Hum. Reprod. Advance Access originally published online on July 22, 2006
Human Reproduction 2006 21(9):2403-2407; doi:10.1093/humrep/del156
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reactive oxygen species level in follicular fluid—embryo quality marker in IVF?
1 School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India and 2 Institute of Reproductive Medicine, Salt Lake, Kolkata, West Bengal, India
3 To whom correspondence should be addressed at: SMST, IIT Kharagpur, Kharagpur, West Bengal, India. E-mail: koel{at}smst.iitkgp.ernet.in
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
BACKGROUND: The impact of oxidative stress in female reproduction is not clear. Contradictory reports on the effect of various oxidative stress markers on follicular fluid, oocytes and embryo quality and fertilization potential exist. The objectives of this study were to examine reactive oxygen species (ROS) levels in follicular fluid of women undergoing IVF and to relate these levels to embryo formation and quality. METHODS AND RESULTS: A total of 208 follicular fluid samples were obtained from 78 women undergoing controlled ovarian stimulation and analysed for ROS and lipid peroxidation (LPO). These samples were divided into groups I and II which represented follicular fluid containing grade III and grade II oocytes, respectively. These groups were further subdivided into groups IA, IB, IIA and IIB according to embryo quality. Subgroups IA and IIA consisted of follicular fluid samples corresponding to grade I/II embryo formation. Subgroups IB and IIB represented fertilization failure/pro-nucleolus (PN) arrest/grade III embryos. No significant correlation was observed in ROS levels on comparing groups I and II (P > 0.05). However, ROS levels were observed to be significantly different on comparing groups IA and IB (P
0.01) and groups IIA and IIB (P 0.05). LPO levels further supported our results. CONCLUSION: ROS levels in follicular fluid appear to play a significant role in embryo formation and quality.
Key words: embryo/follicular fluid/IVF/lipid peroxidation/reactive oxygen species
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
IVF, a popular assisted reproduction technique (ART), is a widely accepted procedure for the treatment of infertility. Unfortunately, the success rate of this technique, measured as the average pregnancy rate per cycle, is only 30–40% (Gerris et al., 1999
). Reactive oxygen species (ROS) has been considered to play a critical role in the success of different ARTs (Sikka, 2004). The impact of ROS on the reproductive potential in men is a subject of extensive research worldwide (Aitken, 1999; Wang et al., 2003; Baker and Aitken, 2005; Nallella et al., 2005). However, there are limited reports about the possible effects of ROS in the female reproductive system (Oyawoye et al., 2003; Pasqualotto et al., 2004; Agarwal et al., 2005).
Follicular fluid may be regarded as a biological ‘window’ reflecting metabolic and hormonal processes occurring in the microenvironment of the maturing oocyte before ovulation and also as a predictor of outcome parameters such as fertilization, embryo cleavage and pregnancy rates in IVF (Wiener-Megnazi et al., 2004). Nevertheless, the relationship between oxidative stress parameters in follicular fluid and IVF outcome has not yet been extensively investigated. Limited knowledge of ROS present in follicular fluid and its subsequent effect on oocyte maturation, fertilization and pregnancy is available. This is attributed to the lack of information on the physiological ROS levels in follicular fluid of normal healthy women in unstimulated ovarian cycles (Attaran et al., 2000). Inconsistent results related to the detrimental and beneficial levels of ROS persist. Whereas Agarwal et al. (2005) suggest that oxidative stress influences the oocyte and embryo quality and thus the fertilization rate, Jozwik et al. (1999) have reported that the concentration of oxidative stress markers such as conjugated dienes, lipid hydroperoxides and thiobarbituric acid-reactive substances in follicular fluid do not reflect the reproductive potential of oocytes. A group of investigators have reported that women who became pregnant by IVF had higher levels of ROS (Attaran et al., 2000) and lipid peroxidation (LPO) (Pasqualotto et al., 2004) than those who did not. On the contrary, Oyawoye et al. (2003) have shown that higher total antioxidant capacity (TAC) level increases fertilization potential in women undergoing IVF. This is in agreement with studies conducted on genetically manipulated mice where a decrease in the litter size and number of litters per month with deficiency of superoxide dismutase (SOD) level has been reported (Ho et al., 1998; Matzuk et al., 1998).
It is evident that the relationship between ROS levels in follicular fluid and oocyte and embryo quality requires further exploration. The objectives of this study were to assess ROS levels in follicular fluid of women undergoing IVF and to relate these levels to embryo formation and quality.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
This study was carried out in the Institute of Reproductive Medicine, Salt Lake, Kolkata, India. A part of the study was done at the Medical Biotechnology Unit, School of Medical science and Technology, IIT, Kharagpur, India. Approval for this study was obtained from the research ethics committee of the Institute. Written informed consent was obtained from women included in this study.
Subject selection
Seventy-eight women with tubal factor infertility reporting for IVF-embryo transfer by controlled ovarian stimulation (long protocol) were included. For the purposes of this study, tubal factor infertility refers to women who had salpingectomy for ectopic pregnancy and proximal tubal obstruction because of low-grade infection or fimbrial occlusion with or without mild peritubal adhesion. Tubal infertility associated with gross hydrosalpingeal changes, dense pelvic adhesion because of endometriosis or pelvic inflammatory disease (PID) has not been included in this study.
These women were down-regulated with a GnRH agonist (Lupride, Sun Pharmaceuticals, Mumbai, India) from mid-luteal phase onwards and, when optimally down-regulated, were stimulated with recombinant FSH (Gonal F, Serono, Geneva, Switzerland). Follicular size was monitored regularly by ultrasound and serum estradiol assays. Subcutaneous hCG (Pregnyl, Organon, the Netherlands) was administered when average diameter of the leading follicles reached at least 18 mm. The oocytes, following retrieval under transvaginal ultrasound guidance, were graded (Veeck, 1999) and subsequently inseminated. Pro-nucleolus (PN) score was noted 16–18 h after insemination. Embryo quality was assessed before embryo transfer (Veeck, 1999), and a maximum of three embryos were transferred to all patients approximately 48 h (4-cell stage) after insemination.
Collection of follicular fluid and processing
Follicular fluid, from 290 follicles (diameter in the range of 15–20 mm) during oocyte retrieval, was carefully aspirated from each follicle and collected in individual tubes. Follicular fluid was not collected from follicles when the follicular diameter <15 mm and when follicular fluid contained more than one oocyte or no oocyte or included an oocyte with abnormal morphology (presence of cytoplasmic droplet or granules). Semen samples from husband/donor were also obtained and analysed according to WHO guidelines (World Health Organization, 1999). Follicular fluid samples were included for only those women whose male partners had sperm with total motility 
50%, morphology 35% and ROS levels in the range of 0.02–0.05 count per minute (cpm) x 106/10 million sperm cells. Furthermore, these semen samples were subjected to hypo-osmotic swelling test (HOST; inclusion criteria 60%) and chromatin decondensation test (inclusion criteria 60%). Follicular fluid contaminated with culture medium or blood was discarded.
Samples of follicular fluid were classified as group I (n = 125; metaphase II and grade III oocyte) and group II (n = 83; metaphase I and grade II oocyte). Group I was further divided into two subgroups based on subsequent embryo formation: group IA represented grade I and grade II embryos, whereas group IB represented fertilization failure/PN arrest/grade III embryo. Similarly, group II was subdivided into group IIA and group IIB.
Follicular fluid samples were centrifuged at 300 x g for 7 min to remove cellular components and the clear supernatant divided into aliquots—one part was frozen in liquid nitrogen (–180°C) and the other was immediately used for the measurement of ROS.
Measurement of ROS
ROS levels in freshly aspirated follicular fluid were evaluated by chemiluminescence assay (Attaran et al., 2000) using luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) as a probe. Four Hundred microlitres of clear supernatant was used in the cuvette of the luminometre (Berthold, Sirius Single tube Luminometer, model no. 0727), and 10 µl luminol (5 mM) in DMSO was added to it. Each sample was scanned for 10 min. ROS values were expressed as counted photons per second (cps).
Measurement of LPO
Malonaldehyde (MDA) was measured using the thiobarbituric acid (TBA) method (Buege and Aust, 1978). MDA reacts with TBA to give a red compound, which has an absorbance at 535 nm.
The frozen aliquot was thawed and immediately used for the estimation of LPO. Two millilitres of stock reagent (12% w/v trichloro-acetic acid, 0.375% w/v TBA and 0.25 mol/l HCl warmed to dissolve the TBA) was mixed thoroughly with 1 ml of the sample and heated for 15 min in a boiling water bath. After cooling, the flocculent precipitate was removed by centrifugation at 1000 x g for 10 min and the optical density of the supernatant determined at 535 nm against a blank containing all the reagents. LPO values were expressed as µM MDA.
Statistical analysis
Data were analysed using analysis of variance (one-way ANOVA) and t-test, as appropriate. All analyses were performed with Ky Plot version 2.0 beta 13 (Koichi Yoshioka, 1997–2000). Statistical significance was defined as P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Two hundred and eight follicular fluid samples from 78 women with tubal factor infertility undergoing IVF were examined for ROS levels and LPO. A linear correlation was observed between these two parameters of oxidative stress in both group I (r = 0.880) and group II (r = 0.954). ROS and LPO levels in the samples of follicular fluid of the two groups are summarized in Table I. No significant differences were observed in both the groups (P > 0.05). ROS and LPO values in follicular fluid of groups IA, IB, IIA and IIB are summarized in Table II. Significant differences were observed in both ROS (P ( 0.01) and LPO (P ( 0.05) levels on comparing groups IA and IB. A similar trend was observed on comparing ROS ((0.05) and LPO values (P ( 0.05) of groups IIA and IIB.
|
|
Embryo formation (%) in each 10 cps interval of follicular fluid ROS (for a range of 41–150 cps) is presented in Figures 1 and 2 for group I and group II, respectively. The bar diagrams clearly define two separate clusters of embryo formation (%) in the range of 41–100 cps and >100 cps, which are tabulated in Table III. The table summarizes that in group I, improved embryo formation (%) and embryo quality are observed below a certain threshold level (
100 cps) as compared with ROS >100 cps. No marked differences were observed on comparing embryo formation (%) for higher and lower ROS levels in group II. It is, however, interesting to note that better embryo quality was formed in the lower range of ROS as compared with ROS >100 cps in group II. Also, embryo formation (%) was found to be higher in group I as compared with group II at low levels of ROS.
|
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
In routine IVF cycles, it is common to see that few morphologically normal metaphase II oocytes obtained from matured follicles remain unfertilized or, if fertilized, result in poor-quality embryo formation, under the same culture conditions. The role of ROS, if any, in such unfertilized oocytes and in poor qualities of embryo formation is explored. Furthermore, effect of follicular fluid ROS levels on fertilization/embryo grade is investigated. This study is expected to help clinicians considerably in identifying potential causes of poor fertilization associated with IVF.
Follicular fluid makes up the actual environment of the mature oocyte before fertilization and may influence IVF outcome parameters such as fertilization, embryo cleavage and pregnancy rates (Agarwal et al., 2003). This environment, in addition to granulosa cells, growth factors and steroids hormones, contains leukocytes, cytokines and macrophages, all of which can produce ROS (Attaran et al., 2000). Hence, ROS may be produced by either the environment or impaired metabolism of the oocyte, or both. Follicular fluid surrounding morphologically poor-quality oocytes or oocytes from follicle with a diameter <15 mm were excluded from the study as it would be difficult to ascertain whether the fertilization failure or poor embryo quality is because of excessive ROS in follicular fluid or because of poor grade of the oocyte, per se.
No significant association was observed between oocyte maturation and ROS levels in follicular fluid for grade II and grade III oocytes. Our findings are in good agreement with the reports of Pasqualotto et al. (2004), where no association between LPO and TAC levels and oocyte maturity is reported. In addition, no association was found between LPO and TAC levels and embryo quality, fertilization and cleavage by the same research group. These observations are in accordance with the results reported by Attaran et al. (2000). Similar observations have also been reported by Jozwik et al. (1999), which suggest that concentration of oxidative stress markers in follicular fluid do not reflect the fertilization potential of oocytes. Interestingly, in this study, significant negative correlation between ROS level in follicular fluid and embryo quality has been observed. This finding is further supported by the estimated LPO values, which also show a significant negative correlation with embryo quality. Our findings are in good agreement with the reports of Oyawoye et al. (2003), who have also observed that lower levels of TAC predict decreased fertilization potential. Deficiency of selenium-dependent glutathione peroxidase (SeGPX) level leading to failure of fertilization is reported by Paszkowski et al. (1995), which also supports our results.
Another interesting finding was observed on plotting a bar diagram with embryo formation (%) on the y axis and ROS (at an interval of 10 cps) on the x axis. In this study, the lower limit of ROS interval was taken to be 41 cps as this value corresponded to the minimum value of ROS recorded. The upper limit was marked at 150 cps as very few samples of follicular fluid exhibited ROS beyond this level. Figures 1 and 2 suggest that ROS levels in follicular fluid, above 100 cps, tend to inhibit embryo formation or fertilization. Subsequently, embryo formation (%) and embryo quality were compared between the two ranges of ROS in follicular fluid, 41–100 cps and >100 cps (Table III). Higher percentage of embryo formation as well as good embryo quality was observed corresponding to follicular fluid with ROS levels <100 cps in both groups (group I and group II). This result is again inconsistent with Attaran et al. (2000), where interference of low ROS with pregnancy outcome is suggested. In this study, association of ROS and LPO levels with pregnancy outcome was not attempted as more than one embryo was transferred (Oyawoye et al., 2003). Figures 1 and 2 clearly indicate that the effect of ROS on embryo formation is favourable up to a certain threshold level of ROS, which appears to be about 100 cps. Detrimental effect of ROS may become significant beyond this range. It was considered worthwhile to report this finding so that various values of ROS in this region are extensively investigated. It is envisaged that the acceptable threshold value of ROS in follicular fluid, beyond which the effect is detrimental for embryo formation, may be established.
The influence of oocyte grade on fertilization rate and embryo quality, though controversial in ICSI, is well established in IVF (Veeck, 1988; Bedford and Kim, 1993). As mentioned earlier, groups I and II consist of metaphase II/mature and metaphase I/immature oocytes, respectively. In accordance with the observations of Veeck (1988) and Bedford and Kim (1993), higher percentage of embryo formation is observed with grade III mature oocytes (group I) as compared with grade II oocytes (group II) for the same levels of ROS. Similar explanation may be put forward for group II where no difference in embryo formation at low and high levels of ROS was observed. Significant contributory effects of ROS in grade II oocytes could not be ascertained, possibly because of the oocyte quality being a major factor for poor percentage of embryo formation. Assessment of ROS levels in all grades of oocytes is suggested to get a clear picture of the oxidative stress impact on oocyte maturity.
In summary, it appears that the role of oxidative stress in relation to female reproduction needs further investigation and evaluation. A better understanding may be achieved by investigating the role of antioxidant activity in follicular fluid. Conflicting reports exist on the way various follicular fluid oxidative stress markers affect the quality of oocytes and embryos and their subsequent association with fertilization and pregnancy. Our results suggest that high levels of ROS in follicular fluid obtained from women with tubal infertility tend to decrease the fertilization potential of oocytes. Future studies on large sample sizes of women with other causes of infertility undergoing IVF are required to highlight the critical role of oxidative stress markers and their optimum levels in female reproduction. This is expected to lead to improved ART success rate and infertility management.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
The authors thank Sunil Kumar, Research Scholar, School of Medical Science and Technology, IIT Kharagpur for assisting in the preparation of the manuscript. Department of Biotechnology, Government of India’s financial support to carry out this study is gratefully acknowledged.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Agarwal A, Saleh RA, Bedaiwy MA. (2003) Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 79:829–843.[CrossRef][Web of Science][Medline]
Agarwal A, Gupta S, Sharma R. (2005) Oxidative stress and its implications in female infertility – a clinician’s perspective. Reprod Biomed Online 11:641–650.[Web of Science][Medline]
Aitken RJ. (1999) The Amoroso Lecture. The human spermatozoon – a cell in crisis? J Reprod Fertil 115:1–7.
Attaran M, Pasqualotto E, Falcone T, Goldberg JM, Miller KF, Agarwal A, Sharma RK. (2000) The effect of follicular fluid reactive oxygen species on the outcome of in vitro fertilization. Int J Fertil Womens Med 45:314–320.[Web of Science][Medline]
Baker MA and Aitken RJ. (2005) Reactive oxygen species in spermatozoa: methods for monitoring and significance for the origins of genetic disease and infertility. Reprod Biol Endocrinol 3:67.[CrossRef][Medline]
Bedford JM and Kim HH. (1993) Sperm/egg binding patterns and oocyte cytology in retrospective analysis of fertilization failure in vitro. Hum Reprod 8:453–463.
Buege JA and Aust SD. (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302–310.[Medline]
Gerris J, De Neubourg D, Mangelschots K, Van Royen E, Van de Meerssche M, Valkenburg M. (1999) Prevention of twin pregnancy after in-vitro fertilization or intracytoplasmic sperm injection based on strict embryo criteria: a prospective randomized clinical trial. Hum Reprod 14:2581–2587.
Ho JS, Gargano M, Cao J, Bronson RT, Heimler I, Hutz RJ. (1998) Reduced fertility in female mice lacking copper-zinc superoxide dismutase. J Biol Chem 273:7765–7769.
Jozwik M, Wolczynski S, Jozwik M, Szamatowicz M. (1999) Oxidative stress markers in preovulatory follicular fluid in humans. Mol Hum Reprod 5:409–413.
Matzuk MM, Dionne L, Guo Q, Kumar TR, Lebovitz RM. (1998) Ovarian function in superoxide dismutase 1 and 2 knockout mice. Endocrinology 139:4008–4011.
Nallella KP, Sharma RK, Allamaneni SS, Agarwal A. (2005) Identification of male factor infertility using a novel semen quality score and reactive oxygen species levels. Clinics 60:317–324.[Medline]
Oyawoye O, Abdel Gadir A, Garner A, Constantinovici N, Perrett C, Hardiman P. (2003) Antioxidants and reactive oxygen species in follicular fluid of women undergoing IVF: relationship to outcome. Hum Reprod 18:2270–2274.
Pasqualotto EB, Agarwal A, Sharma RK, Izzo VM, Pinotti JA, Joshi NJ, Rose BI. (2004) Effect of oxidative stress in follicular fluid on the outcome of assisted reproductive procedures. Fertil Steril 81:973–976.[CrossRef][Web of Science][Medline]
Paszkowski T, Traub AI, Robinson SY, McMaster D. (1995) Selenium dependent glutathione peroxidase activity in human follicular fluid. Clin Chim Acta 236:173–180.[CrossRef][Web of Science][Medline]
Sikka SC. (2004) Andrology lab corner-role of oxidative stress and antioxidants in andrology and assisted reproductive technology. J Androl 25:5–18.
Veeck LL. (1988) Oocyte assessment and biological performance. Ann N Y Acad Sci 541:259–274.[Web of Science][Medline]
Veeck LL. (1999) An Atlas of Human Gametes and Conceptuses: An Illustrated Reference for Assisted Reproductive Technology(Parthenon Publishing, New York).
Wang X, Sharma RK, Sikka SC, Thomas AJ Jr, Falcone T, Agarwal A. (2003) Oxidative stress is associated with increased apoptosis leading to spermatozoa DNA damage in patients with male factor infertility. Fertil Steril 80:531–535.[CrossRef][Web of Science][Medline]
Wiener-Megnazi Z, Vardi L, Lissak A, Shnizer S, Reznick AZ, Ishai D, Lahav-Baratz S, Shiloh H, Koifman M, Dirnfeld M. (2004) Oxidative stress indices in follicular fluid as measured by the thermochemiluminescence assay correlate with outcome parameters in in vitro fertilization. Fertil Steril 82:1171–1176.
Laboratory Manual for the Examination of Human Semen and Sperm–Cervical Mucus Interaction (1999) 4th edn (Cambridge University Press, New York) World Health Organization.
Submitted on February 10, 2006; resubmitted on April 12, 2006; accepted on April 13, 2006.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. Berker, C. Kaya, R. Aytac, and H. Satiroglu Homocysteine concentrations in follicular fluid are associated with poor oocyte and embryo qualities in polycystic ovary syndrome patients undergoing assisted reproduction Hum. Reprod., September 1, 2009; 24(9): 2293 - 2302. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R.W. Browne, W.B. Shelly, M.S. Bloom, A.J. Ocque, J.R. Sandler, H.G. Huddleston, and V.Y. Fujimoto Distributions of high-density lipoprotein particle components in human follicular fluid and sera and their associations with embryo morphology parameters during IVF Hum. Reprod., August 1, 2008; 23(8): 1884 - 1894. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. H. Ruder, T. J. Hartman, J. Blumberg, and M. B. Goldman Oxidative stress and antioxidants: exposure and impact on female fertility Hum. Reprod. Update, June 4, 2008; (2008) dmn011v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. P.A. van Montfoort, J. P.M. Geraedts, J. C.M. Dumoulin, A. P.M. Stassen, J. L.H. Evers, and T. A.Y. Ayoubi Differential gene expression in cumulus cells as a prognostic indicator of embryo viability: a microarray analysis Mol. Hum. Reprod., March 1, 2008; 14(3): 157 - 168. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Tatone, F. Amicarelli, M. C. Carbone, P. Monteleone, D. Caserta, R. Marci, P. G. Artini, P. Piomboni, and R. Focarelli Cellular and molecular aspects of ovarian follicle ageing Hum. Reprod. Update, March 1, 2008; 14(2): 131 - 142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Morimoto, Y. Oku, M. Sonoda, A. Haruki, K. Ito, S. Hashimoto, and A. Fukuda High oxygen atmosphere improves human follicle development in organ cultures of ovarian cortical tissues in vitro Hum. Reprod., December 1, 2007; 22(12): 3170 - 3177. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F.F. Pasqualotto and E.B. Pasqualotto Reactive oxygen species and oocyte fertilization Hum. Reprod., March 1, 2007; 22(3): 901 - 901. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||