Detection of Kisspeptins and miRNAs in Patients With Non-viable Pregnancy (TESTKM)

Ectopic pregnancy (EP) is a pathology that affects 3%-16% of pregnancies in humans, being the main cause of morbidity and maternal mortality in the first trimester of pregnancy worldwide. The relevance of this problem has led to a demand on the scientific community, to obtain specific and early biomarkers in the determination of EP. In this context, the investigator's group has previously confirmed that both kisspeptin 54 and miR-324-3p are specific, selective and precise biomarkers to identify those patients suffering an EP. However, the utility of a diagnostic test using both biomarkers and other related ones in a population with other types of non-viable pregnancies should still be analysed.

Study Overview

• Status: Enrolling by invitation
• Conditions: Non-Viable Pregnancy
• Intervention / Treatment: Diagnostic test: Blood samples collection for analysis

Detailed Description

This is a pilot study in which we intend to determine the moment in which biomarkers begin to be detected in non-viable pregnancies, in order to subsequently be able to develop specific studies focused on that moment. The following visits will be required:

SCREENING VISIT: To be performed before β-hCG test. After recruitment and signing informed consent, patients will be assigned with a three-letter code identifying the clinic and three numbers indicating the order in which the patient is recruited by each clinic.

β-hCG VISIT: To be performed the day of the β-hCG test (week 4):

- Peripheral blood samples will be collected for Progesterone (P4), Kisspeptin 54 and miR-324-3p analysis.

In case of negative result, patient will be out of the study. In case of positive result, β-hCG test > 10 UI/L, patient will continue with the following visits.

SUBSEQUENT BLOOD EXTRACTION VISITS: To be performed on Mondays and Thursdays or Tuesdays and Fridays of each week from β-hCG test until non-viable pregnancy is confirmed or between weeks 6 and 8 when viable pregnancy is confirmed (a maximum of 10 blood sample extractions):

• Peripheral blood samples will be collected Progesterone (P4), Kisspeptin 54 and miR-324-3p will be analysed in these samples. Blood collection will be performed each day in a different arm, allowing a period between extractions in each arm of one week.
• In addition, an optional transvaginal ultrasound scan will be performed before each blood sample if patient wishes.

In case of pregnancy of unknown location (PUL), protocol will continue until VP or NVP is confirmed.

If NVP is confirmed before clinical pregnancy visit, patient will undergo an end of study visit.

CLINICAL PREGNANCY VISIT: To be performed between weeks 5+2 and 5+6, the same day that one of the subsequent blood extractions:

- A transvaginal ultrasound scan will be performed before the blood sample extraction.

In case of non-viable pregnancy, patient will perform the end of study visit. In case of clinical pregnancy, patient will continue with the following visits.

ONGOING PREGNANCY VISIT: To be performed between weeks 6 and 8, the same day that one of the subsequent blood extractions:

- A transvaginal ultrasound scan will be performed before the blood sample extraction. VP will be confirmed with this ultrasound scan.

In case of non-viable pregnancy, patient will undergo the end of study visit. In case of viable pregnancy, patient will undergo the end of study visit in week 10 of pregnancy.

END OF STUDY VISIT: To be performed after an ultrasound scan when NVP is confirmed or in week 10 of pregnancy. The following samples will be collected:

In case of ectopic pregnancy:

- Peripheral blood samples for Progesterone (P4), Kisspeptin 54 and miR-324-3p analysis.

In case of clinical miscarriage:

- Peripheral blood samples for Progesterone (P4), Kisspeptin 54 and miR-324-3p analysis.

In case of Biochemical pregnancy:

• Peripheral blood samples for Progesterone (P4), Kisspeptin 54 and miR-324-3p analysis.
• Blood sample for KIR typing.
• Blood samples of patient and partner for HLA-C typing. If partner does not attend to this visit, his sample may be collected in the following 3 weeks.
• Blood samples for detection of miRNAs related with insulin resistance, miR-424-5p and miR-15b.
• Oral glucose tolerance test (OGTT), including blood sample analysis before 75 g of glucose consumption and after 1h and 2 h.
• Uterine biopsy in the month in which the biochemical pregnancy is confirmed or in the following month (in P + 5) for detection of miRNAs related with insulin resistance, miR-424-5p and miR-15b.

In case of viable pregnancy in week 10 of gestation:

• Peripheral blood samples for Progesterone (P4), Kisspeptin 54 and miR-324-3p analysis.
• Blood sample for KIR typing.
• Blood samples of patient and partner for HLA-C typing. If partner does not attend to this visit, his sample may be collected in the following 3 weeks.
• Blood samples for detection of miRNAs related with insulin resistance, miR-424-5p and miR-15b.
• Data of an oral glucose tolerance test (OGTT) that patients perform routinely in the first trimester of pregnancy will be required.

• Study Type: Observational
• Enrollment (Anticipated): 433

Contacts and Locations

Study Locations

• Spain
  • Madrid, Spain, 28023
    • IVI RMA Madrid
  • Seville, Spain, 41011
    • IVI RMA Seville

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 18 years to 50 years (Adult)
• Accepts Healthy Volunteers: No
• Genders Eligible for Study: Female

• Sampling Method: Probability Sample

Study Population

Those patient that have made a pre-embryonic transfer in the IVI RMA Seville, IVI RMA Madrid, IVI RMA Valencia, and IVI RMA Barcelona clinics will constitute patient population.

Description

Inclusion Criteria:

• Patients who will perform SET.
• Patients using own oocytes.

Exclusion Criteria:

• Complicated uterine cavity.

Study Plan

How is the study designed?

Design Details

• Observational Models: Cohort
• Time Perspectives: Prospective

Number of groups / cohorts

5

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
Non-pregnant patients; Patients with β-hCG test < 10 UI/L.	Diagnostic test: Blood samples collection for analysis; Blood samples: Blood samples will be collected for the determination of the levels of various biomarkers (Kisspeptins and miRNAs), which are molecules that regulate the expression of proteins and can serve as markers for predicting pregnancy viability. Additionally, after biochemical or week 10 of clinical pregnancy confirmation, blood samples will be collected for KIR and HLA-C typing, Oral glucose tolerance test (OGTT) and analysis of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b. Uterine biopsy (only in patients with BP): Uterine biopsy samples will be collected also after biochemical pregnancy confirmation for the determination of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b.; Other Names: Uterine biopsy sample collection (only in biochemical pregnancy patients)
Patients with viable pregnancy; Patients with β-hCG test > 10 UI/L whose pregnancy is confirmed between gestational weeks 6 and 8.	Diagnostic test: Blood samples collection for analysis; Blood samples: Blood samples will be collected for the determination of the levels of various biomarkers (Kisspeptins and miRNAs), which are molecules that regulate the expression of proteins and can serve as markers for predicting pregnancy viability. Additionally, after biochemical or week 10 of clinical pregnancy confirmation, blood samples will be collected for KIR and HLA-C typing, Oral glucose tolerance test (OGTT) and analysis of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b. Uterine biopsy (only in patients with BP): Uterine biopsy samples will be collected also after biochemical pregnancy confirmation for the determination of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b.; Other Names: Uterine biopsy sample collection (only in biochemical pregnancy patients)
Patients with biochemical pregnancy; Patients with β-hCG test > 10 UI/L and without sac observed.	Diagnostic test: Blood samples collection for analysis; Blood samples: Blood samples will be collected for the determination of the levels of various biomarkers (Kisspeptins and miRNAs), which are molecules that regulate the expression of proteins and can serve as markers for predicting pregnancy viability. Additionally, after biochemical or week 10 of clinical pregnancy confirmation, blood samples will be collected for KIR and HLA-C typing, Oral glucose tolerance test (OGTT) and analysis of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b. Uterine biopsy (only in patients with BP): Uterine biopsy samples will be collected also after biochemical pregnancy confirmation for the determination of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b.; Other Names: Uterine biopsy sample collection (only in biochemical pregnancy patients)
Patients with ectopic pregnancy; Patients with β-hCG test > 10 UI/L whose sac is implantated outside the uterine cavity.	Diagnostic test: Blood samples collection for analysis; Blood samples: Blood samples will be collected for the determination of the levels of various biomarkers (Kisspeptins and miRNAs), which are molecules that regulate the expression of proteins and can serve as markers for predicting pregnancy viability. Additionally, after biochemical or week 10 of clinical pregnancy confirmation, blood samples will be collected for KIR and HLA-C typing, Oral glucose tolerance test (OGTT) and analysis of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b. Uterine biopsy (only in patients with BP): Uterine biopsy samples will be collected also after biochemical pregnancy confirmation for the determination of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b.; Other Names: Uterine biopsy sample collection (only in biochemical pregnancy patients)
Patients with clinical miscarriage; Patients with β-hCG test > 10 UI/L whose sac is implanted inside the uterine cavity, but non-viable pregnancy is confirmed before gestational week 8.	Diagnostic test: Blood samples collection for analysis; Blood samples: Blood samples will be collected for the determination of the levels of various biomarkers (Kisspeptins and miRNAs), which are molecules that regulate the expression of proteins and can serve as markers for predicting pregnancy viability. Additionally, after biochemical or week 10 of clinical pregnancy confirmation, blood samples will be collected for KIR and HLA-C typing, Oral glucose tolerance test (OGTT) and analysis of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b. Uterine biopsy (only in patients with BP): Uterine biopsy samples will be collected also after biochemical pregnancy confirmation for the determination of the levels of miRNAs related with insulin resistance, miR-424-5p and miR-15b.; Other Names: Uterine biopsy sample collection (only in biochemical pregnancy patients)

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Level of Kisspeptin 54	Level in peripheral blood of Kisspeptin 54	Mar 2019 - Oct 2021
Level of miR-324-3p	Level in peripheral blood of miR-324-3p	Mar 2019 - Oct 2021

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Level of progesterone	Level in peripheral blood of progesterone	Mar 2019 - Oct 2021
Level of hormone β-hCG	Level in peripheral blood of hormone β-hCG	Mar 2019 - Oct 2021
Level of miRNAs related with insulin resistance, miR-424-5p and miR-15b	Level in peripheral blood of miR-424-5p and miR-15b in patients with viable and biochemical pregnancy	Mar 2019 - Oct 2021
Level of miRNAs related with insulin resistance, miR-424-5p and miR-15b	Level in endometrial sample of miR-424-5p and miR-15b in patients with biochemical pregnancy	Mar 2019 - Oct 2021
KIR typing	Killer-cell immunoglobulin-like receptors typing in patients with viable and biochemical pregnancy	Mar 2019 - Oct 2021
HLA-C typing	HLA (human leucocyte antigens)-C typing in patients with viable and biochemical pregnancy and their partners	Mar 2019 - Oct 2021
Oral glucose tolerance test (OGTT)	Including blood sample analysis before 75 g of glucose consumption and after 1h and 2 h.	Mar 2019 - Oct 2021

Collaborators and Investigators

• Sponsor: IVI Sevilla
• Collaborators: Instituto Valenciano de Infertilidad, IVI VALENCIA; IVI Madrid; IVI Barcelona
• Investigators: Principal Investigator: Manuel Fernández-Sánchez, PhD, MD, IVI RMA Seville

Study record dates

Study Major Dates

• Study Start (Actual): September 15, 2019
• Primary Completion (Anticipated): June 30, 2023
• Study Completion (Anticipated): October 30, 2023

Study Registration Dates

• First Submitted: March 14, 2019
• First Submitted That Met QC Criteria: March 15, 2019
• First Posted (Actual): March 18, 2019

Study Record Updates

• Last Update Posted (Actual): March 31, 2023
• Last Update Submitted That Met QC Criteria: March 30, 2023
• Last Verified: March 1, 2023

More Information

Terms related to this study

• Keywords: biomarkers; miRNA; Ectopic pregnancy; non-viable pregnancy; Kisspeptin; β-HCG
• Other Study ID Numbers: 1801-SEV-009-MF

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No