curcumin influences semen quality parameters and reverses the di(2-ethylhexyl)phthalate...

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Pharmacological Reports xxx (2014) xxx–xxx

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PHAREP 119 1–6

Original research article

Curcumin influences semen quality parameters andreverses the di(2-ethylhexyl)phthalate (DEHP)-inducedtesticular damage in mice

Katarzyna Glombik a, Agnieszka Basta-Kaim a,*, Marta Sikora-Polaczek b,Marta Kubera a, Gabriela Starowicz a, Jozefa Styrna b

a Department of Experimental Neuroendocrinology, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Polandb Department of Genetics and Evolution, Institute of Zoology, Jagiellonian University, Krakow, Poland

A R T I C L E I N F O

Article history:

Received 3 February 2014

Received in revised form 11 April 2014

Accepted 14 April 2014

Available online xxx

Keywords:

Curcumin

DEHP

Semen quality parameters

Testicular damage

A B S T R A C T

Background: Curcumin is a phytochemical derived from rhizome of turmeric Curcuma longa, present in

the curry spice. Recently, it has attracted the attention of researchers and clinicians as an anti-

inflammatory and anti-oxidant agent with a potential use in therapy of many diseases with an

inflammatory component. Interestingly, curcumin despite its very low bioavailability showed protective

activity against many organ lesions.

Methods: In the present study we investigated the effects of curcumin treatment on mice semen quality

parameters in vitro and on semen and testicular damage induced by di(2-ethylhexyl)phthalate in vivo.

Results: The study demonstrated protective effects of low concentrations (1–50 mM) of curcumin on

mouse sperm motility in vitro and on DEHP-induced damage of seminiferous tubules in testes and its

ability to diminish the decrease in sperm motility in vivo. In contrast, curcumin used in high

concentration (100 mM) decreased sperm motility and viability in vitro.

Conclusion: The effects of curcumin were dependent on its concentration. In male germ cells in vivo the

protective effect was seen despite the low bioavailability of curcumin. In contrast, high, unattainable in

the organism, concentration of curcumin had a cytotoxic effect on male reproductive cells in vitro.

Curcumin also had a protective effect against the harmful impact of DEHP on the male reproductive

system.

� 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Institute of Pharmacology,

Polish Academy of Sciences.

Contents lists available at ScienceDirect

Pharmacological Reports

jou r nal h o mep ag e: w ww .e lsev ier . co m / loc ate /p h arep

23242526272829303132333435

Introduction

Curcumin, a natural phytochemical derived from turmeric(Curcuma longa), which is present in commonly used curry spice,draws scientists’ attention due to its multiple biological actions.Numerous pharmacological studies have been performed todescribe biological functions of curcumin. Those studies havedemonstrated its anticarcinogenic [1,2], antioxidant [3–5], reno-protective [6], cardioprotective [3,5] and neuroprotective [4,7]activities. Modulation of cell signaling pathways through thepleiotropic effects of curcumin involved several molecules, like NF-kB, Akt, MAPK, p53, Nrf2, Notch-1, JAK/STAT, b-catenin, and AMPK[8]. Preclinical studies in the last years have indicated that

36373839

* Corresponding author.

E-mail addresses: [email protected], [email protected] (A. Basta-Kaim).

Please cite this article in press as: Glombik K, et al. Curcumin

ethylhexyl)phthalate (DEHP)-induced testicular damage in

j.pharep.2014.04.010

http://dx.doi.org/10.1016/j.pharep.2014.04.010

1734-1140/� 2014 Published by Elsevier Urban & Partner Sp. z o.o. on behalf of Instit

curcumin is a potential therapeutic for many diseases [9].Curcumin exerts powerful oxygen free radical scavenger effectsand increases intracellular glutathione concentration, therebyprotecting lipids from peroxidation. Recent studies have indicatedthat testicular oxidative damage in rats caused by L-thyroxine orcisplatin was attenuated by curcumin treatment [10]. The safety,tolerability, and non-toxicity of curcumin at high doses are wellestablished by human clinical trials [9].

Human population is exposed to numerous chemical andphysical agents that may affect both male and female reproductivefunctions [11]. Recent studies show that occupational andenvironmental exposure to phthalates may have an impact onmale fertility [12,13]. The widespread human exposure tophthalates is an effect of the omnipresence of these chemicalsin customer products, medical devices and food contact applica-tions. The routes of exposure considered to be the most importantfor general population include inhalation, oral administration and

influences semen quality parameters and reverses the di(2-mice. Pharmacol Rep (2014), http://dx.doi.org/10.1016/

ute of Pharmacology, Polish Academy of Sciences.


mailto:[email protected]

mailto:[email protected]



http://www.sciencedirect.com/science/journal/00000000

www.elsevier.com/locate/pharep


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rmal contact [14]. Di(2-ethylhexyl) phthalate (DEHP) is aasticizer used in the production of polyvinyl chloride (PCV). Ita major component of paints, flooring roofing and packaging

aterials, fabrics and also of medical products or toys. Manyudies indicated that DEHP was a developmental toxicant anddocrine modulator in animals and humans. The results showedat DEHP might cause changes in fertility, pregnancy, andenatal and postnatal survival of offspring or might reduceerm counts and cause reproductive organ malformations [15].me data demonstrated that young rats exposed to DEHPhibited the damage of spermatogenic cells which increasednificantly with the increase in DEHP dose and exposure time.HP treatment also significantly increased serum levels of follicle

imulating hormone and luteinizing hormone and decreasedstosterone levels in a dose- and time-dependent manner [16].

In the light of the above-mentioned data, in the present study,e aimed to detect possible preventive effects of curcumineatment on male reproductive function and on DEHP-inducedmages.

aterials and methods

imals

Male 12–36 weeks old mice were obtained from an outbredock (Department of Genetics and Evolution, Institute of Zoology,giellonian University, Krakow). Animals were housed undergulated temperature condition (22 8C) with free access to waterd food and were maintained on 12 h dark-light cycle. Animals

ere killed by cervical dislocation. The experimental proceduresere approved by the Commission of Bioethics at the Jagiellonianiversity.

eparation of solutions of curcumin

Curcumin was obtained from Sigma–Aldrich (Poland) and mM stock solution was prepared in ethyl alcohol. A 200 mMlution of curcumin was prepared from the stock solution usinge G-IVF medium (Vitrolife, Sweden). Then the followingperimental suspensions were prepared: 1 mM, 30 mM, 50 mM,0 mM. The control medium was supplemented with thercumin solvent, i.e. ethanol, used at an appropriate percentagencentration: 0.005%, 0.15%, 0.25% and 0.5%.

erm isolation and incubation

After sacrifice of 2 male mice (for each experiment), the vasferens was dissected. The sperm was collected and gentlyueezed out into 700 ml of culture medium. Sperm suspensionas incubated for 1 h in a 96-well dish under a liquid paraffin at

8C, in an atmosphere containing 5% CO2 in a G-IVF medium withe addition of 1 mM, 30 mM, 50 mM or 100 mM solution ofrcumin and in a control medium supplemented with curcuminlvent at the appropriate concentration.

erm quality parameters

After incubation sperm quality parameters were assessed. Thercentage of motile spermatozoa was determined in a lighticroscope. For evaluation of the percentage of dead sperm cells,

ml of a sperm suspension was taken and placed on a glass slided mixed with 10 ml of 5% aqueous eosin solution (Sigma–drich, USA). Glass slide was covered with a Petri dish lid andcubated on a heating plate for 10 min at 37 8C. The sperm cellsained red (classified as dead) were counted in 200 consecutivells [17].

Please cite this article in press as: Glombik K, et al. Curcuminethylhexyl)phthalate (DEHP)-induced testicular damage in

j.pharep.2014.04.010

Methods used to evaluate the effects of orally administered curcumin

on sperm and testicle damage induced by the phthalate DEHP

Male mice were divided into 4 groups (n = 8) and were treatedfor 21 days with the following compounds: (1) control group(arachidonic oil), (2) curcumin, (3) DEHP phthalate and (4) DEHPwith curcumin.

All solutions were administered orally. DEHP and curcuminwere dissolved in arachidonic oil (Sigma–Aldrich) and adminis-tered orally at a dose of 30 mg/mouse (for DEHP) and a 5 mg/mouse (for curcumin) in a volume of 50 ml/mouse. After 21 days oftreatment mice were killed and weighed. Testicles and vasdeferens were dissected. Testicles were weighed and fixed in 4%formalin solution. The sperm from vas deferens of each male wasgently squeezed out into 300 ml of G-IVF medium. Sperm cellviability and motility were tested and the smear was made toassess the percentage of morphologically abnormal sperm heads.10 ml of sperm suspension was placed on a glass slide to make asmear. Then preparations were dried for 24 h and stained in 1%eosin Y for 1 h. Preparations were rinsed in water and left to dry.200 sperm cell were counted for each individual, and a percentageof morphologically abnormal cells was determined. The classifica-tion was made according to the schedule given by Krzanowska[18].

Morphological analysis of the testes and detection of DNA

fragmentation by labeling the terminal end of nucleic acids using

TUNEL method

Testicles were fixed in 10% formalin solution for 24 h at 4 8C anddehydrated in a graded series of alcohol solutions 70%, 80%, 90%, for24 h in each dilution and in 96% and 100% for 1 h. Then sampleswere exposed to xylene two times for 15 min. Testicles wereembedded in paraffin (24 h, 58 8C). 5 mm thick slices were cutusing a paraffin microtome (Reichert-Jung, Germany) and placedon glass slides (for histological analysis) and on glass slides coatedwith APS-3-aminopropyl-3-oxysilane (for TUNEL method).

Hematoxylin and eosin staining

Slides for histological analysis were immersed in xylene 2 timesfor 10 min, then in 100%, 96%, 80% and 50% ethanol for 5 min pereach change and in distilled water for 12 min. Slides were stainedwith a standard hematoxylin–eosin method. After staining,preparations were rinsed in distilled water and 30%, 50%, 80%ethanol. Then they were dehydrated in 96% ethanol for 3 min, 100%ethanol 2 times for 5 min and in xylene 2 times for 10 min. Theslides prepared in this way were closed with DePeX (Sigma–Aldrich) and examined under a light microscope (Olympus, typeCH2). Morphology of seminiferous tubules and cells involved inspermatogenesis was evaluated. Photos of testicular sections weretaken with a digital camera Olympus C-304 ZOOM, 20� lens.

TUNEL method

Preparations on APS-coated glass slides were dehydrated inxylene, 100%, 95%, 80%, 70% ethanol and rinsed with phosphatebuffer (PBS). Then they were incubated with 20 mg/ml proteinase Ksolution in 10 mM Tris/HCl, pH 7.4–8 (Roche) in a humidifiedchamber for 10 min at 37 8C and rinsed in PBS, incubated with ablocking solution (3% H2O2 solution in methanol). Then sectionswere rinsed twice with PBS, the area around selected sections wasdried and loaded with TdT-terminal deoxynucleotyde transferase(Roche), 50 ml per section. According to the manufacturer’srecommendations, the blank sample was made. Slides were rinsedtwice in PBS and dried. Then 50 ml of horseradish peroxidase (POD,Roche) was added per sample and sections were incubated in ahumidified chamber for 30 min at 37 8C. Samples were rinsed in




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Table 2The effect of curcumin administration on motility, viability and morphology of

sperm’s head in vivo in mice treated with DEHP. The table presents the percentage

of motile, viable and morphologically normal sperm cells in mice treated with

DEHP, DEHP and curcumin in combination, curcumin alone, and in the control

group (8 animals per group, average data).

Mobile sperm

(mean�SD)

Viable sperm

(mean�SD)

Morphologically normal

sperm heads (mean�SD)

Control 80.5 � 2.3 81.2 � 2.2 80.5 � 1.3

Curcumin 78.0 � 2.6 80.9 � 2.4 83.0 � 2.0

DEHP 65.0 � 1.1* 79.1 � 1.8 81.0 � 1.1

DEHP + curcumin 76.0 � 2.8 79.8 � 2.6 82.5 � 2.7

t-Test was performed on the values in arcsin transformation.* Statistically significant differences compared to control, p < 0.001.

K. Glombik et al. / Pharmacological Reports xxx (2014) xxx–xxx 3

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PBS three times and 50 ml of diaminobenzidine (0.05% DAB, Roche)per section was added. Preparations were incubated for 10 min atroom temperature, rinsed with PBS, dehydrated with alcohol andxylene and closed with DePeX. The number of tubules, in which>10 degenerated cells were observed per the number of tubulesvisible in the cross section, was determined. Photos of testicularsections were taken with a digital camera Olympus C-304ZOOM.

Statistical analysis

All data were expressed as the means � SD. The percentage datawere arcsin transformed before statistical analysis. Data of spermquality parameters were pooled for each group. The parameters werecompared by t-test.

Results

Impact of curcumin on the motility and viability of sperm in vitro

Our study showed that a 1-h incubation of sperm in the G-IVFmedium resulted in a significant reduction of sperm cell viabilityand motility. This effect has been commonly observed in this typeof experiments in which sperm was isolated by extrusion from thevas deferens [19]. Despite that fact, a dose-dependent effect ofcurcumin on sperm motility in vitro was observed. There was notoxic effect of curcumin solvent, i.e. ethanol in concentrations of0.005–0.25% on the incubated sperm. Sperm incubated in themedium with 0.005–0.25% ethanol had comparable motility andviability parameters to sperm incubated in an ethanol-freemedium. After a one-hour incubation in the medium with 0.5%alcohol, a 12% decrease in sperm viability (compared to control)was observed. There was no impact of ethanol in this concentrationon motility.

The addition of curcumin in 1, 30 and 50 mM concentration toculture medium inhibited the decrease in sperm motilitycompared to control in a significant manner (p < 0.05). In contrast,curcumin in a concentration of 100 mM had a toxic effect,deepening the toxicity of 0.5% ethanol. Viability of sperm alsosignificantly decreased after a 1 h incubation but no effect ofcurcumin on that parameter was observed. The viability did notsignificantly differ between the experimental and control groups(1–50 mM). Only 100 mM curcumin showed a toxic effect on spermviability. Results are shown in Table 1.

Protective effects of orally administered curcumin on sperm and

testicular damage induced by the phthalate DEHP

Our study showed that sperm motility in the control group wasabout 80%. In mice treated with DEHP we observed a significantdecrease (by about 15%) of sperm motility compared to controlanimals. Furthermore, the present results indicated that

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Table 1The effect of curcumin administration on sperm quality parameters in vitro in four

examined groups. The table presents a percentage of motile and viable sperm cells

after one-hour incubation in the medium with curcumin at a concentration of 1 mM,

30 mM, 50 mM, 100 mM, and in the medium with the appropriate concentration of

ethanol, which was curcumin solvent. For each experiment, sperm was isolated and

pooled from two mice, the data are collected from eight experiments.

Mobile sperm

(mean�SD)

Viable sperm

(mean�SD)

Curcumin 1 mM/control 34.0 � 3.1*/28.0 � 1.5 37.5 � 2.8/36.5 � 1.1



Curcumin 100 mM/control 15.0 � 2.2*/25.0 � 2.5 16.0 � 2.8*/25.0 � 3.5

t-Test was performed on the values in arcsin transformation.* Significant vs. adequate control solution, p < 0.001.



j.pharep.2014.04.010

co-treatment with curcumin reversed the DEHP-induced changesin sperm motility. Administration of curcumin did not affect spermmotility in the curcumin alone-treated group, in which motilitywas at the control level. Curcumin and DEHP had no impact onsperm viability. Average sperm viability in all groups was 69%. Themorphology of sperm heads was also analyzed in all animals. Therewas no significant difference between treated and control groups.Average results with standard deviations from all experimentalgroups are presented in Table 2.

In addition to the analysis of sperm parameters, histologicalanalysis of mouse testicles was performed. There were nostatistically significant differences in the body weight of mice orin testicular weight between the examined groups (Table 3).

In histological preparations, the percentage of degeneratedseminiferous tubes was evaluated. Histological analysis of controlanimals showed normal structure of testicles, seminiferous tubesand Sertoli cells which are located at the base of epithelium. In allpreparations, from the membrane to the tubular lumen, weobserved all four stages of spermatogenesis: spermatogonia,primary spermatocytes, secondary spermatocytes and spermatids.The sperm heads were visible in the lumen of tubules. In theintertubular space, Leydig cells were present (Fig. 1, panel A1) micetreated with curcumin presented identical histological features asin the control group (Fig. 1, panel B1). However, the DEHP-treatedmice demonstrated a significant increase in the number ofdegenerated tubules (Fig. 2, panel C1). Males co-treated withDEHP and curcumin showed an increased number of degeneratedtubules compared to control and a decrease in degenerated tubulescompared to the DEHP-treated males (Fig. 2, panel D1).

The TUNEL method was applied to confirm the apoptosis inseminiferous tubes. Only a few apoptotic cells, no more than 3 inone tube, were observed in control and curcumin-treated group(Fig. 1, panel A2 and B2). Males treated with DEHP showed a highincrease in cell apoptosis in tubes. In degenerated tubes, manydead cells, most often spermatogonia, were observed (Fig. 2, panelC2). Only a few apoptotic cells were also present in the DEHP andcurcumin-treated group (Fig. 2, panel D2). Data are summarized inTable 4.

According to previous data [20–24] DEHP induces death ofspermatogonia. Morphological changes observed as an influence

Table 3The effect of curcumin administration in vivo on body weight and testicular weight

in mice treated with DEHP, DEHP and curcumin in combination, curcumin, and in

the control group (8 animals per group, average data).

Body weight in grams

(mean�SD)

Testicular weight in milligrams

(mean�SD)

Control 25.7 � 1.6 94.5 � 2.3

Curcumin 28.4 � 2.9 100.0 � 2.9

DEHP 26.7 � 2.7 97.0 � 1.4

DEHP + curcumin 30.2 � 3.4 101.0 � 2.6

t-Test was performed on the values in arcsin transformation.




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Fig. 1. Morphology of testes from the control and curcumin-treated mice (panel A1, panel B1). Testis tissue was labeled for apoptosis by TUNEL (A2, B2). Arrows mark normal

tubules in the testis and single apoptotic cells.

FigD2


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DEHP were verified using the TUNEL method, which allows fortection of DNA damage occurring during apoptotic and necroticll death. In testes of control animals and animals treated withrcumin, only sparse dead cells were observed (Fig. 1, panel A2

. 2. Morphology of testes from the DEHP-treated and DEHP + curcumin-treated mice

). Arrows mark representative tubules with strongly degenerated germinal epithel


j.pharep.2014.04.010

and B2). In seminiferous tubules of males treated with DEHP, weobserved numerous dead cells, namely above 10 cells in thetubules considered as degenerated (Fig. 2, panel C2). These resultsconfirmed the histological analysis (Fig. 2, panel C1). In the testes

(panel C1, panel D1). Testis tissue was labeled for apoptosis by TUNEL (panel C2, panel

ium (asterisks) and dead cells.




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Table 4The effect of curcumin administration in vivo on morphology of seminiferous

tubules. The table presents the percentage of degenerated seminiferous tubules in

mice treated with DEHP, DEHP and curcumin in combination, curcumin, and in the

control group (8 animals per group, average data).

Percent of degenerated seminiferous tubules

(mean � SD)

Control 0.00 � 0.00

Curcumin 0.00 � 0.00

DEHP 4.25 � 1.90*

DEHP + curcumin 1.00 � 2.30*

t-Test was performed on the values in arcsin transformation.* Statistically significant differences compared to control, p < 0.001.

K. Glombik et al. / Pharmacological Reports xxx (2014) xxx–xxx 5

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of males treated with DEHP and curcumin simultaneously, weobserved an increase in the percentage of dead cells vs. control,which was, however, significantly lower than that seen in thetestes of males treated only with DEHP (Fig. 2, panel D2).

Discussion

In the present study we have demonstrated that curcumin inlow concentrations have a positive effect on sperm motility in vitroand prevents the decrease in sperm motility in vivo in malestreated with DEHP. Furthermore, we have shown that DEHP causeddegeneration of seminiferous tubes in male testicles and signifi-cantly increased apoptosis in these tubes. Curcumin protectedtesticles against oxidative damage caused by DEHP.

Curcumin appears to be an unusual natural compound. Usedfor thousands of years in natural medicine, now it is considered tobe a potentially therapeutic drug in clinical practice. Until nowonly few studies have been published on the action of curcumin onthe fertility [10,25]. Nowadays infertility prevalence rates arehigh and a lot of couples seek help in infertility diagnostics andtreatment clinics. Very often these clinics have limited treatmentoptions due to reduced semen parameters. Indeed, it has beenestimated that infertility affects both sexes equally. Excessiveexposure to reactive oxygen species (ROS) is often one of thereasons of male infertility. Phthalates are one of many groups ofharmful factors that affect humans which, as previous studiesindicated, caused testicular atrophy in laboratory animals [26],increased ROS production in the testes of rats and inducedspermatocyte apoptosis [27].

The first finding of our study was that curcumin had an impacton semen quality parameters. Sperm motility seems to be the mostcurcumin-sensitive parameter. Curcumin in doses of 1–50 mMshowed a positive effect on sperm motility and no impact on spermviability while a curcumin concentration of 100 mM was found tobe toxic to male gametes. It seems that the mechanism responsiblefor the effect of curcumin on the motility and viability of sperm invitro is related to its antioxidant properties. Low concentration ofreactive oxygen species is necessary for an adequate spermmotility and capacitation. In contrast, cytoplasmic membrane ofsperm cells is rich in polyunsaturated fatty acids, which can easilyundergo peroxidation by ROS which reduces their motility andincreases mortality. ROS affect the sperm directly after ejaculationthus also when it is subjected to cryopreservation. Therefore, acomprehensive study on the impact of curcumin on motility andsurvival of sperm could in the future help improve quality of semenused for in vitro fertilization.

In the next part, we evaluated the effect of curcumin onmale gametes in vivo. Sperm motility was determined afteroral administration of curcumin. There was no difference insperm motility compared to the control but the main result of thispart, was demonstration of curcumin protective activity againstDEHP-induced damage. Our finding was that orally administered



j.pharep.2014.04.010

di(2-ethylhexyl)phthalate significantly decreased sperm motilityin vivo. Moreover, we proved that animals treated with DEHPtogether with curcumin indicated similar level of motility comparedto control and curcumin-treated groups. These data confirm theresults of Farombi [20] who showed that sperm motility of male ratssignificantly increased after co-administration of DBP (dibutylphthalate) and curcumin, compared to DBP only-treated males. Thepotential mechanism is that phthalates cause changes in peroxida-tion of membrane lipids of germinal cells which impairs the spermmotility [20]. Curcumin, as a compound which inhibits the lipidperoxidation of cell membranes can counteract these changes [28].The analysis of sperm viability and sperm head morphology in allgroups showed no differences. This could be caused by too shorttime of DEHP exposure.

The last step of our experiment was to evaluate how DEHP andcurcumin are involved in the formation of histological changes inmice testicles. Comparison of body weight and testicular weight ofmice in all treated groups showed no difference. This is probablyrelated to low toxicity of the phtalate dose. The Kasahara group[27] showed such changes but they used a dose of 2 g/kg andseveral hours of exposure. On the other hand, results of the presentstudy proved that DEHP induced histological changes in testiclesand that curcumin had a protective effect against gonadal injurycaused by DEHP. Structural changes in seminiferous tubules mightbe caused by DEHP-induced oxidative stress. DEHP could also leadto degeneration of Leydig cells and in consequence to a decrease intestosterone level, which is necessary for normal spermatogenesis[28]. Perhaps this was one of the reasons of the degeneration ofseminiferous tubules.

To confirm apoptosis in seminiferous tubules, TUNEL analysiswas performed. DEHP-treated animal showed increased levels ofapoptotic cells. This is probably due to the fact that exposure to thetoxic compound caused DNA damage in spermatogonia and insubsequent cell stages. Apoptosis was not detected in controlanimals or in animals treated with curcumin. Males treated withDEHP and curcumin in combination had only sparse apoptoticcells. This results suggested that curcumin inhibited apoptoticchanges in male gametes. Our studies showed the effect of chronictreatment with phthalate and curcumin at relatively low dosescompared with other reports [27–30]. Curcumin has a very lowbioavailability [31], so getting the positive effects using low dosessuggests its high therapeutic potential.

Conflict of interest

None declared.

Funding

This study was partially supported by the QFunds for StatutoryActivity of the Institute of Pharmacology Polish Academy ofSciences and Qthe project Interdisciplinary PhD Studies ‘‘Molecularsciences for medicine’’ (co-financed by the European Social Fundwithin the Human Capital Operational Programme)

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[4] Lim GP, Chu T, Yang F, Beech W, Frautschy SA, Cole GM. The curry spicecurcumin reduces oxidative damage and amyloid pathology in an Alzheimertransgenic mouse. J Neurosci 2001;21:8370–7.




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curcumin influences semen quality parameters and reverses the di(2-ethylhexyl)phthalate...

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