Reporter and Curator: Dr. Sudipta Saha, Ph.D.
Use of sexed semen in conjunction with in vitro embryo production is a potentially efficient means of obtaining offspring of predetermined sex. Sperm sorting is a means of choosing what type of sperm cell is to fertilize the egg cell. It can be used to sort out sperm that are most healthy, as well as 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. 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).
Sperm sorting utilizes the technique of flow cytometry to analyze and ‘sort’ spermatozoa. During the early to mid 1980s, Dr. Glenn Spaulding was the first to sort viable whole human and animal spermatozoa using a flow cytometer, and utilized the sorted motile rabbit sperm for artificial insemination. Subsequently, the first patent application disclosing the method to sort “two viable subpopulations enriched for x- or y- sperm” was filed in April 1987 and the patent included the discovery of haploid expression (sex-associated membrane proteins, or SAM proteins) and the development of monoclonal antibodies to those proteins. Additional applications and methods were added, including antibodies, from 1987 through 1997. At the time of the patent filing, both Lawrence Livermore National Laboratories and the USDA were only sorting fixed sperm nuclei, after the patent filing a new technique was utilized by the USDA where “sperm were briefly sonicated to remove tails”. USDA in conjunction with Lawrence Livermore National Laboratories, ‘Beltsfield Sperm Sexing Technology’ relies 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.
While highly accurate, sperm sorting by flow cytometry will not produce two completely separate populations. That is to say, there will always be some “male” sperm among the “female” sperm and vice versa. The exact percentage purity of each population is dependent on the species being sorted and the ‘gates’ which the operator places around the total population visible to the machine. In general, the larger the DNA difference between the X and Y chromosome of a species, the easier it is to produce a highly pure population. In sheep and cattle, purities for each sex will usually remain above 90% depending on ‘gating’, while for humans these may be reduced to 90% and 70% for “female” and “male” spermatozoa, respectively. Some approaches to in vitro fertilization involve mixing sperm and egg in a test tube and letting nature take its course. But in about half of all infertility cases, a problem with the man’s sperm may require a more direct method. In these cases, a different process, called intracytoplasmic sperm injection (ICSI), in which a single sperm cell is injected directly into an egg, is sometimes used. With this one-shot opportunity, it’s important to choose a sperm cell with the best potential for success. A team at the University of Edinburgh, Scotland, has now announced a new technique to ensure that the best sperm win: analyzing their DNA for potential damage beforehand, and choosing those that are structurally sound.
To optimize success rates of IVF, selection of the most viable embryo(s) for transfer has always been essential, as embryos that are cryopreserved are thought to have a reduced chance of implanting after thawing. Recent developments challenge this concept. Evidence is accumulating that all embryos can now be cryopreserved and transferred in subsequent cycles without impairing pregnancy rates or maybe even with an improvement in pregnancy rates. In such a scenario no selection method will ever lead to improved live birth rates, as, by definition, the live birth rate per stimulated IVF cycle can never be improved when all embryos are serially transferred. In fact, selection could then only lower the live birth rate after IVF. The only parameter that could possibly be improved by embryo selection would be time to pregnancy, if embryos with the highest implantation potential are transferred first.
In the majority of human IVF cycles multiple embryos are created after ovarian hyperstimulation. The viability of these embryos, and as a consequence the chance for an embryo to successfully implant, is subject to biological variation. To achieve the best possible live birth rates after IVF while minimizing the risk for multiple pregnancy, one or two embryos that are considered to have the best chance of implanting are selected for transfer. Subsequently, supernumerary embryos with a good chance of implanting are selected for cryopreservation and possible transfer in the future while remaining embryos are discarded.
The best available method for embryo selection is morphological evaluation. On the basis of multiple morphological characteristics at one or several stages of preimplantation development, embryos are selected for transfer. However, with embryo selection based on morphological evaluation implantation rates in general do not exceed 35%, although varying results have been reported. This has resulted in a strong drive for finding alternative selection methods.
The best studied alternative selection method is preimplantation genetic screening (PGS). The classical form of PGS involves the biopsy at Day 3 of embryo development of a single cell of each of the embryos available in an IVF cycle and analysis of this cell by fluorescence in-situhybridization (FISH) for aneuploidies, for a limited number of chromosomes. Only embryos for which the analyzed blastomere is euploid for the chromosomes tested are transferred. Although this method of PGS has been increasingly used in the last decade, recent trials show that it actually decreases ongoing pregnancy rates compared with standard IVF with morphological selection of embryos.
In an effort to overcome some of the drawbacks of PGS using cleavage stage biopsy and FISH, new methods to determine the ploidy status of a single cell are developed, such as comparative genomic hybridization arrays or single nucleotide polymorphism arrays. Furthermore, in an attempt to avoid the confounding effects of chromosomal mosaicism, embryos are now biopsied at either the zygote or blastocyst stage. In addition, increasing time and money are invested in the development of high-tech, non-invasive methods to select the best embryo for transfer in IVF.
This Include metabolomic profiling, amino acid profiling, respiration-rate measurement and birefringence imaging.
- In metabolomic profiling, spectrophotometric tests are used to measure metabolomic changes in the culture medium of embryos;
- in proteomic profiling, proteins produced by the embryo and released into the culture medium are identified;
- in amino acid profiling, amino acid depletion and production by the embryo is assessed using the culture medium;
- in respiration-rate measurement, the respiration rate of embryos is assessed; and
- in birefringence imaging, polarization light microscopy is used to assess the meiotic spindle or the zona pellucida.
Embryo donation (also known as embryo adoption) is the compassionate gifting of residual cryopreserved embryos by consenting parents to infertile recipients. At present, only a limited number of such transactions occur. In 2010, the last year for which U.S. data were available, fewer than 1000 embryo donations were recorded. These acts of giving, unencumbered by federal law, are being guided by a limited number of state laws. Moreover, the practice is sanctioned by professional societies, such as the American Society for Reproductive Medicine, subject to the provision that “the selling of embryos per se is ethically unacceptable.” As such, the not-for-profit donation of existing embryos by consenting parents comports with a triad of commonly held ethical attributes. First, donated embryos are not sold for profit. Second, donated embryos are (by original intent) generated for self-use. Third, donated embryos are the product of an unambiguous parental unit and as such are transferable. All told, embryo donation constitutes an established if limited component of present-day assisted reproduction.