The overall objective of this study was to conduct a longitudinal assessment of the association of endogenous hormones with biomarkers of oxidative stress and antioxidant status during the menstrual cycle. There were four main objectives. The first was to study the intra-menstrual cycle variation of various measures of oxidative stress. This objective is intended to assess variation in several measures of oxidative stress during different phases of the menstrual cycle, including F2-8-isoprostanes in serum. Assessment of variation across individuals is planned. The second objective was to determine the relationship between specific reproductive hormone levels and oxidative stress during specific times in the menstrual cycle of premenopausal women. The panel of reproductive hormones in the blood that were of primary interest are oestradiol, progesterone, LH, FSH and sex hormone binding globulin (SHBG). The third objective was to examine the influence of external factors on both oxidative stress and hormone levels, and their interrelation. The study measured various biological factors that might influence oxidative stress, including serum concentration of certain antioxidant vitamin levels (retinoids, tocopherols, carotenoids and ascorbic acid). In addition, the study assessed other factors that might affect oxidative stress such as medication and supplement intake, cigarette smoking, alcohol consumption, dietary intake, physical activity and levels of stress. Lastly, the study was designed to evaluate the validity and reproducibility of the various biological markers included in the BioCycle study.
The study was designed to enrol 250 participants for two menstrual cycles and collect biological samples, conduct physical measurements and administer questionnaires across two complete menstrual cycles. In order to test the feasibility of the study and its measures, a pilot study of nine subjects was conducted for one cycle prior to finalising the study protocol. The full study involved biospecimen collection (blood and urine) at eight key times per cycle for two cycles per participant. The collection times were selected to include points in the menstrual cycle with the most hormonal variation. Sample collection was planned to occur during menstruation, at the middle of the follicular phase, at the time of the oestrogen peak, at the time of the LH and FSH surge, on the day of ovulation, at the time of progesterone elevation and peak and immediately before menstruation. Based on an approximate 28-day cycle length, these would represent approximately days 2, 7, 12, 13, 14, 18, 22 and 27.
Recruitment of subjects
Participants were recruited from regularly menstruating premenopausal female volunteers aged 18–44 years from the western New York state region. We used a variety of recruitment methods including: advertising in clinical practices from the region and the University at Buffalo student health service, placing paid advertisements in local newspapers and other print media, using articles about the study written by the University at Buffalo news bureau, doing radio and television interviews about the study, sending notices about the study to various list serves from the region, and posting flyers at the University and throughout western New York state (mainly in Erie and Niagara counties). These methods were supplemented by our participant web site, which was used as a recruitment tool, but also provided detail about the study that potential participants could access. As potential participants contacted the study, we recorded the methods by which they learned about the BioCycle study.
Initial telephone contact
Regardless of recruitment method, participants were instructed to call our clinical research centre for information and to schedule a screening visit appointment. On these initial calls, participants were given an overview of the study purpose, a description of the baseline enrolment procedures, and a description of the study cycle visit requirements. They also had an opportunity to ask questions about the study. If they were interested in learning more about the study and potentially participating, they were asked to provide contact information (name, address, telephone number) and were scheduled for a screening visit appointment at our clinical research centre. No eligibility screening questions were asked on the telephone call.
Each person scheduled for a visit was mailed a study packet prior to the appointment. The packet contained information describing the study, a copy of the consent form to review before their visit, a parking pass and directions to the clinic. The study was reviewed and approved by the Health Sciences Institutional Review Board (IRB) at the University at Buffalo and was approved by an IRB of the National Institutes of Health. The consent form included detailed information on the study requirements, including frequency and volume of blood and urine collection. Also included in the packet was an instruction sheet detailing what to expect at the screening visit and instructions on what they needed to do for the visit (e.g. fast; bring in all current medications). A reminder call was made the day before each scheduled screening visit.
The screening visit was the first clinic visit. Initial contact included review of the study procedures and review and signing of the informed consent. A copy of the consent was provided to all participants for later review. After a signed consent was obtained, a series of screening questions were used to determine initial eligibility based on the inclusion/exclusion criteria listed in . The full questionnaire was administered to all potential participants in order to determine all reasons for ineligibility. Detailed questions on menstrual and reproductive history were recorded both for determination of eligibility and for use in scheduling. Other questions were asked regarding demographic and life style factors, as well as medical history, for use in characterising the potential and actual participants. In addition, fasting blood (33 mL) and urine specimens were collected (). Blood samples were used as part of screening and eligibility [complete metabolic profile (CMP), total cholesterol, Chlamydia IgG], for detection of trace metals (lead, mercury, cadmium) and for storage for future testing (glass red top for environmental trace elements). A spot urine was collected in the clinic, spun and frozen for future use. After the blood sample and urine collection, subjects were provided a light breakfast.
BioCycle study inclusion/exclusion criteria
Screening visit blood and urine sample collection
A negative blood test for history of Chlamydia infection (IgG) was required for eligibility. Chlamydia infection has been shown to be associated with oxidative stress in several clinical and experimental studies. We therefore excluded individuals with past infection with Chlamydia species. Positive Chlamydia tests and abnormal values of other screening blood tests were referred to the participant’s personal health care provider for evaluation. To further assess eligibility, participants were asked to bring all medications (prescription and over-the-counter) to the clinic where information on type and frequency of use was recorded. Finally, physical measures were obtained, including height and weight using standardised protocols, which were used to determine eligibility with respect to body mass index (BMI).
The screening visit took approximately 2 h to complete. At the end of the visit, an appointment for the baseline visit was made for those who remained interested. The baseline visit was scheduled 1–2 weeks prior to the participant’s next expected bleeding episode.
Baseline (enrolment) visit
At baseline, participants were informed of their final eligibility, including results of their Chlamydia IgG screen. All participants received a handout explaining the Chlamydia test and the meaning of the test results, regardless of whether the result was positive or negative. Those who were positive were advised to discuss these findings with their personal physician. Those who were ineligible for any reason were advised of this and had no other study activities completed.
Participants who were eligible and remained interested had physical/anthropometric measures taken, completed additional questionnaires and provided a urine sample. Physical and anthropometric measures were obtained according to standardised protocols and included blood pressure; pulse; sitting height; knee height; abdominal height; weight; waist, hip, abdominal, arm and thigh circumferences; and skinfolds of the upper arms, thighs, subscapular (shoulder blade) and suprailiac (hip) areas. The urine sample was used to rule out current pregnancy (Quick Vue, Quidel Corporation, San Diego, CA, USA). Excess urine was frozen and stored for future use. One pregnancy was found during the baseline visit screen and no pregnancies occurred during cycle visits.
Participants were asked to complete questionnaires on the following topics (): life style; health history; skin and body hair patterns;18,19
physical activity (IPAQ – The International Physical Activity Questionnaire); family medical history questionnaire; Perceived Stress Scale (14 items);20
Depression Scale (Center for Epidemiologic Studies Depression Scale, CES-D);21–23
and occupational history. Nutrient data were collected using a food frequency questionnaire (FFQ) developed by the Nutrition Assessment Shared Resource of the Fred Hutchinson Cancer Research Center.24
Questionnaires and physical measures by visit type
Participants received both verbal and written instructions in the use of a fertility monitor (Clearblue® Easy Fertility Monitor, Inverness Medical, Waltham, MA, USA). These monitors were used to assist in scheduling visits to correspond with specific phases of the menstrual cycle.25
The monitors assessed fertility based on levels of oestrone-3-glucuronide (E3G, an oestrone metabolite) and LH in urine. Testing began on approximately the sixth day following the start of the woman’s menstrual cycle and continued for 10 or 20 days, depending on the timing of the LH surge and cycle length. For the participants, monitor indications of low, high and peak fertility were used to time mid-cycle visits, with peak day and the following 2 days being those that would approximately represent late follicular, LH surge and ovulation dates. Participants were instructed to bring the monitors to every clinic visit, where we downloaded information using software provided by the manufacturer. Testing history and the corresponding values of E3G and LH were accessed. This information was helpful in assessing participant compliance with daily monitor use, as well as assessing patterns and levels of E3G and LH in participants during the study. Further information about the fertility monitors can be found on the company web site.26
Participants were also provided with a daily diary where they were instructed to record the dates of their bleeding segments, symptoms during these bleeding segments, physical activity, life style habits (alcohol, smoking and caffeine use), sexual activity, stress, hours of sleep, medication intake and findings related to use of their fertility monitor. The participants were instructed to begin their diaries on the first day of their next bleeding segment and to continue entries daily for two complete menstrual cycles. Women were instructed not to start taking any supplements or medications or have X-rays taken during the study, unless medically indicated. Any use was recorded on the daily diaries. Only four women reported taking any medication during the study. The women brought their daily diaries to each clinic visit for review and for recording of information. A calendar of scheduled clinic visits was provided with dates initially based on self-reported cycle length. These calendars were adjusted in an ongoing fashion using information from the fertility monitor.
Cycle-specific visits (16 visits: 8 visits per cycle for 2 cycles)
Participants were asked to contact the clinic at the first sign of bleeding. The first cycle visit was scheduled on the next day (day 2 of their menstrual cycle). Study staff began reminder calls on a daily basis several days before the expected date of their next bleeding episode (based on history) in order to remind participants to call and schedule their first visit as soon as they began menstruating. For subsequent visits, participants received a reminder call 1 day before each scheduled visit to reduce missed appointments. During the call, the women were reminded of their appointment time, of the need to fast, and of the need to bring study forms with them to the clinic. During the first cycle, the visits were scheduled using an algorithm accounting for cycle length, but the mid-cycle visits were adjusted based on the monitor data. If the monitor indicated ‘peak fertility’ on a day without a scheduled visit, the women were asked to come in that morning and the following two mornings. Those not reaching peak by mid-cycle were scheduled according to an algorithm related to their cycle length. A visit calendar for the second cycle was created based on the length of the first cycle and the monitor data from the first cycle. The visit schedule was modified as necessary based on the second cycle monitor readings.
The study included 16 cycle visits (eight per cycle) over two cycles. The timing of these visits was scheduled to correspond with specific times of the menstrual cycle where the most hormonal variation would be expected, approximately days 2, 7, 12, 13, 14, 18, 22 and 27 of a 28-day cycle, adjusted for cycle length ().
Cycle visit schedule by cycle day (based on a 28-day cycle)
Cycle visits were routinely scheduled between 7:00 am and 8:30 am to allow for collection of fasting samples and to reduce diurnal variation. The study clinic was open 7 days per week 363 days per year (no visits Christmas or New Year’s Day). The clinical centre had a ‘no wait’ policy in effect to aid in participant retention. This was accomplished by staggered visit scheduling, adequate staffing and blocking of visit activities. Visits lasted approximately 30 min. At peak capacity, 25 new subjects were enrolled per month, with about 50 subjects seen in any 1 month. Enrolment lasted 12 months, with completion of all visits taking 16 months. provides an overview of the number of subjects screened and enrolled in the BioCycle study.
BioCycle recruitment flow diagram.
Every cycle visit included collection of fasting (12 h overnight) blood and urine, diary review and questions on medication consumption. After specimen collection, participants were given a light breakfast and asked to complete brief questionnaires on recent activity. In addition, data were retrieved from their monitors. On approximately days 2, 7, 14 and 22, participants had longer visits, during which a 24 h dietary recall and an additional clinic visit questionnaire were administered. The questionnaire included questions on physical activity, stress, menstrual symptoms, X-ray exposure, emergency contraception use and illness in the past 7 days. On approximately day 27 of each cycle, participants were asked to complete a 1-month FFQ and a urine-based pregnancy test. During the final visit, after all samples were collected, participants had a dual energy X-ray absorptiometry (DXA) scan (Hologic Discovery Elite, Hologic, Waltham, MA, USA) to determine body composition (fat, lean, bone) and bone mineral density (spine, hip, wrist, whole body).
For the purpose of this study, a completed cycle included a minimum of five visits per cycle, with at least one visit during mid-cycle. Those with less than eight visits per cycle were generally the result of having shorter cycles (e.g. 21 days) or having unexpected events occur (travel, illness). Most women (n = 223) completed the study in two consecutive months, but 27 (10.8%) completed the study in non-consecutive cycles, mostly due to travel or illness. At the end of their participation, monetary reimbursement forms were completed. Participants were provided a modest compensation for their time and travel. Based on the number of visits completed, a participant could receive up to $500 (approximately $25 per visit). Participants were also asked to sign a release if they wanted copies of clinically pertinent tests to be sent to their personal physician (optional).
Specimen collection, processing, testing and storage for cycle visits
Both blood and urine were collected at every cycle visit. A bar code system was developed for labelling all collection tubes, storage vials and data collection sheets. This computer-generated labelling system used a unique participant ID along with the date of collection and specimen type. A database with additional information on the specimen type, time of collection, timing of specimen processing and storage, as well as the location of the stored sample within the biological specimen bank, was developed and used for all biological samples.
Samples were collected at the beginning of each clinic visit Participants were provided a 50mL sterile cup for collecting urine. Capped specimen cups were placed in a cooler and delivered to the processing laboratory within 30 min of collection. At the laboratory, urine was transferred to a 50mL conical tube and cen-trifuged (1500 × g) for 10 min in a 4°C centrifuge. Urine was decanted into 5mL polypropylene storage tubes, being careful not to disturb the sediment. A maximum of seven vials per participant visit were then placed in a −80°C freezer in 7 × 7 storage boxes.
Cycle visit blood samples were collected in a standardised fashion. Each participant was seated in the phlebotomy area by clinic personnel and remained seated for 10 min to allow fluid shifts to equilibrate. A total of up to 50mL of blood was taken at each cycle visit. At the time of collection, blood specimens were immediately wrapped in aluminum foil to protect them from light exposure. Blood tubes with anticoagulant were inverted a minimum of five times after collection. Heparin and EDTA tubes were placed in biohazard bags and put into a cooler with a chiller pack. Serum tubes remained at room temperature for no less than 20 min and no more than 30 min before being delivered to the specimen processing laboratory. At the processing laboratory, specimens were placed in a 4°C centrifuge at 1500 × g for 10 min. Samples for analyses that were processed using fresh blood on a daily basis were placed in a cooler and sent to a local laboratory (Kaleida Center for Laboratory Medicine) for testing (CMP, lipid profile, uric acid, high sensitivity C-Reactive Protein). All other specimens were placed in 9 × 9 boxes according to test and stored frozen at −80°C. The total time between phlebotomy and freezing specimens was recorded and was to be completed within 90 min.
lists the tests that were performed on the samples and the methodology used. Samples not used immediately for tests were frozen and stored for later analysis. Once a complete cycle of samples was obtained from a participant, her frozen samples were sent as a complete cycle set for testing at the specific study laboratories. This helped to reduce batch-to-batch variation within an individual. The storage plan allowed for appropriately sized aliquots for planned tests, which eliminated repeated freeze-thaw cycles for the pre-specified analytes. A mapping system and database were used to track samples.
Cycle visit blood and urine specimen collection
Plasma free F2-8-isoprostanes were measured with a gas chromatography–mass spectrometry-based method27
by the Molecular Epidemiology and Bio-marker Research Laboratory at the University of Minnesota (Minneapolis, MN).
Data management procedures
Databases for information collected were developed in Microsoft® Access. Data entry screens were created to mimic the study forms in order to create a flow for data entry personnel. Tight validation rules and referential integrity were applied to our entry forms to ensure further accurate data entry. Coding of variables was contained in the questionnaire in most instances. In addition, the Access database included variable tables and numeric codes. For all physical measures, questionnaires and laboratory results initially recorded on paper, 100% double entry was performed, and macros were created that produced discrepancy reports in Access. If discrepancies were found, data entry personnel reviewed hard copy records and made corrections. Personal identifiers were maintained in a separate database from the study data, and all data collection was carried out using a unique study ID. A second unique study ID was used for laboratory samples. The laboratory ID and study ID could be linked for data analysis purposes. Data were stored on a secure network server. Data from all sources were converted to SAS® data sets (SAS version 9.1, SAS Institute, Cary, NC, USA).