PRIOR Study (Pre-eclampsia Risk In Oocyte Recipients)

INTRODUCTION AND BACKGROUND Maternal age at conception has increased globally, leading to a higher proportion of pregnancies among women aged ≥35 years. In Denmark, the average maternal age at first birth has risen from 24 years in 1970 to 30 years in 2021. As female fecundity declines markedly after age 35, the need for assisted reproductive technology (ART) is increasing.

The success rate of ART using autologous oocytes decreases significantly after age 40, making oocyte donation (OD) a relevant option for women with diminished ovarian reserve or poor oocyte quality. OD was first introduced in 1984 to treat infertility due to premature ovarian failure and is now used for various indications including Turner syndrome, previous oophorectomy, gonadotoxic therapy, and certain genetic disorders.

OD has been legal in Denmark since 2007 and double gamete donation since 2018. Healthy women under 36 years can donate oocytes anonymously or non-anonymously. The number of OD procedures in Denmark has increased from 124 in 2011 to 2,782 in 2022, resulting in 756 deliveries in 2021. The rise is attributed to delayed motherhood, legal changes, and increased donor compensation.

Several studies have demonstrated that pregnancies following OD are associated with a higher risk of obstetric complications, including pre-eclampsia (PE), intrauterine growth restriction, preterm birth, cesarean section, placental abruption, and postpartum hemorrhage. The risk appears to be even higher in double gamete donation pregnancies. The mechanisms behind these associations remain unclear but are thought to involve immune maladaptation due to exposure to non-self paternal and donor antigens.

PE complicates approximately 3-8% of pregnancies and is defined as new-onset hypertension (≥140/90 mmHg) with proteinuria (≥300 mg/24 h) and/or signs of organ dysfunction after 20 weeks of gestation. Risk factors include prior PE, chronic hypertension, renal disease, diabetes, autoimmune disease, multifetal gestation, nulliparity, advanced maternal age, high BMI, long interpregnancy interval, and family history of PE.

PE and related disorders share a common pathophysiology involving abnormal placentation, impaired trophoblast invasion, and endothelial dysfunction. The etiology is multifactorial and may include genetic, immunological, and vascular components. Recent data indicate that fetal-maternal HLA mismatches, particularly HLA-DR incompatibility, are associated with increased PE risk.

This study hypothesizes that OD pregnancies, known to have elevated risk of PE, provide a relevant model to investigate the pathophysiological mechanisms of the disease.

STUDY OBJECTIVE The aim of this prospective observational cohort study is to investigate the pathophysiological mechanisms and the risk of pre-eclampsia in women who become pregnant following fertility treatment with oocyte donation.

The study population comprises two cohorts. These are a cohort of women pregnant after oocyte donation compared with a cohort of women pregnant after IVF with autologous gametes.

PRIMARY RESEARCH QUESTIONS

• To assess the risk of pre-eclampsia in pregnancies conceived after single or double gamete donation compared with pregnancies achieved using autologous gametes.
• To investigate immunological and angiogenic biomarkers, as well as HLA tissue-type markers, involved in the pathogenesis of pre-eclampsia.
• To evaluate obstetric and neonatal outcomes, including pre-eclampsia, gestational diabetes, preterm birth, placental abruption, intrauterine growth restriction, asphyxia, neonatal morbidity, and mortality among women pregnant after oocyte or double donation in Denmark.
• To establish a comprehensive database and biobank to facilitate future research, including long-term follow-up studies of children born following oocyte donation.

Participants will be monitored throughout pregnancy with serial blood sampling, blood pressure measurements, clinical assessments, and ultrasound examinations. Clinical outcomes will be recorded postpartum.

INCLUSION OF PATIENTS STUDY POPULATION

The study will include two cohorts:

1. Women who achieve pregnancy following oocyte or double gamete donation (OD cohort).
2. Women who achieve pregnancy following IVF treatment with their own (autologous) frozen-thawed blastocysts (IVF control cohort).

Recruitment and Identification of Participants

Participants will be identified early in pregnancy through two recruitment pathways:

1. Fertility Clinic Recruitment (Gestational Week 7-8)

   o Women attending an early pregnancy ultrasound after ART will breifly be informed about the study by fertility clinic staff.

   • If the woman expresses interest, her contact details (name, CPR number, and phone/email) will be forwarded to the principal investigator (PI).
   • The study team will contact the woman with detailed verbal and written information.
   • For women who are patients at Herlev Hospital, information will be provided in person, and a printed information sheet will be handed out.
   • For women treated at collaborating clinics (Rigshospitalet, Sellmer Fertility, Aleris Hamlet, Copenhagen Fertility Center, Trianglen), patient information will be provided via telephone, and written information will be either handed out or sent through secure digital mail (e-Boks).

2. Referral from General Practitioner (Gestational Week 8-10)

   o When women are referred from their general practitioner to the obstetric department, the visitation office will notify the PI of potential participants.

   • Written study information will be sent via secure e-mail (e-Boks), including notification that the woman will receive a phone call from study staff within a few days.
   • The woman will receive verbal information and may schedule an inclusion visit if interested.

   INFORMED CONSENT All participants will receive both verbal and written information about the study. Women will be encouraged to bring a companion or assessor to the inclusion visit. The inclusion visit includes repeated verbal information, opportunity for questions, and a minimum 24-hour reflection period before signing the consent form. Consent covers study participation, access to relevant clinical records, and collection of biological samples as specified in the protocol.

   ELIGIBILITY CRITERIA

   • Inclusion Criteria:

   o Pregnant women who conceived through oocyte donation, double gamete donation, or IVF treatment using their own (autologous) oocytes.

   o Ability to understand study information and provide written informed consent.

   o Willingness to participate in follow-up visits throughout pregnancy.

   • Exclusion Criteria:
   • Withdrawal of consent at any time during the study period.
   • Known multiple pregnancy at the time of inclusion.
   • Chronic medical conditions such as hypertension,

   INCLUSION Inclusion Visit (Gestational Week 10-12) (Visit 1) At the inclusion visit, informed consent will be obtained by the principal investigator or a trained study nurse. Eligibility criteria will be verified, and baseline data will be collected, including demographic information (age, ethnicity, family status), medical and fertility history, hereditary diseases, and current medication. Lifestyle factors such as smoking and alcohol use will be recorded.

   A physical examination will be performed, including height, weight, blood pressure, and heart rate. Routine fertility screening (smear, TSH, TPO, AMH, HIV, hepatitis, syphilis, rubella) will extracted from the patient files. Blood samples for study-specific analyses, including pre-eclampsia biomarkers, will be collected.

   Follow-up Visits (Visit 2-6 and telemedicine)

   Participants will undergo structured follow-up visits throughout pregnancy:

   • Gestational Week 12-14: Nuchal translucency ultrasound and blood sampling for pre-eclampsia markers.
   • Gestational Week 18-20: Malformation ultrasound and blood sampling for pre-eclampsia markers.
   • Gestational Week 28: Initiation of weekly blood pressure measurements sent electronically to the hospital untill delivery. Bloodsampling for pre-eclampsia markers.
   • Gestational Week 34: Clinical visit with ultrasound, obstetric examination, and blood sampling for pre-eclampsia markers.
   • Gestational Week 38: Clinical visit with ultrasound and blood sampling for pre-eclampsia markers.

   Postpartum Data Collection After delivery, clinical outcome data will be obtained from electronic medical records. This includes gestational age at delivery, mode of delivery, birth weight, congenital malformations, and obstetric complications such as pre-eclampsia, gestational diabetes, intrauterine growth restriction, and postpartum hemorrhage.

   OUTCOME MEASURES

   Primary Outcome Measure:

   • Incidence of pre-eclampsia [Time Frame: From gestational week 20 until delivery]

     • Pre-eclampsia is defined as new-onset hypertension (systolic ≥ 140 mmHg or diastolic ≥ 90 mmHg) and proteinuria (≥300 mg/24h) and/or signs of organ dysfunction after 20 weeks of gestation. The primary outcome compares the incidence in pregnancies after oocyte or double gamete donation versus pregnancies after IVF with autologous oocytes.

   Secondary Outcome Measures:

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1. Immunological and angiogenic biomarkers [Time Frame: From inclusion until delivery] o Levels of specific biomarkers measured in maternal blood at multiple time points throughout pregnancy. Below are examples.

   o These are growth markers involved in early pregnancy (PAPP-A, IGF, PlGF, PGF and VEGFR), pro- and inflammatory cytokines (Interleukins, TNF-α and Interferon-γ), complement and thrombocyte activation markers (Selectin and BF-4), and markers for coagulation and fibrinolysis (Thrombin/anti-thrombin complexes and ex vivo fibrinogenesis/fibrinolysis).

2. HLA tissue type markers

   o Analysis of HLA mismatches between fetus and mother and their association with pre-eclampsia risk.

3. Obstetric complications [Time Frame: From inclusion until delivery]

   o Incidence of pre-eclampsia, postpartum hemorrhage, caesarean section, gestational diabetes, preterm birth (\<37 weeks), IUGR, and placental abruption.

4. Neonatal outcomes [Time Frame: Birth until hospital discharge]

   o Incidence of intrauterine growth restriction, birth asphyxia, neonatal morbidity, and neonatal mortality.

5. Establishment of a database and biobank o Collection of clinical data and biological samples to enable future research, including potential long-term follow-up of children born after oocyte donation.

DATA COLLECTION AND HANDLING Signed informed consent allows access to relevant maternal and neonatal medical records for study purposes. All participant data will be collected in a secure electronic data capture system (REDCAP) compliant with local data protection regulations. Each participant will be assigned a unique study ID to ensure confidentiality. Source data will include electronic medical records, laboratory results, ultrasound reports, and telemedicine monitoring data. Only authorized study personnel will have access to identifiable data.

All study data will be recorded in case report forms (CRFs) and reviewed for completeness and accuracy. Queries will be resolved promptly with the study sites. Data will be backed up regularly, and audit trails will be maintained to track any changes to the dataset.

DATA STORAGE AND CONFIDENTIALITY Study data will be stored on secure servers with encryption. Access to identifiable information will be restricted to the principal investigator and designated study staff. Data will be retained for a minimum of 15 years following the end of the study, in accordance with institutional and national guidelines.

STATISTICAL ANALYSIS Data analysis will be performed according to a predefined statistical analysis plan. Descriptive statistics will be used to summarize baseline characteristics and clinical outcomes. Continuous variables will be expressed as means with standard deviations or medians with interquartile ranges, depending on distribution. Categorical variables will be presented as counts and percentages.

Comparisons between groups (e.g., oocyte donation vs. IVF pregnancies) will be conducted using appropriate statistical tests, such as t-tests or Mann-Whitney U tests for continuous variables and chi-square or Fisher's exact tests for categorical variables. Multivariable regression models will be applied to adjust for potential confounders.

All statistical tests will be two-sided, and a p-value \<0.05 will be considered statistically significant. Interim analyses may be conducted if approved by the study steering committee, but final analyses will include all enrolled participants.

RISK MINIMIZATION The study involves minimal additional risk beyond standard clinical care for pregnant women. Blood draws will be performed by experienced personnel to minimize discomfort. Telemedicine monitoring will ensure timely detection of early signs of pre-eclampsia or other complications. Any participant developing severe complications will be referred for immediate clinical management according to standard obstetric protocols.

APPROVALS AND REGISTRATION The study is approved by the National Committee on Health Research Ethics (Approval no. H-22068878). This study was registered retrospectively on clinicaltrials.gov due to an administrative oversight in interpreting the prospective registration requirement. No changes were made to the protocol after study initiation. The research team has implemented revised internal procedures to ensure prospective registration for all future studies.