1 Department of Pediatrics, Obstetrics and Reproductive Medicine, University of Siena, 2 Institute of General Biology, Center for the Study of Germinal Cells, CNR, Siena and Regional Center for Male Infertility and 3 Institute of Obstetrics and Gynecology, University of Siena, Policlinico Le Scotte, Viale Bracci, 53100 Siena, Italy
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Abstract |
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Key words: diabetes/GnRH/hypothalamus/pituitary/testicular axis/infertility/sperm structure
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Introduction |
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It is known that well-controlled diabetes is associated with a physiological endocrine testicular function, as shown by plasma androgen levels in the physiological range (Handselman et al., 1985). However, a large body of evidence has demonstrated that diabetic men appear to be at a disadvantage in terms of sperm quality compared with healthy controls (Handselman et al., 1985; Vignon et al., 1991; Ali et al., 1993a; Niven et al., 1995
)
The objective of the present study was to investigate the hypothalamo-pituitarytesticular axis and sperm quality in men affected by insulin-dependent diabetes.
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Materials and methods |
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Twenty-four healthy men (controls) were selected from a large group of non-diabetic infertile men in our database. They were healthy with a mean age of 37 ± 5 years (range 3146).
Before performing the study, complete history, physical examination and relevant laboratory makeup were performed. Exclusion criteria were history of drug or alcohol abuse, ongoing medical treatment with anabolic steroids and gonadotrophins, heavy smoking habit (>10 cigarettes/day), hypertension, leukocytospermia, varicocele and unilateral testicular atrophy.
Patients and controls underwent a GnRH test and semen examination with optic and electronic microscopy. Written informed consent was obtained from all men. Patients and controls were admitted to the Gynecological Endocrinology Hospital Center of Siena University 2 h before blood sampling was to begin. An indwelling catheter was inserted in the antecubital vein and saline solution was infused slowly to keep the vein patent. All patients did the releasing hormone test (100 µg GnRH; Biochem. Immunosystems, Milan, Italy).
After 23 days of sexual abstinence, semen samples were produced by masturbation, collected into sterile specimen cups and allowed to liquefy at room temperature. Semen volume, sperm concentration and motility were determined according to World Health Organization guidelines (World Health Organization, 1999). Sperm concentration and motility were determined by analysing 5 µl of semen using a Makler chamber (Makler, 1980
).
Transmission electron microscopy (TEM) procedure
Ejaculated sperm were fixed in cold Karnovskys fixative and maintained at 4°C for 2 h. The fixed semen was then centrifuged at 1500g for 15 min. The pellet was removed from the centrifuge tubes, washed in 0.1 mol/l cacodylate buffer (pH 7.2) for 12 h, post-fixed for 1 h at 4°C in 1% buffered osmium tetroxide, dehydrated and embedded in Epon Araldite. The sections, cut with a Supernova ultramicrotome (Reickert Jung Wien, Austria), were collected in copper grids, stained with uranyl acetate and lead citrate, observed and photographed with Philips CM 10 electron microscope (Philips Scientific Instruments, Eindhoven, The Netherlands). For each subject, 300 sperm were randomly observed. Two highly trained evaluators, who were blinded to the aim of the study, performed the TEM analysis. A total of 300 sperm sections were randomly evaluated in at least three different embedded samples for each ejaculate.
TEM data were statistically evaluated by the computerized formula of Baccetti et al. (Baccetti et al., 1995). Briefly the formula evaluates structural and functional sperm integrity by computerizing the submicroscopical characteristics detected by TEM. The formula is valid for a real system of 15 variables and allows a precise simultaneous evaluation of the sperm characteristics and a determination of the total number and percentage of sperm free from ultrastructural defects (Baccetti et al., 1995
).
Hormone assay
Plasma FSH, LH, testosterone and free testosterone levels were assayed by double-antibody radioimmunoassay using commercial kits from Radim (Rome, Italy) for FSH and LH, from Sorin (Saluggia-VC, Italy) for testosterone, and from DPC (Los Angeles, CA, USA) for free testosterone. Samples were assayed in duplicate at two dilutions. Samples from a given subject were analysed for each hormone in the same assay to avoid inter-assay variation. Quality control pools at low, normal and high LH, FSH, testosterone and free testosterone concentrations were present in each assay. The detection limit of the assay was 0.20 IU/l for LH, 0.18 IU/l for FSH, 277 pmol/l for testosterone and 0.5 pmol/l for free testosterone. Intra- and inter-assay variations were 7.8 and 8.2% for LH, 6.2 and 6.5% for FSH, 3.4 and 4.6% for testosterone, and 4.6 and 4.7% for free testosterone.
Statistical analysis
Results are expressed as means ± SD. The total integrated hormonal responses to GnRH were calculated by the trapezoidal method and expressed as the area under the concentrationtime curve (AUC). To compare the differences between the two groups, peak values (the maximum rise above baseline value) and AUC were compared. Comparisons between the two groups were calculated by unpaired t-test if the data were normally distributed and by the MannWhitney U-test because of the small size of the groups. Statistical analysis was performed with the Statsoft software. Statistical significance was set at P < 0.05.
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Results |
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Discussion |
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Insulin has been shown to play a central role in the regulation of pituitary and gonadal function. Indeed, insulin enhances LHRH-induced gonadotrophin secretion in vitro (Adashi et al., 1981). A reduced glucose utilization by the anterior pituitary cells (Goodner and Freinkel, 1961
; Garris et al., 1984
) and a decreased response of FSH and LH to GnRH administration (Kirchick et al., 1979
; Bestetti et al., 1985
; Seethalakshmi et al., 1987
) has been shown in insulin-deficient rats.
Animal experiments have indicated a reduced gonadotrophin secretion in chemically induced diabetes either due to inadequate GnRH release (Johnson and Sidman, 1979; Rossi and Bestetti, 1981
) or reduced pituitary responsiveness to GnRH (Kirchick et al., 1979
; Dong et al., 1991
). Diabetic rats showed an abnormal sexual steroid feedback on the hypothalamo-pituitary axis either due to reduced pituitary sensitivity (Kirchick et al., 1979
; Dong et al., 1991
) or abnormal steroid transport into the effector cells (Gentry et al., 1977
)
The reduced LH and FSH response to GnRH in diabetic men indicated a decreased acute releasable pool of pituitary gonadotrophins. In diabetic women, a reduced LH response to GnRH and alterations in LH secretory activity have been recognized (Djursing et al., 1983, 1984
; la Marca et al., 1999
). Furthermore, increased opioidoergic and dopaminergic tones have been hypothesized. Increased central opioid and dopamine have a deleterious effect on reproductive function by acting mainly on the GnRHLH axis.
We have demonstrated that diabetes in women is associated with a subclinical hypercortisolism, which in turn should have a role on reproductive impairment (la Marca et al., 1999). Whether these mechanisms are functional in diabetic men is not known.
As for hormonal evaluation, discordant data have been published on sperm quality in diabetic men. In the present study, we have found reduced sperm motility in diabetic men by examination with a Makler chamber. For diabetics, a higher sperm count, higher sperm concentration (Padron et al., 1984; Vignon et al., 1991
; Ali et al., 1993
), higher percentage of abnormal sperm (Padron et al., 1984
; Vignon et al., 1991
) and lower motility (Padron et al., 1984
; Handelsman et al., 1985
; Ali et al., 1993
; Niven et al., 1995
) have been reported. Others found reduced sperm volume and normal sperm density, morphology and motility (Handelsman et al., 1985
).
The reasons for the discrepancies between the studies can probably be found, as for the hormonal evaluations, in the limited study population. To our knowledge, this is the first paper to investigate sperm structure in diabetic patients. Although limited to few patients, results of TEM examination showed that sperm from men with diabetes presented severe structural defects. In particular, apoptosis- and immaturity-related defects were recognized. Indeed, alterations in the acrosome, nucleus, mitochondria and plasma membrane were observed. Using the mathematical formula by Baccetti et al. (Baccetti et al., 1995), we found that the percentage of sperm devoid of ultrastructure defects in diabetics was very low (mean 0.4%).
The results of the TEM examination indicate a role for insulin and carbohydrate metabolism in spermatogenesis. The evidence indicates that, at least in animals, insulin plays a role in the maintenance of spermatogenesis and testicular endocrine function. Studies in streptozotocin-induced diabetic rats showed severe dysfunction of the reproductive tract. Diabetic rats had decreased reproductive organ weights as well as diminished sperm counts and motility (Seethalakshmi et al., 1987).
Male diabetic rats showed extensive spermatogenic alterations when electron microscopy was used (Gondos and Bevier, 1995). The same animals, when insulin-treated, showed variable changes ranging from normal spermatogenesis to moderate or severe alterations, indicating that in diabetic rats insulin could, even if not entirely, prevent spermatogenic abnormalities (Gondos and Bevier, 1995
)
Reproductive function damage associated with diabetes is far from understood. A chronic subtle impairment of gonadotrophin secretion may, at least in part, be responsible for defective spermatogenesis. However, the reduction in LH and FSH responses to GnRH administration is too small to be considered as a primary event in causing disturbances in sperm structure. In our opinion, defective spermatogenesis may be the consequence of a direct testicular effect of the disease.
In conclusion, it is possible that the reproductive impairment recognized in men with diabetes could be the result of interferences of the disease on the hypothalamo-pituitarytesticular axis at multiple levels, as indicated by the reduced gonadotrophin response to appropriate stimuli and by the abnormal ultrastructure of ejaculated sperm.
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Notes |
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References |
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Ali, S.T., Shaikh, R.N., Siddiqi, N.A. and Siddiqi, P.Q. (1993) Semen analysis in insulin-dependent/non-insulin-dependent diabetic men with/without neuropathy. Arch. Androl., 30, 4754.[ISI][Medline]
Baccetti, B., Bernieri, G., Burrini, A.G, Collodel, G., Crisa, N., Mirolli, M., Moretti, E. and Piomboni, P. (1995) Notulae seminologicae. 5. Mathematical evaluation of interdependent submicroscopic sperm alterations. J. Androl., 16, 356371.
Bestetti, G. and Rossi, G.L. (1982) Hypothalamic changes in diabetic Chinese hamsters. A semiquantitative, light and electron microscopic study. Lab. Invest., 47, 516522.[ISI][Medline]
Bestetti, G., Locatelli, V., Tirone, F., Rossi, G.L. and Muller, E.E. (1985) One month of streptozotocin-diabetes induces different neuroendocrine and morphological alterations in the hypothalamo-pituitary axis of male and female rats. Endocrinology, 117, 208216.[Abstract]
Daubresse, J.C., Meunier, J.C., Wilmotte, J., Luyckx, A.S. and Lefebvre, P.J. (1978) Pituitarytesticular axis in diabetic men with and without sexual impotence. Diabet. Metab., 4, 233237.[ISI][Medline]
Distiller, L.A., Sagel, J., Morley, J.E. and Seftel, H.C. (1975) Pituitary responsiveness to luteinizing hormone-releasing hormone in insulin-dependent diabetes mellitus. Diabetes, 24, 378380.[Abstract]
Djursing, H., Hagen, C., Nyholm, H.C., Carstensen, L. and Andersen, A.N. (1983) Gonadotropin responses to gonadotropin-releasing hormone and prolactin responses to thyrotropin-releasing hormone and metoclopramide in women with amenorrhea and insulin-treated diabetes mellitus. J. Clin. Endocrinol. Metab., 56, 10161021.[Abstract]
Djursing, H., Carstensen, L., Hagen, C. and Andersen, A.N. (1984) Possible altered dopaminergic modulation of pituitary function in normal-menstruating women with insulin dependent diabetes mellitus (IDDM). Acta Endocrinol. (Copenh.), 107, 450455.[ISI][Medline]
Dong, Q., Lazarus, R.M., Wong, L.S., Vellios, M. and Handelsman, D.J. (1991) Pulsatile LH secretion in streptozotocin-induced diabetes in the rat. J. Endocrinol., 131, 4955.[Abstract]
Garris, D.R., Williams, S.K., Coleman, D.L. and Morgan, C.R. (1984) Glucose utilization by the mouse brain: influence of age and diabetes. Brain. Res., 317, 141146.[Medline]
Gentry, R.T., Wade, G.N. and Blaustein, J.D. (1977) Binding of [3H]estradiol by brain cell nuclei and female rat sexual behavior: inhibition by experimental diabetes. Brain Res., 135, 135146.[ISI][Medline]
Gondos, B. and Bevier, W. (1995) Effect of insulin on testicular alterations in the nonobese diabetic mouse. Ann. Clin. Lab. Sci., 25, 272277.[Abstract]
Goodner, C.J. and Freinkel, N. (1961) Studies of anterior pituitary tissue in vitro: effects of insulin and experimental diabetes mellitus upon carbohydrate metabolism. J. Clin. Endocrinol. Metab., 40, 261272.
Handelsman, D.J., Conway, A.J., Boylan, L.M., Yue, D.K. and Turtle, J.R. (1985) Testicular function and glycemic control in diabetic men. A controlled study. Andrologia, 17, 488496.[ISI][Medline]
Johnson, L.M. and Sidman, R.L. (1979) A reproductive endocrine profile in the diabetes (db) mutant mouse. Biol. Reprod., 20, 552559.[ISI][Medline]
Kirchick, H.J., Keyes, P.L. and Frye, B.E. (1979) An explanation for anovulation in immature alloxan-diabetic rats treated with pregnant mares serum gonadotropin: reduced pituitary response to gonadotropin-releasing hormone. Endocrinology, 105, 13431349.[ISI][Medline]
la Marca, A., Morgante, G. and De Leo, V. (1999) Evaluation of hypothalamicpituitaryadrenal axis in amenorrhoeic women with insulin-dependent diabetes. Hum. Reprod., 14, 298302.
Makler, A. (1980) The improved ten-micrometer chamber for rapid sperm count and motility evaluation. Fertil. Steril., 33, 337338.[ISI][Medline]
Niven, M.J., Hitman, G.A. and Badenoch, D.F. (1995) A study of spermatozoal motility in type 1 diabetes mellitus. Diabet. Med., 12, 921924.[ISI][Medline]
Padron, R.S., Dambay, A., Suarez, R. and Mas, J. (1984) Semen analyses in adolescent diabetic patients. Acta Diabetol. Lat., 21, 115121.[ISI][Medline]
Press, M., Tamborlane, W.V., Thorner, M.O., Vale, W., Rivier, J., Gertner, J.M. and Sherwin, R.S. (1984) Pituitary response to growth hormone-releasing factor in diabetes. Failure of glucose-mediated suppression. Diabetes, 33, 804806.[Abstract]
Rastogi, G.K., Chakraborti, J. and Sinha, M.K. (1974) Serum gonadotropins (LH and FSH) and their response to synthetic LHRH in diabetic men with and without impotence. Horm. Metab. Res., 6, 335336.[ISI][Medline]
Rossi, G.L. and Bestetti, G. (1981) Morphological changes in the hypothalamichypophysealgonadal axis of male rats after twelve months of streptozotocin-induced diabetes. Diabetologia, 21, 476481.[ISI][Medline]
Seethalakshmi, L., Menon, M. and Diamond, D. (1987) The effect of streptozotocin-induced diabetes on the neuroendocrine-male reproductive tract axis of the adult rat. J. Urol., 138, 190194.[ISI][Medline]
South, S.A., Asplin, C.M., Carlsen, E.C., Booth, R.A., Weltman, J.Y., Johnson, M.L., Veldhuis, J.D. and Evans, W.S. (1993) Alterations in luteinizing hormone secretory activity in women with insulin-dependent diabetes mellitus and secondary amenorrhea. J. Clin. Endocrinol. Metab., 76, 10481053.[Abstract]
Tanaka, T., Nagatani, S., Bucholtz, D.C., Ohkura, S., Tsukamura, H., Maeda, K. and Foster, D.L. (2000) Central action of insulin regulates pulsatile luteinizing hormone secretion in the diabetic sheep model. Biol. Reprod., 62, 12561261.
Vierhapper, H. (1985) LH-RH stimulated LH secretion in human endocrine disease. Acta Endocrinol., 269, S3S25.
Vignon, F., Le Faou, A., Montagnon, D., Pradignac, A., Cranz, C., Winiszewsky, P. and Pinget, M. (1991) Comparative study of semen in diabetic and healthy men. Diabete Metab., 17, 350354.[ISI][Medline]
World Health Organization (1999) WHO Laboratory Manual for the Examination of Human Semen and SpermCervical Mucus Interaction. 4th edn, Cambridge University Press, Cambridge, UK.
Wright, A.D., London, D.R., Holder, G., Williams, J.W. and Rudd, B.T. (1976) Luteinizing release hormone tests in impotent diabetic males. Diabetes, 25, 975977.[Abstract]
Submitted on November 15, 2001; resubmitted on May 28, 2002; accepted on July 15, 2002.