Clinical Endocrinology (1 979) 10,383-391.

PROLACTIN C O N C E N T R A T I O N S I N N O R M A L M E N S T R U A L CYCLES A N D CONCEPTION CYCLES ELIZABETH A . LENTON, LINDA M . BROOK, O L A D E L E SOBOWALE A N D IAN D. COOKE

Department o f Obstetrics and Gynaecology, Jessop Hospital f o r Women, Sheffield S 3 7RE (Received 27June 1978; revised 4September 1978;accepted I 1 September 1978) SUMMARY

Plasma prolactin concentrations were measured daily throughout twenty-three menstrual cycles from regularly ovulating women. In five of the cycles conception occurred spontaneously. The frequency distribution of prolactin concentrations was calculated and an appropriate linear transform obtained. Means and 95% confidence limits were determined by inspection of the logarithmic probability plots and these gave mean concentrations (and ranges) of 302 (106-871) mU/1 for the nonconception cycles and 178 (58-550) mU/1 for the conception cycles. Distribution of prolactin concentrations was log-normal in all cases, although the population containing the conception cycles was distinct from the population of non-conception cycles. It is suggested that the normal range found in ovulating women is 80-800 mU/1 but that for conception to occur levels should be slightly lower and approximate to the range 60-600 mU/1. Thus the lower limit of normal is about 60 mU/1 and the upper limit about 800 mU/1. Any concentration over 800 mU/1 should be designated as hyperprolactinaemia. Measurement of plasma prolactin concentration is becoming part of the standard investigation of any patient with a complaint of infertility (Besser et al., 1972; L’Hermite, 1973; Lenton et al., 1977). Although a number of groups have published data on the mean daily prolactin concentration profile throughout the menstrual cycle (see Vekemans et al., 1977), the results are hot in complete agreement. Furthermore, although a number of authors give mean hormone concentration (? SD or SEM) in women with normal cycles (L‘Hermite et al., 1972; Franchimont et al., 1976), 95% confidence limits derived from these data give values which are either close to zero or negative and thus do not conform t o physiological reality. The purpose of this study was to define the distribution and range of plasma prolactin concentrations found in regularly cycling, apparently normal women. Prolactin levels in a number of spontaneous conception cycles were also determined and compared with the larger series of ‘normal’, but non-conception, cycles. Correspondence to: Dr E. A. Lenton, Department o f Obstetrics and Gynaecology, Jessop Hospital for Women, Sheffield S3 7RE. 0 1979 Blackwell Scientific Publications 0300-0664/79/0400-0383$02.00

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Elizabeth A . Lenton et al. SUBJECTS AND METHODS

A total of 525 daily blood samples were obtained from eighteen volunteer women during twenty-three menstrual cycles. All the subjects who participated in this study had a history of regular menstrual cycles (lasting 27-32 days) and had not used oral contraception for at least 3 months. Each cycle had a mid-cycle peak of LH, plasma progesterone levels of at least 13.4 nmol/l (5 ng/ml) on 4 successive days and a luteal phase lasting 12 or more days (Abraham et al., 1972). There was no reason to consider that any of these women had a fertility problem (cf. Discussion in Lenton et al., 1978) although the majority were not married. Samples were obtained in ten cycles from women actively trying to conceive and, in five of the monitored cycles, conception occurred. Blood samples were obtained daily between 08.00 and 10.00 h. Plasma was separated by centrifugation within 60 min and stored at -20°C until assayed for prolactin. LH, FSH, oestradiol and progesterone concentrations in these samples have been reported elsewhere (Sobowale er al., 1978). Prolactiri radioimmunoassay

Prolactin was measured by radioimmunoassay (Reuter et al., 1976) using reagents (antiserum and immunochemical grade prolactin, VLS 3) distributed by the National Institute of Arthritis, Metabolism and Digestive Diseases (National Institute of Health, U.S.A.). Prolactin was labelled with lzSl (Radiochemical Centre, Amersham) using Chloramine-T and purified by filtration on Sephadex G100. Each plasma sample was assayed in triplicate using 50 p1 plasma in a total incubation volume of 350 p1. The antiserum was used at a final dilution of 1:52 000. Separation of bound and free hormone was performed using a second antibody (Wellcome Reagents Limited). All results were expressed in mU/1 of a laboratory standard (a pool of serum from lactating women) which had been calibrated by reference to the Research Standard A7 1/222, distributed by the National Institute for Biological Standards and Control (NIBSC), London. It is now recommended that the results of prolactin radioimmunoassays should be expressed in terms of the 1st International Reference Preparation (1st IRP, 75/504) issued by the NIBSC. This preparation is diluted to give results in mU/1 equivalent to A71/222. Serial dilutions of the laboratory standard exhibited parallelism with A71/222 and the 1st IRP. Interassay error, as determined by eighteen consecutive assays of two control sera, was 10.3% and 9.3% (coefficients of variation) at mean concentions of 213 and 668 mU/1 respectively. The lower limit of sensitivity was defined as 50 mU/1. Calculations

The distribution of prolactin samples was analysed according to the method of Kletzky et al. (1975). After grouping all prolactin concentrations in twenty 50 mU increments ranging from 0 to 1000 mU/l, a frequency histogram was constructed (Fig. la). A cumulative frequency distribution was then obtained (Fig. lb) and the cumulative percentage frequencies calculated. These were converted directly to probit values using scientific tables (Documenta-Geigy Scientific Tables, Ciba-Geigy Limited, Basle). Probit values were plotted against prolactin concentration increments on either linear or logarithmic scales (Fig. 2). The 50% cumulative frequency corresponds t o the sample mean @robit = 5.0) and the range of k1 SD corresponds to probits 4.0-6.0. Similarly the 95% confidence interval corresponds t o probits 3.0-7.0 A number of prolactin concentration differences were tested for statistical significance

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using appropriate tests. In the situation where data from two non-conception cycles from one subject were available, the results were averaged to give only one set of data per individual before any statistical tests were applied. All concentrations were logarithmically transformed before analysis.

RESULTS Prolactin concentrations were found with the greatest frequency in the fourth increment (200-250 mU/l) which represents the mode. Only three samples were recorded with a concentration of less than 50 mU/1 (0.5%) and n o sample had a concentration greater than 950 mU/1. It is clear from Fig. l a that the frequency distribution is skewed to the right and so probit values plotted against concentration also give a curve that is skewed t o the right (Fig. 2a). A normal (Gaussian) distribution would have resulted in a straight line when plotted in this way. The same data plotted on a logarithmic abscissa (Fig. 2b) gave two intersecting

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Fig. 3. After division of the sample into conception ( 0 - 0 ) and non-conception cycles (o---o), the probit-log concentration plot resolves into two separate straight lines (a). Similarly division of the data into follicular phase (A--A) and midcycle plus luteal phases (A-A ) also resolves the multicomponent line of Fig. 2b into two distinct and approximately straight lines (b).

L og-normal p rolac tin distribution

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straight lines. This suggests that the original population was not homogeneous and further division of the sample is necessary (Kletzky et al., 1975). When the prolactin concentrations found in the conception cycles were separated from the non-conception cycles, and cumulative frequencies recalculated, probit analysis gave two separate straight lines (Fig. 3a). Further division of the samples from the non-conception cycles of those subjects who were exposed to the risk of pregnancy but did not conceive, and those who were not exposed, yielded virtually identical distributions. Since the group who failed t o conceive contributed only 105 of 5 2 5 samples, this analysis was not pursued further. On the other hand, division of the data into groups according to the stage of menstrual cycle was also successful in producing two approximately linear plots (Fig. 3b). The follicular phase corresponded to days -14 to -4 before the LH peak, and the ovulatory and luteal phases corresponded t o the remainder of the cycle. These last two parts of the cycle, when analysed separately, yielded superimposable linear transforms and so have been grouped together. Mean hormone concentrations and 95% confidence limits derived from the probability plots are given in Table 1. For comparison the arithmetic mean (kl SD) calculated for all follicular phase samples was 277 k 166mU/1. This gives 95% confidence limits of -55 t o 609 mU/1 which obviously has no physiological meaning. Table 1. Prolactin concentrations (mean * 1 or 2 SD, mU/l) obtained by inspection of logarithmic probability plots Source of samples Conception cycles Non-conception cycles Follicular phase* Ovulatory and luteal phasest

n

-2 SD

-1 SD

Mean

+1 SD

+ SD

129 396 185 340

58 106 63 102

100 178 119 168

178 302 227 282

316 501 427 501

550 871 822 832

* Days -14 to -4 inclusive before the LH peak. t Remainder of the cycle. Although prolactin concentrations vary greatly from day to day and profiles from individuals do not show any consistent trends,mean daily profiles (Fig. 4) of the non-conception and the conception cycles did reveal small differences. Overall mean levels were higher in the non-conception group and this difference was significant (P< 0.05). Similarly increases in prolactin concentration during the ovulatory and luteal phases were more pronounced in the non-conception cycles. Both groups of the cycle, however, did show a gradual increase in prolactin concentration over the 2 8 d a y period. Small but significant differences between 3-day average follicular phase concentrations and the corresponding 3-day mid-cycle or luteal phase concentrations were found using paired t-tests (Table 2).

DISCUSSION The arithmetic mean and range of basal prolactin concentrations gives values for the lower limit of normal that are consistently negative even though, in our series, only 0.5% of the samples were lower than 50 mull. This observation confirms the impression given by Fig. l a and the data illustrated by Ehara et al. (1973), that the distribution of prolactin concentra-

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Fig. 4. Log-mean (+SEM) daily prolactin concentrations throughout the menstrual cycle for all non-pregnant subjects (a) and for the spontaneous conception cycles (b).

Table 2. Prolactin concentration (mean t 1 or 2 SD), mU/l) obtained using logarithmically transformed data over specific days of the menstrual cycle. For each individual (n = 18), an average value representative of the stated period was calculated and these data used to derive the means and ranges Days of cycle

-2 SD

-1 SD

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+1 SD

+2 SD

Early follicular* Days -9, -8, - 7 t Days -1, 0, +1 Days +7, +8, +9

106 83 117 111

151 137 192 183

217 233 317 302

311 383 523 498

446 632 863 8 20

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* Based on two samples obtained within the first 5 days of the cycle (Lenton et al., 1977). t The differences between days -9, -8, -7 and -1, 0, + I and between days -9, -8, -7 and +7, +8, +9 were significant (P< 0.02 and P < 0.01 respectively).

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tions is not normal and the use of an arithmetic mean is inappropriate. Replotting the data on log-probability paper gives a multicomponent linear transform suggesting that the original sample population was not homogeneous. Division of the sample into either conception or non-conception cycles, or into follicular and ovulatory plus luteal phases, results in two separate straight lines. Whichever method of subdividing the data is selected, the distribution of prolactin concentrations remains log-normal. It is not possible to determine which method of grouping the initial data is more correct, as although linearity was slightly better with the first method (i.e. conception versus nonconception cycles), this may have only been a fortuitous reflection of the small number of conception cycles. It is likely that any series of twenty-three cycles may contain a significant number of cycles which would not result in pregnancy even though ovulation occurred apparently normally. In fact at least five of the eighteen non-conception cycles in our subjects fall into this category (Sobowale et al., 1978). The range of individual prolactin concentrations found in our study was very wide (approximately 80-800 mU/l) and was not clearly related t o the stage of the cycle or t o any other of the reproductive hormones (McNeilly e t al., 1973). This makes it difficult either to define the ‘normal’ range precisely or to establish the importance of plalsma prolactin in regulating reproductive function. Our data indicate that prolactin levels should perhaps be lower than the range quoted above for conception to occur and a more apprQpriate normal range may thus be about 60-600 mU/1. Conception frequently follows reduction of circulating prolactin concentrations in regularly ovulating or anovulatory women (Lenton et al., 1977; Pepperell et al., 1977) and it is thought that a gradual elevation in pnolactin concentration leads to cycles characterized by defective luteal phases (Corenblum er al., 1976; Seppala et al., 1976), amenorrhoea (Seppala et al., 1975) and in some patients, galactorrhoea (Varga et al., 1973; Del Pozo et al., 1975). Prolactin depression, on the other hand, is known t o be associated with an increase in FSH secretion (Seki et al., 1975; Penperell et al., 1977). Significantly increased FSH concentrations in the follicular phase were also seen in the five conception cycles reported here (Sobowale et al., 1978). Alternatively, the mall but significant differences between the distribution of prolactin in the follicular phase and the remainder of the cycle (Tables 1 and 2 ) may also be physiologically important and has been observed by others (Franchmont et al., 1976;Vekemans et al., 1977). Great variability between individual cycles was seen (McNeilly & Chard, 1974) with apparently randOm short term fluctuations (Leighton er al., 1976). Our conception cycles showed less vatiability and a smaller change with the duration of the cycle than did the series as a whole (Fig. 4). The profile of these cycles was similar t o the profiles reported by Ehara et al. (1973) and McNeilly & Chard (1974) where no significant differences were found between different phases of the menstrual cycle. In general, when assessing a random basal prolactin concentration, the effect of the stage of the menstrual cycle can be ignored and the normal range taken as about 80-800 mU/1. It is possible that this normal range may differ slightly when prolactin estimations are performed in different laboratories due to variations in technique, reagents and so on. However. if all laboratories expressed their results in terms of the International Reference Preparation (75/504) which has equivalent unitage t o the Research Standard (A71/222), direct interlaboratory comparisons should be possible. Of course any calculation of the normal mean and range which does not take into account the non-Gaussian distribution, will result in upper limits of normal that are too low. From our data, any individual sample with a concentration greater than 1000 mU/1 (and

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probably greater than 800 MU/I) can be considered t o be abnormal and should be investigated further. Elevated prolactin levels may be due t o pathological hyperprolactinaemia, or possibly to the stress of venepuncture (Koninckz, 1978). Any sample that has a concentration between 600 and 1000 mU/1 may or may not be normal (cf the upper limit in the conception cycles was 600 mull). In this situation further prolactin estimations become necessary although interpretation is likely to remain difficult until more information is available on the range of concentrations found in regularly cycling but infertile women, with and without mildly elevated prolactin levels, in relation t o the incidence of pregnancy. At the present time little physiological importance is attached t o the lower limit of normal prolactin concentration but with the widespread use of drugs for suppressing circulating prolactin and the concept of ‘over treatment’ with bromocriptine, this information becomes increasingly relevant. ACKNOWLEDGEMENTS

We are grateful t o Mrs D. Cocker and Miss B. McNeill for technical and secretarial assistance and t o Sandoz Products Limited for financial support for one of us (E.L.). REFERENCES ABRAHAM, G.E., ODELL, W.D., SWERDLOFF, R.S. & HOPPER, K. (1972) Simultaneous radioimmunoassay of FSH, LH, progesterone, 17-hydroxyprogesterone and estradiol-17p during the menstrual cycle. Journal of Clinical Endocrinology and Metabolism, 34, 31 2-31 8. BESSER, G.M., PARKE, L., EDWARDS, C.R.W., FORSYTH, LA. & McNEILLY, AS. (1972)Galactorrhoea: successful treatment with reduction of plasma prolactin levels by brom-ergocryptine. British Medical Journal, iii, 669-672. CORENBLUM, B., PAIRAUDEAU, N. & SCHEWCHUK, A.B. (1976) Prolactin hypersecretion and short luteal phase defects. Obstetrics and Gynecology, 47,486-488. DEL POZO, E., VARGA, L., WYSS, H., TOLIS, G., FRIESEN, H., WENNER, R., VETTER, L. & UETTWILER, A. (1 974) Clinical and hormonal responses to bromocriptine (CB154) in the galactorrhoea syndromes. Journal of Clinical Endocrinology and Metabolism, 39, 18-26. EHARA, Y., SILER, T., VANDENBERG, G., SINHA, Y.N. & YEN, S.S.C. (1973) Circulating prolactin levels during the menstrual cycle: episodic release and diurnal variation. American Journal of Obstetrics and Gynecology, 117, 962-970. FRANCHIMONT, P., REUTER, A,, VRINDTSGEVAERT, Y., VAN CAUWENBERGE, J.R., DOURCY, C., REMACLE, P., LEGROS, J.J., COLIN, C. & GASPARD, U. (1976) In: Radioimmunoassay of Prolactin in Health and Disease. Imprimerie Bietlot Frtres, Brussels. KLETZKY, O.A., NAKAMURA, R.M., THORNEYCROFT, LH. & MISHELL, D.R. (1975) Lognormal distribution of gonadotrophins and ovarian steroid values in the normal menstrual cycle. American Journal of Obstetrics and Gynecology, 121,688-694. KONINCKZ, P. (1978) Stress hyperprolactinaemia. Lancet, i, 273. LEIGHTON, P.C., McNEILLY, A S . & CHARD, T. (1976) Short-term variation in blood levels of prolactin in women. Journal of Endocrinology, 68, 171-1 18. LENTON, E.A., ADAMS, M. & COOKE, I.D. (1978) Plasma steroid and gonadotrophin profiles in ovulatory but infertile women. Clinical Endocrinology, 8, 241-255. LENTON, E.A., SOBOWALE, O.S. & COOKE, I.D. (1977) Prolactin concentrations in ovulatory but infertile women: treatment with bromocriptine. British Medical Journal, ii,1179-1 181. L‘HERMITE, M. (1973) The present status of prolactin assays in clinical practice. Clinics in Endocrinology and Metabolism, 3,423-463, L’HERMITE, M.,DELVOYE, P., NOKIN, J . , VEKEMANS, M. & ROBYN, C. (1972) Human prolactin secretion, as studied by radioimmunoassay: some aspects of its regulation. In: Prolactin and carcinogenesis (ed. by A. R. Boyns and K. Griffiths), pp. 81-97. Alpha Omega Alpha Publishing, Cardiff.

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McNEILLY, A.S. & CHARD, T. (1974) Circulating levels of prolactin during the menstrual cycle. Clinical Endocrinology, 3, 105-1 12. McNEILLY, A.S., EVANS, G.E. & CHARD, T. (1973) In: Human Profactin (ed. by J. L. Pasteels and C. Robyn), pp. 231-232. International Congress Series No. 308. Excerpta Medica, American Elsevier, New York. PEPPERELL, R.J., EVANS, J.H., BROWN, J.B., BRIGHT, M.I., SMITH, M.A., BURGER, H.G. & KEALY, D. (1977) A study of the effects of bromocriptine on serum prolactin, follicle stimulating hormone and luteinising hormone and on ovarian responsiveness to exogenous gonadotrophins in anovulatory women. British Journal of Obstetrics and Gynaecotogy, 84,456-463. REUTER, A.M., KENNES, F., GEVAERT, Y. & FRANCHIMONT, P. (1976) Homologous radioimmunoassay for human prolactin. International Journal of Nuclear Medicine and Biology, 3,21-28. SEKI, K., SEKI, M . & OKUMURA, T. (1975) Effect of CB-154 (2-Br-a-ergocryptine) on serum follicle stimulating hormone, luteinising hormone and prolactin in women with amenorrhoea-galactorrhoea syndrome. Acta Endocrinologica, 79, 25-33. SEPPALA, M., HIRVONEN, E. & RANTA, T. (1976) Hyperprolactinaemia and luteal insufficiency. Lancet, i, 229-230. SEPPALA, M., HIRVONEN, E., RANTA, T., VIRKKUNEN, P. & LEPPALUOTO, J. (1975) Raised serum prolactin levels in amenorrhoea. British Medical Journal, ii, 305-306. SOBOWALE, O., LENTON, E.A., FRANCIS, B. & COOKE, I.D. (1978) Plasma steroid and gonadotrophin profiles in spontaneous human conception cycles. British Journal of Obstetrics and Gynaecology, 85,460-467. VARGA, L., WENNER, R. & DEL POZO, E. (1973) Treatment of galactorrhoea and amenorrhoea with Brergocryptine (CB154): restoration of ovulatory function and fertility. American Journal of Obstetrics and Gynecology, 117, 75-79. VEKEMANS, M., DELVOYE, P., L’HERMITE, M. & ROBYN, C. (1977) Serum prolactin levels during the menstrual cycle. Journal of Clinical Endocrinology and Metabolism, 44,959-1003.