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Time-resolved fluoroimmunoassay compared with radioimmunoassay of luteinizing hormone*
1 Vol. 59, No.2, February 1993
FERTILITY AND STERILITY
Printed on acid·free paper in U.S.A.
Copyright CI 1993 The American Fertility Society
Time-resolved fluoroimmunoassay compared with radioimmunoassay of luteinizing hormone*
Edward C. Ditkoff, M.D.t Jay H. Levin, M.D.:j: Wellington L. Paul, Ph.D.§ Rogerio A. Lobo, M.D. II University of Southern California School of Medicine, Los Angeles, and SmithKline Beecham Clinical Laboratory, Van Nuys, California
Objective: To determine if fluoroimmunoassay (FIA) of serum luteinizing hormone (LH) is more useful clinically than a conventional radioimmunoassay (RIA) because it has been suggested that FIA closely reflects biological activity. Design: Comparison of serum LH measurements by RIA and FIA during various perturbations in normal ovulatory women and in women with polycystic ovarian syndrome (PCDS). Setting: Normal ovulatory subjects were healthy volunteers and women with PCDS were untreated and newly diagnosed outpatients in our Reproductive Endocrinology/Infertility Clinic, Women's Hospital, at the Los Angeles County+University of Southern California Medical Center. Participants: Fifty-three normal ovulatory women, ages 20 to 35, and 27 women with PCDS, ages 21 to 35. All were in good health and received no other medications during the study period. Results: Fluoroimmunoassay of serum LH reflected status of known altered bioactivity better than with a conventional RIA. This was most evident during conditions of gonadotropin suppression and in patients with PCDS. An excellent correlation was found between values of FIA and RIA. Conclusions: The measurement of LH by FIA is clinically useful, specifically when a change in biological activity of LH is sought. Fertil Steril 1993;59:305-10 Key Words: Serum LH, FIA, RIA, biological activity
Since the development of radioimmunoassay (RIA) and related assay techniques by Yalow and Berson (1) and Elkins (2) in the late 1950s, these assays have been extensively applied to numerous Received April 23, 1992; revised and accepted October 28, 1992. * Presented in part at the 47th Annual Meeting of The American Fertility Society, Orlando, Florida, October 19 to 24, 1991. t Present address: Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Columbia-Presbyterian Medical Center, New York, New York. :j: Present address: Center For Reproduction, Hinsdale Hospital, Hinsdale, Illinois. § Present address: SmithKline Beecham Clinical Laboratory. II Reprint requests: Rogerio A. Lobo, M.D., Division of Reproductive Endocrinology, University of Southern California School of Medicine, Women's Hospital, Room 1M2, Los Angeles Courity + University of Southern California Medical Center, 1240 North Mission Road, Los Angeles, California 90033.
Vol. 59, No.2, February 1993
analytes in reproductive endocrinology (3). Several newer immunoassay techniques have been recently developed and among these a fluoroimmunoassay (FIA) has be'come available (4). Fluoroimmunoassay uses a europium chelate as a label with a fluorescence decay time of 10-3 to 10-6 per second, which achieves an excellent sensitivity compared with RIA (3, 5). In addition, it has been suggested recently that fluoroimmunoassay (FIA) reflects biological activity better than conventional RIA and could therefore be more useful clinically (6-10). Our purpose was to determine if FIA is useful clinically and if it reflects trends in bioactivity of serum luteinizing hormone (LH) more than a conventional RIA. We therefore chose to study normal women during various pertubations known to affect
Ditkoff et al.
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biological activity as well as in women with polycystic ovarian syndrome (PCOS). The difference in bioactivity and immunoactivity of serum LH during these perturbations in normal women and in women with PCOS has been described by us and others previously (11-21). Therefore, although we have carefully chosen these conditions for study, we have not repeated the measurements by conventional bioassay. Our comparisons are primarily meant to test the clinical usefulness of the FIA for LH, and information regarding LH bioactivity can only be considered by extrapolation. MATERIALS AND METHODS Subjects
Fifty-three normal ovulatory women, ages 20 to 35 (30.42 ± 0.53), and 27 women with PCOS, ages 21 to 35 (28.1 ± 0.98), were studied. All women had documented ovulatory cycles with lengths of 26 to 32 days and were using a mechanical form of contraception at the time of enrollment. The subjects were in good health and received no medications during the study period. No woman had received any hormonal therapy for at least 2 months before the study. The women with PCOS were 27 consecutive new patients with this diagnosis in our Reproductive Endocrinology/Infertility Clinic, Women's Hospital, at the Los Angeles County+University of Southern California Medical Center. None of these women was taking any medications, and all had perimenarchial onset of oligomenorrhea, chronic anovulation, and elevated androgen levels (testosterone > 80 ng/dL) and/or hirsutism and normal levels of prolactin. No patient was virilized and none had evidence of adrenal enzymatic deficiencies. The project was approved by our Institutional Review Board, and written informed consent was obtained from each subject. Baseline Studies
All sera were collected at 8:00 A.M. for comparing measurements of LH by conventional RIA and by FIA. Nineteen had serum drawn during the early follicular phase (days 1 to 4), and 12 had serum drawn during their midfollicular phases (days 7 to 9). Ten had serum drawn during the late follicular phase when follicles reached a maximum diameter of 16 to 18 mm as measured by a 5.0-MHz Siemens
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Sonoline SL 1 model sonogram (Siemens Medical Systems, Inc., San Ramon, CA). Finally, 12 had serum drawn on day 21 of their cycles. In 27 women with PCOS, serum was also collected at 8:00 A.M. on day 4 after an induced menses after the administration of 150 mg of progesterone (P) in oil intramuscularly. Elevation of LH During the Normal Menstrual Cycle
Five normal women had early follicular phase sampling every 10 minutes for 3 hours to detect at least one LH pulse. Ten normal women had serum drawn at 8:00 A.M. and 4:00 P.M. during the late follicular phase to detect the LH surge. The acute effect of gonadotropin-releasing hormone (GnRH) 100 f.lg IV on LH was also evaluated in 5 normal women during the early follicular phase. Serum was obtained at 0, 10, 20, 30, 60, and 90 minutes after the bolus of GnRH. Suppression of LH
Leuprolide acetate (LA; TAP Pharmaceuticals, Deerfield, IL), a GnRH agonist (1 mg/d SC), was given to 12 normal ovulatory women for 2 weeks from the midluteal phase until estradiol (E 2) levels were <105 pmoljL. Serum LH was again obtained at this time again. For comparison purposes, LA (1 mg/d SC) was given to eight women with PCOS for 2 to 4 weeks until E2 levels were <105 pmoljL. Nal-Glu (50 f.lg/kg 1M), a GnRH antagonist, was given once during the early follicular phase in 7 women and during the late follicular phase to delay the LH surge in 10 women. This protocol was carried out previously for other purposes and has been reported (22). Serum was obtained to measure LH as well as E2 24 hours after each Nal-Glu administration for monitoring purposes. Serum LH was also measured 8 hours after Nal-Glu administration during the late follicular phase. Norethindrone (NET) (10 mg/d) was administered orally to five women during the early follicular phase for a 2-week duration with serum obtained before and immediately after NET administration. Hormonal Assays
All bloods were separated and sera were stored at -20°C until analyzed. Serum LH was measured from identical specimens by a highly specific conventional RIA (23) and an established FIA (3). The time-resolved FIA used europium as a chelate label
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Wi
using Delfia human LH Spec kits (Pharmacia Inc., Piscataway, NJ) according to the method of Lovgren et a1. (3). The sensitivity of the assay was 0.1 mlU/ mL. Serum E2 was measured by RIA (24) (Pantex, Santa Monica, CAl after extraction with hexane: ethyl acetate (2:3). Serum P was measured to confirm the midluteal status of some of the subjects (31.8 to >10 nmol/L) by using a direct RIA kit (Diagnostic System Laboratories, Webster, TX). From each patient, all sera were run in a single assay to reduce assay variation. The mean intra-assay and interassay coefficients of variation (CVs) for the serum RIAs were as follows: LH, 3.2% and 9.2%; E 2, 8.1% and 12.6%; P, 6.6% and 7.2%, respectively. The intra-assay and interassay CVs for LH serum by FIA were 4.4% and 7.6%, respectively. Statistical Analysis
All data are expressed as means ± SEM. Statistical analyses were performed using the two sampled Kolmogro-Smirov test and the Pearson's regression analysis. RESULTS
In comparing LH by FIA and RIA during the early follicular phase, late follicular phase, and midluteal phase samples, FIA was significantly lower than RIA during the early (P < 0.01) and late (P < 0.05) follicular phases. During the midluteal phase, a significant difference was not achieved statistically, although FIA values were lower. There were no significant differences in the FIA:RIA ratios between the various phases of the cycle. After establishing the LH peak by pulse detection during the 3-hour sampling period, LH peak values were compared by RIA and FlA. Serum LH by RIA at the nadir was 7.3 ± 0.8 mIU/mL and at the peak was 11.3 ± 1.1 mlU/mL (P < 0.01). No significant changes occurred in the peak LH FIA:RIA ratio (0.37 ± 0.06) compared with the baseline FIA:RIA ratio (0.35 ± 0.03). Both RIA and FIA values at baseline and during the maximum responses after a bolus of GnRH 100 Ilg IV were evaluated. Both serum LH RIA and FIA values increased significantly (P = 0.01) in response to GnRH. Fluoroimmunoassay values changed from 2.6 ± 0.56 to 17.1 ± 3.72 mIU/mL (598% ± 140% difference), whereas RIA values changed from 7.27 ± 1.16 to 33.82 ± 3.65 mIU/mL (400% ± 82% difference). These changes after GnRH for RIA and FIA were not different statistically. The FIA:RIA
Vol. 59, No.2, February 1993
ratio increased from 0.36 ± 0.05 to 0.50 ± 0.08 after GnRH (P = 0.08). Figure 1 depicted RIA and FIA values in 12 normal women before and during the midcycle surge. During both instances, FIA values were significantly less than RIA values. During the surge, FIA values increased from 15.5 ± 3.5 to 64 ± 7.5 mIU /mL (P < 0.01), whereas RIA values also increased significantly from 31.0 ± 6.0 to 106.2 ± 9.1 mlU/mL (P < 0.01). The FIA:RIA ratio increased from 0.49 ± 0.03 to 0.59 ± 0.04 (P = 0.05). Both RIA and FIA values of serum LH during the midluteal phase and after suppression with LA (1 mg/d SC) are also depicted in Figure 1. Although E2 levels were <105 pmol/L, there was no significant change in RIA values (12.9 ± 2.4 to 16.2 ± 0.7 mIU/mL). The respective FIA values, however, decreased significantly (6.5 ± 1.3 to 1.9 ± 0.3 mIU/mL) (P < 0.01). Subsequently, the FIA:RIA ratio decreased significantly (0.47 ± 0.04 to 0.12 ± 0.01) (P < 0.01). In response to Nal-Glu during the early follicular phase, LH by RIA decreased from 11.5 ± 2.0 to 6.2 ± 0.7 mIU/mL as did FIA measurements 4.5 ± 0.8 to 0.9 ± 0.2 mIU/mL (P < 0.01). Estradiol levels were suppressed to values <105 pmoljL. The administration of Nal-GIu also caused the FIA:RIA ratio to decrease (0.39 ± 0.02 to 0.15 ± 0.03) (P < 0.01). Eight hours after Nal-Glu during the late follicular phase, the RIA values had decreased from 16.6 ± 2.1 12
20
LH
LH
~
mIU
mL
T
0
T
0
0
1.0
LH
Figure 1 Comparison of serum LH by RIA (_), FIA (0), and the FIA:RIA ratio (1!llI) during two situations. First, before and during the midcycJe surge of normal women. *P < 0.01, significant differences in LH by RIA and FIA before and during LH surge. t P = 0.05 for the difference in the FIA:RIA ratio before and during the LH surge. Second, before and after suppression (E2 < 105 pmol/L) with LA (1 mg/d SC). *P < 0.Q1, significant differences in FIA and FIA:RIA values after suppression with LA.
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r
to 8.8 ± 0.8 mlU/mL (P < 0.01) and FIA values from 6.8 ± 1.0 to 2.0 ± 0.2 mlU/mL (P < 0.01). The FIA:RIA ratio also decreased from 0.40 ± 0.02 to 0.22 ± 0.01 (P < 0.01). After NET, there was a significant (P < 0.01) decrease in LH by RIA (7.27 ± 0.78 to 3.55 ± 0.48 mIU/mL) and FIA (2.6 ± 0.38 to 0.48 ± 0.17 mIU/mL). After the administration of NET, the FIA:RIA ratio also decreased significantly from 0.35 ± 0.03 to 0.13 ± 0.04 (P < 0.01). Figure 2 compares RIA and FIA values for LH in 19 women with pcas as compared with 12 normal ovulatory women during the midfollicular phase (days 7 to 9). Serum RIA and FIA values were significantly (P < 0.01) increased in pcas women compared with normal women. Radioimmunoassay values were 17.6 ± 2.1 and 11.0 ± 1.4 mlU/mL (P < 0.01), respectively, whereas FIA values were 9.7 ± 0.9 and 3.8 ± 0.5 mIU/mL, respectively (P < 0.01). The FIA:RIA ratio was also significantly higher in pcas compared with normal ovulatory women (0.62 ± 0.02 versus 0.39 ± 0.02 (P < 0.01). Serum LH RIA and FIA values from women with pcas after suppression with LA (1 mg/d SC) were also evaluated. As stated above, although E2 levels were <105 pmoljL, there was no significant change in RIA values (13.6 ± 1.3 to 14.7 ± 1.2 mIU/mL), whereas the respective FIA values decreased from 8.5 ± 1.2 to 2.3 ± 0.4 mlU/mL (P < 0.01). The FIA: RIA ratio also decreased from 0.6 ± 0.04 to 0.15 ± 0.02 (P < 0.01). Figure 3 illustrates the significant correlations observed between RIA and FIA serum determinations from samples obtained at baseline, stimulated samples (during the midcycle surge and after GnRH), and suppressed samples (after Nal-Glu and LA) in normal women. Also, significant correlations from normal ovulatory women (midfollicular) and women with pcas (day 4 after induced menses) are
20
LH mL
o NORMAL
peDs
Figure 2 Comparison of serum LH by RIA (_), FIA (0), and the FIA:RIA ratio (flIj) in normal women in the midfollicular phase and in women with PCOS. *P < 0.01, significant differences in all three measurements comparing normal women to women with
pcos.
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LH by FIA. and RIA
R-0.96
100
-0:96 '!P.98
FIA LH
mll.! mL
RIA LH mlU/mL
Figure 3 Correlations between serum LH FIA, and RIA among various conditions in normal women and women with pcos. *P < 0.01, significant correlations in all situations noted above. The symbols on each regression line are for illustrating each pertubation and do not represent data points. --e = pcos; --B-- = normal women (midfollicular); = stimulated states; ~ = suppressed states and ~ = baseline states.
+
illustrated. Although all correlations were significant between RIA and FIA, the correlation was weakest in the suppressed state (r = 0.69) in which values of LH by RIA were not suppressed. DISCUSSION
aur data were obtained during conditions of known biological:immunological LH disparity, according to previous data, and suggest that FIA of serum LH reflects trends in bioactivity more than the conventional RIA. These conditions were best reflected by changes in the FIA:RIA ratio. This ratio may be useful for signifying conditions of increased or decreased bioactivity and avoids the difficult, timely, and expensive bioassay methods previously employed. Dufau et al. (14, 18,20,21) studied LH biological and immunological activity during the menstrual cycle under several circumstances. At baseline, the biological:immunological activity ratio remained close to unity throughout the cycle and was not statistically different at any phase of the cycle. After administration of GnRH (100 ILg IV) during the early follicular phase, the bioactive:immunoactive LH ratio was unchanged, although both bioactive and immunoreactive activity increased. However, after the administration of GnRH during the late follicular phase, the bioactive:immunoactive LH ratio did increase significantly. In our study, we found that the FIA:RIA LH ratio was not statistically different between the early, the late follicular phases, or the luteal phases. However, the FIA:RIA LH ratio was increased somewhat after
Fertility and Sterility
GnRH (P = 0.08). We also found a significant difference in the FIA:RIA LH ratio (P = 0.05) in serum values obtained before and during the LH surge. We previously have reported increased biological activity of LH in women with PCDS (12). In this study, women with PCDS had significantly elevated LH by RIA and FIA as well as in the FIA:RIA ratio (P < 0.01). Meldrum et al. (13) and Chiang et al. (15) have studied LH bioactivity after GnRH agonist administration in normal women and women with PCDS and endometriosis. They found that LH bioactivity and the biological:immunological activity ratio decreased after suppression with the agonist. We found no significant change in LH by RIA after 2 weeks of LA therapy in normal women or women with PCDS. However, a very significant decrease was apparent by FIA in both normal women and women with PCDS. Subsequently, a significant decrease in the FIA:RIA ratio occurred, reflecting the known decreased bioactivity. Even without directly assessing bioactivity of LH in these samples, it is obvious that FIA of LH reflects bioactivity better than RIA in that estrogen was suppressed. Mortola et al. (17) showed that Nal-GIu has a maximal effect on bioactivity 5 to 8 hours after administration. In this study, we showed that 8 hours after Nal-Glu LH by RIA and FIA values decreased significantly and that the FIA:RIA ratio was significantly decreased as well (P < 0.01). Dur data confirm that FIA of LH may reflect bioactivity of LH better than a conventional RIA. This is more significant during conditions of gonadotropin suppression (GnRH agonist, antagonist, or progestin) rather than with stimulation (GnRH IV and LH pulses) where only borderline changes in the FIA:RIA ratio were evident. Taken together, our study may only suggest that this measurement of LH is a clinically useful marker, specifically when a change in the biological activity of LH is sought. In addition, because the biological activity of LH is increased in PCDS, FIA measurements of LH may be one of several useful biochemical markers in the study of patients with PCDS. Nevertheless, because we have not directly assessed bioactivity in our sampIes, our contention that FIA reflects bioactivity can only be viewed as being an extrapolation. The measurements of LH by FIA, however, we believe to be useful clinically. REFERENCES 1. Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin in man. J Clin Invest 1960;39:1157-75.
Vol. 59, No.2, February 1993
2. Elkins RP. The estimation of thyroxine in human plasma by an electrophoretic technique. Clin Chim Acta 1960;5:45361. 3. Lovgren T, Hemmila I, Pettersson K, Eskola JU, Bertoft E. Determination of hormones by time-resolved fluoroimmunoassay. Talanta 1984;31:909-16. 4. Barnard G. The development of fluorescence immunoassays. Prog Clin Bioi Res 1988;285:15-37. 5. Gerson B. Fluorescence immunoassay. J Clin Immunoassay 1984;7:73-81. 6. Hughes CL Jr, Dodson WC, Walmer DK, Dixon SB. Utility of fluoroimmunoassay in detecting luteinizing hormone surges in spontaneous and human menopausal gonadotropin-stimulated menstrual cycles. J Reprod Med 1990;35:211-3. 7. Haavisto A-M, Dunkel L, Pettersson K, Huhtaniemi I. LH measurements by in vitro bioassay and a highly sensitive immunofluorometric assay improve the distinction between boys with constitutional delay of puberty and hypogonadotropic hypogonadism. Pediatr Res 1990;27:211-4. 8. Dunkel L, Alfthan H, Stenman U-H, Tapanainen P, Perheentupa J. Pulsatile secretion of LH and FSH in prepubertal and early pubertal boys revealed by ultrasensitive time-resolved immunofluorometric assays. Pediatr Res 1990;27: 215-9. 9. Jaakkola T, Ding Y-Q, Kellokumpu-Lehtinen P, Valavaara R, Martikainen H, Tapanainen J, et al. The ratios of serum bioactive/immunoreactive luteinizing hormone and folliclestimulating hormone in various clinical conditions with increased and decreased gonadotropin secretion: reevaluation by a highly sensitive immunometric assay. J Clin Endocrinol Metab 1990;70:1496-1505. 10. Imoedemhe DAG, Sigue AB, Pacpaco ELA, Olazo AB. Stimulation of endogenous surge of luteinizing hormone with gonadotropin-releasing hormone analog after ovarian stimulation for in vitro fertilization. Fertil Steril 1991;55:328-32. 11. Lobo RA, Shoupe D, Chang SP, Campeau J. The control of bioactive luteinizing hormone secretion in women with polycystic ovary syndrome. Am J Obstet Gynecol 1984;148: 423-8. 12. Lobo RA, Kletzky OA, Campeau JD, di Zerega GS. Elevated bioactive luteinizing hormone in women with the polycystic ovary syndrome. Fertil Steril1983;39:674-8. 13. Meldrum DR, Tsao Z, Monroe SE, Braunstein GD, Sladek J, Lu JKH, et al. Stimulation of LH fragments with reduced bioactivity following GnRH agonist administration in women. J Clin Endocrinol Metab 1984;58:755-7. 14. Dufau ML, Beitins IZ, McArthur JW, Catt KJ. Effects of luteinizing hormone releasing hormone (LHRH) upon bioactive and immunoreactive serum LH levels in normal subjects. J Clin Endocrinol Metab 1976;43:658-67. 15. Chiang RS, Barnes RB, Shoupe D, Lobo RA. Dose-related changes in LH bioactivity with intranasal GnRH agonist administration. Contraception 1985;32:347-57. 16. Veldhuis JD, Beitins IZ, Johnson ML, Serabian MA, Dufau ML. Biologically active luteinizing hormone is secreted in episodic pulsations that vary in relation to stage of the menstrua cycle. J Clin Endocrinol Metab 1984;58:1050-8. 17. Mortola JF, Sathanandan M, Pavlou S, Dahl KD, Hsueh AJW, Rivier J, et al. Suppression of bioactive and immunoassay follicle-stimulating hormone and luteinizing hormone levels by a potent gonadotropin-releasing hormone antagonist: pharmacodynamic studies. Fertil Steril1989;51:957-63.
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,..
18. Dufau ML, Hodgen GD, Goodman AL, Catt JK. Bioassay of circulating luteinizing hormone in the rhesus monkey: comparison with radioimmunoassay during physiological changes. Endocrinology 1977;100:1557-65. 19. Fauser BCJM, Pache TD, Lamberts SWJ, Hop WCJ, de Jong FH, Dahl KD. Serum bioactive and immunoreactive luteinizing hormone and follicle-stimulating hormone levels in women with cycle abnormalities, with or without polycystic ovarian disease. J Clin Endocrinol Metab 1991;73:811-7. 20. Dufau ML, Pock R, Neubauer A, Catt KJ. In vitro bioassay of LH in human serum: the rat interstitial cell testosterone (RICT) assay. J Clin Endocrinol Metab 1976;42:958-69. 21. Dufau ML, Mendelson CR, Catt KJ. A highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonad-
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otropin: testosterone production by dispersed Leydig cells. J Clin Endocrinol Metab 1974;39:610--3. 22. Ditkoff EC, Cassidenti DL, Paulson RJ, Sauer MV, Paul WL, Rivier J, et a1. The gonadotropin-releasing hormone antagonist (Nal-Giu) acutely blocks the luteinizing hormone surge but allows for resumption of folliculogenesis in normal women. Am J Obstet Gynecol1991;165:1811-7. 23. Mishell DR Jr, Nakamura RM, Crosignani PG, Stone S, Kharma K, Nagata Y, et a1. Serum gonadotropin and steroid patterns during the normal menstrual cycle. Am J Obstet Gynecol1971;111:60-5. 24. Stanczyk FZ, Shoupe D, Nunez V, Macias-Gonzales P, Vijod MA, Lobo RA. A randomized comparison of nonoral estradiol delivery in postmenopausal women. Am J Obstet Gynecol 1988; 159: 1540-6.
Fertility and Sterility
FERTILITY AND STERILITY
Printed on acid·free paper in U.S.A.
Copyright CI 1993 The American Fertility Society
Time-resolved fluoroimmunoassay compared with radioimmunoassay of luteinizing hormone*
Edward C. Ditkoff, M.D.t Jay H. Levin, M.D.:j: Wellington L. Paul, Ph.D.§ Rogerio A. Lobo, M.D. II University of Southern California School of Medicine, Los Angeles, and SmithKline Beecham Clinical Laboratory, Van Nuys, California
Objective: To determine if fluoroimmunoassay (FIA) of serum luteinizing hormone (LH) is more useful clinically than a conventional radioimmunoassay (RIA) because it has been suggested that FIA closely reflects biological activity. Design: Comparison of serum LH measurements by RIA and FIA during various perturbations in normal ovulatory women and in women with polycystic ovarian syndrome (PCDS). Setting: Normal ovulatory subjects were healthy volunteers and women with PCDS were untreated and newly diagnosed outpatients in our Reproductive Endocrinology/Infertility Clinic, Women's Hospital, at the Los Angeles County+University of Southern California Medical Center. Participants: Fifty-three normal ovulatory women, ages 20 to 35, and 27 women with PCDS, ages 21 to 35. All were in good health and received no other medications during the study period. Results: Fluoroimmunoassay of serum LH reflected status of known altered bioactivity better than with a conventional RIA. This was most evident during conditions of gonadotropin suppression and in patients with PCDS. An excellent correlation was found between values of FIA and RIA. Conclusions: The measurement of LH by FIA is clinically useful, specifically when a change in biological activity of LH is sought. Fertil Steril 1993;59:305-10 Key Words: Serum LH, FIA, RIA, biological activity
Since the development of radioimmunoassay (RIA) and related assay techniques by Yalow and Berson (1) and Elkins (2) in the late 1950s, these assays have been extensively applied to numerous Received April 23, 1992; revised and accepted October 28, 1992. * Presented in part at the 47th Annual Meeting of The American Fertility Society, Orlando, Florida, October 19 to 24, 1991. t Present address: Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, Columbia-Presbyterian Medical Center, New York, New York. :j: Present address: Center For Reproduction, Hinsdale Hospital, Hinsdale, Illinois. § Present address: SmithKline Beecham Clinical Laboratory. II Reprint requests: Rogerio A. Lobo, M.D., Division of Reproductive Endocrinology, University of Southern California School of Medicine, Women's Hospital, Room 1M2, Los Angeles Courity + University of Southern California Medical Center, 1240 North Mission Road, Los Angeles, California 90033.
Vol. 59, No.2, February 1993
analytes in reproductive endocrinology (3). Several newer immunoassay techniques have been recently developed and among these a fluoroimmunoassay (FIA) has be'come available (4). Fluoroimmunoassay uses a europium chelate as a label with a fluorescence decay time of 10-3 to 10-6 per second, which achieves an excellent sensitivity compared with RIA (3, 5). In addition, it has been suggested recently that fluoroimmunoassay (FIA) reflects biological activity better than conventional RIA and could therefore be more useful clinically (6-10). Our purpose was to determine if FIA is useful clinically and if it reflects trends in bioactivity of serum luteinizing hormone (LH) more than a conventional RIA. We therefore chose to study normal women during various pertubations known to affect
Ditkoff et al.
LH by FIA and RIA
305
biological activity as well as in women with polycystic ovarian syndrome (PCOS). The difference in bioactivity and immunoactivity of serum LH during these perturbations in normal women and in women with PCOS has been described by us and others previously (11-21). Therefore, although we have carefully chosen these conditions for study, we have not repeated the measurements by conventional bioassay. Our comparisons are primarily meant to test the clinical usefulness of the FIA for LH, and information regarding LH bioactivity can only be considered by extrapolation. MATERIALS AND METHODS Subjects
Fifty-three normal ovulatory women, ages 20 to 35 (30.42 ± 0.53), and 27 women with PCOS, ages 21 to 35 (28.1 ± 0.98), were studied. All women had documented ovulatory cycles with lengths of 26 to 32 days and were using a mechanical form of contraception at the time of enrollment. The subjects were in good health and received no medications during the study period. No woman had received any hormonal therapy for at least 2 months before the study. The women with PCOS were 27 consecutive new patients with this diagnosis in our Reproductive Endocrinology/Infertility Clinic, Women's Hospital, at the Los Angeles County+University of Southern California Medical Center. None of these women was taking any medications, and all had perimenarchial onset of oligomenorrhea, chronic anovulation, and elevated androgen levels (testosterone > 80 ng/dL) and/or hirsutism and normal levels of prolactin. No patient was virilized and none had evidence of adrenal enzymatic deficiencies. The project was approved by our Institutional Review Board, and written informed consent was obtained from each subject. Baseline Studies
All sera were collected at 8:00 A.M. for comparing measurements of LH by conventional RIA and by FIA. Nineteen had serum drawn during the early follicular phase (days 1 to 4), and 12 had serum drawn during their midfollicular phases (days 7 to 9). Ten had serum drawn during the late follicular phase when follicles reached a maximum diameter of 16 to 18 mm as measured by a 5.0-MHz Siemens
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Sonoline SL 1 model sonogram (Siemens Medical Systems, Inc., San Ramon, CA). Finally, 12 had serum drawn on day 21 of their cycles. In 27 women with PCOS, serum was also collected at 8:00 A.M. on day 4 after an induced menses after the administration of 150 mg of progesterone (P) in oil intramuscularly. Elevation of LH During the Normal Menstrual Cycle
Five normal women had early follicular phase sampling every 10 minutes for 3 hours to detect at least one LH pulse. Ten normal women had serum drawn at 8:00 A.M. and 4:00 P.M. during the late follicular phase to detect the LH surge. The acute effect of gonadotropin-releasing hormone (GnRH) 100 f.lg IV on LH was also evaluated in 5 normal women during the early follicular phase. Serum was obtained at 0, 10, 20, 30, 60, and 90 minutes after the bolus of GnRH. Suppression of LH
Leuprolide acetate (LA; TAP Pharmaceuticals, Deerfield, IL), a GnRH agonist (1 mg/d SC), was given to 12 normal ovulatory women for 2 weeks from the midluteal phase until estradiol (E 2) levels were <105 pmoljL. Serum LH was again obtained at this time again. For comparison purposes, LA (1 mg/d SC) was given to eight women with PCOS for 2 to 4 weeks until E2 levels were <105 pmoljL. Nal-Glu (50 f.lg/kg 1M), a GnRH antagonist, was given once during the early follicular phase in 7 women and during the late follicular phase to delay the LH surge in 10 women. This protocol was carried out previously for other purposes and has been reported (22). Serum was obtained to measure LH as well as E2 24 hours after each Nal-Glu administration for monitoring purposes. Serum LH was also measured 8 hours after Nal-Glu administration during the late follicular phase. Norethindrone (NET) (10 mg/d) was administered orally to five women during the early follicular phase for a 2-week duration with serum obtained before and immediately after NET administration. Hormonal Assays
All bloods were separated and sera were stored at -20°C until analyzed. Serum LH was measured from identical specimens by a highly specific conventional RIA (23) and an established FIA (3). The time-resolved FIA used europium as a chelate label
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using Delfia human LH Spec kits (Pharmacia Inc., Piscataway, NJ) according to the method of Lovgren et a1. (3). The sensitivity of the assay was 0.1 mlU/ mL. Serum E2 was measured by RIA (24) (Pantex, Santa Monica, CAl after extraction with hexane: ethyl acetate (2:3). Serum P was measured to confirm the midluteal status of some of the subjects (31.8 to >10 nmol/L) by using a direct RIA kit (Diagnostic System Laboratories, Webster, TX). From each patient, all sera were run in a single assay to reduce assay variation. The mean intra-assay and interassay coefficients of variation (CVs) for the serum RIAs were as follows: LH, 3.2% and 9.2%; E 2, 8.1% and 12.6%; P, 6.6% and 7.2%, respectively. The intra-assay and interassay CVs for LH serum by FIA were 4.4% and 7.6%, respectively. Statistical Analysis
All data are expressed as means ± SEM. Statistical analyses were performed using the two sampled Kolmogro-Smirov test and the Pearson's regression analysis. RESULTS
In comparing LH by FIA and RIA during the early follicular phase, late follicular phase, and midluteal phase samples, FIA was significantly lower than RIA during the early (P < 0.01) and late (P < 0.05) follicular phases. During the midluteal phase, a significant difference was not achieved statistically, although FIA values were lower. There were no significant differences in the FIA:RIA ratios between the various phases of the cycle. After establishing the LH peak by pulse detection during the 3-hour sampling period, LH peak values were compared by RIA and FlA. Serum LH by RIA at the nadir was 7.3 ± 0.8 mIU/mL and at the peak was 11.3 ± 1.1 mlU/mL (P < 0.01). No significant changes occurred in the peak LH FIA:RIA ratio (0.37 ± 0.06) compared with the baseline FIA:RIA ratio (0.35 ± 0.03). Both RIA and FIA values at baseline and during the maximum responses after a bolus of GnRH 100 Ilg IV were evaluated. Both serum LH RIA and FIA values increased significantly (P = 0.01) in response to GnRH. Fluoroimmunoassay values changed from 2.6 ± 0.56 to 17.1 ± 3.72 mIU/mL (598% ± 140% difference), whereas RIA values changed from 7.27 ± 1.16 to 33.82 ± 3.65 mIU/mL (400% ± 82% difference). These changes after GnRH for RIA and FIA were not different statistically. The FIA:RIA
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ratio increased from 0.36 ± 0.05 to 0.50 ± 0.08 after GnRH (P = 0.08). Figure 1 depicted RIA and FIA values in 12 normal women before and during the midcycle surge. During both instances, FIA values were significantly less than RIA values. During the surge, FIA values increased from 15.5 ± 3.5 to 64 ± 7.5 mIU /mL (P < 0.01), whereas RIA values also increased significantly from 31.0 ± 6.0 to 106.2 ± 9.1 mlU/mL (P < 0.01). The FIA:RIA ratio increased from 0.49 ± 0.03 to 0.59 ± 0.04 (P = 0.05). Both RIA and FIA values of serum LH during the midluteal phase and after suppression with LA (1 mg/d SC) are also depicted in Figure 1. Although E2 levels were <105 pmol/L, there was no significant change in RIA values (12.9 ± 2.4 to 16.2 ± 0.7 mIU/mL). The respective FIA values, however, decreased significantly (6.5 ± 1.3 to 1.9 ± 0.3 mIU/mL) (P < 0.01). Subsequently, the FIA:RIA ratio decreased significantly (0.47 ± 0.04 to 0.12 ± 0.01) (P < 0.01). In response to Nal-Glu during the early follicular phase, LH by RIA decreased from 11.5 ± 2.0 to 6.2 ± 0.7 mIU/mL as did FIA measurements 4.5 ± 0.8 to 0.9 ± 0.2 mIU/mL (P < 0.01). Estradiol levels were suppressed to values <105 pmoljL. The administration of Nal-GIu also caused the FIA:RIA ratio to decrease (0.39 ± 0.02 to 0.15 ± 0.03) (P < 0.01). Eight hours after Nal-Glu during the late follicular phase, the RIA values had decreased from 16.6 ± 2.1 12
20
LH
LH
~
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mL
T
0
T
0
0
1.0
LH
Figure 1 Comparison of serum LH by RIA (_), FIA (0), and the FIA:RIA ratio (1!llI) during two situations. First, before and during the midcycJe surge of normal women. *P < 0.01, significant differences in LH by RIA and FIA before and during LH surge. t P = 0.05 for the difference in the FIA:RIA ratio before and during the LH surge. Second, before and after suppression (E2 < 105 pmol/L) with LA (1 mg/d SC). *P < 0.Q1, significant differences in FIA and FIA:RIA values after suppression with LA.
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to 8.8 ± 0.8 mlU/mL (P < 0.01) and FIA values from 6.8 ± 1.0 to 2.0 ± 0.2 mlU/mL (P < 0.01). The FIA:RIA ratio also decreased from 0.40 ± 0.02 to 0.22 ± 0.01 (P < 0.01). After NET, there was a significant (P < 0.01) decrease in LH by RIA (7.27 ± 0.78 to 3.55 ± 0.48 mIU/mL) and FIA (2.6 ± 0.38 to 0.48 ± 0.17 mIU/mL). After the administration of NET, the FIA:RIA ratio also decreased significantly from 0.35 ± 0.03 to 0.13 ± 0.04 (P < 0.01). Figure 2 compares RIA and FIA values for LH in 19 women with pcas as compared with 12 normal ovulatory women during the midfollicular phase (days 7 to 9). Serum RIA and FIA values were significantly (P < 0.01) increased in pcas women compared with normal women. Radioimmunoassay values were 17.6 ± 2.1 and 11.0 ± 1.4 mlU/mL (P < 0.01), respectively, whereas FIA values were 9.7 ± 0.9 and 3.8 ± 0.5 mIU/mL, respectively (P < 0.01). The FIA:RIA ratio was also significantly higher in pcas compared with normal ovulatory women (0.62 ± 0.02 versus 0.39 ± 0.02 (P < 0.01). Serum LH RIA and FIA values from women with pcas after suppression with LA (1 mg/d SC) were also evaluated. As stated above, although E2 levels were <105 pmoljL, there was no significant change in RIA values (13.6 ± 1.3 to 14.7 ± 1.2 mIU/mL), whereas the respective FIA values decreased from 8.5 ± 1.2 to 2.3 ± 0.4 mlU/mL (P < 0.01). The FIA: RIA ratio also decreased from 0.6 ± 0.04 to 0.15 ± 0.02 (P < 0.01). Figure 3 illustrates the significant correlations observed between RIA and FIA serum determinations from samples obtained at baseline, stimulated samples (during the midcycle surge and after GnRH), and suppressed samples (after Nal-Glu and LA) in normal women. Also, significant correlations from normal ovulatory women (midfollicular) and women with pcas (day 4 after induced menses) are
20
LH mL
o NORMAL
peDs
Figure 2 Comparison of serum LH by RIA (_), FIA (0), and the FIA:RIA ratio (flIj) in normal women in the midfollicular phase and in women with PCOS. *P < 0.01, significant differences in all three measurements comparing normal women to women with
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LH by FIA. and RIA
R-0.96
100
-0:96 '!P.98
FIA LH
mll.! mL
RIA LH mlU/mL
Figure 3 Correlations between serum LH FIA, and RIA among various conditions in normal women and women with pcos. *P < 0.01, significant correlations in all situations noted above. The symbols on each regression line are for illustrating each pertubation and do not represent data points. --e = pcos; --B-- = normal women (midfollicular); = stimulated states; ~ = suppressed states and ~ = baseline states.
+
illustrated. Although all correlations were significant between RIA and FIA, the correlation was weakest in the suppressed state (r = 0.69) in which values of LH by RIA were not suppressed. DISCUSSION
aur data were obtained during conditions of known biological:immunological LH disparity, according to previous data, and suggest that FIA of serum LH reflects trends in bioactivity more than the conventional RIA. These conditions were best reflected by changes in the FIA:RIA ratio. This ratio may be useful for signifying conditions of increased or decreased bioactivity and avoids the difficult, timely, and expensive bioassay methods previously employed. Dufau et al. (14, 18,20,21) studied LH biological and immunological activity during the menstrual cycle under several circumstances. At baseline, the biological:immunological activity ratio remained close to unity throughout the cycle and was not statistically different at any phase of the cycle. After administration of GnRH (100 ILg IV) during the early follicular phase, the bioactive:immunoactive LH ratio was unchanged, although both bioactive and immunoreactive activity increased. However, after the administration of GnRH during the late follicular phase, the bioactive:immunoactive LH ratio did increase significantly. In our study, we found that the FIA:RIA LH ratio was not statistically different between the early, the late follicular phases, or the luteal phases. However, the FIA:RIA LH ratio was increased somewhat after
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GnRH (P = 0.08). We also found a significant difference in the FIA:RIA LH ratio (P = 0.05) in serum values obtained before and during the LH surge. We previously have reported increased biological activity of LH in women with PCDS (12). In this study, women with PCDS had significantly elevated LH by RIA and FIA as well as in the FIA:RIA ratio (P < 0.01). Meldrum et al. (13) and Chiang et al. (15) have studied LH bioactivity after GnRH agonist administration in normal women and women with PCDS and endometriosis. They found that LH bioactivity and the biological:immunological activity ratio decreased after suppression with the agonist. We found no significant change in LH by RIA after 2 weeks of LA therapy in normal women or women with PCDS. However, a very significant decrease was apparent by FIA in both normal women and women with PCDS. Subsequently, a significant decrease in the FIA:RIA ratio occurred, reflecting the known decreased bioactivity. Even without directly assessing bioactivity of LH in these samples, it is obvious that FIA of LH reflects bioactivity better than RIA in that estrogen was suppressed. Mortola et al. (17) showed that Nal-GIu has a maximal effect on bioactivity 5 to 8 hours after administration. In this study, we showed that 8 hours after Nal-Glu LH by RIA and FIA values decreased significantly and that the FIA:RIA ratio was significantly decreased as well (P < 0.01). Dur data confirm that FIA of LH may reflect bioactivity of LH better than a conventional RIA. This is more significant during conditions of gonadotropin suppression (GnRH agonist, antagonist, or progestin) rather than with stimulation (GnRH IV and LH pulses) where only borderline changes in the FIA:RIA ratio were evident. Taken together, our study may only suggest that this measurement of LH is a clinically useful marker, specifically when a change in the biological activity of LH is sought. In addition, because the biological activity of LH is increased in PCDS, FIA measurements of LH may be one of several useful biochemical markers in the study of patients with PCDS. Nevertheless, because we have not directly assessed bioactivity in our sampIes, our contention that FIA reflects bioactivity can only be viewed as being an extrapolation. The measurements of LH by FIA, however, we believe to be useful clinically. REFERENCES 1. Yalow RS, Berson SA. Immunoassay of endogenous plasma insulin in man. J Clin Invest 1960;39:1157-75.
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2. Elkins RP. The estimation of thyroxine in human plasma by an electrophoretic technique. Clin Chim Acta 1960;5:45361. 3. Lovgren T, Hemmila I, Pettersson K, Eskola JU, Bertoft E. Determination of hormones by time-resolved fluoroimmunoassay. Talanta 1984;31:909-16. 4. Barnard G. The development of fluorescence immunoassays. Prog Clin Bioi Res 1988;285:15-37. 5. Gerson B. Fluorescence immunoassay. J Clin Immunoassay 1984;7:73-81. 6. Hughes CL Jr, Dodson WC, Walmer DK, Dixon SB. Utility of fluoroimmunoassay in detecting luteinizing hormone surges in spontaneous and human menopausal gonadotropin-stimulated menstrual cycles. J Reprod Med 1990;35:211-3. 7. Haavisto A-M, Dunkel L, Pettersson K, Huhtaniemi I. LH measurements by in vitro bioassay and a highly sensitive immunofluorometric assay improve the distinction between boys with constitutional delay of puberty and hypogonadotropic hypogonadism. Pediatr Res 1990;27:211-4. 8. Dunkel L, Alfthan H, Stenman U-H, Tapanainen P, Perheentupa J. Pulsatile secretion of LH and FSH in prepubertal and early pubertal boys revealed by ultrasensitive time-resolved immunofluorometric assays. Pediatr Res 1990;27: 215-9. 9. Jaakkola T, Ding Y-Q, Kellokumpu-Lehtinen P, Valavaara R, Martikainen H, Tapanainen J, et al. The ratios of serum bioactive/immunoreactive luteinizing hormone and folliclestimulating hormone in various clinical conditions with increased and decreased gonadotropin secretion: reevaluation by a highly sensitive immunometric assay. J Clin Endocrinol Metab 1990;70:1496-1505. 10. Imoedemhe DAG, Sigue AB, Pacpaco ELA, Olazo AB. Stimulation of endogenous surge of luteinizing hormone with gonadotropin-releasing hormone analog after ovarian stimulation for in vitro fertilization. Fertil Steril 1991;55:328-32. 11. Lobo RA, Shoupe D, Chang SP, Campeau J. The control of bioactive luteinizing hormone secretion in women with polycystic ovary syndrome. Am J Obstet Gynecol 1984;148: 423-8. 12. Lobo RA, Kletzky OA, Campeau JD, di Zerega GS. Elevated bioactive luteinizing hormone in women with the polycystic ovary syndrome. Fertil Steril1983;39:674-8. 13. Meldrum DR, Tsao Z, Monroe SE, Braunstein GD, Sladek J, Lu JKH, et al. Stimulation of LH fragments with reduced bioactivity following GnRH agonist administration in women. J Clin Endocrinol Metab 1984;58:755-7. 14. Dufau ML, Beitins IZ, McArthur JW, Catt KJ. Effects of luteinizing hormone releasing hormone (LHRH) upon bioactive and immunoreactive serum LH levels in normal subjects. J Clin Endocrinol Metab 1976;43:658-67. 15. Chiang RS, Barnes RB, Shoupe D, Lobo RA. Dose-related changes in LH bioactivity with intranasal GnRH agonist administration. Contraception 1985;32:347-57. 16. Veldhuis JD, Beitins IZ, Johnson ML, Serabian MA, Dufau ML. Biologically active luteinizing hormone is secreted in episodic pulsations that vary in relation to stage of the menstrua cycle. J Clin Endocrinol Metab 1984;58:1050-8. 17. Mortola JF, Sathanandan M, Pavlou S, Dahl KD, Hsueh AJW, Rivier J, et al. Suppression of bioactive and immunoassay follicle-stimulating hormone and luteinizing hormone levels by a potent gonadotropin-releasing hormone antagonist: pharmacodynamic studies. Fertil Steril1989;51:957-63.
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18. Dufau ML, Hodgen GD, Goodman AL, Catt JK. Bioassay of circulating luteinizing hormone in the rhesus monkey: comparison with radioimmunoassay during physiological changes. Endocrinology 1977;100:1557-65. 19. Fauser BCJM, Pache TD, Lamberts SWJ, Hop WCJ, de Jong FH, Dahl KD. Serum bioactive and immunoreactive luteinizing hormone and follicle-stimulating hormone levels in women with cycle abnormalities, with or without polycystic ovarian disease. J Clin Endocrinol Metab 1991;73:811-7. 20. Dufau ML, Pock R, Neubauer A, Catt KJ. In vitro bioassay of LH in human serum: the rat interstitial cell testosterone (RICT) assay. J Clin Endocrinol Metab 1976;42:958-69. 21. Dufau ML, Mendelson CR, Catt KJ. A highly sensitive in vitro bioassay for luteinizing hormone and chorionic gonad-
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otropin: testosterone production by dispersed Leydig cells. J Clin Endocrinol Metab 1974;39:610--3. 22. Ditkoff EC, Cassidenti DL, Paulson RJ, Sauer MV, Paul WL, Rivier J, et a1. The gonadotropin-releasing hormone antagonist (Nal-Giu) acutely blocks the luteinizing hormone surge but allows for resumption of folliculogenesis in normal women. Am J Obstet Gynecol1991;165:1811-7. 23. Mishell DR Jr, Nakamura RM, Crosignani PG, Stone S, Kharma K, Nagata Y, et a1. Serum gonadotropin and steroid patterns during the normal menstrual cycle. Am J Obstet Gynecol1971;111:60-5. 24. Stanczyk FZ, Shoupe D, Nunez V, Macias-Gonzales P, Vijod MA, Lobo RA. A randomized comparison of nonoral estradiol delivery in postmenopausal women. Am J Obstet Gynecol 1988; 159: 1540-6.
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