Infertility affects one in six couples. WHO defines infertility as the inability of a sexually active, noncontracepting couple to achieve pregnancy in 1 year 1. Male and female factors coexist in about one-third of cases, whereas one-third of cases are secondary to male factors only 2.
L-carnitine (β-hydroxy-γ-N-trimethylaminobutyric acid) is an amino acid that plays an important role in energy production by transferring long-chain fatty acids across the mitochondrial membranes, facilitating β-oxidation 3. Approximately 75% body stores of L-carnitine are derived from the diet, whereas only 25% are synthesized de novo from lysine and methionine 4. Meat and milk are the most significant dietary sources of exogenous carnitine for humans 5.
Spermatozoa produced by the testis and secretions from the accessory glands are the two major constituents of semen. Accessory gland secretions make up ∼90% of the semen volume, mainly contributed by epididymis, seminal vesicles, prostate, and bulbourethral glands 6. L-carnitine is concentrated in high-energy demanding tissues such as skeletal and cardiac muscles and in a specialized reproductive tract organ, the epididymis. In addition to transferring long-chain fatty acids across the mitochondrial membranes for β-oxidation, the other functions are modulation of the acyl-coenzyme A/coenzyme A ratio, storage of energy as acetylcarnitine, and reducing the toxic effects of poorly metabolized acyl groups by excreting them as carnitine esters 7.
Free L-carnitine in semen is an epididymal marker of its secretory capacity, which can be used clinically 8. In the epididymis, free L-carnitine is taken up from the blood plasma, transported into the epididymal fluid, and then passively diffused into the spermatozoa, where it accumulates as both free and acetylated L-carnitine 5. Casillas 9 reported that mammalian epididymis is a site for carnitine accumulation by spermatozoa, which is closely related to the development of fertilizing capacity by spermatozoa. The concentration of L-carnitine in epididymal plasma and spermatozoa varies from 2 to 100 mmol/l, which is almost 2000-fold greater than circulating levels (10–50 μmol/l) 5.
Epididymal spermatozoa make use of different substrates as energy sources, but fatty acid oxidation, involving the carnitine-dependent system, seems to be the major energy-supplying process 10. Mazzilli et al.11 found a strict correlation between intrasperm L-carnitine content and motility survival in bovine cervical mucus. This is probably because of the fact that even after ejaculation, lipids present in the cervical mucus are an important energy source for sperm, and to metabolize these lipids, intrasperm L-carnitine is essential. Carnitine also affects testicular sperm maturation in Sertoli cells by indirectly facilitating oxidation of fatty acids 12 and stimulation of glucose uptake by Sertoli cells 13.
L-carnitine is transported by OCTN1, OCTN2, OCTN3, CT2, and ATB+ transporters in mammals 12. There are strong evidences suggesting that mainly OCTN2 and CT2 transport L-carnitine in the epididymis and Sertoli cells influence spermatogenesis. High-affinity sodium-dependent carnitine transporters OCTN2 are localized at the basolateral membrane of epididymal epithelial and Sertoli cells, facilitating supply of carnitine into these cells from the systemic circulation 14, whereas human carnitine transporter CT2 is present selectively in male reproductive tissues, chiefly in Sertoli cells and the luminal membrane of epididymal epithelium, where L-carnitine is secreted into the lumen by an active transport mechanism 15.
The initial endocrine evaluation of the infertile male should include serum testosterone and follicle-stimulating hormone (FSH) levels; if the testosterone level is low, treatment with serum luteinizing hormone (LH) is advised 16. Gonadotrope cells in the anterior pituitary stimulated by gonadotropin-releasing hormone secrete gonadotropic hormones, LH, and FSH. LH is the primary stimulus for the secretion of testosterone by the interstitial cells of Leydig in the testes. FSHs act on Sertoli cells to grow and secrete various spermatogenic substances. To initiate spermatogenesis, both FSH and testosterone are necessary, although once the initial stimulation has occurred, testosterone alone can maintain spermatogenesis for a longer time 17.
The present study aimed to determine whether testosterone, LH, and FSH influence seminal free L-carnitine levels in fertile and infertile men.
Materials and methods
The present study was designed as an observational study to investigate the relationship of seminal free L-carnitine with serum testosterone, LH, and FSH at the Basic Medical Sciences Institute, Jinnah Postgraduate Medical Centre, Karachi, Pakistan. The study was ethically approved by the Basic Medical Sciences Institute and the National Research Institute for Fertility Care. Sixty-one adult men were selected with their consent including fertile and infertile men. Exclusion criteria were; men using drugs, pelvic surgery, current illness, or comorbidities.
Early morning venous blood and semen samples were collected. After centrifugation, serum was separated, stored, and then analyzed for LH, FSH, and total testosterone by enzyme-linked immunosorbent assay technique. Semen was obtained by masturbation following 3–5 days of sexual abstinence. After 30 min of liquefaction at room temperature, samples were immediately divided into two portions, the first for semen analysis and the second for centrifugation for 10 min. Supernatant was kept at −80°C and later analyzed for free L-carnitine using the method developed by Li et al.18 on high-performance liquid chromatography. Semen analysis was carried out immediately and interpreted for its volume, sperm count, motility, and morphology according to the WHO reference values 19.
Statistical analysis Sample size was not determined but was restricted by the number of incident cases over a period of 2 years. However, post-hoc calculations 20 showed adequate power to detect significant correlations. Pearson’s correlation was used to determine the possible relation among the variables. Comparisons among groups were also carried out and significant values were found after the Student t-test was carried out.
Sixty-one men were divided into two main groups, fertile (control) and infertile. Nineteen men were fertile and 42 men were infertile.
Table 1 shows a comparison of the mean (±SEM) values of age, duration of infertility, BMI, ejaculate volume, sperm count, total motility, normal morphology, and white blood cell of the fertile and the infertile group. The sperm count, total motility, and normal morphology were significantly low (P<0.001) in the infertile group compared with the fertile group, whereas other values were nonsignificant after performing the Student t-test.
Table 2 shows a comparison of the mean (±SEM) values of serum testosterone, LH, FSH, and seminal free L-carnitine among the men in the fertile and infertile groups. In the infertile group, the mean value of LH was significantly (P<0.05) higher compared with that of the fertile group. The mean value of testosterone was low and FSH was higher in the infertile group compared with the fertile group, but both were nonsignificant. The mean (±SEM) value of seminal free L-carnitine in infertile men was significantly (P<0.001) lower compared with fertile men.
Seminal free L-carnitine demonstrated significant positive correlation with testosterone (r=0.34, P<0.05). Serum LH was negatively correlated (r=−0.31), attaining statistical significance (P<0.05), whereas serum FSH was also negatively correlated (r=−0.21), but failed to achieve statistical significance.
In this article, we present the correlation of seminal free L-carnitine with serum LH, FSH, and testosterone in fertile and infertile men. This study is an extension of our previously published study 21 that systematically compared seminal free L-carnitine among fertile and infertile participants. We have previously shown that the seminal free L-carnitine was significantly low in azoospermic, asthenospermic, and oligoasthenoteratospermic men compared independently with the fertile (control) group, establishing the fact that L-carnitine in seminal plasma plays an essential role in maintaining male fertility. In the present study, we aimed to investigate the influence of testosterone, LH, and FSH on seminal free L-carnitine levels.
Among infertile men, 43% showed low testosterone levels, whereas 33% had normal testosterone levels, both accompanied by high FSH and LH levels. This may advocate the presence of primary hypogonadism predominantly in this study. However, the mean serum testosterone level in the infertile group was lower than that of the fertile group, but in the normal range, and no significant differences were observed on comparison with each other. However, the mean values of LH and FSH were higher among the men in the infertile group than those in the fertile group, and both were above the normal reference range.
These results are consistent with those of various studies 22–25 reporting that the mean plasma testosterone concentration was in the normal range in infertile men along with high levels of gonadotropins LH/FSH levels on comparing with normal control participants. This phenomenon can be understood by the fact that in primary testicular disorders, high estrogen levels by testosterone aromatization in testes increase serum hormone-binding globulin (SHBG) levels. More testosterone binds to the SHBG and becomes more biologically inactive. Therefore, total testosterone may be in the normal range but circulating free testosterone level could be low 26. The testosterone level will not be enough to inhibit gonadotropin secretions and will mislead the factual diagnosis. To rule out this ambiguity, future studies should analyze SHBG and free testosterone along with other parameters. However, some studies observed low serum testosterone similarly accompanied by high LH and FSH in infertile men 27. In men, elevated serum concentrations of LH and FSH can result from hypergonadotropic hypogonadism because of loss of negative feedback by testicular products 28. Various causes for this include primary testicular failure, seminiferous tubule dysgenesis, and Sertoli cell failure.
The secretory function of epididymis is maintained by the androgens in the male reproductive system 29,30. Androgens are also required to allow maintenance of normal L-carnitine levels in the testes and epididymis. In male rats, castration lowers the epididymal L-carnitine concentration to nondetectable levels but administration of testosterone to the same castrated rats prevented the decrease 31. The uptake of L-carnitine in the epididymis is androgen dependent, supported by studies that report that castration abolishes OCTN2 expression in the rat epididymis, suggesting that OCTN2 expression is regulated by testosterone 32. Our results are in agreement with the above studies, showing that seminal free L-carnitine was significantly correlated with serum testosterone. Another previous study carried out among dairy bulls showed nonsignificant positive correlations between blood plasma testosterone and spermatozoal total carnitine and acylcarnitines, but no correlation with free carnitine 33. Considering this, we may contemplate that decreased serum testosterone may be the main etiological reason for decreased L-carnitine in male infertile.
Several molecular studies suggest that LH and androgens are essential for the regulation of mammalian epididymal function, whereas FSH may play no role 34–37. In our study, LH was negatively correlated with seminal free L-carnitine and attained statistical significance, which may be because of the strong inverse relationship among testosterone and LH in patients with hypergonadotropic hypogonadism. Although the exact mechanism for this association is not completely elucidated, it may be because of loss of negative feedback by low circulating free testosterone; increased LH showed a negative correlation with L-carnitine, whereas a weak negative correlation of seminal free L-carnitine with FSH did not attain statistical significance. This suggests that FSH has no relation with seminal free L-carnitine and merely symbolizes the endocrine status of the patients. However, no supporting study exploring the relationship of LH and FSH with seminal free L-carnitine among humans could be identified after a literature search.
Serum testosterone increases seminal free L-carnitine in humans, whereas serum LH and FSH are inversely correlated with free L-carnitine in seminal fluid. Weak correlations warrant careful interpretation and further larger studies are required to generalize these results. However, this research examines the influence of serum testosterone, LH, and FSH on seminal free L-carnitine and may also reflect the regulation of mitochondrial fatty acid oxidation, which is the major energy-supplying process in sperm by serum testosterone, LH, and FSH implicitly.
Inherent limitations in the study design and small sample size warrant careful interpretation of the results. A prospective study on a larger population is needed to ascertain the external validity of the study.
The authors would like to acknowledge the patients and staff members of the Male Infertility Clinic at the Reproductive Health Centre, Jinnah Postgraduate Medical Centre. They also wish to thank Dr Shakil Ahmed, Incharge, Industrial Analytical Centre, HEJ, University of Karachi; Dr Fatima Bi, Ex-Project Director Pakistan Council of Scientific and Industrial Research (PCSIR), for the immeasurable help in funding, research, and development of the analytical technique on HPLC; and Mr Hermain Kazmi, Editor, Literary and Publication Society, NED University of Engineering and Technology, for manuscript editing.
Conflicts of interest
There are no conflicts of interest.
1. Dohle GR, Jungwirth A, Colpi G, Giwercman A, Diemer T, Hargreave TB.Guidelines on male infertility
.Eur Assoc Urol20076–7.
2. Kolettis PN.Evaluation of the subfertile man.Am Fam Physician2003;67:2165–2172.
3. Jeulin C, Lewin LM.Role of free L-carnitine
in post-gonadal maturation of mammalian spermatozoa.Hum Reprod Update1996;2:87–102.
4. Sigman M, Glass S, Campagnone J, Pryor JL.Carnitine
for the treatment of idiopathic asthenospermia: a randomized, double-blind, placebo-controlled trial.Fertil Steril2006;85:1409–1414.
5. Chiu MN, Blackman MR, Wang C, Swerdloff RS.The role of carnitine
in the male
reproductive system.Ann N Y Acad Sci2004;1033:177–188.
6. .WHO laboratory manual for the examination and processing of human semen2010:5th ed..Geneva:WHO.
7. Vaz FM, Wanders RJA.Carnitine
biosynthesis in mammals.Biochem J2002;361:417–429.
8. Cooper TG.Secretory proteins from the epididymis and their clinical relevance.Andrologia1990;22Suppl 1155–165.
9. Casillas ER.Accumulation of carnitine
by bovine spermatozoa during maturation in the epididymis.J Biol Chem1973;248:8227–8232.
10. Deana R, Rigoni F, Francesconi M, Cavallini L, Arslan P, Siliprandi N.Effect of L-carnitine
and L-aminocarnitine on calcium transport, motility, and enzyme release from ejaculated bovine spermatozoa.Biol Reprod1989;41:949–955.
11. Mazzilli F, Rossi T, Ronconi C, Germini B, Dondero F.Human intrasperm L-carnitine
content related to sperm motility survival.Minerva Ginecol1999;51:129–134.
12. Kobayashi D, Goto A, Maeda T, Nezu JI, Tsuji A, Tamai I.OCTN2-mediated transport of carnitine
in isolated Sertoli cells.Reproduction2005;129:729–736.
13. Agarwal A, Said TM.Oxidative stress, DNA damage and apoptosis in male infertility
: a clinical approach.BJU Int2005;95:503–507.
14. Kobayashi D, Irokawa M, Maeda T, Tsuji A, Tamai I.Carnitine
/organic cation transporter OCTN2-mediated transport of carnitine
in primary-cultured epididymal epithelial cells.Reproduction2005;130:931–937.
15. Enomoto A, Wempe MF, Tsuchida H, Shin HJ, Cha SH, Anzai N, et al..Molecular identification of a novel carnitine
transporter specific to human testis: insights into the mechanism of carnitine
recognition.J Biol Chem2002;277:36262–36271.
16. Shefi S, Turek PJ.Definition and current evaluation of subfertile men.Int Braz J Urol2006;32:385–397.
17. Hall JE.Guyton and Hall textbook of medical physiology2006:11th ed..Philadelphia:Elsevier;996–1010.
18. Li K, Li W, Huang Y.Determination of free L-carnitine
in human seminal plasma by high performance liquid chromatography with pre-column ultraviolet derivatization and its clinical application in male infertility
.Clin Chim Acta2007;3781–2159–163.
19. .WHO laboratory manual for the examination of human semen and sperm–cervical mucus interaction1999:4th ed..Geneva:Cambridge University Press.
20. Faul F, Erdfelder E, Lang AG, Buchner A.G*Power 3: a flexible statistical power analysis program for the social, behavioral, and biomedical sciences.Behav Res Methods2007;39:175–191.
21. Ahmed SDH, Karira KA, Jagdesh, Ahsan S.Role of L-carnitine
in male infertility
.J Pak Med Assoc2011;61:732–736.
22. Khan MS, Ali I, Tahir F, Khan GM.Simultaneous analysis of the three hormones involved in spermatogenesis and their interrelation ratios.Pak J Pharm Sci2008;21:344–349.
23. Stanwell-Smith R, Thompson SG, Haines AP, Jeffcoate SL, Hendry WF.Plasma concentrations of pituitary and testicular hormones of fertile and infertile men.Clin Reprod Fertil1985;3:37–48.
24. Turek PJ, Kim M, Gilbaugh JH III, Lipshultz LI.The clinical characteristics of 82 patients with Sertoli cell-only testis histology.Fertil Steril1995;64:1197–1200.
25. Subhan F, Tahir F, Ahmad R, Khan LU.The study of azoospermic patients in relation to their hormonalprofile (LH, FSH and testosterone
).Rawal Med J1995;221&225–27.
26. .American Association of Clinical Endocrinologists Medical Guidelines for clinical practice for the evaluation and treatment of hypogonadism in adult male
patients – 2002 update.Endocrine Practice: Off J Am Coll Endocrinol Am Assoc Clin Endocrinol2002;8:440–456.
27. Mohammed TG, Ahmed SA, Hussain MK.Relevance of sex hormones levels with spermogram of infertile men.Global J Med Res2012;12:15–18.
28. Ramanujam LN, Liao WX, Roy AC, Ng SC.Association of molecular variants of luteinizing hormone
with male infertility
29. Sharpe RMKnobil E, Neill JD.Regulation of spermatogenesis.The physiology of reproduction1994:2nd ed..New York:Raven Press;1363–1434.
30. Robaire B, Hermo LKnobil E, Neill JD.Efferent ducts, epididymis and vas deferens: structure, functions and their regulation.The physiology of reproduction1988.New York:Raven Press;999–1080.
31. Marquis NR, Fritz IB.Effects of testosterone
on the distribution of carnitine
, acetylcarnitine, and carnitine
acetyltransferase in tissues of the reproductive system of the male
rat.J Biol Chem1965;240:2197–2200.
32. Rodríguez CM, Labus JC, Hinton BT.Organic cation/carnitine
transporter, OCTN2, is differentially expressed in the adult rat epididymis.Biol Reprod2002;67:314–319.
33. Carter AL, Hutson SM, Stratman FW, Haning RV Jr.Relationship of carnitine
and acylcarnitines in ejaculated sperm to blood plasma testosterone
of dairy bulls.Biol Reprod1980;23:820–825.
34. Zhang T, Guo C-X, Hu ZY, Liu YX.Localization of plasminogen activator and inhibitor, LH and androgen receptors and inhibin subunits in monkey epididymis.Mol Hum Reprod1997;3:945–952.
35. Schlatt S, Arslan M, Weinbauer GF, Behre HM, Nieschlag E.Endocrine control of testicular somatic and premeiotic germ cell development in the immature testis of the primate Macaca mulatta.
Eur J Endocrinol1995;133:235–247.
36. Zhou H, Zhang T, Liu Y.Expression and regulation of plasminogen activator and plasminogen activator inhibitor type-1 in rat epididymis.Chin Sci Bull1997;42:779–783.
37. Setty BS, Jehan Q.Functional maturation of the epididymis in the rat.J Reprod Fertil1977;49:317–322.