Non-invasive preimplantation genetic testing for aneuploidy (NiPGT-A) is an emerging approach that analyzes embryonic cell-free DNA (cfDNA) released into the spent culture medium (SCM) to assess chromosomal status without performing trophectoderm biopsy. This technique has the potential to reduce procedural invasiveness and eliminate biopsy-related risks while providing additional information for embryo selection in assisted reproductive technology (ART). However, the clinical value of NiPGT-A remains uncertain because available evidence is limited and largely derived from observational studies, with few studies reporting live birth outcomes.

This prospective observational study aims to evaluate the correlation between NiPGT-A results and clinical outcomes after single blastocyst transfer in patients with a favorable prognosis undergoing IVF/ICSI treatment. In this study, embryos with good morphological quality will undergo non-invasive sampling of spent culture medium for cfDNA analysis. NiPGT-A results will be categorized into four groups: no result, euploid, mosaic, and aneuploid. The study will compare pregnancy and birth outcomes across these groups to determine the clinical utility of NiPGT-A in embryo selection strategies.

Background and Rationale:

Preimplantation genetic testing for aneuploidy (PGT-A) using trophectoderm biopsy has been widely used in IVF to improve embryo selection. However, the invasive nature of embryo biopsy raises concerns about potential impacts on embryo viability and limits its routine use in certain patient populations. Non-invasive preimplantation genetic testing for aneuploidy (NiPGT-A) has been proposed as an alternative approach that analyzes cell-free DNA (cfDNA) released by the embryo into the spent culture medium (SCM) during in vitro development.

Several studies have demonstrated that cfDNA in SCM can reflect embryonic chromosomal status, but the concordance between NiPGT-A results and trophectoderm biopsy remains variable and influenced by factors such as sampling timing, maternal DNA contamination, embryo mosaicism, and laboratory protocols. Additionally, cases have been reported where embryos classified as aneuploid by NiPGT-A resulted in healthy live births, raising concerns about potential misclassification and highlighting the need for further clinical validation.

Recent research has suggested that NiPGT-A may be more valuable when used as a complementary tool to embryo morphology rather than as a direct replacement for biopsy-based PGT-A. Some studies have also reported that embryos with low or undetectable cfDNA levels in the culture medium may have favorable developmental potential, suggesting that cfDNA characteristics themselves could be associated with embryo viability.

Given the limited clinical evidence, particularly regarding live birth outcomes, further prospective studies are needed to clarify the relationship between NiPGT-A results and reproductive outcomes.

Study Objectives:

The primary objective of this study is to evaluate the correlation between clinical outcomes and NiPGT-A results in patients with favorable prognosis undergoing single blastocyst transfer.

A secondary objective is to explore criteria for embryo selection that combine NiPGT-A results with embryo morphological assessment.

Study Design:

This study is a prospective observational study conducted at My Duc Hospital (Tan Binh and Phu Nhuan centers) in Vietnam. A total of approximately 200 patients with favorable prognosis undergoing IVF/ICSI treatment will be enrolled. Eligible patients are women younger than 35 years with good ovarian response and at least four good-quality embryos on day 3.

Procedures:

Controlled ovarian stimulation will be performed using a standard antagonist protocol. Oocyte retrieval will be followed by intracytoplasmic sperm injection (ICSI), and embryos will be cultured to the blastocyst stage.

On day 5, the best-quality blastocyst (grade 1 or 2) from each patient will be selected. Approximately 10 µL of spent culture medium will be collected for NiPGT-A analysis without performing embryo biopsy. The embryo will then be cryopreserved using vitrification according to standard laboratory procedures.

Cell-free DNA extracted from the spent culture medium will undergo whole genome amplification (WGA) followed by next-generation sequencing to detect chromosomal copy number abnormalities. NiPGT-A results will be categorized into four groups: (1)No result (WGA failure); (2) Euploid; (3) Mosaic; (4) Aneuploid.

Importantly, NiPGT-A results will be blinded to clinicians and patients during the study and will not influence clinical decision-making. Embryo selection for transfer will be based solely on morphological assessment according to routine clinical practice. Frozen embryo transfer will be performed in a hormonally prepared cycle. Pregnancy testing will be conducted 10-14 days after embryo transfer. Ultrasound confirmation of clinical pregnancy will be performed approximately 3 weeks later. Participants will be followed throughout pregnancy and delivery using the hospital clinical database.

Outcome Measures:

Primary Outcome: Ongoing pregnancy or live birth rate after single blastocyst transfer, compared across the four NiPGT-A result groups.

Secondary Outcomes: Positive β-hCG rate, Biochemical pregnancy rate, Clinical pregnancy rate, Miscarriage rate, and prenatal ultrasound findings, including nuchal translucency measurement.

Statistical Analysis:

Continuous variables will be summarized using mean ± standard deviation or median with interquartile range. Categorical variables will be presented as percentages. Comparisons between groups will be performed using chi-square tests or Fisher's exact tests where appropriate. Statistical significance will be defined as p < 0.05. Data analysis will be conducted using R software (version 4.3.0).

Ethical Considerations:

The study uses spent embryo culture medium that would otherwise be discarded during routine IVF laboratory procedures. Collection of the medium does not involve embryo biopsy and does not interfere with embryo development, cryopreservation, or embryo transfer procedures.

Participation in the study does not alter standard clinical care, and NiPGT-A results are not used for clinical decision-making. All participants will provide written informed consent, and patient confidentiality will be strictly maintained.

Inclusion Criteria:

• Women undergoing in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI).
• Age younger than 35 years at the time of oocyte retrieval.
• Good ovarian response, defined as having at least four good-quality embryos (Grade 1 or Grade 2) on Day 3 or later.
• Undergoing the first or second IVF cycle.
• Normal semen parameters of the male partner.
• Willing to undergo non-invasive preimplantation genetic testing for aneuploidy (NiPGT-A) using spent embryo culture media.
• Agree to vitrification of a single morphologically best-quality blastocyst and to undergo frozen single embryo transfer.
• Able and willing to provide written informed consent.

Exclusion Criteria:

• Presence of untreated uterine abnormalities, including uterine malformations or significant intrauterine pathology.
• History of uterine surgery.
• History of ovarian surgery.
• Known metabolic disorders.
• Use of donor oocytes.
• In vitro maturation (IVM) cycles.
• Participation in another clinical study during the study period.
• Patients who do not undergo frozen single embryo transfer.

The study population consists of women with good reproductive prognosis undergoing in vitro fertilization (IVF) with intracytoplasmic sperm injection (ICSI) at My Duc Tan Binh Hospital and My Duc Phu Nhuan Hospital. Eligible participants are women aged 18 to 34 years with normal ovarian response, undergoing their first or second IVF cycle, and having at least one morphologically good-quality blastocyst available for frozen single embryo transfer.

All participants receive standard IVF treatment according to routine clinical practice. Non-invasive preimplantation genetic testing for aneuploidy (NiPGT-A) is performed retrospectively using spent embryo culture media collected prior to embryo cryopreservation and is not used for clinical decision-making during the study.

Individual participant data (IPD) will not be shared due to concerns regarding participant confidentiality and the sensitive nature of reproductive health data. Although all data collected in the study will be de-identified, institutional and ethical regulations restrict public access to individual-level clinical data. Aggregated results will be reported in scientific publications and presentations.