Summary
The endocrine disrupting properties of paracetamol have been previously demonstrated in rodent students of abnormal sperm morphology and diminished testosterone production; in addition to epidemiologic studies of diminished couple fecundity. In this study, we examined the relationship between paracetamol and its metabolite p-aminophenol quantified in a single spot urine and semen quality among 501 male partners of couples planning for pregnancy. Men provided a urine specimen and two fresh semen samples collected approximately one month apart and underwent 24-hour analysis for 35 semen quality parameters. Paracetamol and p-aminophenol were quantified in urine by ultra-high performance liquid chromatography coupled with an electrospray triple quadrupole mass spectrometry. The relationship between natural log transformed urinary paracetamol and p-aminophenol rescaled by their standard deviation and 21 Box-Cox transformed, 14 non-transformed semen parameters was assessed using linear mixed-effects models. The median concentrations (IQR) of urinary paracetamol and p-aminophenol were 15.5 ng/mL (5.44, 73.5) and 978 ng/mL (500, 1596), respectively. Following adjustment for creatinine and age, a 1-standard deviation increase in log-transformed urinary paracetamol was associated with a reduction in beat cross frequency and an increase in DNA fragmentation[ β(95% CI): −0.59 Hz (−1.16, −0.03) and 0.05 % (0.01, 0.09), respectively]. These findings were corroborated in models of categorical chemical concentrations; higher concentrations of paracetamol remained associated with reduced beat cross frequency and increased DNA fragmentation. A 1-standard deviation increase in log-transformed urinary p-aminophenol was associated with a reduction in sperm head area [β (95% CI): −0.1 (−0.18, −0.02) and width −0.02 (−0.04, −0.01)]. However, only the association with sperm head area remained statistically significant in models of p-aminophenol quartiles. Our findings suggest that adult male urinary paracetamol is associated with sperm motility and DNA fragmentation, while the metabolite, p-aminophenol, is predominantly associated with sperm head morphometry.
Introduction
Paracetamol, also known as acetaminophen, is found in a large number of over-the-counter (OTC) and prescription drugs given its antipyretic and analgesic properties (Lank 2014). Having a short biologic half-life, 85%-95% of the chemical is excreted by the kidneys within 24 hours post-ingestion exposure (Forrest et al. 1982). Both paracetamol and its primary metabolite, p-aminophenol, are also major metabolites of the environmentally ubiquitous chemical aniline. A chemical used in the synthesis and manufacturing of many industrial products (e.g., dyes (indigo), polyurethane foam) (Agency for Toxic Substances and Disease Registry 2011), aniline is rapidly metabolized and excreted as both p-aminophenol and paracetamol. Biomonitoring data of Danish and German populations suggest sustained levels of paracetamol in the body as the result of aniline exposure (Dierkes et al. 2014; Modick et al. 2014). Common use of paracetamol/acetaminophen in conjunction with the omnipresence of environmental aniline may result in passive and possibly continuous exposure and low-level concentrations in the body (Holm et al. 2015).
The endocrine disrupting properties of paracetamol have been previously demonstrated in rodent fetal studies of increased percentage of abnormal sperm head, reduced sperm count and impaired motility (Ekaluo et al. 2008; Ratnasooriya & Jayakody 2000); reduced anogential distance and decreased testosterone production (Kristensen et al. 2011; Kristensen et al. 2012; Thiele et al. 2013), and epidemiologic research focusing on environmentally relevant concentrations of in utero paracetamol exposure and male reproductive disorders (Jensen et al. 2010; Philippat et al. 2011; Snijder et al. 2012). Moreover, a previous in vitro study with adult human testis cells reported a reduction in testosterone secretion by 18% and 30%, after 24 hours, in response to paracetamol exposure at 10−5 and 10−4 molar concentrations, respectively (Albert et al. 2013). The implications of changes in testosterone levels in the adult male on semen quality (e.g. sperm count) and overall fertility have long been established. In a previous analysis of the Longitudinal Investigation of Fertility and the Environment (LIFE) Study cohort, we found higher concentrations of male urinary paracetamol to be associated with a longer time-to-pregnancy for couples (Smarr et al. 2016). Nonetheless, epidemiologic research of adult male paracetamol exposure and male reproductive health are scarce. Therefore, we examined the relationship between paracetamol and p-aminophenol quantified in urine and semen quality among 501 male partners of couples planning for pregnancy.
Materials and Methods
Study population and participants
Male partners were recruited for participation in the LIFE Study, a prospective cohort of 501 reproductive aged couples from16 counties in Michigan and Texas between 2005 and 2009 who were recruited upon discontinuing contraception for ≤ 2 months prior to enrollment for purposes of becoming pregnant (Buck Louis et al. 2011). Inclusion criteria were: male partners needed to be ≥18 years of age; in a committed relationship; no physician diagnosis of sterility; and an ability to communicate in English or Spanish. Institutional review board approvals were obtained from all collaborating institutions; couples gave written informed consent prior to study participation and any data collection.
Data and Biospecimen collection
Upon enrollment, in-person interviews were conducted with male partners to ascertain lifestyle and reproductive history followed by standard anthropometric assessments to measure body mass index (BMI) (Lohman et al. 1988). During the enrollment home visit, male partners provided a nonfasting baseline urine sample. Additionally,, male partners provided a baseline semen sample, followed by a second semen sample, approximately 1 month apart. Following a 2-day abstinence period after enrollment, males obtained specimens via masturbation, without the use of lubricants, using at-home collection kits (Royster et al. 2000). Collection kits included a glass collection jar with an attached button thermometer to monitor temperature every half hour throughout the process, a glass sperm migration straw (Vitrotubes 3520; VitroCom Inc., Mountain Lakes, NJ) containing hyaluronic acid and plugged at one end, and cold packing materials for shipping. Male partners were instructed to collect the semen sample in the jar, place the sperm migration straw into the jar (as an exploratory marker of sperm motility and viability at the time of collection), and to record on the label the time of last ejaculation and any spillage. Specimens were shipped in insulated shipping containers containing ice packs overnight via Federal Express to the andrology laboratory at the National Institute for Occupational Safety and Health (Cincinnati, OH). Men were instructed to call a toll-free hotline to report the shipment of their semen samples. Semen delivered to a central andrology laboratory by overnight mail in insulated mailing kits have been successful in maintaining specimens for other studies (Luben et al. 2007; Olshan et al. 2007; Royster et al. 2000).
Semen analysis
Following overnight delivery of semen samples, the 24-hour semen analysis comprised inspecting sample integrity and that shipment temperatures were within established ranges (via temperature logging monitor, Maxim Integrated, San Jose, CA, USA), along with general characteristics including turbidity, color, liquefaction, and volume. Samples were then warmed to 37°C and volume was measured to the nearest 0.1 cc. Established laboratory protocols that included ongoing quality assurance and control procedures (American Society of Andrology, 1996). Semen analysis after home collection has been reported to be reliable for all semen parameters with the exception of motility parameters (Stovall et al. 1994). A percentage of sperm are alive after 24 hours and a next-day motility assessment still can be made and may provide important information on sperm function and survivability (Stovall et al. 1994). A total of 35 semen parameters (5 general characteristics: sperm concentration, volume, total count, straw distance, hypo-osmotic swollen; 8 motility measures; 6 sperm head measures; 12 individual and 2 summary morphology measures; and 2 sperm chromatin stability measures)were quantified.. Six parameters are derived from other parameters. Specifically, sperm concentration is equal to total sperm count divided by volume; sperm head area, perimeter, and elongation factor are functions of sperm head length and width; percent linearity is a function of the straight-line and curvilinear velocity; percent straightness is a function of straight-line and average path velocity.
Sperm concentration was assessed using the IVOS system and the IDENT™ stain (all from Hamilton Thorne Biosciences, Beverly, MA)(Zinaman et al. 1996). A migration straw was used so the lab could microscopically assess the distance the vanguard sperm traveled to the nearest millimeter, which indicated sperm motility at the time of collection, in light of using next-day analysis (Turner & Schrader 2006) Sperm viability was determined by hypo-osmotic swelling (HOS assay)(Jeyendran et al. 1992; Schrader et al. 1990) and sperm motility was assessed using the HTM-IVOS computer assisted semen analysis system (CASA) microscope slides were prepared for sperm morphometry. Morphology was assessed via slides prepared by Fertility Solutions® (Cleveland, OH) and was assessed using both traditional (World Health Organization 1992) and strict (Rothmann et al. 2013) classifications. Sperm morphometry was conducted using the IVOS METRIX system (Hamilton Thorne Biosciences). One aliquot of whole semen was diluted in TNE buffer and frozen for the sperm chromatin stability assay (SCSA) (Evenson et al. 2002). SCSA® analysis was conducted by SCSA Diagnostics (Brookings, SD) using a Coulter Epics Elite Flow Cytometer (Coulter, Miami, FL). The SCSA® assay measures sperm DNA damage, which is then quantified as the percentage of separated or damaged DNA (DNA fragmentation index; DFI) and the percentage of highly immature sperm nuclei with abnormal proteins (high stainability)(Evenson 2013).
The second semen sample was assessed to corroborate azoospermia observed in the first sample; in the event of such an observation males were advised to seek clinical care. An abbreviated semen analysis was performed on the second sample (i.e., volume, concentration, next-day motility, and sperm head morphology).
Urinary analysis of chemicals
Chemical analysis of total urinary paracetamol and p-aminophenol was performed at the Wadsworth Center, New York State Department of Health, Albany, NY. Specifically, 300 µL of 1 M ammonium acetate containing 30 U of β-glucuronidase (pH=5.5) was added to 500 µL of urine sample, followed by incubation at 37°C for 12 h. Target analytes were extracted thrice with ethyl acetate and were quantified by ultra-high performance liquid chromatography (Acquity I Class; Waters, Milford, MA) coupled with an electrospray triple quadrupole mass spectrometry (API 5500; AB SCIEX, Framingham, MA) (UPLC-ESI-MS/MS). Separation of target analytes was carried by a Kinetex C18 (1.3 µ, 100A, 50 × 2.1 mm) column (Phenomenex; Torrance, CA) with a SecurityGuard guard column (Phenomenex) with positive ionization, multiple reaction monitoring mode of detection. Quality assurance and quality control parameters included procedural blanks, matrix spikes and duplicate analysis of samples. Labelled internal standards (d7-p-aminophenol and 13C2-15N- paracetamol) were spiked into all samples and quantification was by isotope dilution. The method limits of quantitation (LOQ) for p-aminophenol and paracetamol were 0.25 and 0.5 ng/mL, respectively.
Statistical analysis
Five (1%) men were azoospermic on both samples and excluded from analysis. Univariate analyses assessed the distributions of paracetamol, p-aminophenol and relevant covariates. Male lifestyle characteristics were compared by quartiles of urinary paracetamol using Chi-Square and Wilcoxon non-parametric tests for categorical and continuous covariates. Median and accompanying interquartile ranges (IQRs) of baseline urinary paracetamol concentrations (ng/mL) were calculated; medians were compared between males who did/did not provide a semen sample using Wilcoxon-Mann-Whitney tests.
To avoid biasing regression estimates, instrument-derived values paracetamol were used in all models with urinary creatinine included as a covariate; urinary concentrations below the LOQ were not substituted in any manner (Lubin et al. 2004; Richardson & Ciampi 2003; Schisterman et al. 2006). Urinary creatinine, paracetamol, and p-aminophenol concentrations were natural-log transformed (ln) to normalize distributions; paracetamol and p-aminophenol concentrations were also rescaled by their standard deviations for a meaningful interpretation of regression estimates. Furthermore, 21 of the 35 semen parameters underwent Box-Cox transformations to approach approximately normal distributions prior to regression analyses. Specifically, 14 semen end points required ln transformation; 6 underwent cubic root transformation; and 1 end point, % strict criteria, was required a seventh root transformation.
In linear mixed effects models, accounting for dependence between the two semen samples, urinary concentrations of paracetamol were modeled individually for each semen parameter and adjusted based on a priori selection of covariates: non-time varying age (years) and urinary creatinine (ng/mL). To assess potential non-linear associations, models were performed with both exposures categorized into quartiles. Wald tests were performed to test for nonlinear trend across quartiles of chemical concentrations. Linear and quartile models of summed concentrations of paracetamol and p-aminophenol were also performed given their low correlation (Spearman’s r2 = 0.09, p=0.08), which may be indicative of variation in exposure source. All analyses were performed using SAS (version 9.4; SAS Institute Inc., Cary, NC).
Results
Semen samples were obtained for 468 (93%) non-azoospermic males, urinary chemicals were quantified for 439 (88%); for a total of 414 (83%) male partners with semen and paracetamol data. The average age and body mass index of male partners was 31.8 (SD=4.8) years and 30 (SD=5.7) kg/m2, respectively (Table 1). Males in the present analysis were predominantly overweight, non-Hispanic whites, between 30–40 years of age, with household incomes between $50,000 and $99,000. Socio-demographics of male partners did not tend to differ by quartiles of urinary paracetamol and p-aminophenol concentrations; comparisons by paracetamol quartiles are presented in Table 1. Furthermore, males who did not provide a semen sample were similar to those included in this analysis.
Table 1.
Characteristics of non-azoospermic male partners (n=468).
| Characteristic | n (%) |
|---|---|
| Age categories | |
| <30 | 166 (36) |
| 30–40 | 282 (60) |
| 40+ | 20 (4) |
| Age (years), mean ± SD | 31.8 ± 4.8 |
| BMI categories | |
| Under/healthy (BMI < 25) | 80 (17) |
| Overweight (25 <= BMI < 30) | 191 (41) |
| Obese (30 <= BMI <35) | 121 (26) |
| Morbidly Obese (BMI >= 35) | 71 (15) |
| BMI (kg/m2), mean ± SD | 30.0 ± 5.6 |
| Race/ Ethnicity | |
| Non-Hispanic White | 378 (81) |
| Non-Hispanic Black | 20 (4) |
| Hispanic | 38 (8) |
| Other | 30 (6) |
| Income | |
| < $50,000 | 68 (15) |
| $50,000 - $99,000 | 226 (49) |
| at least $100,000 | 165 (36) |
| Abstinence time (days), mean ± SD | 4.0 ± 4.5 |
| Conditional Parity | |
| No pregnancies | 202 (43) |
| Never fathered a pregnancy | 39 (8) |
| Fathered a pregnancy | 225 (48) |
Data presented are from study baseline. Missing data are not reflected in the table.
The distributions s of urinary paracetamol and p-aminophenol for male partners are displayed in Table 2. Overall, paracetamol and p-aminophenol were readily detected in male urine (93% and 100%, respectively).
Table 2.
Distribution of urinary paracetamol and p-aminophenol
| n | % >LOD | 5th | 25th | 50th | 75th | 95th | |
|---|---|---|---|---|---|---|---|
| Paracetamol (ng/mL) | 414 | 93 | 0.23 | 5.45 | 16.03 | 75.18 | 54959 |
| p-aminophenol (ng/mL) | 414 | 100 | 128 | 493 | 974 | 1605 | 4011 |
Significant associations between continuous and quartile models of urinary paracetamol and semen parameters are presented in Table 3. In crude models, increasing urinary paracetamol concentration was associated with average path velocity (µm/sec), straight-line velocity (µm/sec), curvilinear velocity (µm/sec), beat cross frequency (Hz), straightness (%), and % linear movement. Following adjustment for creatinine and age, only the association with beat cross frequency (Hz) β= −0.59 (95% CI: −1.16, −0.03) remained. Additionally, a 1-SD increase in log-transformed urinary paracetamol was associated with a 0.05% increase in DNA fragmentation (95% CI: 0.01, 0.09). In non-linear models, the second quartile (5.45 – 15.96 ng/mL) of male urinary paracetamol was associated with reduced volume β= −0.19 ml, (95% CI: −0.35, −0.03) and also increased sperm count (* 10^6/ml) β= 0.87 (95% CI: 0.29, 1.46) compared with the lowest quartile (<5.44 ng/ml) after adjustment for age and creatinine. Urinary paracetamol concentrations in the third versus first quartile (16.1 – 74.6 ng/mL) were associated with reductions in beat cross frequency β= −2.13 Hz, (95% CI: −3.74, −0.52), straight motility β=−5.33 % (95% CI: −9.68, −0.99) and % micro head β= −0.16 (95% CI: −0.3, −0.03). Urinary concentrations of paracetamol in the fourth versus first quartile were associated with increased DNA fragmentation β= 0.15 % (95% CI: 0.03, 0.27) (Table 3). Associations between urinary paracetamol concentrations and all 35 semen end points, irrespective of statistical significance are reported in Supplemental Table 1.
Table 3.
Associations between urinary paracetamol and semen parameters a
| Semen parameter by paracetamol category b | Unadjusted | Adjusted c |
|---|---|---|
| β (95% CI) | β (95% CI) | |
| General characteristics | ||
| Volume (ml) | −0.05 (−0.10, 0.00) | −0.04 (−0.10, 0.02) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.21 (−0.36, −0.06) | −0.19 (−0.35, −0.03) |
| 3rd quartile | −0.16 (−0.31, −0.01) | −0.12 (−0.27, 0.04) |
| 4th quartile | −0.16 (−0.31, −0.01) | −0.14 (−0.30, 0.02) |
| p-for linear trend | 0.08 | 0.04 |
| Sperm Count (* 10^6/ml) | −0.09 (−0.29, 0.11) | −0.03 (−0.24, 0.18) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 1.04 (0.48, 1.6) | 0.87 (0.29, 1.46) |
| 3rd quartile | −0.03 (−0.59, 0.54) | 0.03 (−0.56, 0.61) |
| 4th quartile | 0.08 (−0.48, 0.64) | 0.10 (−0.50, 0.69) |
| p-for linear trend | 0.36 | 0.01 |
| Motility | ||
| Average path velocity (µm/sec) | −1.13 (−2.07, −0.19) | −0.84 (−1.81, 0.13) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 0.10 (−2.58, 2.77) | 0.38 (−2.39, 3.15) |
| 3rd quartile | −1.11 (−3.8, 1.58) | −0.48 (−3.27, 2.3) |
| 4th quartile | −2.04 (−4.72, 0.63) | −1.73 (−4.54, 1.08) |
| p-for linear trend | 0.09 | 0.59 |
| Straight-line velocity (µm/sec) | −0.79 (−1.56, −0.01) | −0.53 (−1.34, 0.28) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.25 (−2.47, 1.97) | −0.09 (−2.39, 2.21) |
| 3rd quartile | −1.23 (−3.46, 1.01) | −0.75 (−3.07, 1.56) |
| 4th quartile | −1.38 (−3.6, 0.84) | −1.01 (−3.35, 1.32) |
| p-for linear trend | 0.15 | 1.0 |
| Curvilinear velocity (µm/sec) | −2.02 (−3.63, −0.4) | −1.61 (−3.29, 0.07) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.01 (−4.62, 4.6) | 0.46 (−4.31, 5.23) |
| 3rd quartile | −2.43 (−7.07, 2.21) | −1.61 (−6.4, 3.19) |
| 4th quartile | −4.09 (−8.71, 0.53) | −3.9 (−8.74, 0.94) |
| p-for linear trend | 0.05 | 0.63 |
| Beat cross frequency (Hz) | −0.76 (−1.31, −0.22) | −0.59 (−1.16, −0.03) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.48 (−2.03, 1.06) | −0.19 (−1.79, 1.42) |
| 3rd quartile | −2.27 (−3.82, −0.71) | −2.13 (−3.74, −0.52) |
| 4th quartile | −1.72 (−3.26, −0.17) | −1.53 (−3.16, 0.09) |
| p-for linear trend | 0.01 | 0.65 |
| Straightness (% ) | −1.99 (−3.46, −0.53) | −1.46 (−2.98, 0.06) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −2.27 (−6.45, 1.90) | −1.76 (−6.09, 2.56) |
| 3rd quartile | −6.12 (−10.32, −1.92) | −5.33 (−9.68, −0.99) |
| 4th quartile | −4.13 (−8.31, 0.05) | −3.38 (−7.77, 1.01) |
| p-for linear trend | 0.02 | 0.30 |
| Linearity (% ) | −1.03 (−2.02, −0.05) | −0.64 (−1.67, 0.39) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.90 (−3.71, 1.91) | −0.62 (−3.55, 2.31) |
| 3rd quartile | −2.99 (−5.82, −0.16) | −2.35 (−5.29, 0.59) |
| 4th quartile | −1.85 (−4.66, 0.96) | −1.16 (−4.13, 1.81) |
| p-for linear trend | 0.09 | 0.52 |
| Morphometry | ||
| Megalo head (% ) | 0.03 (−0.02, 0.08) | −0.02 (−0.09, 0.04) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 0.07 (−0.07, 0.21) | 0.05 (−0.09, 0.19) |
| 3rd quartile | 0.08 (−0.07, 0.22) | 0.07 (−0.08, 0.22) |
| 4th quartile | 0.16 (0.02, 0.30) | 0.13 (−0.02, 0.28) |
| p-for linear trend | 0.04 | 0.70 |
| Micro head (% ) | −0.03 (−0.07, 0.02) | 0.03 (−0.02, 0.08) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.05 (−0.17, 0.08) | −0.04 (−0.18, 0.09) |
| 3rd quartile | −0.13 (−0.26, 0.00) d | −0.16 (−0.3, −0.03) |
| 4th quartile | −0.12 (−0.25, 0.01) | −0.13 (−0.26, 0.01) |
| p-for linear trend | 0.03 | 0.43 |
| Sperm chromatin stability | ||
| % DNA Fragmentation | 0.03 (−0.01, 0.07) | 0.05 (0.01, 0.09) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 0.05 (−0.07, 0.16) | 0.04 (−0.08, 0.16) |
| 3rd quartile | 0.05 (−0.06, 0.16) | 0.07 (−0.05, 0.19) |
| 4th quartile | 0.10 (−0.01, 0.21) | 0.15 (0.03, 0.27) |
| p-for linear trend | 0.09 | 0.85 |
Note: Bolded effect estimates and 95% CIs indicate statistical significance.
Restricted to parameters with a significant association in unadjusted and/or adjusted models.
Effect estimates and 95% CIs are presented for regression models of continuous log-transformed paracetamol concentration, unless specified otherwise. Paracetamol quartiles: 1st ( < 5.44 ng/mL, n=103); 2nd (5.45 – 15.96 ng/mL, n=104); 3rd (16.09 – 74.62 ng/mL, n=103) and 4th (≥75.18 ng/mL, n=104).
Models were adjusted for log-transformed creatinine and age (years).
Statistically significant prior to rounding.
Few significant associations were observed between urinary p-aminophenol concentrations and semen parameters (Table 4). Prior to model adjustment for confounders, a 1-SD increase in log-transformed p-aminophenol concentration was associated with reductions in sperm head [area: β = −0.08 µm2 (95% CI: −0.14, −0.02), width: β = −0.01 µm (95% CI: −0.03, −0.002) and perimeter: β = −0.04 µm (95% CI: −0.08, −0.002). Following adjustment for age and urinary creatinine, the significant associations for sperm head area and sperm head width remained [β = −0.10 µm2 (95% CI: −0.18, −0.02) and β = −0.02 µm (95% CI: −0.04, −0.01), respectively]. When urinary p-aminophenol concentrations were modeled as quartiles, the second (493 – 969 ng/mL) versus first (<491 ng/mL) quartile was associated with increased average path velocity β = 2.93 µm/sec (95% CI: 0.09, 5.78) and straight-line velocity β = 2.39 µm/sec (95% CI: 0.04, 4.75) after adjustment for age and creatinine. Positive associations were observed between the 2nd and 3rd quartiles of p-aminophenol and linear motility [β = 3.82 % (95% CI: 0.83, 6.82) and β = 3.19 % (95% CI: 0.14, 6.24), respectively]. Negative associations were observed between the 4th versus 1st quartile and area of sperm head (β = −0.22 µm2 (95% CI: −0.43, −0.02)), in adjusted models. Associations between urinary p-aminophenol concentrations and all 35 semen end points, irrespective of statistical significance are reported in Supplemental Table 1.
Table 4.
Associations between urinary p-aminophenol and semen parameters a
| Semen parameter by p-aminophenol category b | Unadjusted | Adjusted c |
|---|---|---|
| β (95% CI) | β (95% CI) | |
| General characteristics | ||
| Total count (* 10^6 /ml concentration x volume) | −0.22 (−0.52, 0.09) | −0.16 (−0.54, 0.23) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.42 (−1.3, 0.45) | −0.12 (−1.06, 0.81) |
| 3rd quartile | 0.05 (−0.82, 0.92) | 0.33 (−0.63, 1.29) |
| 4th quartile | −1.03 (−1.9, −0.16) | −0.89 (−1.85, 0.07) |
| p-for linear trend | 0.06 | 0.58 |
| Motility | ||
| Average path velocity (µm/sec) | 0.16 (−0.78, 1.10) | 0.57 (−0.59, 1.73) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 2.22 (−0.47, 4.91) | 2.93 (0.09, 5.78) |
| 3rd quartile | 1.81 (−0.88, 4.49) | 2.60 (−0.29, 5.49) |
| 4th quartile | 0.55 (−2.12, 3.22) | 1.01 (−1.9, 3.92) |
| p-for linear trend | 0.77 | 0.02 |
| Straight-line velocity (µm/sec) | −0.05 (−0.83, 0.73) | 0.26 (−0.7, 1.22) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 1.75 (−0.48, 3.98) | 2.39 (0.04, 4.75) |
| 3rd quartile | 1.24 (−0.99, 3.46) | 1.91 (−0.49, 4.31) |
| 4th quartile | −0.02 (−2.23, 2.20) | 0.27 (−2.14, 2.68) |
| p-for linear trend | 0.87 | 0.02 |
| Linearity (% ) | −0.08 (−1.07, 0.90) | 0.50 (−0.72, 1.73) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 2.71 (−0.11, 5.53) | 3.82 (0.83, 6.82) |
| 3rd quartile | 1.82 (−0.99, 4.64) | 3.19 (0.14, 6.24) |
| 4th quartile | −0.30 (−3.11, 2.50) | 0.58 (−2.48, 3.65) |
| p-for linear trend | 0.69 | 0.00 |
| Morphometry | ||
| Sperm head area (µm2) | −0.08 (−0.14, −0.02) | −0.10 (−0.18, −0.02) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.13 (−0.31, 0.06) | −0.13 (−0.33, 0.07) |
| 3rd quartile | −0.08 (−0.27, 0.1) | −0.10 (−0.3, 0.10) |
| 4th quartile | −0.26 (−0.44, −0.07) | −0.22 (−0.43, −0.02) |
| p-for linear trend | 0.01 | 0.41 |
| Sperm head width (µm) | −0.01 (−0.03, 0.00) d | −0.02 (−0.04, −0.01) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.03 (−0.06, 0.01) | −0.02 (−0.06, 0.02) |
| 3rd quartile | 0.00 (−0.03, 0.04) | 0.00 (−0.04, 0.04) |
| 4th quartile | −0.04 (−0.08, −0.01) | −0.04 (−0.08, 0.00) |
| p-for linear trend | 0.09 | 0.54 |
| Sperm head perimeter (µm) | −0.04 (−0.08, 0.00) d | −0.04 (−0.09, 0.00) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.04 (−0.15, 0.06) | −0.05 (−0.17, 0.06) |
| 3rd quartile | −0.06 (−0.17, 0.05) | −0.06 (−0.18, 0.05) |
| 4th quartile | −0.13 (−0.23, −0.02) | −0.10 (−0.22, 0.01) |
| p-for linear trend | 0.02 | 0.51 |
Note: Bolded effect estimates and 95% CIs indicate statistical significance.
Restricted to parameters with a significant association in unadjusted and/or adjusted models.
Effect estimates and 95% CIs are presented for regression models of continuous log-transformed p-aminophenol concentration, unless specified otherwise. p-aminophenol quartiles : 1st ( < 491 ng/mL, n=103); 2nd (492 – 969 ng/mL, n=104); 3rd (978 – 1596 ng/mL, n=103) and 4th ( ≥ 1605 ng/mL, n=104).
Models were adjusted for log-transformed creatinine and age (years).
Statistically significant prior to rounding.
A significant association was only observed between 1-SD increase in log-transformed summed concentrations of urinary paracetamol and p-aminophenol and percent motility (β = −0.21 % (95% CI: −0.42, −0.01), prior to model adjustment; no other signals of association were observed for semen parameters, irrespective of linear model adjustment (Table 5). In models of the sum of urinary paracetamol metabolites categorized as quartiles, the third quartile (1114 – 2029 ng/mL) compared with the first (< 606 ng/mL) was negatively associated with amplitude of lateral head (µm), β = −0.37 (95% CI: −0.68, −0.06) and β = −0.43 (95% CI: −0.77, −0.09) in crude and adjusted models, respectively. Prior to, and after model adjustment, compared with the first quartile, the second quartile (606 – 1113 ng) was associated with a reduction in the percentage of other sperm tail abnormalities β = −0.20 (95% CI: −0.36, −0.05) and β = −0.22 (95% CI: −0.39, −0.05). Additionally, previous findings of increased average path velocity and percent linearity with the second quartile of p-aminophenol concentration (Table 4), were corroborated when paracetamol and p-aminophenol concentrations were summed. Specifically, the second versus first (<491 quartile was associated with increased average path velocity β = 2.87 µm/sec (95% CI: 0.07, 5.68) and percent linear motility β = 3.13 % (95% CI: 0.16, 6.09), in models adjusted for creatinine and age. Associations between the summed concentrations of urinary paracetamol and p-aminophenol and all 35 semen end points, irrespective of statistical significance are reported in Supplemental Table 2.
Table 5.
Associations between the sum of urinary paracetamol metabolites and semen parameters a
| Semen parameter by chemical category b | Unadjusted | Adjusted c |
|---|---|---|
| β (95% CI) | β (95% CI) | |
| General characteristics | ||
| Total count (* 10^6 /ml concentration x volume) | −0.28 (−0.59, 0.02) | −0.18 (−0.51, 0.15) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 0.21 (−0.66, 1.07) | 0.52 (−0.40, 1.45) |
| 3rd quartile | 0.32 (−0.55, 1.19) | 0.61 (−0.36, 1.57) |
| 4th quartile | 1.07 (−1.94, −0.21) | −0.67 (−1.63, 0.28) |
| p-for linear trend | 0.02 | 0.16 |
| Motility | ||
| Average path velocity (µm/sec) | −0.67 (−1.61, 0.27) | −0.41 (−1.41, 0.59) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 2.59 (−0.08, 5.25) | 2.87 (0.07, 5.68) |
| 3rd quartile | 0.18 (−2.49, 2.86) | 0.26 (−2.66, 3.19) |
| 4th quartile | −1.36 (−4.02, 1.29) | −0.52 (−3.41, 2.37) |
| p-for linear trend | 0.13 | 0.34 |
| Amplitude of lateral head (µm) | −0.10 (−0.21, 0.01) | −0.08 (−0.19, 0.04) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.06 (−0.37, 0.25) | −0.05 (−0.37, 0.28) |
| 3rd quartile | −0.37 (−0.68, −0.06) | −0.43 (−0.77, −0.09) |
| 4th quartile | −0.21 (−0.52, 0.10) | −0.20 (−0.54, 0.13) |
| p-for linear trend | 0.06 | 0.08 |
| Linearity (% ) | −0.60 (−1.60, 0.39) | −0.21 (−1.27, 0.85) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 2.26 (−0.54, 5.06) | 3.13 (0.16, 6.09) |
| 3rd quartile | 1.55 (−1.26, 4.37) | 2.50 (−0.59, 5.59) |
| 4th quartile | −1.55 (−4.34, 1.25) | 0.13 (−2.92, 3.18) |
| p-for linear trend | 0.23 | 0.87 |
| Percent Motility (% ) | −0.21 (−0.42, −0.01) | −0.15 (−0.36, 0.07) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | 0.07 (−0.51, 0.65) | 0.23 (−0.39, 0.84) |
| 3rd quartile | −0.13 (−0.72, 0.45) | 0.06 (−0.58, 0.70) |
| 4th quartile | −0.46 (−1.04, 0.12) | −0.15 (−0.78, 0.48) |
| p-for linear trend | 0.09 | 0.52 |
| Morphometry | ||
| Other tail abnormalities (%) | −0.01 (−0.06, 0.04) | 0.00 (−0.06, 0.06) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.20 (−0.36, −0.05) | −0.22 (−0.39, −0.05) |
| 3rd quartile | −0.07 (−0.23, 0.08) | −0.10 (−0.27, 0.07) |
| 4th quartile | −0.07 (−0.23, 0.08) | −0.07 (−0.24, 0.10) |
| p-for linear trend | 0.73 | 0.84 |
| Morphology | ||
| Taper (% ) | −0.01 (−0.07, 0.06) | −0.01 (−0.08, 0.06) |
| 1st quartile | Ref. | Ref. |
| 2nd quartile | −0.17 (−0.35, 0.02) | −0.20 (−0.4, −0.01) |
| 3rd quartile | 0.01 (−0.18, 0.19) | −0.06 (−0.26, 0.15) |
| 4th quartile | −0.03 (−0.22, 0.16) | −0.06 (−0.26, 0.14) |
| p-for linear trend | 0.79 | 0.96 |
Note: Bolded effect estimates and 95% CIs indicate statistical significance.
Restricted to parameters with a significant association in unadjusted and/or adjusted models.
Effect estimates and 95% CIs are presented for regression models of continuous log-transformed sum of urinary metabolites concentration, unless specified otherwise. Summed urinary paracetamol metabolites quartiles : 1st ( < 606 ng/mL, n=103); 2nd (606 – 1113 ng/mL, n=104); 3rd (1114 – 2029 ng/mL, n=103) and 4th ( ≥ 2083 ng/mL, n=104).
Models were adjusted for log-transformed creatinine and age (years).
Discussion
These are the first data known to us that have assessed preconception urinary concentrations of total paracetamol and its metabolite, p-aminophenol, in relation to semen quality that may have further implications for couple fertility. In this exploratory analysis of 35 semen parameters, a total of 11 (31%) semen parameters were associated with increasing paracetamol concentrations and 7 (20%) with increasing concentrations of its metabolite, p-aminophenol, in (un)adjusted linear and non-linear regression models. Overall, we found increasing concentrations of urinary paracetamol to be associated with reductions in sperm volume, motility, and sperm head morphometry and a slight increase in DNA fragmentation. Moreover, increasing concentrations of urinary p-aminophenol tended to be associated with reductions in sperm head morphometry; lower concentrations of p-aminophenol were associated with enhanced sperm motility. When summing urinary paracetamol metabolites, findings of enhanced sperm motility were corroborated. Specifically, increased average path velocity and percent linearity.
In the context of such novel findings, consideration of biologic plausibility is essential for interpretation. One possible explanation includes the possibility that the androgenic properties of paracetamol result in reduced testosterone levels, as reported in an in vitro analysis of analgesics and the adult human testis (Albert et al. 2013). At varying molar concentrations of paracetamol, testosterone secretion was reduced by 18%-30% after 24 hours; such changes in testosterone have implications for spermatogenesis given that high levels of intra-testicular testosterone are required for during this process (Dohle et al. 2003). Our findings corroborate such a relationship as the lowest quartile of urinary paracetamol was associated with increased sperm count.
Interpretation of our findings in relation to other reproductive age male populations is constrained by the lack of epidemiologic assessments of urinary paracetamol or p-aminophenol and semen quality. Still, the findings from the present analysis are strengthened by the home collection of two semen samples from male study participants for 24-hour semen analysis. Nonetheless, while we acknowledge that a sperm motility assessment can be made after 24 hours, we are also aware of the lack of evidence to support the reliability of sperm motility assessed after 24 hours (Stovall et al.1994). An additional strength of the present analysis is the biomarker quantification of paracetamol and its metabolite p-aminophenol that reduces the chance for misclassification on exposure. In LIFE, information on sources of potential paracetamol exposure (e.g., self-report of medication use, occupational or environmental exposures) was not collected. Furthermore, the abundance of paracetamol in contemporary environments beyond medication usage supports quantifying this exposure rather than relying on self -reported medication use to avoid bias. However, we readily acknowledge that a single spot urine collected at study baseline to estimate preconception exposures of paracetamol and its metabolite to be a potential limitation if they are not reflective of those during spermatogenesis. Nonetheless, the generalizability of our findings is supported by the high detection frequency of paracetamol (93%) in our cohort of men from Michigan and Texas, two geographically different states. Furthermore, the common use of paracetamol in medications (Lank 2014) in reproductive aged men and the assumed continuous, passive human exposure to aniline which is used in the manufacturing of various industrial products (Dierkes et al.2014) and rapidly metabolized into both paracetamol and p-aminophenol, speak to the clinical and public health relevance of such findings. Nevertheless, our findings need to be cautiously interpreted until corroborated by future research based on serial measurements of urinary paracetamol.
In conclusion, our findings demonstrate an association between urinary concentrations of paracetamol and metabolite, p-aminophenol and changes in semen quality among reproductive age male partners of couples trying for pregnancy. The widespread exposure and implications for couple fertility warrant future research to further elucidate mechanisms by which adult male paracetamol exposure affects semen quality.
Supplementary Material
Acknowledgements
Funding:
This research was supported by the National Institutes of Health, Intramural Research Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development (contracts N01-HD-3-3355; N01-HD-3-3356; NOH-HD-3-3358; HHSN27500001; HHSN27500006). M.M.S. was supported by the National Institutes of Health Intramural Research Training Award.
Footnotes
Conflict of interest
The authors have no conflicts of interest to declare.
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