Ethics statements
This prospectively, randomized, open-label study was registered in the Chinese Clinical Trial Registry Center (Registration No. ChiCTR1900021269) and approved by the Medical Ethics Committee of Sichuan Provincial Hospital for Woman and Children. Signed informed consent was obtained from all participants. All procedures in this study complied with the ethical standards of the relevant national and institutional committees on human experimentation and with the Helsinki Declaration 1975 (2013 revision).
Sample size of calculation was based on the assumption that clinical pregnancy rate would increase threefold percent after GH pretreatment. The CPR in patients with POR in our center was around 13%, 48 patients were required in each group with α of 0.05 and a β error of 0.1 (power = 90%)
Study subjects
The patients with POR (age 33-43 years) diagnosed according to the Bologna criteria [1], who underwent IVF, were enrolled from Reproductive Medicine Center of Sichuan Provincial Hospital for Women and Children (between Feb. 2019 and Dec. 2019). The patients with POR were randomized 1:1 to either GH pretreatment (POR-GH group) or no (POR-C group) (using computer generated random numbers). The excluded criterion include: (1) hydrosalpinx, congenital uterine malformations and/or endometrial disease as tuberculosis, hyperplasia; (2) basal follicle stimulating hormone (bFSH) ≥15 IU/L; (3) systemic lupus erythematosus, sicca syndrome, polycystic ovarian syndrome; (4) uncontrolled endocrinopathy as diabetes, hyperthyroidism, hypothyroidism, and hyperprolactinemia; (5) underwent IVF-ET treatment within three months; (6) intracytoplasmic sperm injection (ICSI) cycle because of male infertility; (7) supplementation with any antioxidants such as Vitamin E, Vitamin C, CoQ10, beta-carotene and selenium. The tubal factor infertile women (age 20-35 years) with normal ovarian reserve and regular menstrual cycle were recruited as non-POR controls during the same period, who underwent IVF-ET. The exclusion criteria for non-POR group were as the same as POR group.
Medical history of all participants was collected, such as regularity of menstrual cycles, duration of infertility, and history of treatment. Abnormal menstrual cycles included oligomenorrhoea, polymenorrhoea, irregular menstrual cycles, and amenorrhoea. Body weight index (BMI) was calculated by square of body height (M) divided by body weight (Kg). Antral follicle count (AFC) was performed by trans-vaginal ultrasound on day 2-3 of the menstruation or progestin induced withdraw bleeding.
Controlled Ovarian Stimulation (COS) and IVF
All patients underwent Controlled Ovarian Stimulation (COS) with GnRH antagonist protocol. Recombinant follicle stimulating hormone (rFSH) (Gonal-F; Merck-Serono KGaA., Darmstadt, Germany) was injected on day 2 of the menstrual cycle,and rFSH doses were adjusted according to follicle growth and serum hormone levels. In POR-GH group, 4 IU/d recombinant human growth hormone (Jinsai Pharmaceutical Co., Ltd., Changchun, Jilin, China) was injected subcutaneously on day 2 of the previous menstrual cycle before IVF till the trigger day (36-48 days). When a leading follicle reached 12 mm and/or serum E2 levels reached 300 pg/mL, Ganirelix(Merck Sharp & Dohme co., Ltd, Hoddesdon, United Kingdom)was administered. When at least one follicle was above 18mm, recombinant human chorionic gonadotropin (hCG) (Ovitrelle®; Merck-Serono KGaA.) was administered as trigger. When there were no follicles with diameter ≥14 mm after 10 days of gonadotrophin injected or when peak E2 level was below 300 pg/mL, the cycle was cancelled. Ultrasound-guided transvaginal oocyte retrieval was performed 36 hours later, and follicle flushing was not performed. Once oocyte retrieval, only follicle fluid (FF) from follicles with a diameter ≥16mm were collected and immediately centrifuged at 700 g for 5 min in room temperature. The supernatant was stored at -80℃. The precipitates were suspended in 3ml PBS and gently layered in 3 ml 50% Lymphocyte separation medium Beijing Solarbio Science and Technology Corporation, Beijing, Solarbio Science and Technology Co., Ltd, China), then centrifuged at 700g for 10min to remove red blood cells and debris. GC layered at the interface of the gradient were washed twice with 5ml PBS (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, Jiangsu, ChinaKeyGEN Bio TECH Co., Ltd., Jiangsu, China) and was immediately examined ROS using fluorescent microscope and Spectrophotometer. FF or GC from each patient were collected separately and considered as one sample. According to the criteria established by the Istanbul Consensus Workshop on Embryo Assessment, the cultured embryos on day 3 were assessed based on the number of blastomeres and the degree of fragmentation, and higher quality were categorized as grades A/B [19]. One or two embryos with the best morphological grade were selected for transfer. Luteal phase support with intramuscular injection of progesterone 60 mg/d commenced on oocyte retrieval day. Serum hCG was measured 12 days after ET and was considered positive for hCG≥5 IU/mL. The Clinical pregnancy was defined as demonstration of a gestational sac with an embryo showing cardiac activity. Early miscarriage was defined as loss of pregnancy before gestational week 12. The rates of implantation rate, clinical pregnancy and miscarriage were calculated. Ovarian hyperstimulation syndrome (OHSS) was diagnosed according to Navot D et al[20].
We suggest GH 4 IU/d pretreatment on day 2 of the previous menstrual cycle before IVF till the trigger day, because low physiological dose and longer treatment (from the antral follicle stage) might be more beneficial to follicular growth and development[2].
Measurement of endocrine and metabolic parameters
Plasma glucose was measured using the hexokinase method (Beijing Strong Biotechnologies, Inc., Beijing, China). Estradiol (E2), progesterone (P), total testosterone (TT), luteinizing hormone (LH), FSH, and insulin levels were measured using the electrochemiluminescence immunoassay platform (Roche Diagnostics GmbH, Mannheim, Germany). Serum level of anti-Mullerian hormone (AMH) was measured using enzyme linked immunosorbent assay kit (Guangzhou Kangrun Biotech, Co., Ltd, Guangdong, China). Homeostasis model assessment (HOMA-IR) index was calculated as fasting glucose (mmol/l) × fasting insulin (mU/mL)/22.5 [21]. The intra- and inter-assay coefficients of above variation were less than 5% and 10%, respectively.
OS marker in FF assay procedures
FF malondialdehyde (MDA) concentrations (umol/L) were measured using micro-MDA detection kits (NanJing Jiancheng Bioengineering Institute Co. Ltd., Nanjing, Jiangsu, China) and ultraviolet spectrophotometry (Shanghai Meipuda instrument Co., Ltd., Shanghai, China) was performed at 532 nm. Superoxide dismutase (SOD) was determined using SOD kits (NanJing Jiancheng Bioengineering Institute Co. Ltd.) and spectrophotometry at 450nm. Total antioxidant capacity (TAC) (mmol Trolox Equiv./L) and TOS (umol H2O2 Equiv./L) were measured by the semi-automatic microplate colorimetric method,using hydrogen peroxide (H2O2) or Trolox as a standard curve, respectively [22,23] . The oxidative stress index (OSI) expressed as the ratio of TOS to TAC. The detections were duplicated for each OS parameter. Serum samples from healthy volunteers were pooled for quality control. The intra- and inter-assay coefficients of above variations were less than 5% and 10%, respectively.
Detection of intracellular ROS levels
Using a dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe, ROS level in GC was detected by ROS assay Kit (Beyotime Biotechnology Co., Ltd., Shanghai, China), and green fluorescence was examined by fluorescent microscope (Olympus Corporation, Tokyo, Japan). The examination wavelength was 488 nm and the emission wavelength were 525 nm, respectively. The cell nucleus was stained with 4',6-diamidino-2-phenylindole (DAPI) (NeoFroxx, Frankfurt, Germany).
The value of intracellular ROS was measured by NanoDrop UV-Vis Spectrophotometers (Thermo Scientific, Massachusetts, USA), and fluorescence intensities shown as the ratio to the control group (non-POR group).
Statistical Analysis
All data were statistically analysed using SPSS 17.0 software (SPSS Inc., Chicago IL, USA). Continuous variables were expressed as mean ± standard deviation (SD). The Kolmogorov–Smirnov test was used to assess for normality of data distribution. Continuous variables with normal distribution were compared using Student-Newman-Keuls test, and Bonferroni’s test was used as the post hoc test. Categorical data were compared using Chi-squared tests. Two-tailed P values less than 0.05 were considered as statistically significant.