Trial Review

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Trial registered on ANZCTR


Registration number
ACTRN12621001405808
Ethics application status
Approved
Date submitted
3/09/2021
Date registered
18/10/2021
Date last updated
28/09/2022
Date data sharing statement initially provided
18/10/2021
Type of registration
Prospectively registered

Titles & IDs
Public title
Biomarkers predictive of embryo quality and assisted reproductive treatment outcomes
Scientific title
Biomarkers predictive of embryo quality and clinical pregnancy in women prior to undergoing antagonist treatment assisted reproductive therapy
Secondary ID [1] 305085 0
None
Universal Trial Number (UTN)
U1111-1269-1164
Trial acronym
PEQ
Linked study record

Health condition
Health condition(s) or problem(s) studied:
Assisted reproduction 323446 0
Subfertility 323447 0
Infertility 323448 0
Reproductive health 323779 0
Condition category
Condition code
Reproductive Health and Childbirth 320998 320998 0 0
Fertility including in vitro fertilisation

Intervention/exposure
Study type
Observational
Patient registry
False
Target follow-up duration
Target follow-up type
Description of intervention(s) / exposure
Potential participants are identified and recruited consecutively from new bookings, with a note to the site nurse to provide information about the trial either via a phone call or during the patient's orientation and information session.

On day 2 of menses, the consented female participants are scheduled for a blood test as part of routine care, at which point an additional venous blood samples is collected for proteomic biomarker screening.

The following paragraph constitutes routine/standard care for participant at the discretion of the patient's doctor. Female participants undergoing IVF or ICSI by the antagonist treatment protocol consists of follicle-stimulating hormone (FSH) and lutenizing hormone (LH) injections. Starting around the fifth day, the Gonadotropin-Releasing Hormone (GnRH) antagonist is added to prevent premature ovulation. The b-hCG triggered is administers and 36-38 hours later oocytes are collected. Fertilisation occurs by IVF or ICSI, embryos are selected using AI score between days 3 - 5 post-fertilisation and frozen or transferred to female patient. Biochemical pregnancy test is performed after 2 weeks implantation, clinical pregnancy after 6 weeks and miscarriage monitoring up to 20 weeks gestation by obtaining patient records.

Upon receipt of the patient consent form, the female participant receives the questionnaire by email. Questions to ascertain a patient’s probability of a spontaneous pregnancy within 1 year were included as a baseline/benchmark as a comparator to our model prediction. A semi-quantitative food frequency questionnaire (FFQ), was modified to include food groups with know effects on embryo quality and pre-pregnancy supplementation recommendations. In addition, the survey contains questions to refine the clinical utility of a predictive model from the patient's perspective.

Female and male patient records will collected at the periodically throughout the study, including comorbidities, hormonal stimulation, embryological pathologies and pregnancy outcomes. No further active involvement from the patient is required.

With exception of the additional blood collection for proteomic analysis and the completion of a questionnaire, all treatment/procedures are part of routine care
Intervention code [1] 321573 0
Early Detection / Screening
Comparator / control treatment
No control group
Control group
Uncontrolled

Outcomes
Primary outcome [1] 328776 0
Clinical Pregnancy, rate per embryo transfer.
Clinical pregnancy is determined by fetal heartbeat at ultrasound and number of embryos transferred are both obtained from patient records.
Timepoint [1] 328776 0
Six weeks gestation
Secondary outcome [1] 400294 0
Number of usable embryos - Gardner score
Obtained from patient records.
Timepoint [1] 400294 0
Embryo selection, 3 - 5 days following fertilisation
Secondary outcome [2] 400295 0
Artificial Intelligence (AI) embryo quality score, Ivy score.
Obtained from patient records
Timepoint [2] 400295 0
Embryo selection, 3 - 5 days following fertilisation
Secondary outcome [3] 400297 0
Artificial Intelligence (AI) score all usable embryos.
Obtained from patient records
Timepoint [3] 400297 0
Embryo selection, 3 - 5 days following fertilisation
Secondary outcome [4] 400299 0
Miscarriage rate.
Obtained from patient records.
Timepoint [4] 400299 0
before 20 weeks gestation
Secondary outcome [5] 400302 0
Biochemical pregnancy rate.
Determined from b-hCG (approximately greater than 10 U/L) detected in peripheral blood sample and obtained from patient records.
Timepoint [5] 400302 0
between 7 - 10 days post-implantation
Secondary outcome [6] 400303 0
Number of oocyte retrieved at oocyte pickup (OPU).
Obtained from patient records.
Timepoint [6] 400303 0
Oocyte retrieval, 36 - 38h after b-hCG trigger
Secondary outcome [7] 400304 0
Number of oocytes fertilised normally (2PN)
Obtained from patient records.
Timepoint [7] 400304 0
Oocyte retrieval, 36 - 38h after b-hCG trigger
Secondary outcome [8] 401342 0
1536 proteomic biomarkers from peripheral blood.
Relative quantification using proximity extension assay (PEA).
Timepoint [8] 401342 0
Menses day 2

Eligibility
Key inclusion criteria
1. Seeking and undergoing fertility treatment at one of Virtus Health’s three major Sydney clinics (Alexandria, Greenwich and Westmead)
2. Female
3. 18 - 42 years of age
4. Antagonist treatment protocol
5. Autologous oocyte
6. In vitro fertilisation (IVF)
7. Intracytoplasmic sperm injection (ICSI)
8. Scheduled for a blood collection within the first 72h of menses
Minimum age
18 Years
Maximum age
42 Years
Sex
Females
Can healthy volunteers participate?
No
Key exclusion criteria
1. Male-factor infertility defined by Australia and New Zealand Assisted Reproduction Database (ANZARD) in patient history
2. Undertaking assisted reproductive therapy (ART) and oocyte or embryo freeze all for fertility preservation
3. Body Mass Index greater than 35kg.m2 or less than 18.5kg.m2
4. Currently receiving steroid based therapy in the last two weeks
5. Recent antimicrobial treatment n the last two weeks
6. Recent iron infusion OR infusion/transfusion based therapy n the last 12 weeks
7. Genetic disorder(s) diagnosis
8. Any past history of chemotherapy
9. Autoimmune disorder(s) diagnosis
10. Recent surgery within n the last 12 weeks
11. Acute illness, such as an infection, within the last two weeks
12. Renal impairment diagnosis
13. Liver dysfunction diagnosis and or significant hypoalbuminaemic state

Study design
Purpose
Screening
Duration
Longitudinal
Selection
Defined population
Timing
Prospective
Statistical methods / analysis
Sample size
Determination of an appropriate sample size to achieve statistical power, including by simulation, is difficult for the planned regularisation models without substantial prior assumptions, as one would need to assume not only effect sizes and variance, but also covariance and the lambda hyperparameter. Therefore, it is not possible to determine whether the sample size is sufficient for the current statistical plan and should be considered exploratory in nature.

Missing Data
Completeness of data, particularly for confounders, will be an Inclusion criteria. Predictors with greater than 50% missing data will however be dropped. To assess the missing completely at random (MCAR) assumption, a dummy variable of missing data will be tested by logistic regression, using “missing data” as the dependent variable and clinical information as the independent variables. Reasons for missingness and proportion of patients with missing data will be discussed. The multiple imputation method will be used to draw imputed values from a distribution of predicted values, with the estimates combined over m imputed data sets, with linear predictors combined using Rubin’s rule. Demographics of questionnaire non-responders will be compared to responders to assess information bias.

Outliers
Extreme outliers will be detected by Tukey, using the three times the interquartile range rule, and verified where possible. Implausible extreme outliers will be replaced during multiple imputation. Plausible continuous variables adjusted using Winsorizing to replace extreme values with 1 or 99 percentile values.

Testing regression assumptions
Normality of independent variables will be tested using Shapiro-Wilk’s method and transformed using Z-score normalisation. As regularisation techniques like Elastic Net models give weight to the features based on their importance, which handles multi-collinearity issues automatically, highly correlated variables will not be removed. Variables with near zero variance will be removed to ensure variability of independent variables are positive and to increase power by restriction of candidate predictors.
Assumptions of the dependent variable will be checked by plotting the distribution of the resulting residuals with Q-Q plots and Breusch Pagan Test. Alternative transformation methods will be devised if evidence suggests heteroscedasticity.

Confounders
A list of possible confounders in ART, derived from the scientific literature, can be found in the Baseline Data. We have chosen to minimise the influence of confounding variables using a combination of study design and analysis, by restriction and regression, respectively.
Restriction was performed by focusing our study on predominantly healthy women, undergoing Antagonist treatment Protocol for oocyte collection, with healthy (preimplantation genetic diagnosis - PGD, negative) and subject-derived oocytes, of partners with no apparent male-factor infertility, with the intention of minimising the known confounders of ART success associated with comorbidities, hormonal stimulation, embryological pathologies and male partner factors, respectively. The applicability of this study was not expected to be significantly affected by use of restriction as we estimate 43% Virtus’ patients are still eligible despite the Inclusion/Exclusion criteria.
Residual confounding variables may still be present and will be included in the regression analysis and final models. If confounders still require adjustment, stratification may be attempted.

Outcome/Endpoint
The primary and secondary endpoints are embryo quality score and clinical pregnancy. These are objective measures and industry standard and reporting metric for all fertility services regarding ART success. The model for ART success will be developed using clinical pregnancy as the response variable and predictive capabilities will be estimated for the other secondary outcomes using the same model.
Model Development and Feature Selection
For biomarker selection and effect estimation, regularisation (Elastic Net, Ridge Regression or Lasso) will be used. Lambda and alpha will be optimised with 10 fold cross validation. Bootstrapping will be employed to estimate the distribution of regression coefficients, including calculation of 95% confidence intervals. Model development will be reported according to the TRIPOD principles.

Internal Validation and Model Optimism
Internal validation will also be performed via bootstrapping, where 1. The performance of the optimised model (Model) is tested against the full data set used to create the model (apparent performance), 2. A sub-sample (with replacement) from the full data is drawn to construct a bootstrap model (Model*), which is subsequently tested against the sub-sample data set for performance (bootstrap performance) and 3. Performance of Model* against the full data set is determined (test performance). Optimism equals test performance - bootstrap performance and optimism adjusted performance equals apparent performance - optimism. The adjusted performance is what will be reported and the expected performance level for external validation.

Model Performance
R2 will be reported as an overall measure to quantify the amount of information captured by the model for the given data set. The effectiveness of decision-making models is largely dependent on the rigor of model calibration; therefore, the c statistic is the preferred measure of model discrimination, as it is insensitive to miscalibration. Incremental value of a marker will be determined by the net reclassification improvement and used to justify the inclusion of each biomarker in the final model.

Clinical Utility
The clinical utility of a model that predicts embryo quality and clinical pregnancy would be used in future studies for clinical decision making, whereby the model determines if a woman should proceed with oocyte donation and hence delay treatment. Therefore, model accuracy will be determined using a clinically intuitive cutoff agreed upon by both the Chief Investigator and Managing Clinician. The threshold (0% - commence all, 100% - commence none) for determining commencement of ART will be set at a value between the rate of ART success (clinical pregnancy, 35%) and the rate at which women produce high quality embryos (embryo quality greater than 3, 50%). Meaning that between 50 to 65% of patients are assumed to fail ART if proceedings without some kind of intervention. This threshold prioritises model sensitivity (avoiding missed diagnosis) over specificity (avoiding false diagnosis), which is consistent with decision-making models used in a clinical setting.

Recruitment
Recruitment status
Recruiting
Date of first participant enrolment
Anticipated
1/11/2021
Actual
1/11/2021
Date of last participant enrolment
Anticipated
7/09/2023
Actual
Date of last data collection
Anticipated
7/03/2024
Actual
Sample size
Target
400
Accrual to date
191
Final
Recruitment in Australia
Recruitment state(s)
NSW
Recruitment hospital [1] 20602 0
City West Day Surgery - Westmead
Recruitment hospital [2] 20603 0
Alexandria Specialist Day Hospital - Alexandria
Recruitment hospital [3] 20604 0
North Shore Specialist Day Hospital - Greenwich
Recruitment postcode(s) [1] 35394 0
2145 - Westmead
Recruitment postcode(s) [2] 35395 0
2015 - Alexandria
Recruitment postcode(s) [3] 35396 0
2065 - Greenwich

Funding & Sponsors
Funding source category [1] 309482 0
Commercial sector/Industry
Name [1] 309482 0
Drop Bio Pty Ltd
Country [1] 309482 0
Australia
Funding source category [2] 309568 0
Commercial sector/Industry
Name [2] 309568 0
Virtus Health
Country [2] 309568 0
Australia
Primary sponsor type
Commercial sector/Industry
Name
Drop Bio Pty Ltd
Address
PO BOX 6307 UNSW SYDNEY, NSW 1466
Country
Australia
Secondary sponsor category [1] 310454 0
Commercial sector/Industry
Name [1] 310454 0
Virtus Health
Address [1] 310454 0
Level 3, 176 Pacific Highway Greenwich NSW, 2065
Country [1] 310454 0
Australia

Ethics approval
Ethics application status
Approved
Ethics committee name [1] 309269 0
IVF Australia Ethics Committee
Ethics committee address [1] 309269 0
Level 3, 176 Pacific Highway Greenwich NSW 2065
Ethics committee country [1] 309269 0
Australia
Date submitted for ethics approval [1] 309269 0
13/09/2021
Approval date [1] 309269 0
01/10/2021
Ethics approval number [1] 309269 0

Summary
Brief summary
This is a observational prospective cohort study whereby convenient and routine venous bloods are collected prior to commencement (day 2 menses) IVF or ICSI, antagonist treatment and oocyte collection, and analysed for proteomic biomarkers indicative of embryo quality score and clinical pregnancy. The primary objective is to develop a predictive model that facilitates decision-making in the clinic, particularly regarding the choice to proceed to oocyte collection given the subject’s health status and probability of assisted reproduction success.
Trial website
Trial related presentations / publications
Public notes

Contacts
Principal investigator
Name 113566 0
Dr Connor O'Meara
Address 113566 0
Room 403, D26 Bioscience North, UNSW Kensington NSW 2052
Country 113566 0
Australia
Phone 113566 0
+61 468861179
Fax 113566 0
Email 113566 0
[email protected]
Contact person for public queries
Name 113567 0
Dr Connor O'Meara
Address 113567 0
Room 403, D26 Bioscience North, UNSW Kensington NSW 2052
Country 113567 0
Australia
Phone 113567 0
+61 468861179
Fax 113567 0
Email 113567 0
[email protected]
Contact person for scientific queries
Name 113568 0
Dr Connor O'Meara
Address 113568 0
Room 403, D26 Bioscience North, UNSW Kensington NSW 2052
Country 113568 0
Australia
Phone 113568 0
+61 468861179
Fax 113568 0
Email 113568 0
[email protected]

Data sharing statement
Will individual participant data (IPD) for this trial be available (including data dictionaries)?
No
No/undecided IPD sharing reason/comment


What supporting documents are/will be available?

Doc. No.TypeCitationLinkEmailOther DetailsAttachment
13035Informed consent form  [email protected]
13036Study protocol  [email protected]
13037Statistical analysis plan  [email protected]
13062Ethical approval  [email protected]



Results publications and other study-related documents

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