Thesis Statement:

The method of sorting cryopreserved sperm for the purpose of separating the X and Y chromosomes has been an ongoing process for many years. However, recently this method has become a modern preconception technique in which numerous applications for safe and effective selection procedures now exist in clinical practices. I have chosen to explore how currently these uses have broadened to areas where many of us might see as a moral or ethical issue. These uses might even be linked to a larger scheme of pre-embryonic chromosome attenuation such as the Human Genome Project.

Outline:

I General History of the Sperm Sorting Technique
           A. First used in sex selection of farm animals
           B.Currently used with human pregnancy
           C.Two general types of segregation of sperm

II Sex Selection of Spermatozoa by Chromatin Differences
           A.Differences in Chromatin
           B.Genetics and IVF Institute

III Sperm Sorting Technique
           A. Hoechst 33,342
           B. Flow Cytometer
           C. Intra-ovaductal Insemination or In-vitro Fertilization

IV Continuing Research
           A. Embryo biopsy
           B. Sperm Motility
           C. Use of (FISH)
           D. Use of Percoll

V Clinical Example of a Sex-Linked Recessive Trait Disorder

VI Possible Future Repercussions
           A.Trend Toward Parental Control
           B. Implications in the Human Genome Project

VII The Ethical Debate
          A.Who Should Have Access?

VIII The Change from "Treatment" to "Prevention"

IX Conclusion

As we approach the new Millennium, science continues to make greater advancements in the areas of fertility and genetics. Many heated discussions have taken place in this country over the course of the past decade regarding the topic of artificial sex selection. These scientific advancements have grown out of the public’s desire to have more control over creation. It is no longer acceptable to let nature decide crucial factors. Many parents now desire a way to manipulate genetic factors so that they can choose the sex of their unborn child, or possibly even other traits.

In the early 1980’s, the United States Department of Agriculture first introduced sex selection of farm animals. Methods claiming to achieve sex selection have existed for many years. Numerous applications for safe and effective selection procedures exist in current clinical practices, as sex-linked conditions could be theoretically eliminated by use of appropriate sperm for fertilization or insemination. Recently, the sperm-sorting techniques developed by the USDA were refined for use in human pregnancy (3).

There are generally two types of sperm sorting techniques. The first type involves segregating sperm on the basis of physical or kinetic features such as size and weight. A preferable technique is one that relies on distinctive nuclear characteristics unique to either X or Y-chromosomes bearing sperm (9). It is thought that the most logical and cost effective way to separate X and Y sperm on a large scale would be to use a sperm surface marker that could be detected with an antibody (3). However, a surface marker distinguishing X and Y sperm has yet to be found.

Sex Selection of spermatozoa by chromatin differences is the reliance on distinctive nuclear characteristics unique to either X or Y. This technique has been shown to achieve significant enriched populations of X or Y chromosome bearing sperm (9). This is because X-chromosomes contain 2.8 percent more DNA than the Y chromosomes (2). With this information, researchers at the Genetics and IVF Institute in Fairfax, Virginia have announced a sperm-sorting technique that almost guarantees that a girl will be conceived(2 ). This is an improvement over the natural 50/50 chance of conceiving a girl, which would for example decrease the odds for sex-linked disorders.

The current technique used in the separation is to treat the sperm with a fluorescent marker, Hoechst 33,342, which attaches in proportion to the amount of DNA present in the sperm head. Then the sperm are put through an instrument called a flow cytometer where the cells travel through a laser beam that generates ultraviolet light. Excited by the ultraviolet light, the sperm heads give off different amounts of light according to how much dye they have absorbed. The X sperm glow brighter because they have more DNA. Then, based on the brightness, each sperm then gets shunted to the appropriate tube attached to the machine.

Approximately 250,000 sperm can be sorted in one hour. Analysis of the sorted sperm using DNA probes that show the purity of the tube samples can reach 80% (3). Furthermore, in a similar study, researchers found that sorted X and Y bearing spermatozoa were found to maintain their viability for several hours after sorting, which is essential in coordinating the next step of the process, which is either intro-ovaductal insemination or in-vitro fertilization.

In 1998, an important study regarding the results and repercussions of sex selection was conducted. Reproductive biologist, Edward F. Fugger and his team of colleagues at the Genetics and IVF Institute began their study by recruiting 119 couples who wanted a baby girl. Of these women, 29 became pregnant using sorted sperm. Eight of these women had miscarriages. At the time of publication, twelve women had ongoing pregnancies and nine women had already delivered eleven babies, including two sets of twins. Of the fourteen pregnancies in which the gender had been determined, thirteen were girls. The researchers had identified no safety concerns in the published study. "All of the babies born have been healthy," Fugger said. "That doesn’t mean that all of the risk has been excluded. There’s a lot that’s not known" (5). It appears that within this study the only area uncovered is future repercussions. Unfortunately the researchers as well as the public will have to wait some time for those results. Therefore the question remains, do we go ahead and continue with the procedure based solely on available data? Many people are still left disconcerted.

Due to the time pressure and curiosity, researchers have been involved in intense experimentation on cells processed by flow cytometry. A closely related study was recently done to determine the purity and efficiency of this technique in hopes to detect possible future implications. The sperm sorting procedure was followed by in-vitro fertilization in which an embryo biopsy and a nested polymerase chain reaction (PCR) test were conducted to determine the purity and karyotype of the sample. This study found by multiplex PCR analysis that the embryos were female and the embryo transfer resulted in a karyotypically normal female fetus (12).

Before work even began with a human subject, concerns had been raised about using the fluorescent marker and ultraviolet light on human sperm cells. Letters to the editor of the journal Human Reproduction last year suggested that the combination of the two might lead to mutagenesis. Technology developer, Lawrence A. Johnson of the United Stated Department of Agriculture in Beltsville, Maryland comments, "The birth data on cattle, swine, rabbits, and sheep has all been devoid of any hint of abnormality." He added, however, that concerns about mutagenesis "can never be discounted from a theoretical standpoint, because certain cell types studied react differently to the stain than others" (3).

Research is still continuing today to make the process of sperm sorting selection more efficient. Recently, researchers in the Netherlands have tackled one aspect of the sperm sorting technique. It was found that with the use of cryopreserved X and Y sperm during flow cytometric analysis, there is a broad distribution without a distinct X and Y peak (10). Their results indicated that these problems are mainly caused by the large amount of dead sperm normally present in sperm populations. Therefore, these researchers have found that with the addition of the drug Percoll, the fluorescence is quenched in the chromatin stained with Hoechst 33,342 in the dead sperm. Viable populations then improved from 30-45% to 85-92% indicating that cryopreserved sperm can now be used to reach equally high efficiency as noncryopreserved sperm. There have also been many studies done on the effects of sperm sorting. In one study done on the "Comparative motility of X and Y chromosome," researchers used computer assisted sperm analysis (CASA), which measures sperm motility, to determine that the sperm are unaffected by the DNA binding dye Hoechst 33.342, the ultraviolet light, or the physical process of cell sorting (6).

Case Study

In 1962, five year-old Bobby Massie developed uncontrollable bleeding in his left knee, a symptom of his Hemoplilia A, an inherited clotting disorder. It took 30 transfusions of plasma over the next three months to stop the bleeding. Because the knee joint had swelled and locked into place during that time, Bob was unable to walk for the next seven years. Today, Bobby still suffers from painful joint bleeds but he injects himself with factor VIII, the coagulation protein that his body cannot make. The factor VIII soon controls the bleeding. Bobby’s parents decided against having more children due to the possibility of passing on this disorder (8).

Hemophilia is a sex-linked recessive trait defined by the absence of a certain protein required for blood clotting. Hemophiliacs bleed excessively when injured, and the most seriously affected individuals may bleed to death after relatively minor skin abrasions, bruises, or cuts (1).

In humans the term sex-linked usually refers to x-linked characters. In addition to their role in determining sex, the sex chromosomes have genes for many characteristics unrelated to sex. If a sex-linked trait is due to a recessive allele, a female will express the phenotype only if she is a homozogote. Because males have only one locus, the terms homozygous and heterozygous lack meaning for describing their sex-linked genes (the term hemizygous is used in such cases). Any male receiving the recessive allele from his mother will express that trait. For this reason, far more males than females have disorders that are inherited as sex-linked recessives.

With an awareness of the pattern of inheritance in sex-linked recessive disorders, it is understandable why the Massies would have made the decision they did regarding their family planning. However, what if the Massies had the choice of having another natural child- a child that had a natural combination of both the parents’ genetic information but could avert the inheritance of any sex-linked recessive disorders? What decision would the Massies have made then? Perhaps we should change the scenario a little. Let’s change the disorder to Huntington’s disease. Huntington’s disease is a bit more extreme than hemophilia in that it is inevitably fatal. Huntington’s disease is the product of a gene transmitted in an autosomally dominant inheritance pattern ? in other words, a gene that occurs on one of the twenty-two non-sex human chromosomes and whose effect dominates its normal partner (14). It is entirely penetrant, which means that if a gene-carrier lives long enough, the disease is inexorably expressed (14). Clearly the use of sperm sorting could have been an option for the Massies and their child with Hemophilia, but unfortunately the technology was unavailable at that time. What choice would you have made? Unfortunately the sperm sorting technique is not an option for Huntington’s disease but what if there was a similar option available?

Now that this technique is readily available, this procedure is being used for other purposes such as "family balancing." Many people believe that they need to structure the birthing of certain gender children to fit society’s preconceived ideas about family structure, such as having one boy and one girl. The questions are whether or not this is ethical, and who should be allowed to utilize this procedure. This is the debate that rests in the minds of many people today.

The trend toward more parental control over a child’s characteristics will increase in the future, warns biomedical ethicist Thomas H. Murray, the director of the Center for Biomedical at Case Western in Cleveland (5). Murray notes that scientists working on the Human Genome Project will soon have methods of identifying disease-causing genes as well as the DNA that produces such characters as hair color, height, athletic ability, and perhaps some behaviors (5). Where will we draw the line? There are teams developed for the singular purpose of deciding these issues in the Human Genome Project (HGP) however, once you know the facts how will you decide?

With power such as this we have to wonder, what will tomorrow’s society be like? There is a large possibility that when this procedure becomes more public and common, we could alter balances in the population. For example, who will be able to afford a procedure such as this? Perhaps in the beginning only the rich will have access due to the governments’ inability or unwillingness to fund such a treatment for all classes of people. This could create ripple effect in the population where the rich eventually become not only the richest but also the smartest, most athletic, attractive and powerful people on earth. Perhaps this is a little extreme but without regulations and guidelines to procedures such as this, a great advancement in medical science designed to enhance the quality of life could turn into a catastrophic event within the human population.

A second major issue is the most common moral issue for most people being, are we involving ourselves too much in the processes of creation? This question is difficult to answer however, a possible way to understand advancements in science today is to look at advancements in the past. Decades ago the study and practice of medicine was exactly that, a process and goal to treat illness with medication. Did society fear it then? Yes, society did not approach the habit of medication to cure or reliance on surgery to repair their ailments with open arms - it took years to gain acceptance. We continue to look at medical treatment with the intention to medicate for the alleviation of pain from illness. However, we neglect to see the possibility of approaching treatment as prevention. Prevention is simply a state of preparation. As many people have noticed with the example of antibiotics that the development of medicine for treatment is becoming increasingly difficult. Perhaps we will never find a "cure" for diseases such as Hemophilia or Huntington’s disease. Maybe there is no hope for treatment with medication. Perhaps the only hope is finding and eliminating these diseases before they ever evolve in future generations.

Artificial sex selection is certain to be a heated topic in the future. As more advancements continue to be made, no one is able to predict where these types of advancements will lead. These are decisions that we as a society have to weigh carefully to determine what will bring about the most good for the overall population. Perhaps we need to realize the possibility that conventional medicine as we know it could be at its peak. If that is the case, we could be opening a door to a greater era of medical science where the study is no longer treatment but prevention.

As we approach the millennium current techniques are being modified to be safer and more effective while new advancements are being made daily. As medical science continues to more forward, researchers develop more and more unanswered questions which brings about the realization of just how endless the possibilities really are.

The current method of sex selection is only the tip of the iceberg when we are discussing treatment as prevention. As we look into similar research with similar goals we find the Human Genome Project (HGP). Within the Human Genome Project researchers are attempting to identify and map the location of every gene within the human body. The implications of this information are boundless. Within the lifetime of many of us we will see genetherapy occurring in humans at both the adult and preembryonic level. The potential for using genes themselves to treat disease ?known as gene therapy, is the most exciting application of DNA science. It has captured the imaginations of the public and the biomedical community for good reason. Although still in its infancy, this rapidly developing field holds great potential for the treating or even curing genetic and acquired diseases, using normal genes to replace or to bolster immunity to disease. For example, by adding a gene that suppresses tumor growth (13). Recently researches have found that there is a possibility with Huntington’s disease that determining critical chemical pathways affected by the gene defect may prove more amenable to intervention than the HD gene itself (14).

As we have seen, "Artificial Sex Selection" is only a thread of the ribbon that envelops the world of future creation by gene therapy. Nancy S. Wexler has written an excellent article entitled "Clairvoyance and Caution", which deals with possible repercussions from the Human Genome Project. In it she says, "For a while we may have the worst of all possible worlds ? limited or no treatments, high hopes and probably unrealistic expectations ? everything to challenge our inventiveness and stamina. But these ingredients will be, I hope, catalysts for change. The stakes are high, the payoff is high" (14).
 
 

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