This is a multi-center randomized trial involving four treatment groups. The research protocol is compliant with the Consolidated Standards of Reporting Trials (CONSORT) guidelines and Standards for Reporting Interventions in Clinical Trials of Acupuncture (STRICTA), as well as with the Standard Protocol with the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) statement. The SPIRIT checklist is presented in Supplementary file 1.
A total of 1,100 infertile women with PCOS will be recruited from 28 hospitals in China. They will be randomly allocated into four groups: personalized acupuncture, fixed acupuncture, letrozole or placebo letrozole. Women will be recruited through advertisements in local newspapers and bulletin boards in each trial center. Eligible women will be approached and sign the consent form after detailed explanation of the study design and comprehensive counseling.
Inclusion criteria are:
- Age of women between 20 and 40 years.
- Chronic oligomenorrhea or amenorrhea: oligomenorrhea is defined as an intermenstrual interval > 35 days or < 8 menstrual bleedings in the past year. Amenorrhea is defined as an intermenstrual interval >90 days.
- Hyperandrogenism (either hirsutism or hyperandrogenemia) or polycystic ovaries on ultrasound. Hirsutism is determined by a modified Ferriman-Gallwey (FG) Score ≥ 5 at screening examination, and biochemical hyperandrogenism is defined as total testosterone (T) >2.6 nmol/L and free testosterone ≥ 6.0 pg/mL (the cutoff level may differ from each study site). Polycystic ovaries are present when the number of small antral follicles (2–9 mm in diameter) ≥12 or ovarian volume > 10mL on transvaginal scanning .
- At least one patent tube shown by hysterosalpingogram or diagnostic laparoscopy within 3 years if the patient does not have a history of abortion or pelvic operation. If the patient has a history of pregnancy and no history of pelvic operation within the past 5 years, she is not required to undergo a tubal patency test.
- Sperm concentration ⩾ 15 × 106/mL and total motility ⩾ 40% or total motile sperm count ⩾ 9 million in the semen analysis of the husband .
- The couple agree to have regular intercourse i.e. 2-3 times per week during the study period.
Exclusion criteria included:
- Exclusion of other endocrine disorders
a. patients with hyperprolactinemia (defined as two prolactin (PRL) levels at least one week apart ≥ 25 ng/mL)
b. Patients with FSH levels > 15 mIU/mL. A normal level within the last year is adequate for entry
c. Patients with uncorrected thyroid disease (defined as thyroid-stimulating hormone (TSH) < 0.2 mIU/mL or > 5.5 mIU/mL). A normal level within the last year is adequate for entry
d. Patients diagnosed with Type I or Type II diabetes who are poorly controlled (defined as glycated haemoglobin (HbA1c) level > 7.0%), or patients receiving antidiabetic medications such as metformin, insulin, thiazolidinediones, acarbose, or sulfonylureas
e. Patients with suspected Cushing's syndrome.
- Use of other TCM treatments including Chinese herbal prescriptions and acupuncture in the past 3 months.
- Use of other western medications known to affect reproductive function or metabolism in the past 2 months.
- Pregnancy within the past 6 weeks.
- Within 6 weeks postabortion or postpartum.
- Breastfeeding within the last 6 months.
- Not willing to give written consent to the study.
- Additional exclusion criteria are as follows.
a. Patients taking other medications known to affect reproductive function or metabolism. These medications include oral contraceptives, depot progestins, hormonal implants (including Implanon), GnRH agonists and antagonists, antiandrogens, gonadotropins, antiobesity drugs, antidiabetic drugs such as metformin and thiazolidinediones, somatostatin, diazoxide, angiotensin-converting-enzyme inhibitors, and calcium channel blockers. The washout period on these medications will be two months, longer washouts may be necessary for certain depot contraceptive forms or implants, especially where the implants are still in place. A one-month washout will be required for patients on depot progestins.
b. Patients with liver disease defined as aspartate aminotransferase (AST) or alanine aminotransferase (ALT) >2 times normal or total bilirubin >2.5 mg/dL. Patients with renal disease defined as blood urea nitrogen (BUN) > 30 mg/dL or serum creatinine >1.4 mg/dL.
c. Patients with hemoglobin < 10 g/dL.
d. Patients with a history of deep venous thrombosis, pulmonary embolus, or cerebrovascular accident.
e. Patients with known heart disease that is likely to be exacerbated by pregnancy.
f. Patients with a history of suspected cervical carcinoma, endometrial carcinoma, or breast carcinoma. A normal Pap smear result will be required for women 21 years and over.
g. Patients with a current history of alcohol abuse. Alcohol abuse is defined as >14 drinks/week or binge drinking.
h. Patients enrolled into other investigative studies that require medications, prescribe the study medications, limit intercourse, or otherwise prevent compliance with the protocol.
i. Patients who anticipate taking longer than a one-month break during the protocol should not be enrolled.
j. Patients with a suspected adrenal or ovarian tumor secreting androgens.
k. Couples with previous sterilization procedures (vasectomy, tubal ligation), which have been reversed. The prior procedure may affect study outcomes, and patients with both a reversed sterilization procedure and PCOS are rare enough that exclusion should not adversely affect recruitment.
l. Subjects who have undergone a bariatric surgery procedure in the recent past (< 12 months) and are in a period of acute weight loss or have been advised against pregnancy by their bariatric surgeon.
m.Patients with untreated poorly controlled hypertension defined as a systolic blood pressure 160 mmHg or a diastolic 100 mm Hg obtained on two measures obtained at least 60 minutes apart.
n. Patients with known congenital adrenal hyperplasia.7
All participants will be informed of benefits of regular physical exercise and will be instructed to have regular intercourse every 2 to 3 days.
Women in both personalized and fixed acupuncture groups will receive acupuncture treatment three times a week with a maximum of 48 treatment sessions over 16 weeks. Acupuncture treatment will start on day 3 after a spontaneous period or after a withdrawal bleeding following progestin. They will be contacted by phone if they miss the scheduled appointment. If they miss appointments during treatment, missed appointments will be clearly documented in the record for analysis later. They will be treated for up to 16 weeks. If they become pregnant, the acupuncture treatment will be stopped. For personalized and fixed acupuncture group, credibility and expectancy questionnaires will be completed on the third acupuncture treatment and the last acupuncture treatment.
Personalized acupuncture protocol
The rationale of personalized acupuncture protocol is based on Zang-fu organ system, Yin-yang theory and clinical rules for PCOS acupoint selection. Two sets of acupoints will be selected for the two types (Table 1). The basic acupoint-prescription includes the conception vessel (CV) 4, CV 6, CV 12 and spleen (SP) 6 bilaterally, stomach (ST) 25 bilaterally, extra point of chest and abdomen (EX-CA) 1 bilaterally, ST 40 bilaterally and SP 9 bilaterally. Additional point ST 36 bilaterally and moxibustion as adjuvant therapy will be added for the type of yang deficiency of spleen and kidney, while additional points kidney (K) 13, liver (LR) 3 for the type of yin deficiency of liver and kidney. Besides, flexible modifications of 2-3 acupoints will be performed according to patients’ special symptoms.
The additional points ST 36, K 13, LR 3 and all the basic points will be inserted with disposable needles (Huanqiu, Suzhou Acupuncture Goods Co., Suzhou, China. Size: 0.30×25mm and 0.30×40mm). The depth and degree of puncture for each acupoint is described in Table 1, which could be adjusted according to body shape of participants as shallower for thin women and deeper for fat women. The needles for abdominal acupoints should be inserted until resistance without manual stimulation. After inserting the needles, the acupuncturist will rotate the needles to evoke needle sensation (de qi). De-qi (a dull aching and irradiating sensation indicative of effective needle placement) refers to a sensation of numbness, distension, or electrical tingling at the needling site that might radiate along the corresponding meridian. Once de-qi is achieved, further techniques might be utilized which aim to "influence" the de-qi. Simultaneously, the other techniques of acupuncture with thumb and forefinger to tonify or sedate qi. Tonifying is used to reinforce deficiency while sedating is to clear excess, which are the two manners to stimulate qi in the acupuncture in China. The former is the needles rotate slower and gentle in clockwise direction. Conversely, the latter is turning faster and more forcefully in counterclockwise rotation. Furthermore, the points SP 6 to SP 9 bilaterally for both types will be connected to electrical stimulator (SDZ-IIB, Huatuo, China) and stimulated with low-frequency at 2 Hz, continuous wave and the intensity is adjusted to produce local muscle contractions without pain or discomfort.
Abdominal and back moxibustion will be used for the type of yang deficiency of spleen and kidney (Figure 1). Ignited 1.5 moxa sticks (Hanyi, Nanyang, China; Size: 18mm×200mm) in a special moxa box (19.5×29.5×16.5cm) will be used for a treatment session. The moxa box will be placed onto patients’ abdomen with acupuncture treatment simultaneously for 30 minutes. The box of abdominal moxibustion will cover from the midpoint of sternal body xiphoid junction and umbilicus to superior margin of pubic symphysis. After 30 minutes, the moxa box on the back will be continue for another 30 minutes subsequently without acupuncture. And the box will cover from 11th thoracic vertebra to the 4th sacral posterior foramen. Moxibustion will be stopped at any time if the participants feel mouth parched, canker sore and sore throats. Women without moxibustion will be provided local radiation for abdomen by special electromagnetic spectrum therapy apparatus. Needles not connected to the electrical stimulator or not given moxibustion will be manually stimulated to evoke de-qi every 10 minutes, in total four times.
Fixed acupuncture protocol
The fixed acupuncture protocol is based on the previous study. Two sets of acupuncture points will be alternated every second treatment (Table 2). The first set consists of CV 3, CV 6, ST 29 bilaterally, SP 6 bilaterally, SP 9 bilaterally, governor vessel (GV) 20 and large intestine (LI) 4 bilaterally. In total, 11 needles will be placed. The needles for abdominal acupoints should be inserted until resistance without manual stimulation. Other needles will be manipulated manually by rotation to evoke needle sensation, de qi. CV 3, CV 6 (mid-line), ST 29 bilaterally, SP 6 bilaterally and SP 9 bilaterally will be connected to an electrical stimulator (SDZ-IIB, Huatuo, China). The second set consists of 13 needles: ST 25 bilaterally, ST 29 bilaterally, CV 3, CV 6, SP 6 bilaterally, LR 3 bilaterally, pericardium meridian (PC) 6 bilaterally and GV 20. The following points will be connected to an electrical stimulator: ST 25 bilaterally, ST 29 bilaterally, SP 6 bilaterally, LR 3 bilaterally. Electrical stimulation is given with low-frequency electric acupuncture (EA) of 2Hz, continuous wave, with an intensity adjusted to produce local muscle contractions without pain or discomfort. Electromagnetic spectrum therapy apparatus will be used during the treatment. Needles not connected to the electrical stimulator and not for abdominal acupoints will be manually stimulated to evoke de-qi every 10 minutes, in total four times.
Women in the letrozole group will receive letrozole without acupuncture. Letrozole (Femara, Novartis Pharmaceuticals, Basel, Switzerland) 2.5 mg (1 pill) will be given daily from day 3 to day 7 of the spontaneous menstrual cycle or after a withdrawal bleeding following progestin. If there is response with ovulation (ie, the serum progesterone level on day 21 or day 28 of the cycle is higher than 3 ng/mL) this dose will be maintained. In those with no ovulatory response, letrozole tablets of the next ovulation cycle will be taken on the day 28 of the menstrual cycle and the letrozole dose will be increased to 5 mg (2 pills) a day for 5 days. If there is still no response, the dose will be increased to 7.5 mg (3 pills) per day for 5 days in the next cycle. The maximum daily dose of letrozole will be 7.5 mg daily for five days.
Women will receive placebo letrozole with no acupuncture. Placebo letrozole will have the same appearance as letrozole (Dongyangguang pharmaceutical Co. LTD, Guangdong, China). Placebo letrozole will be given in the same way as letrozole, women will receive 1 tablet a day of placebo letrozole from the day 3 to day 7 of the menstrual cycle and placebo letrozole dose will be increased 2 tablets a day in the next cycle if there is no response. The maximum daily dose of placebo letrozole will be 3 tablets daily for five days.
Randomization and patient allocation
An online computerized program of web-based Research Management (ResMan) database system will be used to allocate patients in 1: 1: 1: 1 ratio. The data coordinating center (DCC) statisticians generated and validated the randomization scheme for the study before it was implemented in ResMan database system. When a participant is enrolled, an investigator of participating site will take out sealed envelope and enter the ID number into the ResMan database system, and login a password protected secured website designed by the DCC to find the group allocation.
The acupuncture groups will be known only to the acupuncturists besides the ResMan data manager. The letrozole and the placebo letrozole having the same appearance will be organized in a kit consisting of one cycle tablets for each subject and one subject will at most take 45 tablets in total for 4 cycles. The sealed envelope with 4 cycles tablets will be labeled with an ID number mapped to the letrozole or placebo letrozole known only to the ResMan personnel. Participants and investigators will be blinded to the letrozole or placebo letrozole allocation. All outcome assessors and the statisticians performing data analysis will be blinded to group allocation. Except for emergencies, unblinding of individual study subjects will not take place until all subjects have delivered and reported outcomes to the DCC. In the event that emergency unblinding is needed, only site PI will be able to unblind the participant to treatment by connecting the director of DCC. The site PI and DCC staff will receive notification from the central randomization service when emergency unblinding has occurred.
Study-specific visits and procedures (Figure 2)
Women will attend up to five visits, including the screening visit, baseline visit, treatment visit, pregnancy visit and end of treatment visit (Table 3). Adverse events (AEs) and concomitant medications will be recorded during every visit. Face-to-face adherence reminder sessions will take place at each study visit thereafter.
Women are screened in the morning after an overnight 12-h fast. Detailed information about the study design is given.
Complete physical examinations will be performed including height, weight, hip, waist measurements, and vital signs. Height and weight will be recorded to the nearest 0.1 cm and 0.1 kg respectively. Waist and hip circumference will be recorded to the nearest 1 cm. Hirsutism assessment by FG, acne by standard acne lesion counts.
Transvaginal ultrasound of ovaries
Ovaries, including the ovarian size in three dimensions, the size of the largest ovarian follicle/cyst and size of every follicle with a mean diameter greater than 10 mm, and total antral follicle count (small follicles with mean diameter of 2-9 mm) of each ovary will be obtained through transvaginal ultrasound.
A urine and serum pregnancy test will be performed to exclude pregnancy.
Blood samples will be collected for the following laboratory: T, sex hormone binding Globulin (SHBG), FSH, luteinising hormone (LH), progesterone (P), estradiol (E2), PRL, fasting glucose, fasting insulin, HbA1C, C-peptide, and lipid profile (cholesterol, triglycerides, high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C)). PCOS safety screen: liver profile (ALT, AST, total bilirubin), renal profile (BUN and creatinine), thyroid profile (TSH, T3, free T4), complete blood cell count (CBC), cervical Pap smear or TCT. Every woman should have T and SHBG assay in local labs. Free androgen index will be calculated.
TORCH screening (Toxoplasma, Rubella Virus, Cytomegalo Virus, Herpes Virus), HIV screening and folic acid prescription will be included.
Women without evidence of ovulation on baseline screening will be provided progestin to induce withdrawal bleeding, with instructions to begin medication once eligibility has been determined.
They will sign the consent form after comprehensive counseling.
The baseline visit will be around day 2–5 of a spontaneous period or after a withdrawal bleeding if a woman fulfills the selection criteria and has signed the consent form. Fasting blood samples will be obtained from the patients and will be shipped to the core laboratory for analysis and stored for a minimum of 5 years, and perhaps further use in the future, which is stated in informed consent (supplementary file 2). Moreover, urine or serum pregnancy test will be done at this visit.
Women will fill in the following questionnaires: the short form 36 (SF 36) of health-related quality of life (QOL), the Polycystic Ovary Syndrome Questionnaire (PCOS-QOL), the Chinese Quality of life (ChQOL), Zung Self-Rating Anxiety Scale (SAS) and Zung Self-reported Depression Scale (SDS).
TCM differentiation of syndromes of PCOS is mainly for the deficiency of liver- kidney-yin type, and the deficiency of spleen-kidney-yang type. The TCM syndrome diagnosis will be made by principal investigator and acupuncturist in each participating site according to a standard questionnaire during baseline visit and end of treatment visit. If the diagnosis is differ from each other, they will discuss until reaching an agreement. Syndrome differentiation in TCM is the comprehensive analysis of clinical information gained by the four-main diagnostic TCM procedures: observation, listening, questioning, and pulse analysis.
All baseline measures will be repeated in all subjects at the end of treatment visit.
The women will be instructed to have intercourse once every 2 or 3 days. A serum progesterone level test will be performed in local laboratory on day 21 of the cycle. An elevated level of progesterone >3 ng/mL is considered to indicate evidence of ovulation. If there is no ovulation, serum progesterone level will be checked every week until ovulation. If ovulation, serum pregnancy test will be performed after a week. Women should make a urine pregnancy test at any time if they are suspected to be pregnant.
We will determine whether the women have anovulation response based on the serum progesterone level. There will be three possible scenarios: ovulation, delayed ovulation and no ovulation (1) Ovulation: serum progesterone level on day 21 of the cycle >3 ng/mL. A serum pregnancy test will be performed after a week to exclude pregnancy, letrozole or placebo letrozole will be started on day 3 of the period, and the letrozole or placebo letrozole dose will be maintained. (2) Delayed ovulation: serum progesterone level in the third week <3 ng/mL. In this case, we will wait for one more week, and check serum progesterone level again. A serum pregnancy test will be performed after a week if ovulation to exclude pregnancy. Letrozole or placebo letrozole will be started on day 3 of the period, and the letrozole or placebo letrozole dose will be maintained. (3) No ovulation: It will be defined as negative serum progesterone levels on day 21 and day 28. In the letrozole and placebo letrozole groups, women will take letrozole or placebo letrozole on day 28 of the period and one more tablet per day will be given for 5 days until the maximum daily dose of 3 tablets a day. Day 28 will be defined as day 1 of next cycle, and serum progesterone test will be done every week until ovulation.
Every period will be recorded, including the date, amount and duration of menstruation.
End of treatment visit
End of treatment visit will be performed when women conceive or finish four cycle treatments. Baseline measures will be repeated in all subjects including vital signs, height, weight, hip and waist measurements, hirsutism and acne assessments, sex hormone steroids (FSH, LH, T for women not pregnant, and HCG, P, E2, T for pregnant women), SHBG, metabolic profile (glucose and insulin concentrations, HbA1C, C-peptide, cholesterol, triglycerides, HDL-C, LDL-C), and safety profile (AST, ALT, total bilirubin, BUN, creatinine). Subjects will return menstrual and intercourse journal logs, and their AEs and concomitant medications will be recorded. Fasting blood samples will be obtained, and TCM syndrome diagnosis and questionnaires SF36, PCOS-QOL, ChQOL, SAS and SDS will be repeated.
Pregnancy visit (only with conception)
Serum progesterone and hCG levels will be determined at local sites to document ovulation and pregnancy. Serial serum hCG level will be checked on a weekly basis if pregnant. Serum beta hCG > 10 IU/L indicates pregnancy. Pelvic ultrasound will be arranged to determine the location, number and viability of the pregnancy when serum hCG is 2000-4000 IU/L.
Women who conceive will be followed till around 9 weeks gestational age and will be referred to their prior or referring doctors for antenatal care. Subsequent scans will be performed at week 18–24, 32, and 36 or at the prescription of the obstetrician. In the present trial, pregnant women who present with threatened miscarriage or are considered to have a significant risk for miscarriage can be given oral Duphaston (10 mg three times a day, Solvay Pharmaceuticals B.V.) till 12 weeks of gestation or 1 week after the vaginal bleeding stops. This information will be documented in the record form. No other medications including hCG, herbal medications, and acupuncture will be given.
Follow-up of pregnancies
For those women who have an ongoing pregnancy, arrangements will be made to follow the outcome of the pregnancy at the end of the first trimester and also after delivery or termination of gestation. All pregnancies (including multiples) will be followed to monitor weight, glucose tolerance, blood pressure and fetal growth and to determine the pregnancy outcomes. The glucose tolerance will be measured by oral glucose tolerance test in all pregnancies at 24–28 weeks of pregnancy. Women will be instructed to inform the study personnel of the outcomes of the pregnancy, and we will obtain delivery record from the obstetricians to determine the birth weight, length of gestation, and any prenatal complications of the mother or neonatal complications of the infant. Phone contacts will be initiated if the pregnant woman has not contacted the study personnel within 6 weeks of the original estimated date of childbirth.
We will collect pregnancy outcome data and track the outcomes of all women who have a positive serum pregnancy screen during the course of this study. We will record biochemical pregnancies (defined as positive serum pregnancy screens without ultrasonically detected pregnancies), ectopic pregnancies and all intrauterine pregnancy losses both before and after 20 weeks, including miscarriages, abortions, fetal deaths and stillbirths.