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Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2018, Vol. 12 Issue (5) : 518-524    https://doi.org/10.1007/s11684-017-0575-y
RESEARCH ARTICLE |
Oxidative stress in granulosa cells contributes to poor oocyte quality and IVF-ET outcomes in women with polycystic ovary syndrome
Qiaohong Lai1(), Wenpei Xiang2, Qing Li1, Hanwang Zhang1, Yufeng Li1, Guijin Zhu1, Chengliang Xiong2, Lei Jin1()
1. Reproductive Medicine Center, Tongji Hospital
2. Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
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Abstract

The increased levels of intracellular reactive oxygen species (ROS) in granulosa cells (GCs) may affect the pregnancy results in women with polycystic ovary syndrome (PCOS). In this study, we compared thein vitro fertilization and embryo transfer (IVF-ET) results of 22 patients with PCOS and 25 patients with tubal factor infertility and detected the ROS levels in the GCs of these two groups. Results showed that the PCOS group had significantly larger follicles on the administration day for human chorionic gonadotropin than the tubal factor group (P<0.05); however, the number of retrieved oocytes was not significantly different between the two groups (P>0.05). PCOS group had slightly lower fertilization, cleavage, grade I/II embryo, clinical pregnancy, and implantation rates and higher miscarriage rate than the tubal factor group (P>0.05). We further found a significantly higher ROS level of GCs in the PCOS group than in the tubal factor group (P<0.05). The increased ROS levels in GCs caused GC apoptosis, whereas NADPH oxidase 2 (NOX2) specific inhibitors (diphenyleneiodonium and apocynin) significantly reduced the ROS production in the PCOS group. In conclusion, the increased ROS expression levels in PCOS GCs greatly induced cell apoptosis, which further affected the oocyte quality and reduced the positive IVF-ET pregnancy results of women with PCOS. NADPH oxidase pathway may be involved in the mechanism of ROS production in GCs of women with PCOS.

Keywords PCOS      ROS      granulosa cell      IVF-ET      NADPH oxidase     
Corresponding Authors: Qiaohong Lai,Lei Jin   
Just Accepted Date: 01 November 2017   Online First Date: 26 December 2017    Issue Date: 29 September 2018
 Cite this article:   
Qiaohong Lai,Wenpei Xiang,Qing Li, et al. Oxidative stress in granulosa cells contributes to poor oocyte quality and IVF-ET outcomes in women with polycystic ovary syndrome[J]. Front. Med., 2018, 12(5): 518-524.
 URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0575-y
http://academic.hep.com.cn/fmd/EN/Y2018/V12/I5/518
Parameters PCOS (n = 22) Tubal (n = 25) P value
Age (year) 28.64±3.12 29.96±4.01 NS
Basal FSH (mIU/mL) 5.79±2.65 6.66±2.13 NS
Basal LH (IU/L) 5.61±3.26 5.54±3.17 NS
BMI (kg/m2) 23.21±2.85 22.12±1.89 NS
D3 antral follicle count (AFC) 21.36±8.14 13.27±6.32 <0.05
Duration of infertility (year) 3.29±1.22 3.66±2.35 NS
Tab.1  Baseline characteristics of the PCOS and tubal groups
Parameters PCOS (n = 22) Tubal (n = 25) P value
rFSH dosage (amp) 22.95±8.17 26.37±9.49 NS
rFSH duration (d) 9.86±1.94 9.51±1.88 NS
HCG day
Estradiol (pmol/L) 4873.27±3202.11 3294.87±3018.24 <0.05
Progesterone (ng/mL) 1.34±0.57 1.29±0.94 NS
≥14 mm follicles 20.23±9.18 15.21±8.73 <0.05
Endometrial thickness (mm) 12.51±2.53 11.37±2.14 NS
Oocytes retrieved 15.18±1.36 11.78±1.25 <0.05
Mature oocytes 9.09±1.23 8.57±1.01 NS
Fertilization rate (2PN) (%)
Cleavage rate (2PN) (%)
46.21
87.74
58.26
98.65
NS
NS
Grade I/II embryo formation (%) 39.96 48.72 NS
Clinical pregnancy rate (%) 42.98 50.67 NS
Implantation rate (%) 38.26 46.34 NS
Miscarriage rate (%) 22 15 NS
Tab.2  Ovarian stimulation characteristics and IVF results for the PCOS and tubal groups
Fig.1  Comparison of the GC ROS level and apoptosis between PCOS and tubal factor groups. (A) Isolated GCs were resuspended in DMEM medium and incubated at 25 µmol/L carboxy-H2DCF-DA in 30 min. Green fluorescence was observed under a fluorescence microscope. The GC suspension in each well of the 96-well plates was seeded, and the fluorescent intensity was measured by microplate fluorescence. (B) GC apoptosis was detected by TUNEL assay. White arrows indicate apoptotic cells. Positive cells were counted, and the difference was significant between two groups (P<0.05).
Fig.2  (A) The western blot results of NOX2 subunits in GC. The expression levels of p47phox and gp91phox protein of the two groups are not different. (B) The intracellular ROS fluorescence was detected. GCs were divided into three groups: DMSO group (10 mg/mL), DPI group (5 pmol/L), and apocynin group (20 pmol/L) and were incubated for 1 h. The cells were washed and added with 25 µmol/L carboxy-H2DCF-DA for 30 min, and the ROS level was detected. The DPI and apocynin in tubal factor group slightly inhibited the ROS levels compared with DMSO, and no differences were observed. The ROS levels in PCOS group significantly decreased after adding DPI and apocynin compared with adding DMSO (P<0.05).
1 Lainas TG, Sfontouris IA, Zorzovilis IZ, Petsas GK, Lainas GT, Alexopoulou E, Kolibianakis EM. Flexible GnRH antagonist protocol versus GnRH agonist long protocol in patients with polycystic ovary syndrome treated for IVF: a prospective randomised controlled trial (RCT). Hum Reprod 2010; 25(3): 683–689
https://doi.org/10.1093/humrep/dep436 pmid: 20008886
2 Orvieto R, Meltcer S, Homburg R, Nahum R, Rabinson J, Ashkenazi J. What is the preferred GnRH analogue for polycystic ovary syndrome patients undergoing controlled ovarian hyperstimulation for in vitro fertilization? Fertil Steril 2009; 91(4 Suppl): 1466–1468
https://doi.org/10.1016/j.fertnstert.2008.07.1711 pmid: 18774558
3 Homburg R. Polycystic ovary syndrome. Best Pract Res Clin Obstet Gynaecol 2008; 22(2): 261–274
https://doi.org/10.1016/j.bpobgyn.2007.07.009 pmid: 17804299
4 Pal L, Zhang H, Williams J, Santoro NF, Diamond MP, Schlaff WD, Coutifaris C, Carson SA, Steinkampf MP, Carr BR, McGovern PG, Cataldo NA, Gosman GG, Nestler JE, Myers E, Legro RS; Reproductive Medicine Network. Vitamin D status relates to reproductive outcome in women with polycystic ovary syndrome: secondary analysis of a multicenter randomized controlled trial. J Clin Endocrinol Metab 2016; 101(8): 3027–3035
https://doi.org/10.1210/jc.2015-4352 pmid: 27186859
5 Klevedal C, Turkmen S. Fetal-maternal outcomes and complications in pregnant women with polycystic ovary syndrome. Minerva Ginecol 2017; 69(2): 141–149
pmid: 27310674
6 Sabuncu T, Vural H, Harma M, Harma M. Oxidative stress in polycystic ovary syndrome and its contribution to the risk of cardiovascular disease. Clin Biochem 2001; 34(5): 407–413
https://doi.org/10.1016/S0009-9120(01)00245-4 pmid: 11522279
7 Uyar A, Torrealday S, Seli E. Cumulus and granulosa cell markers of oocyte and embryo quality. Fertil Steril 2013; 99(4): 979–997
https://doi.org/10.1016/j.fertnstert.2013.01.129 pmid: 23498999
8 Huang B, Qian K, Li Z, Yue J, Yang W, Zhu G, Zhang H. Neonatal outcomes after early rescue intracytoplasmic sperm injection: an analysis of a 5-year period. Fertil Steril 2015; 103(6): 1432–7.e1
https://doi.org/10.1016/j.fertnstert.2015.02.026 pmid: 25813286
9 Adashi EY. Endocrinology of the ovary. Hum Reprod 1994; 9(5): 815–827
https://doi.org/10.1093/oxfordjournals.humrep.a138602 pmid: 7929728
10 Jakimiuk AJ, Weitsman SR, Navab A, Magoffin DA. Luteinizing hormone receptor, steroidogenesis acute regulatory protein, and steroidogenic enzyme messenger ribonucleic acids are overexpressed in thecal and granulosa cells from polycystic ovaries. J Clin Endocrinol Metab 2001; 86(3): 1318–1323
pmid: 11238527
11 Sharma RK, Duda T. Ca(2+)-sensors and ROS-GC: interlocked sensory transduction elements: a review. Front Mol Neurosci 2012; 5: 42
https://doi.org/10.3389/fnmol.2012.00042 pmid: 22509149
12 Seino T, Saito H, Kaneko T, Takahashi T, Kawachiya S, Kurachi H. Eight-hydroxy-2′-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilization-embryo transfer program. Fertil Steril 2002; 77(6): 1184–1190
https://doi.org/10.1016/S0015-0282(02)03103-5 pmid: 12057726
13 Jancar N, Kopitar AN, Ihan A, Virant Klun I, Bokal EV. Effect of apoptosis and reactive oxygen species production in human granulosa cells on oocyte fertilization and blastocyst development. J Assist Reprod Genet 2007; 24(2-3): 91–97
https://doi.org/10.1007/s10815-006-9103-8 pmid: 17216562
14 Rajani S, Chattopadhyay R, Goswami SK, Ghosh S, Sharma S, Chakravarty B. Assessment of oocyte quality in polycystic ovarian syndrome and endometriosis by spindle imaging and reactive oxygen species levels in follicular fluid and its relationship with IVF-ET outcome. J Hum Reprod Sci 2012; 5(2): 187–193
https://doi.org/10.4103/0974-1208.101020 pmid: 23162358
15 Huang B, Yang F, Dong X, Zheng Y, Tan H, Ai J, Jin L . Lower limit of antioxidant activity in follicular fluid: relationship to embryo quality in IVF cycle. Int J Clin Exp Med 2016; 9(8): 16346–16352
16 Yang CM, Lee IT, Hsu RC, Chi PL, Hsiao LD. NADPH oxidase/ROS-dependent PYK2 activation is involved in TNF-α-induced matrix metalloproteinase-9 expression in rat heart-derived H9c2 cells. Toxicol Appl Pharmacol 2013; 272(2): 431–442
https://doi.org/10.1016/j.taap.2013.05.036 pmid: 23774252
17 Lambeth JD. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 2004; 4(3): 181–189
https://doi.org/10.1038/nri1312 pmid: 15039755
18 Huang B, Ren X, Wu L, Zhu L, Xu B, Li Y, Ai J, Jin L. Elevated progesterone levels on the day of oocyte maturation may affect top quality embryo IVF cycles. PLoS One 2016; 11(1): e0145895
https://doi.org/10.1371/journal.pone.0145895 pmid: 26745711
19 Liu HC, He ZY, Mele CA, Veeck LL, Davis O, Rosenwaks Z. Human endometrial stromal cells improve embryo quality by enhancing the expression of insulin-like growth factors and their receptors in cocultured human preimplantation embryos. Fertil Steril 1999; 71(2): 361–367
https://doi.org/10.1016/S0015-0282(98)00451-8 pmid: 9988412
20 Opøien HK, Fedorcsak P, Omland AK, Abyholm T, Bjercke S, Ertzeid G, Oldereid N, Mellembakken JR, Tanbo T. Invitro fertilization is a successful treatment in endometriosis-associated infertility. Fertil Steril 2012; 97(4): 912–918
https://doi.org/10.1016/j.fertnstert.2012.01.112 pmid: 22341637
21 Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab 2004; 89(6): 2745–2749
https://doi.org/10.1210/jc.2003-032046 pmid: 15181052
22 Balen AH. Hypersecretion of luteinizing hormone and the polycystic ovary syndrome. Hum Reprod 1993; 8(Suppl 2): 123–128
https://doi.org/10.1093/humrep/8.suppl_2.123 pmid: 8276945
23 Balen AH, Tan SL, MacDougall J, Jacobs HS. Miscarriage rates following in-vitro fertilization are increased in women with polycystic ovaries and reduced by pituitary desensitization with buserelin. Hum Reprod 1993; 8(6): 959–964
https://doi.org/10.1093/oxfordjournals.humrep.a138174 pmid: 8345091
24 Morais RD, Thomé RG, Lemos FS, Bazzoli N, Rizzo E. Autophagy and apoptosis interplay during follicular atresia in fish ovary: a morphological and immunocytochemical study. Cell Tissue Res 2012; 347(2): 467–478
https://doi.org/10.1007/s00441-012-1327-6 pmid: 22314847
25 Nakahara K, Saito H, Saito T, Ito M, Ohta N, Takahashi T, Hiroi M. The incidence of apoptotic bodies in membrana granulosa can predict prognosis of ova from patients participating in in vitro fertilization programs. Fertil Steril 1997; 68(2): 312–317
https://doi.org/10.1016/S0015-0282(97)81521-X pmid: 9240262
26 Das M, Djahanbakhch O, Hacihanefioglu B, Saridogan E, Ikram M, Ghali L, Raveendran M, Storey A. Granulosa cell survival and proliferation are altered in polycystic ovary syndrome. J Clin Endocrinol Metab 2008; 93(3): 881–887
https://doi.org/10.1210/jc.2007-1650 pmid: 18073308
27 Brown ZA, Louwers YV, Fong SL, Valkenburg O, Birnie E, de Jong FH, Fauser BC, Laven JS. The phenotype of polycystic ovary syndrome ameliorates with aging. Fertil Steril 2011; 96(5): 1259–1265
https://doi.org/10.1016/j.fertnstert.2011.09.002 pmid: 21963227
28 Sagle M,  Bishop K,  Ridley N,  Alexander FM,  Michel M,  Bonney RC,  Beard RW,  Franks S. Recurrent early miscarriage and polycystic ovaries. BMJ  1988; 297(6655):1027–1028
pmid: 314259
29 Saller S, Merz-Lange J, Raffael S, Hecht S, Pavlik R, Thaler C, Berg D, Berg U, Kunz L, Mayerhofer A. Norepinephrine, active norepinephrine transporter, and norepinephrine-metabolism are involved in the generation of reactive oxygen species in human ovarian granulosa cells. Endocrinology 2012; 153(3): 1472–1483
https://doi.org/10.1210/en.2011-1769 pmid: 22234472
30 Saller S, Kunz L, Berg D, Berg U, Lara H, Urra J, Hecht S, Pavlik R, Thaler CJ, Mayerhofer A. Dopamine in human follicular fluid is associated with cellular uptake and metabolism-dependent generation of reactive oxygen species in granulosa cells: implications for physiology and pathology. Hum Reprod 2014; 29(3): 555–567
https://doi.org/10.1093/humrep/det422 pmid: 24287819
31 Víctor VM, Espulgues JV, Hernández-Mijares A, Rocha M. Oxidative stress and mitochondrial dysfunction in sepsis: a potential therapy with mitochondria-targeted antioxidants. Infect Disord Drug Targets 2009; 9(4): 376–389
https://doi.org/10.2174/187152609788922519 pmid: 19689380
32 Karuputhula NB, Chattopadhyay R, Chakravarty B, Chaudhury K. Oxidative status in granulosa cells of infertile women undergoing IVF. Syst Biol Reprod Med 2013; 59(2): 91–98
https://doi.org/10.3109/19396368.2012.743197 pmid: 23278116
33 Kim YM, Cho M. Activation of NADPH oxidase subunit NCF4 induces ROS-mediated EMT signaling in HeLa cells. Cell Signal 2014; 26(4): 784–796
https://doi.org/10.1016/j.cellsig.2013.12.005 pmid: 24378533
34 Murillo I, Henderson LM. Expression of gp91phox/Nox2 in COS-7 cells: cellular localization of the protein and the detection of outward proton currents. Biochem J 2005; 385(3): 649–657
https://doi.org/10.1042/BJ20040829 pmid: 15377283
35 Casbon AJ, Allen LA, Dunn KW, Dinauer MC. Macrophage NADPH oxidase flavocytochrome B localizes to the plasma membrane and Rab11-positive recycling endosomes. J Immunol 2009; 182(4): 2325–2339
https://doi.org/10.4049/jimmunol.0803476 pmid: 19201887
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