Amniocentesis

Amniocentesis involves the aspiration of 10 to 20 ml of amniotic fluid through the abdominal wall under ultrasonographic guidance (Figure 21.1). This is usually performed around the 16th week of gestation. The sample is spun down to yield a pellet of cells and supernatant fluid. The fluid can be used in the prenatal diagnosis of neural tube defects by assay of α-fetoprotein (p. 328). The cell pellet is resuspended in culture medium with fetal calf serum, which stimulates cell growth. Most of these cells in the amniotic fluid, that have been shed from the amnion, fetal skin, and urinary tract epithelium, are non-viable, but a small proportion will grow. After approximately 14 days, there are usually sufficient cells for chromosome and DNA analysis, although a longer period may be required before enough cells are obtained for biochemical assays. Increasingly, sensitive polymerase chain reaction (PCR) techniques make direct DNA analysis possible without the need for culture.

FIGURE 21.1 Diagram of the technique of amniocentesis.

When a couple is considering amniocentesis, they should be informed of the 0.5% to 1% risk of miscarriage associated with the procedure, and that if the result is abnormal they will be facing the possibility of having to consider a mid-trimester termination of pregnancy that involves an induction of labor.

Trials of amniocentesis earlier in pregnancy, at 12 to 14 weeks’ gestation, yielded comparable rates of success in obtaining results with a similar risk of miscarriage. However, concerns have been expressed regarding the reduction in amniotic fluid at this early stage of pregnancy, and early amniocentesis is not widely practiced. Although it has the advantage of allowing a result to be given earlier in the pregnancy, a mid-trimester termination of pregnancy is still usually required if the fetus is found to be affected.

Chorionic Villus Sampling

In contrast to amniocentesis, chorionic villus sampling (CVS), first developed in China, enables prenatal diagnosis to be undertaken during the first trimester. This procedure is usually carried out at 11 to 12 weeks’ gestation under ultrasonographic guidance by either transcervical or, more usually, transabdominal aspiration of chorionic villus (CV) tissue (Figure 21.2). This tissue is fetal in origin, being derived from the outer cell layer of the blastocyst (i.e., the trophoblast). Maternal decidua, normally present in the biopsy sample, must be removed before the sample is analyzed. Placental biopsy is the term used when the procedure is carried out at later stages of pregnancy.

FIGURE 21.2 Diagram of the technique of transvaginal chorionic villus sampling.

Chromosome analysis can be undertaken on CV tissue either directly, looking at metaphase spreads from actively dividing cells, or after culture. Direct chromosomal analysis of CV tissue usually allows a provisional result to be given within 24 hours. Nowadays rapid, direct fluorescent in-situ hybridization (FISH) probing (p. 34), or DNA analysis by the multiplex ligation-dependent probe amplification technique (p. 66), is used to test for common chromosome aneuploidies prior to a standard karyotype following culture of CV tissue; this may also detect other chromosome abnormalities and balanced rearrangements. For single-gene disorders, sufficient CV tissue is usually obtained to allow prenatal diagnosis by immediate biochemical assay or DNA analysis using uncultured CV tissue.

The major advantage of CV sampling is that it offers first-trimester prenatal diagnosis, although it has the disadvantage that even in experienced hands the procedure conveys a 1% to 2% risk of causing miscarriage. There is also evidence that this technique can cause limb abnormalities in the embryo if carried out before 9 to 10 weeks’ gestation; for this reason is not performed before 11 weeks’ gestation.

Ultrasonography

Ultrasonography offers a valuable means of prenatal diagnosis. It can be used not only for obstetric indications, such as placental localization and the diagnosis of multiple pregnancies, but also for prenatal diagnosis of structural abnormalities not associated with known chromosomal, biochemical or molecular defects. Ultrasonography is particularly valuable because it is non-invasive and conveys no known risk to the fetus or mother. It does, however, require expensive equipment and a skilled, experienced operator. For example, a search can be made for polydactyly as a diagnostic feature of a multiple abnormality syndrome, such as one of the autosomal recessive short-limb polydactyly syndromes that are associated with severe pulmonary hypoplasia—invariably lethal (Figure 21.3). Similarly, a scan can reveal that the fetus has a small jaw, which can be associated with a posterior cleft palate and other more serious abnormalities in several single-gene syndromes (Figure 21.4).

FIGURE 21.3 Ultrasonographic image of a transverse section of the hand of a fetus showing polydactyly.

FIGURE 21.4 Longitudinal sagittal ultrasonographic image of the head and upper chest of a fetus showing micrognathia (small jaw) (arrow).

Until a few years ago, detailed ultrasonography for structural abnormalities was offered only to couples who had a child with a genetic disorder or syndrome for which there was no chromosomal, biochemical, or molecular marker. Increasingly, however, detailed ‘fetal anomaly’ scanning is being offered routinely to all pregnant women at around 18 weeks’ gestation as screening for structural abnormalities such as neural tube defects or cardiac anomalies. This technique can also identify features that suggest the presence of an underlying chromosomal abnormality. Such a finding would lead to an offer of amniocentesis or placental biopsy for definitive chromosome analysis.

The future of fetal scanning holds the prospect of three-dimensional imaging and magnetic resonance imaging being used more widely and routinely. Although this will clearly enable the unborn baby to be visualized in far greater detail, it will also generate bigger challenges for the dysmorphologist, who might be expected to diagnose serious disorders on the basis of very subtle features.

Nuchal Translucency

In addition, the observation that increased nuchal translucency (NT) is seen in fetuses who are subsequently born with Down syndrome has resulted in the introduction of measurements of nuchal pad thickness (Figure 21.5) in the first and second trimesters as part of screening for Down syndrome (p. 273). In fact, the finding is not specific and may be seen in various chromosomal anomalies as well as isolated congenital heart disease.

FIGURE 21.5 Nuchal thickening—an accumulation of fluid at the back of the neck. The greater the thickness, the more likely there will be a chromosomal abnormality (e.g., Down syndrome) and/or cardiac anomaly. This finding leads to detailed fetal heart scanning and, usually, fetal karyotyping.

(Courtesy Dr. Helen Liversedge, Exeter.)

Fetoscopy

Fetoscopy involves visualization of the fetus by means of an endoscope. Increasingly, this technique is being superseded by detailed ultrasonography, although occasionally fetoscopy is still undertaken during the second trimester to try to detect the presence of subtle structural abnormalities that would point to a serious underlying diagnosis.

Fetoscopy has also been used to obtain samples of tissue from the fetus that can be analyzed as a means of achieving the prenatal diagnosis of several rare disorders. These have included inherited skin disorders such as epidermolysis bullosa and, before DNA testing became available, metabolic disorders in which the enzyme is expressed only in certain tissues or organs, such as the liver—e.g., ornithine transcarbamylase deficiency (p. 172).

Unfortunately, fetoscopy is associated with a 3% to 5% risk of miscarriage. This relatively high risk, coupled with the increasing sensitivity of ultrasonography and the availability of either linked DNA markers or specific mutation analysis, means that fetoscopy is used only infrequently and in highly specialized prenatal diagnostic centers.

Cordocentesis

Although fetoscopy can also be used to obtain a small sample of fetal blood from one of the umbilical cord vessels in the procedure known as cordocentesis, improvements in ultrasonography have enabled visualization of the vessels in the umbilical cord, allowing transabdominal percutaneous fetal blood sampling. Fetal blood sampling is used routinely in the management of rhesus iso-immunization (p. 205) and can be used to obtain samples for chromosome analysis to resolve problems associated with possible chromosomal mosaicism in CV or amniocentesis samples.

Radiography

The fetal skeleton can be visualized by radiography from 10 weeks onwards, and this technique has been used in the past to diagnose inherited skeletal dysplasias. It is now employed only occasionally because of the dangers of radiography to the fetus (p. 26) and the widespread availability of detailed ultrasonography.

Maternal Serum Screening

It has been government policy in the UK since 2001 that antenatal Down syndrome screening be available to all women, though it was introduced in the late 1980s. Where it is standard practice, maternal serum screening is offered for NTDs and Down syndrome using a blood sample obtained from the mother at 16 weeks’ gestation. In this way up to 75% of all cases of open NTDs and 60% to 70% of all cases of Down syndrome can be detected.

Neural Tube Defects

In 1972 it was recognized that many pregnancies in which the baby had an open NTD (p. 258) could be detected at 16 weeks’ gestation by assay of AFP in maternal serum. AFP is the fetal equivalent of albumin and is the major protein in fetal blood. If the fetus has an open NTD, the level of AFP is raised in both the amniotic fluid and maternal serum as a result of leakage from the open defect. Open NTDs fulfil the criterion of being serious disorders, as anencephaly is invariably fatal, and between 80% and 90% of the small proportion of babies who survive with an open lumbosacral lesion are severely handicapped.

Unfortunately maternal serum AFP screening for NTDs is neither 100% sensitive nor 100% specific (p. 319). The curves for the levels of maternal serum AFP in normal and affected pregnancies overlap (Figure 21.6), so that in practice an arbitrary cut-off level has to be introduced below which no further action is taken. This is usually either the 95th centile, or 2.5 multiples of the median (MoM); as a result around 75% of screened open spina bifida cases are detected. Those pregnant women with results that lie above this arbitrary cut-off level are offered detailed ultrasonography; which is usually sufficient to diagnose NTD. In fact, ultrasonography has more or less superceded maternal serum screening as a means of diagnosing NTD. Anencephaly shows a dramatic deficiency in the cranium (Figure 21.7) and an open myelomeningocele is almost invariably associated with herniation of the cerebellar tonsils through the foramen magnum. This deforms the cerebellar hemispheres, which then have a curved appearance known as the ‘banana sign’; the forehead is also distorted, giving rise to a shape referred to as the ‘lemon sign’ (Figure 21.8). A posterior encephalocele is readily visualized as a sac in the occipital region (Figure 21.9) and always prompts a search for additional anomalies that might help diagnose a recognizable condition such as Meckel-Gruber syndrome.

FIGURE 21.6 Maternal serum α-fetoprotein (AFP) levels at 16 weeks’ gestation plotted on a logarithmic scale as multiples of the median (MoMs). Women with a value off or above 2.5 multiples of the median are offered further investigations.

(Adapted from Brock DJH, Rodeck CH, Ferguson-Smith MA [eds] 1992 Prenatal diagnosis and screening. Edinburgh, UK: Churchill Livingstone.)

FIGURE 21.7 Anencephaly (arrow). There is no cranium and this form of neural tube defect is incompatible with life.

(Courtesy Dr. Helen Liversedge, Exeter, UK.)

FIGURE 21.8 The so-called banana sign showing the distortion of the cerebellar hemispheres into a curved structure (solid arrow). The forehead is also distorted into a shape referred to as the ‘lemon sign’ (broken arrow).

(Courtesy Dr. Helen Liversedge, Exeter, UK.)

FIGURE 21.9 Posterior encephalocele (arrow), a more rare form of neural tube defect. This may be an isolated finding or associated with polydactyly and cystic renal changes in Meckel-Gruber syndrome.

(Courtesy Dr. Helen Liversedge, Exeter, UK.)

A raised maternal serum AFP concentration is not specific for open NTDs (Box 21.1). Other causes include threatened miscarriage, twin pregnancy and a fetal abnormality such as exomphalos, in which there is a protrusion of abdominal contents through the umbilicus.

As a result of these screening modalities there has been a striking decline in the incidence of open NTDs in liveborn and stillborn babies. Other contributory factors are a general improvement in diet and the introduction of periconceptional folic acid supplementation (p. 258). In England and Wales the combined incidence of anencephaly and spina bifida in liveborn and stillborn babies fell from 1 in 250 in 1973 to 1 in 6250 in 1993.

Down Syndrome and Other Chromosome Abnormalities

Ultrasonography

Almost all pregnant women are routinely offered a ‘dating’ scan at around 12 weeks’ gestation. At around this time there is a strong association between chromosome abnormalities and the abnormal accumulation of fluid behind the baby’s neck—increased fetal nuchal translucency (NT) (see Figure 21.5). This applies to Down syndrome, the other autosomal trisomy syndromes (trisomies 13 and 18; p. 275), Turner syndrome, and triploidy, as well as a wide range of other fetal abnormalities and rare syndromes. The risk for Down syndrome correlates with absolute values of NT as well as maternal age (Figure 21.10) but, because NT also increases with gestational age, it is more usual now to relate the risk to the percentile value for any given gestational age. In one study, for example, 80% of Down syndrome fetuses had NT above the 95th percentile. By combining information on maternal age with the results of fetal NT thickness measurements, together with maternal serum markers, it is possible to detect more than 80% of fetuses with trisomy 21 if invasive testing is offered to the 5% of pregnant women with the highest risk (see Table 21.3). Some babies with Down syndrome have duodenal atresia, which shows up as a ‘double bubble sign’ on ultrasonography of the fetal abdomen (Figure 21.11).

FIGURE 21.10 Risk for trisomy 21 (Down syndrome) by maternal age, for different absolute values of nuchal translucency (NT) at 12 weeks’ gestation.

FIGURE 21.11 The ‘double bubble sign’, suggestive of duodenal atresia, sometimes associated with Down syndrome.

(Courtesy Dr. Helen Liversedge, Exeter, UK.)

In many centers, it is also standard practice to offer a detailed ‘fetal anomaly’ scan to all pregnant women at 18 weeks. Although chromosome abnormalities cannot be diagnosed directly, their presence can be suspected by the detection of an abnormality, such as exomphalos (Figure 21.12) or a rocker-bottom foot (Figure 21.13) (Table 21.4). A chromosome abnormality is found in 50% of fetuses with exomphalos identified at 18 weeks, and a rocker-bottom foot is a very characteristic, though not specific, finding in babies with trisomy 18 (p. 275), who are invariably growth retarded. The use of other ultrasonographic ‘soft markers’ in identifying chromosome abnormalities in pregnancy is discussed in the following section (p. 335).

FIGURE 21.12 Ultrasonogram at 18 weeks showing exomphalos.

(Courtesy Dr. D. Rose, City Hospital, Nottingham, UK.)

FIGURE 21.13 A, Ultrasonogram at 18 weeks showing a rocker-bottom foot in a fetus subsequently found to have trisomy 18. B, Photograph of the feet of a newborn with trisomy 18.

(Courtesy Dr. D. Rose, City Hospital, Nottingham, UK.)

Table 21.4 Prenatal Ultrasonographic Findings Suggestive of a Chromosome Abnormality

Feature	Chromosome Abnormality
Cardiac defect (especially common atrioventricular canal)	Trisomy 13, 18, 21
Clenched overlapping fingers	Trisomy 18
Cystic hygroma or fetal hydrops	Trisomy 13, 18, 21
Duodenal atresia	45,X (Turner syndrome) Trisomy 21
Exomphalos	Trisomy 13, 18
Rocker-bottom foot	Trisomy 18

Advanced Maternal Age

This has been the most common indication for offering prenatal diagnosis. There is a well-recognized association of advanced maternal age with increased risk of having a child with Down syndrome (see Table 18.4; p. 274) and the other autosomal trisomy syndromes. No standard criterion exists for determining at what age a mother should be offered the option of an invasive prenatal diagnostic procedure for fetal chromosome analysis. Most centers routinely offer amniocentesis or CVS to women age 37 years or older, and the option is often discussed with women from the age of 35 years onward. These risk figures relate to the maternal age at the expected date of delivery. The risk figures for Down syndrome at the time of CVS, amniocentesis, and delivery differ (see Figure 18.1; p. 274) because a proportion of pregnancies with trisomy 21 are lost spontaneously during the first and second trimesters. Interestingly, despite industrial-scale efforts to screen for Down syndrome, there has been a slight rise in the numbers of live births in the United Kingdom since 2000, following a steady decline from the widespread introduction in screening in 1989 (National Down Syndrome Cytogenetic Register). However, the numbers of prenatal diagnoses and terminations for Down syndrome has also increased over this period. Both observations are attributed to the slightly older age at which women are now having children, and there may also be an increasing willingness to raise a child with the condition.

Previous Child with a Chromosome Abnormality

Although there are a number of series with slightly different recurrence risk figures, for couples who have had a child with Down syndrome because of non-disjunction, or a de novo unbalanced Robertsonian translocation, the risk in a subsequent pregnancy is usually given as the mother’s age-related risk plus approximately 1%. If one of the parents has been found to carry a balanced chromosomal rearrangement, such as a chromosomal translocation (p. 44) or pericentric inversion (p. 48), that has caused a previous child to be born with serious problems due to an unbalanced chromosome abnormality, the recurrence risk is likely to be between 1% to 2% and 15% to 20%. The precise risk will depend on the nature of the parental rearrangement and the specific segments of the individual chromosomes involved (p. 48).

Family History of a Chromosome Abnormality

Couples may be referred because of a family history of a chromosome abnormality, most commonly Down syndrome. For most couples, there will usually be no increase in risk compared with the general population, as most cases of trisomy 21 and other chromosomal disorders will have arisen as a result of non-disjunction rather than as a result of a familial translocation, or other rearrangement. However, each situation should be evaluated carefully, either by confirming the nature of the chromosome abnormality in the affected individual or, if this is not possible, by urgent chromosome analysis of blood from the relevant parent at risk. The results of parental chromosomal analysis can usually be obtained within 3 to 4 days; if normal, an invasive prenatal diagnostic procedure is not then appropriate as the risk is no greater than that for the general population.

Family History of a Single-Gene Disorder

If prospective parents have already had an affected child, or if one of the parents is affected or has a positive family history of a single-gene disorder that conveys a significant risk to offspring, then the option of prenatal diagnosis should be discussed with them. Prenatal diagnosis is available for a large and ever-increasing number of single-gene disorders by either biochemical or DNA analysis.

Family History of a Neural Tube Defect

Careful evaluation of the pedigree is necessary to determine the risk that applies to each pregnancy. Risks can be determined based on empiric data (p. 346). In high-risk situations, ultrasonographic examination of the fetus, possibly in conjunction with assay of maternal serum AFP, can be offered. However, even with good equipment and an experienced ultrasonographer, small closed NTDs can still be missed. Fortunately, the latter types of NTD are not usually associated with the serious problems seen with large open NTDs (p. 258).

Family History of Other Congenital Structural Abnormalities

As with NTDs, evaluation of the family pedigree should enable the provision of a risk derived from the results of empiric studies. If the risk to a pregnancy is increased, detailed ultrasonographic examination looking for the specific structural abnormality can be offered at around 16 to 18 weeks’ gestation. Mid-trimester ultrasonography will detect most serious cranial, cardiac, renal and limb malformations. Some couples request detailed ultrasonographic scanning, not because they wish to pursue the option of termination of pregnancy but because they wish to prepare themselves if the baby is found to be affected.

Family History of Undiagnosed Learning Difficulty

An increasingly common scenario is the urgent referral of a pregnant couple who already has a child, or close relative, with an undiagnosed learning difficulty, with or without dysmorphic features. Whereas in the past a standard karyotype and fragile X syndrome test might be carried out, today it is incumbent on geneticists to use the latest technique of microarray-CGH (p. 281). This must first be undertaken on the affected child or individual, of course, and producing an urgent report (2 weeks) is a challenge for the laboratories.

Abnormalities Identified In Pregnancy

The widespread introduction of prenatal diagnostic screening procedures, such as triple testing and fetal anomaly scanning, has meant that many couples unexpectedly present with diagnostic uncertainty during the pregnancy that can be resolved only by an invasive procedure such as amniocentesis or CVS. Other factors, such as poor fetal growth, can also be an indication for prenatal chromosome analysis, as confirmation of a serious and non-viable chromosome abnormality, such as trisomy 18 or triploidy (p. 276), can influence subsequent management of the pregnancy and mode of delivery.

Other High-Risk Factors

These factors include parental consanguinity, a poor obstetric history, and certain maternal illnesses. Parental consanguinity increases the risk that a child will have a hereditary disorder or congenital abnormality (pp. 113). Consequently, if the parents are concerned, it is appropriate to offer detailed ultrasonography to try to exclude a serious structural abnormality. It may also be appropriate to offer to test the couple for cystic fibrosis and spinal muscular atrophy carrier status, and possibly other conditions depending on ethnicity. A poor obstetric history, such as recurrent miscarriages or a previous unexplained stillbirth, could indicate an increased risk of problems in a future pregnancy and detailed ultrasonographic monitoring. A history of three or more unexplained miscarriages should be investigated by parental chromosome studies to exclude a chromosomal rearrangement such as a translocation or inversion (pp. 44, 48). Maternal illnesses, such as poorly controlled diabetes mellitus (p. 235) or epilepsy treated with anticonvulsant medications such as sodium valproate (p. 261), would also be indications for detailed ultrasonography. Both of these factors convey an increased risk of structural abnormality in a fetus.

Failure to Obtain a Sample or Culture Failure

It is important that every woman undergoing one of these invasive procedures is alerted to the possibility that, on occasion, it can prove impossible to obtain a suitable sample or the cells obtained subsequently fail to grow. Fortunately, the risk of either of these events occurring is less than 1%.

An Ambiguous Chromosome Result

In approximately 1% of cases, CVS shows evidence of apparent chromosome mosaicism—i.e., the presence of two or more cell lines with different chromosome constitutions (p. 50). This can occur for several reasons:

In the case of amniocentesis, in most laboratories it is routine for the sample to be split and for two or three separate cultures to be established. If a single abnormal cell is identified in only one culture, this is assumed to be a culture artifact, or what is termed level 1 mosaicism, or pseudomosaicism. If the mosaicism extends to two or more cells in two or more cultures this is taken as evidence of true mosaicism, or what is known as level 3 mosaicism. The most difficult situation to interpret is when mosaicism is present in two or more cells in only one culture, termed level 2 mosaicism. This is most likely to represent a culture artifact, but there is up to a 20% chance that the mosaicism is real and will be present in the fetus.

To resolve the uncertainty of chromosomal mosaicism in cultured CV tissue it may be necessary to proceed to amniocentesis. If the latter test yields a normal chromosomal result, then it is usually concluded that the earlier result was not a true indication of the fetal karyotype.

Counseling couples in this situation may be extremely difficult. If true mosaicism is confirmed, it is often impossible to predict the phenotypic outcome for the baby. An attempt can be made to resolve ambiguous findings by fetal blood sampling for urgent karyotype analysis, but this too is limited in terms of the information it yields about the phenotype. Whatever option the parents choose, it is important that tissue (blood, skin, or placenta) is obtained at the time of delivery, whether the couple elects to terminate or continue with the pregnancy, to resolve the significance of the prenatal findings.

An Unexpected Chromosome Result

Three different types of unexpected chromosome results may occur, each of which usually necessitates specialized detailed genetic counseling.

Ultrasonographic ‘Soft’ Markers

Sophisticated ultrasonography has resulted in the identification of subtle anomalies in the fetus, the significance of which is not always clear. For example, choroid plexus cysts are sometimes seen in the developing cerebral ventricles in mid-trimester (Figure 21.14). Initially, it was thought that these were invariably associated with the fetus having trisomy 18 but in fact they occur frequently in normal fetuses, although if they are very large and do not disappear spontaneously they can be indicative of a chromosome abnormality.

FIGURE 21.14 Ultrasonogram of a fetal brain showing bilateral choroid plexus cysts (arrows).

Increased echogenicity of the fetal bowel (Figure 21.15) has been reported in association with cystic fibrosis—the prenatal equivalent of meconium ileus (p. 301). Initial reports suggested this finding could convey a risk as high as 10% for the fetus having cystic fibrosis, but it is now clear that this risk is probably no greater than 1% to 2%. Novel ultrasonographic findings of this kind are often called soft markers, and their interpretation must be approached cautiously in the effort to distinguish normal from abnormal variation.

FIGURE 21.15 Echogenic bowel. Regions of the bowel showing unusually high signal (arrow). This is occasionally a sign of meconium ileus seen in cystic fibrosis.

(Courtesy Dr. Helen Liversedge, Exeter, UK)

In Vitro Fertilization

Many thousands of babies worldwide have been born by IVF over the past 30 years, when the technique was first successful. The indication for the treatment in most cases is subfertility, which now affects one in seven couples. In some Western countries, 1% to 3% of all births are the result of assisted reproductive technologies (ARTs). The cohort of offspring conceived in this way is therefore very large, and evidence is gathering that the risk of birth defects is increased by 30% to 40% compared with the general population conceived in the normal way and about 50% more children are likely to be small for gestational age (SGA). Specifically, a small increase in certain epigenetic conditions due to defective genomic imprinting (p. 121) has been observed—Beckwith-Wiedemann (p. 124) and Angelman (p. 123) syndromes, and ‘hypomethylation’ syndrome, though the possible mechanisms are unclear. In cases studied, loss of imprinting (LOI) was observed at the KCNQ1OT1 locus (see Figure 7.27; p. 125) in the case of Beckwith-Wiedemann syndrome, and at the SNRPN locus (Figure 7.23; p. 123) in the case of Angelman syndrome. No apparent imprinting differences explain the increase in SGA babies conceived by ICSI.

Epigenetic events around the time of fertilization and implantation are crucial for normal development (p. 103). If there is a definite increased risk of conditions from abnormal imprinting after ARTs, this may relate, in part, to the extended culture time of embryos, which has become a trend in infertility clinics. Instead of transferring cleavage-stage embryos, it is now more routine to transfer blastocysts, which allows the healthier looking embryos to be selected. However, in animal models it has been shown that in vitro culture affects the extent of imprinting, gene expression, and therefore the potential for normal development.

Intracytoplasmic Sperm Injection

As mentioned, this technique is employed as part of IVF when combined with PGD, although the main indication for directly injecting the sperm into the egg is male subfertility because of low sperm count, poor sperm motility, abnormal sperm morphology, or mechanical blockage to the passage of sperm along the vas deferens. Chromosomal abnormalities or rearrangements have been found in about 5% of men for whom ICSI is suitable, and 10% to 12% in those with azoospermia or severe oligospermia. Examples include the robertsonian 13 : 14 translocation and Y-chromosome deletions. For men with azoospermia or severe oligospermia the karyotype should be checked, including the application of molecular techniques looking for submicroscopic Y deletions. In those with mechanical blockage due to congenital bilateral absence of the vas deferens (CBAVD), a significant proportion has cystic fibrosis mutations. ICSI offers hope to men with CBAVD, as well as those with Klinefelter syndrome, following testicular aspiration of sperm.

Some of the chromosomal abnormalities in the men may be heritable—especially those involving the sex chromosomes—and there is a small but definite increase in chromosomal abnormalities in the offspring (1.6%).

Donor Insemination

As a means of assisted conception to treat male infertility, or circumvent the risk of a genetic disease, donor insemination (DI) has been used since the 1950s. Only relatively recently, however, has awareness of medical genetic issues been incorporated into practice. Following the cases of children conceived by DI who were subsequently discovered to have balanced or unbalanced chromosome disorders, or in some cases cystic fibrosis (indicating that the sperm donor was a carrier for cystic fibrosis), screening of sperm donors for cystic fibrosis mutations and chromosome rearrangements has become routine practice in many countries. This was recommended only as recently as 2000 by the British Andrology Society. In the Netherlands, a donor whose sperm was used to father 18 offspring developed an autosomal dominant late-onset neurodegenerative disorder (one of the spinocerebellar ataxias), thus indicating that all 18 offspring were conceived at 50% risk. This led to a ruling that the sperm from one donor should be used no more than 10 times, as against 25 before this experience. In the United Kingdom, men older than age 40 years cannot be donors because of the small but increasing risk of new germline mutations arising in sperm with advancing paternal age.

Of course, it is not possible to screen the donor for all eventualities, but these cases have served to highlight the potential conflict between treating infertility (or genetic disease) by DI and maintaining a high level of concern for the welfare of the child conceived. More high profile in this respect is the ongoing debate about how much information DI children should be allowed about their genetic fathers, and the law varies across the world.

Naturally, all of these issues apply in an equivalent way to women who wish to be egg donors.

Assisted Conception and the Law

In the United States, no federal law exists to regulate the practice of assisted conception other than the requirement that outcomes of IVF and ICSI must be reported. In the United Kingdom, strict regulation operates through the HFEA based on the Human Fertilization and Embryology Act of 1990. The HFEA reports to the Secretary of State for Health, issues licences, and arranges inspections of registered centers. The different licences granted are for treatment (Box 21.2), storage (gametes and embryos), and research (on human embryos in vitro). A register of all treatment cycles, the children born by IVF, and the use of donated gametes, must be kept. The research permitted under licence covers treatment of infertility, increase in knowledge regarding birth defects, miscarriage, genetic testing in embryos, the development of the early embryo, and potential treatment of serious disease. At the time of writing it is not clear what arrangements will be put in place when the HFEA is abolished.