MATERIALS AND METHODS

Transcription

1 Analysis of follicular fluid and serum markers of oxidative stress in women with infertility related to endometriosis Laura Prieto, M.D., a Juan F. Quesada, M.D., a Olivia Cambero, M.D., a Alberto Pacheco, Ph.D., b Antonio Pellicer, M.D., b Rosa Codoceo, M.D., a and Juan A. Garcia-Velasco, M.D. b a Department of Obstetrics and Gynecology, La Paz Hospital; and b IVI-Madrid, Reproductive Endocrinology Section, Rey Juan Carlos University, Madrid, Spain Objective: To study the levels of four markers of oxidative stress in follicular fluid (FF) and plasma of patients with infertility related to endometriosis and controls. Design: Experimental study. Setting: University-affiliated hospital and infertility center. Patient(s): Ninety-one infertile women were included in the study (23 infertile women with endometriosis and 68 controls including infertile women due to tubal factor, male factor, or healthy egg donors). Intervention(s): Blood was obtained at the time of egg retrieval, and FF from the mature follicles of each ovary was centrifuged and frozen until analysis. Main Outcome Measure(s): Vitamin C and E, malondialdehyde, and superoxide dismutase concentrations in plasma and follicular fluid. Result(s): Women with endometriosis showed a lower vitamin C concentration in FF ( vs mg/ml) and lower superoxide dismutase concentration in plasma ( vs U/mL) compared with controls. Vitamin E plasma levels were significantly higher in women with endometriosis ( vs mg/ml). A nonsignificant trend toward a lower plasma concentration of malondialdehyde was found in women with endometriosis. Conclusion(s): These findings suggest a lower antioxidant capacity in infertile women with endometriosis. Although a certain level of reactive oxygen species is required under physiological conditions, an altered balance between pro-oxidant and antioxidant activities may have an impact on folliculogenesis and adequate embryo development. (Fertil Steril Ò 2012;98: Ó2012 by American Society for Reproductive Medicine.) Key Words: Oxidative stress, endometriosis, infertility Patients with endometriosis have worse outcomes in assisted reproduction techniques, specifically IVF, most likely because of lower oocyte quality (1, 2). An explanation for the lower pregnancy rate and poorer egg quality in these patients may be oxidative stress in infertile women with endometriosis. An imbalance between pro-oxidant molecules and antioxidants that are needed for proper cell function could contribute to the development of endometriosis and play a role in the infertility associated with it (3). The end product of oxidative metabolism can induce the formation of molecules in an active electronic state. Overall, these activated molecules derived from oxidative metabolism are called reactive oxygen species (ROS). Reactive oxygen species can directly affect the embryo or the surrounding Received December 7, 2011; revised March 24, 2012; accepted March 27, 2012; published online May 10, L.P. has nothing to disclose. J.F.Q. has nothing to disclose. O.C. has nothing to disclose. A.P. has nothing to disclose. A.P. has nothing to disclose. R.C. has nothing to disclose. J.A.G.-V. has nothing to disclose. Reprint requests: Juan A. Garcia-Velasco, M.D., IVI Madrid, Av del Talgo 68, Madrid, Spain ( juan.garcia.velasco@ivi.es). Fertility and Sterility Vol. 98, No. 1, July /$36.00 Copyright 2012 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert microenvironment (e.g, leukocytes, granulosa cells, endothelial cells) (4). Although controlled production of ROS is necessary for various physiological functions, high levels of ROS can overwhelm the antioxidant capacity and lead to oxidative stress damage (5, 6). To prevent ROS-induced damage, cells have antioxidant systems; thus, a delicate balance exists between ROS production and antioxidant activity, which maintains cellular homeostasis. When this balance is broken by excessive ROS production or a deficit in the antioxidant systems, a state of oxidative stress is created that causes cellular damage and dysfunction. Several studies have focused on the microenvironment surrounding the 126 VOL. 98 NO. 1 / JULY 2012

2 Fertility and Sterility oocyte and ROS and antioxidants found in the follicular fluid (FF) (7, 8). An imbalance between ROS and antioxidant systems in the FF could be responsible for abnormal oocyte development, causing DNA, cytoskeleton, and cell membrane damage, which would result in lower egg quality. Thus, ROS appear to be involved in different facets of endometriosis, peritoneal adhesion formation, and associated infertility, acting at different levels, including fertilization, embryo quality, implantation, and embryonic development (9 11). We studied different markers of oxidative stress in plasma FF from infertile patients with endometriosis and patients with sterility not caused by endometriosis (control) in an attempt to investigate a possible role of oxidative stress. MATERIALS AND METHODS We performed a prospective clinical cohort study from October 2008 to June We enrolled a total of 91 patients being tested and treated for infertility and healthy egg donors at the Hospital La Paz and IVI Madrid. The study received institutional review board approval by the institute's ethics committee. Patients were required to have been infertile for at least a year and diagnosed as sterile, and submitted to IVF or intracytoplasmic sperm injection in order to obtain a sample of FF because the experimental group consisted of patients whose only known cause of infertility was stage III IV endometriosis diagnosed either by ultrasound or laparoscopy and pathological study (n ¼ 23). Patients with other causes of infertility served as controls (n ¼ 68). The controls were grouped according to the cause of infertility: tubal factor (n ¼ 4), male factor (n ¼ 13), unexplained infertility (n ¼ 34), mixed infertility (n ¼ 3), and donors (n ¼ 14). Although laparoscopy remains the gold standard for a definitive diagnosis of endometriosis, transvaginal ultrasound provides a very precise diagnosis of ovarian endometriomas. Endometrioma was diagnosed by transvaginal sonography when an adnexal mass with diffuse low-level internal echoes and absence of particular neoplastic features, if no hyperechoic wall foci are present or features of acute hemorrhage are observed, in order to differentiate it from hemorrhagic cysts or corpus luteum (12). The most common sonographic features of an endometrioma is a cystic, unilocular sonolucent with precise boundaries and content, echoes of sparse, even distribution without septa or papillae, with no or very little Doppler map color inside. Images that are less typical, with alternating hyperechoic areas, hyperechoic areas, or sonolucent areas inside, can be observed, as well as completely cystic images of sonolucent content in which differentiating functional formations is difficult (13). Because diagnostic laparoscopy was not routinely performed in our patients, we cannot rule out the existence of minimal or mild endometriosis. However, careful imaging by experienced sonographers has demonstrated to be sensitive enough to detect small cysts or firm adhesions (13). Plasma samples were obtained from all patients the day of oocyte retrieval, centrifuged at 2,900 rpm for 10 min at room temperature, and the cell-free supernatants stored immediately in aliquots at 30 C until assayed. FF from the first two mature follicles was aspirated into 10 ml tubes. The needle was withdrawn and completely emptied before each puncture, and no culture medium was used in the collection tubes. Blood-contaminated FFs were excluded. After removing the oocytes, follicular aspirates were centrifuged at 2,900 rpm for 10 min and the supernatant stored at 30 C until assayed. Sample size was estimated on the basis of the statistical assumption that cases is a large enough number of cases to draw statistically significant conclusions in experimental settings (14). Antioxidants, consisting of vitamins C and E and the activity of the enzyme superoxide dismutase (SOD), were measured in both plasma and FF using spectrophotometry, high-performance liquid chromatography (HPLC), and ELISA, respectively. The parameter for measuring ROS was lipid oxidation malondialdehyde (MDA) via spectrophotometry. The normal range established in our laboratory for HPLC of serum vitamin E in adults is 8 21 mg/ml. The technique has a detection limit (sensitivity) of 0.8 mg/ml and a specificity of 97.3% (intraassay and interassay coefficient of variation of 2.58 and 2.64, respectively). The normal range established in our laboratory for spectrophotometric determination of serum vitamin C in adults is 6 20 ng/ml. However, no established values are available for vitamin C in FF. The technique has a detection limit (sensitivity) of 0.39 ng/ml, with a specificity approaching 98% (intraassay and interassay coefficient of variation of 2.33 and 2.51, respectively). For MDA, the normal range established in our laboratory is nmol/ml in serum, but no range is available for FF. The technique has a detection limit of 0.1 nmol/ml and specificity greater than 95%. We have not established normal ranges for the detection of SOD in serum or FF by ELISA. The detection limit of the test (sensitivity) is 0.04 ng/ml and the specificity is nearly 100% because no cross-reactions have been described. The intraassay and interassay precision is 5.1% and 5.8%, respectively. Categorical data were expressed as number and percentage, and numerical data were expressed as mean SEM. The statistical analysis used was Mann-Whitney test and Spearman's correlation. P <.05 was considered statistically significant. All statistical analyses were performed with the Statistics Package for Social Sciences version 10.0 (SPSS). RESULTS Cycle outcome and clinical data have been summarized in Table 1. In women with endometriosis, we found lower values of vitamin C in the FF ( vs mg/ml, P¼.003) (Fig. 1) and reduced SOD enzyme activity in the plasma ( vs U/mL, P¼.059) compared with the control group (Fig. 2). Vitamin E plasma levels were significantly higher in women with endometriosis ( vs mg/ml, P¼.001), although no differences were found in FF. The plasma MDA values in the control group were higher compared with the patients with endometriosis, but the difference was not significant ( vs mg/ml). We found a negative correlation between levels of plasma vitamin C and the number of oocytes retrieved, number of VOL. 98 NO. 1 / JULY

3 ORIGINAL ARTICLE: ENDOMETRIOSIS TABLE 1 FIGURE 2 Clinical data and cycle outcome. Endometriosis Controls P value No. of follicles >14 mm 6.0 (2 18) 10.5 (2 25).007 COCs retrieved 8 (4 22) 10 (4 22) NS Metaphase II obtained 7 (2 21) 9 (1 20) NS Fertilized oocytes 7 (1 14) 7 (0 16) NS Embryos obtained 6 (0 14) 7 (1 14) NS GQE 3 (0 9) 4 (0 13) NS Embryos transferred 2 (0 3) 2 (0 3) NS Frozen embryos 2 (0 10) 2 (0 8) NS Twin rate 17.4% 19.3% NS Miscarriage rate 13% 15.9% NS Ectopic pregnancy rate 1.8% 8.7% NS Note: Values within parentheses are ranges (min max). COC ¼ cumulus oocyte complex; GQE ¼ good-quality embryos; NS ¼ not significant. mature oocytes, and number of fertilized oocytes (P¼.036, P¼.007, and P¼.046, respectively). These same parameters showed a positive correlation with FF concentrations of SOD; the correlation was statistically significant for only the number of mature oocytes (P¼.023) but was close to statistically significant in the case of number of oocytes retrieved (P¼.053) and fertilized (P¼.054). As shown in Table 2, we also found a statistically significant positive correlation between the concentrations of lipoperoxides in FF and the implantation rate (P¼.0001). DISCUSSION Patients with endometriosis often have to resort to assisted reproductive techniques to achieve pregnancy. Numerous studies have shown worse outcomes for these techniques in patients with endometriosis compared with those without (1,2,15, 16); nevertheless, other studies have failed to demonstrate differences in IVF outcomes in these patients (1, 3, 17, 18). The role of oxidative stress in the pathophysiology of FIGURE 1 Follicular fluid vitamin C concentrations. Plasma activity of the enzyme superoxide dismutase (SOD). infertility in patients with endometriosis has been of interest in recent years (7, 19 22). An imbalance between prooxidant molecules and antioxidants may partially explain the sterility of these patients, especially in less severe degrees of illness in which the mechanical factor is not fully justified (10). Oxidative stress has been studied in normal ovarian cycles by quantifying various markers (5,7,23), several of which have been found in lower concentrations in plasma than FF, suggesting that FF has a high concentration of antioxidant systems that protect the oocyte from oxidative damage (23). Vitamin C is an important antioxidant system in follicles. The lack of vitamin C results in ovarian atrophy and extensive follicular atresia, demonstrating an important role against oxidative stress (23 25). We found lower vitamin C values in FF from patients with endometriosis compared with infertile patients without the disease. Our results are in favor of the FF having a greater antioxidant capacity in patients without disease. In patients with endometriosis, the levels of vitamin C in the FF would be lower because of excessive consumption of the antioxidant in an attempt to neutralize excess ROS or excess ROS may be present because of initially low levels of antioxidant capacity in the FF of these patients. The higher vitamin E concentration found in the plasma of women with endometriosis is in accordance with Jackson et al. (26), although other groups found discordant results (5, 6). We may hypothesize that this could be due to exogenous administration of vitamin E, either from an antioxidant-rich diet or as a supplement, because women with endometriosis are frequently given this type of supplements. Unfortunately, we did not control our patients for diet or vitamin E supplements. Inconsistent findings in oxidative stress among investigators are not unusual. In fact, there is little agreement in many different oxidative stress markers, and vitamin E may be one of them. Differences in stages of the disease, concomitant disease, specific diet intake, or even methodology and protocols to evaluate vitamin E may account for such 128 VOL. 98 NO. 1 / JULY 2012

4 Fertility and Sterility TABLE 2 Correlation between different cycle parameters and follicular fluid MDA concentrations in women with endometriosis. No. of COCs No. of Metaphase II No. of fertilized oocytes No. of embryos No. of GQE Implantation rate (%) Correlation coefficient P value Note: COC ¼ cumulus oocyte complex; GQE ¼ good-quality embryos. differences, and it is not easy to control for all confounding variables. In our patients, differences were significant, although as previously published (27 29), it does not seem to have an impact on pregnancy rates. Another important antioxidant system is SOD enzyme activity in the follicle. The presence of SOD enzymatic activity in the human follicle suggests that ROS and SOD play a role in the process of ovulation and oocyte development. Superoxide dismutase catalyzes the dismutation of superoxide into hydrogen peroxide and oxygen (30). Less enzyme activity in the plasma of patients with endometriosis compared with the control group suggests decreased antioxidant capacity in patients with endometriosis. The parameter to measure ROS in our study was MDA, which is a good marker of the metabolic activity within the follicle (3). Although we found no significant differences, we did find a trend of lower values for plasma MDA in patients with endometriosis. Similarly, Ota et al. (31) and Polak et al. (32) found that antioxidant enzymes, such as catalase, xanthine oxidase, and SOD, are overexpressed in patients with endometriosis in response to excess free radicals to neutralize the increased oxidative stress in these patients (20). This context would decrease MDA levels to those observed in patients without disease. We found a statistically significant negative correlation between plasma vitamin C levels and the number of oocytes retrieved, number of mature oocytes, and number of fertilized oocytes. Vitamin C is a potent natural antioxidant, so these findings were not surprising and may have a logical explanation. The lower concentrations of plasma vitamin C may be a reflection of excess consumption to neutralize ROS. Another explanation is that certain levels of ROS are necessary for proper oocyte maturation and embryonic development, and an excess of vitamin C would drastically reduce ROS concentrations (5, 6, 30). Similarly, we found a positive correlation between SOD in FF and the number of oocytes retrieved, number of mature oocytes, and number of fertilized oocytes. SOD is an antioxidant enzyme that protects the egg from ROS. SOD has been localized to the ovary, especially in the cells of the theca interna. Thus, the theca interna cells may protect the oocyte from oxidative stress during oocyte maturation (23). Numerous studies support our findings. Pasqualotto et al. (31) and Oyawoye et al. (8) studied the total antioxidant capacity of the FF (including the activity of antioxidant enzymes such as SOD) and found that the total antioxidant capacity of unfertilized oocytes was smaller than that of fertilized oocytes. In addition, total antioxidant capacity values were lower in patients who failed to achieve pregnancy. This finding would favor the idea of a protective role of antioxidants in the FF (32). Thus, the concentration of SOD in FF may be a marker of good prognosis in IVF. Finally, we found a positive correlation between levels of MDA in the FF and the implantation rate, which coincides with other reports (30 32). Patients who became pregnant after IVF or intracytoplasmic sperm injection had higher levels of MDA and total antioxidant capacity in the FF, which positively correlated with the pregnancy rate. In patients who were not pregnant, ROS concentrations were lower, suggesting that ROS occur at low concentrations in the FF and are perhaps even necessary for normal fertilization (33, 34). All of these findings suggest a reduced antioxidant capacity in infertile women with endometriosis. Although certain levels of ROS are required under physiological conditions, and bearing in mind the aforementioned limitations of our population studied, an imbalance between pro-oxidants and antioxidants may have an impact on folliculogenesis and embryonic development, which could contribute to a lower-quality oocyte and embryo in women with endometriosis. REFERENCES 1. Mahutte NG, Arici A. Endometriosis and assisted reproductive technologies: are outcomes affected? Curr Opin Obstet Gynecol 2001;13: Barnhart K, Dunsmoor-Su R, Coutifaris C. Effect of endometriosis on in vitro fertilization. Fertil Steril 2002;77: du Plessis S, Makker K, Desai N. Impact of oxidative stress on IVF. Expert Rev Obstet Gynecol 2008;3: Banwell K, Lane M, Russel D, Kind K, Thompson JG. Oxygen concentration during mouse oocyte in vitro maturation affects embryo and fetal development. Hum Reprod 2007;22: Agarwal A, Saleh R, Bedaiwy M. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003;79: Agarwal A, Gupta S, Sikka S. The role of free radicals and antioxidants in reproduction. Curr Opin Obstet Gynecol 2006;18: Agarwal A, Gupta S, Sharma R. Oxidative stress and its implications in female infertility a clinician's perspective. Reprod Biomed Online 2005;11: Oyawoye O, Abdel-Gadir A, Garner A, Constantinovici N, Perrett C, Hardiman P. Antioxidants and reactive oxygen species in follicular fluid of women undergoing IVF: relationship to outcome. Hum Reprod 2003;18: Van Langendonckt A, Casanas-Roux F, Donnez J. Oxidative stress and peritoneal endometriosis. Fertil Steril 2002;77: Shanti A, Santanam N. Autoantibodies to markers of oxidative stress are elevated in women with endometriosis. Fertil Steril 1999;71: Bedaiwy MA, Falcone T. Peritoneal fluid environment in endometriosis. Clinicopathological implications. Minerva Ginecol 2003;55: VOL. 98 NO. 1 / JULY

5 ORIGINAL ARTICLE: ENDOMETRIOSIS 12. Patel M, Feldstein V, Chen D, Lipson S, Filly R. Endometriomas: diagnostic performance of US. Radiology 1999;210: Garcia-Velasco JA, Cerrillo M, Ornat L. Ultrasonography of pelvic endometriosis. In: Botros Rizk, editor. Ultrasonography in reproductive medicine and infertility. Cambridge: Cambridge University Press; 2010: Pocock SJ. Clinical trials: a practical approach. Somerset, UK: Wiley Science Series; Wardle P, Mitchell J, McLaughlin E, Ray B, McDermott A, Hull M. Endometriosis and ovulatory disorder: reduced fertilisation in vitro compared with tubal and unexplained infertility. Lancet 1985;2: De Ziegler D, Borguese B, Chapron C. Endometriosis and infertility: pathophysiology and management. Lancet 2010;376: Prieto L, Cuadrado C, Ordas J, Gonzalez A. Resultado de las tecnicas de fecundacion in vitro en pacientes con endometriosis. Rev Iberoam Fertil 2005;22: Antoine JM. Endometriosis and fertility: physiopathology and treatment options. Contracept Fertil Sex 1995;23: Szczepanska M, Kozlic J, Skrzypezak J, Skrzypezak J. Oxidative stress may be a piece in the endometriosis puzzle. Fertil Steril 2003;79: Gupta S, Agarwal A, Krajcir N, Alvarez J. Role of oxidative stress in endometriosis. Reprod Biomed Online 2006;13: Foyouzi N, Berkkanoglu M, Arici A, Kwintkiewicz J, Izquierdo D, Duleba A. Effects of oxidants and antioxidants on proliferation of endometrial stromal cells. Fertil Steril 2004;82: Murphy AA, Palinski W, Rankin S, Morales AJ, Parthasarathy S. Evidence for oxidatively modified lipid-protein complexes in endometrium and endometriosis. Fertil Steril 1998;69: Gupta S, Surti N, Metterle L, Chandra A. Antioxidants and female reproductive pathologies. Arch Med Sci 2009;5: Tatemoto H, Ootaki K, Shigeta K, Muto N. Enhancement of developmental competence after in vitro fertilization of porcine oocytes by treatment with ascorbic acid 2-O-alpha-glucoside during in vitro maturation. Biol Reprod 2001;65: Dalvit G, Llanes S, Descalzo A, Insani M, Beconi M, Cetica P. Effect of alphatocopherol and ascorbic acid on bovine oocyte in vitro maturation. Reprod Domest Anim 2005;40: Jackson L, Schisterman E, Dey-Rao R, Browne R, Armstrong D. Oxidative stress and endometriosis. Hum Reprod 2005;20: Andrade A, Rodriguez J, Dib L, Romao G, Ferriani R, Jordao A, et al. Serum markers of oxidative stress in infertile women with endometriosis. Rev Bras Ginecol Obstet 2010;32: Mier-Cabrera J, Genera-Garcia M, De la Jara-Diaz J, Perichart-Perera O, Vadillo-Ortega F, Hernandez-Guerrero C. Effect of vitamins C and E supplementation on peripheral oxidative stress markers and pregnancy rate in women with endometriosis. Int J Obstet Gynecol 2008;100: Mier-Cabrera J, Aburto-Soto T, Burrola-Mendez S, Jimenez-Zamudio L, Tolentino M, Casanueva E, et al. Women with endometriosis improved their peripheral antioxidant markers after the application of a high antioxidant diet. Reprod Biol Endocrinol 2009;7: Agarwal A, Allamaneni S. Role of free radicals in female reproductive diseases and assisted reproduction. Reprod Biomed Online 2004;9: Pasqualotto E, Agarwal A, Sharma R, Izzo V, Pinotti J, Joshi N, et al. Effect of oxidative stress in follicular fluid on the outcome of assisted reproductive procedures. Fertil Steril 2004;81: Attaran M, Pasqualotto E, Falcone T, Goldberg J, Miller K, Agarwal A, et al. The effect of follicular fluid reactive oxygen species on the outcome of in vitro fertilization. Int J Fertil Womens Med 2000;45: Ota H, Igarashi S, Hatazawa J, Tanaka T. Endometriosis and free radicals. Gynecol Obstet Invest 1999;48: Polak G, Koziol-Montewka M, Gogacz M, Blaszkowska I, Kotarski J. Total antioxidant status of peritoneal fluid in infertile women. Eur J Obstet Gynecol Reprod Biol 2001;94: VOL. 98 NO. 1 / JULY 2012