A lifestyle intervention improves sexual function of women with obesity and infertility: A 5 year follow-up of a RCT

Background Obesity and infertility are associated with poorer sexual function. We have previously shown that a lifestyle intervention in women with obesity and infertility reduced weight and improved cardiometabolic health and quality of life, which may positively affect sexual function. We now report on sexual function 5 years after randomization. Methods and findings In total 577 women, between 18–39 years of age, with infertility and a BMI ≥29 kg/m2 were randomized to a six-month lifestyle intervention targeting physical activity, diet and behavior modification or prompt infertility care as usual. Intercourse frequency and sexual function were assessed with the McCoy Female Sexuality Questionnaire (MFSQ), 5.4±0.8 years after randomization. 550 women could be approached for the follow-up study, of whom 84 women in the intervention and 93 in the control group completed the MFSQ. Results were adjusted for duration of infertility, polycystic ovary syndrome and whether women were attempting to conceive. The intervention group more often reported having had intercourse in the past 4 weeks compared to the control group (aOR: 2.3 95% CI 0.96 to 5.72). Among women reporting intercourse in the past 4 weeks, the intervention group (n = 75) had intercourse more frequently (6.6±5.8 vs. 4.9±4.0 times; 95% CI 0.10 to 3.40) and had higher scores for vaginal lubrication (16.5±3.0 vs. 15.4±3.5; 95% CI 0.15 to 2.32) and total ‘sexual function’ score (96.5±14.2 vs. 91.4±12.8; 95% CI 0.84 to 9.35) compared to the control group (n = 72). Sexual interest, satisfaction, orgasm and sex partner scores did not differ statistically between the groups. The intervention effect on sexual function was for 21% mediated by the change in moderate to vigorous physical activity. Conclusion A six-month lifestyle intervention in women with obesity and infertility led to more frequent intercourse, better vaginal lubrication and overall sexual function 5 years after the intervention. (Trial Registration: NTR1530).


Nature and extent of the burden and risks associated with participation, benefit and group relatedness:
The advantages of a weight loss and exercise program in overweight or obese subfertile women have been shown in small intervention studies. Even if the patient does not conceive during the 6 months period in the lifestyle intervention arm, the health benefits of weight loss on the long-term are clearly in the interest of the patient. By starting this project, fertility treatment in the intervention arm will be delayed for a maximum of 6 months, there is no evidence that this delay will cause lower spontaneous or treatment dependent pregnancy chances. A more structured weight loss and exercise program will probably lead to higher spontaneous and therapy induced ongoing pregnancy chances. The Burden: Patients that are randomized in the intervention arm will attend the clinic for six extra visits in order to improve lifestyle, this will take around 280 minutes. In between visits counselling will take place per telephone or mail four times for 15 minutes. During these visits physical examination, measuring weight, WC and WHR in week: one, three, seven, twelve, eighteen and twenty-four will take place. Patients in the intervention group will have to adapt there lifestyle towards a more healthy lifestyle, this will give a certain amount of stress since they will have to adapt their diet and physical activity. Patients will be learned how to use stepcounter PAM and wear it on a daily basis in order to monitor their physical activity during the intervention period and will fill in their activity list.
Patients who have been randomized in the "usual care" group will undergo fertility treatment when indicated by national guidelines (www.nvog.nl). Patients with overweight or obesity have additional risks during pregnancy compared to normal weight women. The risks involve: hypertension (OR: 2.2), preeclampsia (OR: 1.8-2.7), pregnancy-induced diabetes ( OR: 2. 2-3.4), caesarian section (OR: 1. 3-2.4) (see tables of evidence in appendix). These risks are not influenced by fertility treatment. Lifestyle intervention aims to reduce the involved pregnancy risks. An independent Safety Monitoring Board (SMB) will be installed to review major complications in both treatment groups. This board will evaluate complications after every 150 included patients and six months and 12 months after the end of inclusion of the study (6 times during the study). The SMB will differentiate between major en minor complications, blinded for treatment arm. Every case of preeclampsia, eclampsia, HELLP syndrome, maternal or infant mortality will be reported to the SMB. The SMB findings will be reported to the Ethics Committee of the UMCG Questionnaires to be filled in by all participants in the study: SF-36, SQUASH-list, FFQ, DEBQ, concerning eating behaviour at the start, week twelve, twenty-four and fifty-two of the study. A questionnaire for details on direct and indirect costs like transportation, sporting activity and productivity loss due to lifestyle intervention programme and fertility treatment will be filled in by the participants during the study. This will take 30-45 minutes.

INTRODUCTION AND RATIONALE
The evidence for the adverse effects of overweight on women's reproductive health is indisputable. Overweight affects reproductive capacity in all subfertile couples. Moreover, overweight is associated with pregnancy complications and poor neonatal outcome. Pregnancy complications associated with obesity are hypertensive disorders, gestational diabetes, prolonged duration of labour, increased need of operative delivery, macrosomia, shoulder dystocia and increased blood loss (Garbaciak et al.,1985;Edwards et al.,1996). Obesity is associated with an increased risk of adverse pregnancy outcomes such as unexplained still birth Kristensen et al.,2005;Bergstrom et al.,1998;Linne et al.,2004, neonatal admissions (Usha Kiran et al.,2005 and five times higher costs (Linne et al.,2004).
Each year about 15.000 to 20.000 new couples present themselves with subfertility. In a large Dutch cohort study of 3.000 subfertile couples , 19% of the women had a BMI between 25 and 30, 7% had a BMI between 30 and 35, and 4% above 35. Thus, approximately 30% of these couples are overweight or obese.
Obesity has further been associated with reduced fertility in the general population (Ramlau-Hansen et al.,2007). Ovulatory subfertile women with a BMI of 29 kg/m2 or higher have a 4% lower pregnancy rate per kg/m 2 increase ( Van der Steeg et al., 2007). Obese women also have a lower live birth rate after IVF and ICSI Wang et al.,2000;Wang et al.,2002;Fedorcsak et al.,2004, Lintsen et al.,2005. The relationship between BMI and the risk of spontaneous abortion is inconsistent (Lashen et al.,1999;Fedorczak et al.,2000;Wittemer et al.,2000;Wang et al.,2001, Lintsen et al.,2005. A meta-analysis on the effect of overweight and obesity on ART reported a lower chance of pregnancy following IVF (OR 0.71, 95% CI:0.620-0.81) and an increased miscarriage rate (OR 1.3, 95% CI:1.06-1.68). Extrapolating from available data one could argue that the combination of lower chance of pregnancy and higher miscarriage rate in overweight and obese women could result in a reduced live birth rate (Maheshwari et al.,2007).
Obesity is strongly related to polycystic ovary syndrome (PCOS), which is the cause of subfertility in 25% couples. The accompanying insulin resistance and hyperinsulinism mark this syndrome as a prediabetic state (Dunaif, 1997), leading to an increase of neonatal complications.
In reproductive medicine, modest weight reduction (5-10%) with lifestyle intervention is not only associated with restoration of ovulation and fertility in overweight women with PCOS (Hoeger et al.,2004) but is also shown to decrease insulin resistance (Pasquali et al.,1989;Knowles et al.2002). Several studies have shown that even a modest weight loss leading to a 5% loss of central fat, in women with PCOS improves, the menstrual cycle, the rate of ovulation, and the likelihood of a healthy pregnancy (Clark et al.,1995, Norman et al.,2004, Huber-Buchholz et al,.1999, Stamets et al., 2004.
In view of the evidence that overweight has a negative impact on fertility and pregnancy outcome, lifestyle intervention prior to treatment of subfertile couples could improve spontaneous fertility chances, and prevent unnecessary fertility treatment as well as complications of fertility treatment.
Several reviews showed that lifestyle interventions targeted at changing diet and physical activity can succeed in sustained decrease of average bodyweight on the longer term (McTigue, 2004;Franz 2007). A decrease of 3 to 4 kilograms can be achieved after three years of follow-up (Avenell, 2004). The adverse health effects of increased body weight on for instance development of diabetes mellitus type 2, coronary heart disease, musculoskeletal disorders and some types of cancer are well known (WHO-euro document 2007). The cost-effectiveness of lifestyle interventions was below acceptable limits . For the Diabetes prevention project, for instance, the costs to prevent one new case of diabetes mellitus were calculated to be around 16.000 US dollars (Herman, 2005).
The intervention we propose to evaluate is based on dietary therapy, increased physical activity and individualized behavioural modification plan. A combination of these three interventions leads to maximal weight loss and maintenance of weight loss (NIH guideline, CBO guideline 2007, Zelissen et al 2004). This behavioural modification therapy can be delegated to a nurse practitioner or trained nurse or dietician. Such an intervention program can be implemented within gynaecological care.
Although lifestyle modification is advised as a key component for the improvement of reproductive function in overweight women specifically with PCOS (NICE 2004Tang et al.,2006, Pasquali et al.,1989, Hoeger et al.,2004, Clark et al.,1995, the evidence of its effectiveness as demonstrated in clinical studies is limited. It's costeffectiveness has never been assessed in large groups of subfertile women with respect to prevention of unnecessary fertility treatment, prevention of complications of fertility treatment and improvement of perinatal outcome. The cost-effectiveness of such program will be investigated in the this study. (see table Summary of studies assessing outcome after lifestyle intervention).

OBJECTIVES
In view of the lack of convincing evidence from large intervention studies and the strong practice variation in The Netherlands we propose a randomized clinical trial in overweight and obese subfertile women, in which we compare the costs and effects of a six months structured lifestyle program, aimed at weight loss, to "usual care" with the aim to prevent obesity related pregnancy complications, unnecessary fertility treatment and complications associated with fertility treatment, enhance pregnancy chances and improve perinatal outcome. The control group of women will receive fertility treatment "care as usual" without lifestyle intervention.
We aim to estimate the costs and effects of a structured lifestyle program in terms of costs per additional healthy singleton birth rate beyond 37 weeks gestation born after vaginal delivery in a follow up period of 24 months.
The questions that we aim to answer are: Primary Objective: Does a structured lifestyle program aiming at a weight reduction of 5%-10% reduce the need for fertility treatments due to an increase in spontaneous conception rates?

Secondary Objective(s):
Does a structured lifestyle program aiming at a weight reduction of 5%-10% increases the success rates of fertility treatments, including assisted reproductive technology? Does a structured lifestyle program aiming at a weight reduction of 5%-10%, lead to less maternal complications during pregnancy, such as hypertensive disorders, premature birth, macrosomia, shoulder dystocia, operative delivery, and haemorraghia, and multiple pregancies in subfertile women with overweight or obesity? Does a structured lifestyle program aiming at a weight reduction of 5%-10% lead to a lower percentage of women with overweight or obesity and subfertility developing gestational diabetes?
Does a structured lifestyle program aiming at a weight reduction of 5%-10% in subfertile women, lead to a better quality of life, as well as body weight reduction, reduction of waist circumference, behaviour influencing weight, i.e. nutritional habits and exercise pattern, blood pressure, serum glucose/insulin ratio (HOMA) and hormonal profile changes.

STUDY DESIGN
Multicenter randomised clinical trial. Flow chart 1 gives an overview of the study procedure form randomization till end of follow up period. Table 1 is added to give a overview of the lifestyle intervention that subjects will undergo in the course of research.

STUDY MANAGEMENT:
The randomisation is performed by computer at a central randomisation centre in the AMC. Randomisation will be stratified according presence or absence of anovulation and center of treatment. Data will be collected using Oracle Clinical Remote Data Capture (RDC), which is a new generation of application system that enables collection and cleanup of clinical trial data using the Internet. For detailed information on Oracle RDC, please visit the page of Oracle RDC products (http://www.ctc-g.co.jp/en). The expertise for this technology is already used in the study group.

Population (base)
All couples suffering from subfertility visiting the clinics participating in the study will undergo a basic fertility work-up including a semen-analysis, an assessment of tubal patency using CAT or HSG, and monitoring of the cycle to assess ovulation. After the work-up has been completed, a fertility diagnosis will be made and a prognosis for treatment independent pregnancy will be calculated (Hunault et al.,2004), followed by a management proposal for the individual couple. Ovulatory and anovulatory women with a body mass index between 29 kg/m² and 40 kg/m² will be approached to participate in the present study. After informed consent, they will be randomly allocated to an intervention program to improve lifestyle with a duration of six months, or to "usual care".

Inclusion criteria
We aim to include subfertile women with a BMI between 29 kg/m² and 40 kg/m². Women have to be between 18 and 38 years old. Subfertility is defined as failure to conceive within 12 months of unprotected intercourse in couples( where the woman has an ovulatory cycle), as well as couples where the women suffers from chronic anovulation due to WHO I or II or PCO.

Exclusion criteria
Couples suffering from azoospermia, severe endometriosis AFS 3 and 4 (Revised American Fertilty Society classification), chronic anovulation due to WHO III, endocrinopathies (like: cushing syndrome, adrenal hyperplasia, hypothyroidism, diabetes mellitus type I) will not be eligible for the study. Women with preexistent hypertension or diabetes, or pregnancy induced hypertension, preeclampsia, eclampsia or HELLP syndrome in a previous pregnancy are also excluded. Incapacitated adults will be not asked to participate in the study.
Women eligible for the study will de referred to a research nurse. This person is dedicated to counselling eligible patients and will perform randomisation and collect follow-up data, but will not perform the experimental intervention. The lifestyle intervention programme will be done by trained intervention nurses. We will collaborate with the research nurses who are involved in the obstetric and urogynaecological consortium. In this consortium, more than 20 large randomised clinical trials are performed. Information on this infrastructure is obtainable from http://www.studies-obsgyn.nl/

Sample size calculation
SAMPLE SIZE: On the basis of the literature the cumulative live birth rate in a follow up of two years of subfertility treatment will be 45% for the "usual care" group (Dutch OFO-study, unpublished data). The lifestyle intervention group is estimated to have a life birth rate of 60% (Dutch OFO-study, unpublished data) We expect an improvement of healthy singleton of more than 37 week gestation born after vaginal delivery from 45% to 60% in the intervention group. Drop out is expected to be 20% in the intervention arm and is included in the analysis of effectiveness.
To prove that, we need to include two groups of 272 women to be included (alpha 0.05, power 80%). To account for 5% lost to follow up 285 women will be included per group. In total 570 women will be included.

Investigational treatment
THE "USUAL CARE" ARM: In the "usual care" arm treatment will be started if the individual prognosis, based on the Hunault model (Hunault et al.2004) is less than 30% chance of conception within one year or when more than 3 years subfertility (guideline NVOG) justifies starting treatment. Even so, in case of chronic anovulation, ovulation induction will be started. Fertility treatment can either be IUI, IVF or ICSI whatever is indicated according to the Dutch guideline on IVF treatment (NVOG). In anovulatory patients, ovulation induction will be started using clomiphene, or gonadotrofines, which is applicable. When the Hunault model shows a prognosis of more than 30% pregnancy chance in the forthcoming year (and patients are less than three years subfertile), expectant management will be offered.
THE EXPERIMENTAL ARM: The overweight intervention is based on the lifestyle module which is developed in ZONMW project (50-50110-98-078) and will be adapted for the study proposal in this specfic group of subfertile woman for a duration of six months. The software module developed, used and evaluated in this program will be adapted for this subfertile patient group. The use of the software program will reduce practice variation between fertility centres. The lifestyle intervention is based on the recommendations of Dutch CBO guideline "Diagnostiek en behandeling van obesitas bij volwassenen en kinderen" and consists of a structured lifestyle program targeted at: 1. Changing the dietary pattern. 2. Stimulating physical activity of moderate intensity. 3. Changing existing behaviour. 4. Self-monitoring. 5. Involvement of the partner. 6. Referral to psychologist when indicated. ad 1. CHANGING DIETARY PATTERN Women will be advised to adapt their dietary pattern and sustain a healthy diet with a caloric reduction of approximately 600kcal compared to their normal caloric intake (but not below 1200kcal/day). A realistic target weight will be set of 5-10% below their body weight. The diet should be based on the Dutch guideline "Gezonde voeding" in order to achieve a sustainable diet change. ad 2. STIMULATING PHYSICAL ACTIVITY Physical activity is necessary in order to sustain a modest weight loss and increase effect of dietary measures. Patients will use a stepcounter (PAM) to measure daily steps aiming for a daily goal of 10.000 steps. Two to three times a week sporting of at least 30 minutes of moderate intensity (60-85% of maximum hearth frequency) is advised. Sporting advice is individualized taking into account a woman's preference and possibilities (swimming, walking, stepping, cycling, cardio-fitness, etc.). Patients keep a diary to not the result of the stepcounter, and the sporting activities. These results are evaluated by the trained nurses. ad 3. CHANGING EXISTING BEHAVIOUR The motivation to change lifestyle is measured and evaluated during the program with the PACE score measuring stages of change (precontemplative stage, considering -preparatory stage, action stage and maintenance stage). This PACE score is part of the software module "GOAL". Motivational counselling is individualized for these stages of change. Changing existing behaviour is achieved by motivational counselling directed at: -Awareness of actual lifestyle leading to overweight or obesity.
-Counselling healthy lifestyle measures, the effect of healthy lifestyle in relation to subfertility and spontaneous and treatment dependent pregnancy chances as well as pregnancy complications and influence on perinatal outcome. -Individualized goals are formulated and embedded in a "patient contract". "Patient contracting" is a tool to actually individualize the measures that the patient will take and to stick to the goals and targets that are agreed upon. During the intervention individual goals will be evaluated, feedback will be given and goals adapted when necessary. Continuous evaluation and feedback will be maintained focussed on the target bodyweight and long term maintenance of lifestyle changes. ad 4. SELFMONITORING Self-monitoring is an essential tool to improve compliance during a lifestyle program. It will be implemented using a PAM "stepcounter" to measure daily physical activity and the "eetmeter" (www.voedingscentrum.nl). This webbased tool is used to give feedback on food intake, and caloric intake on a daily basis. Patients will be trained to use this device. A diary with sporting activities as well as caloric intake will be evaluated by the dedicated nurses. ad 5. INVOLVEMENT OF THE PARTNER Involvement of the partner and family of the women is important in order to guarantee social support and a "buddy system" during lifestyle changes. ad 6. REFERRAL When indicated, referral to psychologist or intervention available in the general health care system will used.
Patients undergoing the lifestyle program will be guided and supported by trained nurses or dieticians or nurse practitioners, who will be trained prior to the study. Women who miss two consecutive sessions and who are not willing to catch up in another group are excluded from the program.
The standardized lifestyle program consists of four sessions in the first three months, and than a booster of two sessions in the last three months. Telephone consultation or consultation by mail is scheduled in between these sessions four times. (see table 1) FOLLOW-UP: All participating couples (usual care arm and the experimental care arm) will complete several questionnaires, i.e. the SF-36: measuring satifaction, the SQUASH list: for physical activity, the FFQ: food frequency questionnaire for assessing nutrient intake): a structured questionnaire about food pattern (e.g. breakfast yes/no) and important food components (e.g. snacks, fruits and vegetables and meat), the Dutch Eating Behaviour Questionnaire (DEBQ): for assessment of restrained, emotional and external eating behaviour (van Strien 1986). Finally, several questions are completed i.t.o. history of body weight, e.g. the duration of overweight, weight fluctuations. Questionnaires are webbased. In case a patient does not use the computer paper questionnaires will be used. Questionnaires will be completed at start, 12 weeks, 24 weeks and 52 weeks after randomisation. In addition each woman will receive a questionnaire for details on associated direct costs of professional care and on indirect costs like transportation, sporting activity and productivity loss. These questionnaires will be handed out in consultation 2, week 3 of the lifestyle module or at the start of fertility treatment for women that were randomised for "usual care". This will be repeated at the time points of the other questionnaires (12, 24 and 52 weeks after randomisation and at the end of follow-up).
Moreover, we will register reproductive outcome, fertility treatments as well as the course and outcome of subsequent pregnancies, including obstetrical interventions for a period up to 24 months after randomisation.

Study parameters/endpoints
Background and Demographic Characteristics To assess whether the treatment groups were balanced, the study populations will be compared for baseline measurements including female age, type of infertility (primary/secondary), duration of infertility, intoxications, body mass index, as well as sperm analysis according to WHO standards.

Main study parameter/endpoint
The primary endpoint will be a healthy singleton of more than 37 week gestation born after vaginal delivery. The initial analysis, will be done by intention to treat, so we will not make a difference for treatment independent pregnancies and pregnancies that occurred after treatment. Patients who will drop out of the study will by analysed according to their group of randomisation. In a per protocol analysis drop outs will be asked to provide information on the primary outcome, i.e. life birth within two years after randomisation. These drop outs will be analysed as non-compliant.

Secondary study parameters/endpoints
Secondary outcome parameters are: 1. pregnancy outcome and -complications: miscarriage, gestational diabetes, Pregnancy induced hypertension, preeclampsia, HELLP, prematurity<37 weeks, macrosomia> 4200 grams, induction of labour, prolonged duration of labour, operative delivery, and increased blood loss, and multiple pregnancies 2. percentage of women needing fertility treatment in both groups (OI, IUI, IVF, ICSI) and ongoing pregnancy rates in these treatments. 3. perinatal outcome: stillbirth, pregnancy duration, body weight and health of neonate, neonatal admission. 4. quality of life, pre-pregnancy body weight, weight gain during pregnancy, waist circumference, behaviour influencing weight, i.e. nutritional habits and exercise pattern, blood pressure, glucose/insulin ratio (HOMA), hormonal profile (androgens, adipokines and cytokines) and costs.

Other study parameters ECONOMIC EVALUATION:
The aim of the economic evaluation is to compare the costs and health effects of a lifestyle program versus "usual care". The analysis will be a costeffectiveness analysis with the proportion of healthy singleton of more than 37 week gestation born after vaginal delivery as the primary outcome.

Randomisation, blinding and treatment allocation
The randomisation is performed by computer at a central randomisation centre in the AMC. Randomisation will be stratified according to presence or absence of anovulation and center of treatment. Women eligible for the study will de referred to a research nurse. This person is dedicated to counselling eligible patients and will perform randomisation and collect follow-up data, but will not perform the experimental intervention. We will collaborate with the research nurses who are involved in the obstetric and urogynaecological consortium. In this consortium, eight large randomised clinical trials are performed. Information on this infrastructure is obtainable from http://www.studies-obsgyn.nl/

Study procedures TIME SCHEDULE LIFESTYLE PROGRAM
(see flow chart I: TIME SCHEDULE LIFESTYLE PROGRAM) WEEK 1: BASELINE ASSESSMENT. Consultation 1.
Counselling session 45-60 minutes. Motivational counselling will include an extensive history of weight changes, knowledge and insight of weight related health problems. Awareness, actual behaviour and lifestyle leading to overweight and history of body weight and slimming are discussed.
Counselling negative effect of overweight on frequencies of spontaneous and treatment dependent pregnancy chances and increase frequency of complications during pregnancy for mother and foetus. The stages of change can be identified using the PACE score in the GOAL software. DEBQ is evaluated to asses the type of eating behaviour. Counselling and providing knowledge and advice about healthy lifestyle. Evaluating diet questionnaire Evaluating SQUASH score for physical activity Healthy diet is advised aiming for a reduction of 600 Kcal/day ("Energy reduced diet"). Diet should be based on the Dutch guideline "Gezonde Voeding". Food intake should not be reduced below 1200 Kcal/day in order to achieve a sustainable lifestyle change.
Handout of "eetmeter" en stepcounter and food and physical activity diary A blood sample of 10 cc will be taken and serum stored.
CONSULTATIONS VIA TELEPHONE OR MAIL WEEK 5,8,15,AND 21. In between the consultations in the outpatient fertility clinic, consultation via telephone or mail are scheduled at week 5, week, 9, week 15, and week 21 with a duration of 15 minutes. In these consultations evaluation regarding: motivation of weight loss and physical activity are discussed Evaluating results regarding: weight, food diary, eetmeter, physical activity, stepcounter. Feedback on results. Individualized advise for improvement when necessary and adjust agreements when necessary to reach realistic targets. Discuss relapse.
Evaluating the barriers to follow the program when necessary.
Questionnaires for all included women: SF36, FFQ and Squash-list will be evaluated at the start and week 12 and week 24 and 52 weeks after randomisation.
Start of fertility treatment When in spontaneous ovulatory patients in the lifestyle intervention arm: • have finished their six month lifestyle program or, • when they meet their target weight reduction of 5-10% or, • when their weight decreases < BMI 29 kg/m², fertility treatment will be started if the individual prognosis, based on the Hunault model (Hunault et al.2004) if less than 30% chance of conception within one year or when more than 3 years subfertility (guideline NVOG) justifies starting treatment (either IUI, IVF or ICSI (according to national guidelines NVOG).
In case of chronic anovulation, ovulation induction will be started when patients in the lifestyle intervention arm: • have finished their six month lifestyle program or, • when they meet their target weight reduction of 5-10% or, • when their weight decreases < BMI 29 kg/m².

Withdrawal of individual subjects
Subjects can leave the study at any time for any reason if they wish to do so without any consequences. Patients who decide to withdraw from the study will be asked by the responsible investigator to provide information regarding the primary endpoint of the study 24 months after randomisation. Patients who drop out of the study will be treated according to the local protocols for infertility patients. The investigator can decide to withdraw a subject from the study for medical reasons. Women who miss two consecutive counselling sessions and who are not willing to catch up are excluded from the program.

Section 10 WMO event
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects and the reviewing accredited METC if anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly greater than was foreseen in the research proposal. The study will be suspended pending further review by the accredited METC, except insofar as suspension would jeopardise the subjects' health.
The investigator will take care that all subjects are kept informed.

Adverse and serious adverse events
Adverse and serious adverse events as a direct result of the lifestyle intervention are not anticipated in this study. However, patients with overweight or obesity have additional risks during pregnancy compared to normal weight women. As a result of both the intervention arm and of the "care as usual" arm, pregnancies may occur in patients with increased risks. The risks involve hypertension (OR: 2.2), preeclampsia (OR: 1.8-2.7), pregnancy-induced diabetes (OR: 2.2-3.4), caesarian section (OR: 1.3-2.4) (see tables of evidence in appendix). These risks are not influenced by fertility treatment. Lifestyle intervention aims to reduce the involved pregnancy risks. Participants in the study have been informed about these risks. An independent Safety Monitoring Board (SMB) will be installed to review major complications in both treatment groups. This board will evaluate complications after every 150 included patients and six months and 12 months after the end of inclusion of the study (6 times during the study). The SMB will differentiate between major en minor complications, blinded for treatment arm. Every case of preeclampsia, eclampsia, HELLP syndrome, will be reported to the SMB. The SMB findings will be reported to the Ethics Committee of the UMCG Adverse events are defined as any undesirable experience occurring to a subject during a clinical trial, whether or not considered related to the investigation. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded.
A serious adverse event is any untoward medical occurrence or effect that at any dose results in death; is life threatening (at the time of the event); requires hospitalisation or prolongation of existing inpatients' hospitalisation; results in persistent or significant disability or incapacity; is a congenital anomaly or birth defect; is a new event of the trial likely to affect the safety of the subjects, such as an unexpected outcome of an adverse reaction, lack of efficacy of an IMP used for the treatment of a life threatening disease, major safety finding from a newly completed animal study, etc.
All SAEs will be reported to the accredited METC that approved the protocol, according to the requirements of that METC.

Follow-up of adverse events
All adverse events will be followed until they have abated, or until a stable situation has been reached. Depending on the event, follow up may require additional tests or medical procedures as indicated, and/or referral to the general physician or a medical specialist.

Descriptive statistics
To asses whether the groups were balanced the study population will be compared for baseline measurements including female age, type of subfertility (primary or secondary), duration of subfertility, intoxications, smoking, BMI, as well as sperm analysis according to WHO standards, anovulation, and subfertility treatment. Confounding factors, such as smoking will be addressed in the analysis.

Univariate analysis and multivariate analysis
The primary analysis will be by intention to treat.
The primary outcome will be analysed by comparing the pregnancy rates in both groups using a chi-square test. Relative risks and 95% confidence intervals will be calculated. Delivery rates over time will be compared using life table methods with correction for confounders (Cox-regression analysis). Exploratory subgroup analyses will be performed for spontaneous and treatment-induced pregnancies and for women with a BMI below 35 versus above 35, as well as for anovulatory versus ovulatory women and for women with a waist-hip ratio of above 0.8 versus below or equal to 0.8. Additional analysis concerning the influence of HOMA, androgens, adipokines and cytokines on pregnancy chances and recovery of ovulations in anovulatory patients will be conducted. Incidence of complications of treatment and during pregnancy will be compared in both groups using relative risks and 95% confidence intervals.
Quality of life will be analysed using repeated measures analysis of variance A per protocol analysis will also be performed, in which drop out patients will be identified as non-compliant. Information on the primary outcome, i.e.lifebirth within two years after randomisation will be used whenever provided. Patients who drop out of the lifestyle intervention arm will be analysed in the "usual care" arm in this per protocol analysis.
Women who drop out of the study will be asked to provide the reason for dropping out. This reason will be recorded and patients will be asked to provide information regarding the primary outcome, i.e. pregnancy within two years after randomisation. In addition, patients will be offered the possibility to continue to complete cost questionnaires for the remainder of the study duration. For patients dropping out of the lifestyle intervention, further course will be determined by the responsible physician.

Economic evaluation
The aim of the economic evaluation is to compare the costs and health effects of a lifestyle program versus "usual care". The analysis will be a cost-effectiveness analysis with the proportion of healthy singleton of more than 37 week gestation born after vaginal delivery as the primary outcome.
The economic evaluation will be performed from a societal perspective. Direct medical and non-medical costs (intervention costs, time and travel costs) as well as indirect non-medical costs (productivity losses) will be taken into account.
We will build on the work of the ZonMW funded "Paraplu" study, in which cost data data of six cost-effectiveness studies were integrated. Dr H. Groen played an important role in this process.
The time horizon will be from randomisation to the end of follow-up. Resource use will be recorded as individual patient data in the CRF. In addition each woman will receive a questionnaire for details on associated direct costs of professional care and on indirect costs like transportation, sporting activity and productivity loss. These questionnaires will be handed out in consultation 2, week 3 of the lifestyle module or at the start of fertility treatment for women that were randomised for "usual care". This will be repeated at the time points of the other questionnaires (12, 24 and 52 weeks after randomisation and at the end of followup). Resource use will be valued according to Dutch guidelines. Intervention costs will determined based on actual resource use obtained from the centers. Costs will reflect the following resources: staff, materials, equipment, housing, overhead. Productivity loss will be valued by the friction cost method according to Dutch guidelines (Oostenbrink et al. 2004). Outcome of pregnancy will be recorded and valued using tariffs or data from the literature (Lukassen et al, 2004).
Detailed information on maternal complication will be obtained from obstetricians treating the woman concerned using patient medical files. Six weeks after the expected day of delivery all women will be contacted by telephone to ask information on the delivery and on the health of the child. If the child has been hospitalised the paediatrician treating the child will be contacted for further information.
Scenario analysis will be performed to model cost-effectiveness beyond the time horizon of the study. For longer term analyses, costs and effects will be discounted at commonly accepted rates. Sensitivity analysis will be performed on costs, pregnancy rates of the two groups and the valuation of different outcomes (no child, handicapped child, twin, healthy child, obstetric complications). Uncertainty surrounding cost-effectiveness estimates will be explored by bootstrapping. Cost will be presented in euros.

Recruitment and consent
Women eligible for the study will de referred to a research nurse by their treating doctors. This person is dedicated to counselling eligible patients, randomisation and follow-up for data, but not to the experimental intervention. We will collaborate with the research nurses who are involved in the obstetric and urogynaecological consortium. In this consortium, eight large randomised clinical trials are performed. Information on this infrastructure is obtainable from http://www.studies-obsgyn.nl/ The research nurse must explain to each subject the nature of this study, its purpose, procedures, expected duration and the potential risks and benefits involved in study participation along with any discomfort it may entail. Each subject must be informed that participation in the study is voluntary and that withdrawal of consent will not affect his/her right to the most appropriate medical treatment or affect the doctor relationship. This informed consent will be given by means of a standard written statement. It will be written so as to be easily understood by the subject. The subject will be given the time to read and understand the statement herself before signing her consent and dating the document. The subject will receive a copy of the written statement once signed. The informed consent form must be considered as a part of the protocol with which it is to be submitted by the investigator to the IRB/Ethics committee. Patients will be given a maximum of one week to consider their decision to participate in the study.

Objection by minors or incapacitated subjects (if applicable)
Incapacitated adults will be not asked to participating in the study. Minors are excluded as well.

Benefits and risks assessment, group relatedness
The ethical issues pertaining to this project will be discussed by considering the four ethical principles as discussed by Gillon. (37) Autonomy: After the intake consultation and counseling session by the research nurse, the patient is given the choice to participate in the study. This process of informed consent respects her autonomy.
Beneficence: The advantages of a weight loss and exercise program in overweight or obese subfertile women (in order to improving chances of conception and eventually taking a baby home) have clearly been shown. Even if the patient does not conceive during the 6 months period, the health benefits of weight loss on the long-term are clearly in the interest of the patient.
Non-maleficence: In some reproductive medicine units, overweight or obese subfertile women are counselled and advised to loose weight before treatment is started. By starting this project, fertility treatment in the intervention arm will be delayed for a maximum of 6 months. There is no evidence that this short delay will influence the eventual pregnancy chances. The structured weight loss and exercise program will probably lead to higher spontaneous and therapy induced ongoing pregnancy chances. Before giving informed consent all eligible women are informed about the risks mentioned before (in writing in the patients' brochure). Women who are considered to be at the highest risk (i.e. those who have pre-existent hypertension or diabetes, or pregnancy induced hypertension, (pre)eclampsia or HELLP syndrome in a previous pregnancy) are excluded from participation. During the study adverse events will be monitored by a SMB.
Justice: Introducing the study will not infringe on other's rights (rights based justice). The study will mainly be financed through ZonMW and therefore no conflict distribution of scarce funds (distributive justice). Inclusion in the study respects all morally acceptable laws (legal justice). Aan het onderzoek deelnemende proefpersonen zullen schriftelijk worden ingelicht over deze verzekering.

Compensation for injury
Elke aan het onderzoek participerende instelling draagt zorg voor de verzekering van de in de eigen instelling te includeren proefpersonen.

Handling and storage of data and documents
Data will be handled confidentially and anonymously. To be able to trace data to an individual subject, a subject identification code list will be used. Participating subjects will be registered by a 5-digit number that will be used to link the data to the subject. This personal code will be on all forms retrieved from participants. The key to the code is safeguarded by the investigator. The handling of personal data will comply with the Dutch Personal Data Protection Act (in Dutch: De Wet Bescherming Persoonsgegevens, Wbp).
Serum will be stored for 5 years at minus 80 degrees anonymously and coded as mentioned above.

Amendments
Amendments are changes made to the research after a favourable opinion by the accredited METC has been given. All amendments will be notified to the METC that gave a favourable opinion.

Annual progress report
The sponsor/investigator will submit a summary of the progress of the trial to the accredited METC once a year. Information will be provided on the date of inclusion of the first subject, numbers of subjects included and numbers of subjects that have completed the trial, serious adverse events/ serious adverse reactions, other problems, and amendments.

End of study report
The investigator will notify the accredited METC of the end of the study within a period of 8 weeks. The end of the study is defined as the last patient's last visit.
In case the study is ended prematurely, the investigator will notify the accredited METC, including the reasons for the premature termination.
Within one year after the end of the study, the investigator/sponsor will submit a final study report with the results of the study, including any publications/abstracts of the study, to the accredited METC. Subfertility affects approximately one in ten couples planning conception. Among subfertile women, about 30% are overweight or even obese. Epidemiological data suggest that the reduction of overweight will increase the chances of conception, decrease pregnancy complications and improve perinatal outcome. In small intervention studies beneficial reproductive effects of improvement of lifestyle leading to a reduction in body weight have been demonstrated. The British Fertility Society advises that fertility treatment be withheld if the body mass index (BMI) is over 35.

REFERENCES
In the Netherlands, there is at present no agreed standard of care for subfertile women with overweight or obesity. Some centres simply withhold treatment of couples in whom the woman is overweight or obese, but most fertility centres treat overweight or obese women irrespective of their BMI. In a few centres, support is offered to women to help them lose weight.
Objective: In view of this lack of evidence and strong practice variation we propose a randomized clinical trial in overweight and obese subfertile women, in which we compare the costs and effects of a six months structured lifestyle program, aimed at weight loss, to "usual care". The intervention aims to prevent unnecessary fertility treatment, complications associated with fertility treatment and obesity related pregnancy complications, thus improving pregnancy chances and perinatal outcome.

Study design:
Multicenter randomised clinical trial. Study population: Subfertile women (age 18-39) with a BMI of 29 kg/m² or above. Exclusion criteria are: azoospermia or donor semen, severe endometriosis, chronic anovulation due to WHO III and endocrinopathies. Women with an untreated preexistent hypertension or diabetes type 1, or pregnancy induced hypertension, preeclampsia, eclampsia or HELLP syndrome in a previous pregnancy are also excluded.

Intervention:
The intervention consists of a structured lifestyle program of six months, which aims at realistic weight loss of at least 5% to10%, achieved by the combination of a healthy diet (caloric reduction of 600kCal/day), increase of physical activity (aiming at 10.000 steps per day) and two to three times a week sporting activity, and behavioural modification. The structured lifestyle program, in which practice variation is minimized using a structured software program, has been developed, used and evaluated previously (Zon-MW project 50-50110-98-078; the Groninger Overweight And Lifestyle study (GOAL)). Following six months of lifestyle intervention patients will start fertility treatment as indicated in the "usual care" group.
In the "usual care" arm fertility treatment will be started if this is justified by the individual prognosis (guideline NVOG) All randomised women will fill in questionnaires concerning: diet, eating behaviour, physical activity and smoking: SF-36, FFQ, DEBQ, Squash list, at inclusion, and at 12 weeks, 6 months, 12 months and 24 months after randomisation. Blood samples (10 cc) will be taken at the start, 3 months and 6 months after the start of the study. Cord blood will be taken from approximately 150 patients after childbirth to assess the epigenetic and metabolic profile.
The data will also be used in the follow-up study in case women give informed consent to participate in the follow up study of her child: "the preconception weight loss; does it affect infant health study" (METc: 2011/134), (the so-called "Lifestyle-kids" study).

Main study parameters/endpoints:
Primary endpoint is the birth of a healthy singleton after vaginal delivery of at least 37 weeks gestation. Secondary outcome parameters are number of fertility treatments (OI, IUI, IVF, ICSI), quality and amount of oocytes and embryos and cryo embryos derived in an IVF or ICSI procedure, the amount of embryos per transfer, implantation rate of transferred embryos, complications of fertility treatment, clinical and ongoing pregnancy rates, perinatal outcome, complications and quality of life as well as body weight, waist circumference, behaviour influencing weight, i.e. nutritional habits and exercise pattern, smoking habits, blood pressure, glucose/insulin ratio (HOMA), hormonal profile and costs.
DATA ANALYSIS: Analysis will be by intention to treat. Randomisation will be stratified for the presence or absence of anovulation and per treatment center. We expect an improvement in vaginal singleton delivery beyond 37 weeks from 45% to 60%. Anticipating drop out of 20% in the intervention arm ad a 5% loss to follow up in the study, we need to include two groups of 285 women (two-sided test, alpha error.05, beta-error .20).

STUDY DURATION:
Preparation of the study, including the training of the nurses, will take three months. Inclusion of the couples will take 24 months and the lifestyle intervention will take six months. Follow up is continued for 24 months after randomisation. Database cleaning and analysis of the study will take 6 months. Duration of the study: 57 months Nature and extent of the burden and risks associated with participation, benefit and group relatedness: The advantages of a weight loss and exercise program in overweight or obese subfertile women have been shown in small intervention studies. Even if the patient does not conceive during the 6 months period in the lifestyle intervention arm, the health benefits of weight loss on the long-term are clearly in the interest of the patient. By starting this project, fertility treatment in the intervention arm will be delayed for a maximum of 6 months, there is no evidence that this delay will cause lower spontaneous or treatment dependent pregnancy chances. A more structured weight loss and exercise program will probably lead to higher spontaneous and therapy induced ongoing pregnancy chances.
The Burden: Patients that are randomized in the intervention arm will attend the clinic for six extra visits in order to improve lifestyle, this will take around 280 minutes. In between visits counselling will take place per telephone or mail four times for 15 minutes. During these visits physical examination, measuring weight, WC and WHR in week: one, three, seven, twelve, eighteen and twenty-four will take place. Patients in the intervention group will have to adapt there lifestyle towards a more healthy lifestyle, this will give a certain amount of stress since they will have to adapt their diet and physical activity. Patients will be learned how to use stepcounter PAM and wear it on a daily basis in order to monitor their physical activity during the intervention period and will fill in their activity list.
Patients who have been randomized in the "usual care" group will undergo fertility treatment when indicated by national guidelines (www.nvog.nl). Patients with overweight or obesity have additional risks during pregnancy compared to normal weight women. The risks involve: hypertension (OR: 2.2), preeclampsia (OR: 1.8-2.7), pregnancy-induced diabetes ( OR: 2.2-3.4), caesarian section (OR: 1.3-2.4) (see tables of evidence in appendix). These risks are not influenced by fertility treatment. Lifestyle intervention aims to reduce the involved pregnancy risks. An independent Safety Monitoring Board (SMB) will be installed to review major complications in both treatment groups. This board will evaluate complications after every 150 included patients and six months and 12 months after the end of inclusion of the study (6 times during the study). The SMB will differentiate between major en minor complications, blinded for treatment arm. Every case of preeclampsia, eclampsia, HELLP syndrome, maternal or infant mortality will be reported to the SMB. The SMB findings will be reported to the Ethics Committee of the UMCG Questionnaires to be filled in by all participants in the study: SF-36, SQUASH-list, FFQ, DEBQ, concerning eating behaviour at the start, week twelve, twenty-four and fifty-two of the study. A questionnaire for details on direct and indirect costs like transportation, sporting activity and productivity loss due to lifestyle intervention programme and fertility treatment will be filled in by the participants during the study. This will take 30-45 minutes.

INTRODUCTION AND RATIONALE
The evidence for the adverse effects of overweight on women's reproductive health is indisputable. Overweight affects reproductive capacity in all subfertile couples. Moreover, overweight is associated with pregnancy complications and poor neonatal outcome. Pregnancy complications associated with obesity are hypertensive disorders, gestational diabetes, prolonged duration of labour, increased need of operative delivery, macrosomia, shoulder dystocia and increased blood loss (Garbaciak et al.,1985;Edwards et al.,1996). Obesity is associated with an increased risk of adverse pregnancy outcomes such as unexplained still birth Kristensen et al.,2005;Bergstrom et al.,1998;Linne et al.,2004, neonatal admissions (Usha Kiran et al.,2005 and five times higher costs (Linne et al.,2004). Obesity has further been associated with reduced fertility in the general population (Ramlau-Hansen et al.,2007). Ovulatory subfertile women with a BMI of 29 kg/m2 or higher have a 4% lower pregnancy rate per kg/m 2 increase (Van der . Obese women also have a lower live birth rate after IVF and ICSI Wang et al.,2000;Wang et al.,2002;Fedorcsak et al.,2004, Lintsen et al.,2005. The relationship between BMI and the risk of spontaneous abortion is inconsistent (Lashen et al.,1999;Fedorczak et al.,2000;Wittemer et al.,2000;Wang et al.,2001, Lintsen et al.,2005. A meta-analysis on the effect of overweight and obesity on ART reported a lower chance of pregnancy following IVF (OR 0.71, 95% CI:0.620-0.81) and an increased miscarriage rate (OR 1.3, 95% CI:1.06-1.68). Extrapolating from available data one could argue that the combination of lower chance of pregnancy and higher miscarriage rate in overweight and obese women could result in a reduced live birth rate (Maheshwari et al.,2007).
Obesity is strongly related to polycystic ovary syndrome (PCOS), which is the cause of subfertility in 25% couples. The accompanying insulin resistance and hyperinsulinism mark this syndrome as a prediabetic state (Dunaif, 1997), leading to an increase of neonatal complications.
In reproductive medicine, modest weight reduction (5-10%) with lifestyle intervention is not only associated with restoration of ovulation and fertility in overweight women with PCOS (Hoeger et al.,2004) but is also shown to decrease insulin resistance (Pasquali et al.,1989;Knowles et al.2002). Several studies have shown that even a modest weight loss leading to a 5% loss of central fat, in women with PCOS improves, the menstrual cycle, the rate of ovulation, and the likelihood of a healthy pregnancy (Clark et al.,1995, Norman et al.,2004, Huber-Buchholz et al,.1999, Stamets et al., 2004. In view of the evidence that overweight has a negative impact on fertility and pregnancy outcome, lifestyle intervention prior to treatment of subfertile couples could improve spontaneous fertility chances, and prevent unnecessary fertility treatment as well as complications of fertility treatment. Several reviews showed that lifestyle interventions targeted at changing diet and physical activity can succeed in sustained decrease of average bodyweight on the longer term (McTigue, 2004;Franz 2007). A decrease of 3 to 4 kilograms can be achieved after three years of follow-up (Avenell, 2004). The adverse health effects of increased body weight on for instance development of diabetes mellitus type 2, coronary heart disease, musculoskeletal disorders and some types of cancer are well known (WHO-euro document 2007). The cost-effectiveness of lifestyle interventions was below acceptable limits . For the Diabetes prevention project, for instance, the costs to prevent one new case of diabetes mellitus were calculated to be around 16.000 US dollars (Herman, 2005).
The intervention we propose to evaluate is based on dietary therapy, increased physical activity and individualized behavioural modification plan. A combination of these three interventions leads to maximal weight loss and maintenance of weight loss (NIH guideline, CBO guideline 2007, Zelissen et al 2004). This behavioural modification therapy can be delegated to a nurse practitioner or trained nurse or dietician. Such an intervention program can be implemented within gynaecological care.
Although lifestyle modification is advised as a key component for the improvement of reproductive function in overweight women specifically with PCOS (NICE 2004Tang et al.,2006, Pasquali et al.,1989, Hoeger et al.,2004, Clark et al.,1995, the evidence of its effectiveness as demonstrated in clinical studies is limited. It's costeffectiveness has never been assessed in large groups of subfertile women with respect to prevention of unnecessary fertility treatment, prevention of complications of fertility treatment and improvement of perinatal outcome. The cost-effectiveness of such program will be investigated in the this study. (see table Summary of studies assessing outcome after lifestyle intervention).

OBJECTIVES
In view of the lack of convincing evidence from large intervention studies and the strong practice variation in The Netherlands we propose a randomized clinical trial in overweight and obese subfertile women, in which we compare the costs and effects of a six months structured lifestyle program, aimed at weight loss, to "usual care" with the aim to prevent obesity related pregnancy complications, unnecessary fertility treatment and complications associated with fertility treatment, enhance pregnancy chances and improve perinatal outcome. The control group of women will receive fertility treatment "care as usual" without lifestyle intervention.
We aim to estimate the costs and effects of a structured lifestyle program in terms of costs per additional healthy singleton birth rate beyond 37 weeks gestation born after vaginal delivery in a follow up period of 24 months.
The questions that we aim to answer are: Primary Objective: Does a structured lifestyle program aiming at a weight reduction of 5%-10% reduce the need for fertility treatments due to an increase in spontaneous conception rate and treatment dependent or treatment independent life birth rate?

Secondary Objective(s):
Does a structured lifestyle program aiming at a weight reduction of 5%-10% increases the success rates of fertility treatments, including assisted reproductive technology?
Does a structured lifestyle program aiming at a weight reduction of 5%-10%, lead to less maternal complications during fertility treatment or pregnancy, such as hypertensive disorders, premature birth, macrosomia, shoulder dystocia, operative delivery, and haemorraghia, and multiple pregancies in subfertile women with overweight or obesity?
Does a structured lifestyle program aiming at a weight reduction of 5%-10% increases the quality and number of oocytes and embryos and cryo embryos resulting from an IVF or ICSI procedure and increases the amount of embryos per transfer and the implantation rate of transferred embryos ?
Does a structured lifestyle program aiming at a weight reduction of 5%-10% lead to a lower percentage of women with overweight or obesity and subfertility developing gestational diabetes?
Does a structured lifestyle program aiming at a weight reduction of 5%-10% in subfertile women, lead to a better quality of life, as well as body weight reduction, reduction of waist

STUDY DESIGN
Multicenter randomised clinical trial. Flow chart 1 gives an overview of the study procedure form randomization till end of follow up period. Table 1 is added to give an overview of the lifestyle intervention that subjects will undergo in the course of research.

STUDY MANAGEMENT:
The randomisation is performed by computer at a central randomisation centre in the AMC. Randomisation will be stratified according presence or absence of anovulation and center of treatment. Data will be collected using Oracle Clinical Remote Data Capture (RDC), which is a new generation of application system that enables collection and cleanup of clinical trial data using the Internet. For detailed information on Oracle RDC, please visit the page of Oracle RDC products (http://www.ctc-g.co.jp/en). The expertise for this technology is already used in the study group.

Population (base)
All couples suffering from subfertility visiting the clinics participating in the study will undergo a basic fertility work-up including a semen-analysis, an assessment of tubal patency using CAT or HSG, and monitoring of the cycle to assess ovulation. After the work-up has been completed, a fertility diagnosis will be made and a prognosis for treatment independent pregnancy will be calculated (Hunault et al.,2004), followed by a management proposal for the individual couple. Ovulatory and anovulatory women with a body mass index between 29 kg/m² and 40 kg/m² will be approached to participate in the present study. After informed consent, they will be randomly allocated to an intervention program to improve lifestyle with a duration of six months, or to "usual care".

Inclusion criteria
We aim to include subfertile women with a BMI of 29 kg/m² or above. Women have to be between 18 and 39 years old. Subfertility is defined as failure to conceive within 12 months of unprotected intercourse in couples( where the woman has an ovulatory cycle), as well as couples where the women suffers from chronic anovulation due to WHO I or II or PCO.

Exclusion criteria
Couples suffering from azoospermia or donor semen, severe endometriosis AFS 3 and 4 (Revised American Fertilty Society classification), chronic anovulation due to WHO III, endocrinopathies (like: cushing syndrome, adrenal hyperplasia, hypothyroidism, diabetes mellitus type I) will not be eligible for the study. Women with an untreated preexistent Protocol ZonMW 50-50110-96-518 LIFEstyle Vs 5. 25-9-2013 hypertension or diabetes, or pregnancy induced hypertension, preeclampsia, eclampsia or HELLP syndrome in a previous pregnancy are also excluded. Incapacitated adults will be not asked to participate in the study.
Women eligible for the study will be referred to a research nurse. This person is dedicated to counselling eligible patients and will perform randomisation and collect follow-up data, but will not perform the experimental intervention. The lifestyle intervention programme will be done by trained intervention nurses. We will collaborate with the research nurses who are involved in the obstetric and urogynaecological consortium. In this consortium, more than 20 large randomised clinical trials are performed. Information on this infrastructure is obtainable from http://www.studies-obsgyn.nl/

Sample size calculation
SAMPLE SIZE: On the basis of the literature the cumulative live birth rate in a follow up of two years of subfertility treatment will be 45% for the "usual care" group (Dutch OFO-study, unpublished data). The lifestyle intervention group is estimated to have a life birth rate of 60% (Dutch OFO-study, unpublished data) We expect an improvement of healthy singleton of more than 37 week gestation born after vaginal delivery from 45% to 60% in the intervention group. Drop out is expected to be 20% in the intervention arm and is included in the analysis of effectiveness.
To prove that, we need to include two groups of 272 women to be included (alpha 0.05, power 80%). To account for 5% lost to follow up 285 women will be included per group. In total 570 women will be included.

Investigational treatment
THE "USUAL CARE" ARM: In the "usual care" arm treatment will be started if the individual prognosis, based on the Hunault model (Hunault et al.2004) is less than 30% chance of conception within one year or when more than 3 years subfertility (guideline NVOG) justifies starting treatment. Even so, in case of chronic anovulation, ovulation induction will be started. Fertility treatment can either be IUI, IVF or ICSI whatever is indicated according to the Dutch guideline on IVF treatment (NVOG). In anovulatory patients, ovulation induction will be started using clomiphene, or gonadotrofines, which is applicable. When the Hunault model shows a prognosis of more than 30% pregnancy chance in the forthcoming year (and patients are less than three years subfertile), expectant management will be offered.
THE EXPERIMENTAL ARM: The overweight intervention is based on the lifestyle module which is developed in ZONMW project (50-50110-98-078) and will be adapted for the study proposal in this specfic group of subfertile woman for a duration of six months. The software module developed, used and evaluated in this program will be adapted for this subfertile patient group. The use of the software program will reduce practice variation between fertility centres. The lifestyle intervention is based on the recommendations of Dutch CBO guideline "Diagnostiek en behandeling van obesitas bij volwassenen en kinderen" and consists of a structured lifestyle program targeted at: 1. Changing the dietary pattern. 2. Stimulating physical activity of moderate intensity. 3. Changing existing behaviour. 4. Self-monitoring. 5. Involvement of the partner. 6. Referral to psychologist when indicated. ad 1. CHANGING DIETARY PATTERN Women will be advised to adapt their dietary pattern and sustain a healthy diet with a caloric reduction of approximately 600kcal compared to their normal caloric intake (but not below 1200kcal/day). A realistic target weight will be set of 5-10% below their body weight. The diet should be based on the Dutch guideline "Gezonde voeding" in order to achieve a sustainable diet change.
ad 2. STIMULATING PHYSICAL ACTIVITY Physical activity is necessary in order to sustain a modest weight loss and increase effect of dietary measures. Patients will use a stepcounter (PAM) to measure daily steps aiming for a daily goal of 10.000 steps. Two to three times a week sporting of at least 30 minutes of moderate intensity (60-85% of maximum hearth frequency) is advised. Sporting advice is individualized taking into account a woman's preference and possibilities (swimming, walking, stepping, cycling, cardio-fitness, etc.). Patients keep a diary to not the result of the stepcounter, and the sporting activities. These results are evaluated by the trained nurses. ad 3. CHANGING EXISTING BEHAVIOUR The motivation to change lifestyle is measured and evaluated during the program with the PACE score measuring stages of change (precontemplative stage, considering -preparatory stage, action stage and maintenance stage). This PACE score is part of the software module "GOAL". Motivational counselling is individualized for these stages of change. Changing existing behaviour is achieved by motivational counselling directed at: -Awareness of actual lifestyle leading to overweight or obesity.
-Counselling healthy lifestyle measures, the effect of healthy lifestyle in relation to subfertility and spontaneous and treatment dependent pregnancy chances as well as pregnancy complications and influence on perinatal outcome.
-Individualized goals are formulated and embedded in a "patient contract". "Patient contracting" is a tool to actually individualize the measures that the patient will take and to stick to the goals and targets that are agreed upon. During the intervention individual goals will be evaluated, feedback will be given and goals adapted when necessary. Continuous evaluation and feedback will be maintained focussed on the target bodyweight and long term maintenance of lifestyle changes. ad 4. SELFMONITORING Self-monitoring is an essential tool to improve compliance during a lifestyle program. It will be implemented using a PAM "stepcounter" to measure daily physical activity and the "eetmeter" (www.voedingscentrum.nl). This webbased tool is used to give feedback on food intake, and caloric intake on a daily basis. Patients will be trained to use this device. A diary with sporting activities as well as caloric intake will be evaluated by the dedicated nurses.
ad 5. INVOLVEMENT OF THE PARTNER Involvement of the partner and family of the women is important in order to guarantee social support and a "buddy system" during lifestyle changes. ad 6. REFERRAL When indicated, referral to psychologist or intervention available in the general health care system will used.
Patients undergoing the lifestyle program will be guided and supported by trained nurses or dieticians or nurse practitioners, who will be trained prior to the study. Women who miss two consecutive sessions and who are not willing to catch up in another group are excluded from the program.
The standardized lifestyle program consists of four sessions in the first three months, and than a booster of two sessions in the last three months. Telephone consultation or consultation by mail is scheduled in between these sessions four times. (see table 1) FOLLOW-UP: All participating couples (usual care arm and the experimental care arm) will complete several questionnaires, i.e. the SF-36: measuring satifaction, the SQUASH list: for physical activity, the FFQ: food frequency questionnaire for assessing nutrient intake): a structured questionnaire about food pattern (e.g. breakfast yes/no) and important food components (e.g. snacks, fruits and vegetables and meat), the Dutch Eating Behaviour Questionnaire (DEBQ): for assessment of restrained, emotional and external eating behaviour (van Strien 1986). Finally, several questions are completed i.t.o. history of body weight, e.g. the duration of overweight, weight fluctuations. Questionnaires are webbased. In case a patient does not use the computer paper questionnaires will be used. Questionnaires will be completed at start, 12 weeks, 24 weeks and 52 weeks after randomisation. In addition each woman will receive a questionnaire for details on associated direct costs of professional care and on indirect costs like transportation, sporting activity and productivity loss. These questionnaires will be handed out in consultation 1, week 1 of the lifestyle module or at the start of fertility treatment for Protocol ZonMW 50-50110-96-518 LIFEstyle Vs 5. 25-9-2013 Version 5: date 25-9-2013 20 of 43 women that were randomised for "usual care". This will be repeated at the time points of the other questionnaires (12, 24 and 52 weeks after randomisation and at the end of follow-up).
Finally, patients who get pregnant during the period up to 24 months after randomisation will receive a questionnaire in which they can register their (self-reported) weight-gain during pregnancy. To assess gestational diabetes, an oral glucose tolerance test is advised at 28-30 weeks of gestation. Cord blood will be taken from approximately 150 patients after childbirth to assess the epigenetic and metabolic profile. The data will also be used in the follow-up study in case women give informed consent to participate in the follow up study of her child: "the preconception weight loss; does it affect infant health study" (METc: 2011/134), (the so-called "Lifestyle-kids" study).
Moreover, we will register reproductive outcome, fertility treatments as well as the course and outcome of subsequent pregnancies, including obstetrical interventions for a period up to 24 months after randomisation.

Study parameters/endpoints
Background and Demographic Characteristics To assess whether the treatment groups were balanced, the study populations will be compared for baseline measurements including female age, education, ethnicity, type of infertility (primary/secondary), duration of infertility, intoxications, body mass index, as well as sperm analysis according to WHO standards.

Main study parameter/endpoint
The primary endpoint will be a healthy singleton of more than 37 week gestation born after vaginal delivery. The initial analysis, will be done by intention to treat, so we will not make a difference for treatment independent pregnancies and pregnancies that occurred after treatment. Patients who will drop out of the study will by analysed according to their group of randomisation. In a per protocol analysis drop outs will be asked to provide information on the primary outcome, i.e. life birth within two years after randomisation. These drop outs will be analysed as non-compliant.

Secondary study parameters/endpoints
Secondary outcome parameters are: 1. pregnancy outcome and -complications: miscarriage, gestational diabetes, Pregnancy induced hypertension, preeclampsia, HELLP, prematurity<37 weeks, macrosomia (>p90 (Large for Gestational Age), induction of labour, prolonged duration of labour, operative delivery, assisted delivery and increased blood loss, and multiple pregnancies 2. percentage of women needing fertility treatment in both groups (OI, IUI, IVF, ICSI) and ongoing pregnancy rates in these treatments. 3. Quality and number of oocytes and embryos and cryo embryos resulting from an IVF or ICSI procedure and the amount of embryos per transfer and implantation rate of transferred embryos 4. complications during fertility treatment: ovarian hyperstimulation syndrome, infection, tubal pregnancies, torsion (etc). 5. perinatal outcome: stillbirth, pregnancy duration, body weight, Apgar scores, arterial pH, congenital anomalies and neonatal admission to a neonatal medium, high or intensive care unit..
6. quality of life, pre-pregnancy body weight, weight gain during pregnancy, waist circumference, behaviour influencing weight, i.e. nutritional habits and exercise pattern, blood pressure, glucose/insulin ratio (HOMA), hormonal profile (androgens, adipokines and cytokines) and costs.

Other study parameters ECONOMIC EVALUATION:
The aim of the economic evaluation is to compare the costs and health effects of a lifestyle program versus "usual care". The analysis will be a costeffectiveness analysis with the proportion of healthy singleton of more than 37 week gestation born after vaginal delivery as the primary outcome.

Randomisation, blinding and treatment allocation
The randomisation is performed by computer at a central randomisation centre in the AMC. Randomisation will be stratified according to presence or absence of anovulation and center of treatment. Women eligible for the study will de referred to a research nurse. This person is dedicated to counselling eligible patients and will perform randomisation and collect follow-up data, but will not perform the experimental intervention. We will collaborate with the research nurses who are involved in the obstetric and urogynaecological consortium. In this consortium, eight large randomised clinical trials are performed. Information on this infrastructure is obtainable from http://www.studies-obsgyn.nl/

Study procedures
RANDOMISATION BY RESEARCH NURSE Baseline measurement of body weight, length, waist, hip and blood pressure for all patients. A blood sample of 10 cc will be taken and serum stored of all patients.
Individualized advice for improvement when necessary and adjust agreements when necessary to reach realistic targets. Discuss relapse.
Evaluating the barriers to follow the program when necessary. Referral to a dietician or psychologist is done in a standardized way, when appropriate. Measuring waist circumference and weight. A blood sample of 10 cc will be taken and serum stored.
CONSULTATIONS VIA TELEPHONE OR MAIL WEEK 5,8,15,AND 21. In between the consultations in the outpatient fertility clinic, consultation via telephone or mail are scheduled at week 5, week, 9, week 15, and week 21 with a duration of 15 minutes. In these consultations evaluation regarding: motivation of weight loss and physical activity are discussed Evaluating results regarding: weight, food diary, eetmeter, physical activity, stepcounter. Feedback on results. Individualized advise for improvement when necessary and adjust agreements when necessary to reach realistic targets. Discuss relapse.
Evaluating the barriers to follow the program when necessary.
Questionnaires for all included women: SF36, FFQ and Squash-list will be evaluated at the start and week 12 and week 24 and 52 weeks after randomisation.
Start of fertility treatment When in spontaneous ovulatory patients in the lifestyle intervention arm: • have finished their six month lifestyle program or, • when they meet their target weight reduction of 5-10% or, • when their weight decreases < BMI 29 kg/m², fertility treatment will be started if the individual prognosis, based on the Hunault model (Hunault et al.2004) if less than 30% chance of conception within one year or when more than 3 years subfertility (guideline NVOG) justifies starting treatment (either IUI, IVF or ICSI (according to national guidelines NVOG).
In case of chronic anovulation, ovulation induction will be started when patients in the lifestyle intervention arm: • have finished their six month lifestyle program or, • when they meet their target weight reduction of 5-10% or, • when their weight decreases < BMI 29 kg/m².

Withdrawal of individual subjects
Subjects can leave the study at any time for any reason if they wish to do so without any consequences. Patients who decide to withdraw from the study will be asked by the responsible investigator to provide information regarding the primary endpoint of the study 24 months after randomisation. Patients who drop out of the study will be treated according to the local protocols for infertility patients. The investigator can decide to withdraw a subject from the study for medical reasons. Women who miss two consecutive counselling sessions and who are not willing to catch up are excluded from the program.

Section 10 WMO event
In accordance to section 10, subsection 1, of the WMO, the investigator will inform the subjects and the reviewing accredited METC if anything occurs, on the basis of which it appears that the disadvantages of participation may be significantly greater than was foreseen in the research proposal. The study will be suspended pending further review by the accredited METC, except insofar as suspension would jeopardise the subjects' health.
The investigator will take care that all subjects are kept informed.

Adverse and serious adverse events
Adverse and serious adverse events as a direct result of the lifestyle intervention are not anticipated in this study. However, patients with overweight or obesity have additional risks during pregnancy compared to normal weight women. As a result of both the intervention arm and of the "care as usual" arm, pregnancies may occur in patients with increased risks. These risks are not influenced by fertility treatment. Lifestyle intervention aims to reduce the involved pregnancy risks. Participants in the study have been informed about these risks. An independent Safety Monitoring Board (SMB) will be installed to review major complications in both treatment groups. This board will evaluate complications after every 150 included patients and six months and 12 months after the end of inclusion of the study (6 times during the study). The SMB will differentiate between major en minor complications, blinded for treatment arm. Every case of preeclampsia, eclampsia, HELLP syndrome, will be reported to the SMB. The SMB findings will be reported to the Ethics Committee of the UMCG Adverse events are defined as any undesirable experience occurring to a subject during a clinical trial, whether or not considered related to the investigation. All adverse events reported spontaneously by the subject or observed by the investigator or his staff will be recorded.
A serious adverse event is any untoward medical occurrence or effect that at any dose results in death; is life threatening (at the time of the event); requires hospitalisation or prolongation of existing inpatients' hospitalisation; results in persistent or significant disability or incapacity; is a congenital anomaly or birth defect; is a new event of the trial likely to affect the safety of the subjects, such as an unexpected outcome of an adverse reaction, lack of efficacy of an IMP used for the treatment of a life threatening disease, major safety finding from a newly completed animal study, etc.
All SAEs will be reported to the accredited METC that approved the protocol, according to the requirements of that METC.

Follow-up of adverse events
All adverse events will be followed until they have abated, or until a stable situation has been reached. Depending on the event, follow up may require additional tests or medical procedures as indicated, and/or referral to the general physician or a medical specialist.

Descriptive statistics
To asses whether the groups were balanced the study population will be compared for baseline measurements including female age, type of subfertility (primary or secondary), duration of subfertility, intoxications, smoking, BMI, as well as sperm analysis according to WHO standards, anovulation, and subfertility treatment. Confounding factors, such as smoking, education, ethnicity will be addressed in the analysis.

Univariate analysis and multivariate analysis
The primary analysis will be by intention to treat. The primary outcome will be analysed by comparing the pregnancy rates in both groups using Kaplan-Meier analysis of time to pregnancy using the Log-rank test. In this analysis, patients will be censured at the time they discontinue or complete the study without getting pregnant. In addition, pregnancy rates and 95% confidence intervals per group will be calculated based on the Kaplan-Meier estimates at various time points. Further analysis of delivery rates over time will be performed using Cox-regression analysis with correction for the stratification variables ovulatory status and treatment centers as well as for confounders. These analyses will also be performed for spontaneous and treatment-induced pregnancies separately. The same analysis will be performed for the secondary outcome ongoing pregnancies. This will include ongoing pregnancies, that have not yet resulted in the birth of a child. Exploratory subgroup analyses of the primary outcome will be performed for spontaneous and treatment-induced pregnancies and for women with a BMI below 35 versus above 35, as well as for anovulatory versus ovulatory women and for women with a waist-hip ratio of above 0.8 versus below or equal to 0.8 and for women who are 36 years or older versus younger than 36 years based on tests of interaction with treatment group. Additional analysis concerning the influence of HOMA, androgens, adipokines and cytokines on pregnancy chances and recovery of ovulations in anovulatory patients will be assessed using multivariate Cox-regression. Incidence of complications of treatment and during pregnancy will be compared in both groups using relative risks and 95% confidence intervals.
Quality of life will be analysed using repeated measures analysis of variance A per protocol analysis will also be performed, in which drop out patients will be identified as non-compliant. Information on the primary outcome, i.e.lifebirth within two years after randomisation will be used whenever provided. Patients who drop out of the lifestyle intervention arm will be analysed in the "usual care" arm in this per protocol analysis.
Women who drop out of the study will be asked to provide the reason for dropping out. This reason will be recorded and patients will be asked to provide information regarding the primary outcome, i.e. pregnancy within two years after randomisation. In addition, patients will be offered the possibility to continue to complete cost questionnaires for the remainder of the study duration. For patients dropping out of the lifestyle intervention, further course will be determined by the responsible physician.

Economic evaluation
The aim of the economic evaluation is to compare the costs and health effects of a lifestyle program versus "usual care". The analysis will be a cost-effectiveness analysis with the proportion of healthy singleton of more than 37 week gestation born after vaginal delivery as the primary outcome.
The economic evaluation will be performed from a societal perspective. Direct medical and non-medical costs (intervention costs, time and travel costs) as well as indirect non-medical costs (productivity losses) will be taken into account.
We will build on the work of the ZonMW funded "Paraplu" study, in which cost data data of six cost-effectiveness studies were integrated. Dr H. Groen played an important role in this process.
The time horizon will be from randomisation to the end of follow-up. Resource use will be recorded as individual patient data in the CRF. In addition each woman will receive a questionnaire for details on associated direct costs of professional care and on indirect costs like transportation, sporting activity and productivity loss. These questionnaires will be handed out in consultation 2, week 3 of the lifestyle module or at the start of fertility treatment for women that were randomised for "usual care". This will be repeated at the time points of the other questionnaires (12, 24 and 52 weeks after randomisation and at the end of followup). Resource use will be valued according to Dutch guidelines. Intervention costs will determined based on actual resource use obtained from the centers. Costs will reflect the following resources: staff, materials, equipment, housing, overhead. Productivity loss will be valued by the friction cost method according to Dutch guidelines (Oostenbrink et al. 2004). Outcome of pregnancy will be recorded and valued using tariffs or data from the literature (Lukassen et al, 2004).
Detailed information on maternal complication will be obtained from obstetricians treating the woman concerned using patient medical files. Six weeks after the expected day of delivery all women will be contacted by telephone to ask information on the delivery and on the health of the child. If the child has been hospitalised the paediatrician treating the child will be contacted for further information.
Scenario analysis will be performed to model cost-effectiveness beyond the time horizon of the study. For longer term analyses, costs and effects will be discounted at commonly accepted rates. Sensitivity analysis will be performed on costs, pregnancy rates of the two groups and the valuation of different outcomes (no child, handicapped child, twin, healthy child, obstetric complications). Uncertainty surrounding cost-effectiveness estimates will be explored by bootstrapping. Cost will be presented in euros.

Regulation statement
The study will be conducted according to the principles of the Declaration of Helsinki (

Recruitment and consent
Women eligible for the study will de referred to a research nurse by their treating doctors. This person is dedicated to counselling eligible patients, randomisation and follow-up for data, but not to the experimental intervention. We will collaborate with the research nurses who are involved in the obstetric and urogynaecological consortium. In this consortium, eight large randomised clinical trials are performed. Information on this infrastructure is obtainable from http://www.studies-obsgyn.nl/ The research nurse must explain to each subject the nature of this study, its purpose, procedures, expected duration and the potential risks and benefits involved in study participation along with any discomfort it may entail. Each subject must be informed that participation in the study is voluntary and that withdrawal of consent will not affect his/her right to the most appropriate medical treatment or affect the doctor relationship. This informed consent will be given by means of a standard written statement. It will be written so as to be easily understood by the subject. The subject will be given the time to read and understand the statement herself before signing her consent and dating the document. The subject will receive a copy of the written statement once signed. The informed consent form must be considered as a part of the protocol with which it is to be submitted by the investigator to the IRB/Ethics committee. Patients will be given a maximum of one week to consider their decision to participate in the study.

Objection by minors or incapacitated subjects (if applicable)
Incapacitated adults will be not asked to participating in the study. Minors are excluded as well.

Benefits and risks assessment, group relatedness
The ethical issues pertaining to this project will be discussed by considering the four ethical principles as discussed by Gillon. (37) Autonomy: After the intake consultation and counseling session by the research nurse, the patient is given the choice to participate in the study. This process of informed consent respects her autonomy.
Beneficence: The advantages of a weight loss and exercise program in overweight or obese subfertile women (in order to improving chances of conception and eventually taking a baby home) have clearly been shown. Even if the patient does not conceive during the 6 months period, the health benefits of weight loss on the long-term are clearly in the interest of the patient.
Non-maleficence: In some reproductive medicine units, overweight or obese subfertile women are counselled and advised to loose weight before treatment is started. By starting this project, fertility treatment in the intervention arm will be delayed for a maximum of 6 months. There is no evidence that this short delay will influence the eventual pregnancy chances. The structured weight loss and exercise program will probably lead to higher spontaneous and therapy induced ongoing pregnancy chances. Before giving informed consent all eligible women are informed about the risks mentioned before (in writing in the patients' brochure). Women who are considered to be at the highest risk (i.e. those who have pre-existent hypertension or diabetes, or pregnancy induced hypertension, (pre)eclampsia or HELLP syndrome in a previous pregnancy) are excluded from participation. During the study adverse events will be monitored by a SMB.
Justice: Introducing the study will not infringe on other's rights (rights based justice). The study will mainly be financed through ZonMW and therefore no conflict distribution of scarce funds (distributive justice). Inclusion in the study respects all morally acceptable laws (legal justice). Aan het onderzoek deelnemende proefpersonen zullen schriftelijk worden ingelicht over deze verzekering.

Handling and storage of data and documents
Data will be handled confidentially and anonymously. To be able to trace data to an individual subject, a subject identification code list will be used. Participating subjects will be registered by a 5-digit number that will be used to link the data to the subject. This personal code will be on all forms retrieved from participants. The key to the code is safeguarded by the investigator. The handling of personal data will comply with the Dutch Personal Data Protection Act (in Dutch: De Wet Bescherming Persoonsgegevens, Wbp).
Serum and cordblood will be stored for 5 years at minus 80 degrees anonymously and coded as mentioned above.

Amendments
Amendments are changes made to the research after a favourable opinion by the accredited METC has been given. All amendments will be notified to the METC that gave a favourable opinion.

Annual progress report
The sponsor/investigator will submit a summary of the progress of the trial to the accredited METC once a year. Information will be provided on the date of inclusion of the first subject, numbers of subjects included and numbers of subjects that have completed the trial, serious adverse events/ serious adverse reactions, other problems, and amendments.

End of study report
The investigator will notify the accredited METC of the end of the study within a period of 8 weeks. The end of the study is defined as the last patient's last visit.
In case the study is ended prematurely, the investigator will notify the accredited METC, including the reasons for the premature termination.
Within one year after the end of the study, the investigator/sponsor will submit a final study report with the results of the study, including any publications/abstracts of the study, to the accredited METC. In the Netherlands, there is at present no agreed standard of care for subfertile women with overweight or obesity. Some centers simply withhold treatment of couples in whom the woman is overweight or obese, but most fertility centers treat overweight or obese women irrespective of their BMI. In a few centers, support is offered to women to help them lose weight.
In view of this lack of evidence and strong practice variation we propose a randomized clinical trial in overweight and obese subfertile women, in which we compare the costs and effects of a six months structured lifestyle program, aimed at weight loss, to "usual care". The intervention aims to prevent unnecessary fertility treatment and prevent complications associated with fertility treatment and obesity-related pregnancy complications, thus improving pregnancy chances and perinatal outcome.

Study design
The study is a multicenter randomized clinical trial. Subfertile women (age 18-39) with a BMI of 29 kg/m² or above will be included. Exclusion criteria are: azoospermia or donor semen, severe endometriosis, chronic anovulation due to WHO III and endocrinopathies. Women with an untreated pre-existent hypertension or diabetes type 1, or pregnancy-induced hypertension, preeclampsia, eclampsia or HELLP syndrome in a previous pregnancy are excluded.
The intervention consists of a structured lifestyle program of six months, which aims at realistic weight loss of at least 5% to 10%, achieved by the combination of a healthy diet (caloric reduction of 600kCal/day), increase of physical activity (aiming at 10.000 steps per day) and two to three times a week sporting activity, and behavioral modification. The structured lifestyle program, in which practice variation is minimized using a structured software program, has been developed, used and evaluated previously (Zon-MW project 50-  (GOAL)). After six months of lifestyle intervention patients will start fertility treatment as indicated.
In the "usual care" arm fertility treatment will be started if this is justified by the individual prognosis (guideline NVOG) All randomized women will fill in questionnaires concerning: diet, eating behavior, physical activity and smoking: SF-36, FFQ, DEBQ, Squash list, at inclusion, and at 12 weeks, 6 months, 12 months and 24 months after randomization. Blood samples (10 cc) will be taken at the start, 3 months and 6 months after the start of the study.

Study flow chart
LIFEstyle study Statistical Analysis Plan Page 8/14

STUDY OBJECTIVES
The objective of the study is to compare the costs and effects of a six months structured lifestyle program, aimed at weight loss, to "usual care" with the aim to prevent obesity-related pregnancy complications, unnecessary fertility treatment and complications associated with fertility treatment, enhance pregnancy chances and improve perinatal outcome. The control group of women will receive fertility treatment "care as usual" without lifestyle intervention.

General considerations
We aim to estimate the costs and effects of a structured lifestyle program in terms of costs per additional healthy singleton birth rate beyond 37 weeks gestation born after vaginal delivery in a follow-up period of 24 months.

Primary efficacy endpoint
The primary endpoint will be a healthy singleton of more than 37 week gestation born after vaginal delivery. The initial analysis will be done by intention-to-treat, so we will not make a difference for treatment-independent pregnancies and pregnancies that occurred after treatment. Patients who drop out of the study will be analyzed according to their group of randomization. In a per-protocol analysis drop-outs from the lifestyle intervention will be analyzed as non-compliant. For the conventional treatment no difference will be made between the per-protocol and the intention-to-treat analysis.

Secondary efficacy endpoints
Secondary outcome parameters are: 1. Pregnancy outcome and -complications: miscarriage, gestational diabetes, Pregnancy induced hypertension, preeclampsia, HELLP, prematurity<37 weeks, macrosomia (>p90 (Large for Gestational Age), induction of labour, prolonged duration of labour, operative delivery, assisted delivery and increased blood loss, and multiple pregnancies 2. Percentage of women needing fertility treatment in both groups (OI, IUI, IVF, ICSI) and ongoing pregnancy rates in these treatments.

Sample size
On the basis of the literature the cumulative live birth rate in a follow-up of two years of subfertility treatment will be 45% for the "usual care" group. The lifestyle intervention group is estimated to have a live birth rate of 60%. We expect an improvement of healthy singleton of more than 37 week gestation born after vaginal delivery from 45% to 60% in the intervention group. 1,2,3 Drop-out is expected to be 20% in the intervention arm 4 and is included in the analysis of effectiveness. To prove that, we need to include two groups of 272 women to be included (alpha 0.05, power 80%). To account for 5% lost to follow up 285 women will be included per group. In total, 570 women will be included.

Level of significance
For all hypothesis testing performed in the Lifestyle study, a p-value of less than 0.05 will be considered to indicate statistical significance.
The randomization is performed by computer at a central randomization center in the AMC.
Randomization will be stratified according to presence or absence of anovulation and center of treatment. Women eligible for the study will be referred to a research nurse. This person is dedicated to counsel eligible patients and will perform randomization and collect follow-up data, but will not perform the experimental intervention. We will collaborate with the research nurses who are involved in the obstetric and urogynaecological consortium. In this consortium large randomized clinical trials are performed. Information on this infrastructure is obtainable from http://www.studies-obsgyn.nl/ The study analysis will be performed on the final data that will become available after the database is formally locked.

Study analysis
The primary analysis will be by intention-to-treat. The dataset for this analysis includes all randomized subjects not violating any of the in-or exclusion criteria, who participated in study activities, and for whom post-baseline data is present. A per-protocol analysis will also be performed, in which drop-out patients will be identified as non-compliant. Information on the primary outcome, i.e. live birth within two years after randomization will be used whenever provided. Patients who drop out of the lifestyle intervention arm will be analyzed in the "usual care" arm in this per protocol analysis.

Efficacy parameters
Intention-to-treat analysis The outcomes with respect to pregnancy will be tabulated for both groups. Counts will be presented and percentages will be calculated in relation to the number of started participants.
The following outcomes will be described: -Vaginal delivery of a healthy singleton >37 weeks of gestation (primary outcome, twin or higher order births counted as failure).
-Live births irrespective of term or health status (multiples counted as 1).
-Ongoing and clinical pregnancies (multiples counted as 1) -Fetal loss: ectopic pregnancy, miscarriage (<16 weeks), still birth (before or after 24  Per-protocol analysis: The outcomes with respect to pregnancy will be tabulated for both groups. Counts will be presented and percentages will be calculated in relation to the number of started participants.
The following outcomes will be described: -Vaginal delivery of a healthy singleton >37 weeks of gestation (primary outcome, twin or higher order births counted as failure).
-Live births irrespective of term or health status (multiples counted as 1).
-Ongoing and clinical pregnancies (multiples counted as 1) -Fetal loss: ectopic pregnancy, miscarriage (<16 weeks), still birth (before or after 24  Differences between groups will be expressed as relative risks and 95% confidence intervals.
The primary analysis will be by intention-to-treat.
The main analysis of the primary outcome will also consist of a Kaplan-Meier analysis of time to pregnancy leading to a vaginal delivery at term for both groups. The Log-rank test will be used to determine statistical significance. In this analysis, patients will be censured at the time they discontinue or complete the study without getting pregnant.
In addition, pregnancy rates and 95% confidence intervals per group will be calculated based on the Kaplan-Meier estimates at various time points. Further analysis of delivery rates over time will be performed using Cox-regression analysis with correction for the stratification variables ovulatory status and treatment centers as well as for confounders. These analyses will also be performed for spontaneous and treatment-induced pregnancies separately. The same analysis will be performed for the secondary outcome ongoing pregnancies. This will include ongoing pregnancies, which have not yet resulted in the birth of a child. The results of these analyses will be presented in a separate manuscript.
Exploratory subgroup analyses of the primary outcome and ongoing pregnancy rates will be performed for spontaneous and treatment-induced pregnancies and for women with a BMI below 35 versus above 35, as well as for anovulatory versus ovulatory women and for women with a waist-hip ratio of above 0.8 versus below or equal to 0.8 and for women who are 36 years or older versus younger than 36 years based on tests of interaction with treatment group. The results of these analyses will be presented in a separate manuscript.
LIFEstyle study

Statistical Analysis Plan
Page 13/14 Additional analysis concerning the influence of HOMA, androgens, adipokines and cytokines on pregnancy chances and recovery of ovulations in anovulatory patients will be assessed using multivariate Cox-regression. The results of these analyses will be presented in a separate manuscript.

Lifestyle program effectiveness
The effects of the lifestyle program will be presented as the changes in body weight, BMI, systolic and diastolic blood pressure, waist circumference and waist/hip ratio for both groups, presented as mean or median with standard deviation or interquartiele range as appropriate from randomization until the occurrence of a pregnancy. In addition, the change in percentage of ovulatory women will be compared between the groups. The results of these analyses will be presented in a separate manuscript.