Abstract
Purpose
Elevated oxidative stress has been proposed as an important factor in the pathogenesis of polycystic ovary syndrome (PCOS)-related infertility. Our study was aimed at simultaneously exploring local and systemic oxidative stress in PCOS individuals and its relationship with embryo quality.
Methods
We recruited 86 PCOS cases and 60 controls. Five representative oxidative stress markers, namely, total oxidant capacity (TOC), total antioxidant capacity (TAC), malonaldehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD), were measured in both follicular fluid (FF) and serum.
Results
Women with PCOS compared to normal controls had higher levels of TOC in both FF (10.13 ± 2.68 vs.7.03 ± 2.45, P < 0.001) and serum (11.76 ± 2.92 vs. 8.82 ± 2.57, P < 0.001). The oxidative stress index (OSI, the ratio of TOC to TAC) was also higher in PCOS cases. They were still significant after BMI adjustment (Padj<0.01). In addition, the serum OSI level was much higher than the FF OSI level in both groups. Correlation analysis showed that the FF and serum TOC were negatively correlated with the high-quality embryo rate on day 3 and the later blastocyst formation rate in the PCOS group (P < 0.05). The correlation coefficient was higher in FF. Moreover, as the regression analysis data showed, the FF MDA level was significantly associated with embryo quality indicators (P < 0.05).
Conclusions
PCOS was accompanied by elevated oxidative stress in both serum and FF. Even though serum oxidative stress was severe, the study suggested that FF oxidative stress contributed more to embryo quality, to which we should give more attention in the future.
Keywords: Oxidative stress, PCOS, Follicular fluid, Embryo quality
Introduction
Oxidative stress refers to the overproduction of highly oxidative molecules such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) when the body is exposed to various harmful stimuli. The degree of oxidation exceeds that of antioxidation; therefore, excessive ROS or RNS disrupt redox signaling as well as damaging DNA, proteins, and lipids, resulting in tissue damage [1, 2]. Representative oxidative stress (OS) markers include (1) total oxidant capacity (TOC), which reflects the total number of oxidant molecules; (2) total antioxidant capacity (TAC), which reveals the total number of antioxidant molecules; (3) malonaldehyde (MDA), which represents representative lipid peroxidation product; (4) glutathione (GSH), which represents nonenzymatic antioxidant capacity; (5) superoxide dismutase (SOD) activity, which represents enzymatic antioxidant capacity; and (6) the oxidative stress index (OSI, the ratio of TOC to TAC), which indicates the relative level of oxidative stress [3]. Previous studies have confirmed that oxidative stress participates in many diseases, such as type 2 diabetes [4], asthma [5], cardiovascular disease [6], and infertility [7].
Polycystic ovary syndrome (PCOS) is the most common reproductive endocrine and metabolic disease [8]. It is the leading cause of anovulatory infertility due to abnormal follicle development and poor oocyte/embryo quality. It is also associated with long-term metabolic disorders [9, 10], which can aggravate infertility by affecting oocyte/embryo quality. Previous studies have found that OS markers are elevated in the serum of PCOS compared with controls, while few studies have explored whether this condition exists in follicular fluid (FF), the microenvironment most closely related to oocytes [11]. Even fewer studies simultaneously explored the local and systemic OS status of PCOS and their relationship with oocyte/embryo quality. Moreover, markers used in many articles are unilateral, which may only reflect one aspect of total oxidant capacity or total antioxidant capacity and cannot fully reflect the state of oxidation–antioxidant balance.
Our study was aimed at simultaneously evaluating the OS level in FF and serum of PCOS and the controls and further at exploring the relationship between OS and embryo quality.
Materials and methods
Subjects and controlled ovarian stimulation (COS) protocol
Women aged 20–32 years, with or without PCOS, undergoing in vitro fertilization-embryo transfer (IVF-ET) in the Center for Reproductive Medicine, Shandong University, were recruited for our study. Each PCOS subject met the Rotterdam diagnostic criteria, which are as follows: oligomenorrhea (i.e., fewer than eight cycles per year), biochemical and/or clinical hyperandrogenism, and polycystic ovaries confirmed by ultrasound examination. Patients who had at least two of these three characteristics and excluded diseases that mimicked PCOS were eventually diagnosed [12]. Women in the control group had regular menstruation and no PCOS-related signs. All of the participants provided informed consent, and the study was approved by the Ethics Committee.
Patients enrolled in our study received a standardized gonadotropin-releasing hormone (GnRH) agonist long protocol for COS [13]. Under ultrasound monitoring, when two or more follicles developed with diameters up to 18 mm, subcutaneous hCG (recombinant human chorionic gonadotropin injection) was administered to induce follicular maturation and oocytes were taken by ultrasound-guided puncture 36 h later. In vitro fertilization was performed after 4–6 h of culture in vitro, and embryo quality was assessed before embryo transfer.
Collection and processing of serum and follicular fluid
On the first to the third day of the menstrual cycle before ovarian stimulation, about 5 mL of venous blood was drawn by elbow venipuncture. After standing at room temperature for 2 h, blood samples were centrifuged at 2000 rpm for 5 min; then, serum was separated and stored in a refrigerator at minus 80 °C.
FF was aspirated from follicles during oocyte retrieval. After the centrifugation at 300 × g for 10 min, clarified FF without various cellular components were stored at minus 80 °C.
Evaluation of embryo quality
Embryos were observed on the third day after oocyte retrieval, when embryos with cells ≥ 7 and the fragments < 10% are identified as high-quality embryos. The embryo scoring was done by two different embryologists, who were blinded to the treatment groups. Day 3 high-quality embryo rate equals the number of high-quality embryos divided by the total number of zygotes × 100%. Blastocysts were observed on the fifth day after oocyte retrieval, and blastocyst formation rate equals blastocysts divided by the total number of surviving zygotes × 100%.
Measurements of OS markers and clinical indicators
A semiautomatic microplate colorimetric method was used to measure TOC with hydrogen peroxide (H2O2) used as a standard. The assay was performed as previously described [14]. The final results were expressed in micromolar hydrogen peroxide equivalent per liter (μmol H2O2 Equiv./L). TAC, MDA, GSH, and SOD activities were all measured by spectrophotometry using colorimetry kits (Beyotime, Shanghai, China). Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T), and estradiol (E2), as well as plasma insulin, glucose, and the homoeostasis model insulin resistance index (HOMA-IR), were measured or assessed as described previously [15].
Statistical analysis
The Statistical Package for the Social Sciences (SPSS, version 23.0, IBM Corp., Armonk, New York) was used to analyze the results. Differences between PCOS and controls were assessed through Student’s t-test with body mass index (BMI), HOMA-IR, and LH/FSH as covariants. Correlation was used to explore the relationship between embryo quality and oxidative stress markers. To further explore which factors affected embryo quality more in PCOS, multivariate stepwise regression analyses were performed to assess the effect of OS markers and other indicators, including BMI, LH/FSH, HOMA-IR, and T, on embryo quality.
Results
Baseline characteristics of the PCOS cases and the controls
According to the inclusion and exclusion criteria, 86 women with PCOS and 60 control women were included in our study. The baseline information including clinical characteristics, hormonal levels, and metabolic profile is summarized in Table 1. Women with PCOS had higher levels of BMI (23.91 ± 3.73 vs. 21.81 ± 3.09, P = 0.001), hyperandrogenism (T: 32.97 ± 14.62 vs. 23.90 ± 8.05, P < 0.001), and insulin resistance (HOMA-IR: 3.59 ± 2.16 vs. 1.79 ± 0.47, P < 0.001) than women in the control group.
Table 1.
Baseline indicators of PCOS subjects and controls
| Controls (n = 60) | PCOS (n = 86) | P value | |
|---|---|---|---|
| Age* | 28.15 ± 2.69 | 28.43 ± 3.42 | 0.6 |
| BMI (kg/m2)* | 21.81 ± 3.09 | 23.91 ± 3.73 | 0.001 |
| Fasting glucose (mmol/L)* | 5.15 ± 0.5 | 5.4 ± 0.55 | 0.005 |
| Fasting insulin (mIU/L)# | 7.7 (6.14–9.61) | 12.69 (8.66–26.88) | <0.001& |
| HOMA-IR# | 1.75 (1.36-2.22) | 2.89 (1.79–5.34) | <0.001& |
| FSH (mIU/mL)* | 6.56 ± 1.2 | 5.93 ± 1.16 | 0.002 |
| LH (mIU/mL)* | 5.32 ± 1.67 | 6.99 ± 2.82 | <0.001 |
| LH/FSH# | 0.79 (0.63-1.01) | 1.06 (0.78–1.57) | <0.001& |
| E2 (pg/mL)# | 33.20 (26.08–43.48) | 34.90 (27.45–49.65) | 0.193& |
| T (ng/dL)* | 23.90 ± 8.05 | 33.82 ± 13.83 | <0.001 |
PCOS polycystic ovary syndrome, BMI body mass index, HOMA-IR homoeostasis model insulin resistance index, LH luteinizing hormone, FSH follicle-stimulating hormone, E2 estradiol, T total testosterone, SD standard deviation
*Values are presented as the mean ± SD for continuous indicators following normal distribution
#Values are presented as the median (interquartile range) for continuous indicators not following normal distribution
&t-test after variables obeying normal distribution through log transformation
FF and serum oxidative stress levels in the PCOS cases and the controls
As shown in Table 2, after adjustment for BMI, HOMA-IR, and LH/FSH, FF TOC was higher in the PCOS group than in the control group, while TAC in the PCOS group showed little change. The ratio of the above, namely, OSI, was higher in PCOS. Furthermore, MDA, the product of lipid peroxidation, was not significantly increased in PCOS. Nonenzymatic antioxidant and enzymatic antioxidant molecules, namely, GSH and SOD, exhibited no difference between the two groups, which was consistent with the change in TAC levels.
Table 2.
Oxidative stress markers in follicular fluid and serum of PCOS subjects and controls
| Controls (n = 60) | PCOS (n = 86) | P value | P* adjusted | |
|---|---|---|---|---|
| Oxidative stress levels of FF | ||||
| TOC (μM H2O2) | 7.03 ± 2.45 | 10.13 ± 2.68 | <0.001 | <0.001 |
| TAC (μM) | 1.15 ± 0.27 | 1.18 ± 0.28 | 0.724 | 0.953 |
| OSI | 6.50 ± 2.91 | 9.44 ± 4.28 | <0.001 | 0.004 |
| MDA (μM) | 4.81 ± 3.08 | 4.93 ± 2.60 | 0.822 | 0.132 |
| GSH (μM) | 2.43 ± 0.83 | 2.49 ± 0.98 | 0.692 | 0.72 |
| SOD (U/mL) | 37.31 ± 9.06 | 40.30 ± 8.52 | 0.093 | 0.626 |
| Oxidative stress levels of serum | ||||
| TOC (μM H2O2) | 8.82 ± 2.57 | 11.76 ± 2.92 | <0.001 | 0.003 |
| TAC (μM) | 0.65 ± 0.11 | 0.65 ± 0.11 | 0.873 | 0.512 |
| OSI | 15.63 ± 3.70 | 18.39 ± 4.74 | <0.001 | 0.001 |
| MDA (μM) | 5.96 ± 2.84 | 8.04 ± 2.50 | <0.001 | 0.18 |
| GSH (μM) | 1.57 ± 0.35 | 1.63 ± 0.40 | 0.339 | 0.555 |
| SOD (U/mL) | 32.70 ± 6.75 | 32.38 ± 6.94 | 0.811 | 0.599 |
TOC total oxidant capacity, TAC total antioxidant capacity, OSI oxidative stress index, MDA malonaldehyde, GSH glutathione, SOD superoxide dismutase
Values are presented as the mean ± SD
P* adjusted: BMI, HOMA-IR, and LH/FSH are adjusted as covariants
Interestingly, serum presented higher OSI in both PCOS cases and controls than FF. However, similarly, serum TOC, OSI, and MDA were elevated in PCOS, even though the increase in MDA was not significant after adjustment. Total antioxidant capacity, SOD activity, and GSH showed no significant change compared with the controls.
The relationship between oxidative stress and embryo quality
High-quality oocytes/embryos usually indicate good pregnancy outcomes, so the quality of oocytes/embryos is significant for IVF-ET. The blastocyst formation rate or day 3 high-quality embryo rate is a common indicator of oocyte/embryo quality. As shown in Table 3, women with PCOS showed lower embryo quality. On day 3, the high-quality embryo rate was similar between the two groups. On day 5, the blastocyst formation rate of PCOS was slightly lower than that of controls, indicating that the embryo quality of PCOS was decreased. The lower embryo quality in PCOS patients might subsequently lead to a higher miscarriage rate and lower live birth rate.
Table 3.
Embryo quality of the PCOS group and control group
| Controls (n = 60) | PCOS (n = 86) | P value | |
|---|---|---|---|
| Embryo number on D3 | 549 | 795 | |
| D3 high-quality embryo rate (%)# | 53.02 ± 26.62 | 50.66 ± 28.59 | 0.63 |
| Blastocyst formation rate (%)# | 40.11 ± 27.99 | 32.33 ± 24.19 | 0.099 |
| Miscarriage rate (%) | 16.22 | 38.18 | 0.023 |
| Live birth rate (%) | 59.62 | 42.5 | 0.055 |
#Values are presented as the mean ± SD
Correlation analysis outcomes (Table 4) revealed that TOC and OSI were negatively correlated with embryo quality in both FF and serum of PCOS. Furthermore, the above two markers in FF had a stronger correlation with embryo quality than those in serum.
Table 4.
Correlation coefficient between oxidative stress markers and embryo quality in PCOS and controls
| D3 high-quality embryo rate | Blastocyst formation rate | |||
|---|---|---|---|---|
| Controls | PCOS | Controls | PCOS | |
| Markers in FF | ||||
| TOC (μM H2O2) | −0.334** | −0.405** | −0.583** | −0.473** |
| TAC (μM) | 0.131 | 0.093 | 0.018 | 0.285* |
| OSI | −0.313* | −0.297** | −0.459** | −0.416** |
| MDA (μM) | −0.019 | −0.417** | −0.327* | −0.430** |
| Markers in serum | ||||
| TOC (μM H2O2) | −0.253 | −0.362** | −0.444** | −0.292* |
| TAC (μM) | −0.091 | −0.003 | −0.202 | −0.078 |
| OSI | −0.154 | −0.334** | −0.328* | −0.202 |
| MDA (μM) | 0.081 | −0.080 | 0.136 | 0.096 |
*P < 0.05, **P < 0.01
Multivariate stepwise regression analyzed the effect of OS markers and other embryo quality-related indicators, including BMI, LH/FSH, HOMA-IR, and T, on the blastocyst formation rate or day 3 high-quality embryo rate in PCOS. As shown in Table 5, MDA and TOC in FF were significant factors affecting the day 3 high-quality embryo rate of PCOS. MDA in FF and BMI were significant factors affecting the blastocyst formation rate of PCOS. In the non-PCOS group, oxidative stress markers were also related to embryo development. FF TOC and LH/FSH were significant factors affecting the day 3 high-quality embryo rate. FF TOC, T, and OSI in serum were significant factors affecting the blastocyst formation rate (data not shown).
Table 5.
Association of important independent variables with embryo quality indicators by multivariate regression analysis in PCOS
| Independents | Unstandardized coefficients | Standardized coefficients | |||
|---|---|---|---|---|---|
| B | SE | Beta | t | P | |
| Model I: D3 high-quality embryo rate as dependent | |||||
| Constant | 1.365 | 0.253 | 5.398 | <0.001 | |
| FF MDA (μM) | −0.062 | 0.025 | −0.453 | −2.512 | 0.022 |
| FF TOC (μM H2O2) | −0.051 | 0.023 | −0.395 | −2.186 | 0.042 |
| Model II: blastocyst formation rate as dependent | |||||
| Constant | 1.365 | 0.325 | 4.196 | 0.001 | |
| FF MDA (μM) | −0.072 | 0.016 | −0.749 | −4.498 | <0.001 |
| BMI (kg/m2) | −0.030 | 0.013 | −0.390 | −2.340 | 0.031 |
Discussion
In this study, we simultaneously measured representative OS markers in the FF and serum of PCOS and controls to evaluate the OS level and its relationship with embryo quality. We demonstrated that PCOS had higher OS levels in both FF and serum. Furthermore, the OS level in FF was lower than that in the serum. OS markers in the FF of PCOS were found to be more closely correlated with embryo quality than those in the serum, which can predict embryo quality more accurately.
Oxidative stress exerts an important influence at both the local and systemic levels. Follicular fluid provides a local and direct microenvironment for oocyte development, while serum can only partially work indirectly. In recent years, there have been many studies on PCOS and OS. Most of them focused on serum [16], revealing an increase in oxidative markers in PCOS, whereas some of them studied granulosa cells [17] or peripheral mononuclear cells [18], uncovering a similar situation. Although there are a small handful of studies involving follicular fluid, the OS markers they choose are not comprehensive enough [19–22]. In our study, not only did we use MDA, GSH, and SOD, major markers that reflect lipid pro-oxidative levels, nonenzymatic antioxidant capacity, and enzyme antioxidant capacity, respectively, but also we utilized TOC and TAC to reflect overall oxidation and antioxidant levels [23]. Importantly, OSI, an indicator that could provide an overview of the balance between oxidation and antioxidant levels, was also used in our study. Therefore, we can provide a rigorous and comprehensive evaluation of oxidative stress levels in PCOS.
Our work found that both TOC and OSI, the absolute oxidative marker and relative oxidative marker, were higher in the FF of PCOS, which indicated that oxidative capacity was elevated and pro-oxidative capacity exceeded antioxidant capacity in the FF of PCOS. Although total antioxidant capacity showed no significant change compared with the controls, all antioxidant markers, namely, TAC, GSH, and SOD, were slightly increased, which may be due to the compensatory increase to maintain redox homeostasis. High T level, a feature of PCOS, also impacted oxidative stress. There was a trend of positive correlation between T level and oxidative stress, but only the serum MDA and T were significantly correlated (r = 0.265, P < 0.05); others did not reach statistical significance. In non-PCOS women, there was no correlation between T and OS level.
FF showed lower TOC and OSI than serum in both PCOS patients and controls, not only because of the different sample collection times but also because of the different kinds of body liquids. Varnagy et al. reported that serum 8-OHdG (a biomarker of oxidative DNA damage) was slightly lower in serum after COS than in the natural cycle [24], indicating that oxidative stress was decreased after COS. Varnagy et al. also found that oxidative stress in FF was significantly lower than that in serum after COS, which is consistent with our data, implying that FF had lower OS levels and provided a relatively mild environment to protect oocytes from OS damage. This may be due to a series of protective measures, such as the theca interstitial cell barrier, the defensive role of cumulus cells [25], and antioxidative enzymes with high activity.
Our study found that OS markers were correlated with embryo quality in PCOS. Multivariate stepwise regression demonstrated that TOC and MDA in FF were pivotal factors affecting embryo quality, which can be used as predictors of embryo quality in PCOS. There are other methods or biomarkers that predict embryo quality in IVF-ET. Kim et al. [26] and Lin et al. [27] found that AMH in FF was positively correlated with embryo quality in non-PCOS women. However, caution should be used when using AMH as a predictor in PCOS. AMH in both serum and FF is elevated abnormally in PCOS [28], not only because PCOS subjects have more small follicles to secrete AMH but also because granulosa cells in PCOS patients have a stronger ability to produce AMH [29]. As a classic antioxidative molecule, melatonin in FF was also regarded as a predictor of embryo quality [30]. In PCOS subjects, melatonin was decreased in FF [31], but its relationship with embryo quality was unclear. In addition, the mitochondrial DNA copy number of cumulus granulosa cells was also linked to embryo quality [32, 33]. Some cumulus cell genes could also become potential biomarkers of embryo quality [34, 35], but this required multiple genes to be tested simultaneously and conjoint analysis. Moreover, metabolomics [36] and proteomics [37] have also been used in FF or embryo culture media, but the detection cost is relatively high. OS markers in FF are easy to measure and have good ability to predict embryo quality, so they are powerful supplements to current predictors of embryo quality.
Women with PCOS have a high rate of miscarriage, in which maternal uterine insufficiency is an important factor [38]. Hu et al. proved that a pregnant rat suffering from hyperandrogenism and insulin resistance (PCOS-like) exhibited abnormal ROS level and uterine defects, which led to pregnancy loss [39]. In our batch of data, serum OS markers showed little difference between the aborted and nonaborted groups of PCOS. This could be due to that OS level during pregnancy is different from that before pregnancy. Pregnancy is well known to increase the serum oxidative stress [40], and we use OS level before pregnancy, which cannot fully represent the OS level during pregnancy. Therefore, further research is needed on this issue.
The main limitation of the study is that the sample size is not big enough; therefore, the number of indicators that can be included in the regression analysis is limited. Since OS markers are dynamic indicators, our data only represent the OS level at the time of sample collection and do not reflect physiological OS level during the entire menstrual cycle and other special periods like pregnancy.
Conclusions
In conclusion, our data demonstrate that PCOS subjects had elevated oxidative stress in both FF and serum. Oxidative stress in FF was less severe than that in the serum of PCOS. Furthermore, oxidative markers in FF, especially MDA and TOC, were more closely correlated with embryo quality in IVF-ET, which can be accurate markers to predict PCOS embryo quality in the future.
Author contributions
Shigang Zhao was responsible for the conceptualization. Zhiheng Yu was responsible for the methodology. Lei Cheng was responsible for the resources. Han Zhao was responsible for the supervision. Yue Liu wrote the original draft. Yuanyuan Man and Xueying Gao were responsible for the review and editing. All authors have read and agreed to the published version of the manuscript.
Funding information
This study was funded by the National Key Research and Development Program of China (2018YFC1004000), National Natural Science Foundation of China (31871509, 81622021, and 82071606), National Natural Science Foundation of Shandong Province (JQ201816), Innovative Research Team of High-Level Local Universities in Shanghai (SSMU-ZLCX20180401), Key Research and Development Program of Shandong Province (2019GSF108274), and Young Scholars Program of Shandong University.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical approval
The approval was obtained from the ethics committee of the Center for Reproductive Medicine, Shandong University. The procedures used in this study adhere to the tenets of the Declaration of Helsinki.
Statement of informed consent
Informed consent was obtained from all individual participants included in the study.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Zuo T, Zhu M, Xu W. Roles of oxidative stress in polycystic ovary syndrome and cancers. Oxidative Med Cell Longev. 2016;2016:8589318–8589314. doi: 10.1155/2016/8589318. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Glasauer A, Chandel NS. Ros. Curr Biol. 2013;23(3):R100–R102. doi: 10.1016/j.cub.2012.12.011. [DOI] [PubMed] [Google Scholar]
- 3.Lim SS, Davies MJ, Norman RJ, Moran LJ. Overweight, obesity and central obesity in women with polycystic ovary syndrome: a systematic review and meta-analysis. Hum Reprod Update. 2012;18(6):618–637. doi: 10.1093/humupd/dms030. [DOI] [PubMed] [Google Scholar]
- 4.Gerber PA, Rutter GA. The role of oxidative stress and hypoxia in pancreatic beta-cell dysfunction in diabetes mellitus. Antioxid Redox Signal. 2017;26(10):501–518. doi: 10.1089/ars.2016.6755. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Mishra V, Banga J, Silveyra P. Oxidative stress and cellular pathways of asthma and inflammation: therapeutic strategies and pharmacological targets. Pharmacol Ther. 2018;181:169–182. doi: 10.1016/j.pharmthera.2017.08.011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Higashi Y, Noma K, Yoshizumi M, Kihara Y. Endothelial function and oxidative stress in cardiovascular diseases. Circ J. 2009;73(3):411–418. doi: 10.1253/circj.cj-08-1102. [DOI] [PubMed] [Google Scholar]
- 7.Agarwal A, Gupta S, Sharma RK. Role of oxidative stress in female reproduction. Reprod Biol Endocrinol. 2005;3:28. doi: 10.1186/1477-7827-3-28. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Escobar-Morreale HF. Polycystic ovary syndrome: definition, aetiology, diagnosis and treatment. Nat Rev Endocrinol. 2018;14(5):270–284. doi: 10.1038/nrendo.2018.24. [DOI] [PubMed] [Google Scholar]
- 9.Randeva HS, Tan BK, Weickert MO, Lois K, Nestler JE, Sattar N, Lehnert H. Cardiometabolic aspects of the polycystic ovary syndrome. Endocr Rev. 2012;33(5):812–841. doi: 10.1210/er.2012-1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Moran LJ, Misso ML, Wild RA, Norman RJ. Impaired glucose tolerance, type 2 diabetes and metabolic syndrome in polycystic ovary syndrome: a systematic review and meta-analysis. Hum Reprod Update. 2010;16(4):347–363. doi: 10.1093/humupd/dmq001. [DOI] [PubMed] [Google Scholar]
- 11.Dumesic DA, Meldrum DR, Katz-Jaffe MG, Krisher RL, Schoolcraft WB. Oocyte environment: follicular fluid and cumulus cells are critical for oocyte health. Fertil Steril. 2015;103(2):303–316. doi: 10.1016/j.fertnstert.2014.11.015. [DOI] [PubMed] [Google Scholar]
- 12.Revised 2003 consensus on diagnostic criteria and long-term health risks related to polycystic ovary syndrome. Fertil Steril. 2004;81(1):19–25. 10.1016/j.fertnstert.2003.10.004. [DOI] [PubMed]
- 13.Lambalk CB, Banga FR, Huirne JA, Toftager M, Pinborg A, Homburg R, van der Veen F, van Wely M. GnRH antagonist versus long agonist protocols in IVF: a systematic review and meta-analysis accounting for patient type. Hum Reprod Update. 2017;23(5):560–579. doi: 10.1093/humupd/dmx017. [DOI] [PubMed] [Google Scholar]
- 14.Erel O. A new automated colorimetric method for measuring total oxidant status. Clin Biochem. 2005;38(12):1103–1111. doi: 10.1016/j.clinbiochem.2005.08.008. [DOI] [PubMed] [Google Scholar]
- 15.Xu X, Shi Y, Cui Y, Ma J, Che L, Chen ZJ. Endocrine and metabolic characteristics of polycystic ovary syndrome in Chinese women with different phenotypes. Clin Endocrinol. 2012;76(3):425–430. doi: 10.1111/j.1365-2265.2011.04194.x. [DOI] [PubMed] [Google Scholar]
- 16.Hilali N, Vural M, Camuzcuoglu H, Camuzcuoglu A, Aksoy N. Increased prolidase activity and oxidative stress in PCOS. Clin Endocrinol. 2013;79(1):105–110. doi: 10.1111/cen.12110. [DOI] [PubMed] [Google Scholar]
- 17.Lai Q, Xiang W, Li Q, Zhang H, Li Y, Zhu G, et al. Oxidative stress in granulosa cells contributes to poor oocyte quality and IVF-ET outcomes in women with polycystic ovary syndrome. J Hum Nutr Diet. 2017; 10.1111/jhn.12545. 10.1007/s11684-017-0575-y. [DOI] [PubMed]
- 18.Nose E, Azhary JM, Koike H, Kunitomi C, Yoshino O, Izumi G, et al. Plasmatic and intracellular markers of oxidative stress in normal weight and obese patients with polycystic ovary syndrome. Sci Rep. 2017;125(8):506–513. doi: 10.1055/s-0043-111241. [DOI] [PubMed] [Google Scholar]
- 19.Chattopadhayay R, Ganesh A, Samanta J, Jana SK, Chakravarty BN, Chaudhury K. Effect of follicular fluid oxidative stress on meiotic spindle formation in infertile women with polycystic ovarian syndrome. Gynecol Obstet Investig. 2010;69(3):197–202. doi: 10.1159/000270900. [DOI] [PubMed] [Google Scholar]
- 20.Pekel A, Gonenc A, Turhan NO, Kafali H. Changes of sFas and sFasL, oxidative stress markers in serum and follicular fluid of patients undergoing IVF. J Assist Reprod Genet. 2015;32(2):233–241. doi: 10.1007/s10815-014-0396-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Yilmaz N, Inal HA, Gorkem U, Sargin Oruc A, Yilmaz S, Turkkani A. Follicular fluid total antioxidant capacity levels in PCOS. J Obstet Gynaecol. 2016;36(5):654–657. doi: 10.3109/01443615.2016.1148683. [DOI] [PubMed] [Google Scholar]
- 22.Seleem AK, El Refaeey AA, Shaalan D, Sherbiny Y, Badawy A. Superoxide dismutase in polycystic ovary syndrome patients undergoing intracytoplasmic sperm injection. J Assist Reprod Genet. 2014;31(4):499–504. doi: 10.1007/s10815-014-0190-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Murri M, Luque-Ramirez M, Insenser M, Ojeda-Ojeda M, Escobar-Morreale HF. Circulating markers of oxidative stress and polycystic ovary syndrome (PCOS): a systematic review and meta-analysis. Hum Reprod Update. 2013;19(3):268–288. doi: 10.1093/humupd/dms059. [DOI] [PubMed] [Google Scholar]
- 24.Varnagy A, Koszegi T, Gyorgyi E, Szegedi S, Sulyok E, Premusz V, et al. Levels of total antioxidant capacity and 8-hydroxy-2′-deoxyguanosine of serum and follicular fluid in women undergoing in vitro fertilization: focusing on endometriosis. Hum Fertil (Camb) 2020;23(3):200–208. doi: 10.1080/14647273.2018.1535719. [DOI] [PubMed] [Google Scholar]
- 25.Shaeib F, Khan SN, Ali I, Thakur M, Saed MG, Dai J, Awonuga AO, Banerjee J, Abu-Soud HM. The defensive role of cumulus cells against reactive oxygen species insult in metaphase II mouse oocytes. Reprod Sci. 2016;23(4):498–507. doi: 10.1177/1933719115607993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.Kim JH, Lee JR, Chang HJ, Jee BC, Suh CS, Kim SH. Anti-Mullerian hormone levels in the follicular fluid of the preovulatory follicle: a predictor for oocyte fertilization and quality of embryo. J Korean Med Sci. 2014;29(9):1266–1270. doi: 10.3346/jkms.2014.29.9.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Lin WQ, Yao LN, Zhang DX, Zhang W, Yang XJ, Yu R. The predictive value of anti-Mullerian hormone on embryo quality, blastocyst development, and pregnancy rate following in vitro fertilization-embryo transfer (IVF-ET) J Assist Reprod Genet. 2013;30(5):649–655. doi: 10.1007/s10815-013-9973-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Stracquadanio M, Ciotta L, Palumbo MA. Relationship between serum anti-Mullerian hormone and intrafollicular AMH levels in PCOS women. Gynecol Endocrinol. 2018;34(3):223–228. doi: 10.1080/09513590.2017.1381838. [DOI] [PubMed] [Google Scholar]
- 29.Pellatt L, Hanna L, Brincat M, Galea R, Brain H, Whitehead S, Mason H. Granulosa cell production of anti-Mullerian hormone is increased in polycystic ovaries. J Clin Endocrinol Metab. 2007;92(1):240–245. doi: 10.1210/jc.2006-1582. [DOI] [PubMed] [Google Scholar]
- 30.Tong J, Sheng S, Sun Y, Li H, Li WP, Zhang C, Chen ZJ. Melatonin levels in follicular fluid as markers for IVF outcomes and predicting ovarian reserve. Reproduction. 2017;153(4):443–451. doi: 10.1530/rep-16-0641. [DOI] [PubMed] [Google Scholar]
- 31.Kim MK, Park EA, Kim HJ, Choi WY, Cho JH, Lee WS, Cha KY, Kim YS, Lee DR, Yoon TK. Does supplementation of in-vitro culture medium with melatonin improve IVF outcome in PCOS? Reprod BioMed Online. 2013;26(1):22–29. doi: 10.1016/j.rbmo.2012.10.007. [DOI] [PubMed] [Google Scholar]
- 32.Ogino M, Tsubamoto H, Sakata K, Oohama N, Hayakawa H, Kojima T, Shigeta M, Shibahara H. Mitochondrial DNA copy number in cumulus cells is a strong predictor of obtaining good-quality embryos after IVF. J Assist Reprod Genet. 2016;33(3):367–371. doi: 10.1007/s10815-015-0621-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Desquiret-Dumas V, Clément A, Seegers V, Boucret L, Ferré-L'Hotellier V, Bouet PE, Descamps P, Procaccio V, Reynier P, May-Panloup P. The mitochondrial DNA content of cumulus granulosa cells is linked to embryo quality. Hum Reprod. 2017;32(3):607–614. doi: 10.1093/humrep/dew341. [DOI] [PubMed] [Google Scholar]
- 34.Shepel EA, Voznesenskaya ТY, Blashkiv TV, Yanchii RI. Cumulus cell genes as potential biomarkers of oocyte and embryo developmental competence. Fiziol Zh. 2016;62(1):107–113. doi: 10.15407/fz62.01.107. [DOI] [PubMed] [Google Scholar]
- 35.Assou S, Haouzi D, De Vos J, Hamamah S. Human cumulus cells as biomarkers for embryo and pregnancy outcomes. Mol Hum Reprod. 2010;16(8):531–538. doi: 10.1093/molehr/gaq032. [DOI] [PubMed] [Google Scholar]
- 36.Revelli A, Delle Piane L, Casano S, Molinari E, Massobrio M, Rinaudo P. Follicular fluid content and oocyte quality: from single biochemical markers to metabolomics. Reprod Biol Endocrinol. 2009;7:40. doi: 10.1186/1477-7827-7-40. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.Kaihola H, Yaldir FG, Bohlin T, Samir R, Hreinsson J, Åkerud H. Levels of caspase-3 and histidine-rich glycoprotein in the embryo secretome as biomarkers of good-quality day-2 embryos and high-quality blastocysts. PLoS One. 2019;14(12):e0226419. doi: 10.1371/journal.pone.0226419. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 38.Giudice LC. Endometrium in PCOS: implantation and predisposition to endocrine CA. Best Pract Res Clin Endocrinol Metab. 2006;20(2):235–244. doi: 10.1016/j.beem.2006.03.005. [DOI] [PubMed] [Google Scholar]
- 39.Hu M, Zhang Y, Guo X, Jia W, Liu G, Zhang J, Li J, Cui P, Sferruzzi-Perri AN, Han Y, Wu X, Ma H, Brännström M, Shao LR, Billig H. Hyperandrogenism and insulin resistance induce gravid uterine defects in association with mitochondrial dysfunction and aberrant reactive oxygen species production. Am J Physiol Endocrinol Metab. 2019;316(5):E794–E809. doi: 10.1152/ajpendo.00359.2018. [DOI] [PubMed] [Google Scholar]
- 40.Chiarello DI, Abad C, Rojas D, Toledo F, Vazquez CM, Mate A, et al. Oxidative stress: normal pregnancy versus preeclampsia. Biochim Biophys Acta Mol basis Dis. 1866;2020(2):165354. doi: 10.1016/j.bbadis.2018.12.005. [DOI] [PubMed] [Google Scholar]