Midcycle increase of prolactin seen in normal women is absent in subjects with unexplained infertility

FERTILITYAND STERILITY@

Vol. 67, No. 4, April1997

Copyright o 1997AmericanSocietyforReproductive Medicine

Printed

on acid-free paper in U. S. A.

Published by ElsevierScienceInc.

Midcycle increase of prolactin seen in normal women is absent in subjects with unexplained infertility*

Marappa G. Subramanian, Ph.D.t@ Carole L. Kowalczyk, M.D.? Richard E. Leach, M.D.?+ David M. Lawson, Ph.D.* Charla M. Blacker, M.D.?

Kenneth A. Ginsburg, M.D.? John F. Randolph, Jr., M.D.11 Michael P. Diamond, M.D.? Kamran S. Moghissi, M.D.?_

Wayne State University, Detroit, Michigun, and The University

ofMichigan,

Ann Arbor, Michigan

Objective: To compare the bioactive and immunoactive PRL in normal and unexplained infertility subjects. Design: Prospective study. Setting: Department of Obstetrics and Gynecology, Wayne State University and The University of Michigan. Patient(s): Twelve normal, fertile women compared with 12 patients with unexplained infertility. Intervention(s): Serum samples were obtained across the menstrual cycle and for each subject, 5 pools were prepared by combining serum aliquots from the early follicular, late follicular, midcycle, and midluteal and late luteal phases of the cycle. Main Outcome Measure(s): Niobium lymphoma cell bioassay and an immunoradiometric assay were used to quantitate PRL. Result(s): A midcycle increase in PRL was seen in controls by both assays and these levels were greater compared with other cycle stages. Comparison of midcycle PRL between groups showed differences only between bioactive PRL (34.2 2 8.3 versus 19.2 % 3.4 ng/mL [conversion factor to SI unit, 1.001). The ratios between bioactive and immunoactive PRL were comparable. Significant correlation between bioactive and immunoactive PRL was seen for both control (r = 0.616) and unexplained infertility (r = 0.660) groups. Conclusion(s): The midcycle elevations of bioactive and immunoactive PRL seen in normal women were absent in women with unexplained infertility. This alteration in PRL dynamics may be a part of subtle differences in the reproductive hormone profile of women with unex(Fertil Steril@ 1997;67:644-7. plained infertility compared with their fertile counterparts. 0 1997 by American Society for Reproductive Medicine.) Key Words: Prolactin, unexplained infertility, bioactive prolactin, immunoactive prolactin, menstrual cycle

An elevation

in serum PRL is associated

with a

variety of reproductive disorders, including amenorReceivedJuly 8,1996; revisedand acceptedDecember19,1996. * Supportedby grantU54 HD29184 fromthe NationalCooperative Programfor InfertilityResearchand by grantAA07670from the NationalInstitutesof Health,Bethesda,Maryland. t Departmentof Obstetricsand Gynecology,Wayne State University. $ Departmentof Physiology,Wayne State University. QReprintrequests:MarappaG. Subramanian,Ph.D., Departmentof Obstetricsand Gynecology,WayneStateUniversity,C. S. Mott Center,275 East HancockAvenue,Detroit,Michigan48201 (FAX: 313-577-8554).

644

rhea, oligomenorrhea, and anovulation and/or luteal phase defects with subsequent infertility. Little is known, however, regarding the pattern of PRL secretion in patients who are diagnosed with unexplained infertility, specifically those individuals in whom no abnormal fertility factors are identified in their evaluation. It is well known that isoforms of PRL in varying proportions are secreted in different pathophysio-

11 Departmentof Obstetricsand Gynecology,The Universityof Michigan. 00150282/97/$17.00 PI1 SOO15-0282(97)00009-5

logic conditions. Current standard laboratory evaluation of serum PRL is done by RIA. Following the establishment of niobium (Nbz) lymphoma cell bioassay to measure lactogenic activity (l), differences in biologic versus immunologic activity have been reported by this laboratory (2-4) and by a number of other laboratories (5, 61, under a variety of clinical situations. It is thus possible that there is a difference in bioassay versus immunoassay values of PRL in patients with unexplained infertility. This investigation determined the pattern of PRL secretion in patients with unexplained infertility compared with normal fertile control subjects by using both bioassay and immunoassay techniques. MATEXIALS

AND METHODS

This prospective study compared the serum PRL levels of fertile women to those of women with unexplained infertility. Our study population included women participating in the National Center for Infertility Research at Michigan, an affiliate of the National Cooperative Program for Infertility Research, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland. Institutional Review Board approval was obtained from the Human Investigation Committees at Wayne State University and The University of Michigan, and informed consent was given by each subject. The criteria for the diagnosis of unexplained infertility in women consisted of the following: [l] 25 to 40 years of age; 121ovulatory menstrual cycles from 26 to 32 days as determined by urine ovulation predictor kits and basal body temperature charts; [31 endometrial biopsy results confirming normal luteal function; 141normal tubal and peritoneal anatomy confirmed by hysterosalpingography and/or laparoscopy; 151normal baseline serum FSH, LH, PRL, Ez, P, T, DHEAS, and TSH on cycle days 3 to 5; 161 absence of antisperm antibodies in cervical mucus and serum; and 173body mass index (BMI) between 18 and 28 kg/m2. The male partner had no evidence of male factor as determined by: 111two normal semen analyses according to the World Health Organization (WHO) criteria (7); 121 no evidence of anatomic/functional disorders of the reproductive tract; [31 absence of antisperm antibodies in serum and seminal plasma by immunobead testing; and 141normal postcoital test according to the WHO criteria (7). Twelve women with unexplained infertility who met the above criteria were selected from 1,885 women with infertility who requested to participate in the study. Women were identified from the practices of the investigators, referrals from other physicians, and inquiries by telephone from newspaper advertisements. Vol. 67, No. 4, April 1997

The control group of fertile female volunteers (n = 12) met the following inclusion criteria: 11125 to 40 years of age; 121BMI between 18 and 28 (kg/ m2); [33 regular menstrual cycles between 26 and 32 days; 143at least 6 months postpartum and postlactation; 151normal baseline serum screen in the follicular phase for FSH, LH, PRL, TSH, and hematocrit; and [6] free of hormone therapy for 3 months before the study. Blood was drawn, starting with the first day of menses, at 8:00 A.M. every other day from women with unexplained infertility and normal controls. The time for blood withdrawal (8:OOA.M.) was kept constant to minimize the variations in serum PRL levels. Daily blood samples were obtained during the late follicular, midcycle, and early luteal phases of the cycle followed by every other day sampling during the midluteal and late luteal phases until the onset of menses. Serum was separated and stored at -20°C for hormone assays. The day of LH peak was taken as day 0 and other blood sampling days reported as + days from the LH peak as follows: -10 to -6 (early follicular), -5 to -1 (late follicular), 0 (midcycle), +3 to +7 (midluteal), and +8 to end of cycle (late luteal). For PRL bioassay and immunoassay, serum pools for each subject were obtained by combining equal aliquots of sera from the several samples collected across each of the following phases of the cycle: early follicular, late follicular, midcycle, midluteal, and late luteal. These samples were analyzed by using an immunoradiometric assay (IRMA) and the Nb, lymphoma cell bioassay (Nb,BA) (1) to quantitate immunoactive PRL and bioactive PRL, using hPRL (AFP 8982C; National Institute of Diabetes, and Digestive and Kidney Diseases, Bethesda, MD) as the reference preparation. Coat-a-Count PRL IRMA (Diagnostic Products, Los Angeles, CA), used for measuring immunoactive PRL, is a solid-phase immunoradiometric assay. PRL is captured between polyclonal anti-PRL antibody immobilized on the inside surface of polystyrene tubes and radiolabeled monoclonal anti-PRL antibody is added to the tubes. Unbound labeled antibody is removed by decanting the reaction mixture and washing the tube. The PRL concentration is directly proportional to the radioactivity remaining in the tube after the wash step. This IRMA has a detection limit of approximately 0.1 ng/mL. Samples were analyzed in two assays. The intraassay and interassay variabilities, expressed as coefficients of variation, were calculated from values for four serum pools run in duplicates. The intraassay variations for the two assays were 2.24% and 2.30%, and the interassay variation was 1.37%. Subramanian et al. Prolactin in unexplained infertility

645

The proliferation of a rat-node lymphoma cell line in response to PRL is the basis of the PRL bioassay (1). The assay is specific for PRL and other lactogenic hormones, including human growth hormone (hGH) and placental lactogen (hPL). However, hGH concentration in samples similar to these is normally
Table 1 Clinical Characteristics Unexplained Infertility Groups*

Characteristic Age (y) BMI (kg/m’) Cycle length (d) Years of infertility

Control (n = 12)

of Normal Control Versus

Unexplained infertility (n = 12)

32.8 ? 1.2 23.9 2 0.7 28.5 2 0.7 NA

33.2 22.3 28.4 4.4

2 2 + 2

Significance

NW

1.3 0.6 0.4 0.8

NS NS NAS

* Values are means + SEM. t NS, not significant. $ NA, not applicable.

is directly proportional to the 3H-thymidine incorporation in the range of 2.5 pg to 2.5 ng per well (2.5 to 2500 ng/mL by using the equivalent of 1 ,uL of sample per well). All samples were analyzed in one assay and the intraassay variation was 14.0%. The serum PRL data were analyzed statistically by using the two-way ANOVA and Newman-Keul’s post hoc test. Correlations between the bioactive and immunoactive PRL were analyzed by using the z test. In all statistical analyses, a P < 0.05 was considered significant. REMJLTS There were no significant differences in clinical characteristics between the fertile control and unexplained infertility populations with respect to age, BMI, mean cycle length, or the follicular or luteal phases of the menstrual cycle (Table 1).

45 40 sE P

35

Eha -s

25

3s 95 n 5

20

ii

10

to

30

I5

5 EF

LF

MC

ML

LL

0 i

Figure 1 Serum total lactogen in normal women (open bars) and women with unexplained infertility (hatched bars) across the menstrual cycle as measured by Nbz lymphoma bioassay. EF = early follicular; LF = late follicular; MC = midcycle; ML = midluteal; and LL = late luteal. Midcycle PRL (normal) was significantly greater than other phases (normal) by NbzBA and midcycle (unexplained infertility) by NbzBA (P < 0.05). 646

Subramanian et al. Prolactin

in unexplained

infertility

EF

LF

MC

ML

LL

Figure 2

Serum PRL levels in normal women (open bars) and women with unexplained infertility (hatched bars) across the menstrual cycle as measured by IRMA. EF, early follicular; LF, late follicular; MC, midcycle; ML, midluteal; and LL, late luteal. Midcycle PRL (normal) was significantly greater than other phases (normal) by IRMA (P < 0.05).

Fertility

and Sterility@

Bioactive and immunoactive serum PRL values are presented in Figures 1 and 2 for control and unexplained infertility subjects. A midcycle increase in PRL was seen in control subjects by both bioassay (Fig. 1) and immunoassay (Fig. 2) and these levels were different compared with all other cycle stages (P < 0.05). Comparison of midcycle PRL between control and unexplained infertility subjects showed differences only between bioactive PRL (34.2 2 8.3 versus 19.2 + 3.4 ng/mL; P < 0.05), but not immunoactive PRL (26.9 + 4.3 versus 22.1 t 2.6 ng/mL; P > 0.05). The ratios between bioactive and immunoactive PRL showed neither group, time, nor interSignificant correlation beaction effects (P> 0.05). tween bioactive and immunoactive PRL values were seen for both control (r = 0.616; P < 0.001) and unexplained infertility (r = 0.660; P < 0.001) groups and the r values were comparable (P> 0.05).

groups (228 5 25 versus 217 + 30 pg/mL [conversion factor to SI unit, 3.6711, respectively). The correlation coefficients between bioactive PRL and immunoactive PRL for the two groups were similar. Additionally, no differences in the ratios between bioactive PRL and immunoactive PRL were seen. This alteration in PRL dynamics may be a part of other subtle differences in the reproductive hormonal axis of women with unexplained infertility compared with their fertile counterparts. However, it should be emphasized that blood samples for this study were obtained during the morning hours, at which time, the PRL levels are relatively low (10). Studies examining immunoassayable and bioassayable nocturnal PRL levels as well as the pattern of PRL secretion after provocative tests with secretagogues or food intake should be evaluated to identify possible alterations of PRL secretion in women with unexplained infertility.

DISCUSSION

It has been well recognized that although the immunoassay is a convenient and cost effective procedure for measurement of hormones, comparative evaluation of immunoactivity to bioactivity is always important. Until 1980, a number of bioassays for PRL were available, ranging from the classic pigeon crop assay to in vitro assays employing mammary gland explants (8, 9). The major limitation of these earlier bioassays is the lack of adequate sensitivity in measuring serum levels of PRL. Since the NbZ lymphoma cell bioassay for lactogenic hormones was first reported by Tanaka et al. (l), several laboratories have reported comparative studies between bioactive PRL and immunoactive PRL (2-5). Although, in most instances, there was good agreement between the two assay systems, occasional discrepancies in specific pathophysiologic conditions have also been noted (5, 6). In the present study, we evaluated the bioactive PRL and immunoactive PRL in sera of women with unexplained infertility and normal reproductive history. The results showed that midcycle elevation of both bioactive PRL and immunoactive PRL were seen only in control subjects and not in women with unexplained infertility. This midcycle elevation of PRL in the control group compared with the unexplained infertility group is not attributable to differences in midcycle Ez levels. In fact, Ez levels on the day of LH peak were quite comparable for the two

Vol. 67, No. 4, April 1997

REFERENCES 1. Tanaka T, Shiu RPC, Gout PW, Beer CT, Noble RL, Friesen HG. A sensitive and specific bioassay for lactogenic hormones: measurement of prolactin and growth hormone in human serum. J Clin Endocrinol Metab 1980;51:1058-63. 2. Subramanian MG, Gala RR. Do prolactin levels measured by RL4 reflect biologically active prolactin? J Clin Immunoassay 1986;9:42-52. 3. Subramanian MG, Spirtos NJ, Moghissi KS, Magyar DM, Hayes MF, Gala RR. Correlation and comparison of Nbz lymphoma cell bioassay with radioimmunoassay for human prolactin. Fertil Steril 1984;42:870-4. 4. Ataya KM, Subramanian MG, Moghissi KS, Magyar DM, Hayes MF, Lawson DM, et al. Clinical value of prolactin bioassay in euprolactinemic reproductive disorders. Fertil Steril 1988;49:71-5. 5. Love R, Rose D. Elevated bioactive prolactin in women at risk for familial breast cancer. Eur J Cancer Clin Oncol 1986;21:1553-4. 6. Rowe RC, Cowden EA, Faiman C. Correlation of Nbz bioassay and radioimmunoassay values for human serum prolactin. J Clin Endocrinol Metab 1983;57:942-6. 7. World Health Organization. Laboratory manual for the examination of human semen and semen-cervical mucus interaction. 2nd ed. New York: Cambridge University Press, 1992:2-19. 8. Nicoll CS. Bioassay of prolactin: analysis of the pigeon crop sac response to local prolactin injection by an objective and quantitative method. Endocrinology 1967;80:641-55. 9. For&h IA, Myres RP. Human prolactin: evidence obtained by the bioassay ofhuman plasma. J Endocrinol1971;51:15768. 10. Sassin JF, Frantz AG, Kapen S, Weitzman ED. The nocturnal rise of human prolactin is dependent on sleep. J Clin Endocrino1 Metab 1973;37:436-40.

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et al.

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in unexplained

infertility

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