Trans–fatty acid levels in sperm are associated with sperm concentration among men from an infertility clinic
2010; Elsevier BV; Volume: 95; Issue: 5 Linguagem: Inglês
10.1016/j.fertnstert.2010.10.039
ISSN1556-5653
AutoresJorge E. Chavarro, Jeremy Furtado, Thomas L. Toth, Jennifer B. Ford, M. Keller, Hannia Campos, Russ Hauser,
Tópico(s)Aquaculture Nutrition and Growth
ResumoWe measured the sperm fatty acid composition using gas chromatography in anonymized semen samples of 33 men undergoing infertility evaluation at an academic medical center. Trans–fatty acids were present in human sperm and were related inversely to sperm concentration (r = −0.44). We measured the sperm fatty acid composition using gas chromatography in anonymized semen samples of 33 men undergoing infertility evaluation at an academic medical center. Trans–fatty acids were present in human sperm and were related inversely to sperm concentration (r = −0.44). Trans–fatty acids are unsaturated fatty acids with at least one double bond in the trans, instead of the physiologic cis, configuration. There are two sources of trans fats in the diet. Most are found in foods containing partially hydrogenated vegetable oils used in margarines and commercially prepared foods (1Mozaffarian D. Katan M.B. Ascherio A. Stampfer M.J. Willett W.C. Trans fatty acids and cardiovascular disease.N Engl J Med. 2006; 354: 1601-1613Crossref PubMed Scopus (1275) Google Scholar). Smaller amounts are found in meats and dairy products from ruminants (e.g., cattle, goats, and sheep), as a result of bacterial action in the animal's rumen (1Mozaffarian D. Katan M.B. Ascherio A. Stampfer M.J. Willett W.C. Trans fatty acids and cardiovascular disease.N Engl J Med. 2006; 354: 1601-1613Crossref PubMed Scopus (1275) Google Scholar, 2Sommerfeld M. Trans unsaturated fatty acids in natural products and processed foods.Prog Lipid Res. 1983; 22: 221-233Crossref PubMed Scopus (120) Google Scholar). Little is known about the reproductive health effects of trans fats. Their intake has been related to a higher risk of fetal loss (3Morrison J.A. Glueck C.J. Wang P. Dietary trans fatty acid intake is associated with increased fetal loss.Fertil Steril. 2008; 90: 385-390Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and infertility due to anovulation (4Chavarro J.E. Rich-Edwards J.W. Rosner B.A. Willett W.C. Dietary fatty acid intakes and the risk of ovulatory infertility.Am J Clin Nutr. 2007; 85: 231-237PubMed Google Scholar). In addition, rodent models have shown that trans–fatty acid intake impairs spermatogenesis (5Jensen B. Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency.Lipids. 1976; 11: 179-188Crossref PubMed Scopus (26) Google Scholar, 6Hanis T. Zidek V. Sachova J. Klir P. Deyl Z. Effects of dietary trans-fatty acids on reproductive performance of Wistar rats.Br J Nutr. 1989; 61: 519-529Crossref PubMed Scopus (38) Google Scholar, 7Veaute C. Andreoli M.F. Racca A. Bailat A. Scalerandi M.V. Bernal C. et al.Effects of isomeric fatty acids on reproductive parameters in mice.Am J Reprod Immunol. 2007; 58: 487-496Crossref PubMed Scopus (18) Google Scholar), but it is unknown whether the same relation exists in humans.We collected semen samples as part of a pilot study to determine the feasibility of measuring sperm fatty acid composition in large-scale studies. Between September 2008 and February 2009 we collected 33 deidentified semen samples from the same number of men presenting for evaluation of infertility at the Massachusetts General Hospital Fertility Center. Samples from men presenting for postvasectomy semen analysis were not included. Semen samples were produced on-site by masturbation into a sterile plastic specimen cup. Men were instructed to abstain from ejaculation for 48 hours before producing the semen sample. After collection, the sample was liquefied at 37°C for 20 minutes before analysis. All semen samples were analyzed with use of computer-assisted sperm analysis (version 10HTM-IVOS; Hamilton Thorne, Beverly, MA) as previously described (8Duty S.M. Silva M.J. Barr D.B. Brock J.W. Ryan L. Chen Z. et al.Phthalate exposure and human semen parameters.Epidemiology. 2003; 14: 269-277PubMed Google Scholar, 9Duty S.M. Calafat A.M. Silva M.J. Brock J.W. Ryan L. Chen Z. et al.The relationship between environmental exposure to phthalates and computer-aided sperm analysis motion parameters.J Androl. 2004; 25: 293-302Crossref PubMed Scopus (127) Google Scholar). After the computer-assisted sperm analysis assessment, study samples were chosen on the basis of their sperm concentration value by overselecting samples with concentrations <20 × 106 sperm per milliliter to address the goals of the pilot study. Samples from men with azoospermia were not included in the study. For each man we collected up to four 250-μL aliquots in cryotubes from a single leftover unprocessed clinical semen sample scheduled to be discarded. Semen samples then were stored at −80°C until analysis. All clinically identifiable data, with the exception of sperm concentration, were delinked from study samples.Aliquots were thawed, mixed with 500 μL of phosphate-buffered saline solution, and centrifuged at 600 × g for 12 minutes to pellet the sperm. After centrifugation, the supernatant seminal plasma was removed and replaced with 500 μL normal saline solution to wash seminal fluid from cells. Fatty acids were extracted from sperm into isopropanol and hexane containing 50 mg of 2.6-di-tert-butyl-p-cresol as an antioxidant and transmethylated with methanol and sulfuric acid, as previously described (10Zock P.L. Gerristen J. Katan M. Partial conservation of the sn-2 position of dietary triglicerides in fasting plasma lipids in humans.Eur J Clin Invest. 1996; 26: 141-150Crossref PubMed Scopus (30) Google Scholar, 11Zock P.L. Mensink R.P. Harryvan J. de Vries J.H. Katan M. Fatty acids in serum cholesteryl esters as quantitative biomarkers of dietary intake in humans.Am J Epidemiol. 1997; 145: 1114-1122Crossref PubMed Scopus (149) Google Scholar, 12Baylin A. Kim M.K. Donovan-Palmer A. Siles X. Dougherty L. Tocco P. et al.Fasting whole blood as a biomarker of essential fatty acid intake in epidemiologic studies: comparison with adipose tissue and plasma.Am J Epidemiol. 2005; 162: 373-381Crossref PubMed Scopus (196) Google Scholar). After esterification, the samples were evaporated, and the fatty acids were redissolved in iso-octane and quantified by gas-liquid chromatography on a fused silica capillary cis/trans column (SP2560; Supelco, Bellefonte, PA). Peak retention times were identified by injecting known standards (Nu-Chek Prep, Elysian, MN) and analyzed with the ChemStation A.08.03 software (Agilent Technologies, Santa Clara, CA). The fatty acid levels in each sample were expressed as the percentage of total fatty acids. Coefficients of variation (CVs) ranged between 29.1% for 18:2 trans isomers and 43.7% for 18:1 trans isomers. The large assay variability may be due to heterogeneity in sperm concentration throughout a single semen sample because the variability of the same method is substantially lower in blood products and adipose tissue (12Baylin A. Kim M.K. Donovan-Palmer A. Siles X. Dougherty L. Tocco P. et al.Fasting whole blood as a biomarker of essential fatty acid intake in epidemiologic studies: comparison with adipose tissue and plasma.Am J Epidemiol. 2005; 162: 373-381Crossref PubMed Scopus (196) Google Scholar, 13Chavarro J.E. Stampfer M.J. Campos H. Kurth T. Willett W.C. Ma J. A prospective study of trans fatty acid levels in blood and risk of prostate cancer.Cancer Epidemiol Biomarkers Prev. 2008; 17: 95-101Crossref PubMed Scopus (63) Google Scholar).We calculated the median trans–fatty acid level from all of the available aliquots (up to four) from a single semen sample for each man and assumed the median to be the best estimate of fatty acid level for each man (14Rosner B. Fundamentals of biostatistics.5th ed. Duxbury Press, Pacific Grove, CA2000Google Scholar). We calculated Spearman correlation coefficients between sperm trans–fatty acid levels and sperm concentration. We divided men into quartiles according to their levels of individual sperm trans–fatty acids and calculated the median (25th–75th percentile) sperm concentration in each group. We also calculated the relative difference in sperm concentration across quartiles of sperm trans–fat levels using linear regression models where the outcome was log-transformed sperm concentration.The median sperm concentration was 14 × 106/mL, ranging from 0.01 × 106/mL to 400 × 106/mL. Saturated fatty acids were the predominant fatty acid type in sperm, representing 63.8% of all fatty acids, followed by polyunsaturated fatty acids (19.6% of total) and monounsaturated fatty acids (12.8% of total). The observed relative fatty acid composition of sperm is in line with previously observed values (15Conquer J.A. Martin J.B. Tummon I. Watson L. Tekpetey F. Fatty acid analysis of blood serum, seminal plasma and spermatozoa of normozoospermic vs. asthenozoospermic males.Lipids. 1999; 34: 793-799Crossref PubMed Scopus (110) Google Scholar, 16Zalata A. Christophe A. Depuydt C. Schoonjans F. Comhaire F. The fatty acid composition of phospholipids of spermatozoa from infertile patients.Mol Hum Reprod. 1998; 4: 111-118Crossref PubMed Scopus (201) Google Scholar, 17Gulaya N.M. Margitich V.M. Govseeva N.M. Klimashevsky V.M. Gorpynchenko II, Boyko M.I. Phospholipid composition of human sperm and seminal plasma in relation to sperm fertility.Arch Androl. 2001; 46: 169-175Crossref PubMed Scopus (77) Google Scholar, 18Tavilani H. Doosti M. Abdi K. Vaisiraygani A. Joshaghani H.R. Decreased polyunsaturated and increased saturated fatty acid concentration in spermatozoa from asthenozoospermic males as compared with normozoospermic males.Andrologia. 2006; 38: 173-178Crossref PubMed Scopus (79) Google Scholar, 19Aksoy Y. Aksoy H. Altinkaynak K. Aydin H.R. Ozkan A. Sperm fatty acid composition in subfertile men.Prostaglandins Leukot Essent Fatty Acids. 2006; 75: 75-79Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Total trans–fatty acids accounted, on average, for <1% of total sperm fatty acids but were detectable in all men (range 0.14% to 4.43%).There were large differences in sperm concentration between the lowest and highest quartiles of total sperm trans–fatty acids and a statistically significant inverse association between total sperm trans–fatty acids and sperm concentration (Table 1). Similar associations were observed for 16:1n-7t, 18:1n-9t, 18:2n-6ct, and total 18:2t isomers, whereas levels of 18:1n-12t, 18:1n-7t, 18:2n-6tt, and 18:2n-6tc were unrelated to sperm concentration. The correlations between sperm trans–fatty acids and sperm concentration were comparable when the median across all available aliquots or a single random aliquot was used to represent each man's sperm fatty acid levels.Table 1Association between sperm trans–fatty acid levels and sperm concentration (n = 33).Fatty acidQuartiles of sperm fatty acid levelsP value, trendr1aSpearman correlation coefficient between the sperm levels of the specified fatty acid and sperm concentration. Sperm fatty acid level is the median concentration across all aliquots from the same sample for an individual. All r ≥ |0.35| are statistically significant at P<.05.r2bSpearman correlation coefficient between the sperm levels of the specified fatty acid and sperm concentration. Sperm fatty acid level is a random aliquot from all available aliquots for each individual. All r ≥ |0.35| are statistically significant at P<.05.1 (n = 8)2 (n = 8)3 (n = 9)4 (n = 8)Total trans Median fatty acid level (%)0.250.430.611.77 Sperm concentration, ×106/mL (IQR)100 (50–141)14 (3–18)12 (3–49)3 (0.5–51) Relative difference (95% CI)Ref.−89 (34, −99)−91 (0, −99)−96 (−53, −99).05−0.44−0.5016:1 trans 16:1n-7t Median fatty acid level (%)0.020.050.070.12 Sperm concentration, ×106/mL (range)59 (14–119)13 (5–70)15 (12–86)1 (0.3–5) Relative difference (95% CI)Ref.−52 (433, −96)−72 (193, −97)−97 (−66, −99).03−0.44−0.3118:1 trans Total 18:1 trans Median fatty acid level (%)0.100.180.240.78 Sperm concentration, ×106/mL (range)8 (0.4–108)34 (12–89)12 (4–15)9 (0.7–79) Relative difference (95% CI)Ref.212 (4,359, −78)12 (1,386, −92)−30 (907, −95).52−0.16−0.01 18:1n-12t Median fatty acid level (%)0.020.040.060.24 Sperm concentration, ×106/mL (range)16 (0.6–57)13 (6–50)6 (3–93)33 (4–79) Relative difference (95% CI)Ref.−37 (847, −96)−6 (1306, −93)9 (1526, −93).83−0.01−0.01 18:1n-9t Median fatty acid level (%)0.100.180.240.78 Sperm concentration, ×106/mL (range)79 (6–135)16 (2–52)12 (6–15)4 (0.1–59) Relative difference (95% CI)Ref.−57 (453, −97)−60 (374, −97)−93 (−10, −99).04−0.32−0.29 18:1n-7t Median fatty acid level (%)0.020.040.070.27 Sperm concentration, ×106/mL (range)32 (3–98)18 (2–89)12 (4–15)9 (0.7–79) Relative difference (95% CI)Ref.−30 (899, −95)−71 (289, −98)−73 (285, −98).40−0.10−0.1418:2 trans Total 18:2 trans Median fatty acid level (%)0.100.180.240.78 Sperm concentration, ×106/mL (range)75 (59–100)16 (3–75)3 (0.2–10)9 (0.5–72) Relative difference (95% CI)Ref.−87 (24, −99)−98 (−85, −99)−95 (−54, −99).04−0.49−0.50 18:2n-6tt Median fatty acid level (%)0.000.040.070.59 Sperm concentration, ×106/mL (range)35 (5–75)107 (56–141)3 (0.3–12)5 (0.5–15) Relative difference (95% CI)Ref.567 (5,669, −33)−91 (−23, −99)−84 (36, −98).10−0.48−0.08 18:2n-6ct Median fatty acid level (%)0.000.030.060.18 Sperm concentration, ×106/mL (range)75 (35–101)17 (5–78)12 (3–15)1 (0.3–29) Relative difference (95% CI)Ref.−59 (350, −94)−93 (−28, −99)−96 (−54, −99).01−0.45−0.38 18:2n-6tc Median fatty acid level (%)0.000.010.020.06 Sperm concentration, ×106/mL (range)75 (59–100)9 (0.4–15)14 (10–62)2 (0.1–34) Relative difference (95% CI)Ref.−92 (−11, −99)−19 (778, −93)−91 (8, −99).19−0.29−0.30Note: CI = confidence interval. Ref. = reference; IQR = interquartile range.a Spearman correlation coefficient between the sperm levels of the specified fatty acid and sperm concentration. Sperm fatty acid level is the median concentration across all aliquots from the same sample for an individual. All r ≥ |0.35| are statistically significant at P<.05.b Spearman correlation coefficient between the sperm levels of the specified fatty acid and sperm concentration. Sperm fatty acid level is a random aliquot from all available aliquots for each individual. All r ≥ |0.35| are statistically significant at P<.05. Open table in a new tab We also examined whether the associations of other sperm fatty acid levels with sperm concentration were consistent with those previously reported in the literature. Sperm levels of total polyunsaturated fatty acids (r = 0.68) and docosahexaenoic acid (DHA; r = 0.72) were related positively to sperm concentration whereas sperm levels of oleic acid (r = −0.18), total saturated fatty acids (r = −0.42), and total monounsaturated fatty acids (r = −0.11) were inversely related to sperm concentration in agreement with previous reports (15Conquer J.A. Martin J.B. Tummon I. Watson L. Tekpetey F. Fatty acid analysis of blood serum, seminal plasma and spermatozoa of normozoospermic vs. asthenozoospermic males.Lipids. 1999; 34: 793-799Crossref PubMed Scopus (110) Google Scholar, 16Zalata A. Christophe A. Depuydt C. Schoonjans F. Comhaire F. The fatty acid composition of phospholipids of spermatozoa from infertile patients.Mol Hum Reprod. 1998; 4: 111-118Crossref PubMed Scopus (201) Google Scholar, 19Aksoy Y. Aksoy H. Altinkaynak K. Aydin H.R. Ozkan A. Sperm fatty acid composition in subfertile men.Prostaglandins Leukot Essent Fatty Acids. 2006; 75: 75-79Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar).To our knowledge, this is the first study demonstrating the presence of trans–fatty acids in human sperm and a relation between sperm trans–fatty acids and sperm concentration in humans. Because human fatty acid metabolism cannot introduce trans double bonds into a fatty acid chain, the presence of trans–fatty acids in a human cell or tissue implies dietary intake and can serve as a biomarker of diet. Even though we could not evaluate the relation between trans–fat intake and sperm levels, several studies have shown previously that trans–fatty acids in blood, red blood cells, plasma, and adipose tissue are adequate markers of intake (12Baylin A. Kim M.K. Donovan-Palmer A. Siles X. Dougherty L. Tocco P. et al.Fasting whole blood as a biomarker of essential fatty acid intake in epidemiologic studies: comparison with adipose tissue and plasma.Am J Epidemiol. 2005; 162: 373-381Crossref PubMed Scopus (196) Google Scholar, 20Sun Q. Ma J. Campos H. Hankinson S.E. Hu F.B. Comparison between plasma and erythrocyte fatty acid content as biomarkers of fatty acid intake in US women.Am J Clin Nutr. 2007; 86: 74-81PubMed Google Scholar, 21Baylin A. Kabagambe E.K. Siles X. Campos H. Adipose tissue biomarkers of fatty acid intake.Am J Clin Nutr. 2002; 76: 750-757PubMed Google Scholar). Therefore, our results suggest that higher intake of trans–fatty acids is related to a lower sperm concentration. However, it is not possible to know from our data what dietary intake levels are necessary to achieve the sperm trans–fat levels associated with reduced sperm concentration nor the timing between intake and any eventual effects on spermatogenesis. Nevertheless, this finding is in agreement with previous animal models of supplementation with trans–fatty acids. Male rodents fed trans–fats accumulate them in the testis (5Jensen B. Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency.Lipids. 1976; 11: 179-188Crossref PubMed Scopus (26) Google Scholar, 22Privett O.S. Phillips F. Shimasaki H. Nozawa T. Nickell E.C. Studies of effects of trans fatty acids in the diet on lipid metabolism in essential fatty acid deficient rats.Am J Clin Nutr. 1977; 30: 1009-1017PubMed Google Scholar); have decreased fertility, serum T levels, sperm count, motility, and normal morphology; and, in extreme cases, have spermatogenic arrest and testicular degeneration (5Jensen B. Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency.Lipids. 1976; 11: 179-188Crossref PubMed Scopus (26) Google Scholar, 6Hanis T. Zidek V. Sachova J. Klir P. Deyl Z. Effects of dietary trans-fatty acids on reproductive performance of Wistar rats.Br J Nutr. 1989; 61: 519-529Crossref PubMed Scopus (38) Google Scholar, 7Veaute C. Andreoli M.F. Racca A. Bailat A. Scalerandi M.V. Bernal C. et al.Effects of isomeric fatty acids on reproductive parameters in mice.Am J Reprod Immunol. 2007; 58: 487-496Crossref PubMed Scopus (18) Google Scholar). Given the potential clinical and public health implications of our finding it is important that it be replicated in other studies specifically designed to examine this relation.Our study has some limitations that should be considered when interpreting our results. First, this study was designed originally to develop a laboratory method. As a result we used deidentified samples that could not be linked to data on important covariates such as age, body mass index, diet, or lifestyle factors. Therefore, we cannot discount the possibility that sperm trans–fats are a marker of a negative nutritional or lifestyle factor affecting sperm concentration that we could not account for in the analysis. An additional limitation is the small sample size of this study. Nevertheless, the strength of the observed associations suggests that power was not an important issue and that a larger study would be able to detect smaller associations than we did. Last, the variability of fatty acid determinations in sperm, reflected in high CVs, also could have affected our results. However, high CVs usually lead to an attenuation of observed associations suggesting that the association between sperm trans–fats and sperm concentration may be stronger than what we observed. Also, we obtained the median fatty acid level from all the available aliquots (from the same ejaculate) for each man in an attempt to minimize the measurement error due to assay variability. We also replicated previous findings regarding sperm fatty acid composition and sperm concentration, lending support to the validity of our methods and of our findings for trans–fatty acids.In summary, we found that trans–fatty acids were present in human sperm and were related inversely to sperm concentration. Our data are in agreement with experimental data in rodents showing that trans–fatty acids can affect spermatogenesis profoundly. Nevertheless, given the limitations of this study and the potential clinical and public health implications of our findings, it is important that these hypothesis generating findings be reevaluated in larger, better-designed studies and that the relation between intake of trans–fats and sperm levels of these fatty acids be examined closely. Trans–fatty acids are unsaturated fatty acids with at least one double bond in the trans, instead of the physiologic cis, configuration. There are two sources of trans fats in the diet. Most are found in foods containing partially hydrogenated vegetable oils used in margarines and commercially prepared foods (1Mozaffarian D. Katan M.B. Ascherio A. Stampfer M.J. Willett W.C. Trans fatty acids and cardiovascular disease.N Engl J Med. 2006; 354: 1601-1613Crossref PubMed Scopus (1275) Google Scholar). Smaller amounts are found in meats and dairy products from ruminants (e.g., cattle, goats, and sheep), as a result of bacterial action in the animal's rumen (1Mozaffarian D. Katan M.B. Ascherio A. Stampfer M.J. Willett W.C. Trans fatty acids and cardiovascular disease.N Engl J Med. 2006; 354: 1601-1613Crossref PubMed Scopus (1275) Google Scholar, 2Sommerfeld M. Trans unsaturated fatty acids in natural products and processed foods.Prog Lipid Res. 1983; 22: 221-233Crossref PubMed Scopus (120) Google Scholar). Little is known about the reproductive health effects of trans fats. Their intake has been related to a higher risk of fetal loss (3Morrison J.A. Glueck C.J. Wang P. Dietary trans fatty acid intake is associated with increased fetal loss.Fertil Steril. 2008; 90: 385-390Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) and infertility due to anovulation (4Chavarro J.E. Rich-Edwards J.W. Rosner B.A. Willett W.C. Dietary fatty acid intakes and the risk of ovulatory infertility.Am J Clin Nutr. 2007; 85: 231-237PubMed Google Scholar). In addition, rodent models have shown that trans–fatty acid intake impairs spermatogenesis (5Jensen B. Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency.Lipids. 1976; 11: 179-188Crossref PubMed Scopus (26) Google Scholar, 6Hanis T. Zidek V. Sachova J. Klir P. Deyl Z. Effects of dietary trans-fatty acids on reproductive performance of Wistar rats.Br J Nutr. 1989; 61: 519-529Crossref PubMed Scopus (38) Google Scholar, 7Veaute C. Andreoli M.F. Racca A. Bailat A. Scalerandi M.V. Bernal C. et al.Effects of isomeric fatty acids on reproductive parameters in mice.Am J Reprod Immunol. 2007; 58: 487-496Crossref PubMed Scopus (18) Google Scholar), but it is unknown whether the same relation exists in humans. We collected semen samples as part of a pilot study to determine the feasibility of measuring sperm fatty acid composition in large-scale studies. Between September 2008 and February 2009 we collected 33 deidentified semen samples from the same number of men presenting for evaluation of infertility at the Massachusetts General Hospital Fertility Center. Samples from men presenting for postvasectomy semen analysis were not included. Semen samples were produced on-site by masturbation into a sterile plastic specimen cup. Men were instructed to abstain from ejaculation for 48 hours before producing the semen sample. After collection, the sample was liquefied at 37°C for 20 minutes before analysis. All semen samples were analyzed with use of computer-assisted sperm analysis (version 10HTM-IVOS; Hamilton Thorne, Beverly, MA) as previously described (8Duty S.M. Silva M.J. Barr D.B. Brock J.W. Ryan L. Chen Z. et al.Phthalate exposure and human semen parameters.Epidemiology. 2003; 14: 269-277PubMed Google Scholar, 9Duty S.M. Calafat A.M. Silva M.J. Brock J.W. Ryan L. Chen Z. et al.The relationship between environmental exposure to phthalates and computer-aided sperm analysis motion parameters.J Androl. 2004; 25: 293-302Crossref PubMed Scopus (127) Google Scholar). After the computer-assisted sperm analysis assessment, study samples were chosen on the basis of their sperm concentration value by overselecting samples with concentrations <20 × 106 sperm per milliliter to address the goals of the pilot study. Samples from men with azoospermia were not included in the study. For each man we collected up to four 250-μL aliquots in cryotubes from a single leftover unprocessed clinical semen sample scheduled to be discarded. Semen samples then were stored at −80°C until analysis. All clinically identifiable data, with the exception of sperm concentration, were delinked from study samples. Aliquots were thawed, mixed with 500 μL of phosphate-buffered saline solution, and centrifuged at 600 × g for 12 minutes to pellet the sperm. After centrifugation, the supernatant seminal plasma was removed and replaced with 500 μL normal saline solution to wash seminal fluid from cells. Fatty acids were extracted from sperm into isopropanol and hexane containing 50 mg of 2.6-di-tert-butyl-p-cresol as an antioxidant and transmethylated with methanol and sulfuric acid, as previously described (10Zock P.L. Gerristen J. Katan M. Partial conservation of the sn-2 position of dietary triglicerides in fasting plasma lipids in humans.Eur J Clin Invest. 1996; 26: 141-150Crossref PubMed Scopus (30) Google Scholar, 11Zock P.L. Mensink R.P. Harryvan J. de Vries J.H. Katan M. Fatty acids in serum cholesteryl esters as quantitative biomarkers of dietary intake in humans.Am J Epidemiol. 1997; 145: 1114-1122Crossref PubMed Scopus (149) Google Scholar, 12Baylin A. Kim M.K. Donovan-Palmer A. Siles X. Dougherty L. Tocco P. et al.Fasting whole blood as a biomarker of essential fatty acid intake in epidemiologic studies: comparison with adipose tissue and plasma.Am J Epidemiol. 2005; 162: 373-381Crossref PubMed Scopus (196) Google Scholar). After esterification, the samples were evaporated, and the fatty acids were redissolved in iso-octane and quantified by gas-liquid chromatography on a fused silica capillary cis/trans column (SP2560; Supelco, Bellefonte, PA). Peak retention times were identified by injecting known standards (Nu-Chek Prep, Elysian, MN) and analyzed with the ChemStation A.08.03 software (Agilent Technologies, Santa Clara, CA). The fatty acid levels in each sample were expressed as the percentage of total fatty acids. Coefficients of variation (CVs) ranged between 29.1% for 18:2 trans isomers and 43.7% for 18:1 trans isomers. The large assay variability may be due to heterogeneity in sperm concentration throughout a single semen sample because the variability of the same method is substantially lower in blood products and adipose tissue (12Baylin A. Kim M.K. Donovan-Palmer A. Siles X. Dougherty L. Tocco P. et al.Fasting whole blood as a biomarker of essential fatty acid intake in epidemiologic studies: comparison with adipose tissue and plasma.Am J Epidemiol. 2005; 162: 373-381Crossref PubMed Scopus (196) Google Scholar, 13Chavarro J.E. Stampfer M.J. Campos H. Kurth T. Willett W.C. Ma J. A prospective study of trans fatty acid levels in blood and risk of prostate cancer.Cancer Epidemiol Biomarkers Prev. 2008; 17: 95-101Crossref PubMed Scopus (63) Google Scholar). We calculated the median trans–fatty acid level from all of the available aliquots (up to four) from a single semen sample for each man and assumed the median to be the best estimate of fatty acid level for each man (14Rosner B. Fundamentals of biostatistics.5th ed. Duxbury Press, Pacific Grove, CA2000Google Scholar). We calculated Spearman correlation coefficients between sperm trans–fatty acid levels and sperm concentration. We divided men into quartiles according to their levels of individual sperm trans–fatty acids and calculated the median (25th–75th percentile) sperm concentration in each group. We also calculated the relative difference in sperm concentration across quartiles of sperm trans–fat levels using linear regression models where the outcome was log-transformed sperm concentration. The median sperm concentration was 14 × 106/mL, ranging from 0.01 × 106/mL to 400 × 106/mL. Saturated fatty acids were the predominant fatty acid type in sperm, representing 63.8% of all fatty acids, followed by polyunsaturated fatty acids (19.6% of total) and monounsaturated fatty acids (12.8% of total). The observed relative fatty acid composition of sperm is in line with previously observed values (15Conquer J.A. Martin J.B. Tummon I. Watson L. Tekpetey F. Fatty acid analysis of blood serum, seminal plasma and spermatozoa of normozoospermic vs. asthenozoospermic males.Lipids. 1999; 34: 793-799Crossref PubMed Scopus (110) Google Scholar, 16Zalata A. Christophe A. Depuydt C. Schoonjans F. Comhaire F. The fatty acid composition of phospholipids of spermatozoa from infertile patients.Mol Hum Reprod. 1998; 4: 111-118Crossref PubMed Scopus (201) Google Scholar, 17Gulaya N.M. Margitich V.M. Govseeva N.M. Klimashevsky V.M. Gorpynchenko II, Boyko M.I. Phospholipid composition of human sperm and seminal plasma in relation to sperm fertility.Arch Androl. 2001; 46: 169-175Crossref PubMed Scopus (77) Google Scholar, 18Tavilani H. Doosti M. Abdi K. Vaisiraygani A. Joshaghani H.R. Decreased polyunsaturated and increased saturated fatty acid concentration in spermatozoa from asthenozoospermic males as compared with normozoospermic males.Andrologia. 2006; 38: 173-178Crossref PubMed Scopus (79) Google Scholar, 19Aksoy Y. Aksoy H. Altinkaynak K. Aydin H.R. Ozkan A. Sperm fatty acid composition in subfertile men.Prostaglandins Leukot Essent Fatty Acids. 2006; 75: 75-79Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Total trans–fatty acids accounted, on average, for <1% of total sperm fatty acids but were detectable in all men (range 0.14% to 4.43%). There were large differences in sperm concentration between the lowest and highest quartiles of total sperm trans–fatty acids and a statistically significant inverse association between total sperm trans–fatty acids and sperm concentration (Table 1). Similar associations were observed for 16:1n-7t, 18:1n-9t, 18:2n-6ct, and total 18:2t isomers, whereas levels of 18:1n-12t, 18:1n-7t, 18:2n-6tt, and 18:2n-6tc were unrelated to sperm concentration. The correlations between sperm trans–fatty acids and sperm concentration were comparable when the median across all available aliquots or a single random aliquot was used to represent each man's sperm fatty acid levels. Note: CI = confidence interval. Ref. = reference; IQR = interquartile range. We also examined whether the associations of other sperm fatty acid levels with sperm concentration were consistent with those previously reported in the literature. Sperm levels of total polyunsaturated fatty acids (r = 0.68) and docosahexaenoic acid (DHA; r = 0.72) were related positively to sperm concentration whereas sperm levels of oleic acid (r = −0.18), total saturated fatty acids (r = −0.42), and total monounsaturated fatty acids (r = −0.11) were inversely related to sperm concentration in agreement with previous reports (15Conquer J.A. Martin J.B. Tummon I. Watson L. Tekpetey F. Fatty acid analysis of blood serum, seminal plasma and spermatozoa of normozoospermic vs. asthenozoospermic males.Lipids. 1999; 34: 793-799Crossref PubMed Scopus (110) Google Scholar, 16Zalata A. Christophe A. Depuydt C. Schoonjans F. Comhaire F. The fatty acid composition of phospholipids of spermatozoa from infertile patients.Mol Hum Reprod. 1998; 4: 111-118Crossref PubMed Scopus (201) Google Scholar, 19Aksoy Y. Aksoy H. Altinkaynak K. Aydin H.R. Ozkan A. Sperm fatty acid composition in subfertile men.Prostaglandins Leukot Essent Fatty Acids. 2006; 75: 75-79Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). To our knowledge, this is the first study demonstrating the presence of trans–fatty acids in human sperm and a relation between sperm trans–fatty acids and sperm concentration in humans. Because human fatty acid metabolism cannot introduce trans double bonds into a fatty acid chain, the presence of trans–fatty acids in a human cell or tissue implies dietary intake and can serve as a biomarker of diet. Even though we could not evaluate the relation between trans–fat intake and sperm levels, several studies have shown previously that trans–fatty acids in blood, red blood cells, plasma, and adipose tissue are adequate markers of intake (12Baylin A. Kim M.K. Donovan-Palmer A. Siles X. Dougherty L. Tocco P. et al.Fasting whole blood as a biomarker of essential fatty acid intake in epidemiologic studies: comparison with adipose tissue and plasma.Am J Epidemiol. 2005; 162: 373-381Crossref PubMed Scopus (196) Google Scholar, 20Sun Q. Ma J. Campos H. Hankinson S.E. Hu F.B. Comparison between plasma and erythrocyte fatty acid content as biomarkers of fatty acid intake in US women.Am J Clin Nutr. 2007; 86: 74-81PubMed Google Scholar, 21Baylin A. Kabagambe E.K. Siles X. Campos H. Adipose tissue biomarkers of fatty acid intake.Am J Clin Nutr. 2002; 76: 750-757PubMed Google Scholar). Therefore, our results suggest that higher intake of trans–fatty acids is related to a lower sperm concentration. However, it is not possible to know from our data what dietary intake levels are necessary to achieve the sperm trans–fat levels associated with reduced sperm concentration nor the timing between intake and any eventual effects on spermatogenesis. Nevertheless, this finding is in agreement with previous animal models of supplementation with trans–fatty acids. Male rodents fed trans–fats accumulate them in the testis (5Jensen B. Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency.Lipids. 1976; 11: 179-188Crossref PubMed Scopus (26) Google Scholar, 22Privett O.S. Phillips F. Shimasaki H. Nozawa T. Nickell E.C. Studies of effects of trans fatty acids in the diet on lipid metabolism in essential fatty acid deficient rats.Am J Clin Nutr. 1977; 30: 1009-1017PubMed Google Scholar); have decreased fertility, serum T levels, sperm count, motility, and normal morphology; and, in extreme cases, have spermatogenic arrest and testicular degeneration (5Jensen B. Rat testicular lipids and dietary isomeric fatty acids in essential fatty acid deficiency.Lipids. 1976; 11: 179-188Crossref PubMed Scopus (26) Google Scholar, 6Hanis T. Zidek V. Sachova J. Klir P. Deyl Z. Effects of dietary trans-fatty acids on reproductive performance of Wistar rats.Br J Nutr. 1989; 61: 519-529Crossref PubMed Scopus (38) Google Scholar, 7Veaute C. Andreoli M.F. Racca A. Bailat A. Scalerandi M.V. Bernal C. et al.Effects of isomeric fatty acids on reproductive parameters in mice.Am J Reprod Immunol. 2007; 58: 487-496Crossref PubMed Scopus (18) Google Scholar). Given the potential clinical and public health implications of our finding it is important that it be replicated in other studies specifically designed to examine this relation. Our study has some limitations that should be considered when interpreting our results. First, this study was designed originally to develop a laboratory method. As a result we used deidentified samples that could not be linked to data on important covariates such as age, body mass index, diet, or lifestyle factors. Therefore, we cannot discount the possibility that sperm trans–fats are a marker of a negative nutritional or lifestyle factor affecting sperm concentration that we could not account for in the analysis. An additional limitation is the small sample size of this study. Nevertheless, the strength of the observed associations suggests that power was not an important issue and that a larger study would be able to detect smaller associations than we did. Last, the variability of fatty acid determinations in sperm, reflected in high CVs, also could have affected our results. However, high CVs usually lead to an attenuation of observed associations suggesting that the association between sperm trans–fats and sperm concentration may be stronger than what we observed. Also, we obtained the median fatty acid level from all the available aliquots (from the same ejaculate) for each man in an attempt to minimize the measurement error due to assay variability. We also replicated previous findings regarding sperm fatty acid composition and sperm concentration, lending support to the validity of our methods and of our findings for trans–fatty acids. In summary, we found that trans–fatty acids were present in human sperm and were related inversely to sperm concentration. Our data are in agreement with experimental data in rodents showing that trans–fatty acids can affect spermatogenesis profoundly. Nevertheless, given the limitations of this study and the potential clinical and public health implications of our findings, it is important that these hypothesis generating findings be reevaluated in larger, better-designed studies and that the relation between intake of trans–fats and sperm levels of these fatty acids be examined closely.
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