Sperm sorting

Sperm sorting is a means of choosing what type of sperm cell is to fertilize the egg cell. Several conventional techniques of centrifugation or swim-up. Newly applied methods such as flow cytometry expand the possibilities of sperm sorting and new techniques of sperm sorting are being developed. Sperm sorting is a means of choosing what type of sperm cell is to fertilize the egg cell. Several conventional techniques of centrifugation or swim-up. Newly applied methods such as flow cytometry expand the possibilities of sperm sorting and new techniques of sperm sorting are being developed. It can be used to sort out sperm that are most healthy, as well as for determination of more specific traits, such as sex selection in which spermatozoa are separated into X- (female) and Y- (male) chromosome bearing populations based on their difference in DNA content. The resultant 'sex-sorted' spermatozoa are then able to be used in conjunction with other assisted reproductive technologies such as artificial insemination or in-vitro fertilization (IVF) to produce offspring of the desired sex - in farming animals but also in human medical practice. Several methods have been used to sort sperm before the advent of flow cytometry. Density gradient centrifugation (in a continuous or discontinuous gradient) can concentrate semen samples with low concentration of sperm, using the density of sperm as a measure of their quality. Similarly, so-called swim-up techniques apply a centrifugation step and then sperm is allowed to swim up into a medium, thus enriching a subpopulation of motile sperm. However, use of sperm centrifugation is detrimental to the sperm viability and elicits production of reactive oxygen species. Conventional techniques are routinely used in assisted reproductive technology. Flow cytometry is another method used to sort sperm and adaptations of this technique opens new opportunities in sperm sorting. However, because flow cytometry-based sperm sorting often uses fluorescent dyes that often stain DNA, the safety of this technique in human reproductive medicine is a matter of scientific discussion. However, flow cytometry is the only currently used technique able to determine the sex of future progeny by measuring DNA content of individual sperm cells. It evaluates if they contain the larger X chromosome (giving rise to a female offspring) or smaller Y chromosome (leading to male progeny). It then allows separation of X and Y sperm. The so-called Beltsfield Sperm Sexing Technology was developed by USDA in conjunction with Lawrence Livermore National Laboratories, relying on the DNA difference between the X- and Y- chromosomes. Prior to flow cytometric sorting, semen is labeled with a fluorescent dye called Hoechst 33342 which binds to the DNA of each spermatozoon. As the X chromosome is larger (i.e. has more DNA) than the Y chromosome, the 'female' (X-chromosome bearing) spermatozoa will absorb a greater amount of dye than its male (Y-chromosome bearing) counterpart. As a consequence, when exposed to UV light during flow cytometry, X spermatozoa fluoresce brighter than Y- spermatozoa. As the spermatozoa pass through the flow cytometer in single file, each spermatozoon is encased by a single droplet of fluid and assigned an electric charge corresponding to its chromosome status (e.g. X-positive charge, Y-negative charge). The stream of X- and Y- droplets is then separated by means of electrostatic deflection and collected into separate collection tubes for subsequent processing. Another cytometric technique used in sperm sorting is magnetic-activated cell sorting (MACS) which is routinely applied in assisted reproduction hospitals to sort out sperm with fragmented DNA. This is achieved using antibodies to surface markers of programmed cell death (apoptosis) such as annexin V, coupled with magnetic beads. Following the binding of these antibodies, spermatozoa which undergo apoptosis are sorted by applying magnetic field to the sperm suspension. MACS obviates the need for fluorescent DNA binding molecules. DNA damage in sperm cells may be detected by using Raman spectroscopy. It is not specific enough to detect individual traits, however. The sperm cells having least DNA damage may subsequently be injected into the egg cell by intracytoplasmic sperm injection (ICSI). Many other methods for sperm sorting have been proposed or are currently tested.