Andrologia. 202000:e13720. wileyonlinelibrary.comjournaland 1 of 9 [622631]

Andrologia. 2020;00:e13720. wileyonlinelibrary.com/journal/and  |  1 of 9
https://doi.org/10.1111/and.13720
© 2020 Blackwell Verlag GmbH1 |  INTRODUCTION
Of late, infertility disorders in the male population have ascended
(Andersson et al., 2007; Travison, Araujo, O'Donnell, Kupelian, &
McKinlay, 2007). This is credited to myriad aetiological factors, in –
cluding lifestyle practices, genetics and an over-exposure to environ –
mental and industrial toxins. Together, these adversely affect semen quality and production, that is, an abnormal sperm morphology, re –
duced sperm count, motility and vitality (Fleming, Bean, Rudolph, & Hamilton, 1999; Pflieger, Schuppe, & Schill, 2004; Toft, Hagmar, Giwercman, & Bonde, 2004). Phthalate plasticisers are added during the manufacturing of the variety of plastic wares. These impart enhanced flexibility, durability and resistance to the final product. Phthalate esters such as di-n-butyl phthalate (DBP) and diethyl phthalate (DEP) are used in the production of perfumes and cologne, paints, insect repellent, medication coatings, hairspray, shampoo and medical devices (intravenous fluid bags, infusion tubing and di –
alysis bags) (Agency for Toxic Substances & Disease Registry, 1995;
David, McKee, Butala, Barter, & Kayser, 2001; Nässberger, Arbin, &
Ostelius, 1987).
Over the years, there has been a considerable increase in the
production of phthalate esters. It accounts for more than 60% of the total plasticisers produced globally (Sommer, 1985). Indiscriminate
use of phthalates and their environmental persistence is a serious
Received: 26 March 2020  | Revised: 24 May 2020  | Accepted: 28 May 2020
DOI: 10.1111/and.13720
ORIGINAL ARTICLE
A study on the effect of phthalate esters and their metabolites
on idiopathic infertile males
Rashmi Rana1 | Shikha Joon1 | Arun Kumar Jain2 | Nayan Kumar Mohanty3
1Department of Research, Sir Ganga Ram
Hospital, New Delhi, India
2National Institute of Pathology, ICMR,
New Delhi, India
3Department of Urology, Safdarjang
Hospital and VMMC, New Delhi, India
Correspondence
Rashmi Rana, Department of Research,
Sir Ganga Ram Hospital, Delhi, India.Emails: [anonimizat]; [anonimizat]
Funding information
This study did not receive any specific
grant from funding agencies in the public,
commercial or not-for-profit sectors.Abstract
Phthalate plasticisers in medical, cosmetic and consumer products might pose seri –
ous health implications in humans including infertility. We sought to investigate the
correlation, if any, between the phthalates and their metabolites and sperm qual –
ity parameters, and male infertility. Phthalate esters (15) and their metabolites (5)
were estimated in the blood serum and urine samples from the age-matched 152
infertile and 75 fertile males using gas chromatography (GC) and high-performance
liquid chromatography (HPLC). Finally, the data were analysed to correlate phthalate exposure and semen quality parameters in the infertility group. The estimated levels
of DEHP, DBP, DIBP, BEHIP, BPBG, DPP, DIOP, DIHP, DMP, DINP, BIOP, DMOP and
DICHP were significantly higher in the infertile males compared to the fertile males (p < .05 or p < .01). However, these were not found to be associated with the semen
quality parameters (sperm count, motility and sperm morphology). Similarly, HPLC data revealed that the associations between semen parameters (sperm count, sperm motility and sperm morphology) and phthalate metabolite (MEHP and MBP) con –
centrations in urine samples from the infertile males were mostly unremarkable or statistically nonsignificant. Conclusively, environmental exposure to phthalates and their impacts on male infertility were statistically insignificant in our study groups.
KEYWORDS
blood serum and urine, gas chromatography, high-performance liquid chromatography,
idiopathic infertile males, phthalate esters and metabolites

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health concern in humans (Agency for Toxic Substances & Disease
Registry, 1995, 1997, 2001, 2002; Centers for Disease Control & Prevention, 2003). These find their way to the human system through various routes and are detected at considerably high concentrations in the biological fluids (Brotons, Olea-Serrano, Villalobos, Pedraza, & Olea, 1995; Giust, Seipelt, Anderson, Deis, & Hinders, 1990; Kataoka, Ise, & Narimatsu, 2002; Khaliq, Alam, & Srivastava, 1995; Leibowitz, Sarmiento, Sugar, & Ethridge, 1995; Rastogi, 1998). Phthalate esters such as di-(2-ethylhexyl) phthal –
ate (DEHP), dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), benzyl butyl phthalate (BBP) and di-n-octyl phthalate (DOP) are potential carcinogens. Additionally, these might lead to testicular toxicity and endocrine dysfunc –
tion (Harris, Henttu, Parker, & Sumpter, 1997; Jobling, Reynolds, White, Parker, & Sumpter, 1995; Kamendulis et al., 2002). In ani –
mals, toxicological studies have shown that some phthalates cause detrimental effects on their reproductive systems. In 2000, Colon et al reported that there is a probable association between phthal –
ates and premature breast development in the female population (Colón, Caro, Bourdony, & Rosario, 2000). During 1999–2000, the National Health and Nutrition Examination Survey (NHANES) found detectable levels of phthalate metabolites in more than 75% of the urine samples of the individuals aged>/equal to 6 years in
the United States. These included monoethyl phthalate (MEP), monobutyl phthalate (MBP), monobenzyl phthalate (MBzP) and mono-(2-ethylhexyl) phthalate (MEHP) respectively (Centers for Disease Control & Prevention, 2005; Silva et al., 2004a).
Despite the fact that male infertility is an ominous issue, little
attention has been paid to it. Duty et al reported that there is an association between the semen quality and the phthalate metabo –
lites detected in the urine samples of the infertile male population in the United States (Duty et al., 2003). However, substantial evidence
on their association with the semen quality parameters in the infer –
tile male population in India is still lacking (Pant, Kumar, Upadhyay, Gupta, & Chaturvedi, 2014; Pant et al., 2003, 2008; Thurston
et al., 2016; Wang et al., 2015). The present study was designed to measure the phthalate esters and their metabolites in the biological samples (blood serum and urine) of the infertile males and the fertile control males (healthy individuals) and to investigate the correlation, if any, between the phthalates and sperm quality parameters, and male infertility.
2 |  MATERIALS AND METHODS
2.1  | Chemicals and reagents
High-purity grade analytical standards of phthalate esters (DIHP, DMP, BPBG, DDIP, DIBP, DINP, DMOP, BEHIP, DPP, IBCHP, BIOP, DBP, DICHP, DIOP and DEHP) were obtained from Ultra Scientific Analytical Solutions. Chromatography grade solvents ( n-hexane,
acetone, methanol and acetonitrile) were purchased from Merck Limited. Routine solvents were purchased from Sigma-Aldrich. The phthalate metabolites, namely MEHP, MOP, MEP, MBzP and MBP, were purchased from AccuStandard Inc.
2.2 | Study population
The patients, who came for semen analysis as part of infertility treatment to Urology OPD of Safdarjang Hospital, New Delhi, India, were prospectively enrolled in this study. Patients in the reproduc –
tive age group (18 to 45 years) with no known cause of infertility
(idiopathic) were included as the study population. Patients with oc –
cupations involving the manufacturing or use of paint, ink or plastic were specifically included. The selected patients were azoospermia, oligozoospermia, asthenozoospermia, teratozoospermia and oligo-astheno-teratozoospermia. Patients with known causes of infertility, such as genital neoplasm, hydrocele, prostatic diseases, spermatic cord distortion, varicocele, hypopituitarism, hypogonadism, hae –
mospermia, aspermia obstruction and surgical, were excluded from the study. Considering the above selection criteria, 227 total sub –
jects (18 to 45 years, mean ± SD age, 30.38 ± 5.46 years) were taken
in the present study. Out of these, 152 cases of infertile males (18 to 45 years, mean ± SD age, 29.8 ± 5.54 years) were referred from the
Department of Urology (Andrology clinic), Safdarjang Hospital, New Delhi between April 2006 and November 2009. Seventy-five normal, healthy age-matched individuals (range: 18 to 45 years, mean ± SD
age, 31.57 ± 5.14 years) without a previous history of infertility, ge –
netic diseases or other disorders were recruited as ‘controls’ after a thorough clinical examination by the urologist. Specifically, these were having less than one-year-old children. Semen, blood and urine samples were obtained from the recruited subjects with their prior consent.
2.3 | Ethics statement
The study was approved by the Ethics Committee of the Safdarjang Hospital and Vardhman Mahavir Medical College (VMMC), New Delhi, India. Informed consent was obtained from all the participat –
ing patients and healthy donors. The details regarding the patient's age, occupation, physical activity, smoking, family history and part –
ner history (female) were also collected.
2.4 | Sample collection and analysis
Phthalate esters are found in common solvents, glassware and other laboratory equipment. All the samples were collected in phthalate-free propylene vials and stored in phthalate-free environment. Glassware was thoroughly cleansed and rinsed with distilled water and acetone before use. High-purity solvents, reagents and gases were used. Analytical blanks were performed before sample analysis to allow background subtraction whenever needed along with es –
sential quality control standards for data accuracy.

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2.4.1  | Semen
Semen samples were collected from the male patients, who mas –
turbated in the clinic premises. They were specifically instructed
to observe a brief period of sexual abstinence (2–5 days) before their study visit. The time of sampling was accurately recorded. The semen samples were collected in the sterilised glass vials. Samples were analysed within 30 min of collection, and semen quality pa –
rameters (sperm count, motility, morphology, vitality and viscosity) were examined (Stokes-Riner et al., 2007). The semen examinations
were performed according to the guidelines of the WHO (World Health Organization, 1999). All the necessary precautions were ob –
served while performing sample collection and analysis as described under ‘special precautions’. Analytical blanks were performed before sample analysis to allow background subtraction whenever needed along with essential quality control standards for data accuracy.
2.4.2 | Blood
Five ml of peripheral blood was collected from the infertile male pa –
tient ( n = 152) and healthy control individuals ( n = 75). The samples
were stored at −20°C until further use. Next, blood serum was sepa –
rated at 1,500 RPM. The phthalate esters were detected using gas chromatography (GC-2014 system, Shimadzu Corporation). Linear correlation coefficient ( R
2) for the phthalate metabolites was also
determined.
2.4.3 | Urine
Five phthalate metabolites, namely monoethyl-hexyl-phthalate (MEHP), monobutyl phthalate (MBP), monooctyl phthalate (MOP), monoethyl phthalate (MEP) and monobenzyl phthalate (MBzP), were detected in urine samples of both the infertile and the fer –
tile males by high-performance liquid chromatography (HPLC) as described elsewhere (Calafat, Ye, Wong, Reidy, & Needham, 2008; Kato, Hata, Banerjee, Futcher, & Zhang, 2004; Silva et al., 2004b; Silva et al., 2017). Briefly, 1 ml of the urine sample was buffered with
ammonium acetate (25 µl, pH 6.5) and sonicated for 5 min. The en –
zymatic deconjugation of the urine samples with a β -glucuronidase
enzyme (5 µl; 200 units/ml) allowed the release of the free phtha –
late monoesters from their glucuronidated form. Next, automated on-line solid-phase extraction was performed using solid-phase extraction cartridges (Oasis HLB, Waters, and Milford). These were pre-treated with various solvents and buffered aqueous solu –
tions. Further, 20 µl of the urine sample was injected into a high-performance liquid chromatography (Shimadzu LC −20 AD) column
(PrincetonSPHER-100 C18 100A, Betasil, 250 × 4.6 mm, Cranbury, NJ, U.S.A) loaded with PrincetonSPHER C18 100A 5u Guard car –
tridges (4 × 10 mm). The concentration of the individual standards in each sample was calculated using the calibration curve derived from the known standard mixtures. The concentrations of all the analytes were corrected for the concentrations of the same analytes present in the reagent blanks. The concentrations in the blanks in each run were averaged; the average blank concentration was subtracted from the concentration of each sample and the quality control. The limits of detection and the limits of quantification were in the ranges of 15–500 µg/ml and 87– 510 µg/ml respectively.
2.4.4  | Special precautions
Phthalate esters are ubiquitous in our environment, even in the labo –
ratory. They have been detected as interferences in solvents, glass –
ware and other laboratory equipment. For this reason, strict quality control procedures are required to assure the good quality of the data. Glassware was thoroughly cleansed and rinsed with distilled water and acetone before use. High-purity solvents, reagents and gases were used. Analytical blanks were performed prior to sample analysis to allow background subtraction whenever needed. These included solvents, GC system, SPE fibres and matrix blanks.
2.5 | Statistical analysis
The nonparametric Mann–Whitney U test was applied to test the
significant difference of phthalate ester concentrations between in –
fertile patients and controls. Karl Pearson's correlation was used to measure the correlation between phthalate esters and semen qual –
ity parameters (sperm motility, sperm count and sperm morphol –
ogy). A two-sided p ≤ .05 was considered statistically significant.
The data analysis was performed using SPSS 19.0 version software (IBM Corp.).
3 |  RESULTS
3.1  | Analysis of semen parameters
Five semen quality parameters (sperm count, motility, morphology, vitality and viscosity) were observed in both infertile ( n = 152) and
fertile men ( n = 75). These are summarised in Table 1. The major –
ity of the infertile men ( n = 85) had a low sperm count (<20 million/
mL), poor sperm motility ( n = 67 <30 million/mL) and vitality ( n = 78
<30 million/mL) as compared to the fertile healthy male controls (n = 75: sperm count > 20 million/mL; sperm motility < 30 million/
mL; sperm vitality < 30 million/mL). Other semen parameters such
as sperm morphology and viscosity were also impaired as compared to the healthy fertile controls.
3.2 | Detection of phthalate esters in blood samples
The GC detection of 15 pure phthalate compounds standards in pure solution included diisohexyl phthalate (DIHP) dimethyl phthalate

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(DMP), butyl phthalyl butyl glycolate (BPBG), diisodecyl phthalate
(DDIP), diisobutyl phthalate (DIBP), diisononyl phthalate (DINP), dimethoxy octyl phthalate (DMOP), bis(2-ethylhexyl) isophthalate (BEHIP), diphenyl phthalate (DPP), isobutyl cyclohexyl phthalate (IBCHP), butyl isooctyl phthalate (BIOP), dibutyl phthalate (DBP), dicyclohexyl phthalate (DICHP), dioctyl phthalate (DIOP) and dieth –
ylhexyl phthalate (DEHP). We found 13 phthalate compounds in the serum sample of an infertile male patient. For this, we had compared the area under the curve for each phthalate compound in the extract to the area under the curve for standards in a chromatogram. The Semen parametersInfertile
(n = 152) Fertile ( n = 75) p-value
Sperm Count (million/ml) Nil 50 (32.9%) 0 (0%) .001
<20 85 (55.9%) 0 (0%)
>20 17 (11.2%) 75 (100%)
Sperm Motility (million/
ml)Nil 50 (32.9%) 0 (0%) .001
<30 67 (44.1%) 0 (0%)
>30 35 (23.0%) 75 (100%)
Sperm morphology Abnormal 73 (48.0%) 0 (0%) .001
Normal 79 (51%) 75 (100%)
Sperm vitality (million/
ml)Nil 53 (34.9%) 0 (0%) .001
<30 78 (51.3%) 0 (0%)
>30 21 (13.8%) 75 (100%)
Sperm viscosity Low 52 (34.2%) 0 (0%) .001
Normal 75 (49%) 75 (100%)
High 25 (16.4%) 0 (0%)
Abbreviations: ‘ n’, number of subjects (fertile/infertile males); Nil, not detectable.TABLE 1 Analysis of semen
characteristics in fertile and infertile males
Phthalate estersInfertile male
n = 152Fertile (Control)n = 75
p-value Present Absent Present Absent
DIHP 9 143 0 75 .032
*
DMP 21 131 0 75 .001**
DDIP 8 144 1 74 .154
DIOP 19 133 0 75 .001**
IBCHP 4 148 1 74 .531
DINP 14 138 1 74 .025*
DIBP 23 129 0 75 <.001**
BIOP 26 126 2 73 .005*
DBP 73 79 2 73 <.001**
DMOP 18 134 2 73 .022*
DPP 36 116 0 75 <.001**
BPBG 30 122 0 75 <.001**
BEHIP 42 110 1 74 <.001**
DICHP 99 53 33 42 .002*
DEHP 47 105 3 72 <.001**
Note: Significance testing of the results using nonparametric Mann–Whitney U test. Significance level at 5% are denoted as significant (
*) and highly significant (**).
Abbreviations: ‘ n’, number of subjects (fertile/infertile males);BEHIP, bis(2-ethylhexyl) isophthalate;
BIOP, butyl isooctyl phthalate; BPBG, butyl phthalyl butyl glycolate; DBP, di- n-butyl phthalate;
DDIP, diisodecyl phthalate; DEHP, di-(2-ethylhexyl) phthalate; DIBP, diisobutyl phthalate; DICHP, dicyclohexyl phthalate; DIHP, diisohexyl phthalate; DINP, diisononyl phthalate; DIOP, dioctyl phthalate; DMOP, dimethoxy octyl phthalate; DMP, dimethyl phthalate; DPP, diphenyl phthalate; IBCHP, isobutyl cyclohexyl phthalate.TABLE 2 Association between male
infertility and phthalate esters detected in serum samples

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TABLE 3  Association between serum phthalates and semen quality parameters in infertile males
Phthalates DHIP DMP DDIP DIOP IBCHP DINP DIBP BIOP DBP DMOP DPP BPBG BEHIP DICHP DEHPSperm
countSperm motilitySperm morphology
DIHP 1.00 0.465
**0.066 0.495**−0.041 0.498**0.516**0.330**0.261**0.339**0.385**0.296**0.157 0.184*0.315**−0.037 −0.070 −0.019
DMP 1.00 0.503**0.713**0.053 0.598**0.576**0.375**0.264**0.384**0.360**0.376**0.222**0.133 0.392**−0.004 −0.044 0.013
DDIP 1.00 0.267**0.145 0.332**0.312**0.362**0.186*0.370**0.284**0.253**0.118 0.049 0.161*−0.051 −0.044 −0.028
DIOP 1.00 0.062 0.636**0.673**0.357**0.274**0.354**0.351**0.362**0.122 0.151 0.307**−0.0078 −0.073 −0.021
IBCHP 1.00 0.090 −0.069 0.034 −0.076 0.067 0.102 0.022 0.174*0.034 −0.021 −0.070 −0.083 0.034
DINP 1.00 0.627**0.218**0.286**0.517**0.358**0.414**0.210**0.185*0.378**−0.060 −0.041 −0.016
DIBP 1.00 0.296**0.292**0.413**0.413**0.390**0.150 0.232**0.433**−0.073 −0.057 −0.052
BIOP 1.00 0.333**0.536**0.322**0.170*0.110 0.186*0.263**−0.084 −0.120 −0.091
DBP 1.00 0.259**0.363**0.417**0.289**0.565**0.383**−0.052 −0.113 −0.094
DMOP 1.00 0.418**0.330**0.274**0.183*0.283**−0.020 −0.026 0.002
DPP 1.00 0.618**0.452**0.310**0.364**0.060 −0.045 −0.001
BPBG 1.00 0.470**0.328**0.384**0.003 −0.004 0.019
BEHIP 1.00 0.390**0.255**0.118 0.062 0.123
DICHP 1.00 0.370**0.035 −0.016 −0.029
DEHP 1.00 0.001 −0.080 −0.069
Sperm
Count1.00 0.750**0.729**
Sperm
Motility1.00 0.672**
Sperm
Morph1.00
Abbreviations: BEHIP, bis(2-ethylhexyl) isophthalate; BIOP, butyl isooctyl phthalate; BPBG, butyl phthalyl butyl glycolate; DBP, di- n-butyl phthalate; DDIP, diisodecyl phthalate; DEHP, di-(2-ethylhexyl)
phthalateDIBP, diisobutyl phthalate; DICHP, dicyclohexyl phthalate; DIHP, diisohexyl phthalate; DINP, diisononyl phthalate; DIOP, dioctyl phthalate; DMOP, dimethoxy octyl phthalate; DMP, dimethyl
phthalate; DPP, diphenyl phthalate; IBCHP, isobutyl cyclohexyl phthalate.
*Correlation is significant at the 0.05 level (2-tailed).
**Pearson correlation test (2-tailed); correlation is significant at the 0.01 level (2-tailed).

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difference in the mean ranks between infertile and fertile subjects
was statistically significant. Table 2 shows the qualitative differ –
ences in the detected serum phthalates in the infertile and fertile males. The p -value observed for 6 phthalate esters, that is DMP,
DIOP, DIBP, DBP, DPP, BPBG, BEHIP and DEHP, was highly sig –
nificant ( p-value ≤ 0.001) in the infertile male patients as compared
to the healthy controls. Others in the group, namely DIHP, DINP, BIOP, DMOP and DICHP, had significant p -values. DICHP (99/152
of infertile males) and DBP (73/152 of infertile males) were the most prominent amongst the phthalates detected in the serum samples of the infertile men. DDIP and IBCHP (4/152; 2.6%) were rare in the serum of the infertile males (Table 2). In our study, phthalate esters were not detected in the fertile men's serum, except DICHP (33/75 of fertile males) (Table 3). This is in agreement with some studies that reported the presence of some phthalate esters in the semen of the fertile males (Han et al., 2009; Pant et al., 2008). No statistically
significant correlation was observed with phthalate compounds and semen quality parameters in the infertile males (Table 3).
3.3 | Estimation of phthalate metabolites in
urine samples
Different concentrations (5, 10, 25 and 50 µg/ml) of phthalate me –
tabolites viz., mono ethylhexyl phthalate (MEHP) and monobutyl
phthalate (MBP) in urine samples were analysed by high-performance liquid chromatography (HPLC). The concentration of the individual standards in each urine sample was calculated using the calibration curve derived from the known standard mixtures. The concentra –
tions of all the analytes were corrected for the concentrations of the same analytes present in the reagent blanks. The concentrations in the blanks in each run were averaged; the average blank concentra –
tion was subtracted from the concentration of each sample and the quality control. The calibration curve of these phthalate metabolites showed linear correlation coefficients (>0.99) (Table 4).
Out of five metabolites screened, only two metabolites, MEHP
and MBP, were found in urine samples of the infertile males with an average percentage of 16% and 16.5% respectively. The average percentage of these metabolites was quite low in the case of the fertile male controls (MEHP, 2.6%; MBP, 8%) (Table 5).
The urinary phthalate metabolite concentration level (µg/mL) of
MEHP and MBP varied in the infertile males. While the maximum concentration of MEHP was 364 µg/ml, it was found to be 580 µg/ml for MBP. The urinary mean and median concentrations of MBP were higher than those for MEHP. This suggests that the predom –
inant metabolic route for DBP and DEHP is hydrolysis to MBP and MEHP followed by oxidation of MBP and MEHP in the infertile male (Table 6).
3.4 | Associations between semen parameters and
Phthalate Metabolites MEHP and MBP
Associations between semen parameters (sperm count, sperm mo –
tility and sperm morphology) and phthalate metabolites MEHP and
MBP were mostly unremarkable. There were no strong or consistent associations between any of the phthalate metabolites and semen parameters. However, there was weak evidence of an inverse rela –
tionship between MEHP and abnormal sperm morphology (Table 7). Finally, there was also no strong evidence of statistical 2-tailed of MEHP and MBP with semen parameters.
4 |  DISCUSSION
Phthalate esters are chemical plasticisers found ubiquitously in the environment (Arcadi et al., 1998; Hileman, 1996; Kavlock et al., 1996). Their indiscriminate use has raised serious health concerns. In the present study, the quantitative and qualitative estimation of phtha –
late esters (DEHP, DBP, DIBP, BEHIP, BPBG, DPP, DIOP and DICHP) in the serum of the infertile males revealed a high concentration in comparison with the healthy fertile controls (Blount et al., 2000; Gray et al., 2000). This high concentration of the phthalate esters was not found to affect any of the semen quality parameters, that is sperm count, motility, morphology, vitality and viscosity. Similarly, the phthalate metabolites were not found to be associated with any
Phthalate metabolitesConcentration
(µg/ml)Calibration curve range (AUC)Linear correlation coefficients ( R
2)
MEHP 5 1,010 .9977
10 2036
25 3,967
50 7,926
MBP 5 95,173 .9951
10 123,454
25 185,552
50 273,811
Abbreviations: AUC, Area under the curve; MBP, monobutyl phthalate; MEHP, mono-(2-ethylhexyl) phthalate.TABLE 4 Linear correlation coefficient
(R2) for phthalate metabolites ( R2 > .99)

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of the semen quality parameters in the infertile males. Animal stud –
ies with rats have shown that adult rats are less sensitive to MEHP
than are rats exposed in utero or during puberty (Higuchi, Palmer, Gray, & Veeramachaneni, 2003; Park, Habeebu, & Klaassen, 2002). It is, therefore, assumed that, in adult men, environmental background levels of DEHP may not be associated with altered semen quality where MEHP is the metabolite of DEHP.
Many of the phthalate esters detected in study samples have
been reported to possess a low oestrogenic activity compared to oestradiol and other potent oestrogenic substances (Malik, Kenny, & Ahmad, 1993). These may cause endocrine disruption through sev –
eral pathways by acting at critical periods of development (Harris et al., 1997; Jobling et al., 1995). For example, the intrauterine pe –
riod of human embryonic differentiation and development is partic –
ularly sensitive to weak estrogens. Besides their oestrogenic activity, phthalates such as DBP have anti-androgenic activity (Colborn & Clement, 1996). Phthalate esters have the potential to interfere with male fertility, and extensive research is required to address this issue.
To the best of our knowledge, this is the first study on the ef –
fect of phthalate esters and their metabolites in the infertile men in India (Fredricsson, Möller, Pousette, & Westerholm, 1993; Imajima, Shono, Zakaria, & Suita, 1997; Pant et al., 2003, 2008, 2014; Poon et al., 1997; Siddiqui & Srivastava, 1992; Srivastava,
Srivastava, Saxena, Chandra, & Seth, 1990; Thurston et al., 2016; Wang et al., 2015). All the participants were enrolled in the study
following the exclusion and inclusion criteria, and the sperm qual –
ity parameters were examined as per the WHO guidelines to en –
sure the fertility status and provide a generalised comparison in the semen parameters in these study groups. Men who had undergone a clinical examination at the infertility clinic had lower sperm con –
centration, motility and morphology as per the WHO guidelines (World Health Organization, 1999). Although the sperm quality pa –
rameters were impaired in the infertile men in comparison with the fertile healthy controls ( p < .001) (Table 1), no correlation could be
deduced between the deteriorated semen quality and phthalate ex –
posure. These findings are in corroboration with the studies on male
infertility, where phthalate exposure was not found to be associated
with classical semen quality parameters (Han et al., 2009; Thurston
et al., 2016). The findings of this study emphasise the environmental factors, especially those affecting the human male reproductive sys –
tem effectuating male fertility (Sáenz de Rodriguez, Bongiovanni, & Conde de Borrego, 1985; Sjöberg, Lindqvist, & Plöen, 1986). While some researchers have reported an association between the semen quality parameters and phthalates detected in the biological sam –
ples of the infertile males (Duty et al., 2003; Pant et al., 2008), oth –
ers did not find any correlation amongst them as observed in our study (Han et al., 2009; Thurston et al., 2016). At present, it is un –
clear whether phthalate esters and their metabolites by themselves or in association with other endogenous or exogenous oestrogenic compounds are capable of inducing precocious sexual development in humans. It is speculated that these environmental contaminants might cause other deteriorations in the semen quality such as DNA damage. Since this study was conducted between April 2006 and November 2009, it is desirable to evaluate the effect of phthalates on male fertility considering the present human exposure to these environmental contaminants. Conclusively, the physiological effects of phthalate esters and their metabolites must be further investi –
gated concerning male infertility in India.
5 |  CONCLUSIONS
In conclusion, the concentrations of phthalates and their metabo –
lites were statistically significant in the infertile males; however, no TABLE 5 Average percentage of phthalate metabolites in urine
samples of fertile and infertile males
Phthalate MetabolitesInfertile
(n = 152)Fertile (n = 75)Total subjects
MEHP 24 (16%) 2 (2.6%) 227
MBP 25 (16.5%) 6 (8%) 227
Abbreviation: ‘ n’, number of subjects (fertile/infertile males).
TABLE 6 Urinary phthalate metabolite concentrations (µg/mL) in infertile males
Percentile
Phthalate metabolites (µg/mL)No of patients Minimum 10th 25th 50th 75th 90th 95th Maximum Mean
MEHP 24 25 39.50 77.00 112.50 128.50 178.50 321.75 364 113.46
MBP 31 14 17.84 50.00 99.00 157.00 338.00 543.40 580 138.19
Phthalate metabolitesSperm countSperm motilitySperm morphologySperm vitality
MEHP −0.251 −0.146 −0.334
*−0.245
MBP 0.183 0.269 0.227 0.237
*Correlation is significant at the level of 0.05 (2-tailed test). TABLE 7 Association between
phthalate metabolites and semen quality parameters in infertile males

8 of 9  |    
RANA et Al.
association was found between phthalates and semen quality pa –
rameters. Further studies focused on other parameters of semen
quality such as genetic alterations or DNA damage are required to better delineate the correlation of phthalates occurrence in the bio –
logical samples with male infertility.
ACKNOWLEDGEMENTS
Dr. Rashmi Rana was the recipient of a Senior Research Fellowship from Indian Council of Medical Research (ICMR), Delhi, India. The authors are thankful to department of Urology, Safdarjung Hospital, New Delhi, India, and National Institute of Pathology (ICMR) in pro –
viding all the necessary support. We are thankful to Dr. A.K. Mishra, AIIMS, New Delhi for the statistical analysis of the data. We are truly thankful to all the patients involved in the study.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
AUTHOR CONTRIBUTIONS
RR involved in the planning and execution of the study and critical analysis of the clinical data. RR and SJ analysed the data and pre –
pared the manuscript. RR, NKM involved in sample procurement, data interpretation, and critical review of the manuscript for impor –
tant intellectual content. AKJ has guided for successful completion of the study and preparation of the final manuscript. All authors contributed significantly to the study and have approved the final manuscript.
ORCID
Rashmi Rana
https:/ /orcid.org/0000-0003-0071-089X
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How to cite this article: Rana R, Joon S, Kumar Jain A, Kumar
Mohanty N. A study on the effect of phthalate esters and their metabolites on idiopathic infertile males. Andrologia .
2020;00:e13720. https://doi.org/10.1111/and.13720