Long term follow-up study of 60 cases

Transcription

1 INTERNATIONAL JOURNALOFANDROLOGY 8 (1985) Department of Endocrinology', Clinical Research Unit for the Male2, NataonalResearch Institute for Family Planning, Beijing and Depaltment of Urology3, Peoples Hospital, Beijing Medical College, Peeple's Republic of China Dynamic study of serum gonadotrophin and testosterone levels in gossypol-treated men Long term follow-up study of 6 cases BY Zhang Gui-yuan1, Xiao Bilianl, Chen Zhen-wen2, Zhu Ji-chum3 and Meng Guang-dong The endocrine effects of gossypol were studied in 26 men for 52 months before, during and after treatment. A further 34 subjects were studied after cessation of gossypol treatment. A control group of 6 age-matched volunteers were monitored for 1 year. No significant changes in testosterone levels were observed during the course of study. Serum LH levels were, however, significantly higher (P <.1) during earlier phases of gossypol treatment and returned to normal after cessation of treatment except in 14 men with persistent disruption of spermatogenesis. In the latter, serum levels of LH and testosterone were significantly higher (P <.1) than those found in normal subjects and significantly lower (P <.5) than in those subjects in which spermatogenesis had recovered. Serum FSH levels did not rise during the first 8-9 months of treatment with gossypol, although azoospennia generally occurred within 76 days of commencing treatment. After 9 months of treatment, serum FSH levels rose gradually and remained significantly elevated (P <.1) after cessation of treatment in both the azoospermic/ oligospermic group and in the group in which spermatogenesis recovered. In the latter group the serum levels of FSH were correlated significantly with the sperm concentration. Key words: gossypol - FSH - LH - testosterone - spermatogenesis. Received on November 19th, Andrology 8,3 177

2 The antifertility effects associated with the use of crude cotton seed oil was reported in 1957 in China, and it was suggested that the cotton seed oil could be used for contraception (B S Liu 1957). A biphenolic compound, gossypol, was identified as the cause of this antifertility action, and studies on its mode of action were initiated (Natl Coordinating Group on Male Antifertility Agents 1978). On this basis of experiments in animals, and of surveys in the area where crude cotton seed oil was used for cooking it was considered justified to commence clinical studies using purified preparations of gossypol, and studies involving a total of more than 88 subjects have been reported in China (Z Q Liu et al. 1981). When side-effects of gossypol, in particular hypokalaemia, were confirmed most clinical trials were terminated. There have been a number of reviews on gossypol (Z Q Liu et al. 1981; Prasad & Diczfalusy 1982; Sang 1983; Qian & Wang 1984), but reports on its endocrine effects have been contradictory, and it is not clear whether gossypol has a direct action on the secretion of hormones of the pituitary-testicular axis. This paper describes the endocrine changes observed in a group of 6 men during and after cessation of gossypol treatment from a long-term follow-up study. Subjects Materials and Methods A total of 26 volunteers aged years were recruited. All had completed their families, had normal sperm counts, haematological and endocrine parameters. The selection of volunteers fulfilled the ethical criteria in use at the time of the study, and all subjects gave their informed consent. Treatment was terminated in 1981 as suggested by a WHO consulting group of scientists according to the criteria for the evaluation of fertility regulating agents. A treatment dose of 2 mg gossypol/day was given for 6-75 days followed by a maintenance dose of 5 mg/week. Blood samples were taken monthly to provide sera for hormone analysis. Due to non-compliance, not all subjects presented for semen analyses and blood tests at each time point leading to variations in the numbers of volunteers at each time. A further group of 34 gossypol users who had not taken part in the original study were recruited to monitor endocrine changes that occurred after cessation of gossypol treatment. Blood and semen samples collected between 8. and 1. h were.collected at monthly intervals after cessation of treatment until the sperm concentration was greater than 2 x 16/ml in 3 consecutive samples. Hormone levels and the sperm concentrations in a control group of 6 age-matched fertile volunteers were also monitored once a month for a year. Hormone analyses were performed by radioimmunoassay (RIA) using the matched reagents provided by the WHO Special Programme of Research Develop- 178

3 ment and Research Training in Human Reproduction (Sufi et al. 1982). The intra-assay coefficients of variation for FSH levels towards the middle of the response range were 8%, 6.6% and 6.5% for FSH, LH and testosterone, respectively. Equivalent values for inter-assay reproducibility were 196, 9% and 13.5%, respectively (Xiacr et al. 1984). The annual mean values for FSH, LH and testosterone and the testosterone/lh ratio obtained from 6 age-match@ normal Chinese men were 2.42 k 1.33 IU/l (mean SD), 4.64 k 2.3 IU/l, 22.7.k 4.3 nmol/l and 5.72 k 2.36, respectively. Statistical analjses Geometric means and standard deviations for treatment and control groups were calculated for specific time inervals during and after cessation of gossypol treatment. The mean values of the hormone levels obtained during and after cessation of gossypol treatment and those from 14 azoospermic or oligospermic men were compared with the annual means of those from normal subjects using Student s t-test. Linear regression analysis was used to determine whether there was a correlation between serum FSH levels and sperm concentration in men in whom spermatogenesis had recovered and in those with azoospermia or oligospermia. NORMAL DURING TREATMENT T t l5 1 AFTER CESSATION * I * I T (nmol/l) TJLH Fig. I. Serum levels of FSH, LH and testosterone (T) and the testosterone/lh (T/LH) ratio in gossypol-treated men before, during and after cessation of treatment. Bars represent the geometric mean k SEM, and values at the base of each column indicate number of men *

4 Results Dynamic changes in the serum levels of LH, FSH and testosterone before, during and after cessation of treatment are shown in Figs.2 and 3. The levels of testosterone fluctuated within the normal range, whereas LH levels increased during the earlier phase of gossypol treatment. In comparison, serum FSH levels did not change during the first 8-9 months of gossypol treatment although azoospermia generally occurred within 76 days (Fig. 2). After 9 months of treatment, FSH levels rose gradually (Fig. 2) and remained significantly elevated (P <.1) thereafter (Fig. 1). Taking the data as a whole and comparing the hormone changes between normal controls and gossypol users during, and after, cessation of treatment, no significant changes in testosterone levels were observed. However, serum LH levels were significantly higher (P <.1) during gossypol treatment, and this led to a significant lowering (P <.1) of the testosterone: LH ratio. Subsequently, LH * d GOSSYPOL TREATMENT e """ 2 s 3r - I \ 7 I 11 Y 1 I I I I I I,, > >6 BEFORE DURING AFTER CESSATION OF DRUG TREATMENT MONTHS Fig. 2. Relationship between sperm concentration and serum levels of FSH in gossypol-treated men before, during and after cessation of treatment. Values are the geometric mean f SD for hormone levels and the arithmetic mean f SD for sperm concentration. 18

5 GOSSYPOL TREATMENT E ::::;: a OL 25 r I -, I I I I I I I I I I > >6 BEFORE -DURING AFTER CESSATION OF DRUG TREATMENT MONTHS Fig.3. Comparison of the mean serum levels of FSH, LH anf testosterone (T) ratio in normal men and in gossypol-treated men during and after cessation of treatment. Values are the geometric mean f SD, and the number of subjects at each time point are also indicated. *** P <.1, compared to normals. I. levels declined in the post-treatment phase and were then not significantly different from controls. Serum FSH levels also increased significantly (P <.1) and remained elevated when treatment was ceased (Fig. 1). Serum levels of LH and testosterone in men who remained azoospermic or oligospermic weresignificantly different (P <.1 and P <.5, respectively) from those found in normal subjects or in treated men in whom spermatogenesis had recovered. FSH levels in both the azoospermic/oligospermic group and the recovered group were sign6 cantly higher (P <.1) than in control subjects (Fig. 4), although FSH levels in the latter group increased to a much smaller extent than in the oligo/azoospermic men. Linear regression analysis indicated that there appeared to have been hyperbolic relationship between FSH levels and the sperm concentration with a negative correlation (Fig. 5). 181

6 25r ONORMAL OLIGOAZOOSPERMATIC RECOVERED * T * FSH (IU/I) LH (IU/I) T (nrnol/l) T/LH Fig. 4. Comparison of the mean serum levels of FSH, LH and testosterone (T) and the T/LH ratio in normal men and in gossypol-treated oligo/azoo-spermic and recovered men. Bars represent the geometric mean k SEM. * P <.5, *** P <.1, compared with normal men. Men were considered to have recovered when the sperm concentration had reached more than 2 x 16/ml in 3 consecutive samples. UNRECOVERED Y ~ ~ (r:-.434, P>.5) RECOVERED Y= ~ (r:-.382, P<.5) lt.5 l,,,, O,,,,, I,,,,,,, SPERM COUNT (x1o6/rni) Fig. 5. Correlation between the annual mean values for serum FSH and the sperm concentration in gossypol-treated men. 182

7 Discussion Despite the fact that large numbers of subjects have received gossypol there are few reports of its effect on the pituitary-testis axis in men (Wang et al. 1981; Coutinho 1982; Zhang 1983). It has been suggested that there is no effect on Leydig cell function as testosterone levels are not significantly affected by gossypol treatment. Similarly, pituitary responsiveness to LHRH and testicular responsiveness to hcg remain unimpaired (Coutinho 1982). Some studies in rats and rhesus monkeys also suggest that gossypol has no effect on testosterone secretion (Wang 1979), but the majority of studies with animal models indicate that gossypol inhibits the Leydig cell function both in vivo and in vitro (Ma 1983; Lin 1981; Saksena 1982). Our data from the long-term follow-up of gossypol treated men in whom spermatogenesis did not recover indicate that Leydig cell function is affected, and that LH levels in this group of subjects were nearly double those of normal men. The data indicate that higher levels of LH required to maintain normal testosterone levels, representing a state of compensated Laydig cell failure. In this regard it is possible that contradictory findings in the literature might be due to differences in gossypol treatment regimes used. The mechanisms by which Leydig cell function is disrupted are not as yet known. Testicular biopsies from sterile men who have taken cotton seed oil have shown changes in Leydig cell morphology (Y F Wang et al. 1982). Gossypol may inhibit testosterone secretion by affecting the enzymes involved in its biosynthesis (Lin Tu et al. 1981) or by affecting Leydig cell LH reseptors or adenylate cyclase (Lin Tu et al. 1981; Saksena et al. 1981). Alternatively, the lowering of testosterone secretion may be a secondary effect, as de Kretser et al. (1983) have demonstrated that disruption of seminiferous tubules can often lead to a disruption of Leydig cell function. It is well known that damage to seminiferous tubules is also associated with elevated levels of FSH, and our data confirm that there is a negative correlation between serum levels of FSH and sperm density in gossypol-treated subjects. It is unlikely, however, that the mechanism by which gossypol affects spermastogenesis is the same as that which alters serum FSH levels, as a decrease in sperm motility is observed within 2 weeks (Xue et al. 1981) and azoospermia is achieved within 76 days of starting gossypol treatment. FSH levels became elevated 6 months after the onset of azoospermia, so it is likely that this is a consequence of an effect on another element of the seminiferous epithelium. FSH secretion is controlled via negative feedback by inhibin, a peptide secreted by Sertoli cells (de Kretser et al. 1983). It is possible that prolonged gossypol treatment damages the Sertoli cells resulting in a decrease in inhibin secretion and a consequent increase in FSH secretion by the pituitary. While the mechanisms involved in the hormonal control of sprmatogenesis are not fully understood it is generally accepted that spermatogenesis is initiated and 183

8 maintained by FSH and testosterone. Approximately 8% of gossypol treated men recovered both spermatogenesis and normal hormone levels after cessation of treatment. Normal hormone concentrations were not observed in any of the azoospermic or oligospermic subjects, indicating that there is a close relationship between the reversibility of spermatogenesis and the normalization of hormone levels. The severity of disruption of testicular function has already been shown to be correlated with serum FSH levels (Zhang et al. 1983) and the present study confirms our earlier findings. It is probable that the risk of irreversible azoospermia and other side effects could be minimized if the dosage and duration of gossypol treatment could be monitored using parameters other than the onset of azoospermia, and it is clear that a critical review of the usefulness of indicators or testicular cell damage will be needed before gossypol or its derivatives can achieve their potential as male contraceptives. Acknowledgments We are grateful to Mr. S. Sufi, Professor D. M. de Kretser and Dr. E. M. Ritz6n for their help in the preparation of this paper. We would also like to thank the WHO Special Programme of Human Reproduction for providing the matched reagents for radioimmunoassay. The technical help of W. Q. Yan, J. L. Zhao, X. L. Zhang, D. D. Feng, L. Dong, W. M. Zheng, and B. Yang is gratefully acknowledged. References. Coutinho E M (1982): Clinical studies with gossypol. Arch Androl9: 37. de Kretser D M & Kerr J B (1983): The effect of testicular damage on Seroli and Leydig cell function. In: de Kretser D M, Burger H G & Hudson B (eds). The Pituitary and Testes. Clinical and Experimental Studies, pp Springer Verlag, Berlin, Heidelberg, New York, Tokyo. Liu B S (1957): A tentative idea of the use of cooking cotton seed oil for fertility control. Shanghai Chin Med 6: 43. Liu Z Q, Liu G 2, Hei L S, Zhang R A & Yu C Z (1981): Clinical trial on gossypol as a male antifertility agent. In: Chang C F, Griffin D & Wollman A (eds). Recent Advances in Fertility Regulation, pp Proc Symp Beijing, September 198. Atar, Geneva. Lin S X (1981): Radioimmunoassay of serum testosterone and LH levels in rats treated by gossypol. Acta Exp Biol 14: 191. Lin C Y, Hadley M A, Klingener D & Dym M (198): Effects of gossypol on the reproductive system of male rats. Biol Reprod 22. Suppl95A. Lin T U, Murono E P, Osterman J, Nankin H R & Colson P B (1981): Gossypol inhibits testicular steroidogenesis. Fertil Steril35 : 563. Ma X N, Li W J & Sun Y B (1983): Effect of gossypol acetic acid on testosterone, LH and FSH levels of male rats. Acta Pharm Sin 18:

9 Sat1 Coordination Group Male Antifertility Agents (1978): Gossypol, a new male antifertility agent. Clin Med J (Engl) 91: 417. Prasad M R M & Diczfalusy E (1982): Gossypol. Int J Androl. Suppl5: 53. Qian S Z & Wang Z G (1984): Gossypol. A potential antifertility agent for male. Ann Rev Pharm Toxic1 24: 329. Sang G W (1983): Effect of gossypol on male reproduction. In: Fotherby K & Pal S B (eds). Hormones in Normal and Abnormal Human Tissues. Vol 111: 215. Ealter de Grayter & Co., Berlin, New York. Sufi S S, Donaldson A & Jeffcote S L (1982): Methos manual. In: WHO Special Programme of Research, Development and Research Training in Human Reproduction, Programme for the Provision of Matched Assay Reagents for the Radioimmunoassay of Hormones in Reproductive Physiology. 6th edn. Saksena S K, Salmonsen M A, Lan I F & Chang M C (1981): Gossypol: its toxicological and endocrinological effects in male rabbits. Contraception 24: 23. Wang Y E, Luo Y D, Shen L T & Tan X C (1981): Plasma testosterone levels in Chinese normal males, endocrinopathy and gossypol receivers. Reprod Contacept 1: 41. Wang Y E, Luo Y G & Tang X G (1979): Studies on the antifertility actions of cotton seed meal and gossypol. Acta Pharm Sin 14: 663. Wang Y F, Wu M X, Wang Z X, Gu D Q, Gu J Z & Wu Q H (1982): Quantitative histological investigation of testes from normal adults and men infertile after taking raw cotton seed oil. Reprod Contracept 2 : 31. Xiao B L, Zhang X L, Yan W Q, Dong L & Feng D D (1984): Application of quality control in radioimmunoassays. Reprod Contracept 4: 51. Xue S P (1981): Studies on the antifertility effect of gossypol, a new contraceptive for males. In: Chang C F, Griffin D & Wollman A (eds). Recent Advances in Fertility Regulation, pp Proc Symp Beijing, September Atar, Geneva. Zhang G Y, Zhou X Y, Gao S M, Guo Z S, Liu G Z & Cao J (1983): Comparison of the changes of serum gonadotropins and steroid hormones in azoospermic men caused by gossypol and other factors. Reprod Contracept 3 : 31. Author s address: Dr. Zhang Gui-yuan, Department of Endocrinology, National Research Institute for Family Planning, No. 18 Bei Huan Xi Lu, Beijing, People s Republic of China. 185