Preimplantation DNA Methylation Test (PIMT) in ART

Study Overview
=================
Brief Summary
-----------------
The purpose of this clinical trial is to determine the safety and effect of methylation level of DNA methylation in embryos on the outcome of assisted reproductive technology (ART) during blastocyst embryo screening. Subjects with blastocysts on day 5-7 of embryo culture will be biopsied. A Freeze-all strategy and a single frozen blastocyst transfer will be performed till all study-specific embryos have been transferred. Then whole genome bisulfate sequencing will be performed on all cells that obtained from biopsy.

Detailed Description
-----------------
The purpose of this clinical trial is to determine the safety and affect of DNA methylation level on ART. Subjects with blastocysts on day 5-7 of embryo culture will be biopsied. A Freeze-all strategy and a single frozen blastocyst transfer will be performed till all study-specific embryos have been transferred. Then whole genome bisulfate sequencing will be performed on all cells that obtained from biopsy. The investigators will perform whole genome DNA methylation sequence for two to seven blastocysts from one couple. The methylation level and genomic copy number variation will be analyzed by using the methylome data. Embryos with aneuploid chromosomes will be rejected for embryo transfer to uterus. The study will examine which kind of methylation level can produce the best clinical outcome for ART. Biopsied cells will perform with preimplnatation DNA methylation test (PIMT). DNA methylation level and chromosome copy number variation will be calculated by using whole genome DNA methylation sequencing data. As we do not kown which kind of methylation state can produce the best outcome in ART practice. This study will try to find a standard of selection embryos according to DNA methylation information. Under current stage, the selection of embryo is according to chromosome copy number. The embryo selection will not consider DNA methylation information for this study.

Official Title
-----------------
Cumulative Live Birth Rate With eSET After Preimplantation DNA Methylation Test (PIMT) in ART

Conditions
-----------------
DNA Methylation

Intervention / Treatment
-----------------
* Procedure: CNV


Participation Criteria
=================
Eligibility Criteria
-----------------
Inclusion Criteria: Women who are participating in preimplantation screening with PGS indications,defined as maternal age above 38 years, repeated implantation failure (RIF) usually defined as three or more transfers of morphologically high-quality embryos without the establishment of pregnancy, recurrent miscarriage (RM) in patients with normal karyotypes (usually at least three previous consecutive miscarriages) and severe male factor infertility (usually defined as abnormal semen parameters). Women who obtain 2 or more good-quality blastocysts that defined as morphological score of inner cell mass B or A, trophectoderm C or better, and grade 4 or better on Day five of embryo culture will be randomized. Exclusion Criteria: Women with a uterine cavity abnormality, such as a uterine congenital malformation (uterus unicornate, bicornate, or duplex); untreated uterine septum, adenomyosis, submucous myoma, or endometrial polyp(s); or with history of intrauterine adhesions. Women with untreated hydrosalpinx. Women who use donated oocytes or sperm to achieve pregnancy. Women with contraindication for assisted reproductive technology or for pregnancy, such as poorly controlled Type I or Type II diabetes; undiagnosed liver disease or dysfunction (based on serum liver enzyme testing); renal disease or abnormal serum renal function; significant anemia; history of deep venous thrombosis, pulmonary embolus, or cerebrovascular accident; uncontrolled hypertension, known symptomatic heart disease; history of or suspected cervical carcinoma, endometrial carcinoma, or breast carcinoma; undiagnosed vaginal bleeding.

Ages Eligible for Study
-----------------
Minimum Age: 20 Years

Sexes Eligible for Study
-----------------
Female

Accepts Healthy Volunteers
-----------------
No

Study Plan
=================
How is the study designed?
-----------------

Design Details

Primary Purpose: Treatment
Intervention Model: Single Group Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: PIMT methylation<br>Subjects will have blastocyst biopsy and whole genome DNA methylation sequencing done with 2 or 7 good-quality embryos on Day 5 to 7. Principle of freeze-all and single thawed blastocyst transfer will be applied. Only embryos with euploid chromosome will be transfered to the uterus. The outcome of all euploids transfers within 1 year will be followed up. During study, every subject will have at most one live birth. | Procedure: CNV<br>* Embryo with euploid chromosome will transfer to uterus. The order of transfer is accroding to morphological grade.<br>|

What is the study measuring?
-----------------
Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The effect of DNA methylation level on live birth rate | The rate of live birth at different methylation level will be calculated. Live birth is defined as the delivery of any viable infant at 28 weeks or more of gestation, and cumulative live birth rate is calculated by dividing the number of women achieving live birth after transfers (up to 3 transfers of single blastocycst within 1 year). | 22 months | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The effect of DNA methylation level on pregnant rate, pregnant loss rate. | The rate of pregnant and pregnant loss at different methylation level will be calculated. Pregnancy loss refers to a complete spontaneous abortion or a nonviable pregnancy before 28 weeks of gestation. | 22 months | 
| Duration of pregnancy | The time from the first day of last menstrual period to the day of delivery. | 22 months | 
| Birth weight | Weight of newborns at delivery. | 22 months | 
| Cumulative incidence of maternal complications during whole | Number of pregnancies with complications / number of pregnancies over (up to) 3 transfers within 1 year; | 22 months | 
| Cumulative incidence of neonatal complications during whole | Number of live births with neonatal complications / number of live births over (up to)3 transfers within 1 year | 22 months | 
| Number of embryo transfers to achieve live birth | Number of embryo transfers the patients have gone through to achieve live birth. | 22 months | 

 Terms related to the study
=================
Keywords Provided by Centre Hospitalier Valida
-----------------
Preimplantation Methylome Screening, Preimplantation Genetic Screening, single embryo transfer, cumulative live birth rate