Study design

The present study is a retrospective cohort study (Trial registration: ChiCTR-OOC-14005389, 2014). In this study, 321 PCOS cases was conducted from October 2015 to April 2017 to assess the effect of short-term vitamin E administration on infertile PCOS women undergoing ovulation induction with CC and HMG in the Reproductive Medicine Center, Jiangsu Province Hospital of Chinese Medicine, Nanjing, China. This retrospective cohort study was approved by the Institutional Review Board of the Department of Chinese Medicine Hospital of Jiangsu Province.

The inclusion criteria of this study were as follows: (i) Undergoing ovulation induction with CC and HMG; (ii) no previous infertile treatment; (iii) age less than 40 years; (iv) normal in hysterosalpingography; and (v) normal in semen analysis. The diagnostic criteria of PCOS was according to the 2006 Rotterdam criteria [17]: (1) Anovulation or olig-ovulation, (2) Clinical evidence of hyperandrogenism (on the basis of hirsutism or an elevated testosterone level), (3) Polycystic ovaries (a more than 10ml ovarian volume or at least 12 antral follicles with 29mm in diameter). PCOS could be confirmed if any 2 out of the following 3 criteria were met and if any other diseases that caused hyperandrogenism or anovulation could be excluded.

Other disorders that mimic the PCOS, including hyperprolactinemia, thyroid disease, late-onset congenital adrenal hyperplasia, androgen-secreting tumors and Cushings syndrome were ruled out. The PCOS patients with the major myocardial, liver and renal disorders, and taking confounding medications (primarily sex steroids, other infertility drugs, and insulin sensitizers) were excluded. Patients were divided into 3 groups according to the vitamin E used.

In this study, as shown in Fig. 1a, 110 of 321 PCOS cases underwent controlled ovarian stimulation but without vitamin E administration (Group A, n=110). Based on previous clinical medication experience, a dosage of 100mg/day vitamin was selected. Two-hundred eleven of or 321 PCOS cases underwent controlled ovarian stimulation combined with vitamin E administration (100mg/day, p.o.) started from follicular phase (Group B, n=105) and luteal phase (Group C, n=106), respectively. Administration of vitamin E in follicular phase(Group B) began from the 3rd day of the menstrual cycle to 14th day of luteal phase. Administration of vitamin E in luteal phase (Group C) started when ovulation was confirmed, and lasted for 14 consecutive days. After 14days of the HCG administration, serum -HCG was measured. The presence of a gestational sac on ultrasound was performed at 6 and 12weeks of gestational age to determine clinical pregnancy rate and ongoing pregnancy. The women enrolled in this study were followed up until miscarriage or delivery.

Summary of patient flow diagram a and Vitamin E administration and stimulation protocol b. EV=estradiol valerate; HCG=urinary human chorionic gonadotropin; Pg=progesterone;CC=clomiphene citrate;VE=vitamin E;TVU=Transvaginal ultrasonography; HMG=human menopausal gonadotropin

As shown in Fig. 1b, the ovulation was stimulated with CC (Merck Serano, China) at 100mg/day for 5days starting on day 3 of a spontaneous menstrual cycle or withdrawal bleeding. Starting from day 8, HMG (Livzon, China) was injected at 75IU every second day and estradiol valerate (Progynova, Bayer, China) was administered at 2mg/day. Transvaginal ultrasonography was performed from day 10 to adjust the HMG dosage. When at least one follicle had reached a diameter of 18mm, 10,000IU urinary human chorionic gonadotropin (hCG) (Livzon, China) was administered. All patients received luteal phase support by oral administration of progesterone (Dydrogesterone, Abbott Biologicals B.V, China) at 10mg three time a day for 14days starting on the day of ovulation. In each cycle, medroxyprogesterone acetate was used to induce withdrawal bleeding in cases in which there was no response. The complete participation considered as pregnancy or anovulation within a total of 6 cycles.

Body mass index (BMI) was used to evaluate the body weight. According to World Health Organization(WHO) criteria [18], women with a BMI <18.5, 18.525, 2529 and30kg/m2 were defined as underweight, normal weight, overweight and obese, respectively. Scores on the modified FerrimanGallwey scale [19], range from 0 to 36, were used for hirsutism evaluation. Higher scores indicated a greater degree of hirsutism.

Age, height, weight, waist, FerrimanGallwey hirsutism score, age of menarche, incidence of oligomenorrhea and amenorrhea, numbers of previous pregnancies and previous ovarian were obtained from patient medical records.

Levels of estradiol (E2), androstenedione (T), luteinizing hormone (LH), prolactine (PRL) and follicle-stimulating hormone (FSH) were tested by RIA (Beijing North Institute of Biological Technology of China and the CIS Company of France). Peripheral blood samples were taken on the 3th day of menstrual cycle after overnight fasting.

In this study, we measured four oxidative stress serum markers (malondialdehyde (MDA), ischemia modified albumin (IMA), total antioxidant capacity measurements (TAC), and vitamin E) in 3 time points (T0:before stimulation, T1: the day of HCG treatment, and T2: the day of complete participation) to evaluate the levels of oxidative stress. Serum levels of MAD, an end-product formed during lipid peroxidation that is released into the extracellular space and finally appears in the blood [20], were measured using a thiobarbituric acid-reactive commercial kit (Jiancheng Bioengineering Institute, China). Serum TAC, provided better information on antioxidant status than individual antioxidant compounds [21], were tested using an antioxidant assay kit (Jiancheng Bioengineering Institute, China). Serum levels of IMA, a novel marker of oxidative stress, were evaluated by cobalt to albumin binding capacity kit (CUSABIO, China). Serum contents of vitamin E were evaluated by colorimetric method using assay kit (Jiancheng Bioengineering Institute, China).

Statistical analysis was carried out by SPSS (version 23, USA). Data were presented as either median (Min-Max) or meanSD as appropriate. Quantitative data analyses were carried out by independent samples t-test or Mann-Whitney U-test depending on the normality of data. Categorical variables were compared with Chi-Square test. A P value <0.05 was considered as statistically significant.

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Effect of a short-term vitamin E supplementation on oxidative stress in infertile PCOS women under ovulation induction: a retrospective cohort study -...