Vyas S, Whitehead M, Croo k D, Meire H, Campbell S. Pulsatility inde x in internal caro tid artery in relation to tran sde rma l oes tradiol and tim e since menopause. Lancet 1991; 338:839-42. 25. j en sen J, Nilas L, Chr istianse n C. Influ en ce of men op au se on seru m lipid s and lipop rot eins. Maturi tas 1990 ;12:3213 1.
primarily a result of marked hypersecretion of estradiol. Second, a literature search failed to reveal any report of polycystic ovarian disease that resulted in such a massive secretion of estradiol as to mimic an estrogenproducing tumor. The occurrence of this case also suggests that polycystic ovarian disease should be included in the differential diagnosis of estrogen-producing neoplasms, in spite of its being a very rare cause of such excessive estradiol production.
November 1993 Am J Obstet Gynecol
We thank Laura Molho, MD, for the pathology reports and for preparing the photomicrographs. REFERENCES 1. Chang RJ, Mandel FP, LuJK, Judd HL. Enhanced disparity of gonadotropin secretion by estrone in women with polycystic ovarian disease. J Clin Endocrinol Metab 1982;54: 490-4. 2. Hoffman D, Lov K, Lobo RA. The prevalence and significance of elevated DHEA-S levels in anovulatory women. Fertil Steril 1983;39:404-5.
Blood flow variations in internal carotid and middle cerebral arteries induced by postmenopausal hormone replacement therapy Mauro Penotti, MD,· Torquato Nencioni, MD,· Livio Gabrielli, MD,b Massimiliano Farina, MD,b Elisabetta Castiglioni, MD,· and Filippo Polvani, MD· Milan, Italy OBJECTIVE: Our purpose was to clarify the mechanisms by which postmenopausal estrogen replacement therapy exerts its protective effect on cardiovascular risk. STUDY DESIGN: By means of a bidirectional Doppler ultrasonographic system we measured pulsatility index variations the internal carotid artery and middle cerebral artery in 25 early postmenopausal women during a 6-month period of hormone replacement therapy. Transdermal estradiol (50 IJog/day) was continuously administered. A 12-day course of medroxyprogesterone acetate (10 mg/day) was added every second month. RESULTS: The pulsatility index showed a significant (p = 0.0001) reduction in both arteries after 6 weeks. At 22 weeks a 25% reduction was measured. No variation of the estrogen-induced pulsatility index reduction was observed at the end of every cyclic progestogen supplementation. CONCLUSIONS: In early postmenopausal women hormone replacement therapy causes a rapid reduction of pulsatility index in brain arteries. Cyclical progestational supplementation does not modify this positive effect on reactivity of the blood vessels. (AM J OesTET GVNECOL 1993;169:1226-32.)
Key words: Hormone replacement therapy and cardiovascular protection, hormone replacement therapy and blood flow, pulsatility index in brain arteries
Hormone replacement therapy is known to reduce the risk of ischemic heart disease in postmenopausal women.':" although the mechanism by which it exerts this effect is not completely understood. Favorable plasma lipid variations induced by hormone replacement therapy (high-density lipoprotein' increase and From the Second Obstetrical and Gynecological Department' and the Institute of Vascular Surgery,' University of Milan. Received for publication February 28, 1993; revised April l ), 1993; accepted May 26, 1993. Reprint requests: M. Penotti, MD, Second Obstetrical and Gynecological Department, University of Milan, Via Commenda 12, 20122 Milano, Italy. Copyright © 1993 by Mosby-Year Book, Inc. 0002-9378 $1.00 + .20 6/1/48938
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low-density lipoprotein decreasej'" seem to account for about half the effect on cardiovascular mortality risk.' In fact, experimental studies performed on monkeys with food-induced hypercholesterolemia, in which a surgical menopause was obtained, showed that hormone replacement therapy highly reduces both atherosclerotic plaque size? and coronary artery low-density lipoprotein uptake," but that these effects seem to be only partially related to plasma lipoprotein variation. Besides these favorable actions on both lipid profile and atherosclerotic process, evidence exists about the presence and functionality of estrogen receptors on female baboon myocardium and aorta" so that a direct, receptor-mediated effect of estrogens on the cardiovascular
Volume 169, Number 5 Am J Obstet Gynecol
system can be postulated. As for humans, Bourne et al. 10 initially reported on uterine artery pulsatility index variations during postmenopausal hormone replacement therapy. Recently a positive effect of hormone replacement therapy on cardiac function in postmenopausal women has been documented suggesting a combined action on both inotropism and peripheral vasodilatation. II To study the effect of hormone replacement therapy on peripheral blood vessels, in May 1991 we began a study of how it affects blood flow characteristics III internal carotid and middle cerebral arteries. Material and methods
Twenty-five normotensive, postmenopausal Italian women entered the study. They were all within 6 months to 2 years from the last menses. The mean age of patients at enrollment in the study was 51.68 years (± 3.30), mean body mass index was 23.50 (± 3.26). All women were complaining of menopausal symptoms and had gonadotropin and estradiol serum values within the postmenopausal range for our laboratory. All women underwent a pretreatment endometrial evaluation by Vabra curettage. Seven of the 25 patients who participated in the study were smokers. Of these, two smoked 10 to 20 cigarettes per day and five smoked < 10 cigarettes per day. No subject had received any exogenous sex steroid since the time of menopause. Patients were informed of the aims of the study and gave consent. The design of the study was approved by the Ethical Committees of our institutes. Women were given a continuous estradiol transdermal supplementation at a dosage of 50 ILg/day (Estraderm ITS 50, Ciba-Geigy). Patients used patches in the way suggested by the manufacturer, attaching them on the buttocks or on the abdominal wall and replacing them twice weekly. In the first 12 days of every second month we added a 10 rug/day course of medroxyprogesterone acetate (Farlutal, Carlo Erba). Visits were scheduled as follows: two control visits before the beginning of therapy, with a 6-week interval between the first and second visits; 6 weeks after the beginning of therapy (at the end of the estrogen-only period); 8 weeks from the beginning of therapy (at the end of the progestogen supplementation); 22 weeks from the start of therapy (at the end of the estrogen-only period); and at the end of the twenty-fourth week, during the progestogen supplementation. Therapy started at the second control visit. According to our schedule the first estrogen-only control was performed at least 40 days after the beginning of therapy and the second at least 40 days after the end of the previous progestogen supplementation. During therapy patients were studied on the second day of the patch administration. A 24-hour urine collection
Penotti et al. 1227
on the day before each visit was obtained from all patients. We asked the patients to keep the used patches and bring them at the following control visit. Compliance to the therapy was assessed at each control visit by asking the patients, evaluating the bleeding pattern, counting used patches, and assessing estrone3-glucuronide levels on the 24-hour urine collection. Routine blood laboratory examinations and complete urinalysis were performed on each patient before the beginning of the study. No subject had history of significant illness (e.g., diabetes mellitus; hypertension; cardiovascular, pulmonary, peripheral vascular, or renal disease). Estrone-3-glucuronide was measured on a sample from a 24-hour urine collection obtained at each control visit and stored at - 20° C until the assay. All samples were assayed together in duplicate with a radioimmunoassay method using dextran-charcoal for the separation of antibody-bound and free hormone fractions. Antibody was provided by Dr. P. Samarajeewa, Department of Biochemistry, University College, London. Sensitivity was 0.33 pmol/ml. Doppler studies. This study was carried out with a color flow imaging (Philips QAD - PV) system with a 7.5 MHz phased linear-array transducer and a bidirectional Doppler ultrasonographic system (Oxford Sonicaid Vasoflo 4) supplied with a spectrum analyzer and two probes (2 MHz pulsed wave and 4 MHz continuous wave for intracranial and extracranial evaluation, respectively). Both devices were used in the first control, whereas in the following examinations only the Doppler ultrasonography system was used. During the first control in each patient both common carotid arteries, carotid bulbs, and internal and external carotid arteries were scanned with color flow imaging, to identify plaques or stenoses. The presence of parietal alterations excluded subjects from study. Women who passed this preliminary examination were later subjected to the bidirectional Doppler study. The internal carotid artery on both sides within the first centimeter from bifurcation and the middle cerebral artery on both sides to a depth between 40 and 55 mm from the temporal surface were explored. For each evaluation, the pulsatility index on internal carotid and middle cerebral arteries was considered. Carotid blood flow is influenced by noise, ambient temperature, and food intake. 12, 13 Patients fasted for at least 2 hours were studied in a noiseless laboratory with constant temperature and light by the same examinator (M.F.) with earphones connected to the Doppler's audio output at the same time of day. Before examination they rested in a supine position for 15 minutes, until pulse rate varied by < 5 beats/min, and systolic-diastolic blood pressure varied by < 5 mm Hg over two consecutive measurements.
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November 1993 Am J Obstet Gynecol
Penotti et al.
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OL-----,----,------.-------r----r-----.--~ 22w 24w 6w 8w -6w o Time (weeks) Fig. 1. Plots of urinary concentration of estrone-3-glucuronide. Boxes contain 50% of observed values. Dots, Single case values; +, mean; - - -, median; whiskers, upper and lower quartiles of distribution of cases. Outliers (values that exceed 1.5 times the correspondent whisker) are not included.
In our laboratory the coefficient of variation of the pulsatility index on internal carotid and cerebral middle arteries was 1.5% and 2.2%, respectively. It was calculated over twelve Doppler evaluations in each of two premenopausal women taking a combined oral contraceptive (Ginoden, Schering), to exclude the influence of endogenous plasmatic estradiol on the impedance in explored arteries. Doppler evaluations were performed in 2 days, six measurements a day for each of the two subjects, with a 5-minute interval between measurements. Patients who entered the study underwent the bidirectional Doppler evaluation of internal carotid and middle cerebral arteries during each control visit from the first control visit to the end of the twenty-fourth week of the study. Statistical analysis. The statistical analysis of time changes of urinary estrone-3-g1ucuronide and pulsatility index measured in internal carotid and middle cerebral arteries were performed by analysis of variance (PROC GLM, SAS/PC version 6.04) for repeated measurements. A set of pairwise nonorthogonal contrasts between the two measurements before treatment and between each treatment time and baseline was performed. Measurements at each time were also compared with values at each other time. The level of significance of all pairwise contrasts was adjusted according to the Bonferroni procedure" to cope with the inference problem caused by multiple nonorthogonal contrasts.
Results
Twenty-one patients completed the 24-week study period, were compliant, and are still carrying on the therapy scheme. Four patients were lost at follow-up: two at the eighth week and two at the twenty-second week. Of these four patients, three did not want to continue because of personal difficulties in adhering to the close follow-up rules. The fourth patient was switched to different clinical care after the sixth week because of the pathologic result of basal time endometrial sampling. During the period of the study no significant changes took place either in blood pressure or in pulse rate. The results of estrone-3-g1ucuronide assessment in each patient are shown in Fig. 1. Mean values of estrone-3-g1ucuronide (SD) changed as follows: first control visit, 20.66 nmol per 24 hours (16.83); second control visit, 17.26 nmol per 24 hours (10.45); 6 weeks after starting therapy, 40.77 nmol per 24 hours (22.73), p < 0.05 versus basal time values; 8 weeks after starting therapy, 41.11 nmol per 24 hours (21.62), P < 0.05 versus basal time values; 22 weeks after starting therapy, 32.95 nmol per 24 hours (16.96); at the end of 24 weeks, 29.2 nmol per 24 hours (15.11). A preliminary statistical evaluation showed, at each control time, no difference in the results of Doppler evaluations between the right- and left-side arteries. Thus the mean value of the two sides was used in the analyses, both for the internal carotid and the middle cerebral artery. Single patient values of the pulsatility
Penotti et al. 1229
Volum e 169, Number 5 Am J Ob stet Gynecol
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0.5 '----.----r-----.---------r----=--r~---_.__----J 24 w 6w 8w 22w -6w o Tim e (weeks) Fig. 3. Plots of pu lsatility ind ex values in midd le cerebra l artery. Boxes contain 50% of observed values. Dots, Single case values; + , mean ; - - -, median ; whiskers, upper and lower quartiles of distribution of cases.
index in the internal carotid and middle cerebral arteries a re depicted in Figs . 2 and 3. Values of pulsatility index at each assess me nt (me an, SD, m inimum and maxim um value) in the internal caro tid and middle cerebral artery are shown in Tables
I and II , together with the number of measurements (left plus right side) perfor med at each time . No variatio n in pulsatility index was observed between th e two basal assess me nts in both arteries. In the in ternal carotid artery a highly significant
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November 1993 Am J Obstet Gynecol
Table 1. Internal carotid artery pulsatility index: Descriptive statistics Time (wk)
Scans Mean SD Minimum Maximum
- 6
o
6
8
22
24
44 0.833 0.\08 0.590 1.040
38 0.843 0.\09 0.570 1.070
44 0.795* 0.110 0.620 1.190
46 0.735 t 0.096 0.520 0.900
40 0.66 3! 0.076 0.530 0.830
38 0.647 0.067 0.530 0.780
*P = 0.001 versus basal time. t p < 0.01 versus previous time. !P = 0.0001 versus previous time.
-------I
., -
Table II. Middle cerebral artery pulsatility index: Descriptive statistics
Scans Mean SD Minimum Maximum
-6 44 0.873 0.120 0.640 1.130
T_im_e_(W_k_)
-r-r-
_
o
6
8
22
24
38 0.883 0.102 0.660 1.100
44 0.794* 0.12\ 0.600 1.080
46 0.777 0.104 0.620 1.030
40 0.658t 0.097 0.500 0.850
38 0.651 0.084 0.530 0.830
*P = 0.0001 versus basal time. tp = 0.0001 versus previous time.
(jJ = 0.001 ) reduction of pulsatility index was observed at 6 weeks. At 8 weeks, at the end of the progestogen supplementation, pulsatility index value showed a highly significant (jJ < 0.0 I ) reduction versus the previous time assessment. At week 22 a further highly significant (jJ = 0.0001) reduction was observed, when the pulsatility index showed a 25 .3% reduction versus basal time . No variation in pulsatility index was observed at week 24, at the end of the cyclic progestogen supplementation, compared with its value at th e previous time . In middle cerebral artery a highly significant (jJ = 0.000 I) reduction of pulsatility index was ob served at the sixth week . No variation was measured at 8 weeks, at the end of the first cyclic progestogen supplementation, in comparison with the previous value. At week 22 the pulsatility index showed a highly significant (jJ = 0.0001) reduction with respect to its value at the previous time, up to a maximum of 24.5% compared with basal time . At week 24 the pulsatility index value did not show any significant variation compared with the previous time . Co mment
Doppler examination of venous and arterial circulation was introduced in 1970 for a better knowledge of circulatory physiology. Since then new techniques have imp roved analysis of ultrasonic signal and have widened the field of investigation to abdominal an d intra-
cranial vasal diagnostics. Man y parameters automatically calculated by Doppler de vices are used to recognize and quantify hemodynamic changes of blood flow. Among these parameters pulsatility index has been used extensively and has been considered an expression of resistance to blood flow downstream of the point where measurements are performed. Its values vary in relation to age an d exploration site.":" We measured the pulsatility index on internal carotid and middle cerebral arteries to determine the correlations, if any, between hormonal status and blood flow. Our data clearly show a huge effect of estradiol administration in the cerebral blood flow of postmenopausal women. Our results showed no significant variation in pul satility index values between the two pretreatment measurements. These data illustrate the basal situation during postmenopausal estrogen deprivation. After 6 weeks of estradiol supplementation the pulsatility index already showed a significant reduction in the internal carotid arery. This reduction means a blood flow increase in cerebral arteries. This datum is confirmed by the ob servation of a significant reduction of pulsatility index in the middle cerebral artery at 6 weeks . This artery, which has no significant resistance district downstream, is subjected to a local autoregulation related to systemic blood pressure and metabolite change (Pco j, PO z, pH, hormones, tissue lactic acid). " ?" Therefore a middle cerebral artery pulsatility index reduction in po stmeno-
Penotti et al.
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pausal subj ects during hormon e replacement therap y, without blood pressure and metabolite change, confirm s an action of estradiol on middle cerebral artery. We did no t ob serve an y vari ation in the pulsatility index of the middle cerebral artery as mea sured at 8 weeks versus previous time evalu ation . On th e other hand, a further significant reduction in pulsatility index was mea sured in th e int ernal carotid artery at the same time. These facts see m to point out th at no impairmen t of estradiol's dilating effect should be ascrib ed to progestoge n sup pleme n tation. At th e twenty-second week a further significan t de crease in the pulsatility ind ex of both arteries was observed , when the pulsatility index was reduced by > 20% of basal time values . In both arteries no significant variation was measured at th e twenty-fourth week, at the end of the cyclic progestogen sup plementation . This datum again suggests the absence of a worsening effect of progestogen on estradio l-me diated vascular reactivity. Most epide miologic studies report that hormone re placem ent therap y can reduce the card iovascular mortality risk in postmenopausal wome n . A recent report from Stampfer et al." showed a 49% redu ction in the risk of major coronary artery disease among current estr ogen users. In this lar ge coho rt prospe ctive study the autho rs could demonstrate a significant reduction of the risk for this group of patients only. However, the mechanism by which these result s can be obtained remain s cont roversial. Un til now alm ost all epidemiolog ic and clinical studies have de alt with data referring to patients on estrogen repl acem ent therapy onl y, and the favorable effect of estrogen s on seru m lipid profile is the best known mechanism by which the protection ag ainst coronary artery disease is obtained: Furthermore, stud ies on coronary ang iogr aphy showed a redu ction of atherosclerosis in patients on estrogen replacement therapy." : 22 Whether progestogen supplementati on could be a factor th at reduces estrogeninduced protective effect is still open to debate. The reduction in pul satility index cause d by ho rmone replacement therapy that we observed in some arteries of the brain can be con sider ed an expression of a general (perha ps locally differ entiated ) reactivity of arteri es to estrad iol. In fact, in postmenopausal women Bourne et al." reported a 50% reduction in uterine artery p ulsatility index after tran sdermal estradiol admin istrati on. This action on vascular reactivity increases blood peripheral supply and can be considered one of the mechanisms by which estro gens exert their protective effect from cardiovas cular disease. In this regard it sho uld be very in teresting to kn ow whether the pul satility index in internal caro tid and middle cerebral art er ies changes with menop au se (i.e., from lower values to higher ones). Unfortunately, no research has yet
1231
Table III. Internal carotid artery and middle cer ebral artery pul satil ity ind ex in normal fertile and postmenopau sal women
Internal caro tid artery Middle cere bral artery No. of cases Age (yr)
Normal fertile women
Postmenopausal women
0.71 (0.09)
0.83 (0. 10)*
0.74 (0.1 I )
0.88 (0.10)*
15 28. 10 (4.07)
25 5 1.68 (3.30)
Values are mean ± SD. *P < 0.001 versus normal fertil e wom en .
been produced on this . However, the onl y published re sults (as far as we know) on premenopau sal middle cere bra l ar tery pul satil ity ind ex values see m to support th is hypothesis." Our own unpublished results on the pul satility index of these arteries of you ng fertile women are in agreement; mean values seem to be lower in th is group of patients compar ed with postmenopausal basal values (Table III ). Gangar et aJ.2 4 first showed a d irec t effect of estradiol su p pleme ntation on th e reactivity of the internal carotid art ery in postmenopau sal women and demonstra ted a correlation between men opausal age and pulsatility index. These authors studied a gr oup of women with a greater menopau sal age than in our study and used a different therapeutic sche m e in estrogen administr ati on . They did not report any variation in pulsatility index after 6 weeks of therapy. Our re sults show instead th at estrogen administration in early postmenopausal women is able to induce a rapid reduction of blood flow resistan ce in cerebral circulation . It has recently been shown that th e onset of menopause causes a worsening of th e lipid profile." It can be speculated th at the preventi on of thi s worsening exerte d by hormone repl acem ent therap y, together with the protective effect on the progression of atherosclerot ic pro cess, as proved in anima ls," s could be the lasting factor in cardiovascular protective action. A function al, and perhaps tran sient, act ion on vascular reactivity could be the facto r th at makes such a protective effect no longer so evident in past users.' Furthermore, because it seems th at women at lower risk for cardiovascular disease enj oy th e same relative benefit aga inst coronary disease as women in general,' it would ap pear th at such a fun ctional effect of estrogens on blood vessel reactivity could actually playa pivotal role in re alizing this protective action. H owever, a much lon ger follow-up on such gro ups of pat ients is needed to ascertain whether this effect on vascular reactivity is
1232
Penotti et al.
maintained throughout the p eriod of es trogen su p p lementation .
A limit o f o ur study is the lack of information as to the effect of unopposed estrogen supplementation on va scular rea ctivity. In fact , it is possible that the effect we evaluated co u ld be m o re in ten se and rap id without the cyclic proge st o gen su p p le men tatio n. H o wever, it is re co gnized wo r ld wid e that h ormone r epla cement ther apy in wo men who still h ave a n intact uterus sh o u ld b e r ealized with a cycl ic pro ge st ogen su p p le menta t io n to prevent e n dom e tr ia l hyperpla sia ; a ll wo men wh o to ok p art in o ur stu dy h ad n ot und ergone a ny previous gynecolo g ic su r gery. On th e o th er hand, it seem s to u s ve ry interesting that in o ur stu d y no r eduction in the favo r able, estrogen-induced pulsatility in dex variation was ever o b se rved at the e n d o f the cyclic progest o ge n su p p le me n tati ons. Thus in the light of o ur data a n d those of Gangar et al." it seems that progest o gen su p p le me n tation is n ot a b le to wo rse n the estro gen-induced favorable vascu lar m odifications. H o weve r, fu rther stu d ies a re n eeded to con cl u d e that medro xypr o gesterone acetate doc s n ot d e creas e th e beneficial effec t of es trogen on the art erial wall. For instance, a double-blind, crosso ver study with medroxyprogest erone ace ta te al one , es tr o ge n al one, then estrogen-medroxyprogest erone a ce ta te co u ld be carried o u t. We thank E. Kowarich for his wo r k in e strone- 3glucuronide urinary assays a n d Dr. M. Sanarico (N o us tat) for sta tistical evaluatio n o f d ata a n d p r oduction o f grap h ics . REFERENCES
I. Stampfer M], Willett WC, Colditz .lA, et al. A prospective study of postmenopausal estr ogen th erap y and coronary heart disease. N Eng l J Med 1985;313:1044-9. 2. Hen derson BE, Pagan ini-H ill A, Ross RK. Estrogen replacem ent th erapy and prot ection fro m acu te myocardi al infarc tion. Av ] OBSTET GYNECOL 1988;159:31 2-7. 3. Stampfer Mj , Co ld itz GA, Willett WC, et al. Postmen op ausal estrogen therapy an d cardiovasc ular disease. N Eng l J Med 1991; 325:756-62. 4. Bush Tl., Barrett- Connor E,Cowan LD, et al. Cardiovascular mo rta lity and non- con traceptive use of estrogen in wome n: res ults from the Lipid Research Clinics Pro gram follow-up study. Circulation 1987;75:1102-9. 5. Paganini-Hill A, Ross RK, Henderson BE. Postmen opausal estrogen treatmen t and stroke: a pr ospective study. BMJ 1988;295:519-22. 6. Fah raeu s L. The effects of estra dio l on blood lipid s and lipoprotein s in postm en op au sal wome n . Obs te t Gynecol 1988;72:18-22S. 7. Ada ms MR, Kapl an jR, Manuck SB, et al. Inh ibition of
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coro nary artery athero scler osis by 1713-estrad iol in ovariectomized monk eys. Arteriosclerosis 1990;10:105 1-7. 8. Wagner JD, Clark son TB, St. Clair RW, Schwenke DC, Adams MR. Estrogen replace ment th er ap y and coronary artery (CA) atherogen esis in surgically postmenopausal cynomolgus monkeys. Circulation 1989;80 (sppl 2):3 31, A-1320. 9 . Lin AL, Gonzales R, Carey KD, Shain SA. Estradiol-1713 affects estro gen recep tor d istribu tion and elevates pro ge stero ne receptor content in baboon aorta. Arte riosclerosis 1986;6:495-50 4. 10. Bourne T , Hillard TC, Whiteh ead MI, Croo k D, Campbe ll S. Oe strogens, ar te rial sta tu s, and postmenopausal women . Lan cet 1990;335:1470-1. I I. Pines A, Fisman EZ, Levo Y, et al. T he effects of hormon e repl acem ent therap y in normal postm en opau sal wome n: measur em ents of Dop pler- derived parameter s of aortic flow. AM J OBSTET GYN ECOJ. 1991;164 :80 6-12. 12. Meire HB, Kossoff G, Lod ge T. Recent advances in ultrasound. In : Steiner RE, cd. Recent advances in radiology and med ical imag ing. Ed inburgh: Churchill Livingst on e, 1983:63. 13. Grant EG. In: Grant EG, White EM, eds. Dupl ex sonography of th e cerebrovascular system. New York: Springer 1988:37. 14. Kirk RE. Experimental design : procedu res for the beh aviora l sciences . Monter ey, Californ ia: Bro oks Cole, 1982: 105-6. 15. Vr iens EM, Kraaier V, Mussbach M, Wieneke GH, Van Huffel en AC. T ran scranial pul sed Dop pler measur em ents of blood velocity in th e middl e cerebral artery. Ultrasound Med BioI 1989; 15:40 5-8. 16. Otis SM, Rossman ME, Schneid er PA, Rush MP, Ringelstein EB. Relationship of cerebr al blood flow regulation to acute mountain sickness . J Ultrasoun d Med 1989;8: 143-8. 17. Matti e H, Gro limun d P, Hu bert P, Stu rzen egger M, Zurbrugg HR . Transcra nia l Doppler sonogra p hy findings in midd le cerebral artery disease . Arch Neurol 1988;45:28995. 18. Reivich M. Arterial Pco, and cer ebra l haemod ynami cs. Am J Physiol 1964 ;25:206. 19. Bishop CCR, Powell S, Rutt D, Browse NL. Transcra nial Doppler mea surem ent of middle cerebral artery blood flow velocity: a validation study. Stroke 1986;17 :913- 5. 20. Ringelstein EB, Sievers C, Ecker S, Schn eid er PA, Otis SM. Noninvasive assessm ent of CO 2-indu ced cerebral vasom otor respon se in normal indi viduals an d patients with internal carotid artery occlusio ns. Stroke 1988 ;19:963 -9 . 21. Sullivan JM , Zwagg RV, Lemp GF, et al. Postmenop au sal estrogen use and coronary at heros clerosis. Ann Int ern Med 1988;108:358-63. 22. Gro uchow HW, And er son AJ , Barboriak j], Soboci nski KA. Postm en op au sal use of estrogen and occlusio n of coronary art eries. Am Heart ] 1988;115:954-63. 23 . Sha mma FN, Fayad P, Brass L, Sarrel P. Middl e cere bra l artery blood veloci ty during co nt rolled ovarian hyperstimulation . Fertil Steri l 1992;57:1 022 -5. 24. Gangar KF, Vyas S, Whitehead M, Croo k D, Meire H, Campbell S. Pulsatility inde x in internal caro tid artery in relation to tran sde rma l oes tradiol and tim e since menopause. Lancet 1991; 338:839-42. 25. j en sen J, Nilas L, Chr istianse n C. Influ en ce of men op au se on seru m lipid s and lipop rot eins. Maturi tas 1990 ;12:3213 1.