Adenomyosis and Pregnancy Outcomes in Women Undergoing Assisted Reproductive Technology Treatment

The study investigators propose a prospective observational study to determine the association between adenomyosis and pregnancy loss in women undergoing ART treatment using a consistent classification system for adenomyosis diagnosed on ultrasound scan. This will be supplemented with subgroup analyses such as the following: presence vs absence of adenomyosis symptoms; frozen and fresh embryo transfer cycles; and varying severity of adenomyosis. These subgroup analyses will help determine the influence of these factors on the association between adenomyosis and pregnancy outcomes.

AIMS AND OBJECTIVES

AIM OF THE STUDY: To investigate the association between adenomyosis and pregnancy outcomes in women undergoing ART treatment.

PRIMARY OBJECTIVES: To compare the incidence of pregnancy loss in women with and without adenomyosis undergoing ART treatment. The pregnancy loss will include biochemical pregnancy loss, miscarriage, pregnancy of unknown location (PUL) and ectopic pregnancy.

SECONDARY OBJECTIVES:

• To compare rates of treatment outcomes (implantation rate, biochemical pregnancy rate, clinical pregnancy rate, ongoing pregnancy rate, and live birth rate) in women with and without adenomyosis undergoing ART treatment.
• To investigate whether there is an association between the severity of adenomyosis and pregnancy outcomes

STUDY DESIGN AND SETTING

STUDY DESIGN: Prospective multicentre cohort study SETTING The study will be conducted at all main CARE fertility units, one of the largest providers of fertility treatment, in the United Kingdom.

PARTICIPANT POPULATION

• Population: The study cohort will comprise of women diagnosed with adenomyosis on pre-treatment baseline ultrasound scan before ART treatment who satisfy the eligibility criteria and consent to participate in the study.
• Duration of recruitment: Recruitment will take place over the course of 18 months
• Sample size: The total number of participants required to determine the association between women with adenomyosis and the primary outcome (pregnancy loss) will be 750 with 375 participants per arm. This will ensure that the study is powered to 80%. The estimated sample size for a two-sample proportions was calculated using Stata 17.0. The basis for this calculation stems from a recent meta-analysis which showed a miscarriage rate of 31.33% among women with adenomyosis. To detect a 9.53% increase in miscarriage from the baseline risk of 21.8% in women undergoing ART treatment, 337 clinical pregnancies will be needed (alpha error: 0.05, beta error: 0.2, power 80%). Anticipating a 10% loss to follow up and inconclusive USS diagnosis, total number of participants per arm required will be 375 (374.44). This has been further elaborated in the statistics section of this study protocol.
• Control: To ensure adequate comparability of the cohort, women with a normal uterus on baseline ultrasound scan for the duration of the study will be used as controls and will be retrospectively matched for the following variables: age, embryo quality, type of ART cycle (donor or self and IVF or ICSI) and number of embryos transferred. The eligibility criteria will be applicable to the control group as well.
• Blinding: Women will be screened for adenomyosis during the pre-treatment baseline ultrasound scan (USS) and there will be no formal endeavour to blind the USS operators to the baseline characteristics of the women. All the stored ultrasound images of adenomyotic and normal uteruses will be reviewed by a 2nd clinician who will be blinded to the baseline characteristics of the women to avoid identification and recruitment bias. ART cycle management will be as per standard protocol with no blinding of the treating clinician to the diagnosis of adenomyosis. A pre-specified subgroup analysis for study population with different types of stimulation protocol will be performed. Since the primary outcome measurement will be USS based, there will not be any blinding of the outcome assessors.

DIAGNOSTIC TOOL FOR DETECTION OF EXPOSURE

• Diagnostic tool: The pooled sensitivity and specificity of TVUS for the diagnosis of adenomyosis for all combined imaging characteristics is 83.8% and 63.9% for 2D and 88.9% and 56.0% for 3D, respectively. The diagnosis of adenomyosis in the study participants will be made using transvaginal ultrasound scan; 2D and 3D Ultrasound with a 4-9 MHz trans-vaginal probe on Voluson S8.
• Pre-implementation meeting: Preliminary meetings to discuss the sonographic appearance and diagnostic criteria for adenomyosis will be organised with the clinicians and nurse sonographers participating in the study. An operations manual will be disseminated to the participating centres to assure a uniform and standardised approach is followed for the diagnosis of adenomyosis.
• Timing of the diagnosis: At the time of baseline pre-treatment scan
• Image: Images will be printed or stored as 2D and 3D images. Where possible 2D video clips and 3D volumes will be stored as well
• Reliability assessment: Intra-observer reliability will be assessed using test-retest measure
• Diagnostic criteria: The Morphological Uterus Sonographic Assessment (MUSA) criteria will be used to describe the ultrasound features of adenomyosis. The MUSA criteria outlines the ultrasound features of myometrium and myometrial lesions using standardised terms, definitions and measurements. The MUSA criteria for diagnosis of adenomyosis on ultrasound is based on consensus opinion of a panel of clinicians with expertise in gynaecological ultrasonography, fertility treatment, hysteroscopy, general gynaecology and clinical research. A diagnosis of adenomyosis is made if any morphological features of adenomyosis are present. This stems from data demonstrating a strong correlation between the presence of at least one sonographic feature of adenomyosis with histopathological findings of adenomyosis. All the images stored will be re-reviewed by a 2nd clinician specialising in gynaecological ultrasound. Where the two practitioners do not reach a consensus on the diagnosis of adenomyosis, arbitration will be undertaken by a senior third clinician. If there is no consensus reached, the case will be excluded. The following MUSA criteria would be used for diagnosis of adenomyosis
• Anteroposterior asymmetrical myometrial wall: Ratio of two walls above/below 1 or subjective impression. The anterior and posterior myometrial walls are measured from the external uterine serosa to the external endometrial contour. This should include the junctional zone (JZ) but not the endometrium and be perpendicular to the endometrium
• Myometrial echogenicity: Overall heterogenous myometrium with fan shaped shadowing
• Myometrial or sub-endometrial lesions: Myometrial cysts or Hyperechogenic islands or sub-endometrial echogenic lines/buds or fan shaped shadowing due to lesions
• Junctional Zone (JZ): Thickened or irregular or interrupted or ill-defined
• Vascularity of myometrial lesion: Translesional vascularity. In adenomyoma and focal adenomyotic lesions diffuse minimal vascularity seen as diffuse spread of small vessels within the myometrium. Translesional vascularity: defined as vessels perpendicular to the endometrium crossing the lesion.
• Uterine contour: Globally enlarged uterus. Fundus of the uterus appears enlarged
• Classification of severity: A schematic mapping system of adenomyosis severity will be used for determining the severity of uterine adenomyosis. This has been chosen due to reproducibility, substantial to almost perfect interobserver agreement rate and clinical correlation with symptom severity. A score ranging from to 1 to 4 is attributed to each grade, and the sum of the score numbers is used to calculate the extension of the disease: mild (range, 1-3), moderate (4-6), and severe (>7). The investigators will also classify the extent of affected myometrial as a percentage: Mild (<25%), Moderate (25-50%), Severe (>50%). Stored 2D and 3D USS images of patients diagnosed with adenomyosis on USS will be reviewed by two clinicians to determine the severity score.

CONSENT Written informed voluntary consent will be obtained by the investigator for each potential participant meeting the diagnostic and eligibility criteria. Responsibility for taking consent will be either of a clinician or nurse as delegated by the Principal Investigator. To facilitate the consent process, each participant will be provided with a Participant Information Sheet (PIS). Adequate explanation about the purpose of the study, the exposure and possible benefits of the study will be discussed with each participant. The voluntary nature of the participation will be discussed. Participants will be informed that they may decline participation or withdraw from the study at any time without giving any reason. This will not affect their medical care or legal rights. Time will be given to the participants to read the PIS and to discuss their participation with others outside of the site research team. The participants will have the opportunity to ask questions before signing the latest version of the Informed Consent Form (ICF).

After the participant has signed and dated the consent form, the investigator or delegate will also sign and date the ICF. The consent signing will be done online where possible. There will be triplicates of the ICF; one copy will be given to the participant; a second copy will be filed in the woman's medical notes; and the original will be placed in the Investigator Site File (ISF). The participant will have the opportunity to ask questions about the study throughout the study duration. The record of the details of informed consent discussions will be made in the participant's medical notes. This will include the name of the study, date of discussion, summary of discussion, version number of the PIS given to participant and version number of ICF signed and date consent received.

RECRUITMENT AND ENROLMENT

All women undergoing pelvic pre-treatment ultrasound scans before fertility treatment will be screened for adenomyosis at the participating centres by the clinicians and nurses experienced in performing ultrasound scan. The clinicians and nurses will be performing the baseline scan as per the standard protocol and therefore blinding to the baseline characteristics of the women will not be possible. All the stored ultrasound images of adenomyotic and normal uterus will be reviewed by a 2nd clinician who will be blinded to the baseline characteristics of the women to avoid identification and recruitment bias. Women who meet the eligibility criteria will be provided with a PIS describing the study. They will be enrolled in the study after informed consent is obtained. The demographic, clinical and treatment characteristics which will be recorded and used for data analysis are outlined as below:

• Demographics: Age (in years), Ethnicity, BMI (in Kg/m2), Duration of subfertility, Ovarian reserve (AMH and AFC), Smoking/Alcohol intake
• Clinical history: Cause of subfertility, Associated male subfertility, Primary/Secondary subfertility, Presence/absence of symptoms of abnormal uterine bleeding, gynaecological pain symptoms, dysuria, dyschezia, Previous obstetric history (live birth, miscarriage).
• ART: IVF, ICSI, Previous number of IVF/ICSI cycles
• IVF cycle details: Type of ovarian stimulation protocol, Total Gonadotrophin dose and duration of stimulation, Number of mature follicles >14mm, Number of oocytes retrieved Metaphase 2 oocytes, Embryo quality, Fresh/Frozen embryo transfer, Endometrial preparation, Number of Embryos transferred, Day of embryo transfer, Thickness of endometrium

MANAGEMENT OF IVF/ICSI CYCLE FOR PARTICIPANT POPULATION Women recruited for the study will be offered ART treatment according to a standardised clinical protocol at CARE fertility. The starting dose of gonadotrophins and the type of protocol for stimulation will be based on patient's age, body mass index (BMI), AMH and the number of basal antral follicles. Transvaginal ultrasound and oestradiol measurements will be used to monitor follicular growth, and the doses of gonadotrophin will be adjusted accordingly. Trigger in form of HCG/ GnRH agonist will be given once at least two follicles are observed to be greater than 17 mm in diameter on ultrasound examination and oocyte retrieval will be carried out 36 h after injection of trigger. Embryo transfer will be carried out on day 2, day 3 or day 5 depending on the number of good quality embryo and the number of embryos transferred will be 1 or 2 depending on the age of the patient and the quality of embryo.

Since this study also includes frozen embryo transfer cycles, endometrial preparation will be as per FET protocols according to local practice (natural, stimulated, modified natural with HCG trigger, or hormone replacement therapy [HRT] cycles). All embryo transfers will be performed under transabdominal USS guidance using soft Wallace catheter. This will be followed by luteal phase support with progesterone (vaginal capsules at a dose of 400 mg twice daily or combined vaginal and subcutaneous progesterone at a dose of 25 mg once daily) until 8-12 completed weeks of gestation.

OUTCOME All women will be followed up for primary and secondary outcomes. The CARE Fertility electronic patient database will be used to record the outcome data and collated for subsequent analyses.

DATA HANDLING AND RECORD KEEPING Data will be stored in the CARE Fertility electronic patient record system in accordance with the Data Protection Act 2018. Patient data will be anonymised for the purposes of statistical analysis.

QUALITY CONTROL AND QUALITY ASSURANCE Principles of Good Clinical Practice will be followed throughout the study duration.

ADVERSE EVENT REPORTING Considering the observational nature of the study and no additional step involved in the study design other than baseline USS, there are no safety considerations involved.

STATISTICS

• SAMPLE SIZE The total number of participants required to determine the association between adenomyosis and the primary outcome (miscarriage) would be 750 with 375 participant population per arm. This would ensure our study is powered to 80%. The estimated sample size for a two-sample proportions using two-sided test is calculated using Stata 17.0. The following explanation outlines the basis for the calculation: A recent meta-analysis has shown that the miscarriage rate among women with adenomyosis was 31.33%. The baseline risk of miscarriage in women undergoing ART treatment is 21.8%. To detect a 9.53% difference in incidence of miscarriage, 337 clinical pregnancies will be required per arm (alpha error: 0.05, beta error: 0.2, power 80%). Anticipating a 10% loss to follow up and inconclusive USS diagnosis, total number of participants per arm required will be 375 (374.44).
• OUTCOME MEASURE ANALYSIS The demographic, clinical and ART cycle characteristic comparison between group with adenomyosis and the control group will be completed using descriptive statistics. Categorical variables will be reported as frequencies and percentage and continuous variables as mean and standard deviation for normally distributed data. The Shapiro Wilk test will be used to determine the distribution of the data. Univariate analysis will be performed by using the student's t-test or Wilcoxon test for continuous variables and Chi square test or Fisher's exact test for categorical variables where appropriate.

The cohort group will comprise of women with USS diagnosis of adenomyosis and the control group will comprise of women with normal uterus on USS. The cohort and the control group will be matched for the following variables: age, embryo quality, type of ART cycle (donor or self and IVF or ICSI) and number of embryos transferred. Pregnancy outcomes will be reported as dichotomous variables. Generalised linear models will be used to determine the strength of the association between adenomyosis and pregnancy outcomes and will be reported as point estimates (e.g. risk ratios, odds ratios, risk differences) with 95% confidence interval. Generalised linear models with a binomial distribution with appropriate link functions will be used to calculate the adjusted relative risk and the mean differences, accounting for the following variables: BMI, ovarian reserve, previous IVF cycles, stimulation protocol, endometrial preparation and endometrial thickness prior to transfer. Statistical confidence will be determined by using the p value. Statistical analysis will be conducted using Stata 17.0.

• SUBGROUP ANALYSIS

A subgroup analysis for the primary outcome will also be conducted for women with:

• varying severity of adenomyosis
• presence /absence of symptoms of adenomyosis
• frozen vs fresh embryo transfer
• short vs. long vs. ultralong ovarian stimulation protocol
• recurrent miscarriages: loss of two or more pregnancies prior to 24 weeks of gestation as defined in the ESHRE guideline on recurrent pregnancy loss, 2018.
• other associations that may become apparent in post-hoc analyses. The subgroup characteristics will be described in detail and statistical analysis that has been outlined for the primary outcome will be followed for subgroup analysis
• MISSING DATA AND ANALYSES FOR HANDLING MISSING DATA

The investigators aim to collect the baseline characteristics and follow-up parameters on all the participant and control populations in the best possible way. In first instance for prevention of missing data, the missing data will be flagged up in the data entry stage and investigators will try their best to collect this information from the database and patient follow up. The following methods of analyses will be used to handle missing data:

• Complete case analysis: In this analysis the participants with missing data will be excluded from the primary analysis. This will reduce the sample size; however, the observed data will not be biased. The extent of missing data will be reported in the limitations of the study. Complete case analysis will be used as primary analysis in the following situations: Proportion of the missing data are <5% or >40%; certainty that the missing data is completely at random (MCAR) (In MCAR the mechanism causing missing data may depend neither on observed data nor on the missing data); only the dependent variable data is missing and auxiliary variables are not identified
• Multiple imputation: This will be used as primary method to handle the missing data if the above situations are excluded.

CONFIDENTIALITY AND DATA PROTECTION A unique trial identification number will be used for identification of study participants. All the personal data used in the study will be processed in accordance with the Data protection Act 2018 and will be regarded as strictly confidential.

CONFLICTS OF INTEREST There are no conflicts of interest associated with this study.

AMENDMENTS Any amendments to the study protocol or documentation will be initiated by the Chief Investigator. All substantial amendments will be submitted to IRB for approval followed by implementation of the changes in all sites involved in the study. Amendments will be documented in the 'Protocol Amendment' section of the study protocol.

END OF PROJECT DEFINITION Patient recruitment will occur over the course of 18 months. In order to follow up the patients for the outcome of live birth rate, data analysis, manuscript writing, and publication will take place over the subsequent 12 months.

September 2022: Amendments to the protocol: Some of the methodological aspects of the AdAPT-ART study to ensure timely delivery of the study to a full sample size. These amendments have been approved by the ethics committee and are as follows:

1. Removal of patient consent specific to the study and use of HFEA CD form to confirm their consent for participation in research Rationale: A. Data of women undergoing IVF/ICSI who consent for participation in research on the HFEA CD form will be looked at B. Baseline ultrasound scan which is the diagnostic tool for adenomyosis is part of routine management and there is no additional step or intervention involved in the study
2. Eligibility criteria: Amendment added in the eligibility section
3. Sample size: Amendment added in the groups section Rationale: In the preliminary stages we had not realized the uneven balance that exists between patients with normal uterus and those with adenomyosis undergoing ART cycles. Our latest unpublished systematic review on prevalence of adenomyosis in a subgroup of women undergoing ART has concluded the prevalence of 11% which translates to 11 women with adenomyosis to 100 women with normal uterus undergoing ART