**Abstract**

Preimplantation genetic testing (PGT) is widely adopted to select embryos with normal ploidy but requires invasive embryo biopsy procedures. Therefore, noninvasive PGT (niPGT) detection of cell-free DNA (cfDNA) in blastocyst culture medium has gradually become a hot area in the field of assisted reproduction. This chapter will systematically summarize how researchers use embryonic cfDNA to conduct niPGT detection worldwide. It will also thoroughly review the factors that affect the accuracy of the test and its underlying issues, as well as prospective applications. We hope to provide a useful reference for the standardized operation of non-invasive PGT that can be widely applied in clinical practice.

**Keywords:** niPGT, Spent Culture Media, cfDNA, Aneuploid

### **1. Introduction**

In vitro fertilization and embryo transfer (IVF-ET) is an effective method for the treatment of infertility, yet it still has a relatively low success rate [1]. Furthermore, the multiple pregnancy rate remains high as a result of multiple embryos being transferred, which can increase the chances of adverse pregnancy outcomes and affect the health of both the mother and children [2]. Elective single embryo transfer (eSET) is the most effective way to reduce the rate of multiple pregnancies and is increasingly used worldwide [3]. Nonetheless, the success rate of single embryo transplantation is not satisfactory, mainly due to the lack of a systematic approach for evaluating the conceivability of embryonic development. Presently, embryonic morphological assessment is still the most commonly used method, but this method has many problems due to the lack of quantifiable indicators and its susceptibility to subjective factors of laboratory embryologist [4–6].

Studies have shown that there is an approximately 40%–60% risk of chromosomal aneuploidy even before embryo implantation [7–9]. PGT-A can be used to identify embryos with chromosomal aneuploidy, thereby improving clinical outcomes in IVF patients [10–13]. Nonetheless, PGT-A relies on embryo biopsy, and its invasive biopsy approaches may increase its technical limitations for the following reasons: (1) it may have an influence on the quality and the level of development of the embryo [14]; (2) it may increase the chance of abnormal epigenetic modifications [15, 16]; (3) it has high requirements on the environment and operating instruments, and there is a potential risk of sampling failure; and (4) the accuracy of test results may be affected by mosaicism. Consequently, it is necessary to establish an optimal selection procedure in line with the morphological assessment for embryos that truly reflects the chromosome ploidy status of the embryo, avoids invasive operations, and does not require expensive equipment. Recently, several studies have found the presence of cfDNA in the culture medium and blastocyst fluid of embryos [17–21]. Noninvasive PGT-A detection through cfDNA has become a growing niche in the field of assisted reproduction. In this chapter, we will comprehensively review the advancements and attempts at noninvasive PGT-A using foetal cfDNA and discuss the pros and cons as well as insights into its clinical applications in IVF-ET.
