J. Endocrinol. Invest. 1.' 39, 1978

Hypothalamic-pituitary-ovarian function In hyperprolactinemic women

P. Travaglini, B. Ambrosi, P. Beck-Peccoz, R. Elli, M. Rondena, R. Bara, and G. Weber Second Medical Clinic, Endorine Unit, University of Milano, Italy .

ABSTRACT. Hypothalamic-pituitary-ovarian function, as well as other pituitary tropic hormone secretions, was examined in 27 hyperprolactinemic patients (17 with PRL-secreting tumor and 10 with so called functional hyperprolactinemia). Serum PRL levels ranged between 39 and 108 ng/ml in functional cases and between 34 and 2500 ng/ml in patients with pituitary adenoma. Basal serum LH, FSH and 17 /3-estradiol levels did not differ from those recorded between days -14 and -10 in normally cycling women in functional cases, and were lowered in patients with pituitary tumor. LH response to GnRH (25 Jig iv) was normal in 7, impaired in 1 and exaggerated in 2 functional cases, and appeared normal in 9 and impaired in 8 tumoral cases. Two exaggerated FSH responses were seen in functional patients and 4 in patients with pituitary tumor. Estradiol benzoate administration (1 mg im) caused a significant fall in serum FSH and LH within 24 hr, but failed to induce serum LH increase within 96 hr in 11 out of 12 examined patients while a definite LH peak occurred within 48-72 hr in 7 patients retested after serum PRL normalization. Clomiphene citrate (100 mg/day for 5 'days) administration cau~sed a normal rise in serum LH, FSH and 17 /3-estradiol in 5 functional cases and in 1 case with pituitary tumor out of 9 patients tested. Normal pulsatility of serum LH was observed in 3 examined patients. In 3 normogonadotropic patients with PRL-secreting tumor, hCG + hMG administration (5000 + 150 IU im for 3 days) did not cause any significant increase in plasma 17/3-estradiol while inducing a sharp rise in the same patients retested after serum PRL normalization. No abnormal changes in TSH, GH and ACTH secretion both in the basal state ar.d after provocative stimuli were observed in patients with functional hyperprolactinemia, whereas in patients with pituitary tumors TSH secretion was impaired in 53% of cases, GH secretion in 71% and ACTH secretion in 29%. The present data along with previous reports in the literature suggest that high serum PRL can induce anovulation and amenorrhea by acting at roth hypothalamiC and ovarian levels.

INTRODUCTION High serum prolactin (PRL) levels are frequently associated with amenorrhea and anovulation (1-4). However, sites and mechanisms of action of ele­vated serum PRL levels in impairing the hypothalamic-pituitary-ovarian function are still con­troversial. Reduced or absent LH secreting episodes (5, 6) and lack of LH elevation both after estrogens (7, 8) .and clomiphene (2) administration observed in hyperprolactinemic women suggest a site of action at hypothalamic level. On the other hand a site of action at ovarian level is suggested (i) by the demon­stration, both in animals (9,10) and in humans (10) of

Key-words: Prolactin. amenorrhea-galactorrhea. functional hyperprolactinemia. PRL-secreting tumors. estrogen positive feedback. Gn-RH. clomiphene, hypothalamic-pituitary-ovarian axis.

Correspondence: dr, Pietro Travaglini, 2nd Medical Clinic, Endocrine Unit, Pd. Sacco, Ospedale Policlinico Via F. Sforza 35, 20122 Milano, Italy.

Received May 21. 1977; accepted October 31, 1977

39

specific receptors for PRL on the granulosa and corpus luteum cells and (ii) by the finding of reduced in vitro progesterone secretion in incubation medium by human follicles in the presence of high PRL concentration (11, 12). The present paper is based on the results of several tests---of hypofhatamic-pituitary-ovarian function car­ried out in women affected with hyperprolactinemic amenorrhea and on the modifications observed in some tests performed after normalization of serum PRL levels.

MATERIALS AND METHODS Twenty seven hyperprolactinemic women aged 19 -40 yr were examined, Seventeen had radiological abnormalities of the pituitary fossa sufficient to as­sume a pituitary adenoma (confirmed at operation in all cases). In 10 cases, no major sellar abnormalities were noted at linear tomography (cuts interval: 5 mm) and were then classified as functional hyper-

P. Travaglini, B. Ambrosi, P. Beck-Peccoz, R. Elli, M. Rondena, R. Bara, and G. Weber

prolactinemia of unknown etiology though the exis­tence of a microadenoma cannot be excluded. Twenty four cases had amenorrhea, 2 patients had hypo-oligomenorrhea and a single one was pOlymen­orrheic; galactorrhea was demonstrable in 15 pa­tients (7 with pituitary tumor and 8 functional cases): it was spontaneously present in 9 patients and provoked by gentle expression of ihe breast in 6. Onset of the symptoms occurred 6 months to 12 yr prior to investigation. Gonadotropic function was studied by determining serum LH and FSH in basal conditions and after the following tests:

iv injection of 25 ~ GnRH (Relisorm L ® Serono, Roma, Italy), blood being drawn at -30, 0, 20, 30, 60, 90 and 120 min. The results were compared with those obtained in 10 normally menstruating women in the early follicular phase (between days -12 and -10); im injection of 1 mg estradiol benzoate, blood being taken at 0, 24, 48, 72, 96 h. This test was carried out in 12 patients (6 with pituitary ade­noma and 6 functional cases) and was repeated in 7 (3 tumoral and 4 functional cases) after normalization of serum PRL levels obtained by either surgical or bromocriptine treatment; oral administration of clomiphene citrate (100 mgl day for 5 consecutive days). This test was carried out in 3 tumoral and 6 functional cases. LH , FSH, PRL, 17fJ-estradiol and progesterone were frequently determined over a 20 days pe­riod; study of LH secretory episodes: in 3 functional cases blood was withdrawn every 20 min from 08:00 to 12:00;

im administration of associated 5000 IU hCG and 150 IU hMG, daily for 3 days in 3 normogonado­tropic patients with PRL-secreting tumors. The test was repeated after normalization of serum PRL following operation.

Serum LH and FSH were evaluated by radioimmu­noassay (13, 14) using as reference standard the second . international reference preparation for human menopausal gonadotropin (2nd IRP-hMG). In our experimental conditions 1 mlU of 2nd IRP-hMG was equivalent to 4.0 and to 25.0 ng of the human pituitary gonadotrophin reference preparation LER 907 for LH and FSH respectively. Serum PRL was estimated by the radioimmunological method of Sinha et al. (15) using as reference standard hPRL VLS 1 and labelled hPRL and antiserum supplied by Biodata (Roma, Italy). Plasma 17 fJ-estradiol and serum progesterone were-determined by the method of Hotchkiss et al. (16) and by the method of Ab­raham et al. (17), respectively, using specific rea-

40

gents supplied by Dow-Lepetit Ltd. (Milano, Italy). In all patients other pituitary hormone secretions were determined by the following methods: Serum GH (hGH Kit® , Dow-Lepetit Ltd, Milano, Italy) was measured in the basal state and after iv insulin induced hypoglycemia (0.1 - 0.2 U/ Kg BW, blood glucose less than 40 mg / 100 ml). Serum TSH was estimated according to Odell et al. (18) using as standard the reference preparation 68 / 38 (NIMR - Mill Hill, London, GB) and a specific

. antiserum and hTSH for labelling supplied by NIH, Bethesda, Md., USA. Serum TSH was determined in basal conditions and after iv injection of 200 fUJ TRH (Bio'data-Roma, Italy). Serum T4 and T3 were determined by radioimmu­noassay (RIA T4 and RIA T3 Kits® , Dow-Lepetit Ltd. - Milano, Italy) and the resin T3 percent uptake by column (Trilute® Ames Co., Yssum - Jerusalem, Israel). Urinary 17 -hydroxycorticosteroids were measured by the method of Silber and Porter (19) before and after metyrapone administration. Plasma 110H-fluorog­enic steroids were estimated according to Mattingly et al. (20) before and after iv insulin.

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Fig. 1 - Serum LH, FSH, PRL and 17 fJ.-E2 basal values in 10 patients with functional hyperprolactmemla and m 17 pa­tients with prolactin secreting tumor. (Shaded areas re­present the normal range; central long horizonta l lines represent the mean of the values of 10 normal women; short horizontal lines the mean ± SE of the values of the patients).

RESULTS

Basal values

Individual mean basal serum PRL, LH, FSH and 17 B-estradiol values are outlined in Figure 1. Serum 'pRL levels ranged between 39 and 108 ng/ ml (mean 73 ± 7 SO ng/ ml) in so called «functional » cases and between 34 and 2500 ng / ml (mean 591 ± 180 ng/ ml) in patients with pituitary adenoma. I n func­tional cases basal serum LH, FSH and 17 p-estradiol values did not differ from those recorded between days -14 and -16 in normally cycling women. In .patients with pituitary tumor basal serum LH levels were significantly below the normal range in 70% of cases, serum FSH did not appear significantly low­ered and plasma 17 p-estradiol levels were clearly reduced (p < 0.001 vs controls).

GnRH test

Serum LH and FSH maximal net increase over baseline after GnRH administration is shown in Figure 2. In functional cases, LH response was normal in 7 patients, impaired in 1 and exaggerated in 2; FSH increase was normal in 8 cases and exaggerated in 2. In tumoral cases, LH response was normal in 9 patients and impaired in 8; FSH was normal in 7 subjects, impaired in 6 and exaggerated in 4.

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Fig. 2 - Serum LH and FSH maximal net increase after iv injection of 25 f-tIJ of GnRH in 10 patients with functional hyperprolactinemia and in 17 patients with prolactin sec­reting pituitary tumor.

41

PRL and hypotha/amic-pituitary-ovarian function

o .... ,. , u u • ..I Uti

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Fig. 3 - Recovery of serum LH and FSH response to estradiol benzoate (E2 B) administration after normalization of serum PRL levels in hyperprolactinemic women.

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Fig. 4 - Modifications of serum LH, FSH, 17 p-estradiol and progesterone in a case of functional hyperprolactinemia during and after clomiphene citrate administration. Similar patterns of response were observed in 5 other patients.

Estrogen test

Estradiol benzoate administration caused a signifi­cant fall in serum FSH (p < 0.005) and LH (p < 0.05) within 24 h, but failed to induce any Increase In serum gonadotropins in all but one patients (in spite of a plasma 17 p -estradiol peak of 366 ± 21 SE pg/ ml). On the contrary a definite increase in serum LH concentration was obtained in 7 patients retested after serum PRL normalization (9.3 ± 2.7 SE ng / ml) (Fig. 3). Throughout the test no significant modifica­tion in serum PRL occurred.

Clomiphene test

Out of 9 tested patients 5 functional and 1 tumoral cases showed a normal rise of LH , FSH, and 17 p~estradiol on days 2 - 8 followed by a second peak on days 12 - 15 (Fig. 4). Clomiphene adminis­tration induced uterine bleeding after 18 - 20 days in 4 patients despite no change in both basal body temperature (88T) and in serum progesterone. It is noteworthy that all clomiphene-responsive patients did not show any LH increase when tested with estradiol benzoate. No substantial modification in

P. Travaglini, B. Ambrosi, P. Beck-Peccoz, R. Elli, M. Rondena, R. Bara, and G. Weber

,, ~ r. \ 0 0

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Fig. 5 - Serum LH (closeCf circles) and PRL (open circles) fluctuations recorded from 08:00 to 12:00 in 3 cases of func tional hyperprolactinemia.

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- • - • • Fig. 6 - Serum 17 p-estradiol net increase after im injection of hCG (5000 IUlaay) and hMG (150 lUi day) for 3conse­cutive days in 3 patients with pituitary tumor, before (closed bars) and after serum prolactin level normalization (shaded bars). Closed circles represent serum PRL levels.

serum PRL levels occurred during and after clomi­phene administration.

LH secretory episodes

Normal pulsatility of serum LH [according to the criteria reported by Yen et al. (21)] was observed in the 3 examined patients (Fig. 5).

Gonadotropic stimulation

In 3 normogonadotropic patients with PRL-secreting tumor hCG + hMG administration did not cause any significant increase in plasma 17 p -estradiol while inducing a sharp rise (up to 300 - 460 pg/ml) in the same patients retested after serum PRL normaliza­tion (Fig. 6).

Other pituitary hormone secretions

No abnormal changes in the secretion of TSH, GH, and ACTH both in the basal state and after provo-

42

cative stimuli were observed in patients with func­tional hyperprolactinemia. Among patients with pitui­tary tumor, serum GH response to insulin was impaired in 71 % of cases; plasma cortisol and uri­nary 17 -OHCS failed to increase after insulin and metyrapone tests respectively in 29% of patients; serum TSH response to TRH was impaired in 53% of patients .

DISCUSSION It is well recognized that hyperprolactinemia can cause short luteal phases, anovulatory cycles and amenorrhea. However, the problem of the sites and mechanism of action of elevated serum PRL levels in affecting hypothalamic-pituitary-ovarian function is still debated. In fact there is general agreement on the very frequent fin.ding of impaired LH response to estrogen induced positive feedback (7 , 8, 22) while the data on the persistence of LH secretory episodes and on the response to clomiphene are controversjal: Boyar et al. (5) and Bohnet et al. (6) observed absent or reduced LH pulsatility while Gelli et al. (23) and ourselves did not. Bohnet et al. (6) and Thorner et al. (2) reported a high incidence of impaired responses to clomiphene test. On the contrary an increase in serum gonadotropins and 17 p -estradiol was ob­served in most of our patients with functional hyper­prolactinemia in the early phase of clomiphene test in agreement with data previously reported by Mor­timer et al. (24) in 6 galactorrhoic patients, thus suggesting the persistence of some sensitivity · to negative estrogen feed-back. This interpretation re­ceives support by the finding of a reduction in serum gonadotropins following estradiol benzoate adminis­tration. This is consistent with the data previously reported by Glass et al. (25) and by L' Hermite et al. (22). Furthermore in 6 of our patients a second LH peak was noted as already described by Marshall et al. (26) in 2 cases, suggesting a normal response to the positive feed-back mechanism. However estra­diol benzoate administration failed to raise serum LH in our clomiphene responsive patients. This discre­pancy might be explained by the more prolonged exposure to estrogen during the late phase of clomi­phene test. On the other hand no patient showed evidence of ovulation after clomiphene treatment. Finally it should be remembered that the persistence of midcycle LH surge can be observed in women in whom an elevation of serum PRL was induced by « short » term administration of sulpiride (27, 28) and that the mid-cycle LH peak disappeared in 3 out of 4 patients in whom sulpiride treatment was more pro­longed (28). Therefore it can be speculated that the alteration of gonadotropin secretion induced by high serum PRL levels is a graded phenomenon depending on degree and duration of hyperprolactinemia and probably also

on individual sensitivity. This alteration appears to be a reversible phenomenon, since the normalization of serum PRL concentration restored normal response to estrogen positive feedback (29) and, as demon­strated by increasing clinical evidence (1 - 9), it re establishes in several patients regular ovulatory menses. Pituitary gonadotropin reserve, studied by a single GnRH stimulation seems not to be affected by high serum PRL levels, at least in functional cases, being even enhanced in some cases in agreement with the data previously reported by Thorner et al. (2), Glass et al. (8), Jacobs et al. (30) and Asfour et al. (31 ). However, some evidence of reduced pituitary LH reserve has recently been suggested by Lachelin et al. (32) by repeated stimulation with small amounts of GnRH. In patients with pituitary tumor the pituitary gonadotropic reserve was reduced or absent in about 47% of cases. This impairment might be due to the presence of the adenoma per se rather than to the higher degree of hyperprolactinemia. In fact the incidence of impaired secretion of gonadotropins and of other pituitary hormones appears to be the same in the present series of patients with PRL-secreting adenoma and in a series of 22 patients with non­functioning pituitary adenoma we studied (unpu­blished data). Basal plasma 17 ~-estradiol levels observed in our patients with functional hyperprolac­tinemia appeared within the normal range in agree­ment with the data reported by others (2, 8, 30, 33, 34) and rose markedly in those cases who re­sponded to clomiphene. These findings taken together with those previously reported in hyperprolactinemic women showing an increase in urinary estrogen excretion after pro­longed hMG stimulation (35) and an adequate rise in plasma estradiol after GnRH (32), suggest that both estrogen synthesis and ovarian responsiveness to gonadotropins are not affected by elevated serum PRL concentration. Experimental results recently obtained by Mc Natty (personal communication) demonstrating that prolactin added to cultured human ovarian follicles does not influence estrogen secretion are also consistent with this interpretation. On the contrary, basal plasma 17 ~-estradiol levels were definitely low in severely hyperprolactinemic patients with pituitary adenomas and did not show any appreciable increase after heG + hMG adminis­tration while increased markedly after serum PRL normalization. Furthermore, unstimulated follicles were found by Mroueh and Siler-Khodr (36) in hyper­prolactinemic women after prolonged gonadotropic treatment while pregnancy occurred in one of them after serum PRL normalization. These data suggest an influence of serum PRL on ovarian responsiveness to gonadotropins and are conflicting with the above mentioned findings. This apparent contradiction might be explained by the

43

PRL and hypotha/amic-pituitary-ovarian function

degree and duration of prolactin excess and by the different schedule of gonadotropic treatment. It is known that PRL concentration in the liquor folliculi is 6 times higher than in circulating blood (6) and that the production of progesterone by cultured granulosa cells is maximal when. PRL concentration in the incubation medium ranges between 5 and 20 ng/ml, being inhibited at higher PRL levels (12). Such experimental data fit well with the absence of modifi­cations of serum progesterone values and of BBT in our clomiphene-responsive patients. Thus elevated serum PRL levels are confirmed to have a host of effects that appear to be exerted at both hypothalamic and ovarian levels in determining anovulation and amenorrhea. Finally it has to be pointed out that an impairment of pituitary tropic hormone secretions other than gonadotropins is frequently found in patients with pituitary tumor. Such a finding may be of diagnostic value.

ACKNOWLEDGMENTS We are indebted to NIAMDD - NIH (Bethesda, Md, USA) for gift of LER - 907, hPRL VLS 1, TSH antiserum and hTSH for labelling and to NIMR (Mill Hill London, GB) fr gift of the 2nd IRP-hMG and the Reference Preparation 68/38. The skillful technical assistance of Mrs. Francesca Secchi and Mrs. Federica Conti is gratefully acknowledged.

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P. Travaglini, B. Ambrosi, P. Beck-Peccoz, R. Elli, M. Rondena, R. Bara, and G. Weber

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31. Asfour M., L'Hermite M., Hedouin-Quincampoix M., Fossati P. Hypogonadism, galactorrhoea and hyper-prolactinemia: evaluation of pituitary gonadotrophins reserve before and under bromocriptine. Acta Endocrinol. (Kbh) 84: 738, 1977.

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PRL and hypothalamic-pituitary-ovarian function

154) in the galactorrhea syndromes. J. Clin. Endocrinol. Metab. 39: 18, 1 974.

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35. Archer D.F., Josimowich H.B. Ovarian response to exogenous gonadotropins in women with elevated serum prolactin. Obstet. Gynecol. 48: 2, 155, 1976.

36. Mroueh A.M., Siler-Khodr T.M. Ovarian refractoriness to gonadotropin in cases of inappropriate lactation: restoration of ovarian function with bromocryptine. J. Clin. Endocrinol. Metab. 43: 1398, 1976.