{"paper_id":"f7d7dccd-ccc3-48d1-b340-7ee46f085ecc","body_text":"DOI: https://doi.org/10.53350/pjmhs2115123214 \nORIGINAL ARTICLE \n \n3214   P J M H S  Vol. 15, No.12, DEC  2021 \nSerum Diethyl Phthalate Levels in Infertile Females with Endometriosis \n \nSADIA NAZIR1, IRAM NAZIR2, RIDA KHAN3, GULFAM AHMAD3, MAHWISH SHAHZAD4, MUNEER IMRAN5  \n1Associate Professor Physiology, Lahore Medical and Dental College, University of Health Sciences, Lahore \n2Baqai Medical college, Baqai Medical  University, Karachi  \n3Department of Physiology and Cell Biology, University of Health Sciences, Lahore  \n4University of Health Sciences, Lahore. Currently working at Department of Biochemistry, Lahore Me dical and Dental College, Lahore \n5Akhtar Saeed Medical and Dental College, University of Health Sciences, Lahore  \nCorrespondence to Dr. Sadia Nazir,  Email: sadiamunir1980@hotmail.com , Cell: 0321-4713384 \n \nABSTRACT \n \nBackground: Endometriosis has a complex multifactorial pathophysiology and is a leading cause of female infertility. Emerging \nevidence suggests the role of endocrine disrupting chemicals and environmental factors such as Diethyl phthalate (DEP) in the  \npathophysiology of the disease.  \nAim: To investigate the serum DEP levels in females with infertility having endometriosis and normal healthy counter parts.  \nMethods: Married females (n=50) age 20 -40 years, diagnosed with endometriosis and having history of >1 year of infertility, \nwere selected as cases. Age matched women (n=50) with proven fertility and screened negative for endometriosis were included \nas controls. Females on any medicine, having co morbid conditions were also excluded from the study. DEP concentration  in \nserum was determined by using High performance liquid chromatography (HPLC).  \nResults: Significantly higher levels ( p=0.003) of DEP were seen in endometriosis females (3.76± 1.28 ng/ml) as compared to \ncontrols (2.61± 1.72 ng/ml). The comparison of DEP levels between different stages of endometriosis revealed an increasing, \nbut no significant trend with advancement of the disease.  \nConclusion: High serum DEP levels in patients substantiate their role in disease pathophysiology. Therefore, it may be \nadvisable to pay attention while using such compounds. There is an increased need to regulate the levels of such industrial \ncompounds manufactured for daily use of human beings by efficient and judicious quality assurance plastics and by using the \nstandards set by WHO and/or FDA. \nKeywords: Phthalate, Endometriosis, Diethyl phthalate, plasticizers, Infertility \n \nINTRODUCTION \n \nEndometriosis, is disease in which the presence of endometrial \ntissue in ectopic sites, other than uterine cavity and is one of the \ncommonest causes of female infertility 1. About 15% of peoples in \nindustrialized world is consults for infertility 2. The frequency of the \nendometriosis in Pakistan reported to 20.2% in cases of primary \ninfertility3 which is consistence with results of other studies of \ndifferent populations4,5. \nNew evidence propose that possible role of envir onmental \ncontamination of endocrine disrupting chemicals (EDC’s) in the \npathophysiology of endometriosis 6. An endocrine disrupting \nchemical is an exogenous substance that hinder with production, \nbinding, and removal of natural sex hormones 7. Some chemicals \nstimulate the growth of endometrial cells that play an important role \nin endometrial angiogenesis 8,9. Among such EDC’s phthalates \nhave attained special focus due to their existence in everyday life \nproducts10. \nPhthalates are esters of Phthalic acid. A class of organic chemicals \nthat are used as plasticizer to increase the pliability of \npolyvinylchloride (PVC). They are used in immense range of \nproducts for example food packaging materials, medical devices, \nmedicines, kids’ toys as well as beauty and personal care \nbrands11. Out of  the primary phthalate compounds , diethyl \nphthalate (DEP) is most frequently used plasticizer globally. Its \nmost common use is in cosmetics, pharmaceutical and food \npackaging materials12. \nOnce in side the body these phthalates are promptly \nmetabolized to their monoesters after hydrolysis and conju gation, \nand then excreted in urine, feces and other body secretions. \nPrimary and secondary phthalate metabolites are bio -active13,14. \nPublic and scientific concern is being focused to inquire the role of \nphthalate exposure in estrogen related disease such as \nendometriosis. Studies have shown accumulation of these \nsubstances in blood, follicular fluid, urine, saliva and sweat of \ninfertile females 15,16,17. So far, fewer reports  have been published \non the subject and nothing exists from Pakistan. A Few authors \nreported that there is positive association between phthalate  \n----------------------------------------------------------------------------------------- \nReceived on 07-06-2021 \nAccepted on 27-11-2021 \nexposure and endometriosis 18,19, while others reported \ncontradictions20,21. Pakistan is a country where plastic and plastic \nrelated products are very heavily used, particularly by the lower \nmiddle class and modest communities. Plastic containers are even \nused for carrying very hot food items and apart from their industrial \nuse, small factories or cottage industries making plastic utensils \nand shopping bags use recycled plastic materials, which make \nthese items quite hazardous to t he general health of the public. \nAlso there are women of the reproductive age who work in these \nhome-based factories where there is least concept of quality \ncontrol, quality assurance and health hazards. Keeping in view of \nthese facts we conducted the present study to understand whether \nexposure to diethyl phthalate is associa ted with risk of \nendometriosis. \n \nMATERIAL AND METHODS \n \nThe case control study was approved by Ethical review board of \nUniversity. Sample size was calculated by using the following \nformula:   \n \nStudy population comprised of 50 young (20 -40 years) females \nlaparoscopically diagnosed of endometriosis and had a history of \ninfertility (>1 years). Staging of the disease was done according to \nASRM criteria for endometriosis staging 22. A comparable number \n(n=50) of same age group healthy females who gave birth a child \nin last two  years, also negative for endometriosis screened by \nultrasonography followed by detailed clinical evaluation and \nexamination by specialized doctors served as controls. Females on \nany medication especially hormone treatment, history of infertility \nother tha n endometri osis or having co -morbid conditions , were \nexcluded from the study. After taking written well informed consent \nfrom all study participants, a detailed medical, socioeconomic and \noccupational history with detailed general physical and systemic \nclinical evaluation was done by a qualified doctor. \nTen milliliters of blood was drawn under aseptic conditions. \nSerum was separated after centrifugation; and stored in glass vials \nat -80 ◦C for later analysis. High performance liquid \n\n\nS. Nazir, I. Nazir, R. Khan et al \n \n \nP J M H S  Vol. 15, No.12, DEC  2021   3215 \nchromatography (Perkin Elmer series 200 UV pump) was used to \ndetermine diethyl phthalate concentrations. \nReagents and solutions preparation : Stock solution of HPLC \ngrade (Merck chemicals, Germany) 99.99% diethyl phthalate \n(DEP) and its internal standards were prepared in  HPLC grade \n(Fisher scientific, Germany) acetonitrile, and stored at -20˚C to test \nthe linearity. With a DEP concentration of 10, 50, 100, 250, 500, \nand 1000 ng/ml, Calibrators were prepared in serum. A total of 200 \nµL of serum was added with 50 µL of internal standard, 400 µL of 1 \nmol/L NaOH (HPLC grade; Sigma Aldrich, USA), 100 µL of 50 % \nH3PO4 (HPLC grade; Sigma Aldrich USA) and 600 µL of 50 % \nacetonitrile  (Fisher scientific, Germany).  \nHigh Performan ce Liquid Chromatography (HPLC): After 10 \nminutes at 1500 rpm centrifugation , the supernatant was \nseparated, and 10 µL of the supernatant was injected into the \nHPLC glass vials via syringe filter (25mm/ 0.22 micrometer) , and \nput in auto- sampler (Perkin Elmer series 200).  To avoid potential \ncontamination, all glass vials were clean with methanol, acetone \nand acetonitril. HPLC was carried out using Perkin Elmer series \n200 UV pump, attached to a Diode Array UV/ visible Perkin Elmer \nseries 200 detector. A Perkin Elmer 250 x 4.6 mm (internal \ndiameter) Brownlee analytical C18 (5 -µm particle size) column \n(USA) was used. Results were analyzed on Total chrome \nNavigator 3.1.5 software. The mobile phase consisted of a mixture \nof 1ml/L acetic acid (Sigma Aldrich, USA) in water and 1ml/L acetic \nacid in acetonitrile (10/90, vol/vol). Elution was performed with the \nuse of an isocratic mode at 0.5 ml/min. Total chromatographic run \ntime was 10 minutes. Peak for DEP was obtained at 6.43 minutes \n(retention time). Concentration of DEP was calculated by using \nstraight line equation. \nStatistical analysis : By using IBM -SPSS version 20 , data was \nanalyzed. The variables were analyzed and expressed as \nMean(±SD). Two sample Student’s‘t’ test, was applied to compare \nthe phthalate esters concentrations in both cases and controls. A p \nvalue of ≤ 0.05 was considered as statistically significant. To \ncompare the levels of DEP between different stages of \nendometriosis one-way ANOVA was applied. \n \nRESULTS \n \nIndividual values for DEP in endometriosis females and controls \nwere calculated by using straight line equation among peak area of \nstandard and  concentration of standard, and putting individual \npeak areas for cases and control in straight line equation, as \nexpressed in fig. 1. \n \nFig. 1: DEP straight line equation according to least square method for \ncalculating values in both cases and controls. \n \n \nDescription of chromatographs: Fig. 2a, 2b, 3a and 3b describe \nDEP chromatographs , obtained through High performance liquid \nchromatography in cases and controls respectively. The X -axis \nshows the retention time (minutes), and Y -axis shows milli- \nAbsorbance Units (mAU).  \nFigures 2a and 3a are real time chromatographs, showing the \npeak of DEP at its respective retention times in cases an d controls \nrespectively. Fig. 2b and 3b , are reprocess results collected from \ntotal chrome navigator 3.1.5 software , showing DEP peaks in \ncases and controls at their respective retention times. \n \nFig. 2a: HPLC Real time chromatograph of cases showing the DEP peak . \nGenerated from: Total chrome navigator; 3.1.5 software. \n \n \nFig. 2b: HPLC Reprocess Chromatograph of cases showing the DEP peak \narea. Generated from: Total chrome navigator 3.1.5 software.  \n \nFigure 3a: Real time chromatograph of HPLC of control showing the DEP \npeak. Generated from Total chrome navigator (3.1.5 software).  \n    \n Retention time (minutes) \n\n\nSerum Diethyl Phthalate Levels \n \n \n3216   P J M H S  Vol. 15, No.12, DEC  2021 \nFigure 3b:  Reprocess Chromatograph of HPLC of control showing the DEP \npeak area. Generated from Total chrome navigator (3.1.5 software). \n \n \nStatistically significant difference ( p=0.003) in mean DEP serum \nconcentration of cases (3.76±1.28 ng/ml) and controls (2.61±1.72 \nng/ml) was observed (figure 4). \n \nFigure 4: Comparison of Means ±SD between cases and controls for DEP \nstatistics according to the “t” test. \n \n \nBased on the points system as per American Society for \nReproductive Medicine classification of endometriosis, patients \nwere assigned to one of the four stages and serum DEP levels \nwere compared among all stages.  \n \nTable 1: Mean ( ±SD DEP) values in all four  stages of endometriosis, \nstatistics according to one- way ANOVA. \n \n \nThere was an increasing tendency in the M ean values of serum \nDEP as the stage advances, but the difference was insignificant.  \nThe comparison of mean values of demographic factors i.e. age, \nage at menarche, weight and height did not differ significantly \nbetween cases and control groups (Table 2). \n \n \nTable 2: Comparison of Anthropometric factors between cases and controls \nstatistics according to the “t” test. \n \n \nDISCUSSION \n \nEndometriosis is a gynecological disorder,  which is characterized \nby the existence of ectopic endometrial tissue. Regardless of th e \nextensive development of endometriosis , the etiology  and \npathophysiology of the disease is yet to be explicated.  \nEnvironmental hazardous toxins have previously shown to \nbe associated to endometriosis. The aim of this  study was to \nidentify the levels of phthalates, which are comm on endocrine \ndisruptors, in age- matched females with diagnosed endometriosis, \nas well as healthy females. Pakista n lacks a strict quality control \nover the use of common plastic compounds such as plastic food \nwraps, medical devices, beverages containers, and the linings of \nmetal cans. A good number of Pakistani females work in the \npharmaceutical industry and an equally good number uses daily \ncosmetics. Hence, the risk of exposure to phthalates in this region \nis reasonably higher compared to developed coun tries. Although a \nvariety of phthalate compounds has been studied, we selected \nDEP which is a parent compound of phthalate, widely distributed \nand mostly present in plastic, medical devices  and daily use \nproducts. It enter the body through inhalation, cutaneous contact, \nand through food and medicine ingestion23.   \nThe literature for serum DEP levels in infertile females with \nendometriosis is sparse. This study is the first report from Pakistan \ndemonstrating the relationship between serum DEP levels and \ninfertility with endometriosis. Our study recruited endometriosis \npatients diagnosed by laparoscopy. Our results showed \nsignificantly higher (p=0.003) serum DEP (diethyl pht halate) levels \nin females with endometriosis, compared to controls. These results \nare i n accord  with previous study , which shows high DEP levels \nbut detection rate is low in this study (0 -16.4%)24. This difference \nmay be due to more judicial use of DEP products in our country \nespecially hot food packaging in plastic bags and containers and \npoor quality cosmetic products. These results are contrary to \nprevious studies which show undetectable levels of DEP in both \ncases and controls 24-27. This difference in results may be due to \ndifferences in the techniques, as Reddy et al (2003) used gas \nchromatography and in Rozati et al (2008) (28) HPLC techniqu e,  \ncontrol and cases criteria was different from ours. DEP used as a \nsolvent and fixative for fragrance , cosmetic ingredients , \nsubsequent contact with skin may lead to high levels of DEP. In \norder to show correlation between DEP concentrations and \nseverity of the disease, we analyzed the mean DEP values \nbetween stages (I, II, III and IV) of  endometriosis. This analysis \nshowed no significant diffe rence in mean DEP le vels among the \nstages of endometriosis. However, there was an increasing trend  \n \n \n\n\nS. Nazir, I. Nazir, R. Khan et al \n \n \nP J M H S  Vol. 15, No.12, DEC  2021   3217 \nof mean values of DEP with advancement of stage (Table 1). \nThese results were in contrary to previous study24 which showed \nincrease phthalate levels with advancement of disease. We \ncompared the anthropometric parameters , i.e. age at menarche, \nheight, weight and age with serum DEP levels , but no statistical ly \nsignificant difference was observed between cases and c ontrols \n(Table 2). Therefore, no association was ascertained among the \nanthropometric factors and endometriosis which is in consistency \nwith previous results by Rozati et al28. \nOur results favor the previous studies showing positive \nassociation of Diethyl phthalates with endometriosis. Unfortunately, \nbecause of  our financial limitations , we could not expand our \nexperiments, which may provide more elaborated understanding \nabout the possible role of these phthalate compounds in the \npathophysiology and etiology of endometriosis. \n \nCONCLUSION \n \nFurther studies are necessary to answer the mechanistic pathway \nthrough which these compounds alter the normal physiology and \nlead to disease development. We proposed that future studies \nshould be conducted on c omparison of serum DEP levels in \ninfertile females with endometriosis with infertile females without \nendometriosis.  \n \nREFERENCES \n \n1. Parasar P, Ozcan P, Terry KL. Endometriosis: Epidemiology, \nDiagnosis and Clinical Management. Curr Obstet Gynecol Rep. 2017; \n6(1):34–41.  \n2. Reid, R., Steel, A., Wardle, J. et al. The prevalence of self -reported \ndiagnosed endometriosis in the Australian population: results from a \nnationally-representative survey. BMC Res Notes, 2019; 12, 88.  \n3. 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