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	Guest Editorial Obstetric Ultrasound: Setting the Standard for 2001 Jo-Ann M. Johnson - Toronto, Canada
	Medical Genetics: An Overview David Chitayat - Toronto, Canada
	Sonography of the Early First Trimester Shia Salem– Toronto, Canada
	Screening Between 12-20 Weeks: Ultrasound and Biochemistry Jo-Ann M. Johnson - Toronto, Canada
	The 18 Week Scan and Fetal Anatomical Survey Ants Toi - Toronto, Canada
	The Fetal Urogenital Tract Katherine Fong - Toronto, Canada
	Adnexal Masses in Pregnancy Anthony Hanbidge - Toronto, Canada
	Fetal Cardiac Screen Shi-Joon Yoo - Toronto, Canada
	Ultrasound and Multiple Pregnancies Pran Pandya - London, United Kingdom
	Bad News in the Scanning Room Rory Windrim and others – Toronto, Canada
end of Obstetric Ultrasound presentations
	Letter to the Editor: Meckel Syndrome - Ductal Plate Malformation Christian Dugauquier (Belgium), Nowaczyk & Mohide (Canada)
	Molecular Diagnosis of Smith-Lemli-Opitz Syndrome: Current Applications Malgorzata Nowaczyk - Hamilton, Canada
	An Unusually High Prevalence of Neural Tube Defects in South Bulgaria E. Simeonov and others – Sophia, Bulgaria
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Obstetric Ultrasound: "Setting the Standard for 2001"

Toronto, Ontario, CANADA – February 23-25, 2001

The 3^rd "Obstetric Ultrasound: Setting the Standard" course was held in Toronto, Ontario on February 23^rd-25^th, 2001. The main objective of the course was to provide participants with the most up-to-date practice standards in obstetric ultrasound and image capturing and to explore the latest technological and clinical advances. This was achieved through lectures, workshops and debates, and "The 11-14 week Nuchal Translucency Course", which was offered in conjunction with the meeting.

The course was a collaborative effort between the Departments of Obstetrics and Gynaecology and Medical Imaging at the University of Toronto and the Department of Paediatric Cardiology at The Hospital for Sick Children. By drawing on the expertise of these various disciplines and emphasizing the integration of ultrasound into current clinical management, the course took obstetric ultrasound to "the next step", demonstrating its evolution from an imaging tool to a multidisciplinary subspecialty.

This issue of Frontiers contains a representative sample of lectures from the well-renowned national and international faculty who spoke at the meeting.

Jo-Ann M. Johnson, M.D.
Course Director
Department of Obstetrics & Gynaecology
University of Toronto, CANADA

Medical Genetics – An Overview

David Chitayat, M.D.

ASSOCIATE PROFESSOR, DEPARTMENTS OF PAEDIATRICS, OBSTETRICS & GYNAECOLOGY, MEDICAL GENETICS AND MICROBIOLOGY, LABORATORY MEDICINE & PATHOBIOLOGY, UNIVERSITY OF TORONTO, TORONTO, CANADA

Human somatic cells contain 46 chromosomes and are organized into 22 autosomal pairs plus the sex chromosomes. The basic haploid set (n=23) is present in the gametes. After fertilization, the zygote contains a diploid set of chromosomes (2n=46); one of each pair is maternal in origin, the other paternal.

During meiosis, which is the nuclear division that gives rise to the gametes, recombination occurs between homologous parental chromosomes. The exchange of chromosomal material leads to the separation of genes originally located on the same chromosome, and gives rise to genetic variation within families.

Each chromosome can be identified by light microscopy with staining techniques that highlight the characteristic pattern of alternating light and dark bands. During metaphase, the two chromatids of each chromosome are joined at the centromere. The short arm of the chromosome is designated ‘p’ and the long arm, ‘q’. Each arm is subdivided numerically into a number of bands, which permits the precise localization of a structural abnormality. High-resolution cytogenetic techniques facilitate the identification of small interstitial chromosome deletions in recognized disorders of previously unknown origin, such as Prader-Willi and Angelman’s syndrome. Deletions too small to be detected by microscopy may be amenable to diagnosis by DNA techniques.

Fluorescence in situ hybridization (FISH) is a recently developed molecular cytogenetic technique that involves hybridization of a DNA probe to a metaphase chromosome spread. Single stranded probe DNA is fluorescently labeled using biotin and avidin and hybridized to the denatured DNA of intact chromosomes on a microscope slide. The resultant DNA binding can be seen direct using a fluorescence microscope. Alternatively, a single DNA probe that corresponds to a specific locus can be used. Hybridization reveals a fluorescent spot on each chromatid of the relative chromosome. This method is used to diagnose a syndrome that is caused by sub-microscopic deletions, or to identify carriers of a single gene defect due to large deletions, such as Duchenne muscular dystrophy.

It is possible to use several separate DNA probes, each of which is labeled with a different fluorochrome, to analyze more than one locus or chromosome region in the same reaction. Another application of this technique is in the study of interface nuclei, which permits the study of non-dividing cells. Thus, rapid results can be obtained for the diagnosis or exclusion of Down syndrome in uncultured amniotic fluid samples using chromosome 21 specific probes.

Chromosome Abnormalities

In liveborn infants, the incidence of chromosomal abnormalities is 0.6%. The incidence of abnormalities of the autosomes and sex chromosomes is approximately the same. The effect of these abnormalities on the child will depend on the type of abnormality. Abnormalities do not occur in balanced rearrangements and are mild in disorders of most of the sex chromosomes. Unbalanced autosomal abnormalities cause disorders with multiple congenital malformations, almost invariably associated with mental retardation.

Chromosome abnormalities are particularly common in spontaneous abortions. Approximately 8% of the sperm, 32% of the oocytes, 10% of all conceptions lost spontaneously and 5% of all stillbirths are chromosomally abnormal

Mendelian Inheritance (Single Gene Disorders)

Single gene defect disorders follow a pattern of inheritance originally described by Mendel. Autosomal dominant disorders affect both males and females and can be traced through many generations of a family. Affected people are heterozygous for the abnormal allele and will transmit the gene for the disease to half of their offspring, whether male or female. However, autosomal dominant conditions have both intra-familial and inter-familial variability in the severity of the manifestations and the age of onset. New mutations may account for the presence of dominant disorder in a subject who does not have a family history of the disease. When a disorder arises by a new mutation, the risk of recurrence in future pregnancies for the mother of the affected child is low, but not zero, due to gonadal mosaicism. However, care must be taken to exclude a mild form of the condition in one of the parent before giving reassurance. New mutations account for most cases of achondroplasia, but only for 50% of the cases of neurofibromatosis and tuberous sclerosis. A dominant disorder in a person with a negative family history may alternatively indicate non-paternity.

A few dominant disorders show a lack of penetrance; that is, a person who inherits the gene but does not develop the disorder. In this case, people who are not affected cannot be completely reassured that they will not transmit the disorder to their children. The risk is, however, fairly low.

An autosomal recessive disorder occurs in a person whose healthy parents both carry the same recessive gene. The risk of recurrence for future offspring of such parents is 25%. Unlike autosomal dominant disorders, there is generally no family history of the condition. In northern Europe, the commonest autosomal recessive disorder is cystic fibrosis. Approximately 1 in 20 people in this population is a carrier. Consanguinity increases the risk of a recessive disorder because both parents are more likely to carry the same defective gene, inherited from the same common ancestor. However, the increased risk for parents who are first cousins of having a child with severe abnormality is low, approximately 2% to 3% above the general population risk.

Autosomal recessive conditions are commonly severe and many of the inborn errors of metabolism follow this type of inheritance.

In X-linked recessive conditions, the disorder is transmitted through healthy female carriers and only males are affected. A female carrier of an X-linked recessive disorder will transmit the condition to half her sons whereas half her daughters will be carriers. An unaffected male does not transmit the disorder. An affected male will transmit the mutant gene to all his daughters who must inherit his X chromosome, but none of his sons, who must inherit his Y chromosome, will be affected. The absence of male-to-male transmission is a hallmark of X-linked inheritance. Many X-linked recessive disorders are severe or lethal during early life, however, so that the affected males do not reproduce.

An X-linked recessive disorder should be considered when the family history indicates maternally related affected males in different generations of the family. Family history is not always positive, however since new mutations are fairly common. Recognizing X-linked recessive inheritance is important because many female relatives may be at risk of being carriers and of having affected sons, irrespective of whom they marry.

An X-linked dominant gene will give rise to a disorder in both hemizygous males and heterozygous females. The gene is transmitted in families in the same way as X linked recessive genes, giving rise to an excess of affected females. In some disorders the condition is lethal in hemizygous males. In this case there will be a fewer males than expected in the family, all of whom will be healthy, and an excess of females, half of whom will be affected.

In Y-linked disorders, only males are affected, with transmission being directly from father to son with the Y chromosome. This pattern of inheritance has been suggested for such conditions as porcupine skin, hairy ears, and webbed toes. In most conditions in which Y-linked inheritance has been postulated, the actual mode of inheritance is probably autosomal dominant, with other factors causing sex limitation.

Multi-factorial Disorders

The genetic contribution to disease varies. Some disorders are entirely environmental while others are wholly genetic. Many common disorders, however, have an appreciable genetic contribution but do not follow simple patterns of inheritance within a family. The terms, multi-factorial or polygenic inheritance, have been used to describe the etiology of these disorders. Normal traits inherited in this manner include height and intelligence.

The concept of multi-factorial inheritance implies that a disease is caused by the interaction of several adverse genetic and environmental factors. The liability of a population to a particular disease follows a normal distribution curve and most people will show only moderate susceptibility and remain affected. Only when a certain threshold of liability is exceeded will the disorder manifest. Relatives of an affected person will show a shift in liability, with a greater proportion of them being beyond the threshold. Familial clustering of a particular disorder may therefore occur.

The risk of recurrence for a multi-factorial disorder within a family is generally low and mainly affects first degree relatives. In many conditions family studies have reported the rate with which relatives of the proband have been affected. This allows empirical values for risk of recurrence to be calculated, which can be used in genetic counseling. Many of the common congenital abnormalities have multi-factorial mode of inheritance and the risk of recurrence depends on the specific malformation, its severity, and the number of affected people in the family.

A rational approach to preventing the disease is to modify known environmental triggers in genetically susceptible subjects. Folic acid supplementation in pregnancies at increased risk of neural tube defect and modifying diet and smoking habits in coronary heart disease are examples of affective intervention, but this approach is not currently possible for many other disorders.

Non-Mendelian Mechanisms

Unstable Mutations

The recent discovery of an unstable mutation mechanism involving a trinucleotide repeat has identified a new cause of some human genetic diseases that are inherited in Mendelian fashion. The three common disorders caused by trinucleotide repeat expansion are myotonic dystrophy, fragile X syndrome and Huntington’s disease. All three share unusual characteristics of inheritance, which can be explained by the instability of the mutation. These include the phenomenon of anticipation, in which the disorder becomes more severe in successive generations of a family, and the striking parental sex bias in the inheritance of the most severe forms of the disorder – maternal in myotonic dystrophy and fragile X syndrome and paternal in Huntington’s disease. Instability of the mutation usually generates larger expansion in offspring, but reduction in size of expansion is also documented.

Imprinting

It has been observed that some inherited traits do not conform to the expected pattern of classical Mendelian inheritance, in which genes inherited from either parent have an equal effect. The term, imprinting, is used to describe the phenomenon by which certain genes function differently, depending on whether they are maternally or paternally derived. The mechanism of DNA modification involved in imprinting is unknown, but it confers functional change in particular alleles at the time of gametogenesis, as determined by the sex of the parent. The imprint lasts for one generation and then disappears, so an appropriate imprint can be re-established in the germ cells of the next generation.

The effect of imprinting can be observed at several levels: at the whole genome level, at that of particular chromosomes or chromosomal segments, or at the level of individual genes. For example, the effect of triploidy in human conceptions depends on the origin of the additional haploid chromosome set. When paternally derived, the placenta is large and cystic with molar changes and the fetus has a large head and small body. When the extra chromosome set is maternal, the placenta is small and underdeveloped without cystic changes and the fetus is noticeably underdeveloped.

One of the best examples of imprinting in human disease is demonstrated by the deletions in the q11-13 region of chromosome 15, which may cause either Prader-Willi syndrome or Angelman’s syndrome. The features of Prader-Willi syndrome are severe neonatal hypotonia and failure to thrive with later onset of obesity, behaviour problems, mental retardation, small hands and feet, facial dysmorphism and hypogonadism. Angelman’s syndrome is quite distinct and is associated with severe mental retardation, microcephaly, ataxia, and epilepsy and absent speech.

The genes for Prader-Willi and Angelman’s syndrome are both situated within the 15q11-13 region. Similar de novo cytogenetic or molecular deletions can be detected in both conditions. In Prader-Willi syndrome, the deletion always occurs on the paternally derived chromosome 15, whereas in Angelman’s syndrome, the deletion is always on the maternally derived chromosome. In patients with Prader-Willi syndrome who do not have a chromosome deletion, both chromosomes 15 are maternally derived. This phenomenon is called uniparental disomy. When it involves imprinted regions of the genome, it has the same effect as a chromosomal deletion that arises from the opposite paternal chromosome. In Prader-Willi syndrome, both isodisomy (inheritance of identical chromosome 15s from one parent) and heterodisomy (inheritance of different chromosome 15s from the same parent) have been observed. The origin of this uniparental disomy is probably the loss of one chromosome 15 from a conception that was initially trisomic. Uniparental disomy is rare in Angelman’s syndrome, but when documented has involved disomy of the paternal chromosome 15.

Imprinting has been implicated in other human diseases, notably in some forms of cancer, such as familial glomus tumours, Wilm’s tumour, and Beckwith-Wiedemann syndrome.

Mitochondrial Disorders

Mitochondria have their own DNA, which consists of a double-stranded circular molecule. This mitochondrial DNA consists of 16,567 base pairs that constitute 37 genes. There is some difference in the genetic code between the nuclear and mitochondrial genomes, and mitochondrial DNA is almost exclusively coding DNA, the genes containing no introns.

A diploid cell contains two copies of the nuclear genome, but there may be more than 1,000 copies of the mitochondrial genome, as each mitochondrium contains up to 10 copies of its circular DNA and each cell contains hundreds of mitochondria. Mutations within mitochondrial DNA appear to be 5 or 10 times more common than mutations in nuclear DNA. This accumulation of mitochondrial mutations with time has been suggested to have some role in ageing.

The mitochondrial genome encodes 22 types of transfer RNA molecules that are involved in mitochondrial protein synthesis, as well as 13 polypeptides involved in the respiratory chain system. Nuclear genes encode the remaining respiratory chain polypeptides. Diseases that affect mitochondrial function may therefore be controlled by nuclear gene mutation and follow Mendelian inheritance, or may result from mutations within the mitochondrial DNA. Since the main function of mitochondria is to synthesize ATP by oxidative phosphorylation, mitochondrial disorders are most likely to affect tissues such as the brain, skeletal muscle, cardiac muscle, and eye, all of which contain abundant mitochondria and rely on aerobic oxidation and ATP production.

Mutations in mitochondrial DNA have been identified in a number of diseases, notably Leber’s hereditary optic neuropathy, MERRF (myoclonic epilepsy with ragged red fibers), MELAS (mitochondrial myopathy with encephalopathy, lactic acidosis, and stroke-like episodes), and progressive external ophthalmoplegia, including Kaerns-Sayre syndrome. Disorders due to mitochondrial mutations often appear to occur sporadically. When they are inherited, however, they demonstrate maternal transmission. This is because only the egg contributes cytoplasm and mitochondria to the zygote. All offspring of a carrier mother will carry the mutation whereas all offspring of a carrier father will be normal.

The pedigree pattern in mitochondrial inheritance may be difficult to recognize, however, because some carriers remain asymptomatic.

In Leber’s hereditary optic neuropathy for example, half the sons of a carrier mother would be affected, but only 1:5 of the daughters will be symptomatic. Nevertheless, all daughters would transmit the mutation to their offspring. The descendants of affected fathers are never affected.

Because multiple copies of mitochondrial DNA are present in the cell, mitochondrial mutations are often heteroplasmic – that is, a single cell will contain a mixture of mutant and wild-type mitochondrial DNA. With successive cell divisions, some cells will remain heteroplasmic but others may drift towards homoplasmy for the mutant or wild-type DNA. Large deletions, which make the remaining mitochondrial DNA appreciably shorter, may have a selective advantage in terms of replication efficiency, so that the mutant genome accumulates preferentially. The severity of disease caused by mitochondrial mutations probably depends on the relative proportions of wild-type and mutant DNA present, but is very difficult to predict in a given subject.

Sonography of the Early First Trimester

Shia Salem, M.D.

ASSOCIATE PROFESSOR, DEPARTMENT OF MEDICAL IMAGING, UNIVERSITY OF TORONTO, TORONTO, CANADA

Most patients referred for sonography in the first trimester present with vaginal bleeding or pain. The referring physician usually requests the sonographic examination to exclude early embryonic demise or an ectopic pregnancy.

The goals of first trimester sonography include:

• Visualizing and localizing the gestational sac - is it intrauterine or extrauterine?
• Estimating gestational age.
• Identifying embryonic demise and an anembryonic pregnancy.
• Identifying embryos still alive but at increased risk for demise.
• Determining the number of embryos and the chorionicity and amnionicity in multifetal pregnancies.
• Early diagnosis of fetal abnormalities.

Transvaginal sonography (TVS) has radically changed sonographic practice in the first trimester. Because of its enhanced resolution, TVS has resulted in earlier visualization of the gestational sac and its contents and earlier identification of embryonic cardiac activity. These structures can be seen approximately 1 week earlier than if one were to use transabdominal sonography (TAS). TVS also improves the visualization of embryonic and fetal structures. Reliable indicators of early pregnancy failure have been identified by TVS which makes it unnecessary to do serial examinations in the majority of patients. This results in decreased morbidity and patient anxiety.

NORMAL SONOGRAPHIC APPEARANCES

The first reliable sonographic evidence of an intrauterine pregnancy is the visualization of the gestational sac within the thickened decidua. The gestational sac, which represents the chorionic cavity, is a small anechoic collection of fluid surrounded by an echogenic ring that represents trophoblasts and decidual reaction. The gestational sac does not lie within the endometrial canal, but rather is intradecidual in its location and abuts the endometrial canal. With TVS, it is usually possible to identify the gestational sac by about 4.5 weeks of menstrual age, when the mean sac diameter (MSD) is approximately 2mm to 3mm.

The yolk sac is usually the first structure seen within the gestational sac. The presence of a yolk sac within the gestational sac is diagnostic of an intrauterine pregnancy. The yolk sac is normally round or oval and has a uniformly thick, echogenic wall. TVS can often demonstrate it when the MSD is 5mm to 6mm and the yolk sac is frequently seen before the embryo or amnion. The yolk sac should always be visualized by TVS when the MSD is 8mm or greater.

The yolk sac plays an important role in early embryonic life. It is involved in the transfer of nutrients to the embryo, hematopoiesis and formation of the primitive gut. The yolk sac remains connected to the midgut by the vitelline duct, which can often be demonstrated sonographically.

The amnion is a thin, rounded membrane that surrounds the embryo. In turn, the amnion is completely surrounded by the thick, echogenic chorion. The yolk sac is situated between the amnion and chorion. The amnion develops at about the same time as the yolk sac (5 to 6 weeks). However, because it is very thin, it is more difficult to visualize and is seen only when it lies perpendicular to the ultrasound beam.

Unlike the yolk sac, the amnion grows rapidly during pregnancy. The growing amniotic membrane begins to fuse with the chorionic membrane by the middle of the first trimester and fusion is not complete until at least the 12^th week and often as late as the 16^th week. By 16 weeks, the amnion has fused with the chorion, thus obliterating the chorionic cavity. The chorionic cavity is more echogenic than the amniotic cavity due to its thicker, stickier consistency. Sonographic differentiation of the amnion and chorion is usually not difficult in the first trimester, thus permitting the reliable determination of amnionicity and chorionicity in multi-fetal pregnancies.

With TVS, embryos as small as 1mm to 2mm can be routinely identified. Cardiac activity, an indicator of embryonic viability, may be seen immediately adjacent to the yolk sac. The absence of cardiac activity however, does not indicate embryonic demise. Using TVS, absent cardiac activity may be normal in embryos up to 5mm. With TAS absent cardiac activity may be normal in embryos up to 9mm.

Using TVS, the threshold level (lowest number at which a finding is present) for cardiac activity is 40 days menstrual age (MSD = 10mm) and the discriminatory level (number at which a certain finding should always be present) is 46 days, menstrual age (MSD = 16mm). However, in using sonographic landmarks as predictors of outcome, it is best to use internal controls (i.e. MSD) rather than menstrual age.

When the crown-rump length (CRL) reaches 12mm, the head can be differentiated from the trunk. The head and trunk are about equal in size. Progressively, the limb buds, umbilical cord and then the primary ossification centres of the maxilla, mandible and clavicle can be visualized. By the 11^th week, the embryo becomes a fetus.

Normal embryologic development in the first trimester may mimic the sonographic appearance of a fetal anomaly. The embryonic rhombencephalon, which later forms the fourth ventricle, appears as a cystic structure in the posterior fossa at about 7 weeks to 8 weeks and should not be misdiagnosed as an intracranial cyst or hydrocephalus.

Physiologic midgut herniation is often seen as a small (less than 1cm) echogenic mass protruding into the umbilical cord at approximately 8 weeks and may still be present up to 12 weeks. This should not be confused with an abdominal wall defect, such as omphalocele or gastroschisis. Small umbilical cord cysts that measure up to 8mm, may be seen in up to 0.4% of normal first trimester pregnancies. They are usually seen at 8 weeks and disappear by 12 weeks and are of no clinical significance.

ESTIMATION OF GESTATIONAL AGE

TVS can identify the gestational sac between 30 days to 35 days menstrual age, the yolk sac at approximately 36 days and the embryo at about 40 days. One measures the gestational sac by the following equation: MSD (length + width + height) divided by 3. These measurements are obtained from the chorionic tissue - fluid interface. The width is measured on the transverse scan and the length and height on the sagittal scan. The gestational sac grows approximately 1mm per day up to 8.5 weeks and the gestational age in days can be estimated during this time by adding 30 to the mean sac diameter. Once the embryo is visualized, the measurement of the CRL is more accurate. The CRL between 6.5 weeks and 10 weeks is the single most accurate method of pregnancy dating.

Sonographic Correlation and hCG Level

Bree correlated the presence of the gestational sac, yolk sac and embryo by TVS with hCG levels using the first International Reference Preparation (IRP). He found the discriminatory level for an intrauterine gestational sac was 1,000 mIU/mL. Absence of a visible gestational sac above this discriminatory level may indicate an ectopic pregnancy or, less likely, a recent spontaneous abortion or molar pregnancy. The discriminatory level for a yolk sac was 7,200 mIU/mL and for an embryo, 10,800 mIU/mL.

EARLY PREGNANCY FAILURE

Threatened abortion is a clinical term used to describe women who have vaginal bleeding with closed cervical os during the first 20 weeks of pregnancy. This occurs in about 25% of pregnancies and of these approximately 50% will subsequently abort. Abnormal intrauterine pregnancies are referred to by various terms depending on the stage of embryological development and the time of sonographic evaluation, relative to the abortion process. Embryonic demise refers to a discrete embryo with clearly documented lack of cardiac activity. Blighted ovum or anembryonic pregnancy refers to a gestational sac in which the embryo failed to develop or died at a stage too early to visualize.

These terms are preferred to the clinical term "missed abortion", because the latter does not adequately describe the development of the pregnancy. It is important to remember that menstrual dates may be inaccurate. Therefore, sonographic findings should be evaluated independent of menstrual history.

The sonographic diagnosis of embryonic demise can be made with certainty by visualizing a discrete embryo that is greater than 5mm (by TVS) or 9mm (by TAS) and documenting the lack of cardiac activity. It is important to remember that an embryo smaller than this, without demonstrable cardiac activity, may be normal. However, demonstrating cardiac activity in early pregnancy does not guarantee a normal outcome. Filly¹ (Table 1) summarized a number of articles on early pregnancy failure following sonographic demonstration of an embryo with cardiac activity, according to the time of demonstration and whether the patient was bleeding.

If an embryo is not visible at the time of sonographic examination, the diagnosis of pregnancy failure is based on characteristics of the gestational sac. The major criteria for an abnormal gestational sac includes a large sac size without a yolk sac and/or embryo, a grossly abnormal sac shape and visualization of the amnion without an embryo.

As early gestational sac growth parallels embryonic growth, an embryo should always be seen in normal pregnancies when the gestational sac exceeds a certain critical size. Using TAS, an MSD <20 mm without a yolk sac and a MSD <25mm without an embryo is diagnostic of an anembryonic pregnancy. Using TVS, the most widely accepted criteria are a MSD <8mm without a yolk sac and a MSD <16mm without an embryo. As it is always best to give the pregnancy the benefit of the doubt, one should probably allow a 1mm to 2mm leeway in the MSD measurements.

Although the shape of a normal gestational sac may vary, grossly aberrant shapes are readily recognized and will reliably predict an abnormal outcome.

After six weeks, an embryo should always be present when one visualizes the amnion. Amnion without an embryo is also diagnostic of early pregnancy failure. At times, it may be difficult to distinguish an abnormally large yolk sac from the amnion. However, the clinical significance is the same, as this appearance is diagnostic of an abnormal pregnancy.

SONOGRAPHIC PREDICTORS OF POOR OUTCOME

Certain sonographic findings have been reported to predict abnormal outcome in the presence of a live embryo or prior to visualization of the embryo. They include embryonic bradycardia, small sac size relative to the embryo, enlarged or abnormal yolk sac and subchorionic hemorrhage. These findings can be used to identify a subgroup of embryos that are at risk for embryonic demise or subsequent diagnosis of fetal anomaly and these pregnancies require close follow-up.

Embryonic Bradycardia

The normal embryonic heart rate in a gestation of up to 6.2 weeks (CRL < 5mm) is equal or greater than 100 beats per minute (bpm). This increases to about 120bpm between 6.3 weeks to 7 weeks (CRL 5mm to 9mm) and to about 140bpm by 8 weeks.

Laboda et al. described five embryos between 5 and 8 weeks with heart rates less than 85bpm, all of whom subsequently aborted. May et al. described six embryos between 5 and 7.3 weeks with heart rates less than 85 bpm, all of whom spontaneously aborted.

In a larger series, Benson and Doubilet followed 37 embryos with an embryonic heart rate less than 90bpm scanned prior to 8 weeks. Thirty-two of the embryos died before the end of the first trimester. More specifically, all 7 embryos with heart rates less than 70bpm died, as did 10 of 11 with heart rates between 70bpm and 80bpm and 15 of 19 with heart rates between 80bpm and 90bpm. In a subsequent series, they determined that embryos between 7weeks and 8 weeks with a heart rate less than 110bpm had a very poor prognosis.

Embryos with a rapid embryonic heart rate (> 134bpm up to 6.2 weeks and > 154bpm between 6.3 and 7 weeks) have a good prognosis, and a high likelihood of a normal outcome.

Small Sac Size

The MSD should be at least 5mm greater than the CRL. Bromley et al. found that 15 of 16 embryos between 5.5 and 9 weeks, in which the MSD was <5mm greater than the CRL, were spontaneously aborted.

Enlarged or Abnormal Yolk Sac

Lindsay et al found that a yolk sac diameter greater than 5.6 mm between 5 and 10 weeks was always associated with an abnormal outcome, either embryonic demise or fetal anomaly. Yolk sac calcification is also associated with embryonic demise.

Subchorionic Hemorrhage

Subchorionic hemorrhage is similar to placental abruption occurring later in pregnancy, and results in elevation of the chorionic membrane. This is a common finding in the first trimester and may be associated with vaginal bleeding. Up to 18% of women with vaginal bleeding in the first half of pregnancy have a subchorionic hemorrhage as the etiology.

The sonographic appearance is that of an extrachorionic fluid collection which tends to conform to the shape of the adjacent gestational sac. Acute hemorrhage is hyper- or iso-echoic relative to the placenta. The hemorrhage gradually becomes hypo-echoic within 7 to 10 days. Occasionally, hemorrhage may be difficult to distinguish from a blighted twin.

There is no consensus as to the prognosis of subchorionic hemorrhage. Some studies have reported a higher risk of spontaneous abortion, whereas most studies have found a greater risk only if the hemorrhage is large. Bennett also found that in addition to size, a higher spontaneous abortion rate in women 35 years or older compared with younger women and in women with bleeding at or before 8 weeks compared with those after 8 weeks. Most small hemorrhages resolve without clinical sequelae.

References

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22. Pederson JF, Mantoni M. Prevalence and significance of subchorionic hemorrhage in threatened abortion: a sonographic study. AJR 1990;154:535-7.
23. Sauerbrei EE, Pham DH. Placental abruption and subchorionic hemorrhage in the first half of pregnancy: US appearance and clinical outcome. Radiology 1986;160:109-12.
24. Bennett GL, Bromley B, Lieberman E, Benacerraf BR. Subchorionic hemorrhage in first-trimester pregnancies: prediction of pregnancy outcome with sonography. Radiology 1996;200:803-6.

Screening Between 12-20 Weeks: Ultrasound and Biochemistry

Jo-Ann M. Johnson, M.D.

ASSOCIATE PROFESSOR, DEPARTMENT OF OBSTETRICS & GYNAECOLOGY, UNIVERSITY OF TORONTO, TORONTO, CANADA

Prenatal genetic screening for chromosome abnormalities has traditionally relied upon simple maternal age-based screening. Now, a number of screening options are or are becoming available within the 12- to 20-week window. These are based on combining maternal age with various maternal biochemical and fetal ultrasound data to provide a woman with a more accurate and personalized estimate of her risk.

Chromosome Defects

Every woman has a risk that her fetus will have a chromosome abnormality. In order to determine a woman’s individual risk, one must consider the background risk that depends on the following variables:

The background risk can be further refined by adding information obtained through prenatal screening tests. Each time a test is carried out, the background risk is multiplied by the test result to calculate the "adjusted risk" which then becomes the background risk for the next test. Additional tests include:

ULTRASOUND

The 11-14 week scan and chromosome defects

Most fetuses with Down’s syndrome (DS) and some normal fetuses have an increased collection of fluid behind the neck in early pregnancy. In the first trimester, this is defined as nuchal translucency (NT). During the second trimester, the translucency usually resolves but in some cases evolves into either nuchal edema/thickening or cystic hygroma, with or without generalized hydrops.

Recently, it has been shown that screening for chromosome abnormalities, by measuring the fetal NT thickness between 11 weeks to 14 weeks gestation and combining it with maternal age, will identify 70% to 80% of fetuses with DS and approximately 70% of other chromosome abnormalities (trisomies 13, 18 and Turner’s syndrome) at an invasive rate of 5% to 8% (Snijders et al, 1998).

In addition, increased NT thickness (³ 3.0mm) in the presence of normal chromosomes is associated with an increased incidence of certain birth defects, including cardiovascular (cardiac septal defects), pulmonary (diaphragmatic hernia), renal and abdominal wall defects, as well as certain genetic syndromes, particularly hypokinesia disorders (Souka et al, 1998).

Screening using NT measurements is also useful in multiple gestation, where conventional methods (eg, maternal serum screening, [MSS]) are not applicable. In dichorionic gestations, discordancy for NT thickness is a useful marker for chromosome and other abnormalities. In monochorionic gestations, it appears to be a useful marker twin-twin transfusion syndrome (Sebire et al, 1996).

Calculating the NT-adjusted risk

The NT thickness normally increases with increasing gestational age. The measurement is also subject to both inter- and intra-observer variability. To reduce the impact of these factors,

1. Guidelines for NT measurement technique have been described (see below).
2. A certification process in nuchal scanning has been established (Fetal Medicine Foundation [FMF], UK and Canada) (see below).
3. Median NT measurements have been established for each gestational age between 11 weeks to 13.6 weeks (CRL 45mm to 84mm).
4. The difference from the normal median for gestation ´ the background risk = the "NT-adjusted risk".
5. The calculation is performed using software that is made available to individuals who have been certified in nuchal scanning.

Certification in Nuchal Scanning

A certification process and quality assurance program has been established at The Fetal Centre at The Hospital for Sick Children, Toronto, Ontario (the Canadian branch of the FMF, UK). The certification process has 2 components: the theoretical and the practical.

• Theoretical Certificate: To receive the theoretical certificate, one must attend the 11-14 week scan course and write an examination. All persons involved in prenatal screening, including geneticists, genetic counsellors, midwives, family physicians, obstetricians, maternal fetal medicine specialists, radiologists and sonographers are invited to take the course.
• Practical Certificate: The practical component is relevant to those individuals who perform sonography and involves submitting a log of 50 nuchal images for audit. If the images fulfil the audit criteria, the individual is eligible to receive the risk-adjustment software provided he/she agrees to comply with an ongoing audit program.

The course is offered every 4 to 6 months in London, UK and has been offered 9 times in Canada. To date, more than 500 individuals from across Canada have participated in the local courses. An ongoing audit mechanism is built into the risk software for quality assurance.

More details can be obtained through The Fetal Centre office or web site (see references).

Current Status of NT-Screening

NT screening is presently NOT a standard of care in Canada. However, if an obvious nuchal or other abnormality is seen at the 11-14 week scan, the patient should be referred promptly for genetic counselling. Using a biometric cut-off is not a recommended screening strategy due to the variation of NT thickness with gestational age. However, until a formal screening policy is adopted, a biometric cut-off of 3mm (95^th percentile) may be used. Since unification of technique and ongoing audit are critical to the performance of NT as a screening test, most centres do rely on the certification and audit process described above.

NT screening is a PROGRAM, not a measurement in isolation. If NT screening is offered, it should be done so in the context of a comprehensive program that offers:

• Pre-ultrasound counselling,
• Ultrasound by trained sonographers (with ultrasound quality assurance program),
• Post-ultrasound counselling and risk-interpretation,
• Immediate access to genetic counselling and invasive testing if an abnormality is detected, and
• Appropriate follow-up.

The 18-20 week scan and chromosome defects

The 18 week scan is not generally considered a screening test for chromosome abnormalities. However, there are an increasing number of subtle abnormalities, commonly referred to as "ultrasound markers", that are associated with an increased risk for chromosome defects. (Benacerraff et al, 1987, 1990, 1991, Snijders and Nicolaides et al, 1992, 1994, 1995, Nyberg et al, 1993, 1998)

Approach to ultrasound markers

1. Perform a detailed scan to exclude additional abnormalities.
2. The risk of a chromosome abnormality depends on

• The type of abnormality

• ie, soft, medium, strong markers (Snijders et al, 1998)

• The number of abnormalities.

• ie, the risk of a chromosome abnormality increases with more than 1 abnormality.

• The mother’s background risk

Examples of markers associated with an increased risk of Trisomy 21*

No abnormalities

Close to two-thirds of fetuses with Down’s syndrome will have a structural defect or at least one of the above "markers" when examined by ultrasound between 16-22 weeks. (Nyberg 1998, Johnson 1998) Consequently,

• If there are no defects, the risk for trisomy 21 is about ½ times the background risk.

QUESTION

A 34 years-old woman (G₁ P₀) at 18 weeks gestation (baseline risk 1:342) has the finding of echogenic bowel at the time of her routine scan. What is her adjusted risk based on this finding? What would the management be? What would her risk be if her ultrasound examination were completely normal? How would you explain it to her?
(Answer at end of article)

In another approach, described by Benacerraf et al, "points", based on specific ultrasound findings, are assigned. For example, structural malformations are assigned 2 points and "markers", such as pyelectasis, 1 point. Genetic counselling and amniocentesis are then offered to any patient with a score of 2 or more. In a recent modification of this approach, points are also assigned for advanced maternal age with 1 point for women aged 35 to 40 years inclusively, and 2 points for women older than 40.

The advantages of ultrasound screening using either system is that it permits improved individual counselling following a second trimester sonogram regarding the risk of fetal DS.

A major disadvantage of assigning risk factors for ultrasound markers at the 18 week scan are the lack of clear definitions of the markers, and the lack of prospective data verifying their significance. Over-interpretation may lead to major patient anxiety, yet failure to report even minor findings can have medico-legal consequences.

MATERNAL SERUM BIOCHEMISTRY AND CHROMOSOMAL DEFECTS

Serum markers at 15-20 weeks gestation

In the late 1980s, a new method of screening for DS was introduced that takes into account not only maternal age, but also the concentrations of various feto-placental products in the maternal circulation. At 16 weeks gestation, the maternal serum concentrations of human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP) and unconjugated estriol (uE3) in DS are sufficiently different from the median in unaffected pregnancies to allow the use of combinations of some or all of these substances to select a "high risk " group for whom, invasive testing (eg, amniocentesis) is then offered.

The method, known as the "triple test" or "maternal serum screening (MSS)"is associated with a detection rate (DR) of fetal DS of ~ 60% at an invasive rate of 5%. In Ontario, the cut-off for offering invasive testing is 1:385 and this is associated with a DR of 75% at an invasive rate of 7% to 8%.

Serum markers at 9-14 weeks gestation

Most of the second trimester biochemical markers are ineffective in the first trimester, but 3 markers have been found to be effective in the first trimester - maternal serum placental-associated plasma protein-A (PAPP-A), free ß-hCG and total hCG. A combination of PAPP-A and free-ß hCG has been shown to be associated with a DR of 60% at a false positive rate (FPR) of 5%, comparable to second trimester screening.

Combining results from screening tests

Ultrasound markers and biochemical markers appear to be independent predictors with respect to DS risk. It is possible to combine them to achieve a higher DR than with each test alone.

Comparison of detection rates for DS by combination of parameters

* False positive rate and invasive rate are used interchangeably.

It is important to note that screening in the first trimester (ultrasound with or without biochemistry) will alter the predictive value of biochemical screening in the second trimester (Kadir et al, 1998, Thilaganathan et al, 1998), and presumably the value of "markers" detected at the 18 week scan as well. Thus, if second trimester MSS (or ultrasound) is performed following first trimester NT screening (+/- biochemistry), one should adjust the MSS risk accordingly. It is possible to combine the 2 risks by deriving a likelihood ratio from the nuchal scan (NT adjusted risk/ baseline risk) and multiplying it by the MSS risk. For example, if the baseline risk is 1/250 and the NT-adjusted risk is 1/500 then the LR is 2. If the MSS is 1/200, the risk adjusted by the previous NT would be 1/400.

Current standard of care in Canada

1. All pregnant women ³ 35 years of age (late maternal age) at the time of delivery should be offered genetic counselling and option of invasive testing.
2. All pregnant women should be offered a screening ultrasound scan between 18-20 weeks of pregnancy.
3. Maternal serum screening (15-20 weeks): Currently, provincially-funded screening programs exist in Ontario, Nova Scotia, Manitoba. Due to significant regional variations in access, MSS is not currently considered standard of care in Canada.
4. The 11-14 week scan is currently NOT a standard of care.

Answer to Question

• The finding of echogenic bowel would increase her risk of fetal DS from a baseline of 1:342 (based on age alone) to 1:62 (1/342 x 5.5). This would result in her being offered genetic counselling and the option of amniocentesis.

• Conversely, a normal ultrasound examination would substantially reduce her age-related risk by approximately 50 percent (ie, 1:342 to 1: 686). While this may be reassuring, she should be informed that ultrasound may miss up to one in every three DS fetuses (about 1/3 of DS fetuses will have a normal ultrasound examination).

References

• Benacerraf BR, Gelman R, Frigoletto FD. Sonographic identification of second trimester fetuses with Down’s syndrome. N Engl J Med 1987:317:1371-6.
• Benaceraff BR, Mandell J, Estroff JA, et al. Fetal pyelectasis: a possible association with Down syndrome. Obstet Gynecol 1990;76:58-60.
• Benaceraff BR, Neuberg D, Frigoletto FD. Humeral shortening in second trimester fetuses with Down syndrome. Obstet Gynecol 1991;164:863-7.
• Kadir RA, Economides DL. The effect of nuchal translucency measurement on second trimester biochemical screening for Down's syndrome. Ultrasound Obstet Gynecol. 1997;9:244-7.
• Nicolaides KH, Azar G, Snijders RJM, et al. Fetal nuchal oedema:associated malformations and chromosomal defects. Fetal Diagn Ther 1992;7:123-131.
• Nicolaides KH, Brizot ML, Snijders RJM. Fetal nuchal translucency thickness: ultrasound screening for fetal trisomy in the first trimester of pregnancy. Br J Obstet Gynaecol 1994;101:782-6.
• Nyberg DA, Resta RG, Luthy DA, et al. Prenatal sonographic findings of Down syndrome: review of 94 cases. Obstet Gynecol 1990;76:370-7.
• Nyberg DA, Luthy DA, Resta RG, et al. Age-adjusted ultrasound risk assessment for fetal Down’s syndrome during the second trimester: description of the method and analysis of 142 cases. Ultrasound Obstet Gynecol 1998;12:8-14.
• Orlandi F, Damiani R, Hallahan TW, Krantz DA, Macri JN. First-trimester screening for fetal aneuploidy: biochemistry and nuchal translucency.Ultrasound Obstet Gynecol 1997; 10: 381-386.
• Pandya PP, Kondylios A, Hilbert L, et al. Chromosomal defects and outcome in 1,015 fetuses with increased nuchal translucency. Ultrasound Obstet Gynecol 1995;5:15-9.
• Sebire NJ, Noble PL, Psarra A, et al. Fetal karyotyping in twin pregnancies: selection of technique by measurement of fetal nuchal translucency. Br J Obstet Gynaecol 1996;103:887-90.
• Sebire NJ, Snijders RJM, Hughes K, et al. Screening for trisomy 21 in twin pregnancies by maternal age and fetal nuchal translucency thickness at 10-14 weeks of gestation. Br J Obstet Gynaecol 1996;103:999-1003.
• Snijders RJM, Holzgreve W, Cuckle H, et al. Maternal age-specific risks for trisomies at 9-14 weeks of gestation. Prenat Diag 1994;14:543-52.
• Snijders RJM, Noble P, Seibre N, et al. UK Multicentre project on assessment of risk of trisomy 21 by maternal age and fetal nuchal-translucencky thickness at 10-14 weeks of gestation. Lancet 1998;352:343-6.
• Snijders RJM, Sundberg K, Holzgreve W, et al. Maternal age and gestation specific risk for trisomy 21. Ultrasound Obstet Gynecol (in press).
• Thilaganathan B, Slack A, Wathen NC. Effect of first trimester nuchal translucency on second-trimester maternal serum biochemical screening for Down's syndrome. Ultrasound Obstet Gynecol 1997;10:261-4.

For information on nuchal screening:

Fetal Medicine Foundation, Canadian Branch
The Fetal Centre at The Hospital for Sick Children
555 University Avenue, Toronto, Canada
Phone: (416) 813-8228
Fax: (416) 813-7880
Web site: http://www.hscFetalCentre.org

The Fetal Medicine Foundation UK
8 Devonshire Place
London, England
For information and course material:
Web site: http://www.fetalmedicine.com

The 18 Week Scan and Fetal Anatomical Survey

Ants Toi, M.D.

ASSOCIATE PROFESSOR, DEPARTMENT OF MEDICAL IMAGING, UNIVERSITY OF TORONTO, TORONTO, CANADA

Who is the patient?

Patients with indication

• About 50% (20-70) of patients have an indication for obstetrical ultrasound (OBUS).
• When ultrasound (US) is indicated, outcomes are improved.
• Indications include:
  • Dates unknown or need for certainty
  • Prior problems
  • Size/dates discrepancy
  • Any clinical concern
  • Procedure guidance

Patients without indication = ROUTINE SCAN GROUP

Who scans?

• Appropriately trained operator
• Suitable equipment
• Standard protocol.^1,2
• Experienced physician supervision.

Why?

The purpose of routine OBUS is to improve outcome for:

• fetus and mother
• family and society

Outcomes with routine scanning

• Improved maternal health consciousness.³
• Decreased scans per pregnancy.^Eik-Nes
• Easier management of problems in late pregnancy.^Eik-Nes
• Reassurance and anxiety reduction.
• Perinatal mortality reduced: 4.6/1,000 versus 9/1,000.⁴
• US detected
  • Wrong dates
  • Anomalies (average 53% [range, 17% to 85%], high risk 80% [range, 27% to 99%])
  • Twins
  • Intrauterine growth retardation

• Perinatal morbidity not proven to be altered.^5,6

Issues with routine ultrasound and outcomes

• Lesions that appear late.
• Insignificant findings.
• False positives and negatives.
• "Markers" that need managing.
• US itself does not change pregnancy.
• US only detects, but does not fix, problems.
• Improvement requires appropriate management.
• Good US is useless without good management, and vice versa.
• Patients who cannot act on the information.

When to scan?

10 to 14 weeks

In Israel, some operators use transvaginal scanning at 12-16 weeks for the routine anatomy survey. While many anomalies can be detected, overall it is too early to examine the fetus consistently and adequately. Some high-risk patients may benefit from such early scans.

18 to 20 weeks

Probably the best time to evaluate fetal anatomy is 24 weeks, but this is too late to manage any detected problems effectively and easily. Many feel that the routine 18 week scan is a good compromise. Under 18 weeks, the fetus is too small for consistent anatomic evaluation. Dates assignment is still accurate. Two weeks remain for diagnostic evaluation prior to 20 weeks when unchallenged pregnancy termination is permitted.

What to scan?

• All structures related to pregnancy are evaluated: uterus, cervix, placenta, fluid, and fetus.
• Important to use standard protocol and refer promptly if any deviation from normal.
• Similar examination guidelines published by the Canadian Association of Radiology (CAR), The Society of Obstetricians & Gynaecologists of Canada (SOGC) and others. ^1,2
• Uterus, cervix
• Adnexa (1^st trimester
• Amniotic fluid volume (volumes determined visually, by maximum vertical pocket or amniotic fluid index [AFI]
• Biometry
  • Accurate biometry has become most important with the advent of serum screening and nuchal translucency screening
• Anatomy
• Multiple pregnancy (determine type of twins at first opportunity)

Limitations of routine scan

• Ultrasound is good, but not perfect.^4-7
• Late developing problems.
• Chromosomal abnormality.
• Lesion size/visibility issues.
• Physician and patient should be aware that there are limitations.

Anomaly detection with routine scan (RADIUS) ⁵

Biometry

Need for accuracy.

• Follow pregnancy progress
• Interventions

Accuracy of dating⁸

• IVF, ovulation induction, artificial insemination, single intercourse record (<3 days)
• Ultrasound, any gestation, (8% of estimate)
• Clinical exam:
  • T1 (2 weeks)
  • T2 (4 weeks)
  • T3 (6 weeks)

• BPD at 15-22 weeks is a better predictor of spontaneous labor than certain dates.^9,10

Which nomograms?

I have found these to be reliable.

BPD	Hadlock.	J Ultrasound Med 1982;1:97
HC	Hadlock	J Ultrasound Med 1982;1:281
AC	Hadlock	AJR 1982;139:367
Fem	Jeanty, P	J Ultrasound Med 1984;3:75

Role of biometry with maternal serum screening program (MSS or triple screen):

MSS detects

• Error in dates.
• Trisomy 21 (60%) [Down syndrome]
• Trisomy 18 (50%)
• Open neural tube defects (80%)

Different age nomograms in Ontario

For standardization, MSS program needs raw measurement CRL or BPD. The MSS computer converts the raw measurement to gestation age with Daya¹¹ for CRL , Hadlock (1982) for BPD.

What is Measured at Routine 18 Week Scan

1. BPD
2. OFD (Occipito frontal diameter)
   

   Corrected BPD

   Adjusts BPD to allow for moulding effects.
   It is as effective as head circumference.
   Corrected BPD = Square root of (BPD x OFD/1.265)
   Head Circumference (HC)
   
   Direct
   Ellipse: - direct measurement
   Formula: - all measurements taken OUTSIDE
   - HC = 1.57 x ([outer side-side] + [outer front-back])
   
   Alternate calculation
   Use BPD and middle-middle OFD
   Convert to "all around outside" measurements with formula:
   HC = 1.62 x (BPD+OFD)

3. Abdominal circumference (AC)
4. Femur
5. Other

• Cerebellum
• Orbits
• Other bones

Ultrasound gestation age calculation

• US measures size, age is assumed.
• Multiple parameters give best estimate.
• Corrected BPD or HC, F, AC
• Additional parameters not helpful for averaging
• Averaged age is as good as weighted composite age.

Earliest measurement is best

• Once dates are established on first good early scan, they never change.
• Subsequent scans only evaluate fetal size and growth.
• A good scan under 20 weeks is better than a certain LMP at predicting delivery date.^9,10

How to decide if one should include a parameter?

• Structures must be normal
• Measurements correctly obtained.
• Size estimates within 2 standard deviations of expected (rule of thumb = within 8% of expected age)
• Difficult in third trimester.

• If limbs are too short - evaluate for dwarfism
• If abdominal circumference is too small - consider IUGR, aneuploidy. Follow growth, health.
• Head small/large - consider anomaly, refer to experts.

When is LMP (last menstrual period)/EDC (expected date of confinement) rejected and replaced by ultrasound dates?

• If no clinical reason to reject measurements.
• If MA is outside 95% limits from US measurements.
• 95^th percentile is 8% of estimate (10% for easy bedside calculation purposes) ⁸

Once Dates are Established, They NEVER change - Ultrasound Follows Growth

What Anatomy is Evaluated?

If you can’t see a structure, it may be abnormal. Re-scan as high risk promptly or refer.

Amniotic Fluid Volume

• Normal can be determined visually or by measurement.
• Oligohydramnios
  • Visual or maximum pocket < 2cm.
• Polyhydramnios
  • Visual or maximum pocket > 8cm or AFI > 24.

Placenta

Relationship to cervix.

Cervix

• Look for shortening [< 2.5cm], funneling

Multiple pregnancy

About 1/90 pregnancies (becoming 1/40 with IVF according to Dr. John Hobbins)

• 70% dizygotic
• 30% monozygotic
  • 25% dichorionic diamniotic
  • 75% monochorionic diamniotic
  • 2% monochorionic
  • monoamniotic
  • rare conjoined twins

Morbidity and mortality is 2 to 3 that of singletons (miscarriage, prematurity, growth restriction, death).

Monochorionic twins have more problems and limited treatments since they share circulation.

Determine chorionicity at first visit using:

• Placental number
• Gender
• Membrane character: "T" vs "Y" insertion, thickness

Naming twin fetuses:

• A and B may change places.
• Better to use specific characteristics (eg. gender) or state which sac fetus is in (eg. Male fetus in upper right sac, and female fetus in lower left sac)

What do you tell the patient about the scan?

As a Technologist:

• Things look good to me, but my radiologist will review the films and report to your physician.
• If problems are seen, it is better not to discuss with patient, but inform radiologist.

As a Supervising Physician:

• Everything looks good.
• Remember that ultrasound is a good test, but it is not perfect.
• While it can find most major problems, it cannot detect every abnormality.
• If you are certain of an abnormality, divulge to the patient and inform her that you will talk to her physician who will help her manage things further.
• If you are not certain, say you are not sure, inform her physician and suggest further prompt consultation.

Routine scan summary

• Should be offered to pregnant women at 18 – 20 weeks.
• Standard protocol.
• Awareness of limitations of study: both patient and physician.
• Prompt management and referral plans in place.
• (Dating scan for MSS 8-12 weeks)
• (NT scan 10 – 14 weeks)

References

1. CAR Ultrasound guidelines: Antepartum obstetric ultrasound examinations. 1993.
2. SOGC Clinical Practice Guidelines: Policy statements 64, 65. Obstetric/Gynaecologic Ultrasound. July 1997.
3. Campbell S. J Psychosom Obstet Gynaecol 1982;1:57.
4. Saari-Kemppainen A, Karjalainen O, Ylostalo P, et al. Ultrasound screening and perinatal mortality: controlled trial of systematic one-stage screening in pregnancy. The Helsinki Ultrasound Trial. Lancet 1990;336:387-91.
5. Ewigman BG, Crane JP, Frigoletto FD, et al. Effect of prenatal ultrasound screening on perinatal outcome. RADIUS Study Group. N Engl J Med 1993;329:821-7.
6. Bucher HC, Schmidt JG. Does routine ultrasound scanning improve outcome in pregnancy? Meta-analysis of various outcome measures. Br Med J 1993;307:13-7.
7. Nelson NL, Filly RA, Goldstein RB, et al. The AUIM/ACR antepartum obstetrical sonographic guidelines: expectations for detection of anomalies. J Ultrasound Med 1993;12:189-96.
8. Hadlock FP. Ultrasound determination of menstrual age. In Ultrasound in Obstetrics and Gynecology. Ed. Callen. 3^rd Edition. Saunders. 1994.
9. Tunon K, Eik-Nes SH, Grottum P. A comparison between ultrasound and a reliable last menstrual period as predictors of the day of delivery in 15,000 examinations. Ultrasound Obstet Gynecol 1996;8:178-85.
10. Mongelli M, Wilcox M, Gardosi J. Estimating the date of confinement: ultrasonographic biometry versus certain menstrual dates. Am J Obstet Gynecol 1996;174:278-81.
11. Daya S. Accuracy of gestational age estimation by means of fetal crown-rump length measurement. Am J Obstet Gynecol 1993;168:903-8.

The Fetal Urogenital Tract

Katherine Fong, M.B., B.S.

ASSISTANT PROFESSOR, DEPARTMENT OF MEDICAL IMAGING, UNIVERSITY OF TORONTO, TORONTO, CANADA

Abnormalities of the urogenital tract account for 30% of all malformations detected in utero by ultrasonography. Accurate and early prenatal diagnosis is important, as this may influence obstetric and neonatal management.

A systematic approach to the prenatal diagnosis of urinary tract abnormalities includes:

1. assessment of amniotic fluid volume (AFV)
2. localization and characterization of urinary tract abnormalities
3. search for associated abnormalities

After the 16^th week of gestation, fetal urine production is the major source of amniotic fluid. Therefore, normal AFV implies the presence of at least 1 functioning kidney and a patent urinary conduit to the amniotic cavity. If oligohydramnios is present (without a history of ruptured membranes or evidence for intrauterine growth restriction), urinary tract anomalies must be strongly suspected. In the setting of a urinary tract abnormality, normal AFV indicates a good prognosis. Severe oligohydramnios in the second trimester carries a very poor prognosis because of the associated pulmonary hypoplasia. Occasionally and paradoxically, polyhydramnios may occur, especially with unilateral obstructive uropathy, mesoblastic nephroma, or when there are concomitant abnormalities of the central nervous system or gastrointestinal tract.

The following questions are helpful in defining and characterizing the urinary tract abnormality:

1. Is the bladder identified and normal in appearance?
2. Are kidneys present? Are they normal in position, size and echogenicity? Are renal cysts identified?
3. Is the urinary tract dilated? If so, to what degree, at which level, and what is the cause?
4. Is the involvement unilateral or bilateral, symmetric or asymmetric?
5. What is the fetal gender?

Prenatal detection of a genitourinary anomaly may indicate the presence of associated abnormalities, a syndrome, or chromosomal abnormality. Hence, a thorough sonographic search is necessary. Renal anomalies may be part of the VACTERL syndrome (vertebral, anal, cardiac, trachoesophageal, renal and limb). When there are additional malformations, the risk for chromosomal abnormalities is significantly increased: x 30 for multiple defects versus x 3 for isolated renal defects.

In addition, renal ultrasound is recommended for parents (and siblings) of fetuses suspected to have certain renal abnormalities (polycystic kidney disease, bilateral renal agenesis),

since it may help to diagnose the type of polycystic kidney disease in the fetus and/or detect asymptomatic renal pathology in parents and siblings.

Specific lesions and their sonographic appearance will be discussed, including renal agenesis, horseshoe kidney, bladder exstrophy, hydronephrosis, duplication anomalies, urethral obstruction, renal cystic disease and neoplasm. Abnormalities of the adrenal gland and the genital tract will also be included in this presentation.

References

• Fong KW, Ryan G. The fetal urogenital tract. In: Rumack CM, Wilson SR, Charboneau JW. Diagnostic Ultrasound, Second edition, Mosby-Year Book, 1997.
• Filly RA, Feldstein VA. Fetal genitourinary tract. In: Callen PW. Ultrasonography in Obstetrics and Gynecology, Fourth edition, W.B. Saunders Company, 2000.
• Moore TR, Cayle JE. The amniotic fluid index in normal human pregnancy. Am J Obstet Gynecol 1990;162:1168-73.
• Nicolaides KH, Cheng HH, Abbas A, et al. Fetal renal defects: associated malformations and chromosomal defects. Fetal Diagn Ther 1992;7:1-11.
• Snijders RJM, Sebire NJ, Faria M, et al. Fetal mild hydronephrosis and chromosomal defects: relation to maternal age and gestation. Fetal Diagn Ther 1995:10:349-55.
• Corteville JE, Gray DL, Crane JP. Congenital hydronephrosis: Correlation of fetal ultrasonographic findings with infant outcome. Am J Obstet Gynecol 1991;165:384-8.
• Fernbach SK, Maizels M, Conway JJ. Ultrasound grading of hydronephrosis: introduction to the system used by the Society for Fetal Urology. Pediatr Radiol 1993;23:478-80.
• Wickstrom E, Maizels M, Sabbagha R, et al. Isolated fetal pyelectasis: assessment of risk for postnatal uropathy and Down syndrome. Ultrasound Obstet Gynecol 1996;8:236-40.
• Persutte WH, Koyle M, Lenke RR, et al. Mild pyelectasis ascertained with prenatal ultrasonography is pediatrically significant. Ultrasound Obstet Gynecol 1997;10:12-8.
• Dremsek PA, Gindl K, Voitl P, et al. Renal pyelectasis in fetuses and neonates: diagnostic value of renal pelvis diameter in pre- and postnatal sonographic screening. AJR 1997;168:1017-9.
• Anderson NG, Abbott GD, Mogridge N, et al. Vesicoureteric reflux in the newborn: relationship to fetal renal pelvic diameter. Pediatr Nephrol 1997;11:610-6.
• Wong DC, Anderson PAM. Congenital hydronephrosis who requires intervention? The Canadian Journal of Urology 1999;6:812-8.
• Persutte WH, Hussey M. Chyu J, et al. Striking findings concerning the variability in the measurement of the fetal renal collecting system. Ultrasound Obstet Gynecol 2000;15:186-90.

Adnexal Masses in Pregnancy

Anthony E. Hanbidge M.B., B.Ch.

ASSISTANT PROFESSOR, DEPARTMENT OF MEDICAL IMAGING, UNIVERSITY OF TORONTO, TORONTO, CANADA

Introduction

The management of adnexal masses in pregnancy presents a difficult clinical decision. Abdominal surgery during pregnancy is risky to both the mother and fetus. On the other hand, conservative management may result in spread of cancer or complications such as torsion or rupture of ovarian cysts. Large masses may obstruct labor. Furthermore, as ultrasonographic examination has become a routine component of obstetrical management, the number of adnexal masses detected during pregnancy has increased.

Small ovarian cysts (<6cm) in pregnancy are extremely common, are usually functional and can be managed expectantly. With the widespread use of ultrasound, approximately 1 in 100 gravid women, imaged in the first trimester, will have an adnexal mass greater than 6 cm in size. Of these, 10% will persist beyond 16 weeks gestation. Mature teratomas, cystadenomas and corpus luteal cysts account for most of these masses. Only 5% will represent ovarian cancer. This gives an overall incidence of ovarian cancer in pregnancy of approximately 1:20,000.

Elective surgery for adnexal masses is delayed until the second trimester, a time associated with a reduced risk of spontaneous abortion and hormonal independence of the corpus luteum of pregnancy. Generally, the only type of adnexal mass necessitating surgery in the first trimester is torsion of a corpus luteum. Torsion of an adnexal mass is more common in the first trimester. Elective surgery is thus rarely performed today to prevent this complication. Similarly removing an adnexal mass to prevent obstruction of labor is uncommon.

Role of Ultrasound

Ultrasound plays an important role in detecting and characterizing adnexal masses in pregnancy. Endovaginal sonography substantially improves the diagnostic accuracy in assessing these masses. Most masses detected in the first trimester will be within reach of the transvaginal probe. Not only will it allow optimal localization of the mass to the ovary, fallopian tube or uterus but it also allows optimal characterization of the mass as cystic or solid and if cystic allows characterization of the cyst wall, the cyst content and the nature of solid components.

Later in pregnancy, adnexal masses are more difficult to assess with ultrasound. They are frequently beyond the reach of the transvaginal probe and we must therefore use the less reliable, lower frequency, suprapubic probes to characterize them. It is often difficult to visualize the ovaries later in pregnancy, so it may also be difficult to say with certainty whether the mass is arising from the ovary or not.

While colour flow and spectral Doppler techniques continue to evolve, basic sonographic morphology coupled with the clinical context remains the mainstay of the sonographic interpretation.

Sonographic Interpretation

Once the clinical background has been established, the next most important step in the evaluation is establishing if the mass is intra or extra-ovarian. The mass is then characterized in terms of its size, is it cystic or solid and if cystic, what are the characteristics of its wall and contents?

Most cystic ovarian masses in early pregnancy are functional. This is particularly true for simple or hemorrhagic cysts that are less than 6 cm in size. On the contrary, solid ovarian masses should be regarded as being malignant. One exception is the ovarian fibroma, which often has a characteristic sonomorphology, appearing as a hypoechoic solid mass that attenuates the sound beam. Most solid adnexal masses, however, are not ovarian in origin but are in fact fibroids arising from the uterus or broad ligament. In this setting a diligent search should be made for normal ovaries separate from the mass. It is sometimes helpful to produce motion of the uterus by gentle external abdominal pressure, with motion of the mass in concert with the uterus suggesting a uterine origin. Similarly, manipulating the endovaginal probe may show that the ovary moves separately from the mass, suggesting its extra-ovarian origin. In difficult cases where the ovary cannot be visualized, magnetic resonance imaging may be helpful.

If a mass is cystic, establishing that it is extra-ovarian is also important, as extra-ovarian cystic masses are virtually always benign. Also, asymtomatic simple ovarian cysts with smooth walls and no intra-cystic septa, nodules or echoes have little or no malignant potential. Any degree of intra-cystic complexity, especially mural nodularity, may represent malignancy. Low malignant potential epithelial tumours of the ovary in particular may have only very minimal complexity.

Certain masses have a more specific appearance. This is true of many dermoid cysts and many endometriomas. Dermoid cysts (mature teratomas) are benign and their management can be deferred until after the pregnancy. These neoplasms derive from ectodermal differentiation of totipotential cells and generally occur in young women during the active reproductive years. They are bilateral in 10 to 15% of cases. They characteristically appear as well-circumscribed, largely cystic masses with an echogenic mural nodule ('dermoid plug'), which may produce shadowing. The 'dermoid plug' is usually produced by a focus of hair, teeth or fat; echogenic hair floating on anechoic liquid sebum produces a characteristic 'hair-fluid level'. Echogenic hair or calcium may shadow all of the tissue deep to it, resulting in a curvilinear interface referred to as "the tip of the iceberg". During pregnancy, MRI may be a helpful adjunct to ultrasound at confirming a mass as a dermoid cyst if there is difficulty. The rare immature (malignant) teratoma consists of a wide variety of tissue elements in varying stages of differentiation. Most malignant teratomas arise in prepubertal adolescent and young women. These tumours grow rapidly with early penetration of the capsule, followed by contiguous spread or distant metastases. Sonographically these tumours appear largely solid.

Endometriosis is a leading cause of infertility. Nonetheless women with endometriosis will present in pregnancy with adnexal masses. It results from the implantation of endometrial tissue outside the uterus. Up to 70% of cases of endometriosis go undetected with ultrasound and only the hemorrhagic collections ('chocolate cysts') are seen. Endometriomas manifest as well-circumscribed cystic masses with widespread low-level internal echoes resulting from the breakdown of blood products. Other characteristics include smooth walls and through transmission, although some irregularity of the wall is possible. It is often possible to suggest the diagnosis of an endometrioma from the characteristic sonomorphology, obviating surgery at least until the pregnancy is complete.

A particularly challenging problem is differentiating between benign and malignant epithelial tumours of the ovary. The three major types include serous, mucinous and endometrioid forms. Greater amounts of solid tissue, thickened and irregular walls, mural nodularity and greater size all increase the possibility of malignancy. These criteria are often difficult to apply to individual cases. The presence of particulate ascites in an asymtomatic patient in association with an ovarian mass is concerning for malignancy.

Serous tumours are common and account for 30% of all ovarian tumours. In all age groups, 60% are benign, 15% borderline and 25% malignant. More will be benign in the reproductive years. They account for 40% of all cancers of the ovary. On sonograms, benign serous cystadenomas appear as sharply marginated anechoic masses that may be large and are usually unilocular. Internal, thin-walled septations may be seen and occasionally papillary projections. Serous cystadenocarcinomas have a more complex appearance, are usually multilocular containing multiple papillary projections and septations with echogenic material occasionally present within locules. Cystadenocarcinomas may be quite large, >15 cm in diameter in some patients. Ascites is common in serous cystadenocarcinomas but uncommon in cystadenomas. These tumours are more likely to be bilateral than the mucinous counterparts.

Mucinous cystadenomas and cystadenocarcinomas are slightly less common than the serous forms, constituting about 20 to 25% of all ovarian neoplasms. Roughly 80% are benign, 10 to 15% are borderline and 5% are malignant. Mucinous cystadenocarcinomas represent only 10% of all ovarian cancers. They are usually unilateral. On sonograms, mucinous cystadenocarcinomas usually appear as large, multiloculated cystic lesions containing fine gravity-dependent echoes and papillary excrescences. They have papillary projections less frequently than the serous type. A unique feature of the malignant form of the mucinous tumour is its tendency to metastasize or rupture to cause pseudomyxoma peritonei, which may resemble ascites sonographically or manifest itself as multiple clusters of anechoic spaces filling much of the pelvis and abdomen.

Ovarian neoplasms of low malignant potential (borderline tumours) behave much less aggressively than frank carcinomas but they are malignant. Serous borderline tumours constitute 10 to 15% of ovarian carcinomas and the mucinous variety 5 to 10%. Imaging features of this group are nonspecific and may resemble the sonographic features of their benign counterparts.

Proposed Management of Adnexal Mass in Pregnancy

Acute complications such as torsion and rupture are treated emergently at presentation. Ovarian torsion most commonly occurs secondary to an underlying ovarian abnormality such as an ovarian cyst or tumour. It can occur in normal ovaries. The end result of ovarian torsion is interruption of arterial and venous circulation with vascular engorgement that may ultimately lead to hemorrhagic infarction. Common clinical symptoms include an intermittent pattern of nausea, vomiting and severe abdominopelvic pain. A specific sonographic sign of ovarian torsion includes the finding of an enlarged ovary with multiple cortical follicles. There may be associated free fluid in the pelvis. In the right clinical context the presence of blood flow on Doppler in a unilaterally enlarged ovary should not negate the diagnosis.

The traditional and historic teaching has been that an asymptomatic adnexal mass > 5cm to 6 cm diagnosed in pregnancy should be surgically removed. This surgery was generally performed on an elective basis in the second trimester when the risk of complication to the mother and fetus is lowest. It may be that with technologic advances this approach can be modified (Figure 1).

References

1. Platek DN, Henderson CE, Goldberg GL. The management of a persistent adnexal mass in pregnancy. Am J Obstet Gynecol 1995;173:1236-40.
2. Whitecar P, Turner S, Higby K. Adnexal masses in pregnancy: A review of 130 cases undergoing surgical management. Am J Obstet Gynecol 1999;181:19-24.
3. Boulay R, Podczaski E. Ovarian cancer complicating pregnancy. Obstet and Gynecol Clinics of North America 1998;25:385-97.
4. Bromley B, Benacerraf B. Adnexal masses during pregnancy: accuracy of sonographic diagnosis and outcome. J Ultrasound Med 1997;16:447-52.
5. Sutton CL, McKinney CD, Jones JE, et al. Ovarian masses revisited: radiological and pathologic correlation. Radiographics 1992;12:853-77.
6. Fried AM, Kenney CM, Stigers KB, et al. Benign pelvic masses: sonographic spectrum. Radiographics 1996;16:321-34.
7. Atri M, Mazarnia S, Bret PM, et al. Endovaginal sonographic appearance of benign ovarian masses. Radiographics 1994;14:747-60.
8. Eyeh HC, Sutterweit W, Thornton JC. Polycystic ovarian disease: ultrasound features in 104 patients. Radiology 1987;163:111-6.
9. Graif M, Itzchak Y. Sonographic evaluation of ovarian torsion in childhood and adolescence. AJR 1988;150:647-9.
10. DePriest PD, Shenson D, Fried AM, et al. A morphology index based on sonographic findings in ovarian cancer. Gynecol Oncol 1993;51:7-11.
11. Peterson WF, Prevost EC, Edmunds FT, et al. Benign cystic teratomas of the ovary: a clinicostatistical study of 1,007 cases with review of the literature. Am J Obstet Gynecol 1955;70:368-82.

Fetal Cardiac Screen

Shi-Joon Yoo, M.D.

CARDIAC IMAGING, DEPARTMENT OF DIAGNOSTIC IMAGING, THE HOSPITAL FOR SICK CHILDREN, TORONTO, CANADA

Fetal cardiac screening is an important part of fetal sonography because congenital heart disease is the most common congenital anomaly and responsible for more than 50% of childhood deaths. It is also important because the newborns with congenital heart disease often require urgent medical or surgical treatment immediately after birth.

The heart consists of three distinct segments; namely, the atriums, the ventricles and the great arteries. In congenitally malformed hearts, these three segments can be abnormally related and connected. Therefore, chamber morphologies, relations and connections are considered as three facets of the make-up of the heart. The congenitally malformed heart should be evaluated in terms of these three facets in a sequential segmental manner. After identification of the morphologies of and relationships between the cardiac chambers and great arteries, the heart should be evaluated according to the following key steps;

1. Determination of the arrangement of the visceral organs and the atriums.
2. Determination of the connection between the atriums and ventricles.
3. Determination of the connection between the ventricles and great arteries.
4. Evaluation of the associated malformation at each cardiac segments.

For sequential segmental approach to the fetal heart, the following views should be obtained (Figures 1 to 7).

1. Transverse view of the upper abdomen.
2. Four-chamber view.
3. Three-vessel view.
4. Left ventricular outflow tract view.
5. Right ventricular outflow tract view.
6. Basal short axis view.
7. Aortic arch view.

As the above list is a rather long list, it is important to develop a standard technique for each view to increase the reproducibility and to reduce the time required (Yoo et al. Cardiol Young 1999;9:430-44).

Before obtaining the above views, we first define the right and left sides of the fetus in its own right. The right and left sides of the fetus should not be defined according to the position of the stomach, which is a common mistake. The stomach can be in a wrong side. As the first three views are orthogonal transverse view, they do not require any specific maneuvers. The transverse view of the upper mediastinum is obtained as for measurement of the abdominal circumference. We then slide the transducer cranially along the long axis of the fetal body to produce the four-chamber view, and then further cranially to produce the 3-vessel view. We obtain the ventricular outflow tract views by using a specific maneuver. We start the maneuver from the four-chamber plane. The transducer is then moved radially around the fetal thorax, a maneuver which also moves around the maternal abdomen in most cases with a vertically oriented fetus, keeping the four-chamber plane in view until the ventricular septum is aligned perpendicular to the sonographic beam. In this particular position, the left ventricular outflow tract view can be obtained simply by rotating the transducer through 20-30 degrees clockwise or counterclockwise towards the cardiac apex. By sliding the transducer upward towards the fetal head from this plane, the right ventricular outflow tract can be obtained. It is important to note that the left and right ventricular outflow tracts cross each other and can not be imaged in a single plane. To obtain basal short axis view, the heart is scanned in an oblique plane by placing the transducer to connect the right lobe of the liver and the left shoulder. Then the transducer is moved upward or downward along the fetal thorax with some cranial or caudal tilt until the aortic valve is imaged in cross-section. The basal short axis view should be adjusted so that the aortic valve is encircled by the right atrium, right ventricle, main pulmonary artery and right pulmonary artery. To visualize the aortic arch, we use another specific maneuver. We start from the three-vessel view. The transducer is then moved radially around the fetal thorax, keeping the three-vessel plane until the cross-sections of the ascending and descending aorta are aligned with the sonographic beam. In this position, the aortic arch can be demonstrated by rotating the transducer 90 degrees towards the feet or head of the fetus. Transverse view of the aortic arch can also be obtained by sliding the transducer upward towards the fetal head from the three-vessel plane. In our experience, it usually takes 5 to 15 minutes to obtain these views for cardiac screening.

For each scanning plane, the items listed in the Table should be searched. If any abnormality is found in these basic screening views, detailed study should be performed with Doppler and M-mode interrogation.

Table. Checklist of clues to the abnormality in each sonographic view.

Basic Sonographic Views.

Ultrasound and Multiple Pregnancies

Pran Pandya, M.D.

CONSULTANT IN OBSTETRICS AND FETAL MEDICINE, UNIVERSITY COLLEGE HOSPITAL, LONDON, UNITED KINGDOM

Almost every maternal and fetal problem is increased in multiple gestations. This fact justifies such a pregnancy being labelled high-risk status and justifies its need for specialized management.

Epidemiology

• Dizygous (DZ, non-identical) twinning has a heritable component and varies with maternal age, race and nutrition.
• Monozygous (MZ, identical) twining rates are constant throughout the world at 3 to 5 per 1,000 births.
• Multiple birth rates have increased significantly. One-third of the increase is due to delayed child bearing, one-third to induction of ovulation and the remaining third due to in vitro fertilisation.
• The distribution of higher order gestations is dependent on the number of embryos transferred. Replacing 3 embryos instead of 2 increases the risk of triplets by a factor of 15, from 0.4% to 6%, with no increase in take-home-baby rate. The British Fertility Society recommends transferring a maximum of 2 embryos in each cycle.
• Assisted conception also increases the incidence of monozygous twinning 2- to 8-fold.
• Perinatal mortality (PNM) rates are 37, 52 and 371 per 1,000 live and stillbirths for twins, triplets and higher order multiple births, respectively.
• The risk per pregnancy of producing a child with cerebral palsy is 8-times greater in twin pregnancies and 47-times greater in triplet pregnancies compared to singletons.

Chorionicity

• Determination of chorionicity is essential in the management of multiple pregnancies. Importance:

1. Miscarriage rate is 13% in MC twins 3% in DC twins
2. PNM in MC twins is 3 to 4 times greater than DC twins
3. Counselling regarding risk of genetic or structural abnormality
4. Invasive testing and management of discordant abnormality
5. Risk of sequelae in the presence of fetal compromise
6. Early detection and management of complications of MC twins

• Dizygous twins can only be dichorionic diamniotic (DCDA).
• Monozygous twins may be DCDA, monochorionic diamniotic (MCDA) or monochorionic monoamniotic (MCMA) depending on the timing of embryonic splitting.

Ultrasound determination of chorionicity

1. Fetal gender (two-thirds of twins are same sex and may be MZ or DZ)
2. Number of placentas
3. Characteristics of the membrane between the two amniotic sacs (DC twins have thicker membrane because of chorion but poor reproducibility)
4. Number of yolk sacs and coelomic cavities. 2 coeloms = DC. 1 coelom = MC. 1 yolk sac = monoamniotic
5. Sonographic examination of the base of the inter-twin membrane.

• 6 to 9 weeks thick septum
• 10 weeks to 14 weeks triangular tissue projection = lambda sign in DC twins.
• after 16 weeks, less reliable.

Structural anomalies

• These may be similar to those that occur in singletons or specific to the twinning process.
• Overall 50% increase in structural anomalies, most of the excess risk in MZ twins. Anencephaly, ventriculomegaly, esophageal atresia and cardiac defects.

Twinning related abnormalities

• Twin to twin transfusion syndrome (TTTS).
• Twin reversed arterial perfusion sequence (TRAP). Acardiac twinning. The acardiac ‘perfused’ twin receives arterial blood supply parasitically via a large arterio-arterial anastomosis from a normal ‘pump’ co-twin.
• Conjoined twins.

Screening for chromosomal abnormalities

Maternal age

In DZ twins, the risk of a chromosomal abnormality for each twin is the same as for a singleton, therefore the risk that at least one fetus is affected is twice as high as in singletons. The risk that both fetuses are affected is derived by squaring the singleton risk.

In MZ twins, the risk for a chromosomal abnormality is the same as a singleton pregnancy and in the vast majority both fetuses are affected. (There are reported cases of discordant chromosomal abnormalities.)

Maternal serum screening

• Poor detection rates for trisomy 21.
• 45% detection 5% false positive rate.

Fetal nuchal translucency (NT) thickness

• NT > 95^th centile in 7.3% fetuses including 88% of fetuses with trisomy 21.

• DC twins sensitivity and false positive rate similar to singleton pregnancies. Therefore when quoting adjusted risks for trisomy 21 use individual risks for each fetus. The risk that at least one fetus is affected is the summation of the two individual risks.
• MC twins higher false positive rate. This is possibly due to an early manifestation of TTTS. When quoting adjusted risk, use lower NT measurement for calculating risk of trisomy 21.

Prevalence of fetal nuchal translucency thickness (NT) above the 95^th percentile (for crown–rump length in singletons), in singleton, monochorionic twin pregnancies and dichorionic twin pregnancies.¹

Fetal Karyotyping

Selection of appropriate invasive technique: no randomized controlled studies in multiple pregnancies

1. Accuracy of obtaining a result from both fetuses
2. Procedure related risk of fetal loss
3. Risks of selective feticide for discordant abnormality.

Amniocentesis

• From 15 weeks of gestation
• Single or double uterine entry
• Pregnancy loss rates 3.5% up to 28 weeks, similar to singleton pregnancies

Chorionic Villus Sampling (CVS)

• From 10+ weeks of gestation
• 5% cases uncertainty if both placentas are sampled
• Sample at extreme ends of placenta.
• 2 uterine entries.
• Transabdominal + transcervical approach.
• Advantages are earlier diagnosis and in the event of a discordant abnormality earlier feticide, which is associated with lower procedure related fetal loss rates.

Which Invasive Test?

• Choice of invasive technique should be based on individual risks calculated by the combination of maternal age and fetal NT. When the risk for a chromosomal defect in at least one of the fetuses is greater than 1 in 50, it may be preferable to perform CVS. For pregnancies with a lower risk amniocentesis after 15 weeks may be more appropriate.

• In general, sample both twins unless structurally normal MC twins.

Late Karyotyping

• Invasive testing ³ 32 weeks
• Risks are of pre-term labour, not fetal loss
• Allows for spontaneous loss of aneuploid fetuses

Management of Discordant Anomalies

Aim is to maximize the chances of survival for the normal twin.

1. Expectant management
2. Selective feticide – risk of miscarriage and pre-term delivery. Risks of selective feticide are greater after 16 weeks (5% versus 16%)

Type of chromosomal abnormality

• trisomy 21 selective feticide, because the majority of babies will survive
• trisomy 18 expectant management.

In MC twins spontaneous death in utero is associated with a 25% risk of intrauterine death of the healthy co-twin and similar risk of neurological and renal lesions. The mechanism of co-twin death is acute haemodynamic imbalance giving rise to hypotension resulting in ischaemia, as the healthy twin transfuses blood into the dead twin’s vasculature.

Procedure

• DC twins feticide can be performed by intra-cardiac injection of potassium chloride.
• MC twins due to placental vascular anastomoses feticide would necessitate cord occlusion. A number of techniques have been described e.g. ultrasound guided monopolar and bipolar diathermy, endoscopic laser coagulation.

Twin to Twin Transfusion Syndrome (TTTS)

Vascular anastomoses within the placenta are present in all monochorionic pregnancies. The anastomoses are either superficial arterio-arterial and veno-venous allowing bi-directional flow or deep arterio-venous with unidirectional flow. TTTS occurs in up to 25% of MC pregnancies. Recent studies suggest a primary vascular basis in which a paucity of superficial bi-directional anastomoses is unable to compensate for the haemodynamic imbalance resulting from unidirectional transfusion along the deep anastomoses. If untreated, TTTS results in fetal mortality in over 80% of cases.

Traditional Diagnosis of TTTS

• Prenatal ultrasound features: large bladder and polyhydramnios in recipient and growth restricted fetus with an ‘absent’ bladder and anhydramnios (stuck twin) in donor twin.
• Neonatal period: inter-twin difference in birth weight of ³ 20% and difference in hemoglobin concentrations of 5g/dL or more.

Early Prediction of TTTS

• Increased fetal NT, > 95^th percentile at 11-14 weeks was associated with a four-fold increase in the risk of subsequent development of TTTS. Increased NT in the recipient may be a manifestation of heart failure due to hypervolemia. NT subsequently resolves due to diuresis.
• Inter-twin membrane folding may be an early manifestation of disparity in amniotic fluid, because of oliguria and collapsed amniotic sac in donor twin.
• Inter-twin folding is seen in about 25% of MC pregnancies and TTTS develops in about 50% of cases.
• Doppler detection of superficial arterio-arterial anastomoses.

References

• Sebire NJ Snijders, Hughes K, et al. Screening for trisomy 21 in twin pregnancies by maternal age and fetal nuchal translucency thickness at 10-14 weeks of gestation. Br J Obstet Gynaecol 1996:103:999-1003.
• Sepulveda W, Sebire NJ, Hughes K, et al. The lambda sign at 10-14 weeks of gestation as a predictor of chorionicity in twin pregnancies. Ultrasound Obstet Gynaecol 1996:7:421-3.
• Taylor MJO, Fisk NM. Prenatal diagnosis in multiple pregnancy. Balliere’s Clinical Obstetrics and Gynaecology 2000:14:663-75.

Bad News in the Scanning Room

Rory Windrim, M.D.¹, Jennifer Johannesen, Diane Savage, M.S.W.²

1 ASSISTANT PROFESSOR, DEPARTMENT OF OBSTETRICS & GYNAECOLOGY, UNIVERSITY OF TORONTO, TORONTO, CANADA
2 DIRECTOR OF SOCIAL WORK, MOUNT SINAI HOSPITAL, TORONTO, CANADA

Introduction

Giving bad news is one of the most difficult tasks faced by caregivers in daily practice.¹ Research suggests that patients are dissatisfied with the information they receive from their physicians² and that only a small percentage of doctors feel competent with their skills in giving bad news.³ The increased acuity of ultrasound machines and recognition of "markers of aneuploidy" have increased the frequency and complexity of this problem.

Potential for Discovering "Bad News" in the Scanning Room

1st Trimester

• Non-viable pregnancy complicates 20% of "normal" pregnancies.
• Ectopic pregnancy found in 1.2% of the normal population.
• Nuchal translucency screening….

2nd Trimester

• Major structural anomaly found in 1.1% of the normal population.
• Isolated aneuploidy ‘markers’ found in 3.6% of the normal population.
• The abnormal cervix….

3rd Trimester

• Major/minor structural anomaly not previously seen.
• Fetal death after 20 weeks found in 1% of the normal population.
• Hydrops fetalis; polyhydramnios or oligohydramnios; intrauterine growth retardation; breech, etc.

Strategies when an Abnormal Scan is Found

Approaches to the communication of the problem vary from lab to lab:

"Contact your physician" « Detailed information, counseling and management options.

Clearly, immediate detailed counseling is impractical in the majority of cases. The referring caregiver is usually in a better position to provide the more detailed information that the patient requires, formulate a care plan and arrange follow- up of unresolved issues.⁴

Real-time scanning, however, often results in the almost immediate realization of a problem ‘in front of’ the parents. In Canada, sonographers are able to provide interpreting physicians with oral or written summaries of their real-time findings, but it is beyond the scope of their practice to provide this information to a referring physician or to the patient. In those instances when the finding is obvious, but the imaging physician is not on hand to inform the patient, the sonographer is placed in a very difficult position. He or she cannot confirm the finding (e.g. pregnancy loss) to the patient, even if both parties are aware of the finding. In this and similar circumstances (under strict policy control), there may be a need to delegate to the sonographer the responsibility of informing the patient of what was seen on the sonogram.⁴

In any event, when little or no information about the scan findings is combined with the advice to "contact your physician," this in itself represents bad news to many anxious parents. Irrespective of the amount of information given at the primary scan, therefore, labs may choose to consider guidelines for the communication of bad news.

The following is a brief background in some of the theory of perinatal loss issues, prior to a presentation of practical "tips" for dealing with these issues.

Perinatal Loss

Perinatal loss is a unique parenting and bereavement experience.

Psychological response to this kind of loss is not dependent on the gestational age of a baby. Of far greater significance, is the meaning of the pregnancy and the degree of attachment experienced by each parent. This is relevant whether ultrasound reveals abnormalities that lead to "chronic grief" or the absence of a fetal heartbeat.

Patients and families will also respond differently depending on their preparedness, cultural background and coping skills.

Parents have access to information, testing, and procedures earlier than ever before. Ultrasounds and fertility treatments can promote attachment earlier and in more concrete ways. However, these same opportunities can represent the end of a life or a dream. Members of the ultrasound team can play a pivotal role in the unfolding of a process of grieving for many couples, by what is said and by what is left unsaid.

"Grief is experienced in relation to the significance of the attachment".⁶

It can be difficult for caregivers in any setting to determine the degree of attachment the patient has to their pregnancy. This can lead to uncertainty about how to refer to the 'baby' or 'fetus'. One study tells us that approximately 75% of people view their pregnancy loss as the loss of a "baby", while 25% are likely to view this as the loss of a 'pregnancy' or 'part of life'.

What can be helpful to patients and families encountered in this setting? First, a definition of bad news...

Bad news definition⁵

Situations where there is either a feeling of no hope, a threat to a persons mental or physical well being, a risk of upsetting life- style or where a message is given which conveys to an individual fewer choices in life.

Determination that the event entails bad news lies for the most part in the mind of the receiver.

Buckman’s six step protocol:⁶

• Step 1: Start off well
• Step 2: Assess the patient’s knowledge
• Step 3: How much does the patient want to know?
• Step 4: Share the information
• Step 5: Respond to the patient’s feelings
• Step 6: Planning and follow-through

Main reactions to bad news:⁶

The presentation of anger, denial, and crying can present challenges in knowing what might be helpful….even deciding what may be normal.

Bad news transmission stress⁶

Transmission of bad news from one person to another may be potentially stressful for either the giver or the receiver. Buckman suggests that physicians experience anticipatory stress in breaking bad news.

Sources of pressure include:

• Fear of causing pain
• Fear of being blamed

• Sympathetic pain

• Fear of therapeutic failure
• Medico-legal issues
• Fear of eliciting a reaction
• Fear of saying "I don’t know"

However, all of these stresses continue to exist, even if the bad news is not disclosed.

Practical Tips for Conveying Bad News

Renee Louise Franche and Ian Hammond, in an editorial in the Journal of Ultrasound in Medicine in 1997 made the following statements:

In those ultrasound units developing quality assurance standards to meet these challenges, the following suggestions from the extensive literature on bad news might be considered:

Resources

"Perinatal Bereavement Resource Guide" - 254 services compiled and published by The Fetal Centre at The Hospital for Sick Children, Toronto, Canada is available on http://www.hscFetalCentre.org.

References

1. Steinert Y. Giving bad news. Canadian Journal of CME January 1999.
2. Lloyd GC, Parker AC. Emotional impact of diagnosis and early treatment of lymphomas. J Psychosom Med 1984;28:157-62.
3. Eggly S, Afonso N, Rojas G, et al. An assessment of residents’ competence in the delivery of bad news to patients. Acad Med 1997;72:397-9.
4. Franche RL, Hammond I, Black D, et al. First trimester pregnancy loss –hHow can we help our patients? J Ultrasound Med 1997;16:513-4.
5. Ptacek JT, Eberhardt TL. Breaking bad news: a review of the literature. JAMA 1996;276:496-502.
6. Buckman R: How to Break Bad News: A Guide for Health Care Professionals. Baltimore, Johns Hopkins University Press, 1992.

Letter to the Editor:
Meckel Syndrome – Ductal Plate Malformation

To the Editor,

I read with interest the article of Drs. Nowaczyk and Mohide about the Meckel Syndrome (MKS).¹ It is a good summary of this syndrome. I would just add that is they mention the liver fibrosis, they did not speak about the bile ducts anomaly. However, ductal plate malformation of the liver is a constant finding in MKS. I personally consider this anomaly as a pathognomic marker for MKS, even if some other genetic syndromes show the same kind of changes (see article by Sergei C et al.²). I have seen MKS without polydactyly or even without CNS anomaly, but until now, I have never seen MKS without bile duct anomaly. I have included a drawing of the typical change of the portal tract in MKS (Figure 1) and images of one recent case (Figures 2 and 3).

References

1. Nowaczyk MJM, Mohide, PT. McMaster University Prenatal Diagnosis Rounds: Meckel Syndrome (MKS). Frontiers in Fetal Health 2000;2(4):18-9.
2. Sergi C, Adam S, Kahl P, Otto HF. Study of the malformation of ductal plate of the liver in Meckel syndrome and review of other syndromes presenting with this anomaly. Pediatr Dev Pathol 2000;3:568-83.

CHRISTIAN DUGAUQUIER, MD
Institut de Pathologie et de Genetique
Alee des Templiers 41
Loverval, Belgium

Authors’ Response,

We extend our thanks to Dr. Dugauquier for his favorable reception of our publication. Our brief review of MKS was intended to highlight the sonographically detectable anomalies that are characteristic of MGS and present in the prenatal period, rather than to provide an exhaustive presentation of all anomalies of the syndrome.

While liver fibrosis, together with cystic dysplasia of the kidneys, and occipital encephalocele or some other central nervous system malformation have been proposed to be minimal diagnostic criteria by Salonen et al.,¹ no specific mention of bile duct proliferation was made at that time. However, in a review of the pathologic findings in 9 cases, Blankenberg et al.² concluded that a hepatic lesion is a consistent feature. A feature in which there is arrested development of the intrahepatic biliary system at the stage of biliary cylinders with varying degrees of reactive bile duct proliferation, bile duct dilatation, portal fibrosis and portal fibrous vascular obliteration.

Of note, on postmortem examination of the fetal liver we did observe the histopathologic findings that Dr. Dugauquier described.

References

• Blankenberg TA, Ruebner BHm Ellis WG, et al.Pathology of renal and hepatic anomalies in Meckel syndrome. Am J Med Genet 1987;Suppl.3:395-410.
• Salonen R. The Meckel syndrome: clinicopathological findings in 67 patients. Am J Med Genet 1984;18:671-89.

MALGORZATA J.M. NOWACZYK, MD¹
PATRICK T. MOHIDE, MD²
Departments of ¹Pathology & Molecular Medicine and Paediatrics and ²Obstetrics & Gynaecology, McMaster University and Hamilton Health Centre Corporation,
Hamilton, Ontario, Canada

Molecular Diagnosis of Smith-Lemli-Opitz Syndrome: Current Applications

Malgorzata J.M. Nowaczyk, M.D.

DEPARTMENTS OF PATHOLOGY & MOLECULAR MEDICINE and PAEDIATRICS, McMASTER UNIVERSITY, THE HAMILTON REGIONAL LABORATORY PROGRAM, HAMILTON, CANADA

Smith-Lemli-Opitz syndrome (SLOS) is caused by an inherited deficiency of 7-dehydrocholesterol reductase (7DHCR).¹ The enzymatic defect in SLOS leads to a generalized cholesterol deficiency, and to an accumulation of the immediate precursor, 7-dehydrocholesterol (7DHC), in all body tissues.^1,2 Clinically, SLOS ranges from mild facial dysmorphism with mental retardation and significant behavioral abnormalities to severe congenital malformation with intrauterine lethality. Before the discovery of the underlying biochemical defect,³ the diagnosis of SLOS was based on the recognition of the pattern of malformations. Currently, SLOS is diagnosed by demonstration of elevated levels of 7DHC in plasma or amniotic fluid. Since the widespread use of the biochemical diagnostic test a number of patients with previous clinical diagnosis of SLOS were found to be phenocopies of different or unknown etiologies.¹

DHCR7, the gene for SLOS, has been mapped in 1998. Numerous mutations in patients with SLOS have been described,^4-6 and genotype-phenotype correlation is emerging.⁷ Since July 2000, the Molecular Diagnostic Laboratory at McMaster University has performed molecular analysis of DHCR7 mutations in 23 patients, and confirmed the status of 15 carrier couples. Two of the latter were families where no samples were available from the probands. We have also performed mutation analysis in four prenatal cases: two postmortem specimens obtained from fetuses with multiple anomalies who were found to be affected, and two fetuses at 25% risk of SLOS who were found to be unaffected. We present here a selection of cases from our experience to illustrate the usefulness of molecular diagnosis in situations where conventional biochemical analyses were not possible, and in comparison to standard biochemical analysis in two prenatal cases.

Family 1

This couple had two children with SLOS type II born in late 1980s. Both children had normal male karyotypes in the setting of characteristic facial features, cleft palate, female external genitalia, polydactyly and 2-3 toe syndactyly; death occurred as a result of pneumonia in the first year of life. With the permission and the knowledge of the parents of the children, another family member contacted us in1996 to inquire if biochemical testing on stored newborn screening blood samples from the deceased children was possible to confirm the diagnosis of SLOS. The woman, a maternal aunt of the affected children, was considering pregnancy at that time, and was also interested in carrier testing. Unfortunately, the stored samples were not suitable to confirm the diagnosis of SLOS in the deceased children.

The family was contacted in 2000 and molecular testing of the parents was discussed. Following genetic counselling which centered on the possibility that the children did not have SLOS, but another condition, and that in such a case molecular testing will not reveal mutations, blood samples were obtained from both parents. Molecular analysis showed that the mother was heterozygous for the common null mutation, and the father was heterozygous for a known missense mutation, thereby providing indirect evidence that the deceased children were affected with SLOS. Carrier testing for interested relatives is now possible in this family.

Family 2

This case was referred because of the prenatal sonographic detection of multiple anomalies, including oligohydramnios, at 18 weeks of gestation. The fetus had CNS anomalies, ambiguous genitalia, cleft lip and palate, postaxial polydactyly of the feet; at autopsy abnormal lobation of the lungs and a complex congenital heart defect were detected. Chromosomal analysis on tissue obtained at that time showed a normal karyotype, 46,XY. Because of the pattern of malformation and lack of plasma and amniotic fluid samples, we performed mutation analysis of DCHR7 in the parents and found the mother to be a carrier of the common null mutation, and the father a carrier of another null mutation. The phenotype of the fetus was in keeping with the known genotype-phenotype correlation of an individual with 0/0 genotype, providing indirect evidence that the fetus was affected with SLOS.⁷ The couple were counselled about the likely diagnosis of SLOS in their child, and about the mode of inheritance and the risks of recurrence. Prenatal diagnosis for SLOS in now available for this couple’s subsequent pregnancies, either by molecular or biochemical analysis.

Family 3

This woman had undergone a termination of pregnancy in a fetus with multiple congenital anomalies. The clinical diagnosis of severe SLOS was confirmed by the demonstration of elevated 7DHC in amniotic fluid obtained at the time of the procedure. Molecular analysis showed that the fetus was homozygous for the common null mutation; the woman and her partner were heterozygous for this mutation.⁸ In the subsequent pregnancy, chorionic villus sampling was performed at 11 weeks of gestation and the fetus was found to be heterozygous for that mutation, and therefore unaffected. This finding was confirmed by biochemical analysis performed at the same time.

Family 4

A third world country family with a child with a clinical diagnosis of SLOS was referred for biochemical and molecular analysis. The mother was 14 weeks pregnant at the time. Amniotic fluid was obtained at 16 weeks of gestation and it was shipped together with blood samples from the parents and the presumed affected child. Biochemical analysis showed elevated 7DHC in the blood sample obtained from the affected child, confirming the clinical diagnosis of SLOS. Molecular analysis showed that the proband was a compound heterozygote for two known SLOS missense mutations; the parents were heterozygous for these mutations. Molecular analysis of DNA obtained from amniocytes showed that the fetus was heterozygous for the parental mutation. Measurement of 7DHC level in the AF confirmed that the fetus was not affected.

Discussion

In 1998, the human DHCR7 gene was cloned and mapped to 11q12-13. Sixty-seven DHCR7 mutations were found in the almost 200 SLOS patients who have been genotyped (our unpublished results).^1,4-7 Missense mutations comprise the vast majority of mutant alleles (85%), followed by a small number of nonsense mutations, small deletions or insertions, and mutations that affect splicing or translation initiation. Extensive gene deletions or rearrangements are yet to be encountered. In all but a few patients, both mutant alleles were identified. Molecular diagnosis has been reported in two prenatal cases where the fetus was at 25% risk of recurrence,⁹ and in one case where no viable tissue was available for biochemical analysis.¹⁰

Diagnosis of SLOS by demonstration of DHCR7 mutations may be the only method available in cases where amniotic fluid, or tissue or plasma samples from the proband are not available for 7DHC measurements, or for 7DHCR enzyme assays. Such situation may arise in cases of stillbirths, macerated fetuses, intrauterine demise, or pregnancy termination in cases of oligohydramions, or when appropriate samples were simply not obtained. In clinically warranted situations, molecular analysis of parental DNA may lead to the diagnosis of the deceased patient as in Family 1 and 2 reported herein.

Currently, DHCR7 mutation analysis is the only feasible method of carrier detection. Carrier detection by enzymatic studies by measurement of the rate of reduction of the ergosterol C-7 double bond in cultured skin fibroblasts from putative carriers, although reliable, is invasive and time-consuming.¹¹ However, caution in interpreting results of molecular carrier testing must be exercised in cases where clinical diagnosis of SLOS has not been confirmed biochemically or molecularly. It is possible that the child previously diagnosed with SLOS was affected with a condition that was a phenocopy of SLOS and negative results of DHCR7 mutation analysis may be falsely reassuring. To date phenocopies of SLOS have been reported in children with deletion 2q32.1q35,¹² with deletion 4pter detectable by FISH,¹³ and with uniparental disomy 16 with mosaic partial trisomy 16(pter-p13),¹⁴ as well as in children with other inborn errors of metabolism or autosomal recessive conditions.² Whenever possible, patients with previous diagnosis of SLOS should have the diagnosis confirmed biochemically. In cases of negative diagnostic test for SLOS, a genetic dysmorphology re-evaluation is indicated. Karyotyping should be performed to rule out the possibility of a cryptic chromosomal rearrangement with its inherent risks of recurrence, and should be considered especially in families with more than one affected individual, or if previous karyotyping was performed more than ten years ago. Subtelomeric microdeletion analysis as well as spectral karyotyping may be necessary in these situations. The consultant dysmorphologist may also begin an investigation for other inborn errors of metabolism.

Summary

Molecular testing of DHCR7 is rapid and reliable. In certain situations it may be the only method of diagnosis of SLOS. Patients previously diagnosed with SLOS based on clinical findings alone require biochemical confirmation for appropriate genetic counselling. Genetic dysmorphology consultation is warranted in patients with phenocopies of SLOS. Carrier testing by DHCR7 molecular analysis in families with known mutations is currently available.

For further information about molecular testing of DHCR7 please contact Dr. Marg Nowaczyk, MD, FRCPC, FCCMG, at <nowaczyk@hhsc.ca>.

References

1. OMIM (Online Mendelian Inheritance in Man™); Baltimore, MD: http://www.ncbi.nih.gov/omim/.
2. Opitz JM. RSH (so-called Smith-Lemli-Opitz) syndrome. Curr Opin Pediatr 1999;11:353-62.
3. Tint GS, Irons M, Elias ER, et al. Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome. N Engl J Med 1994;330:107-13.
4. Moebius FF, Fitzky BU, Lee JN, et al. Molecular cloning and expression of the human delta7-sterol reductase. Proc Natl Acad Sci 1998;95:1899-902.
5. Wassif CA, Maslen C, Kachilele-Linjewile S, et al. Mutations in the human sterol D⁷-reductase gene at 11q12-13 cause Smith-Lemli-Opitz syndrome. Am J Hum Genet 1998;63:55-62.
6. Waterham HR, Wijburg FA, Hennekam RCM, et al. Smith-Lemli-Opitz syndrome is caused by mutations in the 7-dehydrocholesterol reductase gene. Am J Hum Genet 1998;63:329-38.
7. Witsch-Baumgartner M, Fitzky BU, Ogorelkova M, et al. Mutational spectrum and genotype-phenotype correlation in 84 patients with Smith-Lemli-Opitz syndrome. Am J Hum Genet 2000;66:402-12.
8. Nowaczyk MJM, Farrell SA, Sirkin WL, et al. Smith-Lemli-Opitz Syndrome: Holoprosencephaly and IVS8-1G® C/IVS8-1G® C Genotype. Am J Med Genet (submitted).
9. Bzduch V, Kozak L, Francova H, et al. Prenatal diagnosis of Smith-Lemli-Opitz syndrome by mutation analysis. Am J Med Genet 2000;95:85.
10. Löffler J, Trojovsky A, Casati B, et al. Homozygosity for the W151X mutation in the D7-sterol reductase gene (DHCR7) causing a lethal form of Smith-Lemli-Opitz syndrome: retrospective molecular diagnosis. Am J Med Genet 2000;95:174-7.
11. Honda M , Tint GS, Shefer S, et al. Accurate detection of Smith-Lemli-Opitz syndrome carriers by measurement of the rate of reduction of the ergosterol C-7 double bond in cultured skin fibroblasts. J Inher Metab Dis 1998;21:761-8.
12. Lavoie J, Gros-Louis F, Bronsard M, et al. Sexual amibiguity in a child with a karyotype 46,XY,del(2)(q32.1q35) and phenotype resembling Smith-Lemli-Optiz syndrome. Am J Hum Genet 2000;67;Suppl.2:A772.
13. Ginsburg C, Fokstuen S, Schinzel A. The contribution of uniparental disomy to congenital development defects in children born to mothers at advanced childbearing age. Am J Med Genet 2000;95:454-460.
14. Hill S, Creasy M, Bundey S. A family with three sisters with the 4p- syndrome, originally reported as suffering from the Smith-Lemli-Opitz syndrome. J Ment Def Res 1991;35:76-80.

An Unusually High Prevalence of Neural Tube Defects in South Bulgaria

Simeonov E, Kutzeva K,* Brankova M, Staikov K,* Liharska K.

GENETIC CENTRE, MEDICAL FACULTY, SOFIA; *GENERAL HOSPITAL, RUDOZEM, BULGARIA

Table 1. Main Demographic Characteristics of the Rudozem Population (1991).

Population	13,135
Liveborn	158
Birth rate	12.0%
Stillbirths	12.5%
Infant mortality rate	12.7%
Mortality rate	6.2%

The observation of an increased incidence of congenital anomalies in newborn babies in 1987 was the reason to undertake retrospective and prospective registration of congenital anomalies in newborns. It also prompted us to provide genetic services to the community, such as satellite genetic counselling clinics, mass alpha-feto-protein (AFP), prenatal diagnosis and ultrasound screening of pregnant women.

Results

Forty-eight newborn babies and fetuses with major isolated or multiple congenital anomalies have been registered retrospectively (from 1983-1987) and prospectively (from 1988-1992). The type and number of anomalies detected at birth in liveborn and in stillborn babies, as well as in aborted fetuses is presented in Table 2.

Table 2. Congenital Anomalies Registered Prenatally And After Birth in Rudozem Area (1983-1992).

Type of Defect	Number (prevalence)
Chromosomal abnormalities	2 (1.65)
Single gene defects	2 (1.10)
Multifactorial disorders	30 (16.57)
Neural tube defects (NTDs) Anencephaly Anencephaly + spina bifida Spina bifida Meningoencephalocele	20 (11.08) 10 5 4 1
Congenital heart defects	4 (2.20)
Cleft lip/palate	3 (1.66)
Terminal transverse defect	2
Exomphalos	1
Craniofacial malformations	3 (1.65)
Multiple congenital anomaly syndromes	6 (3.32)
TOTAL	48 (26.52)

Table 3 shows the annual and overall prevalence of NTDs during the same period. Twenty newborn babies and fetuses were diagnosed with NTD; 10 with anencephaly, 5 with anencephaly and spina bifida (SB), 4 with spina bifida and 1 with meningoencephalocele. Only one child with SB, who was born in 1988 survived. This child was operated on and is still alive in a home for mentally retarded children.

Prenatal diagnosis by ultrasound examination, mass AFP screening and amniocentesis was introduced in 1988. As a result, 6 NTDs were detected prenatally and 5 of these pregnancies were terminated. One family refused termination for religious reasons, but the pregnancy ended spontaneously at 24 weeks gestation in the stillbirth of a male, anencephalic fetus. The preventive activities should have been stopped in 1991 by financial and organizational reasons and two anencephalic babies were born in this period (Table 3).

Table 3. Prevalence of Neural Tube Defects in Rudozem Area.

			NTDs
Year	Live born infants	Newborns with congenital anomalies	Live born infants	Terminated	Total
1983	187	4	-	0	-
1984	207	2	1	0	1
1985	209	3	1	0	1
1986	207	5	-	0	-
1987	200	15	7	0	7
1988	198	3	1	0	1
1989	162	3	1	1*	2
1990	153	3	0	2*	2
1991	158	2	2	-	2
1992	123	8	1	3*	4

* Detected by prenatal diagnosis

The overall prevalence of NTDs in this community for a 10-year period was found to be as high as 11.08%. The corresponding figures before and after the introduction of prenatal diagnosis were 8.91% (9/1,010) and 13.85% (11/794) respectively. The preventive measures undertaken in the last years reduced the incidence of NTD almost twice from 13.85% to 6.30%.

Discussion

The data in Table 2 indicate a total incidence rate of congenital anomalies (26.6%), which is similar to the average for the whole country (2% to 4%), as reported by several studies in different regions of Bulgaria.^1,2,3 At the same time, a significant difference was found in the incidence of NTD, which ranged from 1.5% to 2.0% for the country, but reached 11.08% in the Rudozem area for the 10-year period. A high number of new cases (11/794 = 13.85%) was documented over the prospective period (1988-1992), compared to 9/1,010 (8.91%) during the retrospective period (1983-1987), although the difference was statistically non-significant (p>0.05).

Obviously, there could not be doubt about the higher baseline prevalence of NTD in this area, as well as the high number of new cases in the last few years. The latter phenomenon is quite different from the tendency of decreasing occurrence of NTD, independent of the introduction of prenatal diagnosis, as reported in some European countries⁴ and USA.⁵

In an attempt to explain these results, we must consider 3 groups of facts should be taken into consideration:

• There is a higher rate of inbreeding in this community. In a village near Rudozem we found that 1/10 of the marriages have various degrees of consanguinity.
• There is a high prevalence rate of congenital dislocation/dysplasia of the hip; in up to 10 % of newborn babies.
• There is generally poor nutrition in this area, characterized by a deficiency of fresh fruits and vegetables. Even in the summer time, the total consumption of fruits and vegetables in this region is less than 50%, when compared to other parts of the country.⁶

The following conclusions with regional and maybe wider significance can be made from this study:

• The prevalence rate of NTD in the Rudozem area is the highest ever registered in Europe. It would be interesting to look at the situation in the neighbouring regions of Bulgaria and Greece.
• The cause of the permanently high rate of these defects in the recent years remains unclear and requires further investigation. It may be due to better prospective ascertainment in a community where both genetic and environmental factors, such as high inbreeding rate and deficient nutrition, are operating.
• Preventive measures, such as registration of congenital anomalies, mass screening of pregnant women, providing genetic information to families at risk, prenatal diagnosis and probably folic acid food fortification, could substantially reduce the occurrence of congenital anomalies, if properly used according to the specific regional characteristics. From this point of view, the dissemination of appropriate information to the medical professionals and the community seems to be of primary importance.

References

1. Simeonov E, Boyadjiev S, Ninjo A, et al. Registration of congenital anomalies in newborn babies. Ped.(Sofia) 1991;30:13-20.
2. Simeonova M, Kovacheva K, Angelova L. Registration of congenital anomalies in newborn babies - 2 year experience of the genetic centre in Pleven. Ped (Sofia) 1993;1:13-6.
3. Mumdjiev Ch, Petrova N, Dobreva A. Prevalence of congenital anomalies in the region of Stara Zagora for a 3 year period. Ped (Sofia) 1994;3:12-6.
4. EUROCAT Working Group. Prevalence of neural tube defects in 20 regions of Europe and the impact of prenatal diagnosis,1980-1986. J Epidemiol Com Health 1991;45:52-8.
5. Feldkamp M, Carey JC. Decreasing prevalence of neural tube defects in Utah,1985-1999. Frontiers in Fetal Health 2000;2(9):11,13.
6. Demireva M, Milev N. Nutritional characteristics of the population in the district of Smolian. Bul Academy of Sciences, Proc of the Institute of Nutrition 1967;6:5-20.