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6 Progress in Research The absence, since 1980, of the Ethics Advisory Board has, in effect, put a moratorium on federal funding for research on many aspects of reproduction. As a result, basic research studies in this field are few and many questions remain unanswered. As noted in earlier chapters, the lack of basic information has slowed the development of contraceptives and the improvement of infertility treatments. We still do not understand important aspects of the human reproduction system, the process of fertilization, the type of gene activity that occurs as a fertilized egg divides, and the best way to assay embryonic cells for the range of chromosomal and genetic defects that cause disease. In this chapter are described three examples of research areas that hold much promise: the role of the brain in reproduction, new methods of diagnosing genetic diseases, and research on embryo health. ROLE OF THE BRAIN TN REPRODUCTION Research makes it clear that the brain governs most aspects of repro- ductive functioning. The hypothalamus appears to be the command center that governs the action of several hormone-driven loops that are part of the operation of the reproduction system. The reproduction processes of both sexes in all vertebrates, down to the fishes, are the consequence of a cascade of neuroendocrine events initiated in the hypothalamus. 126

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PROGRESS IN RESEARCH 127 The discovery that ovulation depends on the regularity of the hy- pothalamic stimulation of the pituitary gland has had immediate applica- tion to patient treatment. Working with monkeys, Dr. Ernst Knobil, of the University of Texas, found that the ovulatory cycle is controlled by the regularity with which the hypothalamus stimulates the pituitary gland to release gonadotropins into the circulatory system. If the pattern is altered in any way, ovulation stops within a week or two. If the regular pulses of hormone are re-established, normal ovulation begins again. Today ovulation is being restored to an increasing number of women by means of the timed delivery of a key hormone. The hypothalamus is affected by nerve and hormonal inputs from other parts of the body. When these inputs are in the normal range, the hypothalamus responds by producing pulses of gonadotropin-releasing hormone (GnRH), which stimulate the pituitary gland into releasing luteinizing hormone (LH) and follicle-stimulating hormone (ESH), the gonadotropin hormones. The gonadotropins, in turn, act on either the testes in the male, regulating sperm production, or the ovaries in the female, regulating the ovulatory cycle. The hypothalamus controls the body's ability to reproduce. In all ani- mals the timing of pregnancy is tailored by biological evolution. In some mammals, including humans, the hypothalamus integrates information about body weight, stress, exercise, and overall health. If these inputs indicate that a female is well nourished and able to sustain the stress of pregnancy and lactation, the hypothalamus secretes gonadotropin- releasing hormone in regular pulses, and regular ovulatory and menstrual cycles begin. Many modern women experience amenorrhea, the absence of menstruation, as a result of physical and psychological stresses. A study in Sweden found that 3 to 4 percent of women suffer amenorrhea for three months or longer during the course of a year in response to venous stresses. Amenorrhea also occurs when the female body becomes too lean through starvation, rigorous exercise, or eating disorders. Through its control of important hormones, the hypothalamus is the ultimate controller of the onset of egg and sperm development. In the male and in the female, this part of the reproductive system is complete and functional at birth but soon afterward becomes dormant. Then, some months before puberty, a still unknown mechanism gradually activates the reproduction-controlling function of the hypothalamus. When the pulses of gonadotropin-releasing hormone reach the pituitary at the proper

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128 SCIENCE AND BABIES frequency, the pituitary responds by producing gonadotropins. Menstrual cycles begin in girls, and in boys sperm production gets under way. Dr. Knobil noted that a variety of factors can negatively influence the pulses of GnRH. Opiates like morphine can slow or stop the pulses entirely. Stress, habitual strenuous exercise, and malnutrition, including anorexia nervosa, also affect it, halting menstruation and ovulation. Research by Drs. Stephanie Jofe and William F. Crowley, of the Harvard Medical School, found that in 1,600 women (physicians, post- doctoral researchers, and medical students) the incidence of amenorrhea was approximately 3 percent, the same as that for women in other occu- pations. The shutdown of hypothalamic function in most of these women could be explained by a poor state of nutrition, a recent illness, a program of rigorous exercise, or grief. The ordinary stresses and strains of life did not appear to be a major factor in causing hypothalamic amenorrhea, Dr. Crowley notes. According to Dr. Crowley's studies, the hypothalamus produces bursts of GnRH in a pattern that varies during the ovulatory cycle. In women whose cycles are normal, the rate of these pulsations speeds up before ovulation and then slows down dramatically until menstru- ation, when it largely ceases. To restore normal ovulation to women with hypothalamus-based amenorrhea, Dr. Crowley and his team treated them with round-the-clock doses of GnRH. The hormone was given in- travenously in 4- or 5-microgram pulses via a beeper-size pump worn by the patient. The pump was programmed to deliver GnRH in pulses that ranged in frequency from one every 60 or 90 minutes or once every 4 to 6 hours, a pattern that mimics the natural timing of the hypothalamus. By copying the normal rhythm, Dr. Crowley achieved a 93 to 95 percent success rate in restoring normal ovulation to more than 100 women with amenorrhea. At present, this treatment is available from medical centers only for primary amenorrhea; approval for treatment of other forms of the disorder is expected from the Food and Drug Administration by late 1990. Not all scientists agree about the importance of keying the changes in the pulsing rate exactly to those that occur naturally, but they do agree that the critical feature in the reproductive hormone network is the delivery of GnRH into the blood circulation in small regular bursts. Some clinicians are able to restore ovulation by administering GnRH every 60 or 90 minutes; the results improve, according to Dr. Crowley, when the treatment most closely follows the innate rhythm of the hypothalamus.

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PROGRESS IN RESEARCH 129 Males have the same network of neurons in the hypothalamus that integrate nerve and hormone signals from the rest of the body and pro- duce GnRH in a regular pulsating pattern, stimulating the pituitary to release the LH and FSH gonadotropins. In males these gonadotropins drive the production of the male sex hormones and, in turn, of sperm. If the hypothalamus is influenced to stop secreting pulses of GnRH en- tirely or if secretion becomes irregular, sperm development is stopped or reduced. Tony M. Plant, of the University of Pittsburgh, is looking for an association among a particular pulsatile pattern of gonadotropin secre- tion, a change in the important ratio between LH and FSH, and reduced sperm count. What remains to be known is the exact morphologic and physiologic nature of this neuronal system: what starts the firing, what controls its duration, and what stops it as abruptly as it began. DIAGNOSING GENETIC DISEASES The increasing ability of medical scientists today to diagnose genetic disorders is making it possible for more couples who may carry the genes for an inherited disease to give birth to healthy babies. Before medical scientists were able to detect the presence of such genes, couples who risked passing on an inherited disease often chose not to have children. Today, thanks to tests that reveal whether a person is or is not a carrier for a genetic disorder or whether or not a fetus has the disease, couples can make informed decisions about having a child. Genetic diagnosis includes identifying adults who might pass along inherited disorders to their children, screening newborn babies for con- genital diseases that would be disabling without an intervention, and detecting genetic disorders in the fetus. Tests include examining chro- mosomes for structural abnormalities and identifying genetic diseases in infants by detecting the presence or absence of certain chemicals in the body. An example of the latter is the use of a blood test to identify ba- bies born with phenylketonuria (PKU). Such children cannot metabolize the amino acid phenylalanine, which accumulates in the blood and pre- vents the brain from developing normally. A special diet averts mental retardation. More recently, genetic diseases are being identified more directly with analyses of DNA, that can reveal the absence of a gene or the presence of a faulty gene. In PKU, for instance, the defective gene is located on chromosome 12. These new techniques are making it increasingly possible to diagnose genetic diseases prenatally.

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., ~ :~: ~ ~ ~~ . *~ . ~ . .~ it:: ~ - :: ~:~ ~ ~ ~ ~ S:,ik ~ ~~ ~ `^v<~ :~ ~ :: ~ , In: t: : A ~ ,,, 6 ::~} , - ,~ ~ :: .. : ~ ~ .~ . ~~ : . A_ ~6 , .: My,,, .:. 'I ? . ^~ .,., :.t SCIENCE AND BABIES :0 i .. .~.. . : - ?.. ... Ad AX . . Researchers study the results of a DNA analyzing technique to find a defective gene sequence. Credit: National Institute of Child Health and Human Development On the horizon are techniques for examining embryos before they are artificially implanted for chromosomal abnormalities and defective genes. If procedures to diagnose genetic and chromosomal defects in early-stage embryos are developed successfully, couples at risk for passing on a genetic disease could be offered in vitro fertilization as a way to have a healthy child. If such technology becomes available, an embryo's chromosomal makeup could be analyzed in advance and only embryos with normal chromosomes and genes would be transferred to the uterus. HOW GENETIC DISEASES OCCUR In the cells of every living thing are genes that, working together, direct the structure and function of every type of cell. Genes are the code, or set of instructions, by which an individual cell replicates and produces certain enzymes and other chemicals vital to the development and function of each individual living thing, whether it is a corn plant or a human being. Although cells may have specialized functions intestinal cells make mucus and heart cells contract rhythmically their basic components are similar. Each cell has an operating center, or nucleus, and in that nucleus

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PROGRESS IN RESEARCH 131 are the chemical compounds we know as chromosomes, the genetic material we inherit from our parents that determines how the cell will function. The most important component of a chromosome is DNA, deoxyribonucleic acid. DNA is a long, twisted, double chain of chemical compounds called nucleotides. Genes are sequences of nucleotides. Some genes are formed of relatively short sequences; other genes are very long. The DNA of each chromosome is composed of thousands of genes. Human cells contain 46 chromosomes that look like short bits of fine thread; during cell division the chromosomes form into 23 pairs. Half of each pair contains genes from the mother; the other half comprises genes inherited from the father. As a cell divides, the chromosomal DNA replicates, so the new cell will contain the same genetic instructions as the original. Sometimes as a cell divides, one of the gene sequences is deleted or altered. Scientists suspect that every human being inherits about. 20 altered, or mutated, genes that may have the capacity to harm. A single altered or missing gene may not cause a noticeable problem, because the child has a normally functioning gene on the companion chromosome inherited from his other parent. But if the mutation is transmitted to a child and that child also receives the same mutated gene from the other parent, severe and sometimes fatal diseases can result. This rarely occurs in a heterogeneous population, but in small closed societies where there is a lot of intermarriage, the odds are substantially increased and a gene mutation can eventually become quite prevalent. Today a number of inherited diseases are associated with certain ethnic groups. Thalassemia, for instance, is a hereditary, often fatal, blood disease common to people of Mediterranean descent, chiefly Italian and Greek. It includes different forms of anemia. In severe cases, children appear healthy at birth but soon become listless, pale, and prone to infections. They grow slowly. The only treatment is frequent blood transfusions, which eventually lead to iron accumulations in the organs and early heart failure. A person who carries the gene for thalassemia will have one normal gene and one thalassemia gene. He or she generally will have no symptoms or only mild symptoms of the disease. But if both parents are thalassemia carriers, there is a two in four chance that their child will inherit one normal and one thalassemia gene and be a cattier for the disorder. Moreover, there is a one in four chance the child will inherit either both normal genes or both thalassemia genes. If the latter occurs, the child will have a severe form of thalassemia.

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132 SCIENCE AND BABIES DIAGNOSIS BY ANALYZING DNA In recent years, as scientists have learned how to locate defective genes among the thousands that compose DNA, a highly sophisticated field of diagnostic testing has come into existence. One of the many hereditary biochemical disorders of metabolism is Lesch-Nyhan syn- drome, in which the body does not produce an essential enzyme. This syndrome is characterized by severe mental retardation, stiff limbs, and self-mutilation. Although the boys (Lesch-Nyhan is a sex-linked disease) born with this disease may live for many years, they often must be re- strained to prevent them from severely hurting themselves. Today cells taken from the amniotic fluid during pregnancy can be examined for the gene mutation responsible for this disease, allowing parents to decide whether they want to carry the pregnancy to term. Most genetic tests are performed on an individual basis for persons who know or suspect they are at risk for passing along a disease. Or such tests may be part of a hospital-based genetic counseling service. Like genetic tests, prenatal tests are done on an individual basis when parents fear their offspring might be born with a serious disability or fatal illness. For some diseases, however, technology makes broad screening programs of adults and newborns possible. Adult Screening If the genetic test for a disorder is fast, accurate, reliable, and inex- pensive and if the population at risk can be defined, wide-scale screening for carriers is feasible. Tay-Sachs disease is an example in which rel- atively inexpensive screening has markedly reduced its incidence. The disease is found chiefly among Jews of European ancestry; screening pro- grams for Tay-Sachs have detected more than 15,000 carriers and have identified some 800 couples at risk for having a baby with the disease. Prenatal tests give such at-risk couples the option of carrying to term only normal children. In 1970 between 50 and 100 babies with this fatal disease were born; today fewer than 10 are born each year. Similar screening for carriers has been attempted for sickle cell ane- mia and thalassemia. In some community-based screening programs, a blood sample for testing was taken when a couple applied for a mar- riage license. The programs were never successful, largely because of inadequate educational efforts, lack of confidentiality, and concern about stigmatization.

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PROGRESS IN RESEARCH 133 Screening Newborns Approximately 12 percent of inherited metabolic diseases can be treated successfully. In order to find the infants who could benefit from such therapies before long-lasting damage can occur, screening programs for these diseases are mandatory in many countries and in most parts of the United States. The tests require small samples of blood and urine; a few drops of blood taken from the baby's heel shortly after birth are all that is necessary to test for PKU and more than 10 other disorders. If PKU is identified in an infant immediately after birth, for example, the baby can be put on a special low-protein diet right away to prevent phenylalanine accumulation. Urine is collected by putting a piece of filter paper in the baby's diaper three or four weeks after birth and sending the paper to a laboratory. Among the diseases identified this way are homocystinuria (high levels of homocystine in the blood), galactosemia (an accumulation of galactose), and maple syrup urine disease, a derangement of amino acid metabolism that gives a maple syrup odor to the baby's urine. Like PKU, these metabolic disorders cause mental retardation and other serious problems; if treated promptly, they can be completely or partially controlled by diets designed to circumvent the child's inability to metabolize the particular chemical. Because the tests are simple and relatively inexpensive, it is feasible to do them for every newborn. The cost of the test is outweighed by the savings in medical care that would be required if the disease were not arrested at a very early stage. Prenatal Testing Techniques Many serious birth defects occur without any known cause, and no prenatal test is available to detect them early in a pregnancy. For some of the most common, however, such as Down syndrome and certain neural tube defects, prenatal tests do exist. The majority of these prenatal tests depend on biochemical assays to detect the evidence of a genetic disease. For several genetic diseases, however, new techniques that analyze fetal DNA to find the markers closely linked to the diseases are now available at some medical centers. If an abnormality is detected, the couple can decide if they want to abort the fetus. Today the following prenatal tests are available:

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134 SCIENCE AND BABIES Amniocentesis is a technique in which a small amount of the am- niotic fluid surrounding the fetus is withdrawn via a needle. The test is used primarily to examine the chromosomes for evidence of Down syndrome and other disorders caused by errors in the chromosome struc- ture. The amniotic fluid also can reveal the presence of by-products of disease, such as abnormal enzymes or abnormal protein molecules. One drawback of conventional amniocentesis is that detection is limited to those diseases that leave evidence in the amniotic fluid itself or in the cells that can be found in the amniotic fluid. Another drawback is that the fluid cannot be withdrawn until after the 14th week of pregnancy, when the womb can be easily felt. Also, the fetal cells must grow in the laboratory for 1 to 3 weeks in order to have enough to analyze. The results of an amniocentesis generally are not known for several weeks, at which point an abortion can be physically and emotionally difficult. About 1 in 200 women miscarry after amniocentesis. Fetal blood testing involves drawing blood from the umbilical cord via a needle guided by ultrasound through the mother's abdomen. Fetal blood can be tested for infectious diseases, for evidence of abnormal metabolism, and for defective genes that account for blood diseases such as thalassemia, sickle cell disease, hemophilia, and chronic granuloma- tous disease, a disorder that affects males, making them very vulnerable to bacterial infections. Umbilical blood cannot be sampled until the blood vessel has grown large enough, which is around the 16th week of preg- nancy. The risk of pregnancy loss for this test is not yet fully known; at some medical centers with much experience with fetal blood sampling, the risk appears to be about 1 percent; otherwise it may vary from center to center. Chorionic villus sampling (CVS) makes it possible to test fetal cells between the 9th and 11th weeks, much earlier than with an amniocentesis or fetal blood test. The chorionic villus is part of the developing placenta and in most cases possesses the same genetic information as the fetus. A snippet of this tissue is taken either via a catheter that is inserted through the birth canal into the uterus or with a needle through the mother's abdomen. Because the procedure collects more live cells than does amniocentesis, the cells do not have to be grown in a culture, so CVS requires less laboratory time. Parents receive results much earlier in the pregnancy, when an abortion is physically less draining. CVS does have a disadvantage, however: It does not always carry the same genetic information as the fetus during early development.

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PROGRESS IN RESEARCH 135 A multicenter study that compared CVS with amniocentesis found that the rates of fetal loss and failure for CVS vary widely from hospital to hospital. The investigators suggest the technique be performed only at medical centers that commit adequate resources to perform the procedure and that perform a substantial number of them. The study found that in experienced hands the spontaneous abortion rate for chorionic villus sampling is 0.S percent higher than for amniocentesis. Ultrasound technology has vastly improved since its debut in the 1970s, and, in the hands of an experienced physician with a good knowl- edge of fetal anatomy, it is a useful diagnostic tool. The clear, real-time images of today's ultrasound can reveal a number of skeletal disorders, defects in the central nervous system such as anencephaly Earl extremely small head indicating defective brain development) and hydrocephalus (an excessive accumulation of fluid in the brain). Urinary tract obstruc- tions and kidney defects can also be detected with ultrasound. Maternal serum alpha-fetoprotein screening (MSAFP) is used to measure the levels of alpha-fetoprotein (AFP) in the blood of pregnant women. AFP is produced by the fetal liver; a small amount passes into the maternal blood circulation, with the concentration gradually increasing until late in the pregnancy. A high level of APP early in a pregnancy can indicate that a woman is carrying a fetus with spine bifida, in which the spinal column has not closed, and other related defects of the central nervous system. Very low levels of AFP are associated with a somewhat increased chance of Down syndrome, but the extent of the risk depends on the mother's age. At present, the MSAFP screen is performed between the 16th and lSth weeks; research is under way to make it possible to do this important assay between the 9th and 12th weeks. Alpha-fetoprotein is a screening mechanism, not a precise diagnostic test. The finding of an unusually high or low level of AFP primarily indicates a need for additional evaluation. The test must be done by a laboratory experienced in MSAFP testing and the results correlated with the age and race of the mother and the week of the pregnancy. Experience with AFP screening programs demonstrates that of 1,000 pregnant women tested, 50 may have high levels of AFP in their blood. Of those 50 women, further testing is likely to reveal that two have fetuses with structural defects of some kind. About one-half of the women with high AFP levels may be at higher risk for complications later in their pregnancies and should be closely monitored by their physician or clinic.

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136 SCIENCE AND BABIES This sonogram of a 21-week-old fetus was one of a series made to evaluate potential kidney problems before birth. There is a history of kidney disease in this family. A healthy baby boy was born in July 1989. New Techniques for Diagnosing Genetic Diseases Conventional prenatal tests are useful only if the affected cells are accessible to a sampling method and if scientists know what biochemical changes or products to look for. Today, in addition to detecting diseases by means of biochemical assays of the disease by-products, scientists are using recombinant DNA technology to analyze fetal cells for certain defective genes that lead to disease. This rapidly developing technology is the most important advance in the field of prenatal diagnosis over the past decade. Recombinant DNA techniques are particularly valuable in detecting inherited diseases that cannot be discovered by biochemical tests. For example, PKU cannot be diagnosed before birth by a biochemical as- say because the abnormal enzyme associated with the disease can be found only in liver cells. Fetal liver cells are not collected by amnio- centesis, fetal blood sampling, or chorionic villus sampling. However, because all cells carry all the genes of a particular individual, PKU and a continually growing number of other genetic diseasescan be diag- nosed prenatally by using DNA analytical techniques to examine fetal skin or blood cells. Today it is possible to identify prenatally the genes that account for cystic fibrosis, sickle cell anemia, and thalassemia. Sickle

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PROGRESS IN RESEARCH 137 cell anemia is a blood disorder that affects large populations, chiefly of African origin; cystic fibrosis causes chronic lung infection that often leads to an early death. DNA analytic techniques show promise for distinguishing even elu- sive genetic alterations. For example, although the gene mutation that leads to Lesch-Nyhan syndrome is highly variable in its mutations, this technology enables researchers to locate it. Dr. C. Thomas Caskey, a molecular geneticist at the Baylor College of Medicine, has studied 50 Lesch-Nyhan patients thus far and no two families have had the same gene mutation. The techniques are becoming more automated and simplified, making it possible to scan a region where a gene sequence may occur in hours, rather than days. One method can find a Lesch-Nyhan gene deletion in approximately 4 hours; the previous test required 7 days. A second method uses direct sequencing of amplified DNA to detect a single base pair mutation in 2 days, replacing a more complicated process that requires several months. Gene mutations associated with human disorders are identified by comparing normal DNA patterns with the chromosome known to have the mutated gene. Often before the gene itself is pinpointed, chromosomal landmarks, or markers, are used to locate its approximate position. Mark- ers are variations in DNA that are easier to identify and close enough to the gene to be inherited with it. Much medical research is devoted to finding markers linked to specific inherited disorders. When such a marker is identified, it can be used to distinguish healthy chromosomes from those carrying the defective gene. Finding the defective genes themselves remains important, however, because only in a given family may markers be reliable indicators of the presence of a gene. Moreover, identifying the faulty gene is the necessary first step toward developing a treatment to replace it or to alleviate its disease-causing activity. DNA analytical methods that detect such markers are being improved and broadened. Dr. Caskey notes that new technologies being used to distinguish the genetic mutations for Lesch-Nyhan syndrome also make it possible to detect with great efficiency the many deletions that can occur in the extensive Duchenne muscular dystrophy gene. Duchenne muscular dystrophy is characterized by progressive muscle weakness, usually ending in death before age 20. The test is done prenatally, using tissue from the chorionic villi, and results are available to the parents in 2 or 3 days.

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138 SCIENCE AND BABIES RESEARCH ON EMBRYO HEALTH Although physicians and researchers have made remarkable advances in their ability to diagnose genetic diseases in human fetuses, only in the last few years have human eggs, sperm, and Reimplantation embryos been studied for abnormal genes and chromosomes. Such studies in this and other countries have been limited by ethical concerns regarding the sources of embryos used for research. Flawed Chromosomes and Pregnancy Spontaneous abortion, especially early in a pregnancy, is a common event. Based on studies made between 1938 and 1953 by Arthur Hertig and John Rock at Harvard, scientists have calculated that one-half to two- thirds of all conceptions do not result in a live birth. Geneticist Aubrey Milunsky, of Boston University, observes that as many as one-third of all recognizes] pregnancies end in spontaneous abortion and another 22 percent are lost before or at the time of the first menstrual period after conception. In his book, Choices, Not Chances, Dr. Milunsky also notes: Among the most common causes of miscarriage are disorders of the chro- mosomes of the developing embryo or fetus. Recent estimates imply that 1 in 10 sperm or eggs cames a chromosome abnormality. In 1978 a method was developed for analyzing the chromosomal complement in human sperm. Sperm from healthy men were found to have abnormalities at an average frequency of ~ to 9 percent. More re- cently, investigators in Italy, England, Sweden, France, and Canada found that substantial percentages of both eggs and embryos exhibit chromo- somal defects. At his IVF clinic in Cambridge, England, Dr. Robert G. Edwards and his co-workers found that 30 percent of the embryos they examined microscopically before implantation had defective chro- mosomes. When Michelle Plachot and her colleagues in Paris analyzed both eggs and embryos, they found that 30 percent of the eggs and 26.1 percent of Reimplantation embryos were chromosomally abnormal. In a Swedish study of infertile women undergoing ovulatory stimulation, a team led by Dr. Hakan Wramsby found that nearly 50 percent of the eggs recovered were abnormal. In almost every instance the eggs and embryos being studied were from infertile women and had been donated for study by infertility clinics because they were no longer needed. Because they came from infertile, often older women, the eggs and embryos may have had a higher than

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PROGRESS IN RESEARCH 139 average rate of abnormalities. In turn, Dr. Plachot believes the high rate of chromosome defects in those embryos may account for the high level of implantation failures and the later fetal losses experienced by women receiving I~F treatment. Not only are chromosome defects common in eggs, sperm, and embryos, but the genetic flaw may prevent the embryo from developing and adjusting to the uterine environment, explaining in part the high rate of embryo loss before implantation. In addition, after embryos become implanted and the pregnancy is recognized, 12 to 15 percent spontaneously abort. As described earlier in this chapter, when a gene is altered or missing, the result usually is a biochemical disorder leading to illness or mental retardation and, frequently, early death. When the chromosome itself is affected, the baby often is born with defects that can range from mild to severe. If a large segment of the chromosome is abnormal, it usually means that more than one gene is involved and the baby is likely to have multiple defects. Chromosome defects include too many or too few chromosomes, as well as chromosomes that are flawed because the end sections of two of them changed places, usually at the time of conception. Parts of chromo- somes can break off and disappear or become reattached upside down. The pair of chromosomes that determines the sex of the embryo appears to be more susceptible to damage than the other 22 pairs; aberrations in the sex chromosomes may affect intellectual development and function. Diagnosing Genetic Diseases in Embryos A procedure for examining the genes of an embryo has been de- veloped. Investigators in England reported early in 1989 that they had removed single cells from 30 three-day-old embryos and had successfully tested the cells for a gene sequence found only on the male-determining Y chromosome. The research demonstrated that it was possible to diagnose genetic diseases in embryos, and it is expected that the test for gender will be useful to women who carry the genes for male-linked diseases such as Duchenne muscular dystrophy, hemophilia, and Lesch-Nyhan syndrome. The test results were confirmed by conventional chromosome testing techniques, which are slower and generally require more genetic material. To find the gender-determining gene sequence in the cell of the embryo, Dr. Alan H. Handyside, of Hammersmith Hospital in London,

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140 SCIENCE AND BABIES and his colleagues used the new, highly sensitive, rapid technique called polymerase chain reaction (PCR) for amplifying gene sequences. PCR has been used on adult cells to identify gene sequences responsible for certain inherited diseases. As PCR tests are developed for specific genetic mutations, scientists believe they could also be used to examine embryos for the diseases. The British group, in fact, has begun to use PCR methodology to test embryos for cystic fibrosis and Duchenne muscular dystrophy. Current practice with in vitro fertilization indicates that the human embryo develops naturally, even if one or more cells are lost at the eight- cell stage. For this reason, Dr. Handyside believes that removing just one cell will not cause any specific defects if embryos analyzed this way are returned to the mother and developed into fetuses. Dr. Handyside and Professor Robert Winston have received ethical approval from British authorities to transfer tested embryos to their mothers. Clinical trials are already in the early stages; if pregnancies result, chorionic villus sampling will be used to confirm the accuracy of the embryo analyses. If further research enables scientists to develop tests that can detect both chromosomal abnormalities and gene defects in embryos, it will be possible to choose only healthy embryos for transfer to the uterus during in vitro fertilization. Moreover, couples at risk for giving birth to a child with a hereditary disease could use embryo testing and in vitro fertilization as a way to have a healthy baby a more desirable alternative to becoming pregnant, testing the fetus, and facing the prospect of having an abortion if the fetus inherited the disease. Observers believe research on embryos is likely to lead to more wanted and healthy children and to a reduction in the number of abortions. Preserving Embryos by Freezing For ethical reasons research on normal reproductive processes in laboratory and farm animals has been more acceptable than in human beings, and such widely used procedures as artificial insemination and in vitro fertilization were first performed in animal husbandry. Similarly, the freezing of animal embryos has been carried out successfully since the early 1970s, and today scientists are continuing their attempt to achieve the same good results with the human embryo. The Office of Technology Assessment (OTA) found that about 60 children have been born after frozen and thawed embryos were transferred to their mothers' wombs.

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PROGRESS IN RESEARCH , a,.:: 141 This embryo was frozen shortly after fertilization, at the two-cell stage, as part of an in vitro fertilization treatment for infertility. Implanted in the uterus of its mother months later, it developed into a healthy infant. Credit: Genetics & IVF Institute/Fairfax Cryobank There are several reasons why preserving human embryos is desir- able. In IVF treatment most or all of the eggs retrieved after ovulatory stimulation might be fertilized. To transfer them all to the mother's uterus could result in a multiple pregnancy, which often is hazardous to the mother as well as to the fetuses. Research has demonstrated that embryos not transferred to the uterus during in vitro fertilization can be preserved by freezing and storing, ready to be used if the first embryo transfer is not successful. Cryopreservation makes it possible to use fewer embryos per transfer and to transfer embryos during a reproductive cycle that has not been stimulated by drugs. Some researchers believe stimulatory drugs may make the uterus less receptive to the implanting embryo. Cryopreserva- tion also substantially reduces the number of retrieval procedures, which means a reduction in cost and patient time. Approximately half the patients having IVF treatment have their extra embryos frozen. Research at the Genetics & IVF Institute and Fairfax Hospital, in Fairfax, Virginia, found little difference in ability to grow between frozen and thawed embryos and fresh early embryos from

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142 SCIENCE AND BABIES the same mother. Almost all (92.7 percent) of the frozen embryos were morphologically intact after thawing and 81.6 percent of them divided, compared to 89.4 percent of the fresh zygotes. The pregnancy rates achieved with the two groups of sibling embryos were also similar. Improving Embryo Development Most of the culture media used to nourish eggs and embryos in IVF centers are based on media developed for mouse eggs. If there are significant differences in metabolism between a human egg and a mouse egg, a culture medium tailored more closely to the needs of the human ovum might improve the results of in vitro fertilization. The metabolic characteristics of the human egg can indicate what enzyme levels it requires in order to survive and develop into an embryo. Oliver H. Lowry and his colleagues at Washington University have de- veloped a method for analyzing a single human egg for its enzymes and metabolites. Their research uncovered enormous differences in enzyme levels between human eggs and mouse eggs. Certain enzyme levels were consistently higher in the human ovum than in the mouse ovum, indicating that the human egg has a greater capacity to burn lipids. The St. Louis scientists note that the capacity to measure several metabolites and enzymes on the same specimen also may offer oppor- tunities beyond that of describing the normal constitution and energy metabolism of the human egg. The noninvasive test they designed, they note, may provide a way to pinpoint the cause of developmental failures in the eggs themselves, rather than in the culture medium or culture conditions. The Embryo Research Vacuum Because there is no public forum for discussing the ethical concerns that surround research on reproduction, very little basic clinical research on human eggs and Reimplantation embryos is being carried out in the United States. Without first undergoing consideration by this forum the defunct Ethics Advisory Board (EAB)such basic research and clinical studies cannot be funded by the federal government. When the OTA researched its report on infertility, it learned that the National Institutes of Health expected to receive over 100 grant applications for reproductive research if the EAB were ever revived. Current research on sperm, eggs, and Reimplantation embryos uses

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PROGRESS IN RESEARCH 143 specimens discarded from infertility programs. As a result, it is not known whether information based on such material will hold true for sperm and eggs produced by couples who do not have fertility problems. Also not known is the effect on human eggs and sperm of environmental influences, such as infection, drugs, radiation, and toxins. There are two sources of embryos for research: those produced in the laboratory from eggs and sperm donated for research and unused embryos that remain after a successful IVF procedure. Only in Great Britain are both types permitted for study; other countries allow research only with post-IVF embryos. In the United States no laws or federal regulations specifically prohibit research on post-IVF embryos. However, the unset- tled nature of the question of federal support for embryo research creates among scientists an uncertainty and reluctance to proceed. Research is limited to observations or measurements rather than interventions. Until the ethical concerns about such studies are addressed in the United States and guidelines developed, there will be no federally funded, organized program for basic embryo research. For carriers of genetic diseases who want to have children and for infertile couples for whom IVF treatment fails, the lack of a policy regarding embryo research means a substantial delay in medicine's ability to help them have healthy children. John Fletcher, of the University of Virginia, has said that the ab- sence of federal support for research on reproduction means the scientific basis for the practice of reproductive medicine and medical genetics "is stunted." Dr. Fletcher also noted that: Genetic disorders account for one-third of all admissions to pediatric units in hospitals and for almost one-fourth of neonatal mortality.... If the pre-embryo is diagnosable, not only couples undergoing infertility treatment could avoid harm to their families and future by embryo diagnosis and selection, but families at higher genetic risk could choose this early form of diagnosis and avoid the emotional and moral suffering of abortion of a wanted pregnancy. CONCLUSION Technology makes it increasingly possible to test adults, newborns, and fetuses for genetic diseases. The tests use a variety of approaches, the most recent being the procedures that survey DNA to locate genetic flaws known to account for inherited disorders. Furthermore, research makes it clear that it is possible to detect chromosome abnormalities

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144 SCIENCE AND BABIES and genetic errors in the human egg and embryo, making it feasible to implant only healthy embryos during in vitro fertilization. In the future, U.S. couples at risk for having a child with a hereditary disease could be offered embryo testing and in vitro fertilization as a way to have a healthy child. In another approach toward solving infertility problems, researchers are finding that important aspects of reproductive functioning are gov- erned by the brain. The hypothalamus appears to control the action of the several hormone systems that regulate sperm production and ovu- lation. Increased understanding of this delicately balanced system has made possible a new treatment for the absence of ovulation, a common cause of female infertility. Further research may identify a relationship among patterns of hormone production and a normal sperm count. These are important studies, and their results hold the promise of improving the reproductive outcomes of many families. However, a de facto moratorium on federal funding of any research involving fertilized eggs and early embryos cannot be overlooked. It has slowed scientific progress toward a better understanding of normal human reproductive functioning and toward developing procedures that would help couples have healthy children. ACKNOWLEDGMENTS This chapter is partially based on presentations by Ernst Knobil, C. Thomas Caskey, Neal First, and John Fletcher. REFERENCES Role of the Brain in Reproduction Fries, H., S.J. Nillius, and F. Pettersson. 1974. Epidemiology of secondary amenorrhea: II. A retrospective evaluation of etiology with special regard to psychogenic factors and weight loss. American Journal of Obstetrics and Gynecology. lS:February:473- 479. Gompel, A., and P. Mauvais-Jarvis. 1988. Induction of ovulation with pulsatile GnRH in hypothalamic amenorrhoea. Human Reproduction. 3~4~:473-477. Knobil, E. 1987. A hypothalamic pulse generator governs mammalian reproduction. News in Physiological Sciences. April:42-43. Knobil, E. 1988. The neuroendocrine control of ovulation. Human Reproduction. 3(41:469-472. Marx, J.L. 1988. Sexual responses are almost all in the brain. Science. 241:903-904.

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PROGRESS IN RESEARCH 145 Pohl, C.R., and E. Knobil. 1982. The role of the central nervous system in the control of ovarian function in higher primates. Annual Review of Physiology. 44:583-593. Pettersson, F., H. Fries, and S.J. Nillius. 1973. Epidemiology of secondary amenorrhea: I. Incidence and prevalence rates. American Journal of Obstetrics and Gynecology. Sept. 1:80-86. Santoro, N., M. Filicori, and W.F. Crowley, Jr. 1986. Hypogonadotropic disorders in men and women: diagnosis and therapy with pulsatile GnRH. Endocrine Review. Vol. 7:11-23. Santoro, N., M.E. Wierman, M. Filicori, J. Waldstreicher, and W.F. Crowley, Jr. 1986. Intravenous administration of pulsatile gonadotropin-releasing hormone in hypothalamic amenorrhea: effects of dosage. Journal of Clinical Endocrinology and Metabolism. 62~1): 109-116. Diagnosis of Genetic Diseases Appelman, Z., and M.S. Golbus. 1987. Uses of fetal tissue sampling. Contemporary Ob/Gyn: Special Issue: Update on Surgery. 42-49. Caskey, C.T. 1987. Disease diagnosis by recombinant DNA methods. Science. 236: 1223-1229. Kolata, G. 1988. Fetuses treated through umbilical cords. New York Times. March 29, C-1. Kolata, G. 1989. Scientists pinpoint genetic changes that predict cancer. New York Times. May 16, C-1. "Genetic Series." 1985-1989. Pamphlets produced by the March of Dimes Birth Defects Foundation. Milunsky, A. 1989. Choices, Not Chances. Boston: Little Brown and Company. Nichols, E. 1988. Human Gene Therapy. Institute of Medicine, National Academy of Sciences. Cambridge, MA: Harvard University Press. The New Human Genetics: lIow Gene Splicing Helps Researchers Fight Inherited Disease. 1984. Written by Maya Pines. Bethesda, MD: National Institute of General Medical Sciences, National Institutes of Health. NIH Publication 84-662. Rhoads, G.G., et al. 1989. The safety and efficacy of chorionic villus sampling for early prenatal diagnosis of cytogenetic abnormalities. New England Journal of Medicine. 320:609-617. Thomas, P. 1988. Fetuses treated by cordocentesis. Medical World News. June 13, 95-96. Research on Embryo Health Chi, M.M., J. Manchester, V.C. Yang, A.D. Curato, R.C. Strickler, and O.H. Lowry. 1988. Contrast in levels of metabolic enzymes in human and mouse ova. Biology of Reproduction. 39:295-307. Fletcher, J.C. 1989. How abortion politics stifle science. The Washington Post. February 5, D-3.

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146 SCIENCE AND BABIES Fugger, E.F., M. Bustells, L.P. Katz, A.D. Dorfman, S.D. Bender, and J.D. Schulman. 1988. Embryonic development and pregnancy from fresh and cryopreserved sibling pronucleate human zygotes. Fertility and Sterility. 50~2):273-277. Martin, R.H., M.M. Mahadevan, P.J. Taylor, K. Hildebrand, L. Long-Simpson, D. Peterson, J. Yamamoto, and J. Fleetham. 1986. Chromosomal analysis of unfertilized human oocytes. Journal of Reproductive Fertility. 78:673-678. McLaren, A. 1988. The IVF conceptus: research today and tomorrow. In In Vitro Fertilization and Other Assisted Reproduction. Edited by H.W. Jones and C. Schrader. Annals of the New York Academy of Sciences. 541:639-645. McLaughlin, L. 1982. The Pill, John Rock, and the Church. Boston: Little Brown and Co. Office of Technology Assessment. Infertility: Medical and Social Choices. 1988. Washington, D.C.: U.S. Congress, OTA-BA-358. Plachot, M., et al. 1988. Chromosomal analysis of human oocytes and embryos in an in vitro fertilization program. In In Vitro Fertilization and Other Assisted Reproduction. Edited by H.W. Jones and C. Schrader. Annals of the New York Academy of Sciences. 541:384-397. Wallach, E.E. 1988. Hail to the animal kingdom. Fertility and Sterility. 50(41:552-554. Weiss, R. 1989. Test screens live "test tube" embryos. Science News, March 4, 135:132. Wood, E.C. 1988. The future of in vetro fertilization. In ln Vitro Fertilization and Other Assisted Reproduction. Edited by H.W. Jones and C. Schrader. Annals of the New York Academy of Sciences. 541:715-721. Wramsby, H., K. Fredga, and P. Liedholm. 1987. Chromosome analysis of human oocytes recovered from preovulatory follicles in stimulated cycles. New England Journal of Medicine. 3 16(3~: 121-124.