This cross-sectional study was conducted at the Infertility Clinic, Reproductive Medicine Division, Department of Obstetrics and Gynecology, King Chulalongkorn Memorial Hospital, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand from February 1, 2017 to April 30, 2018. The study was approved by the Institutional Review Board, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand on January 27, 2017 (IRB No.688/59). Written informed consent were obtained from all participants. This research has been registered at clinicaltrials.gov (NCT03155438).
Fifty-six infertile women, aged between 25-49 years old who underwent IVF were enrolled into this study with 28 participants in normal ovarian response group (NOR) and 28 participants in poor ovarian response group (POR). Patients were categorized to the normal and the poor ovarian response groups according to the number of retrieved oocytes at ovum pick-up (OPU) day.
Poor ovarian response in the present study was defined as 1-4 retrieved oocytes. The control group or normal ovarian response group, age-matched with POR group, consisted of women with 5-15 retrieved oocyte. Exclusion criteria were women with active ovarian pathologies such as ovarian endometrioma or basal follicle stimulating hormone (FSH) level more than 20 IU/L or women with ovarian hyperstimulation syndrome. None of the participants were taking any antioxidant and vitamin supplements other than folic acid.
Sample size calculation
Sample size calculation was analyzed according to the previous study by Donabela et al  with mean of SOD1 relative gene expression in healthy women control group; 2.06 2-Δ ΔCT (SD = 0.53). To compare two different means, the sample size calculation was performed with the following expectation; SOD1 gene expression level in POR was lower than NOR by 20%, resulting in a number of 25 women for each study group. With an addition of 10% probability for data loss, a total of 56 women were finally included in the present study, 28 patients with NOR and 28 women with POR.
Ovarian stimulation protocol
As the patients were categorized into normal responders and poor responders according to their results on the OPU day, ovarian stimulation protocol for each patient was managed and adjusted to patient baseline characteristics under judgement of attending physicians. Both patient groups received flexible gonadotropin-releasing hormone (GnRH) antagonist protocol. Ovarian stimulation initiated on day 3 of menstruation cycle with 200-375 U/day of recombinant follicle stimulating hormone (rFSH; Follitropin alpha; Gonal-f®, Merck Serono SA, Switzerland), Follitropin beta (Puregon®, MSD, France) or human menopausal gonadotropin (hMG; Menopur®, Ferring Pharmaceuticals, St Prex, Switzerland). Dosages were adjusted according to the ovarian response. The GnRH antagonist; Ganerelix (Orgalutran®, N.V. Organon, The Netherlands), was given daily at a dosage of 250 mcg starting when leading follicles reached 14 mm. Finally, 250 mcg of recombinant human chorionic gonadotropin (hCG) (Ovidrel®, Serono, Rockland, MA, USA) was administered when the presence of at least three leading follicles (or most of leading follicles in the poor ovarian response group) reached more than 17 mm in diameter. The OPU was performed at 36-38 hours after hCG administration.
Oocyte and cumulus cell retrieval
Oocyte retrieval was carried out using 16-gauge double lumen and ovum aspiration needle (Cook Medical, Queensland, Australia) under transvaginal ultrasound guidance. Nutrient Mixture (Ham) F-10 (1X) (Gibco®, Life Technologies, New York, USA), containing 25 mM HEPEs (Sigma, St. Louis, MO, USA) and L-Glutamine (Sigma), was used as flushing media. Only oocyte with cumulus cells (cumulus cell oocyte complex; COCs) from first punctured follicle exceeding 18-20 mm in diameter of each patient was collected for further mRNA gene expression analysis.
Cumulus cell retrieval protocol is described briefly as the following: the collected COCs were stored in fertilization media (LifeGlobal, Guilford, CT, USA) containing 10% human serum albumin (LifeGlobal) and incubated at 37°C in 5% CO2 for 2 to 3 hours. The COCs were then exposed to 70 IU hyaluronidase (type VIII from bovine testes, Sigma). The cumulus cells were mechanically separated from oocyte under stereo-microscopy (2X magnification). The oocyte from the first punctured follicle was further cultured separately from other sibling oocytes. To avoid vaginal cell and white blood cell contamination, cumulus cells were washed with phosphate-buffered saline solution and assessed under light microscope (10X magnification). Each sample was stored in 15 mL-tube and further processed for the mRNA gene expression study.
Evaluation of antioxidative enzyme-related genes (SOD1, SOD2 and GPx4) mRNA expression
The cumulus cell total RNA extraction from each individual COCs was performed using ReliaPrep™ RNA Miniprep System (Promega, Madison, WI, USA). RNA concentration was then measured with NanoDrop 1000 Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE, USA). The first-strand complementary DNA (cDNA) was generated from 50 ng total RNA by using Reverse Transcription System (Promega). The cDNA concentration was again measured with Nanodrop spectrophotometer. Gene-specific primers for the subject sequences, housekeeping gene (GAPDH) and target genes (SOD1, SOD2 and GPx4) were predesigned by integrated DNA technologies (PrimeTime Mini qPCR Assay, Integrated DNA Technologies, Singapore) (Table1). Conventional PCR was carried out according to the manufacturer’s instruction (Multiplex PCR kit, Qiagen, Valencia, CA, USA) as following: Taq activation (95°C, 15 min) and 38 cycles of denaturation (94 °C, 30 sec), annealing for 30 sec at a different temperature for each primer (GAPDH, 52 °C; SOD1, 52 °C; SOD2, 56 °C; GPx4, 52 °C) and then extension at 72°C for 20 sec. The final extension was performed at 72 °C for 1 min. Visualization of the PCR product was evaluated using UV light (Gel Documentation System, Bio-Rad, Hercules, CA, USA). Only samples with confirmation of GAPDH expression were proceeded to quantitative real-time polymerase chain reaction (qPCR) with total volume of 20 µl (consisted of cDNA 50 ng, 10 µl qPCR master mix (Luna Universal Probe qPCR master mix, New England Biolabs, Ipswich, MA, USA) and 500 nM of qPCR Primer and probes (PrimeTime Mini qPCR Assay) (Table 1). All reactions were performed in duplicate and processed with qPCR (StepOnePlus Instrument, Applied Biosystem, Foster City, CA) according to following conditions: 95 ºC for 60 sec, followed by 45 cycles at 95 ºC for 15 sec and 56 ºC for 15 sec. Two replications were calculated for each gene expression. Relative quantification of mRNA expression was calculated using 2-Δ ΔCT method .
Baseline characteristics, in vitro fertilization (IVF), embryo transfer and clinical pregnancy outcomes
Patients were determined to undergo fresh or frozen-thawed embryo transfer on the basis of their clinical indication. Clinical pregnancy was defined as the presence of intrauterine gestational sac 4 weeks after embryo transfer. Clinical data such as baseline characteristics, infertility history, basal hormonal profiles, ultrasonography result, IVF and embryo profiles were obtained from medical records.
All data analyses were performed using SPSS statistic software package version 22 (SPSS Inc, Chicago, IL, USA). The nonparametric Mann-Whitney test was used for data analysis to compare variables between normal and poor responders when data was absent of normal distribution. Independent t-test for continuous variables and Chi-square test for categorical variables were performed when data distributed normally. The level of significance was set at P < 0.05.