Study design and randomization
This was a prospective randomized controlled study, conducted at the Reproductive Medical Center of the Peking University Third Hospital, a tertiary university hospital and a center of excellence in Reproductive Medicine in China. The study is reported according to the CONSORT guidelines. The flow of the patients in this study is presented in Fig. 1.
All the participants were randomized 1:1 to either CoQ10 treatment (study group) or no treatment (control group) followed by an ART cycle. The randomization was performed over the period of 14 months (between June 2, 2015 and July 31, 2016) by using the computer-generated randomization codes, which were then placed in the sealed, opaque sequentially numbered envelopes by a third party (nurse practitioner) who was not directly involved in the patient management or in the randomization process. The envelopes were handed out to the participants upon completing the informed consent. The study participants and the investigators were not blinded to the patient grouping. The participants were followed through one completed ART cycle until all frozen embryos generated from the index cycle were used or until delivery in those who achieved pregnancy.
Study population
All consecutive women who were found to have POR and were referred to IVF-ET cycle in our institution were approached. POR was defined according to the ESHRE Bologna criteria [7]. The study inclusion criteria were: age < 35 years, anti-Mullerian hormone (AMH) < 1.2 ng/ml, and antral follicle count (AFC) < 5, the parameters that corresponded to a low prognosis group 3 as per the POSEIDON stratification [9]. Exclusion criteria were: age ≥ 35 years, history of ovarian surgery, endocrine or autoimmune disease (e.g. diabetes, thyroid disease or presence of anti-thyroid antibodies or PCOS), chromosomal abnormality, uterine malformations, more than 3 previous IVF cycles, treatment with cholesterol-lowering drugs, previous treatment with anti-oxidants (last 5 years) or known allergy to CoQ10 or ubiquinol (the water-soluble isoform of CoQ10). All the participants completed the questionnaire with demographic, medical and reproductive information and underwent clinical examination, pelvic ultrasound, chromosome analysis, AMH test, reproductive endocrine profile and thyroid studies. All the included women were specifically asked about any previous treatment with anti-oxidants such as CoQ10, ubiquinol, vitamin A, vitamin E, vitamin C, beta-carotene or selenium, including the duration and time of treatment.
Treatment protocols
The intervention in the study group included oral administration of CoQ10 (GNC Holdings Inc., Pittsburg, PA, USA) 200 mg three times a day, for a period of 60 days in an open label fashion. The ART treatment (in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI)) was commenced in the first menstrual cycle upon completion of CoQ10 treatment. The control group commenced ART (IVF or ICSI) after enrollment without any additional treatment.
Ovarian stimulation and oocyte retrieval
All participants underwent ovarian stimulation with the short GnRH-antagonist protocol. A combination of recombinant follicle stimulating hormone (FSH) (Gonal-F, 225 IU/day, Merck Serono SA Aubonne Branch) and human menopausal gonadotrophin (Menotropins for injection FSH 75 IU: LH 75 IU, 225 IU/day, Livzon Pharmaceutical Group Inc.) in a fixed-dose was started on Day 2 of the menstrual cycle with the option to adjust dose according to response after 4 days of stimulation (Day 6 of menstrual cycle). GnRH antagonist (Cetrorelix 250 μg/day, Merck Serono, Darmstadt, Germany) was started when a leading follicle of 12 mm was achieved. Recombinant human chorionic gonadotrophin (hCG) trigger (Ovidrele 250 μg; Merck Serono S.p.A, Rome, Italy) was administered when at least one follicle was above 18 mm. The cycle was cancelled when there were no follicles with diameter ≥ 14 mm after 8–9 days of gonadotrophin therapy or when peak E2 level was below 250 pmol/l.
Ultrasound-guided transvaginal oocyte retrieval was performed 36–38 h after the trigger injection by using a 17-gauge double-lumen needle (Cook Medical) and a vacuum pump (Cook Medical) under pressure at 125 mmHg. Each follicle sized above 12–14 mm was drained, and follicle flushing was not performed. The cumulus-oocyte complexes (COCs) were removed from the collection fluid using a sterile glass pipette and washed in G-IVF Plus media (Vitrolife, Sweden) and transported to the laboratory.
Oocyte insemination and embryo culture
Oocytes were inseminated either by conventional IVF or by ICSI depending on sperm quality. Oocytes undergoing IVF insemination were placed into a dish with G-IVF (Vitrolife) covered in mineral oil. Oocytes undergoing ICSI were denuded and injected if maturation status was confirmed by the presence of the first polar body (PB). Fertilization was assessed 17–19 h after insemination and was defined by the presence of two pronuclears (2PN) and two PBs. All embryos were transferred to GM medium (G-M, Life Global, CT, USA) for a further 48 h of culture. Embryo development and quality were assessed 68–72 h (day 3) after insemination, based on the number of blastomeres, blastomere symmetry, percentage of fragmentation, and quality of cytoplasm according to the criteria established by the Istanbul Consensus Workshop on Embryo Assessment [26]. All supernumerary day-3 embryos were cryopreserved by vitrification (JIEYING laboratory Inc., Canada) for future use.
Endometrial preparation and embryo transfer
All patients underwent transfer of day-3 embryos in a fresh cycle and subsequent frozen embryo transfer (FET) when the cryopreserved embryos generated from the index stimulation cycle were available. The embryos with the best morphological grade were selected for transfer. In absence of high-quality embryos, transfer of any embryo quality was considered after careful patient counselling.
In a fresh cycle, the luteal phase was supported with progesterone intravaginal gel (Crinone 8% 90 mg/day, Merck-Serono) commenced on the day of oocyte retrieval until 14 days after embryo transfer. In women with positive pregnancy test, luteal support was continued until 8 weeks gestation. The protocols used for FET utilized either natural cycle or artificial estradiol and progesterone endometrium priming in normo-ovulatory and oligo-ovulatory women, respectively. In natural cycle, ovulation was tracked with transvaginal ultrasound and urine LH kit. Oral dydrogesterone (Duphaston, 20 mg daily for 7 days; Abbott Biologicals B.V.) was commenced for luteal phase support 3 days after LH surge on the day of embryo transfer until 8 weeks gestation. In artificial FET protocol, oral estradiol valerate (Progynova 6 mg/day, Schering, Berlin, Germany) was initiated on the third day of the menstrual cycle and endometrial thickness was monitored with transvaginal ultrasonography. When the endometrial thickness exceeded 8 mm, luteal support with progesterone intravaginal gel (Crinone 8% 90 mg, daily; Merck-Serono), combined with oral dydrogesterone (Duphaston, 20 mg daily for 7 days; Abbott Biologicals B.V.) was added and embryo transfer was performed after 5 days. Hormonal treatment was stopped if pregnancy test was negative or continued until 11 weeks gestation with tapering off after 10 weeks. Single or double cleavage-stage embryo transfer were performed by using a soft catheter (K-Soft 5100; Cook, Queensland, Australia) without ultrasound guidance. Serum hCG was measured 14 days after embryo transfer and was considered positive for hCG level ≥ 10 IU. Transvaginal ultrasonography at 30 days after transfer was used to confirm clinical pregnancy.
Hormone assay procedures
All the hormonal assays were performed at the endocrine laboratory of the Peking University Third Hospital Reproductive Centre by using commercially available kits. Serum concentrations of hCG were determined by using the commercially available ELISA kit (Beckman DXI800, Beckman, USA) according to the manufacturer’s instructions. Serum levels of anti-Mullerian hormone (AMH) were measured by automated assays using commercially available kit (Ashlab, USA). Serum luteinizing hormone (LH), FSH, estradiol (E2), and Progesterone (P) were tested using the Immulite 1000 assay based on chemiluminescence (DPC, Poway, CA).
The lower detection limit of the hCG and the AMH assays was 0.5 IU/L and 0.06 ng/ml respectively. The intra- and inter-assay coefficient of variation (CV) for hCG activity was 5% and for AMH was 8%. The lower detection limit of LH, FSH, E2 and P was 0.05 IU/L, 0.12 IU/L, 73.4 pmol/L, 0.64 nmol/L, respectively. The CV of LH and FSH was 6% and of E2 and P was 10%.
Outcome measures
The primary outcome measure was the number of high quality day-3 embryos generated from one stimulation cycle. High quality embryos were defined as embryos that reached 6 to 8-cell stage with cytoplasmic fragmentation occupying less than 10% of the embryo surface and had equal size blastomeres.
The secondary outcomes included ovarian response parameters (duration of stimulation, total dose of gonadotrophins, peak E2 level and endometrial thickness on the day of hCG trigger), embryological parameters (number of oocytes retrieved, fertilization rate, number of patients with frozen embryos and number of patients who did not achieve embryo transfer) and clinical parameters (miscarriage, clinical pregnancy and live birth rate). Fertilization rate was defined as the number of 2PN embryos divided by the number of inseminated oocytes. Clinical pregnancy was defined as a presence of intrauterine gestational sac observed on ultrasound after 30 days of embryo transfer. Miscarriage was defined as a loss of clinical pregnancy before 24 weeks of gestation. Live birth was defined as the birth of at least one living child, irrespective of the duration of gestation. Clinical pregnancy and live birth rate were calculated per embryo transfer cycle as number of pregnancies/ live births divided per number of women who had transfer. Cumulative pregnancy and live birth rate were defined as the number of clinical pregnancies/live births generated from the index ART cycle following fresh or frozen embryo transfer divided by all women who received treatment. In addition, markers of ovarian reserve, including AMH, day 3 FSH and AFC were evaluated before and after CoQ10 treatment in the participants from the intervention (study) group.
Sample size calculation
The sample size calculation for this study was based on the number of high quality embryos as primary outcome. In our center women with poor response have an average 0.6–0.8 high quality embryos per woman. To detect a difference of 50% in primary outcome measure (from 0.6–0.8 to 1.0–1.2 embryos per woman) with alpha 0.05 and power 0.80, the required sample size was estimated at 76 women in each arm. When accounted for a drop out rate of 20%, each arm required 92 women.
Statistical analysis
The Student’s t-test or Mann-Whitney U test were used for comparisons of continuous variables between the groups depending on the distribution of the data. The chi-squared test or Fisher’s exact test, where appropriate, were used for comparisons of categorical variables. Results are presented as mean ± standard deviation (SD), median and interquartile range (IQR) or as percentages. Statistical significance was set at a probability (p) value < 0.05. All statistical analyses were performed using SPSS 22.0 software (IBM Corp., Armonk, NY, USA).