Original Article
Assessment of spermatozoa in different age groups Avaliação dos espermatozóides em diferentes faixas etárias Andy Petroianu1, Marco Antônio Barreto de Melo2, Luciana Magalhães de Almeida3, Luiz Ronaldo Alberti4, Denny Fabrício Magalhães Veloso5
ABSTRACT Objective: To verify sperm disorders in different age groups through analysis of seminal fluid. Methods: The semen of 80 healthy adult men, with age range of 21 to 60 years, was studied after a five-day sexual abstinence. Volunteers were distributed into four age-groups (n = 20): 21 to 30, 31 to 40, 41 to 50 and 51 to 60 years. Results: No differences regarding the physical and chemical aspects of semen were found (smell, color, volume, viscosity, and pH), nor were there differences related to sperm concentration and percentage of mobile forms. Therefore, a non-significant increase in spermatozoa concentration in the semen of men aged over 41 years was observed. In the 41 to 50 years-old Group, the sperm morphology was predominantly oval (p = 0.03) and there were less tapered sperm as compared to other groups (p = 0.02). Conclusions: It was observed that there is no reduction in sperm production up to 60 years and that there is an increase in better shape for fertility, in the age of 41 to 50 years-old. Keywords: Sperm count; Spermatozoa/anatomy & histology; Semen; Infertility, male; Age effect
RESUMO Objetivo: Verificar, por meio da análise do líquido seminal de diferentes faixas etárias, a ocorrência de alterações nos espermatozóides. Métodos: Foi estudado o líquido seminal de 80 homens adultos hígidos, com idade entre 21 e 60 anos, após um período de abstinência sexual de cinco dias. Os voluntários foram distribuídos em quatro grupos etários (n = 20): 21 a 30 anos, 31 a 40 anos, 41 a 50 anos e 51 a 60 anos. Resultados: Não foram encontradas diferenças entre os grupos quanto aos aspectos físico-químicos dos sêmens (odor, cor, volume, viscosidade e
pH). Também não houve diferença entre as concentrações ou porcentagem de formas móveis nos grupos estudados. No entanto, embora não significativo, verificou-se um aumento da concentração de espermatozóides no líquido seminal a partir dos 41 anos. A morfologia dos espermatozóides mostrou maior percentual de formas ovais (p = 0,03) e menor de fusiformes entre 41 e 50 anos em relação aos demais grupos (p = 0,02). Conclusões: Percebeuse não apenas que não ocorre uma redução na produção de espermatozóides até os 60 anos, mas também que há um aumento das melhores formas para fecundação. Descritores: Contagem de espermatozóides; Espermatozóides/ anatomia & histologia; Sêmen; Infertilidade masculina; Efeito idade
INTRODUCTION Infertility is defined as the inability to generating children. It is found in approximately 15% of couples with more than one year of active sexual life without contraceptives. The male partner is affected in 40% of cases, the female in 40% and both partners are affected in 20%(1). Men present sperm deficiencies in 30% of cases(2). Several studies stated that during ageing process there is a decrease of daily sperm production despite an increase in serum gonadotrophin and drop in testosterone levels(3-4). Testicular biopsies and radioimmunoassay assessment of gonadotrophic hormones showed reduced function of Sertoli cells, decreased cytoplasm mass of Leydig cells, degeneration and thickening of the lamina propria of seminal tubules(4-6).
Study carried out at Department of Surgery of Faculdade de Medicina of Universidade Federal de Minas Gerais – UFMG, Belo Horizonte (MG), Brazil. 1
Post-doctorate degree; Full professor at Department of Surgery of the Faculdade de Medicina da Universidade Federal de Minas Gerais – UFMG, Belo Horizonte (MG), Brazil.
2
Physician at Universidade Federal de Minas Gerais – UFMG, Belo Horizonte (MG), Brazil.
3
Physician at Universidade Federal de Minas Gerais – UFMG, Belo Horizonte (MG), Brazil.
4
PhD; Adjunct professor at, Universidade Federal de Minas Gerais – UFMG, Belo Horizonte (MG), Brazil.
5
MD; Universidade Federal de Minas Gerais – UFMG, Belo Horizonte (MG), Brazil.
Corresponding author: Andy Petroianu – Avenida Afonso Pena, 1.626 – apto. 1.901 – Centro – CEP 30130-005 – Belo Horizonte (MG), Brasil – Tel.: 31 8884-9192 – e-mail:
[email protected] Received on Jan 14, 2008 – Accepted on Sep 17, 2008
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Petroianu A, Melo MAB, Almeida LM, Alberti LR, Veloso DFM
Due to scarce and controversial information about seminal fluid of middle-aged men, this paper considers morphological aspects of spermatozoa in different age groups.
METHODS This study was performed according to the Helsinki Declaration and to the Resolution 196/96 of the Brazilian Health Department concerning research on humans. It was also approved by the Research Ethics Committee of Universidade Federal de Minas Gerais (UFMG). All patients agreed to participate in the study and signed the informed consent form(7). The semen of 80 healthy men aged between 21 and 60 years were studied after sexual abstinence of five days. This time period was established because sperm reserves in the epididymis are renewed every four to six days(8). Volunteers were distributed in four age-groups: 21 to 30 years (n = 20); 31 to 40 years (n = 20); 41 to 50 years (n = 20); 51 to 60 years (n = 20). The participants were selected by directed anamnesis referring to sexual history (intercourse frequency, erectile or ejaculation disorders and previous paternity). The individuals with previous urological diseases, diabetes mellitus or on drugs were excluded. Family history of infertility was also studied. Sperm was collected into a sterile flask which was right away hermetically closed. Macroscopic and microscopic analysis was performed within one hour. The macroscopic assessment detected the physical and chemical properties (volume, aspect, odor, viscosity, and pH). The microscopic evaluation included spermatozoa motility and morphology. After diluting semen in 1:20 solution (0.1 ml sperm in 1.9 ml of saline solution 0.9%) sperm cells were counted in the Neubauer chamber. These counts included the five chamber quadrants, the four lateral quadrants normally used for leukocyte count plus the central area used for erythrocyte count. The total was multiplied by one million so the exact number of spermatozoa per ml could be obtained. According to morphology, sperm cells were classified as normal (oval) or abnormal (tapered, round, amorphous, immature, double-head, doubletail, macrocephalic/large head or microcephalic/small head)(8). The descriptive method for mean and mean standard error was used for statistical analysis. Results were compared using analysis of variance (ANOVA) followed by Tukey-Kramer test for multiple comparisons. The differences were considered significant for values amounting to p < 0.05
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RESULTS All patients agreed to participate in this study. All volunteers had negative family and sexual histories as to erectile dysfunction and infertility as well as to disorders increasing risks of having these dysfunctions, such as diabetes mellitus and previous urologic disease. No differences were found among semen features: characteristic odor, light gray color, viscosity within the normality limits and pH of 7. The ejaculated volume varied between 2.0 and 3.2 ml (mean = 2.5 ± 0.2). Additionally, there were no differences between the semen concentrations of the four groups, although higher concentration was found in the group aged 41 years or more. The percentage of mobile spermatozoa observed during the first hour did not vary among the different age groups (Table 1). Table 1. Percentage of the spermatozoids morphological assessment in men aged 21-60 years old Age group (years old) Morphology Oval Tapered Round Immature Amorphous Double-head Double-tail Macrocephalic/large head Microcephalic/small head
21 to 30
31 to 40
41 to 50
51 to 60
% 27.8 24.4 22.4 7.2 9.2 1.2 0.7 4.7 2.4
% 28.2 23.7 23.3 8.4 8.6 0.3 0.4 3.6 3.5
% 38.0* 16.1** 25.7 7.4 7.8 0.5 2.0 2.5
% 30.4 21.3 24.6 7.7 9.1 4.62.3
* percentage greater than in other age groups (p = 0.03; ANOVA followed by Tukey-Kramer test for multiple comparisons); ** percentage smaller than in other age groups (p = 0.02; ANOVA followed by Tukey-Kramer test for multiple comparisons)
After sexual abstinence of five days, which all participants complied to, microscopic analysis showed that the normal oval shape was predominant and was more common in the 41 to 50 year group (p = 0.03). In this age group, on the other hand, the abnormal tapered shape was less frequent than in the other groups (p = 0.02). No differences among the four groups studied were found in other spermatozoon shapes (Table 2). Table 2. Mean concentration spermatozoids and their percentage of mobile shapes found in men aged 21-60 years old Age group (years old)
Concentration (M ± SEM)
% of mobile shapes
(number x 10 /ml) 83 ± 49 76 ± 46 105 ± 49 173 ± 25
70.0 69.1 76.7 75.4
21 to 30 31 to 40 41 to 50 51 to 60 M = mean; SEM = standard error of mean
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Assessment of spermatozoa in different age groups
DISCUSSION Some comparative studies with healthy subjects showed that spermatogenic capacity is higher after five days of sexual abstinence. Shorter intervals showed more immature shapes with decreased concentration and motility. The literature on daily spermatozoon production by middle-aged men is scarce(9). In this study it was observed that, at least in men aged under 60 years, there is no alteration in the concentration of spermatozoa in the seminal fluid. According to Dakouane-Giudicelli et al., atherosclerotic vascular degeneration can decrease testicular blood supply thus causing parenchyma loss and sclerosis of the seminiferous tubules. This sequence of events would be the main cause of reduced spermatogenesis(4). Buwe et al. considered atherosclerosis the main cause of decreased libido and potency(10). Prostatic and urinary disorders, as well as depression and anxiety, are part of the “male climacteric syndrome”, or andropause, and could be complications due to vascular degeneration(11). Johnson et al described a possible autoimmune etiology to explain the parenchyma atrophy and decreased testicular function. The presence of mononuclear inflammatory cells around the seminiferous tubules of elderly men and of antibodies against their own spermatozoa strengthens this hypothesis(12). In this study no abnormalities in physical and chemical characteristics of the seminal fluid or spermatozoon concentration suggesting atherosclerotic or autoimmune alterations or other disorders due to decreased number of Sertoli and Leydig cells were found. The normal (oval) shape is considered ideal for fecundation. It is common to find many tapered sperms when testis temperature and infections of the male genitalia are present. Immature spermatozoa are more commonly found in the seminal fluid of men in sexual abstinence for less than five days(4).
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CONCLUSIONS In the present study no reduction in spermatozoon production was found in the age groups ranging from 21 to 60 years. The normal (oval) shape was found in higher amounts in the 41 to 50 years group. Additionally, in this age group the lowest rate of abnormal shapes was found suggesting higher fertility in this group. REFERENCES 1. Practice Committee of the American Society for Reproductive Medicine. Definition of “infertility”. Fertil Steril. 2006;86(5 Suppl):S228. 2. Tesarik J, Mendoza C. Treatment of severe male infertility by micromanipulationassisted fertilization: an update. Front Biosci. 2007;12:105-14. 3. Schlosser J, Nakib I, Carre-Pigeon F, Staerman F. Male infertility: management strategies. Ann Urol. 2007;41(1):6-11. 4. Dakouane-Giudicelli M, Bergere M, Albert M, Serazin V, Rouillac-Le Sciellour C, Vialard F, et al. Late paternity: spermatogenetic aspects. Gynecol Obstet Fertil. 2006;34(9):855-9. 5. Gleicher N, Barad D. DHEA and testosterone in the elderly. N Engl J Med. 2007;356(6):636-7. 6. Lacombe A, Lelievre V, Roselli CE, Salameh W, Lue YH, Lawson G, et al. A neuropeptide at the origin of testicular aging? Med Sci (Paris). 2006;22(10):809-11. 7. Petroianu A. Pesquisa em Medicina. In: Petroianu A, editor. Ética, moral e deontologia médicas. Rio de Janeiro: Guanabara Koogan; 2000. p.174-8. 8. World Health Organization. WHO laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4 ed. Cambridge: Press Syndicate of the University of Cambridge; 1999. 9. Richthoff J, Spano M, Giwercman YL, Frohm B, Jepson K, Malm J, et al. The impact of testicular and accessory sex gland function on sperm chromatin integrity as assessed by the sperm chromatin structure assay (SCSA). Hum Reprod. 2002;17(12):3162-9. 10. Buwe A, Guttenbach M, Schmid M. Effect of paternal age on the frequency of cytogenetic abnormalities in human spermatozoa. Cytogenet Genome Res. 2005;111(3-4):213-28. 11. Homonnai ZT, Fainman N, David MP, Paz G. Semen quality and sex hormone pattern of 29 middle aged men. Andrologia. 1982;14(2):164-70. 12. Johnson L, Abdo JG, Petty CS, Neaves WB. Effect of age on the composition of seminiferous tubular boundary tissue and on the volume of each component in humans. Fertil Steril. 1988;49(6):1045-50.
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