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. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Fertil Steril. 2010 Nov 11;95(5):1794–1797. doi: 10.1016/j.fertnstert.2010.10.039

Trans fatty acid levels in sperm are associated with sperm concentration among men from an infertility clinic

Jorge E Chavarro a,b, Jeremy Furtado a, Thomas L Toth c, Jennifer Ford c,d, Myra Keller c,d, Hannia Campos a, Russ Hauser c,d,e
PMCID: PMC3062652  NIHMSID: NIHMS248775  PMID: 21071027

Abstract

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 inversely related to sperm concentration (r = − 0.44).

Keywords: fatty acids, diet, sperm, semen analysis, infertility


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 (1). 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 (12). Little is known about the reproductive health effects of trans fats. Their intake has been related to a higher risk of fetal loss (3) and infertility due to anovulation (4). In addition, rodent models have shown that trans fatty acid intake impairs spermatogenesis (57) 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 de-identified semen samples from the same number of men presenting for evaluation of infertility at the Massachusetts General Hospital (MGH) Fertility Center. Samples from men presenting for post-vasectomy 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 37ºC for 20 minutes before analysis. All semen samples were analyzed using Computer-Assisted Semen Analysis (CASA; Hamilton-Thorn Version 10HTM-IVOS) as previously described (89). Following the CASA assessment, study samples were chosen based on their sperm concentration value by over-selecting samples with concentrations below 20×106 sperm/mL in order to address the goals of the pilot study. Samples from azoospermic men were not included in the study. For each man we collected up to four 250μL aliquots in cryotubes from a single left-over unprocessed clinical semen sample scheduled to be discarded. Semen samples were then stored at −80°C until analysis. All clinically identifiable data, with the exception of sperm concentration, were de-linked from study samples.

Aliquots were thawed, mixed with 500μL of phosphate-buffered saline, and centrifuged at 600g for 12 minutes to pellet the sperm. After centrifugation, the supernatant seminal plasma was removed and replaced with 500μL normal saline 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 (1012). 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, Belafonte, PA). Peak retention times were identified by injecting known standards (NuCheck Prep, Elysium, MN) and analyzed with the ChemStation A.08.03 software (Agilent Technologies). The fatty acid levels in each sample were expressed as the percentage of total fatty acids. Coefficients of variation 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 since the variability of the same method is substantially lower in blood products and adipose tissue (1213).

We calculated the median trans fatty acid level from all of the available aliquots (up to 4) from a single semen sample for each man and assumed the median to be the best estimate of fatty acid level for each man (14). 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. SFAs were the predominant fatty acid type in sperm representing 63.8% of all fatty acids, followed by PUFAs (19.6% of total) and MUFAs (12.8% of total). The observed relative fatty acid composition of sperm is in line with previously observed values (1519). Total trans fatty acids accounted, on average, for less than 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 1.

Association between sperm trans fatty acid levels and sperm concentration (n = 33).

Quartiles of sperm fatty acid levels P, trend r1* r2**
1 (n = 8) 2 (n = 8) 3 (n = 9) 4 (n = 8)
Total trans
 Median fatty acid level, % 0.25 0.43 0.61 1.77
 Sperm concentration (×106/mL) 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) 0.05 −0.44 −0.50
16:1 trans
16:1 n−7 t
 Median fatty acid level, % 0.02 0.05 0.07 0.12
 Sperm concentration (×106/mL) 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) 0.03 −0.44 −0.31
18:1 trans
Total 18:1 trans
 Median fatty acid level, % 0.10 0.18 0.24 0.78
 Sperm concentration (×106/mL) 8 [0.4 – 108] 34 [12 – 89] 12 [4 – 15] 9 [0.7 – 79]
 Relative Difference (95% CI) Ref. 212 (4359, −78) 12 (1386, −92) −30 (907, −95) 0.52 −0.16 −0.01
18:1 n−12 t
 Median fatty acid level, % 0.02 0.04 0.06 0.24
 Sperm concentration (×106/mL) 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) 0.83 −0.01 −0.01
18:1 n−9 t
 Median fatty acid level, % 0.10 0.18 0.24 0.78
 Sperm concentration (×106/mL) 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) 0.04 −0.32 −0.29
18:1 n−7 t
 Median fatty acid level, % 0.02 0.04 0.07 0.27
 Sperm concentration (×106/mL) 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) 0.40 −0.10 −0.14
18:2 trans
Total 18:2 trans
 Median fatty acid level, % 0.10 0.18 0.24 0.78
 Sperm concentration (×106/mL) 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) 0.04 −0.49 −0.50
18:2 n−6 t,t
 Median fatty acid level, % 0.00 0.04 0.07 0.59
 Sperm concentration (×106/mL) 35 [5 – 75] 107 [56 – 141] 3 [0.3 – 12] 5 [0.5 – 15]
 Relative Difference (95% CI) Ref. 567 (5669, −33) −91 (−23, −99) −84 (36, −98) 0.10 −0.48 −0.08
18:2 n−6 c,t
 Median fatty acid level, % 0.00 0.03 0.06 0.18
 Sperm concentration (×106/mL) 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) 0.01 −0.45 −0.38
18:2 n−6 t,c
 Median fatty acid level, % 0.00 0.01 0.02 0.06
 Sperm concentration (×106/mL) 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) 0.19 −0.29 −0.30

CI: Confidence Interval

*

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 statically significant at p<0.05.

**

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 statically significant at p<0.05.

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 PUFAs (r = 0.68) and DHA (r = 0.72) were positively related to sperm concentration whereas sperm levels of oleic acid (r =− 0.18), total SFAs (r =− 0.42) and total MUFAs (r =− 0.11) were inversely related to sperm concentration in agreement with previous reports (1516, 19).

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 previously shown that trans fatty acids in blood, red blood cells, plasma and adipose tissue are adequate markers of intake (12, 2021). 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 (5, 22), have decreased fertility, serum testosterone levels, sperm count, motility and normal morphology and, in extreme cases, spermatogenic arrest and testicular degeneration (57). Given the potential clinical and public health implications of our finding it is important that it is replicated in other studies specifically designed to examine this relation.

Our study has some limitations which should be considered when interpreting our results. First, this study was originally designed to develop a laboratory method. As a result we used de-identified 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. Lastly, the variability of fatty acid determinations in sperm, reflected in high CVs, could also 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 inversely related to sperm concentration. Our data is in agreement with experimental data in rodents showing that trans fatty acids can profoundly affect spermatogenesis. 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 are re-evaluated in larger, better designed studies and that the relation between intake of trans fats and sperm levels of these fatty acids is closely examined.

Acknowledgments

Financial Support:

Supported by National Institute of Environmental Health Sciences (NIEHS) grants ES009718 and ES00002, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) grant P30 DK46200-18 and departmental start-up funds to JEC.

Results from this manuscript were presented in part at the 65th Annual Meeting of the American Society of Reproductive Medicine, Atlanta, GA, October 17–21, 2009.

Abbreviations

DPA

docosapentaenoic acid

DHA

docosahexaenoic acid

MUFA

mono-unsaturated fatty acid

PUFA

poly-unsaturated fatty acid

SFA

Saturated fatty acid

CI

confidence interval

SD

standard deviation

Footnotes

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