This is a single-blinded randomized control trial testing the efficacy of a dietary and exercise intervention for women with overweight and obesity undergoing infertility treatment. Ethical approval was obtained from the UAMS Institutional Review Board (UAMS IRB-260113), version 10 of the protocol. This article follows the SPIRIT (Standard Protocol Items: Recommendations for International Trials) guidelines for reporting clinical trial protocols . Table 1 and Fig. 1 detail the time schedule of enrollment, interventions, and assessments. Findings will be disseminated via peer-reviewed manuscripts and presentations at scientific conferences, at which time protocol amendments will be discussed.
Table 1 Schedule of enrollment, interventions, and assessments at specific time pointsFig. 1
Study Flow Chart. Protocol schematic diagram including informed consent, visits, randomization, intervention and control groups, fertility clinic procedures, and cross over design. AF&GA: Arkansas Fertility and Gynecology Associates, BMI: Body Mass Index
This multi-component intervention will be conducted primarily via telemedicine, thus the location will vary and may include places like the participants’ homes or a local gym. Participants will be undergoing fertility treatments at Arkansas Fertility and Gynecology Associates (AF&GA), which serves Arkansas and surrounding states. Data collection will take place within AF&GA and the Arkansas Children’s Nutrition Center (ACNC), both of which are located in Little Rock, Arkansas.
Eligibility and recruitment
Criteria for study inclusion are women with a BMI between 25 and 45 kg/m2, age of 21 years or over, and planning to undergo in vitro fertility treatment. Participants reporting the following will be excluded from the study: pre-existing conditions (e.g. sexual transmitted diseases) that will affect the outcomes of the study as determined by the principal investigator; current use of recreational drugs, tobacco, nicotine, or alcohol; food allergies, food intolerances, or food preferences which would interfere with compliance to the meal plan; and contraindications to exercise or already meeting the Physical Activity Guidelines for Americans  (150 min moderate activity/week or 75 min of vigorous activity/week with resistance exercise on 2 or more days/week.
Recruitment of participants will be conducted by the AF&GA team using IRB-approved tools. Advertisements and information sheets will be distributed to eligible participants electing to undergo fertility treatments. Women who are interested in participating in the study can inform the AF&GA team, call the ACNC recruitment line, send an e-mail expressing their interest, or express their interest through the ACNC website. To establish eligibility for participation in the study, a standardized questionnaire will be administered by phone.
Consent and randomization
The informed consent process will educate potential participants about the research study through IRB-approved methods. Eligible participants will be contacted, and a copy of the informed consent will be provided to the participant prior to signing consent documents. A research team member will reach out to potential participants 2 days prior to the scheduled visit to perform a COVID-19 screening and answer questions on the intervention components of the study. At the first visit, the research team will review all study procedures and the informed consent documents with the potential participant. Participants will be given sufficient time to review the consent form, ask questions, and receive clarification prior to signing any documents. If eligible and interested in enrollment, informed consent will be obtained from the participants and documented.
Women will be randomized, with computer generated randomization, to a standard of care group (N = 60) and an intervention group (N = 60). Due to the high incidence of obese women with Polycystic Ovarian Syndrome (15–30%) , compared with other infertility diagnoses, women will first be classified into one of two strata based on the presence or absence of PCOS. Within each stratum, the proposed study will randomize the enrolled participants into either the intervention group (N = 30) or the standard of care group (N = 30). Thus, the potential for confounders of infertility, besides PCOS, should be equally distributed between the groups and not affecting the main outcome of interests. The study will have four experimental arms: PCOS-standard of care, PCOS-intervention, no PCOS-standard of care, no PCOS-intervention. Allocation will happen at the second visit (after consent and the collection of baseline measures) by providing the participant with an opaque, sealed envelope containing their allocation.
The intervention, combining an exercise and a dietary component along with the standard practice from AF&GA, will take place during the weeks leading up to oocyte retrieval and continue until 2 weeks after implantation. We expect the intervention to last 8–12 weeks, depending on the participant’s specific fertility treatment plan.
The intervention will be delivered by trained research team members who will meet with participants twice weekly and serve as their health coach in order to track compliance and support adherence to the meal and exercise plan. This contact will occur via their preferred contact method (e.g., telephone, text messages, email, or using applications such as FaceTime). All interventionists will complete an intensive training on the intervention protocol and motivational interviewing techniques [9, 27]. They will be provided with detailed counselor guides to follow. Prior to conducting a coaching session with a participant, they will be required to be certified by one of the study investigators in providing both initial and subsequent intervention sessions. All sessions will be video or audio recorded; 15% of sessions will be randomly selected, reviewed, and scored on a fidelity rating scale to ensure that the protocol is being implemented as intended. Fidelity ratings will include building and maintaining rapport; discussion of daily meal plan adherence and compliance; discussion of weekly exercise adherence and compliance ; use of SMART goal setting techniques ; engagement with participant in problem solving as needed; and use of reflective listening, positive reinforcement, and summarizing techniques . Feedback will be provided to the coach on every session reviewed.
The exercise intervention will be based on the Physical Activity Guidelines for Americans for exercise prescription (150 min of moderate exercise per week and muscle strengthening exercises on two or more days per week). The exercise program will be customized to the participant and delivered using a web- and mobile-based application. The exercise program will be built for 8 to 12 weeks, depending on study length, with increasing intensity of combined aerobic and resistance exercises over the course of the study. The goal is for participants to perform at least 3 routines per week at their desired location. Total session time may vary by participants depending on number of breaks and rest periods. Exercise routines will combine aerobic, resistance and stretching exercises. In addition, participants will be encouraged to walk daily and increase their daily steps by 500 steps per day every week until they reach 10,000 steps per day. They will be able to monitor their steps with provided pedometers (Fitbit, Garmin, Apple Watch, etc.). Participant’s adherence to their exercise prescription and walking goal will be assessed weekly by the coach.
The dietary intervention is based on the Mediterranean diet. The meal plan was designed by a Registered Dietitian and follows the 2015 Dietary Guidelines for Americans healthy Mediterranean-style eating pattern (Tables 2 and 3) . Participants will be provided with all meals (3 meals/day) throughout the 8–12-week intervention that are in compliance with the Mediterranean diet in the form of refrigerated packaged meals from a company with the capacity to provide made-to-order meals and deliver them to the participant’s home address. The meals will be heated using conventional equipment found in most households and require no further preparation. Depending on calorie and macronutrient requirements, as assessed by the coach in accordance with participant feedback, snacks that adhere to the Mediterranean meal plan may be added. Snacks will include nutrient dense foods such as whole grains, fruits, nuts, low-fat milk, yogurt, and/or cheese. This nutrition intervention is not designed for participants to lose weight, however with the meal plan that could occur. Participants will be provided with a detailed weekly meal plan and will be asked to keep record of what they eat daily, on paper and/or using RedCap surveys.
Table 2 Dietary Guidelines for Americans Mediterranean-Style Meal Pattern
Standard of care
Participants in the standard of care group will be provided with dietary and physical activity recommendations from the Dietary Guidelines for Americans at their first study visit and will follow the standard of care practice at AF&GA up to their retrieval procedures. Participants will not be required to wait 8 weeks prior to their retrieval procedures since no intervention is needed. If participants in the standard of care group plan to undergo a second oocyte retrieval cycle, they will be offered to join the intervention group at that time as a cross over design.
Compensation and incentives
Participants will be compensated for their time spent at the research facility, their traveling costs and efforts with a monetary value of $50 per visit. Participants randomized to the intervention group will also receive all free meals for a period of 8 to 12 weeks depending on their fertility treatment plan. They will also receive training assistance, coaching, and reimbursement for monthly fees to a gym of their choice up to $50/month for the duration of the intervention. As an incentive to keep up with the exercise intervention, participants will be offered $10/week for meeting their step and exercise goal. A bonus of $80 will be provided to participants who completed all weekly exercise sessions prescribed. Participants in the standard of care group will receive a completion bonus with a monetary value of $200 at their last visit. Total compensation for the standard of care group (all paid by checks or gift cards) will be up to $350 dollars.
Data collection, management, and analysis
Data collection methods
Participants will attend three research study visits at the ACNC, two at the beginning of their fertility procedures and the last within 5 days of their oocyte retrieval procedures. Table 1 and Fig. 1 detail the time schedule of enrollment, participant visits, and the intervention. On average, we anticipate that there will be 8–12 weeks between visit 1 and 3 depending on the participant’s fertility treatment plan. Questionnaires will be administered on paper or via online platforms such as RedCap.
Background information and releases
Women who agree to participate will provide background information including their name, address, date of birth, education, number of people living in their household, marital status, and health history. Participants will be required to obtain an exercise release form from their physician at AF&GA prior to the study visit. They will also be required to sign a consent for release of medical information in order to obtain data relating to the clinical results from the fertility procedures as well as pregnancy and delivery outcomes.
Anthropometrics and vitals
Anthropometric data will include height, weight, and waist and hip circumferences. All measures will be repeated in duplicate or triplicate in order to ensure measures fall within stated tolerance ranges. Height will be measured to the nearest 0.1 cm with a wall-mounted stadiometer (tolerance of +/− 1 cm). Weight will be measured to the nearest 0.1 kg on a scale that has been tared with clothing the participant is wearing (tolerance of +/− 0.1 kg). Waist and hip circumferences will be measured to the nearest 0.1 cm using a tape measure against bare skin or form fitted shorts (tolerance of +/− 1 cm).
Vitals will include blood pressure, pulse, and temperature. Blood pressure and pulse will be measured with a digital blood pressure monitor twice (with 5 min in between each measurement); a third measurement will be taken if the two measures have a difference of 6 mmHg for systolic or 4 mmHg for diastolic blood pressure. Temperature will be taken upon arrival for each visit with an infrared non-contact forehead thermometer in accordance with COVID-19 screening procedures. Participants with a temperature ≥ 100.4 °F will be asked to reschedule their visit while those with a temperature below 100.4 °F will have it taken again orally with a SureTemp Plus thermometer for data collection purposes.
Body composition and bone mineral content will be measured using dual energy X-ray absorptiometry (DXA; Hologic Horizon A, Bedford, MA). This technology involves very small amounts of radiation and yields a very important measure of body composition and bone mineralization . Quality control calibrations will be performed each day and subjects will be positioned according to the manufacturer recommendations for the whole body scan while wearing light clothing without metal.
Resting and activity energy expenditure
Resting energy expenditure will be measured using indirect calorimetry (MOXUS, AEI technologies, IL) following an overnight fast while wearing a heart rate monitor (Polar, Finland) . The instrument measures the flow of oxygen consumed and the flow of carbon dioxide produced to calculate energy expenditure. The system will be calibrated daily. Activity energy expenditure will be assessed using the ActiGraph 9XLink accelerometer (ActiGraph, Pensacola, FL) worn for 7 days and a previously validated physical activity questionnaire (International Physical Activity Questionnaire) .
Fitness and leg strength tests
A cardiorespiratory fitness test will be performed on a treadmill following the guidelines for exercise testing from the American College of Sports Medicine. After evaluating the participant at rest and during the 3–5 min warm up, speed and/or incline will be increased in periods until exhaustion. During the entire test, breath composition will be sampled and measured using a metabolic cart (Medgraphics Ultima PFX® system, MGC Diagnostics Corporation, St. Paul, MN, USA). Leg strength will be measured by an isokinetic dynamometer (Humac Norm, Computer Sports Medicine Inc., Stoughton, MA). The Humac dynamometer will be used for testing of right and left legs for flexion (hamstrings) and extension (quadriceps) at certain angular velocities.
Blood samples, up to 35 ml, will be collected from the participants at visits 1 and 3 by a trained phlebotomist under overnight fasted conditions. During the entire study, we will collect up to 70 ml total. Blood samples will be centrifuged to separate plasma, serum, and red blood cells. Blood samples will be used to assess glucose, insulin, HOMA-insulin resistance score, interleukin-6, C-Reactive Protein, tumor necrosis factor, leptin, lipid profiles or any other biomarkers or analytes of interest using ELISA, electrochemiluminescence (ECL), colorimetric assays, metabolomics or any other appropriate methodology. Urine and stool collection will be obtained to evaluate metabolites, hormones or bacteria/microbes. Samples will be obtained using sterile collection cups. All biological samples will be aliquoted and stored at − 80 °C until further analyses.
Depression and perceived stress
Maternal depression will be evaluated using the Beck Depression Inventory-II, which has been shown to have high reliability and validity . Participants’ level of perceived stress will be evaluated through the administration of the Cohen’s Perceived Stress Scale which is a validated 14-item questionnaire examining the perception of stressful life events and levels of experienced stress over the course of the 4 weeks prior to the completion of the survey . Participants with elevated scores will be provided with mental health resources and referred to their primary care provider.
Baseline dietary intakes will be assessed using 3-day food records. Dietary data will be evaluated by a trained research assistant for completeness and analyzed with the Nutrition Data System for Research (NDSR, Nutrition Coordinating Center, University of Minnesota, MN). Data entry into this software will be randomly audited (10%) by a trained research assistant to ensure quality control.
Oocyte, cumulus cell, and follicular fluid data collection
AF&GA will provide the research team with oocytes (eggs), cumulus cells (cells surrounding the egg), and follicular fluid (liquid surrounding the egg that also contains granulosa cells) that are not needed for the IVF procedures (e.g., too many eggs retrieved). These samples will be collected during the standard oocyte retrieval procedures performed as part of standard care for the participants undergoing fertility treatment. Participants will undergo standard procedures at AF&GA for ovarian stimulation, which include hormonal superovulation and oocyte retrieval 36 h after human gonadotropin injection. Any deviations from the standard clinic procedures will be documented and sensitivity analyses will be performed to determine whether deviations to stimulation protocol yielded in significant differences. It is possible that the research team will not obtain samples in the case that the retrieval yields only enough eggs for the IVF procedures. In this case, only follicular fluid will be obtained. The research team will not obtain samples that could have been fertilized or frozen as agreed with the participant prior to the retrieval procedure. The research team will obtain only samples that are unneeded by the clinic. Thus, no additional treatment or clinical procedures will be required for the participants. The embryologist at the clinic will collect the cumulus cells during the processing of the oocytes. Granulosa cells will be isolated from the follicular fluid by centrifugation at 4000 rpm for 10 min. Follicular fluid samples contaminated with blood will not be used for analysis. Samples will be placed on ice and transported by the research team to the ACNC where they will be stored in messenger RNA lysis buffer and frozen at − 70 °C until processing. Oocytes and cumulus cells will be used to study gene expression while follicular fluid will be used to measure metabolites and hormones that may be influenced by diet and exercise. Both mRNA and genomic DNA will be isolated from individual oocytes as previously described . cDNA libraries will be prepared and fragmented cDNA will be evaluated to determine size distribution of the libraries. Samples will be sequenced using the NextSeq 500 System (Illumina). Individual libraries will be generated and RNA-Seq will be conducted. Sequencing reads from each sample will be trimmed and filtered before being aligned to the human genome (hg19). All data will be analyzed using SeqMonk and R software. Differentially expressed gene expression will be identified using the DESeq2 package which will include multiple testing corrections. Serum and follicular fluid glucose, total cholesterol, and triglyceride levels will be assessed using colorimetric or electrochemiluminescence assays. Serum and follicular fluid insulin, leptin, tumor necrosis factor (TNF)-α, C-reactive protein (CRP), and interleukin-6 will be measured using enzyme-linked immunosorbent or electrochemiluminescence assay kits. Serum lipid profiles—non-esterified fatty acids, high-density lipoprotein, and low-density lipoprotein— will be analyzed by enzymatic methods on a clinical analyzer. All assays will be performed in accordance with the manufacturers’ instructions.
Feasibility and acceptability
Feasibility and acceptability will be measured primarily through structured qualitative interviews that will take place after intervention completion. These interviews will be conducted by trained team members in-person or virtually through video conferencing and are expected to last approximately 60 min. They will be audio recorded and transcribed before being coded and analyzed to identify key factors contributing to feasibility and acceptability of the intervention. Compliance with the intervention provides another measure of feasibility and acceptability. Participants in the intervention group will track their daily dietary intake for the duration of the intervention using paper and/or RedCap surveys. These surveys will be reviewed weekly by the health coach for completeness before being analyzed with NDSR (NDSR, Nutrition Coordinating Center, University of Minnesota, MN). To account for deviations from the meal plan, overall dietary compliance will be evaluated by comparing participant’s Healthy Eating Index (HEI) score to that of the standard meal plan. Participants will track their adherence to the exercise intervention (150 min/wk. exercise) using personal training software (FitSW, FitSW Inc., Colorado Springs, CO) and their steps will be continuously monitored by a pedometer (Fitbit, Garmin, Apple Watch, etc.) provided by the research team. Compliance with the exercise prescription (150 min/week) and walking goal (500 extra steps/week) will be assessed weekly by the health coach. Coaches will document the average number of weekly steps and total exercise minutes.
All investigators and research team members will complete and maintain appropriate CITI training. All data will be entered into the ACNC clinical relational database and/or RedCap. The database will be housed on a shared drive that is backed up nightly off-site Monday through Friday. Access to the server is password protected, as is access to the study database. Data and study samples will be labeled with a unique identifier (e.g., MB-101). This identifier will be in no way associated with the participant’s personal information, such as name or date of birth. The key to the coded information, all identifiers, and biological specimens will be destroyed 7 years after the study close date. Data and samples will not be used for future research, either with or without identifiers. Participant names, contact information, and address will be shared with the meal preparation and delivery company. Data and samples will only be accessible by investigators, research assistants, and institutional oversight offices (e.g., IRB).
Prior to inferential analyses, exploratory data methods will be used to check for potential outliers and aberrant observations and measurements. The following will be tested between the PCOS and no PCOS groups to assess potential differences: fertility diagnosis (male factor, ovulatory disorder, tubal factor, endometriosis or unexplained), pregnancy, live birth, age and race using Mann-Whitney U nonparametric tests for each strata. Follicular fluid and serum metabolites will be analyzed using a one factor Analysis of Covariance (ANCOVA) at four levels because of the potential unbalanced data structure. Pair-wise comparisons using Tukey-Kramer post-hoc tests will be used to identify groups whose means differ significantly while retaining the family-wise error rate at 5%. If the distributional assumptions for ANOVA are not met, and if a suitable data transformation is not found, a Kruskal-Wallis equality-of-populations rank test will be used, followed by Dunn’s test of multiple comparisons. When the design is balanced, two-way ANOVA or non-parametric Friedman test will be used in data analysis. As alternatives, generalized linear models may be also used to test the effects of treatment and presence of PCOS and ANCOVA may also be used to control for additional confounding factors Correlations will be determined using the Pearson Product Moment or the Spearman Rank Order for non-parametric tests. Significance will be set at p < 0.05. RNA-seq data will be analyzed using SeqMonk and R software. Gene expression levels will be expressed as raw read counts for differential expression analysis and as log-transformed normalized reads per kilobase per million mapped reads for visualization. Differentially expressed gene expression will be identified using the DESeq2 package and filtered at ± two-fold change and statistical significance with a p value ≤0.05. Gene Ontology (GO), transcription factor (TF) target analysis, and pathway analyses will be conducted with DAVID bioinformatics and/or WebGestalt, which include multiple testing corrections. For participants assigned to the standard of care whose fertility treatments were unsuccessful, they will be offered to re-enroll and be directly assigned to the intervention group. The data from these participants will be analyzed separately, using paired t-tests or non-parametric equivalent to evaluate the effect of the intervention (pre and post).
Based upon existing data in the literature, a power of 0.8, and a p-value set at 0.05, with 20 women per group, we will be able to detect a mean difference of at least 0.37 standard deviation units in follicular levels of C-reactive protein and of insulin. In our previous study, we found follicular fluid CRP differences of 3.2 μg/ml (2.7 vs. 5.9 μg/ml, respectively) between normal weight and obese women . Thus, we are confident that this sample size will be sufficient to detect clinically meaningful differences. Based on our experience, we expect that we will be unsuccessful in obtaining a good quality oocyte from approximately 33% of women enrolled. Therefore, we will enroll 30 women in each of the 4 groups (PCOS- standard of care, PCOS-intervention, no PCOS- standard of care, no PCOS-intervention) to assure that we will have at least 20 oocytes per group.
A Data and Safety Monitoring Plan is in place for this study. Adverse events, enrollment numbers, lost to follow up counts, and withdrawals will be monitored by the Clinical Coordinator. A continuing review will also be completed annually. Any adverse events and deviations will be reported to the IRB and included in the continuing review. Any adverse effects or unanticipated problems will be reported to the study PI, the IRB, and the study sponsor in accordance with IRB policies. At all times, the IRB will be promptly provided with the information from the Clinical Coordinator as needed.