Subjects in both arms had 12 acupuncture/sham session: twice each week for the first four weeks

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Colleen Roberts
5 years ago
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1 1 1 SUPPLEMENT Materials and Methods Interventions Intervention Protocol Details Common to Both Arms Subjects in both arms had 12 acupuncture/sham session: twice each week for the first four weeks followed by once per week for an additional four weeks. This treatment regimen was the same as used previously by other researchers(1). The needle retention time was 20 minutes. For all sessions with the acupuncturist, the participant was in the prone position with her face in a cradle and her upper limbs supported on an armrest situated beneath the cradle. If this was particularly uncomfortable for the participant, she was allowed to have her treatment in a forward sitting position with her head resting on her arms on the side of the massage table. The participants were not physically able to observe the treatments. As described elsewhere [reference is under review], there were four study acupuncturists. All acupuncturists had more than 5 years of experience, were trained in Traditional Chinese Medicine (TCM), and remained as a study practitioner until the end of the study. Two were female professional non-md acupuncturists and two were male MD/acupuncturists. The intervention sessions were held in the acupuncturist s private office, with the exception of the lead acupuncturist (JJ) who saw participants at an academic medical center. The study practitioners met at least once per year to review the protocol and related details; the lead acupuncturist was available for questions throughout the year from any of the other study acupuncturists. Protocol Details Specific to One Intervention Arm The true acupuncture treatment used Seirin disposable needles of 40 mm length and 0.20 mm (Japanese No. 3 gauge) diameter. EA needles were stimulated at a frequency of 2 Hz for a total of twenty minutes per session. The hertz setting was low for the initial visit, as most participants were acupuncturenaïve, and increased at subsequent sessions as needed until visual or palpable muscle contraction was

2 achieved. For the forearm points only, a sham guide tube was left in place over the verum needle and taped to the skin (with double-sided, donut-shaped sticky tape) to mimic the sham treatment protocol. For the sham acupuncture, the acupuncturists applied the electric stimulation alligator clips on all four sham points and turned the machine on at an intensity of zero (i.e. no active current) in order to mimic the EA verum treatment. Additional Study Clinic Protocol Details For the first 2.5-hour study visit, there was no attempt to time the study visit for the early follicular phase. The pre-intervention serum samples for LH and FSH were collected within 7 days of the self-reported last menstrual period (LMP) for 22% of the study population; For the remaining 78% of the cohort, the median menstrual cycle day (CD) was 39 with an interquartile range of days. The postintervention and post-follow-up 2.5-hour study visit was timed to CD 2-7 for women who were known to be ovulating (based on serum progesterone) or menstruating; due to illness, vacation, or other scheduling conflicts, the visits did not always occur in that time window. Additionally, for women providing urine samples (see ovulation assessment below), those samples were batch analyzed months after the samples were collected so that timely knowledge of ovulation was not possible. More than half of the cohort (54%) provided post-intervention LH and FSH serum samples within 7 days of their LMP; for the remaining 46% of the cohort (73% of who were amenorrheic or anovulatory), the median CD was 36 with an interquartile range of days. For the post-follow-up LH and FSH samples, 41% of the samples were collected within 7 days of their LMP; for the remaining 59% of the cohort (54% of who were amenorrheic or anovulatory), the median CD was 44 with an interquartile range of days. Ovulation Assessment The participants provided weekly blood samples for serum progesterone measurement, or collected first-void urine samples at home (stored in their home freezer), for the entire 5 month protocol. The initial protocol requested once-per-week urine samples. This was revised to thrice-per-week samples upon further review of the pregnanediol glucuronide (PDG) research(2, 3) to ensure the ovulation peak was captured. Approximately one-quarter of the participants were enrolled prior to this protocol revision.

3 The definition of ovulation using the urine samples (peak urinary PDG to the basal PDG level in the follicular phase > 4.0 ) was extrapolated a priori from prior research on the relationship between serum progesterone and urinary PDG (3), as well as evidence that PDG > 3 ug/ml predicted serum progesterone > 2.5 ng/ml with 78% sensitivity and 100% specificity across 5 bands of specific gravity (2). PDG is an indirect and accurate measure of progesterone levels in the body(4) and has been validated (3, 5). Creatinine correction is not necessary, and there is little difference in first morning vs. random urine sampling (3). Individual graphs of serum progesterone, urinary PDG results and self-reported menses were created for each participant (coded without identifiers) for a confirmatory assessment of ovulation by the PI prior to unblinding the treatment arm. A sample graph is displayed in Figure 1. Serum Assays The University of Virginia laboratories performed all assays with the exception of urinary PDG, which were run by the University of Wisconsin Human Primate Lab. FSH, LH, total testosterone, DHEAS, and SHBG were directly measured using standard chemiluminescent enzyme-linked immunoassays on the Immulite 2000 instrument. The analytical sensitivity of FSH was 0.1 miu/ml with a coefficient of variation (cv) of 5.6% among normally ovulating females (mean 4.85 miu/ml) and 6.3% cv for perimenopausal females (mean 15.4 miu/ml). The analytical sensitivity of LH was 0.05 miu/ml with a 7.4% cv at normal follicular phase levels (mean 11.3 IU/L). The analytical sensitivity of testosterone was 15 ng/dl with a 13.0% cv among high-normal female levels (mean 86.1 ng/dl). For DHEAS, the analytical sensitivity was 3 ug/dl with a cv=9.8% at mean DHEAS levels of For SHBG, the analytical sensitivity was 0.02 nmol/l with a cv=5.2% at a mean range of nmol/l. Venous blood glucose values were assayed by the glucose-oxidase method (Yellow Spring Instruments, YSI 2300), and subsequently converted to plasma glucose. Serum insulin samples were stored at -80 C and subsequently batch assayed with a chemiluminescent immunoassay (Immulite 2000 Automated Immunoassay Analyzer). The minimum detection for insulin was 2.0 miu/ml. Statistical Analyses: Potential Predictors

4 For the secondary aim to determine predictors of treatment success with acupuncture, the potential predictors evaluated were: number of menses in the most recent 12 hormone-free months prior to enrollment, age, BMI, percentage of weight change after the intervention compared with baseline (6, 7), pre-intervention insulin (fasting and area-under-the-curve [AUC]), pre-intervention glucose (fasting and AUC), androgens (free testosterone, total testosterone, DHEAS, SHBG) at enrollment, preintervention pituitary gonadotropin hormones (LH and FSH), and pre-intervention depression score (selfreported response to the Quick Inventory of Depressive Symptomatology - Self Report 16(8)) References 1. Stener-Victorin E, Waldenstrom U, Tagnfors U, Lundeberg T, Lindstedt G, Janson PO 2000 Effects of electro-acupuncture on anovulation in women with polycystic ovary syndrome. Acta Obstet Gynecol Scand 79: Sauer MV, Paulson RJ 1991 Utility and Predictive Value of a Rapid Measurement of Urinary Pregnanediol Glucuronide by Enzyme-Immunoassay in an Infertility Practice. Fertil Steril 56: Cekan SZ, Beksac MS, Wang E, Shi S, Masironi B, BM. L, Diczfalusy E 1986 The prediction and/or detection of ovulation by means of urinary steriod assays. Contraception 33: Stanczyk FZ, Gentzschein E, Ary BA, Kojima T, Ziogas A, Lobo RA 1997 Urinary progeterone and pregnanediol use for monitoring progesterone treatment. J Reprod Med 42: Kesner JS, Knecht EA, Krieg J, Edward F., Barnard G, Mikola HJ, Kohen F, Gani MM, Coley J 1994 Validations of time-resolved fluoroimmunoassays for urinary etrone 3-glucuronide and pregnanediol 3-glucuronide. Steroids 59: Guzick DS, Wing R, Smith D, Berga SL, Winters SJ 1994 Endocrine consequences of weight loss in obese, hyperandrogenic, anovulatory women. Fertility & Sterility 61: Clark AM, Thornley B, Tomlinson L, Galletley C, Norman RJ 1998 Weight loss in obese infertile women results in improvement in reproductive outcome for all forms of fertility treatment. Hum Reprod 13:

5 Rush AJ, Trivedi MH, Ibrahim HM, Carmody TJ, Arnow B, Klein DN, Markowitz JC, Ninan PT, Kornstein S, Manber R, Thase ME, Kocsis JH, Keller MB 2003 The 16-Item quick inventory of depressive symptomatology (QIDS), clinician rating (QIDS-C), and self-report (QIDS-SR): a psychometric evaluation in patients with chronic major depression. Biol Psychiatry 54:

6 6 111 FIG 1. Sample urinary PDG graph ID = pdg menses 0 7/26/2009 8/9/2009 8/23/2009 9/6/2009 9/20/ /4/ /18/ /1/ /15/ /29/ /13/ /27/2009

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