The Clinical Trial of Acupuncture Pre-treatment on PCOS
The Clinical Trial of Acupuncture Pre-treatment on PCOS
This is a study protocol for a multicenter, randomized, and controlled trial. In this
 protocol, we present a randomized controlled trial comparing acupuncture pretreatment
 followed by letrozole vs letrozole alone in polycystic ovary syndrome (PCOS) women with
 anovulatory infertility. The high prevalence of insulin resistance (IR) in women with PCOS
 women is considered to be one of the major pathophysiological changes of PCOS, leading to
 anovulatiory infertility. A study has shown that electro-acupuncture could significantly
 improve insulin sensitivity. The effect of acupuncture pretreatment on anovulatory PCOS women
 followed by ovulation induction has not been investigated before. A total of 384 patients
 enrolled in this study will be randomized into one of two groups. The treatment group: a 16
 week acupuncture pretreatment followed by 4 cycles of letrozole and the control group: 4
 cycles of letrozole alone. The primary outcome is the live birth rate. We postulate that
 acupuncture pretreatment followed by letrozole results in a higher live birth rate when
 compared with letrozole alone.
1. Study content 1.1 Study design This is a multi-centered, randomized, clinical trial. 1.2
 Subject selection and exclusion

 Initially, 384 PCOS women anovulatory infertility will be recruited from four hospitals in
 mainland China: Department of Traditional Chinese Medicine in the first affiliated hospital
 of Guangzhou Medical University, Department of Gynecology in Xuzhou maternity & child health
 hospital, Department of Reproduction in Dalian Municipal Women and Children's Medical Center,
 and Department of Traditional Chinese Medicine in Hexian memorial affiliated hospital of
 Southern Medical University if they meet the inclusion criteria and do not have the exclusion
 criteria. Eligible subjects will be approached and sign the consent form after detailed
 explanation and counseling. Recruited subjects will be randomized to one of two groups:

 Treatment group: a 16 week acupuncture pre-treatment followed by LE and Control group: LE
 alone 1.3 Screening visit 1.3.1 Obtain signed informed consent 1.3.2 Complete physical
 examination Perform complete physical examination including height, weight, hip and waist
 measurement. 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. And
 assessment of hirsutism by Ferriamn Gallwey score (FG), acne standard acne lesion counts, and
 pelvic exam with Pap smear or TCT.

 1.3.3 Perform transvaginal ultrasound of the uterus and ovaries

 1. Uterus, including the uterine dimensions, endometrial thickness and echo type, other
 uterine abnormalities, presence and size of leiomyoma will be obtained through
 transvaginal ultrasound.

 2. 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 (small follicles with mean diameter < 10 mm) count of each ovary
 will be obtained through transvaginal ultrasound.

 1.3.4 Check urine pregnancy test 1.3.5 Laboratory Examination

 1. Serum levels of sex hormone steroids including blood follicle-stimulating hormone (FSH),
 luteinizing hormone (LH), estrogen (E2), prolactin (PRL) and T, free testosterone, sex
 hormone binding globulin (SHBG), and thyroid stimulating hormone (TSH) will be evaluated
 on the second day of the spontaneous period or a withdrawal bleeding.

 2. Fasting blood was taken to determine the examination of metabolic and safety profile,
 including fasting blood glucose, insulin, C-peptide, HbA1c, triglycerides, total
 cholesterol, high density lipoprotein (HDL-C), low density lipoprotein (LDL-C), CBC,
 Renal, and Liver Profile.

 3. The oral glucose tolerance test (OGTT) with 75 g glucose will be performed in all
 subjects after an overnight fast. Blood samples will be obtained to measure plasma
 glucose and serum insulin at 0, 60, and 120 min during the OGTT.

 1.3.6 Complete questionnaires Quality of life will be assessed by the short form 36(SF-36),
 the Chinese Quality of Life (ChiQOL), sleeping questionnaires and the Polycystic Ovary
 Syndrome Questionnaire (PCOS-QOL). Investigators will also assess symptoms of anxiety and
 depression by the Zung SAS and Zung SDS questionnaires. And complete the quantization table
 of traditional Chinese medicine (TCM) syndromes about PCOS.

 1.3.7 Exclusion of other infertility factors of the couple The semen analysis of the husband
 and tubal patency of the patient will be assessed.

 1.3.8 Preconception counseling Complete the questionnaire of risk factors for genetic
 disorders, TORCH screening (Toxoplasmosis, Rubella, Cytomegalo Virus, and Herpes Virus) and
 HIV screening, and folic acid prescription.

 1.3.9 Progestin withdrawal Provide progestin prescription to induce withdrawal bleed, with
 instructions to begin medication once eligibility is determined.

 1.4 Baseline visit 1.4.1 Distribute home pregnancy test 1.4.2 Distribute folate 1.4.3
 Distribute intercourse and menstrual journal logs 1.4.4 Take blood sample for repository and
 DNA Collect 20ml blood, and distributed store 10ml blood after serum separation. Other 10ml
 blood is for determination of DNA.

 1.5 Intervention arms

 Eligible patients will be randomized into one of the two arms:

 Treatment group: i.e. a 16 week acupuncture pretreatment followed by LE. Acupuncture
 treatment will start on day 3-5 after a spontaneous period or after a withdrawal bleeding
 following progestin. All subjects will be requested to use contraception during the 16 week
 acupuncture pretreatment. They will receive acupuncture treatment three times a week. Each
 treatment session lasts for 30 minutes and can be separated by an interval of 1-3 days, with
 a maximum of 48 treatment sessions during 16 weeks. After 16 weeks of acupuncture treatment,
 LE will start on day 2-3 after a spontaneous period or after a withdrawal bleeding after
 progestin administration. The subjects will be instructed to have intercourse on a regular
 basis during the cycles.

 Control group: i.e. LE alone. LE will be started on day 3-5 after a spontaneous period or a
 withdrawal bleeding following progestin. The subjects will be instructed to have intercourse
 on a regular basis during the cycles.

 1.5.1 Acupuncture protocol Investigators base the rationale of acupuncture protocols on
 Western Medical theories and the study protocol follows the CONSORT and STRICTA
 recommendations with detailed descriptions of the treatment including number of needle used,
 how needles will be stimulated (manual, electrical), frequency of sessions, and length of
 treatment period. Investigators will use fixed acupuncture protocols.

 Disposable, single-use, sterilized needles made of stainless steel, 0.25 x 30 mm and 0.30 x
 40/50 mm (Hwoto, Suzhou Medical Appliance Fact. 215005 Suzhou, China) will be inserted to a
 depth of 15-35 mm in segmental acupuncture points located in abdominal and leg muscles with
 innervations corresponding to the ovaries. Two sets of acupuncture points will be alternated
 every second treatment. The first set consists of conception vessel (CV) 3, CV 12, and
 stomach (ST) 29 bilaterally and in the muscles above the knee, ST 34, and ST 33 bilaterally
 and below the knee, spleen (SP) 6 and ST 36. Needles will also be placed in extra segmental
 acupuncture points that do not innervate the ovaries large intestine (LI) 4 bilaterally. In
 total 14 needles will be placed and all will be stimulated manually by rotating the needle to
 evoke needle sensation (de qi) once when inserted. The following points will be connected to
 an electrical stimulator (Export Abteilung, Schwa-Medico GmbH, Wetzlarer Str. 41-43; 35630
 Ehringshausen): CV 3 to CV 12, ST 29 bilateral, and ST34 to ST 33 bilateral. Stimulation are
 given as low-frequency EA of 2Hz, 0.3 ms pulse length and with an intensity adjusted to
 produce local muscle contractions without pain or discomfort. Needles not connected to the
 electrical stimulator will be manually stimulated to evoke needle sensation every 10 min, in
 total 4 times. The second set consists of 14 needles placed in segmental abdominal points and
 stimulated electrically: ST 27 bilaterally, CV6 connected to CV10; and leg points: SP10
 connected to a non-acupuncture point located 6 cun proximal of patellas medial border
 (electrical stimulation), and SP 6 and liver (LR) 3 bilaterally (manual stimulation). Extra
 segmental points are pericardium (PC) 6 bilaterally (manual stimulation).

 1.5.2 Needle insertion technique Needle insertion should be gentle. Tighten the skin by
 pressing around the area of needle insertion and gently insert the needle. Don't do it too
 quickly, be gentle.

 1.5.3 Needles and stimulation Needles are for single use. Needle size: 0.25x30 mm or 0.30 x
 40 or 0.30 x 50 mm. Select needle length with respect to the patients BMI. If the patient has
 a low BMI and is thin use the 0.25 x 30 mm needle. If the patient is overweight, use the 0.30
 x 40 mm needles. Obese patients, use needle with the size 0.30 x 50 or 75 mm. Depth of
 insertion may vary from patient to patient. They should be placed with a depth deep enough to
 reach muscle/fibrous tissue. When needle are inserted, stimulate the needle gently until de
 qi (needle sensation reflecting activation of sensory afferents). As soon as de qi has been
 reached, make sure that the needle doesn't hurt or cause any pain and discomfort.

 Attach the electrodes according to the protocol. Turn on the stimulator (program 10) and
 increase the intensity. Instructions to the patient: "As high as possible but with no pain or
 discomfort". Don't start with too intensive stimulation. After 10 minutes, stimulate needles
 without electricity and ask if the intensity of the electrical stimulation is ok, or if it
 should be increased/decreased. Repeat after 20 minutes and again after 30 minutes. Turn off
 the stimulator and release the patient.

 1.5.4 Acupuncture treatment

 1. Record time of the day and acupuncturist name when the patient receives acupuncture.

 2. Note the intensity of stimulation (mA). It may vary between the different electrodes.
 Note the range e.g. 1.2 - 3.0 mA.

 3. Note any other events that may affect the treatment (positive or negative).

 4. Note concomitant medications.

 5. Collect menstrual and intercourse journal logs at the end of every cycle.

 6. Check urine pregnancy test before each acupuncture treatment. If pregnancy test is
 positive, no acupuncture treatment should be performed.

 1.5.5 Letrozole After 16 weeks of acupuncture pretreatment, which the patients in the
 intervention group and the patients in the control group will receive LE (Femara, Novartis
 Pharmaceuticals), starting from 2.5mg daily from day 2-3 for 5 days after a withdrawal
 bleeding. Participants are treated for up to 4 cycles. If the patient is pregnant, the
 treatment should be stopped.

 If there is evidence of ovulation (i.e. serum progesterone level on the 3th week was higher
 than 3 ng/mL) or delayed one week, this dose will be maintained. In patients with no
 ovulatory response, the dose will be increased to LE 5mg a day for five days until LE 7.5 mg
 per day for five days will be reached. The same LE dose will be continued in the next cycle
 if the patients are shown to be ovulating. And the maximum daily dose of LE is 7.5 mg (three
 pills) daily.

 1.6 Monitoring and examination during the treatment And all subjects will be instructed to
 have intercourse on a regular basis during the entire study period. The optimal frequency
 will be once every 2-3 days. Urine pregnancy test will be checked before each acupuncture
 treatment of ovulation. And serum progesterone level test will be checked in the local lab at
 day on the 3th week of the first cycle and every week of the last three cycles.

 The transvaginal ultrasound exam will take place in the anticipated luteal phase of the
 cycle, and initially will be scheduled 3 weeks after the initiation of medication, with a
 window of 4 days on either side of this day (i.e. Day 17-25, assuming a Day 3 medication
 start, or Day 19-27, assuming a Day 5 start). The ultrasound includes the endometrial
 thickness and echogenicity, as well as the number, size, and echogenic characteristics of
 follicles/cysts on the ovary (including an antral follicle count).

 Investigators can decide whether the patients have the ovulation respond based on the results
 of serum progesterone level and transvaginal ultrasound. There will be three possible
 scenarios: ovulation, ovulation delay and non-ovulation. 1) Ovulation, i.e. serum
 progesterone level on the 3th week was higher than 3 ng/mL, and ultrasound may provide
 presumptive evidence of ovulation. Check urine pregnancy test to exclude pregnancy and start
 with LE tablet on day 3-5 of menstruation, and the dose of which will be maintained; 2)
 Ovulation delay, P<3 ng/mL, but the ultrasound will show there is evidence of follicular
 development, (i.e. a follicle with a mean diameter ≥12 mm). Investigators can wait for one
 more week and continue the acupuncture treatment. Check urine pregnancy test to exclude
 pregnancy and start with LE tablet on day 3-5 of menstruation, and the dose of which will be
 maintained; 3) Non-ovulation, P<3 ng/mL, and the ultrasound will not found any follicle with
 a mean diameter ≥12 mm. The patient will also receive LE tablet on day 3-5 of her cycle, and
 she will be instructed to increase her study drug by one tablet per day for five days.

 Every menstruation should be detailed recorded, including the date, volume and duration of
 menstruation.

 1.7 End of pre-treatment visit

 1) Repeat OGTT. 2) Collect menstrual journal logs. 1.8 Pregnancy visit(only with conception)

 1. Complete adverse event query.

 2. Take blood for serum quantitative hCG levels until the ultrasound can observe the
 gestational sacs (1500-2000IU/ml).

 3. Perform transvaginal ultrasound for the number of gestational sacs, location,
 dimensions, presence and size of fetal parts, and documentation of visualization of
 fetal heart motion, documentation of any pregnancy related abnormalities when hCG levels
 or clinical manifestations appear as pregnancy.

 1.9 End of treatment visit

 1. Perform physical examination, including vital signs, height, weight, hip and waist
 measurements as well as repeating hirsutism and acne assessments after the end of
 treatment or pregnancy.

 2. Repeat the serum levels of sex hormone steroids and metabolic profile.

 3. Repeat OGTT.

 4. Collect 20ml blood, and distributed store 10ml blood after serum separation. Other 10ml
 blood is for determination of DNA.

 5. Repeat QOL, sleeping, anxiety/depression questionnaires, and the quantization table of
 TCM.

 6. Collect menstrual and intercourse journal logs.

 7. Record adverse events and concomitant medications.

 8. Arrange follow up for subjects who have conceived and obtain release of records for
 pregnancy and neonatal records.

 1.10 Follow up visit For those women who have an ongoing pregnancy, arrangements will be made
 to follow the outcome of the pregnancy at the end of first trimester and also after delivery
 or termination of gestation. All pregnancies (including multiples) will be followed to
 monitor the weight, glucose tolerance, blood pressure and fetal growth and to determine the
 abortion rate, complication rates and pregnancy outcomes. The glucose tolerance will be test
 by glucose screening test with 75 g glucose performed in all pregnancies at 24-28 weeks of
 pregnancy. Patients will be informed to notify study personnel of the outcome of the
 pregnancy and investigators will obtain release of record forms from treating physicians to
 obtain copies of relevant medical records. Delivery records of both mother and newborn will
 be requested to determine the birth weight, length of gestation, and any prenatal
 complication of mother or neonatal complication of the infant. Phone contacts will be
 initiated if the patient has not contacted study personnel by six weeks beyond the original
 estimated date of confinement.

 And investigators will collect pregnancy outcome data. Investigators will track the outcomes
 of all subjects who have a positive serum pregnancy screen during the course of this study.
 Investigators 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 missed abortions,
 spontaneous abortions, elective abortions, fetal demises, and stillbirths. Investigators will
 review pregnancy and birth records of the mother and of the fetus to establish neonatal
 morbidity and mortality and the presence of fetal anomalies. Investigators will extract from
 these records concomitant medical and obstetrical conditions, exposure information on all
 other medical products used, including prescription products, over-the-counter (OTC)
 products, dietary supplements, vaccines, and insertable or implantable medical devices.
 Investigators will file individual case reports for all congenital anomalies, which will be
 considered a serious adverse event.

 1.11 Safety analysis Adverse events will be categorized and percentage of patients
 experiencing adverse events and serious adverse events during the treatment period will be
 documented. Chi-square tests will be performed to examine differences in the proportion of
 total and categories of adverse events.

 1.12 Statistical tests One sample of the Kolmogorov-Smirnov test will be used to test the
 normal distribution of continuous variables. Continuous variables will be shown as means ±
 standard deviations if they are normally distributed or as medians with interquartile ranges
 if they are not normally distributed. Statistical comparison will be carried out according to
 the intention to treat by Student's t-test, Mann-Whitney U-test, and Wilcoxon signed ranks
 test for continuous variables and by χ2 tests for categorical variables where appropriate.
 All statistical analyses of the data will be performed using the SPSS program version 21.0
 (SPSS Inc., Chicago, IL, USA), and a P-value < 0.05 will be considered statistically
 significant.
Inclusion Criteria:

 1. Women aged between 20 and 40 years.

 2. Confirmed diagnosis of PCOS according to 2003 modified Rotterdam criteria

 3. According to World Health Organization standards (2010), semen analysis of the husband
 must meet ① or ②. ①sperm concentration ≥15*106/ml and total motility ≥40% . ② Total
 motile sperm count ≥9 million, i.e. semen volume*sperm density*percentage of motile
 sperm ≥9 million.

 4. At least one patent tube shown by hysterosalpingogram or diagnostic laparoscopy. The
 results will be valid for 3 years if the patients do not have a history of abortion or
 pelvic operation.

 Exclusion Criteria:

 1) Exclusion of other endocrine disorders:

 -  Patients with hyperprolactinemia (defined as two prolactin levels at least one week
 apart 25 ng/mL or greater or as determined by local normative values). The goal of
 eliminating patients with documented hyperprolactinemia is to decrease the
 heterogeneity of the PCOS population. These patients may be candidates for ovulation
 induction with alternate regimens (dopamine agonists). A normal level within the last
 year or on treatment is adequate for entry.

 -  Patients with FSH levels > 15 mIU/mL. A normal level within the last year is
 adequate for entry.

 -  Patients with uncorrected thyroid disease (defined as TSH < 0.2 mIU/mL or
 >5.5 mIU/mL). A normal level within the last year is adequate for entry.

 -  Patientsdiagnosed with Type I or Type II diabetes who are poorly
 controlled (defined as a Hb A1c level > 7.0%), or patients receiving
 antidiabetic medications such as insulin, thiazolidinediones, acarbose,
 or sulfonylureas likely to confound the effects of study medication;
 Patients currently receiving metformin XR (extended release) for a
 diagnosis of Type I or Type II diabetes or for PCOS are also
 specifically excluded.

 ⑤Patients with suspected Cushing's syndrome. 2) Use of hormonal or
 other medication including Chinese Herbal prescriptions which may
 affect the outcome at least in the past 2 months.

 3) Acupuncture the last 2 months. 4) Pregnancy within the past 6 weeks.
 5) Within 6 weeks post-abortion or postpartum. 6) Breastfeeding within
 the last 6 months. 7) Not willing to give written consent to the study.
 8) Additional exclusion criteria

 1. Patients with a suspected adrenal or ovarian tumor secreting
 androgens. 2. 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.

 3. 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.

 4. Patients with untreated poorly controlled hypertension defined as a
 systolic blood pressure 160 mm Hg or a diastolic 100 mm Hg obtained on
 two measures obtained at least 60 minutes apart.

 5. Patients with known congenital adrenal hyperplasia. 6. Patients on
 oral contraceptives, depot progestins, or hormonal implants (including
 Implanon). A two month washout period will be required prior to
 screening for patients on these agents. 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 oral cyclic progestins.

 7. Patients with liver disease defined as AST or ALT > 2 times normal
 or totalbilirubin >2.5 mg/dL. Patients with renal disease defined as
 BUN > 30 mg/dL or serum creatinine> 1.4 mg/dL.

 8. Patients with significant anemia (Hemoglobin < 10 g/dL). 9. Patients
 with a history of deep venous thrombosis, pulmonary embolus, or
 cerebrovascular accident.

 10. Patients with known heart disease that is likely to be exacerbated
 by pregnancy.

 11. Patients with a history of, or suspected cervical carcinoma,
 endometrial carcinoma, or breast carcinoma. A normal Pap smear or TCT
 result will be required for women 21 and over.

 12. Patients with a current history of alcohol abuse. Alcohol abuse is
 defined as >14 drinks/week or binge drinking.

 13. Patients enrolled simultaneously into other investigative studies
 that require medications, proscribe the study medications, limit
 intercourse, or otherwise prevent compliance with the protocol.

 14. Patients who anticipate taking longer than a one month break during
 the protocol should not be enrolled.

 15. Patients taking other medications known to affect reproductive
 function or metabolism. These medications include oral contraceptives,
 GnRH agonists and antagonists, antiandrogens, gonadotropins,
 anti-obesity drugs, Chinese herbal formula, anti-diabetic drugs such as
 metformin and thiazolidinediones, somatostatin, diazoxide, ACE
 inhibitors, and calcium channel blockers. The washout period on all
 these medications will be two months.