Oxidative stress in granulosa cells contributes to poor oocyte quality and IVF-ET outcomes in women with polycystic ovary syndrome

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

Introduction

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder that affects 5%–10% of reproductive-age women [¹]. The clinical presentation varies from oligo and/or anovulation, clinical and/or biochemical signs of hyperandrogenism, and polycystic change in each ovary [²]. PCOS patients are frequently associated with low fertilization rate and high miscarriage rate even when undergoing in vitro fertilization and embryo transfer (IVF-ET) therapy. The reason for this phenomenon is debatable [³–⁵]. In addition to endocrinopathy, PCOS is also associated with oxidative stress (OS) that impairs female fertility [⁶].

Granulosa cells (GCs) are a type of steroidogenic cells tightly wrapped outside the oocytes; thus, their metabolic activity is assumed to have an important effect on the quality of oocyte [⁷, ⁸]. GCs play an important role in oocyte development, differentiation, ovulation, fertilization, and subsequent implantation [⁹]. GC dysfunction leads to abnormal folliculogenesis in women with PCOS [¹⁰].

Reactive oxygen species (ROS) are oxygen-derived molecules that include superoxide anion (O²⁻), hydrogen peroxide (H₂O₂), and hydroxyl radical (‒OH) [¹¹]. ROS at low doses are important signaling molecules for the maintenance of cell metabolic activity. However, excessive ROS that accumulate in GC during hyper-stimulation cause OS and affect the IVF-ET clinical outcomes [¹²]. Jancar reported that the increased ROS concentrations in GC would reduce the number of retrieved oocytes [¹³]. Rajani and Huang reported that the elevated ROS levels in follicular fluid correlate with poor quality of oocytes and embryos [¹⁴, ¹⁵].

NADPH oxidase is the key enzyme for intracellular ROS production. This enzyme binds other protein subunits to create the NADPH oxidase 2 (NOX2) complex [¹⁶]. The NOX2 complex is composed of six subunits, including gp91phox, p22phox (transmembrane proteins), p47phox, p40phox, p67phox, and Rac (cytosolic proteins) [¹⁷]. In the active state, the cytosolic subunit p47phox first translocates to p91phox and thus activates the NOX complex formation.

In this study, we compared the IVF-ET clinical results between patients with PCOS and those with tubal infertility. In addition, we checked the ROS level and apoptosis expression of GCs between the two groups and further found that the NOX pathway might be involved in the ROS production mechanism in patients with PCOS.

Materials and methods

Patients and IVF-ET protocol

Forty-seven patients undergoing IVF-ET treatment participated in this study from September 2015 to December 2015 in our Reproduction Center, Tongji Hospital, Wuhan, China. Patients were assigned to two groups: 25 had tubal infertility (tubal group) and 22 had tubal infertility combined PCOS (PCOS group). Both groups underwent IVF treatment. According to Rotterdam criteria, PCOS is diagnosed by the presence of at least two of three features: anovulation, clinical, or biochemical sign of hyperandrogenism, and the presence of 12 or more antral follicles in each ovary by supersonic inspection. The inclusion criteria were:≤35 years, body mass index (BMI) 18–25 kg/m², and baseline follicle stimulating hormone<10 IU/L. The exclusion criteria included the following: presence of congenital uterine malformations, hydrosalpinx, ovarian cyst, endometrial tuberculosis, and other metabolic, hepatic, and cardiovascular disorders. Consent forms were obtained from all patients, and studies were approved by the Tongji Hospital Human Assurance Committee.

All patients received oral contraceptive pills for 21 days prior to the treatment cycle. The administration of gonadotropin-releasing hormone (GnRH) agonist triptorelin (0.1 mg/d; Ferring, Sweden) started from day 21 of the menstrual cycle. After the conventional 14 days of injection, pituitary down-regulation was achieved (serum estradiol<50 pg/mL, serum FSH<5.0 IU/L, endometrial thickness<5 mm, and antral follicle size<8 mm). The administration of recombinant FSH (rFSH, Merck-Serono, Switzerland) was initiated, whereas the triptorelin dose was reduced to 0.05 mg/d until the administration day of human chorionic gonadotropin (HCG).

The follicular size and serum hormone levels were monitored regularly based on ultrasound examination. Recombinant HCG (6500 IU, Merck Serono) was administered when the two leading follicle diameters reached 20 mm. Oocytes were retrieved 34–38 h after administering HCG and were fertilized in vitro according to the standard procedures. D3 embryos were assessed and transferred. If the HCG day progesterone level reaches above 1.5 ng/mL, then the fresh cycle transfer is cancelled [¹⁸]. In brief, 60 mg of progesterone oil was administered every day for luteal phase support. After 4 weeks of embryo transfer, the gestational sac(s) with pulsating fetal heartbeats was detected by ultrasonography and defined as clinical pregnancy.

Isolation of GCs

Mural and cumulus GCs were collected from each patient. Each aspirated follicular fluid was placed in a tube containing mural GCs. The tube was left standing for several minutes, and the mural GCs were isolated, washed, and precipitated three times in phosphate-buffered saline (PBS). Oocyte–corona cumulus complex was washed two times and transferred to another culture dish. Cumulus GCs were carefully separated with needle from the oocytes in the culture medium. Isolated mural and cumulus GC were mixed together and resuspended in Dulbecco’s modified Eagle medium (DMEM) (Gibco, USA).

Measurement of intracellular ROS

Intracellular free radicals were detected by  5-(and 6-)-carboxy-2',7'-dichlorodihydrofluorescein diacetate (carboxy-H2DCF-DA) (Invitrogen, USA), a ROS-sensitive fluorescent probe. H2DCF-DA permeates into the GCs, becomes trapped in the cell compartments, and hydrolyzes to DCF. Free radicals in the cells can oxidize DCF and produce green fluorescence, which can be measured by fluorescent microscope and microplate reader.

The mixed mural and cumulus GCs were washed with PBS, resuspended in the Hank’s balanced salt solution with calcium and magnesium (Gibco, USA), and incubated with 25 µmol/L carboxy-H2DCF-DA for 30 min. In brief, 100 µL of GC suspension was seeded in each 96-well plates with 1 × 10⁵/mL density. GC generated the green fluorescent intensity, which was measured by POLARstar automatic multifunctional microplate fluorescence (Omega, German).

TUNEL assay for GC apoptosis detection

GCs were dispersed with 0.1% hyaluronidase by pipetting repeatedly for several minutes and centrifuged for 5 min at 480 g. The cell pellet was subsequently suspended in Bouin’s solution and mounted on a glass slide coated with poly-L-lysine overnight. After allowing the Bouin’s solution to dry, the glass slides were immersed in 50% and 70% ethanol for 24 h each time to remove the picric acid. After washing the GCs on the glass slides in PBS, the samples were prepared for TUNEL staining (Apoptag plus Fluorescein in Site Apoptosis Detection Kit, Chemicon International, USA). In brief, 55 µL /5 cm² of working strength TdT enzyme was applied and incubated at 37 °C for 1 h. The stop/wash buffer was added for 15 s. GCs were incubated with warm anti-digoxigenin conjugate for 30 min, and the slides were washed and applied with DAPI. Finally, GCs were observed and photographed under a fluorescence microscope.

Western blot for NADPH oxidase subunit detection

The concentrations of p47phox and gp91phox in GCs were measured. Samples containing 30 µg of protein were submitted to sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protein bands were transferred onto nitrocellulose membranes. Transferred protein membranes were incubated with 1:2000 p47phox or gp91phox primary antibody (Cell signaling, USA) overnight at 4 °C. The membranes were washed and incubated with secondary antibody. A chemiluminescent substrate was used to detect the peroxidase complex.

NADPH oxidase inhibitor reduced the ROS level

GCs were dispersed with 0.1% hyaluronidase, washed, resuspended in HBSS/Ca/Mg (Gibco, USA), and seeded in six-well plate with 1.5 × 10⁶ cells in each well. The seeded GCs were divided into three groups, namely, DMSO group (added control DMSO 10 mg/mL), DPI group (added DPI 5 pmol/L), and apocynin group (added apocynin 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.

Statistical analysis

Data were presented as the mean±SD and analyzed by SPSS 15.0 software (IBM, New York, USA). Data were analyzed by Student’s t-test and Chi-square exact test. P<0.05 was considered as significant difference between two groups.

Results

Demographic information for the two group subjects

All demographic information for the tubal factor and PCOS subjects is summarized in Table 1. The population of this study is 22 patients with PCOS patients and 25 with tubal factor. No significant difference was detected in the age, basal FSH, BMI, and duration of infertility of the two groups. However, the menstrual day 3 antral follicle count (AFC) in PCOS group (21.36±8.14) was significantly higher than that in tubal factor group (13.27±6.32).

Ovarian stimulation characteristics and IVF results in the two group subjects

All clinical and laboratory results for PCOS and tubal factor patients are summarized in Table 2. The rFSH duration, rFSH dosage, progesterone hormone levels, and endometrial thickness on HCG day were not different between the two groups. The E2 level, large follicles on HCG administration day, and the number of retrieved oocytes were significantly higher in PCOS group than in tubal group (P<0.05). Fertilization, cleavage, grade I/II embryo formation, clinical pregnancy, and implantation rates were lower in PCOS group than in tubal group, but no significant difference was observed. The miscarriage rate in PCOS group was slightly higher than that in tubal group.

Comparison of the ROS level and apoptosis of GC

We detected the intracellular ROS levels of GC between the two groups. The ROS fluorescence of GC in PCOS group was 33.45 × 10³ RLU, which is significantly higher than the 9.21 × 10³ RLU in tubal factor group (P<0.05) (Fig. 1A).

We further detected GC apoptosis by TUNEL assay. Nearly 44.85% TUNEL stain positive GCs (white arrow showed) were detected in PCOS group, whereas only 18.63% TUNEL positive GCs were found in tubal factor group. This difference is considered significant (P<0.05) (Fig. 1B).

NOX2 pathway involved in ROS production

NOX2 is a key enzyme for ROS production. This enzyme binds other protein subunits to comprise the NOX complex, which is the main source of intracellular ROS production. Gp91phox and p47phox protein bands were obtained by Western blot in GC of patients with PCOS and tubal factor (Fig. 2A). Results showed that NOX2 complex was expressed in GC and may induce protein activity to produce ROS. DPI and apocynin are both NOX2 specific inhibitors. We added DPI, apocynin, and control DMSO in cultured GCs and detected the change in intracellular ROS fluorescence. The presence of DPI and apocynin in tubal factor group slightly inhibited the ROS levels compared with that in DMSO group, and no differences were observed between these groups. However, the ROS levels in PCOS group significantly decreased after adding DPI and apocynin compared with adding DMSO (P<0.05) (Fig. 2B). These results showed that NADPH oxidase pathway may be involved in the mechanism of ROS production in GCs of patients with PCOS.

Discussion

As the most common endocrine disease, PCOS affects 5%–10% of reproductive-age women [¹⁹]. This disorder is characterized by hyperandrogenism, elevated LH levels, obesity, chronic anovulation, and reduced fertility. Patients with PCOS generally have low fertilization and high miscarriage rates because of the impaired oocyte quality [²⁰–²²].

In the present study, we compared the demographic information between patients with tubal factor and PCOS. The mean age, basal FSH, BMI, and duration of infertility are similar between the two groups. Many small antral follicles were observed in the PCOS ovaries [²³]; thus, the AFC of PCOS group was significantly higher than that of tubal factor group. Moreover,≥14 mm follicles and estradiol level on HCG administration day in PCOS group were also significantly higher than those in tubal factor group. Although the patients with PCOS retrieved more oocytes, their 2PN fertilization rate, cleavage rate, and grade I/II embryo formation rate of the mature oocytes were lower than those with tubal factor. Clinical pregnancy, implantation, and miscarriage rates were high in patients with PCOS. These results were similar to previous reports [²⁰–²²].

GCs are tightly wrapped around the oocyte. Thus, a large number of apoptotic GCs will affect the quality of oocyte, and accumulated ROS in GCs may be one of the causes of leading GC apoptosis. Apoptosis is closely related to follicular atresia [²³]. Apoptosis of GCs is also associated with empty follicle syndrome and few oocytes retrieved, low fertilization and cleavage rates, and low pregnancy rate in patients undergoing IVF/ICSI therapy [²⁴, ²⁵].

Das et al. reported that PCOS patients have substantial apoptotic GCs in retrieved oocytes [²⁶].

We detected the intracellular ROS level and found that the ROS fluorescence of GC in PCOS group has a nearly fourfold increase compared with that in tubal factor group, and the apoptotic GC in PCOS group was also significantly higher than that in tubal factor group (P<0.05). Therefore, our study revealed that the accumulated ROS in GCs might be one of the leading causes of GC apoptosis.

However, the mechanism involved in ROS generation in GCs of PCOS is not yet revealed [²⁷]. The excessive ROS accumulation in cells might impair the function of mitochondrial oxidative metabolism and lead to morphologically abnormal GCs [²⁸]. Saller et al. found that neurotransmitter norepinephrine (NE) and dopamine (DA) are linked to ROS-regulated events in GCs. NE robustly induces ROS generation, which in turn was prevented by the blockers of NE transporters [²⁹]. A high concentration of DA induces PCOS-derived GC apoptosis [³⁰].

NADPH oxidase is the key enzyme for intracellular ROS production. NOX family includes NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2, and seven other members [³¹]. Only NOX2 is expressed in the female ovary and endometrium [³²]. NOX2 complex is composed of six subunits, gp91phox (NOX2), p22phox, p47phox, p67phox, p40phox, and Rac1 [³³]. In the role of signaling molecules, cytosolic subunits and membrane subunit (gp91phox and p22phox) combine to form active oxidase complex, induce transmembrane movement of electrons, and generate ROS [³⁴,³⁵]. Gp91phox and p47phox protein bands were obtained by Western blot in the GCs of patients with PCOS and tubal factor. Furthermore, NOX specific inhibitors (DPI and apocynin) were found to effectively inhibit the ROS levels in GCs. These results indicate that NOX pathway may be involved in GC ROS production in patients with PCOS. However, the specific role of NOX in GC ROS production is still poorly understood. We will further investigate these mechanisms and processes in the future.

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