Testicular secretion after pulsatile human menopausal gonadotropin therapy in gonadotropin-releasing hormone agonist desensitized dysspermic men *

; Vol. 62, No.1, July 1994

FERTILITY AND STERILITY Copyright

©

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

1994 The American Fertility Society

Testicular secretion after pulsatile human menopausal gonadotropin therapy in gonadotropin-releasing hormone agonist desensitized dysspermic men*

Dimitrios A. Adamopoulos, M.D,t Stamatina Nicopoulou, M.D. Niki Kapolla, B.S.

Panayiotis Vassilopoulos, M,D, Maria Karamertzanis, M,D.

Endocrine Department, Elena's Hospital, Athens, Greece.

Objective: To evaluate Leydig and Sertoli cell response to prolonged pulsatile stimulation with hMG after pituitary desensitization with the GnRH agonist (GnRH-a) triptoreline in normogonadotropic men with abnormal semen analyses. Design: A group of four oligozoospermic men were investigated in the following manner: [1] basal and GnRH-hCG stimulated activity were assessed in all volunteers; [2] a long-acting form of the GnRH-a triptoreline (3.75 mg every month for 3 months) was given, and its effectiveness was evaluated on day 20; and [3] on that day hMG pulsatile administration was introduced (150 IV per 24 hours in 90-minute pulses) with serial hourly sampling (6 to 7 hours) for measurement of FSH, LH, T, E 2 , and inhibin on days 20, 41, and 90 from the first GnRH-a injection. Results: Initial evaluation showed normal basal, GnRH, and hCG-stimulated hormone concentrations. Pituitary and gonadal activity were effectively suppressed by GnRH-a when tested on day 20. Pulsatile hMG had no immediate stimulatory effect on gonadal activity (day 20). However, on middle and final evaluations (days 41 and 90), basal T, E 2 , and inhibin had risen to pre-GnRH-a levels, and, moreover, distinct secretory pulses were seen for these hormones. Conclusion: These findings indicate that suppression of pituitary gonadotropin activity with triptoreline combined with pulsatile hMG stimulation offers a new, useful tool for investigation of the male reproductive system in oligozoospermic men. Fertil Steril1994;62:155-61 Key Words: Inhibin, sex steroids, GnRH-a, hMG, pulsatility

The inter-relationship between the hypothalamic-pituitary complex and the gonads is mainly determined by the central pulse generator unit with proper tropic hormone secretion in association with testicular endocrine activity in periphery. Disturbances at any level of the axis may lead to impairment of reproductive function.

Received September 27, 1993; revised and accepted February 21,1994. * Supported by grants from by the Hellenic Society of Andrology, and Bodossakis Foundation, Athens, Greece. t Reprint requests: Dimitrios A. Adamopoulos, M.D., Endocrine Department, Elena's Hospital, 2 Venizelou Square, GR11521 Athens, Greece (FAX: 30-1-641-1156). Vol. 62, No.1, July 1994

In a clinical context, idiopathic hypogonadotropic hypogonadism (IRR) is a condition associated with disturbed activity of the pulse generator (1, 2). Activation or proper substitution of its function offers a unique opportunity for the study of central-peripheral relationships under prearranged conditions (3, 4). Moreover, in IRR syndromes exogenous pulsatile GnRR-a administration is associated with maximal therapeutic effectiveness both in men and women (5,6). On the other hand, in a subgroup of men with idiopathic oligozoospermia, disturbed frequency and amplitude of gonadotropic secretory pulses has been observed (7). Therapeutic intervention with pulsatile gonadotropin administration has been

Adamopoulos et al.

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155

proposed as a suitable treatment for restoration of endocrine activity and the defective spermatogenesis (Aulitzky W, Chandra I, Frick J, abstract). Furthermore, also on the clinical side, in the polycystic ovarian syndrome (PCOS) the hypothalamic-pituitary-ovarian complex operates in a manner different from that of the normal ovary (8, 9), and suppression of endogenous gonadotropic activity in association with exogenous ovarian stimulation results in high rates of ovulation induction and pregnancy (9). Based on this evidence, a working hypothesis was made proposing that suppression of pituitary gonadotropic secretion in combination with pulsatile testicular stimulation may be a suitable model for the study of central-peripheral inter-relationships in dysspermic men. Furthermore, this combined approach may offer the possibility of a novel therapeutic intervention in selected cases of men with idiopathic oligozoospermia if it can reproduce effectively conditions of normal testicular function and be properly evaluated. To this end, a group of four oligozoospermic men were investigated under conditions of prolonged desensitization of pituitary gonadotropic activity using a long-acting GnRH agonist (GnRH-a) and stimulation of testicular endocrine secretion after achieving effective pituitary suppression with pulsatile administration of hMG and evaluation of Leydig and Sertoli cell activity.

MATERIALS AND METHODS Patients

Four normogonadotropic oligozoospermic men, aged 36 to 45 years, volunteered for this study after the protocol proposed was scrutinized and approved by the hospital's ethical committee. Written consent was obtained from all participants. Oligozoospermia in those men was established by at least two to three consecutive semen analyses according to the criteria set by the World Health Organization (10) and with no relevant treatment for approximately 6 months before the study. Detailed history and complete physical examination revealed no systemic, endocrine, or genital abnormalities, including varicocele, in any of the patients. The number of spermatozoa (mean, 3.9 X 106/mL; range, 1.7 to 6.9 X 106/mL) and their good forward motility (mean, 13%; range, 8% to 21 %) was indicative of severe oligozoospermia. 156

Adamopoulos et al.

EXPERIMENTAL DESIGN

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Protocol of the Study

Phase I

In this initial stage, basal and stimulated pituitary gonadotropin and testicular endocrine activity were evaluated. For this purpose a standard GnRH test (100 fJ,g Relefact GnRH; Hoechst, Berlin, Germany) was performed with sampling at 0, 30, and 60 minutes after the IV injection and measurement of FSH and LH (day 0, Fig. 1). Immediately after the GnRH test, a single dose of 1,500 IV of hCG was given 1M (Pregnyl; Organon, Oss, The Netherlands) with sampling before and 24 hours later and estimation of T, E 2 , and inhibin. Phase II

This started after the post-hCG sampling with administration of the long-acting GnRH -a triptoreline (Arvecap, Ipsen/Biotech, Marseilles, France) at a dose of 3.75 mg given 1M every month for 3 months. Triptoreline is a GnRH -a with the glycine at position 6 replaced by D-tryptophane (D-Trp6 GnRH) and with biological activity 25 to 100 times higher than that of the native hormone. In the preparation used, 3.75 mg of triptoreline is combined with 170 mg of the polymeric dllactide-coglycolide. Maximal blood concentration of the analogue is reached in 2 days, and suppression of gonadotropins and sex steroids to hypogonadal levels is achieved within 7 to 10 days from injection. Evaluation of its effectiveness was made with basal hormone estimations on day 20 from the first injection. Phase III

After sampling on day 20, hMG administration (Humegon; Organon) started via a portable pump (MP-30; Micrel, Athens, Greece). This pump was programmed to deliver a total of 150 IV of hMG per

Stimulation of GnRH-a-suppressed testes

Fertility and Sterility

24 hours in equal 90-minute pulses. This pulsatile treatment lasted for a total of 70 days. Evaluation during the combined treatment was made on days 20, 41, and 90 after introduction of GnRH-a therapy and included serial hourly sampling (6 to 7 sampIes each day from each subject). After termination of hMG treatment with disconnection of the pump, basal endocrine activity was evaluated after 8 weeks (washout samples). Furthermore, postinvestigation follow-up included semen evaluations immediately, 3, and 6 months after termination of the study and with no additional medication. Methods

The concentrations ofFSH, LH, T, E 2, and inhibin were estimated using commercially available kits. To eliminate interassay variations, all samples were run in large, single assays. In particular, FSH and LH were measured using an immunoradiometric assay (CIS; Biointernational, Paris, France). In these systems, the intra-assay coefficient of variation (CV) of FSH-LH is 4.7% and 1.8% for low and 3.2% and 1.7% for high values, with sensitivity at 0.20 and 0.15 U/mL, respectively. Double-antibody techniques were used for determination of T and E2 (Sorin, Saluggia, Italy). In these systems, sensitivities of 0.05 ng/mL and 12 pg/mL have been established for each steroid. The CV for these assays are 10.9 and 16.6 for low and 7.2 and 5.2 for high T and E2 concentrations. Inhibin was measured using a two-site immunoenzymetric assay (CASIA; Medgenix Diagnostics, Fliurus, Belgium), in which two antibodies recognizing distinct epitopes of the a-subunit were used. As it is known, the epitomic specificity of the antibody for human sperm inhibin is localized entirely in the a-subunit (11). All samples were run in a single assay with new standards placed at positions 25, 50, and 75 of the sample series. The sensitivity of the system used was 0.6 U /mL, and the intra-assay CV was 1.9% for low (1.60 ± 0.03 U/ mL) and 2.9% for high (7.70 ± 0.22 U/mL) inhibin concentrations.

phase was tested using Student's t-test. Analysis for the presence of nonrandom pulses in the serial hormone estimations on days 20, 41, and 90 was carried out using the cluster analysis technique introduced by Veldhuis and Johnson (12). With this method, individual peak episodes of each hormone are identified using a dose-dependent variance function in which the relationship between intersample variance and mean hormone concentration is assessed by a power function fit in each subject using all samples assayed. On the other hand, temporal coupling analysis among identified pulses of different hormones was carried out using a recently proposed approach (13). In this, the possible nonrandom associations among three or more distinct hormone series consisting of episodic pulses are assessed. RESULTS

The concentration of different hormones throughout the three phases of the study showed marked fluctuations in all subjects (Figs. 2 and 3). At initial evaluation, basal concentrations were within the normal range in all cases, whereas

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Stimulation of GnRH-a-suppressed testes

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GnRH- and hCG-stimulated values ofFSH, LH, T, and E2 were within the expected range of responses. With regard to inhibin, there was practically no change after GnRH stimulation, but there was a distinct rise in three of the four cases with a mean 63% increase over basal in the post-hCG value of the group. Suppression of pituitary and gonadal endocrine activity was very effective when examined on day 20 after the first triptoreline injection in all four subjects. On that day, hormone concentrations were well within the hypogonadal range of values. Gonadal stimulation with pulsatile hMG administration produced time-related changes on the suppressed testicular secretion. Thus, on day 20 of GnRH-a suppression, very small, if any, fluctuations of gonadal hormones were observed in all subjects, mean values being very low (Table 1). The occasional pulses noted (3 in all) were all for inhibin and none for gonadal steroids. On the other hand, a number of FSH pulses were observed (6 in all) and were apparently related to exogenous hMG administration. Gonadal hormone concentrations were markedly raised on day 41 of evaluation, the mean value of the group being significantly higher than that ofthe previous evaluation (Table 1). Furthermore, a large 158

Adamopoulos et aI.

number of pulses (a total of 14) were observed during that period. The total and mean (±SD) number of pulses per study period were 4 and 1.00 ± 0.69 for T, 4 and 1.0 ± 0.70 for E 2 , and 5 and 1.25 ± 0.85 for inhibin. On final evaluation (day 90), gonadal steroids, but not inhibin, showed a further small rise, significant only in the case ofT (P < 0.001), in relation to their concentrations on day 41, and they were markedly higher than those on day 20 (Table 1). Moreover, a large number of pulses (14 in total) were also discerned, the total and mean (±SD) number being 2 and 0.60 ± 0.45 for T, 5 and 1.25 ± 1.00 for E 2 , and 5 and 1.75 ± 0.50 for inhibin. The difference between means for days 20 and 90 was significant for the peptide hormone (P < 0.05). The mean (±SD) number of pulses for all three hormones were 0.25 ± 0.16 (day 20),1.08 ± 0.45 (day 41), and 1.18 ± 0.42 (day 90), the difference between initial (day 20), intermediate (day 41), and final evaluation (day 90) being significant (initial versus day 41, P < 0.01; initial versus day 90, P < 0.001). Hormone evaluation 8 weeks after discontinuation of treatment showed basal values similar to those of initial evaluation. One-way ANOVA in individual cases and the whole group showed significant differences among the three phases for all gonadal hormones and FSH but not for LH (Table 2). Temporal coupling analysis of the pulses discerned showed no significant correlations either among them or between hMG and gonadal hormone pulses. Significant correlations among hormone concentrations were found only on a few occasions (day 20, FSH to E 2 , P < 0.02, FSH to T, P < 0.05, and day 90, inhibin to LH, P < 0.01). On the other hand, concordance of secretory bursts was seen very occasionally. On a clinical level, triptoreline administration was initially associated with hot flushes (2 cases), irritability (2 cases), and some loss of sexual interest (3 cases), or reduced satisfaction from the activity (3 cases). However, there was a partial recovery after introduction of hMG treatment in all four men and total restoration before the end of the investigation in three of them. One of the subjects (no. 1) experienced a prolongation of this compromising effect on his libido and was given hCG treatment (3,000 IV every 4 days for 4 times) after the end of the study period. Soon after the second injection, the patient reported complete recovery. It is noteworthy that T concentration in this patient did not markedly change after pulsatile hMG treatment during the period of investigation.

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Fertility and Sterility

Table 1

Concentrations and Differences Among Evaluations in the Serially Assayed Hormones in the Group Differences Day 20 (n = 27)

Hormone FSH (U/L)* LH (U/L) T (ng/mL) E2 (pg/mL) Inhibin (U/mL)

5.41 0.52 0.11 10.66 0.78

± ± ± ± ±

0.44t 0.05 0.05 0.45 0.11

Day 41 (n = 26) 7.81 0.49 3.79 18.04 2.76

± ± ± ± ±

0.65 0.03 0.36 1.94 1.01

Day 90 (n = 25)

Day 20 versus 41

Day 20 versus 90

Day 41 versus 90

± ± ± ± ±

P < 0.001 NS:j: P < 0.001 P < 0.001 P < 0.001

P < 0.001 NS P < 0.001 P < 0.001 P < 0.01

P < 0.05 NS P < 0.001 P < 0.001 NS

8.51 0.49 4.71 25.70 2.15

1.34 0.04 0.59 2.12 1.25

* Conversion factors to SI units for FSH, 1.0; LH, 1.0; T, 3.4 70; E 2, 3.671. Inhibin: 1.0 U /L is the average concentration in 20 healthy, eugonadal men. There is no conversion factor for inhibin up to now.

t Values are means ± SD. :j: NS, not significant.

Semen analysis immediately after the end of hMG treatment showed no appreciable changes in comparison with initial evaluation either in the number (mean, 4.6 X 106 /mL; range, 1.3 to 8.7 X 106 /mL) or in the motility of the group (mean, 15%; range, 9% to 28%). A similar picture emerged at the evaluations performed 3 and 6 months later.

throughout the study, but androgen deficiency symptoms, requiring hCG boasting, remained after the end of investigation. However, these symptoms were promptly and in short time completely reversed. Under conditions of endogenous gonadotropin suppression, pulsatile stimulation of Leydig and Sertoli cells with hMG resulted in complete (3 cases) or partial (1 case) restoration of their secretory activity. This effect was relatively delayed and less pronounced for T and E2 and more evident and early in the case of inhibin secretion. This differential response could be attributed to a number of factors, including the dual origin of inhibin from Sertoli and Leydig cells (14, 15), the relatively stronger LH suppression obtained, the direct inhibitory effect of the analogue on Leydig cells (16), the inhibition of steroidogenic enzymes (17), a more pronounced hMG effect on Sertoli cells or a combination of all or some of these factors. Furthermore, the possibility that urinary hMG exerts a biological action different from that of the native gonadotropins cannot be ruled out. Interference of endogenous FSH and LH in our cases was not only minimal but was probably characterized by an unchanged bioactivity: immunoactivity ratio (18). Ad-

DISCUSSION

Pulsatile administration of hMG reversed effectively the suppression of Leydig and Sertoli cell activity induced by pituitary desensitization with the GnRH-a triptoreline. This agonist reduced pituitary gonadotropins and subsequently testicular endocrine activity to hypogonadallevels when evaluated on day 20 from its initial administration. Restoration of gonadal activity after hMG substitution was achieved in a progressive manner since serial measurements on the 1st day of pulsatile treatment showed no immediate secretory response, as judged by the low hormone concentrations and the paucity of distinct pulses. A degree of suppression appeared to persist, despite gonadal stimulation, in one case (subject 1) and perhaps is not accidental that not only T and E2 concentrations remained low

Table 2

One-way ANOVA in the Individual Cases and the Whole Group Between the Three Study Periods (Days 20, 41, and 90) Subjects

Hormone FSH LH T E2 Inhibin

1

2

3

4

Group

P < 0.05

NS* NS P < 0.001 P < 0.001 P < 0.005

P < 0.001 NS P < 0.001 P < 0.001 P < 0.001

P < 0.001 NS P < 0.001 P < 0.01 P < 0.001

P < 0.001 NS P < 0.001 P < 0.001 P < 0.001

NS NS NS NS

* NS, not significant. Vol. 62, No.1, July 1994

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ministration of hMG and hCG has been reported to increase the low inhibin secretion in hypo gonadal men, and certainly our observations lend support to those observations (3). Furthermore, it has been proposed that FSH and hCG exert an independent action, the former on Sertoli and the latter on Leydig cells (3). This action is probably influenced by the sex steroid millieu prevailing because inhibin's response to those stimulants is different in eugonadal men (4). Many endocrine signals occur in pulse trains, as pulsatile hormone time series, in which the occurrence of secretory events over time is random or episodic, and this temporal pattern may convey important information to the target cells. In this study, a relatively large number of pulses was noted for both steroids and inhibin, and this observation indicates that restoration of GnRH-a-suppressed gonadal activity is mediated through a resumption of pulsatile Leydig and Sertoli cell secretion. A lack of correlation among the pulses of T, E 2 , and inhibin concentration was observed either in individual cases or in the group. This was probably due to the sampling schedule (every 60 minutes), to the rather small number of samples (6 to 7 per subject), or to both factors. Other studies have shown concurrent secretory pulses of inhibin and T but in spermatic and not in cubital vein blood (19). As demonstrated by this study, restoration of Leydig and Sertoli cell secretion with hMG was achieved both with an increase ofthe GnRH-a-suppressed T, E 2 , and inhibin concentrations and the appearance of secretory pulses in increased frequency. By and large, the secretory pattern obtained showed some similarities to that observed in normal men (20) or induced in patients with IHH (3, 19), except that in our cases, this was exogenously modulated. Therefore, our findings indicate that an approach similar to that employed in men with IHH can also be used for the study of pituitary-gonadal interactions in patients with impaired spermatogenesis. Suppression of the altered hypothalamic-pituitary-ovarian axis with subsequent gonadal stimulation has been widely practiced in PCOS cases with an ovulation resulting in high pregnancy rates (9). Furthermore, in some men with idiopathic oligozoospermia, therapeutic intervention with pulsatile GnRH administration has been proposed for normalization of the altered pattern of gonadotropin secretion, and this resulted in a significant improvement of dysspermia (Aulitzky W, Chandra I, Frick J, abstract). In view of these results and the find160

Adamopoulos et al.

ings of our study, it seems that a new therapeutic approach could be the combination of pituitary gonadotropin suppression with pulsatile gonadal stimulation in properly selected oligozoospermic men. In summary, effective suppression of pituitary gonadotropin secretion by a GnRH -a followed by pulsatile hMG administration restored Leydig and Sertoli cell activity in normogonadotropic oligozoospermic men. This combined approach offers a useful tool for the investigation of pituitary-testicular pathophysiology in normozoospermic and dysspermic men and also opens a therapeutic possibility for evaluation in selected cases of oligozoospermia. Acknowledgments. We express our deep gratitude to the four volunteers for their participation in the study. We also acknowledge gratefully the skilled technical assistant of Mrs. Maria Sakelaris, the secretarial help of Mrs. Maria Botis, and the statistical advice of Miss Maria Pitenis.

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tion of inhibin subunit messenger ribonncleic acids in human fetal testes. J Clin Endocrinol Metab 1992;75:806-11. Sandow J, Engelbart K, Rechenberg W. The different mechanisms for suppression of pituitary and testicular function. Med Bioi (Helsinki) 1986;63:192-9. Petersson F, Hammar M, Mathson K, Hjertberg H, Varenhorst E. Influence of continuous luteinizing hormone-releasing hormone agonist treatment on steroidogenic enzymes in the human testis. Scand J Urol Nephrol 1987;71:267-71. Pavlou SN, Dahl KD, Wakefield G, Rivier J, Vale W, Hsueh AJ, et al. Maintenance of the ratio ofbioactive to immunoreactive follicle-stimulating hormone in normal men during chronic luteinizing hormone-releasing hormone agonist administration. J Clin Endocrinol Metab 1988;66:1005-9. Winters SJ. Inhibin is released together with testosterone by the human testis. J Clin Endocrinol Metab 1990;70:54850. Crowley WF Jr, Filicori M, Spratt D, Santoro N. The physiology of gonadotropin releasing hormone (GnRH) secretion in men and women. Rec Prog Horm Res 1985;41:473-82.

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