CORRELATION OF SEMINAL BIOCHEMICAL MARKERS WITH FERTILITY HORMONES AMONG SUDANESE INFERTILE PATIENTS

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Author: Abdelmula M. Abdella, MSc, PhD

INTRODUCTION

Male fertility depends on proper function of a complex system of organs and hormones. The male epididymis synthesizes neutral alpha glucosidase that is secreted in semen (1). Spermatozoa form about 5¡V10% of the semen volume. 40-80% of semen is produced by the seminal vesicles which secrete fructose for sperm nutrition. Fructose is the main energy source of sperm cells. A reduction in fructose level is often due to an obstruction of the ejaculatory ducts or absence of the seminal vesicles, especially when associated with a low ejaculate volume (2). Fructose secretion is under an androgenic regulation. Other factors such as frequency of ejaculation, blood glucose level and nutritional status can also affect the seminal plasma fructose concentration (1) Zinc is secreted by the epithelial glands of the prostate and dihydrotestosterone is required for the accumulation of zinc within the prostatic cells and lumen. It is important to emphasize, the relationship between the germ cells and the follicle stimulating hormone (FSH) that is essential for the successful development of normal spermatogenesis (3). This study was the first work in this field in Sudan. It aims to correlate the concentrations of the biochemical markers (semen fructose, citric acid, zinc, and neutral alpha- glucosidase) to fertile hormones in infertile Sudanese males, and highlight their importance in male reproductive disorders.

MATERIALS AND METHODS

500 infertile males were for this study in Khartoum State (Sudan). It was carried out between July 2004 and October 2006. The subjects were males attending the fertility clinics complaining of inability to achieve pregnancy for at least one year after marriage and whom their wives had shown no diagnosed causes of infertility. The infertile patients studied were divided into four groups according to World Health Organization (WHO) standards for semen quality: 1. Azoospermic patients (n=150), 2. Oligospermic patients (n=150), 3. Asthenozoospermic patients (n=100), 4. Patients with abnormal sperm morphology (n =100 ) The control group included 100 fertile males who had fathered a child during the last two years. A consent for the study was obtained from all enrolled subjects. Demographical data were collected via a structural interview that was conducted during the first visit. A basic medical, surgical, reproductive and family history was recorded. A complete physical and genital examination was carried out. All subjects submitted a semen specimen and a blood sample. Venous blood was collected in a plain tube for estimating the fertility hormones: follicle stimulating hormone (FSH), luteinising hormone (LH), prolactin, and total testerone. The separated serum was assayed by the enzyme-immunolinked assay (ELISA). Semen samples were obtained by masturbation and collected in sterile polystyrene containers after a 3-5 days abstinence. Samples were analyzed according to the WHO criteria (4). Seminal plasma was separated from the spermatozoa and divided into two containers: 1. One metal-free polypropylene container for the estimation of zinc by atomic absorption spectrometry (AAS) with the Zeeman background correction method, and using an analytical quality control for accuracy (5). 2. The second container for the estimation of: „Ð Neutral alpha- glucosidase (NAG) according to Cooper, et al (6) and Guerin, et al (7). „Ð Citric acid according to Turkes et al (8). „Ð Fructose according to WHO Manual (4). Analytical photometers and Jenway 6305 spectrophotometer were used for the estimation.

Statistical analysis:

Data were expressed in mean, standard error, and standard deviation. They were analyzed by student t-test, one-way analysis of variance (ANOVA), and a coefficient of correlation (r) with a significance fixed at p = 0.05.

RESULTS

500 Sudanese infertile males were studied. Duration of infertility was 2-15 years in azoospermic patients, 4-18 years in oligospermic patients, 3-14 years in asthenozoospermic patients, and 2-11 years in patients with abnormal sperm morphology. The control group constitutes 100 fertile males with normal spermogram.

Clinical examination and laboratory investigations revealed: „Ð 2.7% of azoospermic patients were suffering from Sertoli-cell syndrome. „Ð 13% of the infertile patients had varicocele. „Ð 9.6% of the infertile patients had sexual dysfunction.

Seminal NAG, zinc, and citric acid levels were significantly reduced in azoospermic and oligospermic patients. Seminal fructose was slightly increased in oligospermic patients and subjects with abnormal sperm morphology. However, it was decreased in azoospermic and asthenozoospermic patients (Table I). Dunnetts T3 multiple analysis of seminal biochemical markers in infertile patients and control group is shown in Table II.

Table III and Table IV show the correlation of seminal biochemical markers with fertility hormones in infertile patients studied. In azoospermic patients a slight correlation was evident between NAG and testosterone; and between fructose and citric acid with prolactin. Furthermore, a significant correlation had been recorded between NAG and FSH and LH in this group of patients. In oligospermic patients correlation was clear between citric acid and FSH, LH, prolactin, and NAG. In addition, a significant correlation was also noticed between seminal zinc, LH, and prolactin in such patients. On the other hand, fructose correlates well with prolactin in asthenozoospermic patients.

Table I Descriptive analysis of seminal biochemical markers in infertile patients and control group

Seminal biochemical markers Azoospermic patients (m¡Ó.se) n = 150 Oligospermic patients (m¡Ó.se) n = 150 Asthenozoospermic patients (m ¡Ó.se) n = 100 Abnormal sperm morphology patients (m¡Ó.se) n = 100 Control Group (m¡Ó.se) n = 100 NAG (mIU/ml) 9.6 ¡Ó 0.22 12.0 ¡Ó 0.28 14.2 ¡Ó 0.56 13.3 ¡Ó 0.47 26.8 ¡Ó 0.59 Fructose (mmol/l) 12.9 ¡Ó 0.73 13.2 ¡Ó 0.69 12.1 ¡Ó 0.81 13.4 ¡Ó 0.87 13.0 ¡Ó 0.39 Citric acid (mmol/l) 1.9 ¡Ó 0.09 2.2 ¡Ó 0.15 2.6 ¡Ó 0.19 2.8 ¡Ó 0.20 3.3 ¡Ó 0.16 Zn (mg/dl) 11.5¡Ó 0.25 11.3¡Ó 0.27 12.6¡Ó 0.45 12.8¡Ó 0.40 19.5¡Ó 0.6

Table II Dunnetts T3 multiple analysis of seminal biochemical markers in infertile patients and control group

Seminal biochemical markers

Statistics Azoospermic patients versus control Oligospermic patients versus control Asthenozoospermic patients versus control Abnormal sperm morphology patients versus control

NAG Mean Difference 17.3 14.8 12.7 13.6 Significance 0.0 0.0 0.0 0.0 Fructose Mean Difference 0.07 - 0.2 0.83 - 0.46 Significance 1.0 1.0 0.98 1.0 Citric Acid Mean Difference 1.4 1.1 0.7 0.5 Significance 0.0 0.0 0.07 0.5 Zinc Mean Difference 7.6 8.0 6.7 6.6 Significance 0.0 0.0 0.0 0.0

Table III Statistical correlation of seminal biochemical markers with fertility hormones in azoospermic and oligospermic patients

Azoospermic patients: Seminal biochemical markers Statistics Testosterone FSH Prolactin LH NAG Person correlation Sig (2-tailed) 0.14 0.1 - 0.25 0.02 0.09 0.3 - 0.20 0.01 Fructose Person correlation Sig (2-tailed) -0.01 0.9 0.02 0.8 0.15 0.08 - 0.02 0.8 Citric Acid Person correlation Sig (2-tailed) - 0.03 0.7 - 0.1 0.2 - 0.11 0.19 0.03 0.76 Zinc Person correlation Sig (2- tailed) 0.07 0.4 0.05 0.5 - 0.1 0.2 0.04 0.7

Oligospermic patients: Seminal biochemical markers Statistics Testosterone FSH Prolactin LH NAG Person correlation Sig (2-tailed) 0.11 0.2 0.05 0.6 0.16 0.05 0.05 0.5 Fructose Person correlation Sig (2-tailed) 0.03 0.7 0.03 0.7 0.08 0.3 0.07 0.4 Citric acid Person correlation Sig (2-tailed) 0.07 0.3 - 0.11 0.17 0.07 0.4 - 0.25 0.02 Zinc Person correlation Sig (2-tailed) - 0.03 0.7 - 0.08 0.3 0.2 0.02 0.20 0.03

Table IV Statistical correlation of seminal biochemical markers with fertility hormones in asthenozoospermic patients and abnormal sperm morphology patients

Asthenozoospermic patients: Seminal biochemical markers Statistics Testosterone FSH Prolactin LH NAG Person correlation Sig (2-tailed) - 0.04 0.7 0.06 0.5 0.08 0.4 - 0.09 0.4 Fructose Person correlation Sig (2-tailed) 0.1 0.3 0.03 1.0 0.17 0.08 0.09 0.4 Citric acid Person correlation Sig (2-tailed) 0.01 1.0 0.01 1.0 - 0.05 0.6 0.1 0.3 Zinc Person correlation Sig (2- tailed) 0.14 0.5 0.15 0.6 0.07 0.5 - 0.13 0.2 Abnormal sperm morphology patients:

Seminal biochemical markers Statistics Testosterone FSH Prolactin LH NAG Person correlation Sig (2-tailed) 0.01 0.9 0.01 1.0 - 0.1 0.3 - 0.08 0.4 Fructose Person correlation Sig (2-tailed) 0.16 0.12 0.13 0.2 0.16 0.12 0.1 0.2 Citric acid Person correlation Sig (2-tailed - 0.07 0.5 0.16 0.12 0.08 0.4 0.06 0.5 Zinc Person correlation Sig (2-tailed 0.14 0.2 0.02 0.8 0.07 0.5 0.1 0.3

DISCUSSION

Some workers (8) measured the neutral alpha-glucosidase (NAD) activity in seminal plasma of infertile patients. They found an inverse correlation of NAD activity with sperm abnormal forms. Semen NAG level in this study was significantly reduced among infertile patients and this result agrees with the findings of these workers (8). Since NAG is secreted by the epididymis that is under the control of the testosterone hormone, the high significant reduction of NAG level recorded in azoospermic patients may be due to obstruction of the first part of the ejaculatory duct next to the epididymis (1).

Seminal fructose level was found increased in oligospermic and abnormal sperm morphology patients. This may has been resulted from the reduced sperm count and activity leading to low consumption of the synthesized fructose. On the other hand, seminal fructose level was decreased in azoospermic and asthenozoospermic patients. This is explained by partial or complete obstruction of the seminal ducts or ageing of the accessory glands that secrete (5).

Seminal citric acid level was slightly reduced in asthenozoospermic patients and significantly reduced in azoospermic and oligospermic patients. Since seminal citric acid is secreted mainly from the prostate gland, any partial or complete obstruction of the ejaculatory ducts may reduce its level in semen. Abnormal testosterone secretion and accessory glands infection may also lead to reduction of seminal citric acid level.

Generally zinc and citric acid concentrations as well as NAG activity were found to decrease significantly with increase in seminal abnormalities. This is quite obvious in infertile azoospermic patients. NAG level is considered the most important marker because it affects the maturation and the acquisition of spermatozoa motility (1).

Seminal NAG, fructose, citric acid, and zinc are usually affected by the testosterone level and accessory glands secreting these markers. This explains the weak relation between NAG, fructose, citric acid, and zinc in azoospermic patients. The secretory activity of the accessory glands is decreased by the level of seminal zinc. This phenomenon was demonstrated by the strong relation between the zinc level and seminal volume and liquefaction in azoospermic and oligospermic patients. Testosterone is essential for all steps of spermatogenesis, hence any reduction in testosterone concentration may affect sperm quality. Hunt et al (1992) deduced the important role of zinc in testosterone production and its need in the spermatogenesis process. They also observed the positive relation between testosterone and zinc in healthy male volunteers fed on zinc-restricted diet. As a consequence of zinc deficiency, serum testosterone concentration and seminal volume per ejaculate were reduced (8).

The fertility hormones levels in infertile patients reflected a relationship with seminal zinc level. The positive correlation of prolactin hormone with seminal zinc indicates the stimulating effect of prolactin on the prostate gland that secretes zinc. The inverse correlation of LH with seminal zinc in oligospermic patients suggests the feed-back stimulation of zinc deficiency on LH secretion by the pituitary gland.

Conclusion:

Seminal NAG and zinc levels are reduced in the infertile male patients studied. Seminal citric acid level is reduced in azoospermic and oligospermic patients. However, seminal fructose level is within normal range. In addition, seminal NAG level has a significant correlation with spermogram abnormality.

Acknowledgement: We gratefully acknowledge the assistance of technologists in the Sudanese Saudi Laboratory, the Central Medical Laboratory Services, and the Military Hospital Laboratory (Khartoum) for their valuable cooperation. Our thanks also go to Al-Neelain University for its financial support.

REFERENCES

1) Vaclav Insler, Bruno Lunenfeld. Infertility: Male and Female. 2nd. ed., London, Churchill Livingstone, p. 739, 1993.

2) Setchell, B.P, Waites G.M.H. Changes in the permeability of the testicular capillaries and of the "blood-testis barrier" after the injection of cadmium chloride in the rat. J. Endocrinol., 47:81- 86, 1970.

3) Carol Mothson. Structure of male sperms. In: Porth Pathophysiology. 5th ed, London, Lippincott, pp. 1149-1155, 1998.

4) World Health Organization (WHO) Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction. Cambridge University Press, 1992.

5) Michael L. Bishop, Janet L. Duben-Engelkirk, Edward P. Fody. Clinical Chemistry: Principle, Procedure, Correlation. 3rd. edition, New York, Lippincott. Pp. 279-288, 1996.

6) Cooper, T. G. , Yeung, C. H. , Nasnan, C. J. Improvement in the assessment of human epididymal function by the use of inhibitors in the assay of alpha-glucosidase in seminal plasma. Int. J. Androl. 13: 297-305, 1990.

7) Guerin, J. F. et al Alpha-glucosidase as a specific epididymal enzyme marker: Its validity for the etiologic diagnosis of azoospermia. J. Androl, 7:156, 1986.

8) Hunt, C. D. , Johrison, P. E. , Herbel, J. , Mullen, L. K . Effect of dietary zinc depletion on seminal volume and zinc loss on serum testosterone concentration and sperm morphology in young men. Am. J. Clin. Nutr. , 56:148-157, 1992.