Non-invasive prenatal testing (NIPT)
Sperm chromatin fragmentation tests (TUNEL and SCSA)
With the advancement of the ICSI technique, the crucial role of sperm in embryonic development became evident. Extensive research in this area has led to the use of functional sperm tests and the correlation of their results with fertility. The status of sperm chromatin is one of the topics that has been studied more extensively, as sperm not only plays a role in egg activation but also contributes half of the genome to the next generation. The health of this genome is of paramount importance for embryonic development. In this context, assessing sperm health is of particular significance. Half of the reasons for this importance can be attributed to the following
- There is an inverse relationship between sperm parameters and DNA damage
- Increased DNA damage in infertile individuals
- There is an inverse relationship between fertilization rates and DNA damage
- Increased miscarriage rates in cases with high DNA damage
Therefore, given the importance of sperm DNA health in fertility, this section will discuss methods of examining chromatin structure.
SCSA technique
The SCSA technique is based on the sensitivity of chromatin and DNA structure when exposed to acid and the metachromatic properties of acridine orange. This fluorochrome intercalates between the two DNA strands and emits green fluorescence when the DNA is intact. However, when the fluorochrome binds to single-stranded DNA, it emits red-orange fluorescence. Stained sperm are then analyzed using flow cytometry. Researchers frequently employ this method in clinical studies to determine an individual’s fertility potential.
TUNEL technique.
This method was initially employed to detect apoptotic sperm populations in ejaculate samples. It can identify breaks in either one or both DNA strands. In this technique, labeled nucleotides target the end nucleotides of broken DNA strands. This reaction is catalyzed by a terminal transferase enzyme that incorporates modified deoxyuridine with biotin or digoxigenin at the 3′-OH end of the broken strand. Subsequently, the modified nucleotides, which are linked to a fluorescent marker, become visible. The nucleotide is directly labeled with a fluorophore. To detect fluorescence in sperm, a fluorescent microscope or flow cytometer can be used. The observed fluorescent color in each sperm indicates a break in one or both DNA strands.