Human Reproduction vol.6 no 8 pp.1070-1073, 1991

Human prolactin release induced by follicle stimulating hormone, luteinizing hormone and human chorionic gonadotrophin

P.G.Crosignani1, M.Carena Maini, E.Negri2 and G.Ragni II Department of Obstetrics and Gynaecology, University of Milano, Via Commende, 12-20122 Milano and 2Mario Negri Institute, 20157 Milan, Italy 'To whom correspondence should be addressed

Plasma prolactin levels rise in stimulated cycles. To clarify the effects of gonadotrophin on the lactotrophs, three studies were performed. First, plasma concentrations of prolactin during clomiphene citrate (CC)- human menopausal gonadotrophin (HMG)-human chorionic gonadotrophin (HCG) treatment of women enrolled for in-vitro fertilization (TVF) were compared with those during HMG-HCG administration while under pituitary suppression with a gonadotrophin releasing hormone (GnRH) analogue (buserelin). Women suppressed with buserelin had higher basal levels of PRL in plasma (14.4 ± 4.3 ng/ml versus 6.9 ± 1.4 ng/ml, P < 0.001). Only buserelin-suppressed women showed a significant rise in plasma prolactin before HCG administration, while both patient groups had marked prolactin peaks after HCG injection. This peak was higher in the buserelin group (71.9 ± 50.7 ng/ml versus 52.6 ± 29.7 ng/ml). The second study showed that plasma levels of prolactin of 6 postmenopausal women were significantly increased 48 h after an injection of 5000IU HCG, i.m. (24.9 ± 17.4 ng/ml versus 12.4 ± 6.2 ng/ml P < 0.05). Third, plasma prolactin was studied in 5 women over 30 days after surgical castration. An upward trend was observed similar to that of endogenous gonadotrophin, with the change in prolactin values closely correlating with the change in concentrations of follicule stimulating hormone (P < 0.005). All these findings suggest that human gonadotrophins stimulate lactotrophs. Key words: subunit/human gonadotrophin/human prolactin

Introduction Plasma prolactin is transiently elevated in women undergoing ovarian stimulation with human gonadotrophins (Healy and Burger, 1983; Collins et al., 1984; Kauppila et al., 1988; Tippet et al., 1989). It may begin to rise during the follicular phase, but a distinct pronounced peak appears almost invariably after HCG injection (Kauppila et al., 1988; Tippet et al., 1989; Gonen and Casper, 1989; Crosignani et al., 1990). This indicates that administration of exogenous gonadotrophins evokes a transient hyperprolactinaemia which might affect the ovarian response and the success rate of in-vitro fertilization (IVF), since superovulation 1070

induced with human menopausal gonadotrophin (HMG) and human chorionic gonadotrophin (HCG) is the first step in IVF programmes. Improved ovarian responses and better fertilization rates have been reported after lowering PRL by treatment with bromocriptine (Reinthaller et al., 1988), but another recent study has questioned the clinical importance of the effects of transient hyperprolactinaemia on outcome (Hofmann et al., 1989). To clarify the underlying causes of the transient hyperprolactinaemia during ovarian stimulation, three approaches were used. First, the rise in prolactin during HMG —HCG administration in cycling women and in women suppressed by a gonadotrophin releasing hormone (GnRH) analogue was studied to investigate the influence of the different basal oestrogen concentrations of the two groups and the direct action of the analogue on the lactotrophs. Second, because of the distinct rise in prolactin after HCG injection, to look for a specific effect of HCG, plasma prolactin was measured in a group of postmenopausal women after an HCG bolus dose. Finally, to see if there is paracrine stimulation of prolactin by endogenous gonadotrophins, plasma prolactin profiles were studied after surgical castration. Patients and methods Ovarian stimulation Twenty-two normoprolactinaemic patients who were enrolled in an FVF programme were treated with either (group A) clomiphene-HMG-HCG or (group B) HMG-HCG while on pituitary suppression with the GnRH agonist, buserelin. To group A patients, 100 mg/day clomiphene was administered p.o. on days 3 - 7 of the cycle, and 2 - 3 ampoules/day HMG (Pergonal) from day 6 onwards. In the cycle preceding ovarian stimulation, group B patients were treated with buserelin, 200 /tg nasally, 5 times per day, starting on days 16—18. Buserelin was continued until the day of HCG administration (see below). On day 2 of the following cycle, ovarian stimulation was induced with 5 ampoules/day of FSH (Metrodin) on days 2 — 5, then with 3 ampoules Metrodin and 1 —4 ampoules Pergonal per day on days 6 - 1 0 . A dose of 5000 IU HCG, i.m., was injected when the leading follicle had a diameter of 16-17 mm. Echoguided oocyte retrieval was performed 35 h after HCG injection. Plasma oestrogen and prolactin levels were measured daily during ovarian stimulation, from day 2 until day 4 after oocyte collection. HCG test With their consent, 5000 IU HCG was given i.m. to 6 postmenopausal women. Prolactin was measured in plasma samples © Oxford University Press

Prolactin release induced by gonadotrophin

Tabte I. Plasma prolactin concentrations before and during ovarian stimulation of normoprolactinaemic women for IVF Stimulation regimen


No. of subjects

Plasma prolactin (ng/ml, mean ± SD) Basal

24 h before HCG

36 h after HCG

84h after HCG

10 12

6.9 ± 1.4 14.4 ± 4 3*

7.7 ± 3.2 20.9 ± 5.8**

52.6 ± 2 9 . 7 " ' 71.9 ± 50.7***

5.7 ± 2. 1 21.4 ± 7. 1

•Student's Mest, P < 0.001 (B versus A). ••Bonferroni's (test, P < 0.05 (versus basal). •••Bonferroni's (-test. P < 0 01 (versus 24 h before HCG)

obtained before and on each of the 2 days after HCG administration.

Table II. Plasma prolactin concentrations (mean ± SD) before and after i.m administration of HCG (5000 IU) to post-menopausal women


Patient no.

Plasma concentrations of follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin in 5 women undergoing complementary oophorectomy for benign uterine diseases were measured before and on days 2, 4, 6, 14 and 30 after surgery.


Prolactin level (ng/ml) Before HCG

12.4 ± 6 2

After HCG day 1

day 2

18.9 ± 1 3 2

24 9 ± 17.4*

•Student's /-test. P - 0 05. Wilcoxon test, P = 0 03

Hormone measurements Hormone measurements were carried out on duplicate daily samples and all samples were assayed at the same time. For all the radioimmunoassays, Biodata S.p.A. kits were used. Statistical methods In the ovarian stimulation experiment, the means of the basal plasma prolactin and oestradiol concentrations for women treated with C C - H M G - H C G and buserelin-HMG-HCG were computed and the significance of the difference was assessed by the Student's Mest. In addition, for both groups, the means of the difference between basal and pre-HCG plasma prolactin concentrations and of the difference between pre-HCG and postHCG plasma prolactin concentrations were computed. To see if these two variables were significantly different from zero, while controlling the rate of error and taking into account the problem of multiple comparisons, Bonferroni's Mest (which provides very conservative results) was used (Miller, 1981). The Student's Mest and the Wilcoxon rank test for paired samples were used to evaluate the significance of differences in the plasma prolactin concentrations before and after HCG administration to post-menopausal women. To compare the profiles of FSH and prolactin in women who had undergone ovariectomy, the differences between days 2, 4, 6, 14, 30 and day 0 were computed and the correlation between these two variables was evaluated by calculation of parametric (Pearson) and non-parametric (Spearman and Kendall) correlation coefficients. The corresponding P-values on the assumption of no correlation were also calculated. Results The rises of plasma prolactin in the 10 patients treated with C C - H M G - H C G and in the 12 who received HMG-HCG treatment during GnRH analogue suppression are summarized in Table I. Plasma oestrogen concentrations (mean ± SE) under basal conditions, 24 h before and 36 h after HCG were 18 ± 2, 1151 ± 118 and 1410 ± 157 pg/ml in the 'buserelin' group

and 45 ± 6, 1275 ± 260 and 1542 ± 323 pg/ml in the 'CC —HMG —HCG' group. The buserelin group had higher plasma prolactin and lower plasma oestradiol levels (f < 0.001). Only this group of women had significant rises in plasma prolactin before HCG administration. Both patient groups had marked prolactin peaks only after HCG injection. This peak was higher in the buserelin-treated women, but was not significantly different from that observed in the CC-HMG-HCG treated patients. The post-HCG rise in prolactin returned to normal invariably within 3 - 5 days. Table II lists the plasma prolactin levels in 6 post-menopausal women before and after a dose of 5000 IU, i.m., HCG. On the second day after HCG, there were significant rises in plasma HCG levels in these subjects. Table HI shows FSH and prolactin plasma concentrations after surgical castration. Both hormones tended to increase after oophorectomy. A comparison of the changes in FSH, LH and prolactin values over 30 days showed a close correlation, supported by the values of three different correlation coefficients: Pearson, 0.60 (P = 0.002); Spearman, 0.52 (P = 0.001); and Kendall, 0.41 (/> = 0.006). Discussion In the study of ovarian stimulation, only the women pretreated with buserelin had slightly high prolactin levels before HCG treatment, probably linked to the greater increase in oestrogen in these women (Shupnik et ai, 1979). It is notable, however, that the more pronounced elevation of prolactin took place only after the injection of 5000 IU HCG and the timing of the prolactin peak was essentially the same as the HCG clearance time. In the postmenopausal group of women, HCG had a similar stimulatory effect on prolactin secretion. The lower prolactin release after HCG administration to these subjects is probably due to the lesser reactivity of postmenopausal lactotrophs. On the contrary, in pregnancy, a synergistic stimulation of lactotrophs is likely to occur in the presence of opposite endocrine conditions: high levels of HCG and oestrogen. Interestingly, HCG also 1071

P.G.Crosignani et al.

Table i n . Plasma FSH and prolacun levels (mean ± SD) in 5 healthy women after oophorectomy

FSH (m]U/ml) Prolactin (ng/ml)



2.9 ± 1.2 7.8 ± 3.5

6.3 ± 6.5 20.1 ± 18.6

92 ± 7 6 18.1 ± 19.4

stimulates prolactin synthesis by decidual cells (Rosenberg and Bhatnagar, 1984) and, in turn, prolactin inhibits HCG production by the trophoblast (Ho Yuen et al., 1980, 1986). The parallel rises of FSH, LH and prolactin after castration support the hypothesis of a paracrine activity of endogenous gonadotrophin. The relatively small release of prolactin induced by oophorectomy is, again, probably due to the low level of circulating oestrogen and its occurrence at variable times might be related to the different times of the gonadotrophin surge after castration. Paracrine stimulation of prolactin by endogenous gonadotrophin is suggested by several types of physiological data. Synchronous pulsatility of circulating LH and prolactin levels has been reported for normal (Baeckstroem et al., 1982; Braund et al., 1984; Clifton et al., 1988) and hypogonadal women (Cetel and Yen, 1983). Prolactin release after GnRH stimulation has already been observed in several studies (Yen etai, 1972; Catania et al., 1976; Giampietro et al., 1979; Barbarino et al., 1982; Tan et al., 1986) and the close relationship of the LH and prolactin responses to GnRH has been explained as being due to a common neuroendocrine releasing mechanism (Mais and Yen, 1986). A similar pattern of differences in responsivity to GnRH of lactotrophs and gonadotrophs during the menstrual cycle has also been described, with LH and prolactin release higher in the periovulatory phase (Mais et al., 1986). Animal experiments further support the clinical findings. In ovariectomized rats, the administration of sheep anti-GnRH serum markedly reduced both basal and stimulated prolactin levels, suggesting that there is paracrine interaction between pituitary gonadotrophs and lactotrophs (Debeljuk et al., 1985a,b). In-vitro studies have provided evidence for paracrine stimulation of prolactin by pituitary gonadotrophs. Denef and Andries (1983) added GnRH to gonadotroph cultures and found in the culture medium an unidentified substance that could induce prolactin release from lactotrophs. Begeot et al. (1984) suggested that the paracrine factor is the free LH alpha subunit, and in fact they showed that free LH alpha subunit does stimulate the differentiation of rat fetal lactotrophs. Interestingly, a GnRH analogue, after a long incubation with rat pituitary cells in culture, induced 2- to 3-fold increases in the synthesis of LH alpha subunit (Hubert et al., 1988). This observation can provide an explanation for the high plasma levels of free alpha subunits found in patients chronically treated with GnRH agonists. Data concerning the direct effect of GnRH analogues on prolactin secretion are still discrepant. An inhibitory activity of the analogues has been reported in hyperprolactinaemic rats (Lamberts et al., 1981; Torres-Aleman et al., 1985) and in men with sulpiride-induced hyperprolactinaemia (Rubio et al., 1987) and with prolactinoma (Rubio et al., 1989). On the contrary, this prolactin-lowering effect was not observed in normoprolactinaemic cycling and menopausal women (Golan et al., 1989; 1072

Days after surgery 6


15.4 ± 14.9 20.9 ± 24 .5



38.9 ± 22 .6 29.6 ± 31 .9

50.5 ± 19.5 29.0 ± 340

Urban et al., 1990). The discrepancies could be due to the different oestrogen status as well as to the acute administration of GnRH analogues. The reported paracrine activity of the gonadotrophin alpha subunit on pituitary lactotrophs and its secretion induced by GnRH agonists indicate a new way by which they can influence prolactin secretion. In the literature, the existing evidence for paracrine interaction between gonadotrophs and lactotrophs is mainly based on in-vitro studies. However, the following findings of our clinical study support this hypothesis: (i) the occurrence of the greater rise in prolactin after HCG during ovarian stimulation; (ii) the time-pattern of the prolactin peak; (iii) the higher basal and stimulated plasma levels of prolactin in buserelin-treated women, because of increased alpha subunit production, the greater rise in oestrogen concentration and the larger dose of HMG needed by these subjects; (iv) the prolactin release induced in post-menopausal women by HCG; and (v) the hyperprolactinaemia occurring after oophorectomy. The physiological role of the stimulatory effects of gonadotrophin on prolactin release is completely unknown. Nevertheless, early in-vitro experiments (McNatty et al., 1974) and recent clinical data (Kauppila et al., 1988) have shown the detrimental effect of hypoprolactinaemia on the steroidogenic activity of the ovary. These findings indicate interesting directions for future investigation.

Acknowledgement National Research Council (CNR), targeted project Prevention and Control Disease Factors, Subproject 5 (Fertility Control), n.91.00131. PF41.115.05532.

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Human prolactin release induced by follicle stimulating hormone, luteinizing hormone and human chorionic gonadotrophin.

Plasma prolactin levels rise in stimulated cycles. To clarify the effects of gonadotrophin on the lactotrophs, three studies were performed. First, pl...
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