Vasomotor flushes and the release of peripheral immunoreactive luteinizing hormone-releasing hormone in postmenopausal women*

FERTILITY AND STERILITY Copyright

©

Vol. 41, No.6, June 1984 Printed in U.SA.

1984 The American Fertility Society

Vasomotor flushes and the release of peripheral immunoreactive luteinizing hormone-releasing hormone in postmenopausal women*

Veronica Ravnikar, M.D.t Karen Elkind-Hirsch, Ph.D. Isaac Schiff, M.D. Kenneth J. Ryan, M.D. Dan Tulchinsky, M.D. Division of Fertility, Endocrine and Menopause, Department of Obstetrics and Gynecology, Harvard Medical School, Brigham and Women's Hospital, Boston, Massachusetts

The plasma concentrations of immunoreactive luteinizing hormone-releasing hormone (lR -LH -RH) and LH and the peripheral skin temperature of symptomatic menopausal women were recorded continuously and compared with those of asymptomatic menopausal women. These studies were performed by an intermittent withdrawal technique (n = 13) and a continuous withdrawal technique (n = 3). The mean plasma concentration of IR -LH -RH was much higher in symptomatic than in asymptomatic women by both methods of analysis. Large episodic secretory spikes of IR -LH -RH and a temporal correlation between the IR -LH -RH spikes and temperature elevations was found (P ~ 0.025) only in symptomatic women. Fertil Steril41:881, 1984

Menopausal vasomotor flushes, which occur after cessation of ovarian function, are considered to be a result of thermoregulatory instability.1, 2 Since vasomotor flushes and luteinizing hormone (LH) surges have been shown to occur concurrently, interest has been raised as to whether there is an association between these two phenomena of the menopause. 3 -5 However, a causal link between pituitary LH secretion and vasomotor flushes (VMF) has not been established. Flushes occur in subjects with pituitary insufficiency6 and

in patients whose pituitary secretion of LH has been ablated with a long-acting luteinizing hormone-releasing hormone (LH-RH) agonist. 7 Therefore, it may be erroneous to compare vasomotor instability, a central hypothalamic event,8 with pituitary secretion of hormones. We designed a study to compare the concentrations of endogenous, immunoreactive LH-RH (IRLH-RH)9 in the peripheral plasma in symptomatic (S) menopausal women and asymptomatic (NS) menopausal women and to correlate them with fluctuations in peripheral temperature (.IT) during VMF.

Received November 23, 1983; revised and accepted February 27, 1984.

*Presented in part at the Sixty-Third Annual Meeting ofthe Endocrine Society, June 17 to 19, 1981, Cincinnati, Ohio. tReprint requests: Veronica Ravnikar, M.D., Harvard Medical School, Division of Fertility, Endocrine and Menopause, Brigham and Women's Hospital, Room 3-069A, 75 Francis Street, Boston, Massachusetts 02115. Vol. 41, No.6, June 1984

MATERIALS AND METHODS INTERMITIENT WITHDRAWAL GROUP (IWG)

Thirteen postmenopausal patients, including 7 Sand 6 NS women, were studied. No significant Ravnikar et al. IR-LH-RH during vasomotor flushes

881

differences were present between the mean ages of the two groups, 45.14 ± 6.26 (S) and 51.17 ± 0.426 (NS); the mean heights, 63.21 ± 3.34 (S) and 62.00 ± 1.26 inches (NS); or the mean weights, 137.57 ± 15.86 (S) and 141.83 ± 23.57 pounds (NS). However, the mean years since menopause initiation was shorter in the S group (1.13 ± 0.62 years), compared with the NS group (5.0 ± 3.22 years) (P < 0.0096). Approximately half of the 13 women underwent natural menopause and the other half surgical castration. None of the patients received any medication for at least 6 months prior to the study. Plasma 17~­ estradiol and serum gonadotropins were in the menopausal range in all patients studied. At 8:00 A.M. on: the day of the study, patients reported to our research unit and signed an informed consent approved by the Human Subjects Review Committee. After being admitted in the fasting state to a quiet room with an ambient temperature of 70° to 75° F, they remained recumbent throughout the procedure. Skin temperatures were recorded from the dorsum of the proximal phalanx of the third finger using a thermistor probe (Yellow Springs Instrument Co., Inc., Yellow Springs, OH), a telethermometer, and a potentiometric strip recorder. In the brachial vein of the opposite arm a heparin lock was inserted, and 10-ml blood samples were drawn every 10 minutes for 3 to 4 hours. During this study period, patients were asked to indicate the immediate onset of a subjective feeling of a hot flash. During the VMF, blood samples were drawn every 1 to 5 minutes until symptoms abated. Seven milliliters of the 10-ml blood samples were aliquoted and placed into chilled heparinized tubes and centrifuged immediately. The plasma was then immediately extracted, lyophilized, and stored, as previously described. 9 The remaining 3-ml serum samples for LH were collected and stored at - 20° C until assayed. Measurements for plasma IRLH-RH and serum LH were done by radioimmunoassay as previously reported. 9 -11 Antiserum of LH-RH, R42, is sensitive to and specific for the entire decapeptide. 9 LH was measured by a double-antibody radioimmunoassay technique. 10, 11 The intraassay coefficients of variation were 5% for ffi-LH-RH and 8% for the LH assay. - The lower limit of detectability for ffi-LH-RH was 0.3 pg/ml, and a fourfold rise from baseline to levels > 1.2 pg/ml was defined as a spike. Temperature elevations exceeding 1°C were considered significant,2-5 and a rise of LH that exceeded 882

Ravnikar et al. IR -LH -RH during vasomotor flushes

baseline by 20% was considered a spike. 11 Time 0 was the onset of a flush; LH and IR-LH-RH levels were correlated with these temperature changes. CONTINUOUS WITHDRAWAL GROUP (CWG)

In order to determine whether different techniques of blood sampling provided additional data, blood samples were collected continuously over 1O-minute intervals in three patients via a B. Braun Perfusor pump (Quigley-Rochester, Inc., Rochester, NY) (total of 10 ml drawn every 10 minutes). Three menopausal patients were studied in this group: two S patients (G. H. and R.C.) who had recently experienced menopause, one by surgical castration and the other naturally. The third patient (S. C.) had been menopausal for 4 years. STATISTICAL METHODS

Statistical methods used for both groups (lWG and CWG) were standard unpaired and paired two-tailed t-test analyses. Linear relationships between different analyses were defined by using the Pearson product-moment correlation test. Fischer Z transformation on Pearson's coefficients was also used to determine the significance of correlation.

RESULTS INTERMITl'ENT WITHDRAWAL GROUP

Flush

All VMF were associated with significant dermal temperature changes. Significant fluctuations in aT > 1°C were identified 19 times in the S group (frequency of one every 1.05 hours) and were absent in the NS group (compare Figs. 1 and 2). The average duration of the flush episode was 8.17 ± 1.51 (standard error [SE]) minutes. LH

The numbers of pulses per hour of LH between Sand NS groups did not differ: 1.1 pulseslhour (S) versus 1 pulselhour (NS). Also, when the LH levels (mIU/ml) of the two groups were compared, no difference in the mean levels was observed: 51.75 ± 4.6 (SE) in the S group and 55.23 ± 6.3 (SE) in the NS group (P = 0.66). There were 19 recorded temperature spikes, and 15 of these were associated with an LH spike. There were 18 indepenFertility and Sterility

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In the S group the mean IR-LH-RH level prior to a vasomotor episode was 3.38 ± 3.1 pg/ml (SE), which was significantly higher than the levels obtained + 5 minutes after the temperature rise (mean LH-RH, 1.562 ± 0.406 pg/ml) (P < 0.05) and + 10 minutes after a flash (mean LH-RH, 1.2146 ± 0.551 pg/ml) (P < 0.01). Furthermore, there was a statistically significant correlation between the -15-minute LH-RH value prior to a flush episode and the + 5-minute ~LH value (r = 0.799; P < 0.05). However, for each point in time, absolute values of IR-LH-RH did not show a linear correlation with LH values even though the mean spike frequency was comparable: 1.1 pulses/hour for LH and 1.3 pulses/hour for IR-LH-RH, respectively. Sixteen of the 19 vasomotor episodes were accompanied by an IR-LH-RH peak. Of the IR-LHRH peaks, 15 of 16 occurred from -15 minutes to + 5 minutes proximal to the vasomotor episode. The IR-LH-RH secretory pattern in the S group demonstrated a central tendency with the onset of a VMF, and this correlation was greater than that expected by chance alone (X 2 = 5.28; P ~ 0.025), i.e., if the IR-LH-RH pulses were to occur randomly at a frequency of one per hour. Finally,

dent LH spikes, for a total of 33 LH pulses in the S patients. When the absolute ~LH values from pre-flash baseline were graphed (Fig. 3) and compared with temperature changes (~T) proximal to a VMF, a nadir in LH levels occurred immediately after the temperature rise. This was followed by a significant LH peak at + 10 time interval (P < 0.0006). In the entire S group, there was a linear correlation noted between the temperature rise (0 F) and actual LH values (r = 0.4; P < 0.003).

IR-LH-RH The two groups differed markedly in their IRLH-RH levels (Figs. 1 and 2). In the S group, the amplitude of the spikes in IR-LH-RH levels was much greater than in the NS group. The mean IR-LH-RH (pg/ml) values were S, 2.66 ± 0.6 (SE) and NS, 0.56 ± 0.14 (SE) (P < 0.0096). Levels> 2 pg/ml were observed in six of the seven S women and in none of the NS women. The number of IR-LH-RH spikes was also markedly different; 1.26 pulseslhour (S) and 0.4 pulseslhour (NS), respectively (P < 0.0001).

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Figure 2 IWG: IR-LH-RH (upper panel) and LH (lower panel) secretory patterns in six NS patients.

Ravnikar et al. IR -LB -RB during vasomotor flushes

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Flush Episode In this group seven flush episodes were recorded in the S group (Figs. 4 and 5), and there were no flushes recorded in the NS group (Fig. 6). The onset of the flush episode is noted by an asterisk (Figs. 4 and 5). The mean duration of the episodes was 10.2 ± 1.64 minutes (SE).

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LH The mean LH values (mean of -10 minutes to ± 2.8 (SE) and (mean of 0 to + 10 minutes) of 76.5 ± 3.7 (SE) were not different from the mean of all samples, 80.3 ± 1.4 (SE) . Again, there was a slight nadir in LH values immediately after the hot flash, although it was not as pronounced as that of the IWG. No correlation by linear regression analysis was achieved between IR-LH-RH and LH at each time (r = 0.189) or staggered at ± 10-minute intervals.

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Figure 3 IWG: Correlation of the net change in temperature after a vasomotor episode and the net change in LH from the preflash baseline. Zero time indicates the onset of the temperature rise associated with a VMF. Mean increment changes of LH are mean values obtained for each 5-minute time interval. The increment change of LH at + 10 minutes is significantly different (P < 0.0006) from the preflash baseline.

IR-LH-RH

there was also an inverse correlation detected between the absolute level of IR-LH-RH and the change in temperature 5 minutes hence (r = - 0.66; P < 0.05), again indicating that the pulse of IR-LH-RH is centered prior to the vasomotor episode. An analysis of the sample means in the IWG should take into consideration the uneven sampling technique; therefore a CWG was studied.

In the first patient (Fig. 4), a strikingly vivid correlation ofVMF (asterisk) and IR-LH-RH was seen. In the second patient, four VMF were recorded, and there were several independent pulses ofIR-LH-RH in proximity to the flush episodes (Fig. 5). The NS patient (Fig. 6) had no IR-LH-RH pulses. In the S patients, the mean IR-LH-RH secretion prior to a flush was 2.8 ± 0.6 pg/ml (SE), compared with an overall mean of 1.5 ± 0.2 pg/ml (SE) (P < 0.02).

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Ravnikar et a1. IR-LH-RH during vasomotor flushes

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DISCUSSION

This study demonstrates that IR-LH-RH levels are higher in S menopausal women than in NS women, and that they are secreted in an episodic fashion. Moreover, IR-LH-RH spikes appear to correlate with the onset of a vasomotor episode. In the IWG, we counted 33 LH pulses with 19 recorded hot flushes; 15 of these LH spikes were associated with significant temperature rises. In comparison, Casper et al. 3 recorded 66 LH pulses, only 55 of these associated with hot flashes. Tataryn et al. 4 demonstrated 31 pulses, 26 of these in close temporal relationship to the temperature elevations. Our withdrawal technique was not similar. In the IWG, we drew specimens every 10 minutes followed by every 1 to 5 minutes at the onset of a flush. In the CWG, we withdrew evenly every 10 minutes. This is compared with a sampling taken every 15 minutes punctuated by samplings every 5 minutes at the time of a flush in other studies. 3 -5 ,12 Seven millimeters of whole blood was required for each IR-LH-RH sample; the study period for each one of our patients was, of necessity, shorter. Some of our patients (i.e., J. P. and R. D., in the IWG) also tended to have clusters of hot flashes and not an even hourly distribution. Therefore, in the IWG we were withdrawing more frequently in a shorter time frame and may have demonstrated many independent LH pulses. In order to pursue this discrepancy and to establish a more even picture of IR-LH-RH secretion, we turned to the CWG. However, this technique probably obliterated our ability to demonstrate the LH pulses which occur with the vasomotor episode. Along with this lack of a persistent correlation between LH and hot flushes, we found no difference in LH secretory activity between the Sand Vol. 41, No.6, June 1984

NS groups. Previous researchers 13 have shown that absolute levels of LH do not differ between S and NS patients. There are no data available comparing IR-LH-RH levels between Sand NS groups, and this is the first study to do so. In the IWG, a linear association between IRLH-RH and LH is not seen. However, there is a correlation between the -15-minute IR-LH-RH level and the peak, i.e., + 5-minute LH value after the flush episode. In the IWG, there is an ~ssociation found between the increment change m LH from preflash baseline and the vasomotor temperature rise (Fig. 3) which we previously desc~ibed. 14 Prior to this LH peak and immediately pnor to the temperature peak, there is a nadir in the LH concentration, as others have shown. 4 , 12 It is both around this nadir and prior to the onset of the flush that we found the IR-LH-RH peaks cluster. This same descriptive relationship in IRLH-RH secretion can be demonstrated in the S patients of the CWG. However, in the CWG, no relationship between the hot flash episodes and an LH spike could be demonstrated. Although the sample size is limited here, it does suggest that a different sampling technique obliterates the LH pulses that occur with flushes. J:

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Figure 6 CWG: IR-LH-RH (upper panel) and LH (lower panel) in an NS patient. Ravnikar et al. IR-LH-RH during vasomotor flushes

885

A major question is whether or not it is possible to measure hypothalamic IR-LH-RH by peripheral sampling. We have reported our technique previously; the antibody R42 used is specific to the entire decapeptide. 9 Other than localization ofIRLH-RH-like substances from the pancreas,15 there is no other known source of LH-RH in humans. We cannot state that the IR-LH-RH we are measuring approximates hypothalamic ~ pituitary signals. However, we do demonstrate episodic secretion that centers around a vasomotor episode. In the S perimenopausal patients, therefore, this is most likely "spillover" from either another source in the brain or the portal system itself. Since IR-LH-RH and LH have discrepant half-lives, we cannot expect to find a direct linear correlation between IR-LH-RH and LH. Although IR-LH-RH is considered to be a neurotransmitter for sexual arousal, it is not known to be a neurotransmitter for thermoregulation. 16 However, the organum vasculosum lamina terminalis (OVLT), which contains nuclei for IR-LHRH, and the center for hypothalamic temperature control in the ventral preoptic area are in close proximityP Indeed, Lomax et al. 18 showed that infusion of LH-RH in the preoptic area of rats caused a aT rise, whereas a saline control infusion does not. When an LH-RH agonist was given to nine healthy males, producing a reversible decline in aLH, follicle-stimulating hormone, and testosterone secretion, in four of the nine "menopausal type" hot flashes developed. 19 When a similar potent LH-RH agonist was used to abolish pituitary LH secretion in menopausal women, VMF continued unabated. 7 Finally, hot flashes have been induced in premenopausal women treated with long-acting gonadotropin-releasing hormone agonists. 20 We can therefore postulate that cessation of ovarian function creates a transient increase in central LH-RH secretion, which is then reset in the later menopausal period. This either causes, or is a result of, central neuroendocrine instability, of which one major manifestation is aberrant temperature control in the immediate perimenopausal phase. The mean IR-LH-RH secreted in the Sand NS patients is lower than that found in the periovulatory period during a similar period of LH hypersecretion. 21 The sensitivity of the anterior pituitary in ovariectomized rats actually increases, and they are more efficient in releasing LH with smaller amounts of LH-RH.22 Furthermore, immediately after ovariectomy in rats, a differential 886

Ravnikar et al. IR -LH -RH during vasomotor flushes

reorganization of hypothalamic LH-RH release between the median eminence and OVLT has been demonstrated. 23 Also, absolute levels ofLHRH in the human hypothalamus are generally lower in menopausal women. 24 Our studies in S subjects were done (1.13 ~ 5 years) after the initiation of the menopause. We cannot, therefore, comment on the immediate changes in peripheral IR-LH-RH in the perimenopausal period. It appears that very little LHRH is required to maintain the long-term hypergonadotropic state in hypogonadal women. 22 , 24 However, the increased secretion of IR-LH -RH in the perimenopausal patients may be related to a reorganization of hypothalamic secretion, which by proximity affect the thermoregulatory center. In summary, we can hypothesize that the excess pulses ofIR-LH-RH in S women which center around the onset of a hot flush are markers of thermoregulatory instability in the early perimenopausal period. We can speculate that our inability to adequately define a postflash LH peak in all groups lies in our method and frequency of sampling. Finally, if the secretion of LH-RH is related to the genesis of a VMF, it may provide help in determining the more proximate factors which generate the symptom.

Acknowledgments. Antiserum to LH was generously provided by the National Pituitary Agency (NIAMDP). Antiserum R42, specific to the entire decapeptide of LH-RH, was generously provided by Drs. Nett and Niswender. Instrumentation used included: (1) B. Braun Perfusor No. 2310 Model # 71103 and (2) Yellow Springs Instrument Co., Inc., thermistor probe and telethermometer Model 41TA, Serial 476. We would like to acknowledge the assistance of Denise Lung and Anna Laffond for preparation of the manuscript.

REFERENCES 1. Molnar GW: Body temperatures during menopausal hot

flash. J Appl Physiol 38:499, 1975 2. Meldrum DR, Shamonke 1M, Frumar AM, Tataryn IV, Chang RJ, Judd HL: Elevations of skin temperature of the finger as an objective index of postmenopausal hot flashes: standardization of the technique. Am J Obstet Gynecol 135:713, 1979 3. Casper RF, Yen SSC, Wilkes MM: Menopausal flushes: neuroendocrine link with pulsatile LH release. Science 205:823, 1979 4. Tataryn IV, Meldrum DR, Lu KH, Frumar AM, Judd HL: LH, FSH, and skin temperature during the menopausal hot flash. J Clin Endocrinol Metab 49:152, 1979 Fertility and Sterility

5. Meldrum DR, Tataryn IV, Frumar AM, Erlik Y, Lu KH, Judd HL: Gonadotropins, estrogens and adrenal steroids during the menopausal hot flash. J Clin Endocrinol Metab 50:685, 1980 6. Meldrum DR, Erlik Y, Lu JKH, Judd HL: Objectively recorded hot flushes in patients with pituitary insufficiency. J Clin Endocrinol Metab 52:584, 1981 7. Casper RF, Yen SSC: Menopausal flushes: effect ofpituitary gonadotropin desensitization by a patent luteinizing hormone-releasing factor agonist. J Clin Endocrinol Metab 53:1056, 1981 8. Cox B, Lomax P: Pharmacologic control of temperature regulation. Annu Rev Pharmacol Toxicol 17:341, 1977 9. Elkind-Hirsch K, Ravnikar V, Schiff I, Tulchinsky D, Ryan KJ: Determinations of endogenous immunoreactive luteinizing hormone-releasing hormone in human plasma. J Clin Endocrinol Metab 54:602, 1982 10. Odell WD, Ross GT, Rayford PL: Radioimmunoassay for luteinizing hormone in human plasma or serum: physiologic studies. J Clin Invest 46:248, 1967 11. Santen RJ, Bardin CW: Episodic luteinizing hormone secretion in man. J Clin Invest 52:2617, 1973 12. Tataryn IV, Meldrum DR, Frumar AM, Lu KH, Judd HL, Bajorek JG, Chesarek W, Lomax P: The hormonal and thermoregulatory changes in postmenopausal hot flushes. In Thermoregulatory Mechanisms and Their Therapeutic Implications. Presented at the Fourth International Symposium on the Pharmacology of Thermoregulation, Oxford, 1979. Basel, Karger, 1979, p 202 13. Campbell S: Intensive steroid and protein hormone profiles en p'>stmenopausal women experiencing hot flushes and a group of controls. In The Management of the Menopause and Menopausal Years, Edited by S Campbell. Baltimore, University Park Press, 1976, p 63 14. Albrecht BH, Schiff I, Tulchinsky D, Ryan KJ: Objective evidence that placebo and oral medroxyprogesterone acetate therapy diminish menopausal vasomotor flushes. Am J Obstet Gynecol 139:631, 1981 15. Seppala M, Wahlstrom T, Leppaluoto J: Luteinizing hormone-releasing factor (LRF)-like immunoreactivity in rat pancreatic islet cells. Life Sci 25:1489, 1979

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16. Pfaff DW: Luteinizing hormone-releasing factor potentiates lordosis behavior in hypophysectomized ovariectomized female rats. Science 182:1148, 1973 17. Sampson WK, Snyder G, Fawcett CP, McCann SM: Chromatographic and biologic analysis of ME and OVLT LHRH. Peptides 1:97, 1980, 18. Lomax P, Bajorek JG, Chesarek W, Tataryn IV: Thermoregulatory effects of luteinizing hormone-releasing hormone in the rat. In Thermoregulatory Mechanisms and Their Therapeutic Implications. Presented at the Fourth International Symposium on the Pharmacology of Thermoregulation, Oxford, 1979. Basel, Karger, 1980, p 208 19. Linde R, Doelle GC, Alexander N, Kirschner F, Vale W, Rivier J, Rabin D: Reversible inhibition of testicular steroidogenesis and spermatogenesis by a patent gonadotropin-releasing hormone agonist in normal men. N Engl J Med 305:663, 1981 20. DeFazio J, Meldrum DR, Laufer L, Vale W, Rivier J, Lu JKH, Judd HL: Induction of hot flashes in premenopausal women treated with a long acting GnRH agonist. J Clin Endocrinol Metab 56:445, 1983 21. Elkind-Hirsch K, Ravnikar V, Tulchinsky D, SChiff I, Ryan KJ: Episodic secretory patterns of immunoreactive luteinizing hormone-releasing hormone (IR-LH-RH) in the systemic circulation of normal women throughout the menstrual cycle. Fertil Steril 41:56, 1984 22. Porter JC, Ben-Jonathan N, Oliver C, Eskay RL, Winters AJ: Interrelationship of CSF, hypophyseal portal vessels, and hypothalamus and their role in the regulation of anterior pituitary function: neuroendocrine regulation of fertility. International Symposia Semla 1974. Basel, Karger, 1976, p 71 23. Kobayashi RM, Lu KH, Moore RY, Yen SSC: Regional distribution of hypothalamic luteinizing hormone-releasing hormone in proestrous rats: effects of ovariectomy and estrogen replacement. Endocrinology 102:98, 1978 24. Parker CR, Porter JC: Luteinizing hormone-releasing hormone and thyrotropin-releasing hormone in the hypothalamus of women: effects of age and reproductive status. J Clin Endocrinol Metab 58:488, 1984

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