CHAPTER

31

Cytogenetics in Reproduction
Cynthia C. Morton and Charles Lee

Cytogenetics is the area of biology that deals with the Organization of Human
study of chromosomes. Classic cytogenetics provides
both an overview of the genome and the ability to identify ­Chromosomes and
chromosome rearrangements and numeric abnormalities. Methods of Study
Molecular cytogenetics permits a finer examination of spe-
cific areas of the genome and has been a powerful tool The human chromosome is a complex structure that con-
in gene mapping. Application of molecular cytogenetic sists of deoxyribonucleic acid (DNA), ribonucleic acid
techniques, particularly fluorescence in situ hybridization (RNA), and protein. Each normal human chromosome
(FISH) and array-based comparative genomic hybridiza- has one centromere, the site at which the kinetochore
tion (array CGH), now makes possible routine diagnosis forms, allowing for attachment of the mitotic spindle and
of microdeletion syndromes and cryptic chromosome rear- proper segregation of the chromosome during cell divi-
rangements in the clinical laboratory. Furthermore, FISH sion. The centromere also divides the chromosome into
provides a means to assess ploidy of any chromosome both two arms that are identified as p (petit) for the short arm
in uncultured cells (largely for rapid diagnosis of trisomy) and q for the long arm. In human chromosomes, the posi-
and in archival materials, which previously were not eas- tion of the centromere can be central (metacentric), distal
ily evaluated. The use of FISH with uncultured cells also ­(acrocentric), or somewhere in between (submetacentric),
­allows assessment of a single blastomere, permitting ge- providing a useful landmark in the identification of a par-
netic screening of preimplantation embryos in a procedure ticular chromosome. At the tip of the short arms of the
commonly termed preimplantation genetic diagnosis (see acrocentric chromosomes (chromosomes 13, 14, 15, 21,
Chapter 30). and 22) are satellites, variably sized structures composed
Cytogenetics is a critical component of reproductive of heterochromatin. Satellites are attached to the chromo-
endocrinology. Chromosome abnormalities are a common some through a secondary constriction known as a satel-
cause of infertility and pregnancy loss, as well as of sexual lite stalk, which contains the genes for 18S, 5.8S, and 28S
dysmorphology. Most chromosomally abnormal concep- ribosomal RNA. At each end of every chromosome arm
tions arise de novo after a meiotic nondisjunction event, is a telomere, a structure composed of tandemly repeated,
but chromosomal anomalies in some conceptuses are in- short nucleotide sequences (Fig. 31-1).
herited from a chromosomally abnormal parent. The chro- Human chromosomes vary in size, with the largest chromo-
mosome abnormality in these conceptuses may or may not some designated as chromosome 1. In 1956, Tjio and ­Levan1
be identical to that of the parent, depending on recom- determined the total normal number of chromosomes in
bination and segregation of chromosomes during game- each human somatic cell to be 46, with 22 pairs of ­autosomes
togenesis. Although some chromosome aberrations cause and 1 pair of sex chromosomes. For each chromosome pair,
overt phenotypic abnormalities, other aberrations have no one chromosome is inherited from the father and one chro-
apparent phenotypic effect. Carriers of these apparently mosome is inherited from the mother. With the exception
phenotypically neutral chromosome aberrations may still of chromosome pairs 21 and 22, the numeric designation
produce chromosomally unbalanced gametes and, as such, reflects the size of the chromosome (e.g., chromosome 4 is
may be identified after multiple pregnancy losses or the the fourth largest chromosome in the human complement).
birth of an abnormal child. ­Although chromosome 21 is smaller than chromosome 22,
777
778 PART III  Reproductive Technologies

Telomere with a normal male karyotype being described as 46,XY.

Excluding chromosome abnormalities and variants, the
karyotypes of two unrelated males or females may ap-
pear virtually identical. Furthermore, excluding sex chro-
mosomes, karyotypes of males and females are usually
Short arm microscopically indistinguishable. However, there are
(p) ­submicroscopic differences that will be discussed later.
Satellite
METHODS IN CLASSIC CYTOGENETICS
Stalk
Centromere Metaphase Preparations of Cultured Cells
Most cytogenetic analyses are performed on chromosomes
that are in mitotic metaphase. The metaphase cell can
show both numeric and structural chromosomal abnor-
Long arm malities. At this stage of the cell cycle, condensation of
(q) chromosomal DNA results in distinguishable entities (i.e.,
that can ultimately be visualized as specific patterns of
alternating dark and light bands, characteristic for each
species). To examine a particular chromosomal region
at higher resolution, less condensed chromosomes (e.g.,
Telomere those found at prometaphase) may be useful.
The most frequently used tissues for cytogenetic evalua-
Metacentric Submetacentric Acrocentric
tion, primarily because of their accessibility, are peripheral
Figure 31-1.  Schematic diagram of human metacentric, submetacen-
tric, and acrocentric chromosomes. The locations of the telomere, cen-
blood lymphocytes, amniotic fluid cells, chorionic villi,
tromere, short arm, and long arm are shown, as are the locations of the skin fibroblasts, bone marrow, lymph nodes, and solid tu-
satellite and stalk on the acrocentric chromosome. mors. The process of preparing chromosomes for analysis
is known as a harvest. Chromosomes are harvested either
this inconsistency in nomenclature has been perpetuated to after culture of the tissue or after direct harvest of mitotic
avoid confusion due to historical references to Down syn- cells, as in the cases of bone marrow, some solid tumors,
drome as trisomy 21. cytotrophoblasts, and cord blood. The harvest itself may be
A karyotype is a display of chromosomes in numeric performed in solution, as is commonly done for lymphocyte
order, with the chromosomes oriented such that the p cultures and for other cultures in which attached cells have
arm is on top (Fig. 31-2). In humans, the female is the been released from the tissue culture vessel, or it may be
homogametic sex, with a normal female karyotype being performed in situ, that is, attached to the tissue culture sur-
designated as 46,XX. The male is the heterogametic sex, face. The in situ procedure is frequently used for amniotic

1 2 3 4 5

Figure 31-2.  Representative nor-

mal human male GTG-banded
karyotype (46,XY). 6 7 8 9 10 11 12

13 14 15 16 17 18

19 20 21 22 X Y
 CHAPTER 31  Cytogenetics in Reproduction 779

fluid cultures when it is desirable to evaluate colonies of commonly used techniques, historically, for identifying
cells to ascertain possible chromosomal ­mosaicism. chromosomal heteromorphisms associated with the cen-
In all cases, dividing cells must be arrested during mi- tromeres of chromosomes 3, 4, 13, and 22; the short arms
tosis. This is accomplished by the addition of colchicine and satellites of acrocentric chromosomes 13, 14, 15, 21,
(or colcemid), which destroys the mitotic spindle and thus and 22; and the distal region of the Y chromosome. In
arrests cells in metaphase. After incubation, the harvest general, these polymorphisms are stable heritable markers
continues with a hypotonic treatment that swells the cells. without clinical significance.7-9 In the past, they were valu-
Several rounds of fixation (usually methanol/glacial acetic able tools in the determination of both paternity10 and the
acid in a 3:1 ratio) complete the harvest. Metaphases will parental origin of the extra chromosome in a trisomy,11,12
now be either dropped onto slides if they have been har- but much more informative molecular markers are now
vested in solution or spread on a coverslip or slide if they used to make these determinations. Nonetheless, chromo-
have been harvested in situ. Preparation of slides or cov- some polymorphisms are still useful, particularly because
erslips after the harvest is one of the most crucial steps in they may indicate maternal cell admixture in a culture of
obtaining quality material for analysis. Factors that influ- amniotic fluid cells or a tissue sample from an abortus.13
ence surface tension, such as temperature and humidity, are Reverse banding, also known as R-banding, produces
doubtless critical variables in slide making. a banding pattern that is essentially the opposite of that
seen with G-banding or Q-banding. In other words, a light
G-band will be a dark R-band, and vice versa. Terminal re-
Banding Techniques
gions of many chromosomes tend to be pale by G-banding
Before the development of banding techniques, not all or Q-banding, and small deletions or rearrangements in
chromosomes could be distinguished, and they were these regions may be difficult to detect. In such cases, an
grouped according to their size and centromere posi- R-banded preparation may make such an aberration visible,
tion. Today, several different banding techniques are used but molecular methods have largely replaced this use of
­routinely in cytogenetics to allow the unequivocal identi- R-banding in the analysis of telomeric regions (described in
fication of each chromosome. The most popular banding the section “Molecular Cytogenetic Methods”). Protocols
techniques are GTG- and reverse-banding (Fig. 31-3). exist for both bright-field and fluorescent R-banding.14
In the United States, GTG-banding, also known as
G-banding, is the most commonly used method. This method Special Stains
of banding produces a pattern of alternating light and dark
bands that allows identification of each chromosome pair Special staining techniques have been used to assist in the
by bright-field microscopy. Investigations of chromosome analysis of particular chromosomes or regions of particular
banding patterns with antinucleotide antibodies indicate chromosomes. For example, C-banding (also referred to as
that light G-bands are predominantly GC-rich and that constitutive heterochromatin or centromere banding) has been
dark G-bands are AT-rich.2 More recently, sequence analysis useful in the analysis of centromeres.15 This technique in-
of the human genome confirmed suggestions that large GC- volves acid treatment of metaphase chromosomes, followed
poor regions are strongly correlated with dark G-bands.3 by incubation in alkali (e.g., barium hydroxide) and then
Light G-bands are believed to contain more euchromatin, staining in Giemsa. The basis of C-banding is that a different
DNA that is replicated early in S-phase of the cell cycle and type of chromatin, known as constitutive heterochromatin, is
is transcribed. In contrast, dark G-bands are believed to be present in the centromeric regions of all normal chromosomes
composed of a larger percentage of heterochromatin, DNA and the distal portion of the Y chromosome. Constitutive het-
that is more condensed and late replicating. Housekeeping erochromatin consists of DNA that is believed to remain con-
genes reside in the light G-bands, and most tissue-specific densed and genetically inactive. On the other hand, facultative
genes appear to be situated in the dark G-bands.4-6 heterochromatin (such as on the inactive X chromosome) is
Q-banding, a fluorescent staining procedure, produces a specifically inactivated in certain cell types or at certain phases
pattern similar to that of G-banding. Brightly fluorescent of development, but still has the potential to decondense and
bands correspond to G-dark bands, and dull Q-bands cor- become genetically active. Chromosomes 1, 9, and 16 tend
respond to G-light bands. Q-banding was one of the most to have larger amounts of C-banded material in the pericen-
tromeric region, which can vary considerably in size among
the human population.16 C-banding may also be used to show
an inversion in the pericentromeric region of chromosome 9,
a common polymorphism limited to heterochromatin.17
Although C-banding was used in the past to elucidate the
­origin of unidentified marker chromosomes or derivative chro-
mosomes, molecular methods using centromere-specific DNA
GTG QFQ Reverse CBG DA/DAPI Rx - FISH probes are less subjective and are now the preferred method
Figure 31-3.  Chromosome 1 shown after various banding techniques: (described in the ­section “Molecular Cytogenetic Methods”).
GTG, QFQ, reverse, CBG, DA/DAPI (distamycin A/diamidinophenylin- Other special stains include silver staining, also known
dole), and Rx-FISH (cross-species color banding). The three-letter code as NOR staining,18 which identifies active nucleolar
banding designations refer to the type of banding, the method used, and
the stain employed (e.g., GTG = G-banding by trypsin with ­ Giemsa).
­organizer regions (i.e., sites of the ribosomal RNA genes)
(From Shaffer LG, Tommerup N (eds). ISCN 2005: An International Sys- on acrocentric chromosomes, and distamycin A/diamid-
tem for ­Human Cytogenetic Nomenclature. Basel, S. Karger, 1995.) inophenylindole (DA/DAPI) staining,19 which reacts with
780 PART III  Reproductive Technologies

centromeres of chromosomes 1, 9, and 16; the short arm of 36.3

chromosome 15; and the distal end of the Y chromosome. 36.2
36.1
More recently, banding patterns have been produced with 35
a combination of molecular genetic and cytogenetic tech- 3
34.2
1
niques. Chromosomes can also be enzymatically digested 33
and stained20 or hybridized with specific repetitive DNA 32

sequences21 to produce banding patterns reminiscent of

p 31
G- and C-bands and R-bands, respectively.
22
High-Resolution Banding
21
In general, clinical laboratories perform banding tech-
niques on chromosomes in midmetaphase, at which time 13
400 to 550 bands can be resolved. Other techniques that 12
11 11
enrich for chromosomes in earlier stages of metaphase, or 12
in prophase, provide higher-resolution banding methods
and are useful with targeted analysis of a particular region 21
of a chromosome. High-resolution methods in practice in- 22
clude amethopterin synchronization of cell cultures with 23
24
thymidine release22 and addition of actinomycin D (dacti- 25
nomycin)23,24 or ethidium bromide25 during the ­ final
hours of culturing, before harvest. q 31

32
Idiograms and Chromosome Nomenclature
41
An idiogram is a schematic standardized karyotype that is
42
derived from measured band sizes (Fig. 31-4). Chromo- 43
some idiograms and nomenclature have been designed by 44
an international committee that has met periodically since 1
1960 to establish uniform language for describing chromo-
Figure 31-4.  Standardized idiogram of human chromosome 1 at the
some bands and chromosome aberrations.26 Chromosome 400-band stage. p, short arm; q, long arm.
regions are subdivided into bands and, at higher resolu-
tion, into sub-bands. For example, the designation 14q24
indicates chromosome 14, the long arm, region 2, band 4. a growing number of microdeletion and microduplica-
On the idiogram, chromosome bands are numbered in an tion syndromes on interphase and metaphase cells. This
ascending manner from the centromere and toward the ­method is also applied regularly on metaphase chromo-
telomere of each chromosome arm. somes to characterize subtle chromosome rearrangements
A fairly sophisticated set of rules governs the descrip- and identify marker chromosomes, which are by ­definition
tion of chromosome abnormalities. These rules are set unidentifiable by classic cytogenetics (Fig. 31-5).
out in the ISCN 2005: An International System for Human Although in situ hybridization was first developed with
Cytogenetic Nomenclature.27 Basically, the total number of isotopic probes, clinical laboratories now use commercially
chromosomes is specified first, followed by the sex chro- available nonisotopic fluorescent probes. This method
mosomes (e.g., the normal male karyotype is given as has become widely known as FISH (fluorescence in situ
46,XY). Chromosome aberrations are listed with sex chro- hybridization). In contrast to conventional cytogenetic
mosome aberrations first (in the position of the normal sex analysis, in which chromosomal abnormalities are ­globally
chromosome), followed by abnormalities of the autosomes assessed directly with a microscope, most FISH assays
in numeric order, irrespective of aberration type. For each are considered targeted approaches that require a priori
chromosome, numeric abnormalities are listed before knowledge of a suspected aberration (Table 31-1). Probes
structural changes. Multiple structural changes of homol- may be labeled with biotin or digoxigenin, which requires
ogous chromosomes are presented in alphabetical order, fluorochrome-conjugated antibodies for detection, or with
according to the abbreviated term of the abnormality. fluorochromes directly conjugated to the DNA. Multicolor
FISH methods (e.g., spectral karyotyping [SKY], multi-
MOLECULAR CYTOGENETIC METHODS plex FISH [M-FISH], and cross-species color banding [Rx-
FISH]), are genome-wide, FISH-based technologies that
Molecular biologic methods have revolutionized cytoge- can readily show complex chromosomal rearrangements
netic studies and will continue to change the discipline. in a single hybridization experiment. Chromosomal CGH
Chromosomal in situ hybridization was first used to deter- is classically a variation of the FISH method that is used
mine the map location of particular DNA sequences and is to identify imbalances in cells being tested at a resolu-
employed routinely in the clinical cytogenetics laboratory. tion of approximately 5 to 10 Mb for one-copy gains or
It has become an invaluable method to screen rapidly for losses. More recent applications of CGH on a microarray
aneuploidy in uncultured, interphase cells and to detect platform (array CGH) containing spotted genomic clones ­
 CHAPTER 31  Cytogenetics in Reproduction 781

A B
Figure 31-5.  A, Metaphase chromosome spread showing a microdeletion in one chromosome 15 in the Prader-Willi/Angelman syndrome region.
Metaphase chromosomes were hybridized with a fluorescently labeled gamma-aminobutyric acid A receptor, beta 3 (GABRB3) probe and, as a con-
trol for chromosome 15, a fluorescently labeled promyelocytic leukemia (PML) probe. After hybridization, chromosomes were counterstained with
diamidinophenylindole (DAPI). Chromosome 15 showing hybridization with only the PML probe has a deletion of the GABRB3 probe, which maps
to the Prader-Willi/Angelman syndrome region (arrow). B, Interphase amniotic fluid cells with trisomy 21. Cells were hybridized with a fluorescently
labeled probe to chromosome 21 and counterstained with DAPI. Three signals indicate the presence of three copies of chromosome 21, consistent
with a clinical diagnosis of Down syndrome.

(e.g., bacterial artificial chromosomes [BACs]) or short cell hybrids.29 Hybridization with these DNA probes
DNA sequences (oligonucleotides) permit identification ­results in fluorescent staining of the entire chromo-
of genomic gains and losses at as much as 30- to 40-kb res- some (hence, the term “chromosome painting”). These
olution. Array CGH is becoming a significant technologic probes are available for each human chromosome, and
improvement in clinical cytogenetics, and implementation are particularly useful for determining interchromosomal
of this technology in studies of human disease is bringing ­rearrangements (i.e., chromosomal rearrangements that
new insights into the pathogenesis of disease. involve nonhomologous chromosomes) that cannot be in-
A variety of types of probes are used to detect chro- terpreted by conventional chromosome banding analyses.
mosomal abnormalities by FISH.28 Repetitive DNA Painting probes are not particularly useful in interphase
probes, whole-chromosome–painting probes, and unique analysis because the hybridization signals are large and
­sequence probes can be visualized. In general, repetitive diffuse, compared with probes developed for specific chro-
DNA probes (e.g., alpha-satellite and beta-satellite se- mosomal regions (e.g., alpha-satellite centromeric probes
quences) hybridizing to particular centromere sequences or unique sequence probes).
are usually used in chromosome enumeration, whereas A third type of FISH probe hybridizes to unique ­locus-
whole-­chromosome paints are applied to show cryptic specific DNA sequences. These probes are usually ­genomic
rearrangements and decipher the origin of marker chro- clones and typically vary in size from approximately 40 kb
mosomes. Unique sequence probes are employed to de- to hundreds of kb. These probes have become the standard
tect microdeletions, certain microduplications, and other in evaluating clinical specimens for microdeletion disorders
chromosomal rearrangements. (e.g., Prader-Willi and X-linked Kallmann syndrome).
Chromosome-specific centromeric probes are ­available
for most chromosomes. However, the centromeric se- Array-Based Comparative Genomic
quences of chromosome pairs 13 and 21 and pairs 14 and Hybridization
22 are similar, and a commercially available centromeric
probe has not yet been developed that selectively hybrid- As mentioned earlier, array CGH is becoming an extremely
izes to only one of the two chromosomes. Thus, to ­detect important molecular cytogenetic method for clinical cyto-
aneuploidy of these chromosomes, unique sequence probes genetic diagnostics.30 This technique is a modification of
are used. The major disadvantage of these probes relative to a procedure initially introduced in 199231 for surveying
the centromeric repetitive probes is that the intensity of the genomic DNA imbalances (i.e., DNA gains and losses) in
signal is generally weaker and some marker chromosomes cancer specimens, but it is now being applied to all areas
may be missing distally located unique ­sequences, making of cytogenetic diagnostics and research. In this procedure,
these probes less informative in certain clinical cases. thousands (or even millions) of DNA fragments, each
Chromosome-painting probes may be developed from representing a specific region of the human genome, are
DNA libraries from flow-sorted human chromosomes or first attached to a glass slide. Then, a patient’s DNA is
DNA amplified from human monochromosomal somatic ­labeled with one fluorescent color/dye, combined with
782 PART III  Reproductive Technologies

TABLE 31-1 Array CGH platforms that are now typically used in
Reasons to Request a Patient’s Karyotype cytogenetic diagnostics have an effective genome-wide
or Fluorescence in Situ Hybridization resolution of as low as 30,000 bp (30 kb) to 40,000 bp
(40 kb). This is a substantial improvement over the
Possible Chromosome 3,000,000-bp (3 Mb) to 5,000,000-bp (5 Mb) resolution
Clinical Indication Test Abnormality associated with detecting gains and losses by GTG-banded
karyotypic analysis, and is associated with less subjective
Couple with ≥3 spontaneous K Balanced structural
abortions ­rearrangement interpretation. Some of the array CGH platforms used for
Parent of child with structural K Balanced structural cytogenetic diagnostics also have an increased number of
chromosome abnormality ­rearrangement probes at clinically informative sites of the human genome
First-degree relative with K Balanced structural (including the pericentromeric regions, subtelomeric re-
­structural chromosome ­rearrangement gions, and known critical regions for microduplication
­abnormality and microdeletion syndromes).
Female expressing X-linked K X-autosome Array CGH offers a less subjective and cost-effective
­disorder ­translocation way to detect DNA gains and losses in a patient’s genome
or 45,X at unprecedented high resolution. Therefore, it is possible
Female <10th percentile in K 45,X; possibly
to detect whole-chromosome aneuploidy, subtelomeric im-
height mosaic
Female with elevated K 45,X; possibly balances, microdeletions/microduplications, and the origin
­gonadotropins mosaic of marker chromosomes (e.g., Fig. 31-6). However, as with
Female with infertility/ovarian K Structural any technology, array CGH also has specific limitations.
dysgenesis ­rearrangement of X First, it is not possible for current array CGH strategies to
Male with elevated detect truly balanced chromosomal rearrangements, such
­gonadotropins, whether as inversions, insertions, and translocations. However, re-
Tall K 47,XXY cent higher-resolution analyses are finding that as many
Short K 45,X/46,XY as 30% to 40% of these chromosomal rearrangements that
Normal height K 46,XX (follow up were previously believed to be balanced at the GTG-banded
with PCR for SRY)
resolution are actually unbalanced.32 Second, because array
Gynecomastia K 47,XXY
azoospermia or oligospermia K Balanced structural CGH specifically detects genomic imbalances, it is unable
rearrangements, to identify “copy-neutral” chromosomal aberrations (e.g.,
­particularly uniparental disomy [UPD]). For detecting such aberrations,
involving sex single-nucleotide polymorphism–detecting­ arrays may be
­chromosomes; informative (e.g., Affymetrix 6.0 arrays [­Affymetrix, Santa
­deletions in Clara, CA]; Illumina 1 million feature arrays [Illumina, San
Yq (PCR for Yq Diego, CA]). Third, the level of mosaicism that is detect-
microdeletions) able can be less than that of classic cytogenetic analysis.
Sexual ambiguity K Current estimates are that whole-chromosome mosaicism
associated with Wilms’ F 11p13 deletion
of less than 10% and segmental genomic imbalances of less
tumor, aniridia
Hypogonadism and other F 15q11q13 deletion than 30% could go undetected by array CGH.
features of Prader-Willi Along with the increased resolution of array CGH test-
syndrome ing comes another level of complexity in the clinical in-
terpretation of the data obtained. Much of this complexity
F, fluorescence in situ hybridization; K, karyotype; PCR, polymerase stems from the recent realization that healthy individuals
chain reaction.
harbor hundreds to thousands of genomic imbalances that
do not appear to cause early-onset, highly penetrant dis-
g­ enomic DNA from a normal control subject labeled with eases or genomic disorders.33,34 These copy number vari-
a different fluorescent color/dye, and then applied to the ants can range in size from 1000 bases (1 kb) to more than
glass slide containing the DNA fragments that recapitu- 2,000,000 bases (2 Mb).35 Several criteria have been pro-
late the human genome at a certain resolution. The flu- posed for differentiating a “benign” copy number variant
orescence intensity ratio of the two labeled dyes at each from a genomic imbalance that is pathogenic,36 but the
DNA fragment “spot” on the glass slide reflects the copy most significant criterion appears to be whether the im-
number ratio of that DNA sequence in the patient’s DNA balance is de novo (i.e., both healthy parents do not carry
compared with the normal control subject. For example, if the specific copy number variant detected in the affected
the patient’s DNA is labeled with cy5 fluorescence and the offspring).
control DNA is labeled with cy3 fluorescence, a significant
increase in cy5 fluorescence, compared with cy3 fluores-
cence, for a particular DNA fragment may be indicative Chromosome Aberrations
of a gain of that DNA sequence in the patient, compared and Reproduction
with the control subject. Conversely, a significant decrease
in cy5 fluorescence, compared with cy3 fluorescence, for Chromosome aberrations are common in humans and ac-
the same DNA fragment, may be indicative of a loss of that count for a large proportion of pregnancy loss and congen-
DNA sequence in the patient. ital malformations. Chromosome aberrations, especially
 CHAPTER 31  Cytogenetics in Reproduction 783

Figure 31-6.  An example of the utility of array comparative genomic hybridization (CGH) in clinical cytogenetic diagnostics. A, A GTG-banded
karyotype obtained from amniotic fluid taken at 15 weeks’ gestation from a 39-year-old woman carrying a pregnancy achieved through in vitro
fertilization. Of 49 cells examined, 13 carried a marker chromosome of unknown origin. GTG-banded karyotype analysis from cord blood taken at 
21 weeks’ gestation showed similar results. B, Array CGH testing, using a 244K oligo-based platform (Agilent, Santa Clara, CA) with an approximately
10-kb effective resolution, showed a significant gain of genomic DNA from an approximate nucleotide position of 15,927,891 to 24,158,055, at the
proximal short arm of chromosome 19. Taken together, these results suggest that the marker chromosome is composed of approximately 8.2 mil-
lion bases of DNA from the proximal short arm of chromosome 19. (Images kindly provided by Chun Hwa Ihm, MD, PhD, of Eulji University Hospital
[Daejeon, South Korea], and Ji Hyeon Park, MD, of CHA General Hospital [Seoul, South Korea]).

aberrations of the X and Y chromosomes, can also be a GAMETOGENESIS ERRORS

cause of infertility. Common clinical practice for couples
Review of Oogenesis and Spermatogenesis
who have experienced three or more spontaneous abor-
tions includes full cytogenetic analysis. For the reproduc- Meiosis, the cell division process that produces haploid
tive specialist, chromosome abnormalities can be divided germ cells from diploid germ cell precursors, is the funda-
into two clinically distinct groups: (1) normal findings on mental event in both oogenesis and spermatogenesis. The
G-banded karyotypic analysis, in which a reproductive overall sequence of meiotic events is the same in oogenesis
­error results in a chromosomally abnormal conceptus; and and spermatogenesis, but several key differences exist.
(2) a recognized de novo or inherited constitutional chro- In both female and male gametogenesis, the chromo-
mosome anomaly associated with reproductive implica- some number is reduced by half, producing haploid gametes
tions and perhaps other phenotypic effects. with 23 chromosomes through a process in which diploid
Chromosome abnormalities arising during reproduc- germ cell precursor cells replicate their DNA and then un-
tion are fairly common. An estimated 8% of concep- dergo two successive cell divisions. The first cell division,
tuses possess a chromosome abnormality.37-39 Most of meiosis I, is termed the reduction division, because it is here
these chromosomally abnormal products of conception that the number of chromosomes is halved. Homologous
are not viable, but an estimated 0.7% of liveborn infants chromosomes pair or synapse and then exchange material
are chromosomally abnormal.40 Errors in gametogenesis by recombination or crossing over. Recombination greatly
are the most common cause of chromosomally abnormal increases the genetic diversity in gametes by reassorting
conceptuses, but fertilization and postfertilization errors paternally and maternally inherited genetic information.
also occur. After recombination, homologous chromosomes, whose
784 PART III  Reproductive Technologies

chromatids now contain segments of both paternally and region 1 (PAR1). DNA in this region can be transferred
maternally derived DNA, segregate to opposite poles and from one sex chromosome to the other,44,45,48,49 and none
form two cells with 23 chromosomes. At this point, each of the at least 24 genes mapped to this region appears to
chromosome is composed of two sister chromatids that be required for specific male or female sexual differentia-
remain together until meiosis II, during which time they tion.50-53 This region is probably important for initiating
separate in a manner analogous to a mitotic division. The chromosome pairing and forming a synaptonemal complex
result of meiosis in both spermatogenesis and oogenesis in male meiosis, and it appears that one recombination
is the production of haploid germ cells with 23 chromo- event is required in this region for proper disjunction of
somes composed of one chromatid each. the XY bivalent.54,55 The X and Y chromosomes also occa-
Although meiosis produces haploid germ cells in both sionally pair and recombine at a second region on the long
female and male gametogenesis, these two processes dif- arm of each chromosome containing at least five genes, at
fer in several critical ways. One crucial difference is in a region termed pseudoautosomal region 2 (PAR2).46,47,53
the timing of events in gametogenesis. In spermatogen-
esis, the production of mature haploid sperm from diploid Meiotic Nondisjunction
spermatogonia does not begin until puberty, but continues
throughout life. Production of haploid sperm from dip- Meiotic nondisjunction errors are common in humans,
loid spermatogonia requires approximately 64 days and resulting in aneuploidy, a term used when the total num-
involves a continuing series of both mitotic and meiotic ber of chromosomes in a cell is not an exact multiple of
divisions. In oogenesis, the mitotic divisions that precede the haploid number. Aneuploidy usually involves a single
meiosis are completed during fetal development and do chromosome, but in rare circumstances, may involve more
not continue throughout life, as in spermatogenesis. The than one. Aneuploidy is present in approximately 0.6% of
onset of meiosis in females occurs during fetal develop- newborns56 and nearly 70% of spontaneous abortions.57
ment, but is arrested before birth, at the end of prophase I. Trisomy for all chromosomes has been observed in spon-
Meiosis does not proceed again until ovulation, at which taneous abortions, indicating that nondisjunction for each
time one oocyte completes meiosis I and proceeds to mei- chromosome does occur.58-60
osis II. Meiosis II is completed only if fertilization occurs. Meiotic nondisjunction can involve only one chromo-
The difference in the timing of both mitotic and meiotic some or the whole chromosome set. Nondisjunction of
divisions is believed to play a significant role in the sus- a single chromosome will produce germ cells that have
ceptibility of oocytes and spermatocytes to mutation and either two (disomy) or zero (nullisomy) copies of the spe-
reproductive errors. Spermatogenesis, which involves con- cific chromosome. If a germ cell with an extra chromosome
tinual cell division and hence replication of DNA, is more is combined with a chromosomally normal germ cell, the
vulnerable to DNA damage and replication errors. Thus, product will be trisomic (i.e., having 47 chromosomes).
de novo point mutations are more commonly of paternal If a germ cell missing a chromosome is combined with
than maternal origin. Conversely, the protracted meiotic a chromosomally normal germ cell, the product will be
arrest in oogenesis is believed to contribute to nondisjunc- monosomic (i.e., having 45 chromosomes). Nondisjunc-
tion, and the extra chromosome in trisomies is usually of tion of the entire chromosome set will lead to either germ
maternal origin. cells with two copies of every chromosome or germ cells
The manner in which cytoplasm is divided during mei- with no chromosomes. The clinical phenotype and the
osis also differs in female and male gametogenesis. In sper- histopathology of conceptuses that can result from numer-
matogenesis, a primary spermatocyte divides its cytoplasm ically abnormal gametes depend on both the total number
equally to produce four functionally equivalent sperm. By of chromosomes and the relative number of ­paternal ver-
contrast, a primary oocyte divides its cytoplasm unequally sus maternal chromosomes.
in the first meiotic division, producing one
�������������������
polar body ����
and Nondisjunction can take place in either meiosis I or
one secondary oocyte that retains most of the cytoplasm�����
. If meiosis II. If nondisjunction occurs in meiosis I, all four
the secondary oocyte is fertilized, it will complete meiosis products of meiosis will be chromosomally abnormal. Two
II, generating a second polar body and a fertilized ovum of the four products of meiosis will have two copies of the
that again retains most of the cytoplasm. This unequal di- chromosome involved in the nondisjunction event, and
vision of cytoplasmic contents is important because the two of the four products of meiosis will have no copies of
ovum contributes most of the non-nuclear cytoplasmic that particular chromosome. Of further note, in germ cells
contents to the fertilized product. Thus, mitochondria and with two copies of the chromosome, the copies, ­although
other cytoplasmic components are essentially exclusively homologous, will not be identical. Homologous chromo-
of maternal origin. somes do not separate in nondisjunction errors in ­meiosis
A third important difference between oogenesis and I, but sister chromatids separate properly in meiosis II.
spermatogenesis is the synapsis configuration of sex chro- Thus, each of the germ cells with an extra chromosome
mosomes during meiosis. In female meiosis, the two X will have a maternally derived chromosome and a pater-
chromosomes pair and recombine over the entire length nally derived chromosome. In the absence of recombi-
of the chromosomes.41,42 In male meiosis, by comparison, nation, one chromosome would be entirely of maternal
the morphologically dissimilar X and Y chromosomes pair origin and the other entirely paternal.
and recombine in only two regions.43-47 The first of these If nondisjunction occurs in meiosis II, two of the four
regions identified is located on the distal short arms of the products will be unaffected by the event and two of the
X and Y chromosomes and is known as pseudoautosomal products will be abnormal. One abnormal product will
 CHAPTER 31  Cytogenetics in Reproduction 785

Nondisjunction Meiosis I Normal

Normal Meiosis II Nondisjunction Normal

Gametes: a b c d e f g h

If fertilized: Monosomy Monosomy Trisomy Trisomy Monosomy Trisomy Normal Normal

Figure 31-7.  Comparison of nondisjunction in meiosis I and meiosis II. With an error in meiosis I, all four gametes (a, b, c, and d) are aneuploid.
Two gametes (a and b) are nullisomic, potentially resulting in a monosomic conceptus. Two gametes (c and d) are disomic, potentially resulting in
a trisomic conceptus. The two homologous chromosomes in the disomic gametes (c and d) are heterodisomic. With an error in meiosis II, only two
gametes (e and f) are aneuploid. One gamete (e) is nullisomic, and the other aneuploid gamete (f) is disomic. The two homologous chromosomes in
the nonreduced gamete (f) are isodisomic.

have an extra chromosome, and the other abnormal prod- nondisjunction seen in trisomic conceptuses does indeed
uct will be missing that chromosome. With nondisjunction reflect differences in the rate of nondisjunction in oocytes
errors in meiosis II, homologous chromosomes separate and spermatocytes. It is still possible, however, that selec-
properly in meiosis I, but sister chromatids do not separate tion against aneuploid sperm occurs after spermatogenesis
in meiosis II. Thus, in contrast to meiosis I nondisjunc- as well.
tion errors, the two nondisjoined chromosomes would be The rate of aneuploidy is higher in oocytes than in sper-
genetically identical in the absence of recombination (Fig. matocytes, and it also increases markedly with maternal
31-7). This apparently trivial difference between errors in (but not paternal) age.69,70 Although the relationship be-
meiosis I and errors in meiosis II can have important clini- tween increased maternal age and Down syndrome was first
cal consequences that are discussed later. Furthermore, described in 1933,71 the mechanism for this aging effect
the study of the parental origin of chromosomes involved remains to be fully elucidated. Most nondisjunction errors
in aneuploidies has led to important observations about in oocytes occur in meiosis I, and it has been hypothesized
the origin and meiotic stage of nondisjunction. that the prolonged arrest in meiosis I ­contributes to these
It is difficult to study meiotic nondisjunction for all errors.72
chromosomes directly in gametes and even indirectly in Paternal nondisjunction is more common in cases of
products of conception because many aneuploid products aneuploidy involving sex chromosomes than in cases
of conception are lost early in pregnancy and are never ­involving autosomes. It has been hypothesized that the
brought to clinical attention. However, conceptuses with XY bivalent is more susceptible to nondisjunction than
trisomies for some chromosomes survive long enough to are the homologous bivalents. FISH studies on sperm
be clinically recognized, and several studies have used have supported this hypothesis, showing rates of sex chro-
DNA polymorphisms to analyze the parental origin of the mosome disomy to be two to four times higher than di-
extra chromosome in these cases of trisomy. These stud- somy for particular autosomes.66,73 Eighty percent of 45,X
ies have shown that maternal nondisjunction ­ accounts karyotypes can be attributed to paternal nondisjunction,
for significantly more cases of autosomal trisomy than ­although some of these cases may be caused by early loss
does paternal nondisjunction. For trisomies 13, 14, 15, of the Y chromosome through mitotic nondisjunction
16, 18, 21, and 22, maternal nondisjunction accounted in the zygote.74,75 Cases of 47,XXY are divided approxi-
for 88%, 83%, 88%, 100%, 93%, 91%, and 89% of cases, mately equally between maternal and paternal nondis-
respectively.54,61-65 Direct studies of aneuploidy in gam- junction.76,77 However, as with the autosomal trisomies,
etes give estimates of 2% to 4% meiotic nondisjunction the extra X chromosome is of maternal origin in 90% of
in sperm58,66,67 and 13% to 18% meiotic I nondisjunc- cases of 47,XXX.77
tion in oocytes.68,69 These findings indicate that the ex- Studies have shown that recombination is crucial for
cess of maternal ­ nondisjunction relative to paternal proper segregation of homologous chromosomes. Yeast
786 PART III  Reproductive Technologies

experiments have shown that recombination is required i­ncidence of approximately 1:800. Estimates of trisomy 18
for formation of the synaptonemal complex and for com- range from 1:3500 to 1:7000 newborns, and estimates of
plete pairing of homologous chromosomes. From this, trisomy 13 range from 1:7000 to 1:21,000.87-89
it has been suggested that, in the absence of pairing and Trisomy 21, the least severe of the autosomal trisomies,
­recombination, nondisjunction would be increased.78 In results in Down syndrome, a well-defined and familiar
humans, studies of trisomies 15, 16, 18, and 21, as well disorder. Down syndrome was first described by Langdon
as of XXY and XXX, have shown that, on average, the Down in 1866 and is the single most common cause of
­particular chromosomes involved in a specific nondis- moderate mental retardation. In addition to mental im­
junction event participated in fewer recombinations than pairment, individuals with Down syndrome frequently
usual.54,79-83 Presumably, the limited region of homology in have other serious clinical phenotypes, including cardiac
which recombination occurs in the XY bivalent ­accounts defects and increased risk of both childhood leukemia
for its increased susceptibility to nondisjunction. Interest- and early-onset Alzheimer disease. Approximately 95%
ingly, the overall rate of recombination is higher in female of cases of Down syndrome result from the presence of
gametogenesis than in male gametogenesis, although some three separate copies of chromosome 21 (i.e., 47,XX,+21
specific chromosomal regions, including the telomeric re- or 47,XY,+21), reflecting a parental nondisjunction event
gions of many chromosomes, have higher recombination involving chromosome 21. Approximately 3% of cases of
rates in males.84,85 Down syndrome result from a chromosomal transloca-
tion in which one of the three copies of chromosome 21 is
joined in a “head-to-head” manner to another acrocentric
Clinical Outcomes of Meiotic Nondisjunction
chromosome, usually through repetitive sequences in the p
Most numeric chromosome abnormalities caused by mei- arms. Such chromosomal rearrangements are referred to as
otic nondisjunction errors result in spontaneous abortion. robertsonian translocations. The remaining 2% result from
As a group, chromosome abnormalities are the most com- a mosaic chromosome constitution in which one normal
mon cause of pregnancy loss, accounting for 50% to 60% cell line has 46 chromosomes and a second abnormal cell
of spontaneous losses. Triploidy, tetraploidy, trisomy, and line carries an additional copy of chromosome 21. In these
monosomy have all been reported in spontaneous abor- cases, the original conceptus may have been trisomic for
tions. The most common chromosome abnormalities in chromosome 21 with a postzygotic mitotic nondisjunction
chromosomally abnormal spontaneous abortions are 45,X event, resulting in an additional cell line disomic for chro-
(20%), trisomy 16 (16%), and triploidy (16%). Trisomy 21 mosome 21. Another possibility is that the original con-
accounts for approximately 5% of chromosomally abnor- ceptus may have been disomic for chromosome 21 with a
mal spontaneous abortions.39 postzygotic mitotic nondisjunction event, resulting in an
The overall incidence and distribution of numeric chro- additional cell line trisomic for chromosome 21.
mosome abnormalities differ in newborns and spontane- Trisomy 18, described by John Edwards in 1960, also
ous abortions, reflecting the differential viabilities of the has a severe clinical phenotype. Only 10% of these individ-
abnormalities. Trisomies 13, 18, and 21 are ­ compatible uals survive beyond the first year of life.90 Failure to thrive
with life, although most of these conceptuses result in and mental retardation are seen in all individuals who are
spontaneous abortion (Table 31-2). No other nonmosaic trisomic for chromosome 18. Prominent occiput, recessed
autosomal trisomy or autosomal monosomy (with the jaw, short sternum, low-set and malformed ears, clenched
exception of monosomy 2186) has ever been reported in fists with a characteristic overlapping of the fingers, and
a liveborn infant. In newborn surveys, trisomy 21 is the rocker-bottom feet are features of this syndrome. Approxi-
most common autosomal aneuploidy, with an overall mately 80% of cases of trisomy 18 result from the presence
of three separate copies of chromosome 18, approximately
10% are mosaic, and the remaining cases primarily involve
a chromosomal translocation.91
TABLE 31-2
Trisomy 13, first described by Klaus Patau in 1960,
Probability of Survival to Birth for Viable is much less common than trisomy 21 and is clinically
Aneusomic Conceptuses much more severe. Approximately 45% of liveborn infants
Probability of
die within the first month after birth and 90% die by the
Karyotype Survival to Birth (%)* age of 6 months. The phenotype of trisomy 13 involves
severe central nervous system malformations, includ-
47,+13 2.8 ing severe mental retardation, holoprosencephaly, and
47,+18 5.4 arrhinencephaly. Cleft lip and cleft palate are frequently
47,+21 22.1 seen. Eye anomalies, ranging from microphthalmia to
45,X 0.3 anophthalmia, are also common. Postaxial polydactyly,
47,XXX 70.0
47,XXY 55.3 clenched fists, and rocker-bottom feet are further features
47,XYY 100.0 of this syndrome. As with trisomy 21, trisomy 13 occurs
as a result of the presence of three separate copies of a
*Basedon the incidence of chromosome abnormalities in spontaneous ­chromosome (chromosome 13) or in conjunction with a
abortions, stillbirths, and livebirths, assuming an overall 15% rate of
spontaneous abortion and 1% rate of stillbirth in the population.
robertsonian translocation.
Adapted from Jacobs PA, Hassold TJ. The origin of numerical Recurrence risks in cases of trisomy in which there are
­chromosome abnormalities. Adv Genet 33:101-133, 1995. three separate copies of a given autosome (i.e., not due to a
 CHAPTER 31  Cytogenetics in Reproduction 787

chromosomal translocation) can be calculated on the basis locus and thus developed from duplication of the haploid
of empirical data for trisomy 21. However, minimal empir- paternal genome in an egg containing no maternal chro-
ical data exist for trisomies 13 and 18 because of the rarity mosomes.94-96 Duplication of the haploid paternal genome
of these conditions. For mothers younger than 35 years, must be caused either by nondisjunction in meiosis II or by
the recurrence risk is approximately 0.5% for a subsequent endoreduplication of a haploid sperm after fertilization.
trisomy 21 and 1% for any chromosome abnormality. For The mechanism by which an egg becomes devoid of ma-
mothers older than 35 years, in whom the age-related ternal chromosomes is unclear. Possibilities include meiotic
risk of trisomy 21 is equal to or greater than 0.5%, the nondisjunction, enucleation of the oocyte, and exclusion
recurrence risks are equivalent to the general population of the maternal chromosomes after fertilization.97 An esti-
age-related risks.92 The reasons for the additional risk in mated 4% to 15% of complete moles are 46,XY and are be-
younger mothers are not entirely clear and may be due lieved to arise from fertilization of a chromosomally empty
to individual differences in rates of nondisjunction or to egg with two sperm.95,98 This same mechanism is believed
cases of parental gonadal mosaicism (discussed in further to account for the 5% of complete moles that are 46,XX,
detail later) for trisomy 21. In such instances, a phenotypi- but are not genetically identical at every locus.95 Note that
cally normal individual has a cell line that is trisomic for 46,YY moles are not seen, presumably because this chromo-
chromosome 21 in gonadal tissue, but not somatic tissue. somal constitution is not compatible with development.
Recurrence for trisomies 13 and 18 is rare, but the risk Comparing the pathophysiology of benign cystic tera-
of trisomy 21 may be increased after the occurrence of tomas and complete hydatidiform moles provided some of
one of these other trisomies. All of these recurrence risks the first evidence for imprinting, the differential effect of
are based on a liveborn index case. As already mentioned, genetic material depending on whether the inheritance is
nonviable autosomal trisomies are common in spontane- of maternal or paternal origin. Although both benign cys-
ous abortions, and it is unclear whether such an event in- tic teratomas and complete hydatidiform moles are diploid
creases the risk of trisomy in subsequent pregnancies. If a entities with genetic contribution from only one parent,
trisomy is detected in a late abortion or in a stillbirth, it is their development is dissimilar. Benign cystic teratomas
probably prudent to use the same risk figures as for a live have little trophoblast development, but do have some
birth.92 Finally, recurrence risks after a chromosomal trans- embryonic development. In contrast, complete moles have
location-based trisomy (i.e., not a result of meiotic nondis- no embryonic development, but do have placental tropho-
junction) depend on several factors, including whether the blast development, albeit abnormal.
translocation was inherited or occurred de novo, the par- Another outcome involving unreduced gametes is trip-
ticular chromosomal translocation involved, and the sex of loidy. Although dispermy is believed to be the most common
the carrier parent (discussed later; see also Table 31-4). cause of triploidy, fertilization with an unreduced (diploid)
Trisomies of the sex chromosomes resulting in XXX, egg or sperm also leads to triploidy. Triploidy almost always
XXY, and XYY are viable and have a much lower rate of results in spontaneous abortion, although it is seen in ap-
spontaneous abortion than do the autosomal trisomies proximately 1:57,000 births. Most of these newborns die
(see Table 31-2). In newborn surveys, 47,XXX, 47,XXY, within the first day. However, in some isolated cases, pa-
and 47,XYY are all found at an approximate incidence of tients reportedly have survived for several months.99,100
1:1000.40 Monosomy for the X chromosome, unlike auto- The phenotype of triploidy differs, depending on
somal monosomy, can be viable. However, more than 99% whether the origin of the extra chromosome set is pater-
of conceptuses with monosomy X result in spontaneous nal (diandric) or maternal (digynic). Although both dian-
abortion, and its frequency of approximately 1 affected dric and digynic triploid products can be associated with
newborn in 10,000 is much lower than that of the other a fetus, the diandric triploid is associated with a less de-
numeric sex chromosome abnormalities.40 In general, phe- veloped fetus, and no diandric triploid surviving to term
notypes of sex chromosome aneuploidies are more subtle has ever been reported. Diandric triploids are associated
than those of the autosomal aneuploidies (discussed fur- with excessive placental growth and partial hydatidi-
ther in the section on sex chromosome abnormalities). form moles. Digynic triploids are associated with smaller,
Recurrence of any of the sex chromosome abnormalities nonmolar placentas. Thus, diandric triploids have in-
is exceedingly rare.92 creased placental growth but decreased fetal development
Infrequently, meiotic nondisjunction for the complete ­compared with digynic triploids.101 These differences in
chromosome set can occur. One outcome of such an event placental and fetal development between diandric and di-
is a benign cystic teratoma. In the absence of fertilization, gynic triploids are similar to the developmental differences
an unreduced egg with 46 chromosomes can develop into between complete moles and benign cystic teratomas, in
a benign cystic teratoma. Benign cystic teratomas repre- which paternal chromosomes are associated with placental
sent a duplication of the maternal genome with no contri- development, but no embryonic development. In contrast,
bution from a paternal genome. These teratomas can also maternal chromosomes are associated with embryonic de-
arise after endoreduplication of a haploid egg. velopment, but little placental development.
As with benign cystic teratomas, complete hydatidiform
moles are diploid. However, with rare exceptions, they are Errors of Recombination
composed of chromosomal DNA solely of paternal ori-
gin,93,94 although the mitochondrial DNA is of maternal or- As already mentioned, not only is meiotic recombination
igin. Studies of molecular polymorphisms have shown that crucial for genetic diversity, but its frequency also appears
75% to 85% of complete moles are homozygous at every to influence rates of meiotic nondisjunction. Accuracy of
788 PART III  Reproductive Technologies

TABLE 31-3
Microdeletions and Microduplications: Chromosomal Location and Availability
of Commercial Fluorescence in Situ Hybridization Probes
Commercially Available
Syndrome Location Deletion or Duplication FISH Probe*
Monosomy 1p36 1p36 Deletion Yes
Sotos 5q35 Deletion Yes
Williams 7q11.23 Deletion Yes
Langer-Giedion/trichorhinophalangeal 2 8q24.1 Deletion No
DiGeorge/velocardiofacial (DGS2) 10p14 Deletion Yes
WAGR† 11p13 Deletion No
Retinoblastoma 13q14 Deletion Yes
Angelman 15q11q13 Deletion Yes
Prader-Willi 15q11q13 Deletion Yes
15q13.3 15q13.3 Deletion No
α-Thalassemia/mental retardation 16p13.3 Deletion No
Rubinstein-Taybi 16p13.3 Deletion No
Smith-Magenis 17p11.2 Deletion Yes
Miller-Dieker 17p13.3 Deletion Yes
Neurofibromatosis type 1 17q11.2 Deletion No
17q21.3 17q21.3 Deletion No
Alagille 20p11.23p12.2 Deletion Yes
DiGeorge/velocardiofacial (DGS1) 22q11.2 Deletion Yes
Steroid sulfatase deficiency Xp22.32 Deletion Yes
Duplication 1p36 1p36 Duplication Yes
Beckwith-Wiedemann 11p15 Duplication No
Charcot-Marie-Tooth 1A 17p11.2p12 Duplication No
Cat-eye 22q11 Duplication No
*Also
available are fluorescence in situ hybridization probes for the usually classically detectable deletions associated with cri du chat (5pl5) and
Wolf-Hirschhorn (4pl6) syndromes.
†Wilms’ tumor, aniridia, genital abnormalities, and mental retardation.

FISH, fluorescence in situ hybridization.

recombination is no less important and is critical in main- most ­notably, triploidy. As previously mentioned, fertiliza-
taining the fidelity of genetic information. Recombination tion of one egg by two sperm is the most common cause
between mispaired chromosomes or misaligned chroma- of triploidy and results in a partial hydatidiform mole. Fer-
tids, known as unequal crossing over, can lead to duplication tilization of the first polar body or retention of the second
or deletion of genetic material. On occasion, the resulting polar body may also result in triploidy.
rearrangements are large enough to be detectable through Postfertilization errors occur as well. Cleavage errors,
conventional cytogenetic studies, but more commonly, resulting in chromosome duplication without subsequent
they can be detected only through high-resolution cytoge- cell division, produce tetraploidy. If a cleavage error oc-
netic methods, such as FISH or array CGH. The number curs during the first division, the result is nonmosaic
of genetic diseases known to be caused by microdeletions tetraploidy. With rare reported exceptions,102,103 this con-
or microduplications is growing, as is the number of com- dition is incompatible with life. A cleavage error at a later,
mercially available probes used for their detection. Cur- multicellular stage produces mosaicism for tetraploid and
rently commercially available FISH probes include those diploid cell lines, which can be observed in spontaneous
for the detection of Prader-Willi syndrome, Angelman syn- abortuses and rarely in liveborn infants.
drome, DiGeorge/velocardiofacial syndrome, and ­Williams
syndrome (Table 31-3). Mosaicism
Just as cleavage errors at a multicellular stage produce mo-
FERTILIZATION AND POSTFERTILIZATION
saicism for tetraploidy and diploidy, mitotic nondisjunc-
ERRORS
tion errors at a multicellular stage produce mosaicism for
In addition to meiotic errors of gametogenesis, a variety individual chromosomes. Theoretically, mitotic nondis-
of errors can occur at the time of fertilization. Normally, junction should create two new cell lines, one monosomic
penetration of the zona pellucida by one sperm triggers for the chromosome that underwent nondisjunction and
a series of events leading to prevention of penetration by one trisomic for the same chromosome. However, not all
other sperm, resumption of meiosis II in the egg, and ex- cell lines are compatible with development; therefore, the
trusion of the second polar body. Failure of any of these monosomic cell lines, which are frequently inviable, are
processes can lead to numeric chromosome aberrations, not usually observed.
 CHAPTER 31  Cytogenetics in Reproduction 789

The three autosomal trisomies, trisomies 13, 18, and is inherited from the other parent. Thus, if one of the three
21, which can result in liveborn infants, are also observed chromosomes is randomly lost in the mitotic nondisjunc-
as mosaics in conjunction with a diploid cell line. Two ad- tion event, the remaining chromosomes will be of biparental
ditional autosomal trisomies, trisomies 8 and 9, are only inheritance two thirds of the time, but of uniparental inheri-
viable as mosaics. The presence of a normal cell line can tance one third of the time.
allow the otherwise lethal trisomies 8 and 9 to be viable, Two types of UPD occur: heterodisomy, in which the two
and may, in some cases, improve the phenotype for triso- chromosomes, although homologous, are not genetically
mies 13, 18, and 21. However, in the setting of prenatal identical, and isodisomy, in which the two chromosomes
diagnosis, a mosaic result for trisomy 13, 18, or 21 is usu- are identical. Because of recombination, both heterodiso-
ally given a prognosis equivalent to that for a complete mic and isodisomic regions may be present. Centromeric
trisomy. Mosaic trisomy for the other autosomes, such as heterodisomy results when the nondisjunction error oc-
the respective complete trisomy, almost always results in curs in meiosis I, and centromeric isodisomy results when
spontaneous abortion, although rare cases of trisomy mo- the error occurs in meiosis II.
saicism have been confirmed postnatally for all autosomes
except chromosomes 1 and 11.89,104-109 The prognosis Clinical Outcomes of Confined Placental
for mosaicism involving sex chromosomes, particularly
Mosaicism and Uniparental Disomy
45,X/46,XY, is more variable (discussed in the section on
sex chromosome abnormalities). A wide range of clinical phenotypes of CPM occur, de-
pending on the extent of aneuploidy in the placenta, the
Tissue-Limited Mosaicism particular chromosome involved in the aneuploidy, the
and Uniparental Disomy presence or absence of UPD, and the type of UPD. Isodi-
somic UPD always carries the risk of “unmasking” a reces-
Tissue-limited mosaicism is a specialized form of mosa- sive gene and in fact was first identified in an individual
icism in which some but not all tissues in an ­individual with cystic fibrosis and short stature who had inherited
have two or more cell lines. This type of mosaicism arises two identical maternal copies of chromosome 7.112 Both
when mitotic nondisjunction occurs in a cell type that is heterodisomic UPD and isodisomic UPD are problems for
a precursor for a subset of tissues. Gonadal mosaicism chromosomes with imprinted regions, areas in which gene
occurs when the abnormal cell line is present in the go- expression is not equivalent for maternally and paternally
nadal tissue. This type of mosaicism becomes apparent inherited genes.
only when an individual has multiple offspring with the Uniparental disomy for chromosome 15 has been stud-
same chromosome abnormality, and it is one reason why ied extensively. Maternal disomy results in Angelman
recurrence risks are increased after diagnosis of an ab- syndrome, and paternal disomy results in Prader-Willi
normality. Confined placental mosaicism (CPM) occurs syndrome. Imprinted genes also exist on chromosomes 6,
when the abnormal cell line is confined to the placenta, 7, 11, and 14. Established phenotypic effects result from
with the fetus being karyotypically normal. CPM is sus- maternal UPD for chromosomes 7 and 14 and from pater-
pected most often after cytogenetic studies of ­tissue ob- nal UPD for chromosomes 6 and 11.113 Paternal UPD for
tained from chorionic villus sampling indicate a mosaic chromosome 21 and maternal UPD for chromosome 22
karyotype, possibly reflective of an abnormal placenta do not appear to have any phenotypic effect. Additional
and a normal fetus. Subsequent chromosome studies of information on human imprinted regions and genes has
amniotic fluid cells may be performed to confirm that been published.114,115
the abnormal cell line is most likely confined to the
placenta.
Theoretically, CPM could arise through at least two Constitutional Chromosome
possible mechanisms. Because the embryo derives from a Anomalies Affecting Reproduction
relatively small number of progenitor cells,110,111 one pos-
sibility is that CPM could occur after a postzygotic non- Constitutional chromosome anomalies affecting reproduc-
disjunction event in a cell lineage that contributes only tion can be divided into two classes: abnormalities involv-
to extraembryonic tissues. Alternatively, CPM may arise ing autosomes and abnormalities involving one or more of
through a mechanism in which the original conceptus is the sex chromosomes. Within each of these groups, chro-
trisomic, but then undergoes a nondisjunction event, lead- mosome abnormalities can be numeric or structural.
ing to loss of the aneuploid chromosome in some but not In practice, numeric abnormalities affecting repro-
all daughter cells. Those pregnancies with a resulting dip- duction are limited to sex chromosome abnormalities,
loid fetal karyotype may be more likely to progress; this ­although issues of reproduction do affect individuals with
mechanism is known as trisomy rescue. trisomy 21. Males with trisomy 21 are usually subfer-
To complicate matters further, CPM resulting from tri- tile or sterile and rarely reproduce, although exceptions
somy rescue places the fetus at risk for UPD, a condition have been reported.116,117 Females with trisomy 21 are fer-
in which both homologues of a given chromosome are in- tile, but they also rarely reproduce. Therefore, data on the
herited from one parent. This risk of UPD arises because risk of trisomy 21 in offspring are limited. According to
the three chromosomes involved in the original trisomy are Harper,92 a risk of 1:3 is most likely.
not equivalent. Two of the three chromosomes are inher- A variety of structural chromosome rearrangements
ited from one parent, and one of the three chromosomes can affect reproductive outcome. Structural chromosome
790 PART III  Reproductive Technologies

Normal Translocation Normal

chromosome chromosomes chromosome

Figure 31-8.  Meiotic segregation

Pairing at
with a reciprocal translocation. Of six
meiosis
possible gametes after 2:2 segrega-
tion, two will be balanced (a and b)
and four will be unbalanced (c, d, e,
and f). After fertilization with a normal
haploid gamete, gamete a will produce
a chromosomally normal conceptus
and gamete b will produce a balanced Alternate Adjacent 1 Adjacent 2 2:2 segregation
carrier of the translocation.

a b c d e f
Gametes: Balanced Unbalanced Unbalanced
If fertilized: Normal translocation
carrier

r­ earrangements can be considered either balanced or unbal- the risk of both spontaneous abortions, which are presum-
anced. In balanced rearrangements, chromosomes contain ably chromosomally unbalanced, and male infertility.118-120
the normal complement of genetic information. In unbal-
anced rearrangements, genetic information is either dupli- STRUCTURAL CHROMOSOME
cated or missing. As with numeric abnormalities, unbalanced REARRANGEMENTS
rearrangements with significant reproductive concerns are Translocations
basically limited to sex chromosome rearrangements because
unbalanced autosomal rearrangements produce fairly severe Translocations (an exchange of chromatin between two or
phenotypes or are nonviable. Balanced chromosome rear- more chromosomes) are classified as reciprocal and rob-
rangements usually do not have a phenotypic effect because ertsonian. Reciprocal translocations, as the name implies,
essentially all of the genetic information is present, although involve presumably reciprocal exchange of a segment of
the rearrangement may occasionally disrupt a gene or result in one chromosome with a segment of another chromosome.
a cryptic duplication or deletion. Recent studies have shown Robertsonian translocations, the most common chro-
that a substantial number (reported to be as large as approxi- mosome rearrangements in humans, involve essentially
mately 40% of cases) of apparently balanced chromosomal whole-arm exchange of the acrocentric chromosomes.
rearrangements (as defined by G-banded karyotypic analy- More specifically, most robertsonian translocations involve
sis) actually have gains or losses at or near the breakpoints or two nonhomologous chromosomes and appear to result
elsewhere in the genome.31 However, even in the absence of from recombination between homologous DNA sequences
any associated phenotype, carriers of presumably balanced present in the proximal short arms of the acrocentric chro-
structural rearrangements can produce unbalanced gametes, mosomes.121-123 The resulting rearrangement is a dicen-
that is, gametes in which genetic information is duplicated or tric chromosome composed of the two long arms of the
deleted. Because of this, carriers of balanced rearrangements acrocentric chromosomes, with loss of most of the short
are at increased risk for having abnormal offspring with un- arm material. One of the two centromeres is inactivated,
balanced karyotypes. Balanced ­rearrangements also increase allowing the robertsonian fusion to be stable throughout
 CHAPTER 31  Cytogenetics in Reproduction 791

rob(14;21) 14 21
2:1 segregation
at meiosis
Figure 31-9.  Meiotic segregation in a robertsonian
t(14;21)(q10;q10) carrier. Of six possible gametes after
2:1 segregation, two (a and b) will be balanced and four
(c, d, e, and f) will be unbalanced. Of the unbalanced
gametes, only one (c) has the potential to result in a live-
born offspring.

Gamete: a b c d e f
Balanced Unbalanced Unbalanced

If fertilized: Normal rob(14;21) Trisomy 21 Monosomy 21 Trisomy 14 Monosomy 14

carrier

the cell cycle. Homologous robertsonian translocations is likely to include some, if not all, of the features of the
are much less common than nonhomologous robertsonian complete trisomy. Some translocations undergo 3:1 segre-
translocations and, in fact, are usually isochromosomes, gation more often than others; one common translocation,
cytogenetically indistinguishable from homologous trans- t(11;22)(q23;q22), frequently segregates in a 3:1 manner.
locations.124-127 Carriers of either homologous or non- Finally, for reasons that remain to be elucidated, female
homologous robertsonian translocations have a balanced carriers of chromosome rearrangements are more likely
karyotype containing 45 chromosomes. than male carriers to have unbalanced offspring.
Carriers of reciprocal translocations can produce both In carriers of robertsonian translocations, the risk of
balanced and unbalanced gametes. Because homologous having unbalanced offspring depends both on the specific
chromosome segments pair in meiosis, the two translo- translocation and on the sex of the carrier. Presumably,
cated chromosomes and the two normal chromosomes unbalanced gametes produced by carriers of robertsonian
form a quadrivalent figure (Fig. 31-8). If the chromo- translocations lead to conceptuses that are either mono-
somes segregate in such a way as to keep the two normal somic or trisomic for one of the acrocentric chromosomes
­chromosomes together and the two translocation chromo- involved in the translocation. However, in assessing the
somes together, the resulting gametes are either karyotypi- risk of unbalanced offspring, only viable outcomes need
cally normal or balanced. Of normal offspring, 50% will to be considered. For example, carriers of the most com-
be karyotypically normal and 50% will be translocation mon robertsonian translocation involving chromosome
carriers. On the other hand, if a normal chromosome seg- 21, rob(14;21), theoretically would produce, in equal pro-
regates with a translocation chromosome, the resulting portion, six types of gametes, two of which would be bal-
gametes will be unbalanced. Chromosomes from these anced. Of the other four gametes, two would be nullisomic
quadrivalent figures can also undergo abnormal 3:1 segre- for either chromosome 14 or 21, and two would be disomic
gation, which always produces unbalanced gametes. for one of these chromosomes. The two nullisomic gam-
In reciprocal translocation carriers, the risk of having an etes would lead to nonviable monosomy, whereas the two
unbalanced liveborn offspring depends on several factors. disomic gametes would lead to trisomy for either chromo-
Clearly, some unbalanced conceptuses are viable, whereas some 14 or 21. Because trisomy 14 is nonviable, only three
others are not. As a result, the risk of having a chromosom- of the gametes could develop into viable offspring, and the
ally abnormal offspring is different for carriers identified by risk of Down syndrome might be assumed to be approxi-
chance and for those identified through a previous unbal- mately 1:3 (Fig. 31-9). In actuality, empirical risks to carri-
anced liveborn offspring. In general, viability is more likely ers of rob(14;21) and other nonhomologous robertsonian
if the chromosome duplication or deletion is small, and translocations are much lower (Table 31-4). The risks of
duplicated material is usually more easily tolerated than unbalanced offspring are greater for female carriers of rob-
deleted material. If the imbalance is a subset of a known vi- ertsonian translocations than for male carriers.
able imbalance, for example, duplication of a chromosome The risk of an abnormal trisomic conceptus increases to
21 segment, viability is a possibility and the ­ phenotype virtually 100% when a translocation involves whole-arm
792 PART III  Reproductive Technologies

TABLE 31-4 an increased risk of abnormal gametes in the carrier. Two

Empiric Probabilities of Unbalanced types of inversions exist: paracentric inversions, in which
and Balanced Carrier Offspring for the inverted segment does not include the centromere, and
Robertsonian Carriers pericentric inversions, in which the inverted segment in-
cludes the centromere. These two types of inversions carry
Empiric Empiric different risks for chromosomally unbalanced offspring.
Probability Probability In both types of inversions, the risk of chromosome im-
(Risk) of of Balanced balance is the result of meiotic recombination within
Sex of Unbalanced Carrier the inverted segment. For a paracentric inversion, struc-
Translocation Carrier Offspring (%) Offspring (%) tural rearrangement resulting from recombination will
rob(13;14)* Female <1 50 lead to a dicentric chromosome and an acentric chromo-
Male Very low 50 some fragment. With rare exceptions, these recombinant
rob(13;13) Either sex 100 0 chromosomes are not stable and will not lead to viable
rob(14;21)* Female 10 50 offspring. Pericentric inversions are more problematic.
Male 2.5 50 Recombination can ­ produce monocentric chromosomes
rob(21;22) Female 6.8 50 with duplicated and deleted material. These recombinant
Male <2.9 50 chromosomes are stable and can be found in unbalanced
rob(21;21) Either sex 100 0 offspring. For two reasons, carriers of large pericentric in-
*The risks for rob(13;15) are probably similar to those for rob(13;14); versions are at higher risk for having unbalanced offspring
the risks for rob(13;21) and rob(15;21) are probably similar to those than are ­ carriers of small pericentric inversions. First, a
for rob(14;21). These translocations are much less common, so risk larger inverted chromosome segment is more likely than a
data are less extensive. small inverted segment to be involved in a recombination
Adapted from Therman E, Susman M. Human Chromosomes:
Structure, Behavior, and Effects, 3rd ed. New York, Springer-Verlag, event. Second, the resulting duplication and deletion will
1993, p 295. be smaller if the inverted segment is larger, and thus, the
offspring is more likely to be viable.
exchanges between two homologous acrocentric chro-
mosomes. Most of these conceptuses end in spontaneous Insertions
abortion. However, because trisomies 13 and 21 are viable,
carriers of rob(13;13) and rob(21;21) can have liveborn off- Balanced insertions, another type of rearrangement, rarely
spring with trisomy. Thus, for genetic counseling purposes, have a phenotypic effect on the carrier, but portend an
a carrier of a homologous robertsonian translocation will increased risk of abnormal gametes. If the two chromo-
have only spontaneous abortions or abnormal offspring somes involved in the insertion do not segregate together
with rare exceptions of trisomy rescue. in meiosis, gametes with duplication or deletion of the in-
Rare exceptions include offspring carrying the same ho- serted segment will result. Meiotic recombination can also
mologous robertsonian translocation without an additional produce recombinant chromosomes by crossing over be-
copy of the chromosome inherited from the other parent. tween a specific region of a rearranged chromosome with
These exceptions arise either from fertilization with a nul- the normal homologue. The risk of unbalanced liveborn
lisomic gamete or from a postzygotic loss of the normal, offspring depends on the viability of the duplication or
nontranslocated chromosome. These rare individuals, al- deletion.
though karyotypically identical to the phenotypically nor-
mal carrier parent, have UPD because both copies of the SEX CHROMOSOME ABNORMALITIES
chromosome have been inherited from the parent with the
robertsonian translocation. Some of these individuals are In humans, as in all mammals, the Y chromosome is the
phenotypically normal. Maternal UPD for chromosome 22 key determinant of sex. In the embryo, the human gonad
and paternal UPD for chromosome 21 appear to have no is undifferentiated until approximately 6 to 7 weeks of
unusual phenotype.124 However, UPD for chromosome development. The presence of a normal Y chromosome
15, whether maternally or paternally inherited, has severe causes the indifferent gonad to develop into a testis. Tes-
phenotypic consequences, resulting in either Prader-Willi tes produce two effectors responsible for subsequent male
syndrome or Angelman syndrome, respectively. sexual differentiation, testosterone and anti-müllerian
In addition to the risk of producing unbalanced gametes, hormone. In the absence of a Y chromosome, the male dif-
male carriers of balanced translocations are at increased risk ferentiation pathway is not initiated, the indifferent gonad
for infertility. The incidence of balanced autosomal translo- develops into an ovary, and female differentiation ensues.
cations is approximately sixfold higher in infertile men than The Y-encoded gene critical for testes development, the
in the general population,119 and translocations involving SRY gene, has been cloned and characterized,110-113,131-134
the X or Y chromosome frequently cause sterility.128-130 but the complete story of sexual differentiation remains
to be elucidated. Clearly, additional gene products are re-
quired for normal sexual differentiation in both males and
Inversions
females. This observation has been evident for a long time
Apparently balanced inversions, similar to apparently bal- on the basis of phenotypes of individuals with sex reversal
anced translocations, most often have no phenotypic ef- and individuals whose chromosomes differ from the nor-
fect. However, like translocations, they are associated with mal 46,XX or 46,XY.
 CHAPTER 31  Cytogenetics in Reproduction 793

PAR1 Figure 31-10.  Idiograms of G-banded human X

2.7 Mb Recurrent deletions
and Y chromosomes at the 450-band stage. Approxi-
associated with
mate locations of the two pseudoautosomal regions
azoospermia/oligospermia
(PAR1 and PAR2) are indicated on the X and Y chro-
X-inactivation
mosomes. On the X chromosome, the location of the
center (XIST) AZFa-0.8 Mb
SRY X-inactivation center that contains the XIST gene is
also indicated. On the Y chromosome, the locations
AZFa-0.8 Mb of the heterochromatic region (diagonally hatched
AZFb-3.2 Mb P5/proximal-P1-3.2 Mb lines), the azoospermic factors AZFa-c, and the tes-
AZFc-3.5 Mb
tis-determining gene SRY, and the locations of recur-
gr/gr-1.6 Mb rent deletions associated with either azoospermia or
AZFc-3.5 Mb oligospermia (black vertical bars) are indicated.
PAR2
0.33 Mb P5/distal-P1-7.6 Mb

X Y

The Y Chromosome
conditions and disturbed bone development.139-143 Of the
The structure of the Y chromosome has been studied ex- five genes mapped to PAR2, the two most proximal, SPRY3
tensively at both cytogenetic and molecular levels (Fig. and SYBL1, undergo X-inactivation.144
31-10). The Y chromosome is easy to distinguish cytogeneti­ Just proximal to PAR1 on Yp, the short arm of the Y
cally on the basis of its small size and its extraordinarily chromosome, is the SRY gene. In rare cases, recombination
bright fluorescence with quinacrine staining. This bright outside of PAR1 occurs and can produce XX males and XY
staining represents a heterochromatic region present in the females. XX males carry SRY on one of their X chromo-
distal segment of the long arm, which varies greatly in size somes, whereas XY females have a Y chromosome deleted
in the human population without any apparent phenotypic for SRY. DNA analysis of these sex-reversed individuals
effect. An essentially complete physical map of the Y chro- was instrumental in mapping SRY to an approximately
mosome was constructed in 1992 and estimated that the 140-kb segment of the Y chromosome.145 The SRY gene
chromosome contains 58 to 60 Mb DNA.3,135 More recent- was cloned several years later.131 Its identity was verified
ly, an almost complete sequence of the euchromatic region by molecular analysis of female 46,XY individuals with
of one Y chromosome was reported. This report shows the point mutations in SRY.146,147
euchromatic region to be approximately 23 Mb, including Between the two PAR regions is the bulk of the Y chro-
8 Mb on the short arm and 14.5 Mb on the long arm.136 mosome, termed the male-specific region, or MSY.136 An
Euchromatin in the Y chromosome is derived from several essentially complete sequence of the Y chromosome, with
million base pairs of DNA from autosomes, and represents discussion of its salient features, has been published.136
a majority of ­ noncoding genes and a minority of coding This region accounts for 95% of the chromosome and con-
gene families predominantly expressed in testis.137 Large tains a combination of heterochromatic sequences plus
palindromic repeats in this region maintained by intra- three categories of euchromatic sequences: X-degenerate,
chromosomal gene conversion lead to predictable patterns X-­transposed, and ampliconic. The X-degenerate se-
of DNA loss and spermatogenic failure.138 More than 100 quences reflect a common autosomal ancestor from which
Y-linked, testis-specific transcripts have been identified. the X and Y chromosomes have diverged, whereas the
Two regions on the Y chromosome can pair and re- X-­transposed sequences are the result of a more recent
combine with the X chromosome during male meiosis. transposition event that occurred after the divergence of
These two homologous regions are known as pseudoauto- humans and chimpanzees.136,148 The ampliconic sequences
somal regions because DNA sequences in these regions un- are composed of long repeat units with greater than 99.9%
dergo homologous recombination and hence do not show identity between repeat pairs, which is hypothesized to be
strictly sex-linked inheritance.53 These two regions, PAR1 maintained by gene conversion. The ampliconic sequences
and PAR2, are found at the tips of the short and long arms, contain 64 of the 83 MSY genes.
respectively. PAR1 is the major pseudoautosomal region The ampliconic and X-degenerate sequences represent
and the site of an obligate crossing over between X and most of the genes on the Y chromosome. Genes within
Y chromosomes during male meiosis. PAR2 is the minor the ampliconic sequences have testis-specific expression.
pseudoautosomal region. Crossing over in this chromo- Most are members of Y chromosome gene families and
some segment is neither necessary nor sufficient for suc- do not have homologous copies on the X chromosome.
cessful male meiosis. Genes within the X-degenerate sequences have homolo-
Genes within PAR1 have homologous copies on the X gous X chromosome copies, and most have ubiquitous tis-
chromosome and escape X-inactivation in females. The 24 sue expression. One notable exception is SRY, which has
PAR1 genes have no known sex-specific functions. SHOX, predominantly testis-specific expression.
located in PAR1, is the only known disease gene within The idea that genes involved in spermatogenesis and
PAR1 and PAR2 and plays a role in various short stature growth control are present in the euchromatic portion
794 PART III  Reproductive Technologies

of the long arm of chromosome Y was first hypothesized of Y material is important because these females are at
more than 30 years ago because of the association of cy- increased risk for gonadoblastoma.
togenetically visible Yq deletions with azoospermia and Structural aberrations of the Y chromosome with no
short stature.149-151 Seven recurrent deletions in Yq have phenotypic effect exist as well. Large portions of the het-
been defined, AZFa (azoospermic factor a), P5-proximal erochromatic region can be deleted without any pheno-
P1 (AZFb), P5-distal P1, AZFc (b2/b4), gr/gr, b1/b3, and typic effect, and large variations in the size of this ­region
b2/b3, and several are known to affect spermatogenesis.152 are common polymorphisms. Also fairly prevalent is a
Y chromosome microdeletions can lead to azoospermia or pericentric inversion that is present in approximately
severe oligospermia, with an estimated incidence of a min- 1:200 males and moves the centromere to the border of the
imum of 15% of cases of idiopathic male infertility.153-155 heterochromatic region. More rare (but still considered a
AZFc deletions are the most commonly diagnosed molec- normal variant) is the presence of satellites on the long
ular cause of spermatogenic failure and are associated with arm of the Y chromosome, resulting from a translocation
a variable phenotype. Furthermore, gr/gr deletions (some- between the Y chromosome and an acrocentric chromo-
times referred to as “DAZ1/DAZ2”—deleted in azoosper- some (usually chromosome 15).
mia), a subset of AZFc (b2/b4) deletions, have an even
more variable spermatogenic failure phenotype and have The X Chromosome
been associated with susceptibility to testicular germ cell
tumors.156 Diagnostic testing includes screening for dele- The structure of the X chromosome has also been studied
tions in AZFa, P5/P1, and AZFc, and in some cases, for extensively at the cytogenetic and molecular levels. The
gr/gr.152 There is extremely little chance of finding sperm X chromosome is estimated to be 185 cM165 and contains
during a testicular sperm extraction procedure in the pres- approximately 152 Mb of DNA.3,137 Approximately 1100
ence of either an AZFa or a P1/P5 deletion, whereas men genes are annotated on the X chromosome, and at least
with AZFc deletions may have sperm in their testis. 800 encode proteins.166
Genes within AZF regions have been identified and The presence of two X chromosomes in normal mam-
include USP9Y (ubiquitin-specific peptidase 9, Y-linked) malian females compared with a single X chromosome in
and DDX3Y (DEAD [Asp-Glu-Ala-Asp] box polypeptide 3, normal mammalian males demands a mechanism for dos-
Y-linked) in AZFa and RBMY (RNA-binding motif protein age compensation of X-linked genes. This is accomplished
on the Y chromosome) in AZFb. The DAZ genes, present through inactivation of essentially one X chromosome in
in a cluster of four copies within AZFc, are predicted to be females. The theory of X-inactivation167 was developed
RNA-binding proteins by DNA sequence153,157 and have from studies in cats in which condensed chromatin (Barr
been shown to have testis-limited expression.153 Although body) was detected in females, but not in males. Subse-
no point mutations have been identified within these quently, it was shown that humans with numeric sex chro-
genes, approximately 6% to 13% of men with oligospermia mosome abnormalities always had one fewer Barr body
or azoospermia have deletions of most or all of this gene than the number of X chromosomes, resulting in a single
cluster. No deletions have been seen in men with normal active X chromosome.
sperm counts.153,157-159 The process of X-inactivation is believed to involve
In both Drosophila and mice, loss of function of the three mechanisms: a counting mechanism that determines
DAZ gene homologues leads to infertility, further support- X chromosome number, a choice mechanism that ran-
ing a critical role for the human DAZ genes in spermato- domly selects an active and an inactive X chromosome,
genesis. Similar to the DAZ genes, RBMY is a member of a and a silencing mechanism that causes transcriptional in-
multicopy gene family.160,161 Its gene product localizes to activation of most of the genes along the inactive X chro-
the nucleus of spermatogenic cells, and deletions of this mosome. The silencing mechanism is further subdivided
gene have been documented in infertile males.162,163 A into three separate processes: initiation of inactivation,
point mutation in USP9Y, one of two genes located within spreading of inactivation along the length of the X chro-
AZFa, has been associated with spermatogenic failure.164 mosome, and lastly, maintenance of inactivation through-
Unlike DAZ and RBMY, which are members of multigene out subsequent cell divisions. Inactivation requires, in cis,
families residing in ampliconic sequences with testis- the X-inactivation center (XIC), which maps to chromo-
limited expression, USP9Y is a single-copy gene, with an some region Xq13.168,169 XIST, the critical gene within the
X-linked homologous gene and tissue-ubiquitous expres- X-inactivation center that initiates inactivation, encodes a
sion.136 functional, non–protein-encoding RNA that is transcribed
In addition to sterility, structural aberrations of the Y from the inactive X chromosome.170-174 This RNA coats
chromosome can have other phenotypic effects, including the inactive X chromosome, which also acquires other
intersex and females with symptoms of Turner syndrome. features associated with transcriptional inactivation (e.g.,
Correlations between specific structural aberrations and late replication, hypoacetylation, and hypermethylation).
phenotypes have been difficult to make because the ab- The product of XIST, although critical for initiation of
normal Y chromosome is frequently present in one of mul- X-­inactivation, does not appear to be required for main-
tiple cell lines within an individual. The second cell line tenance of inactivation.175 X chromosome inactivation is
may be normal male, normal female, or 45,X, or may have opposed by TSIX, an anti-sense transcript of XIST that reg-
an additional abnormal sex chromosome. Whenever the ulates XIST in cis and determines X chromosome choice
possibility of Y chromosome material in a phenotypic fe- without affecting silencing.176 Recently, it has been deter-
male exists, molecular testing to determine the presence mined that murine (and presumably human) Xist and Tsix
 CHAPTER 31  Cytogenetics in Reproduction 795

form duplexes in vivo that are processed to small RNAs, function, and both X-linked and non–X-linked factors
most likely on the active X chromosome in a Dicer-depen- are likely to contribute to premature ovarian failure. The
dent manner, implicating RNA interference (RNAi) in X most common cause of premature ovarian failure is an
chromosome inactivation.177 FMR1 premutation. Unlike fragile X syndrome, in which
X-inactivation occurs early in development and is con- full mutations of FMR1 result in the absence of FMR1
sidered random for maternal and paternal X ­chromosomes. protein, in cells with premutation alleles, FMR1 mes-
However, nonrandom X-inactivation is seen in females senger RNA levels are elevated and reduced translational
with structural abnormalities and with single-gene dis- efficiency of the premutated alleles results in decreased
orders of the X chromosome (e.g., Duchenne muscular FMR1 protein.182
dystrophy). In females carrying a structurally abnormal X X-autosome translocations have a variety of pheno-
chromosome, cells with the normal active X may survive types. They may occasionally disrupt an X-linked gene,
preferentially. Conversely, in females with balanced trans- resulting in X-linked recessive diseases both in males and
locations between an X chromosome and an autosome, the in females. Males manifest the recessive disease because
normal X chromosome may appear to be inactivated pref- they are hemizygous for the X chromosome. Females may
erentially. Presumably, this reflects survival of cells with be affected because of preferential inactivation of the nor-
active autosomal material because inactivation can spread mal X chromosome. X-autosome translocations may also
into autosomal material in X-autosome rearrangements. impair fertility in both sexes, and as with any transloca-
Once X-inactivation occurs, it appears to be irreversible, tion, they may segregate improperly and result in unbal-
except in oogenesis, during which time reactivation of anced offspring. Individuals with translocations between
the inactive X chromosome occurs at some point before an X and a Y chromosome can be phenotypically female or
­meiosis. male, depending on the breakpoints of the translocation,
Inactivation does not abrogate transcription of all genes the pattern of X-inactivation, and the remaining chromo-
on the X chromosome. The first genes discovered to es- some constitution.
cape X-inactivation were located within PAR1, and it was
once believed that all genes escaping inactivation would
Numeric Abnormalities of Sex Chromosomes
be located in this region, thus maintaining equal dosage
between males and females for genes with functional cop- Numeric abnormalities of sex chromosomes are much more
ies on the Y chromosome. Now genes escaping inactiva- prevalent than numeric abnormalities of autosomes, with an
tion have been identified throughout the X chromosome. overall frequency of 1:500 births. Individuals with as many
Some of these genes have functional copies on the Y chro- as five sex chromosomes (penta-X syndrome) have been
mosome outside the pseudoautosomal regions (e.g., ZFX identified, but the most common sex chromosome abnor-
and RPS4X), and thus, as with genes in the pseudoauto- malities are the trisomies XXX, XXY, and XYY. The viability
somal region, equal dosage between males and females is of those affected by these abnormalities is good, as evidenced
maintained by the absence of X-inactivation in females. by the fairly high frequency among liveborn infants and the
However, some of the genes escaping X-inactivation do low frequency among spontaneous abortions. Conversely,
not have homologues on the Y chromosome (e.g., UBA1 monosomy for the X chromosome is significantly less com-
and SMC1A). These genes may have higher expression in mon among liveborn infants, but is the most frequent chro-
females than in males. Most likely, the activity of genes mosomal abnormality in spontaneous abortions.39,74 For
escaping X-inactivation leads to some of the abnormali- all sex chromosome abnormalities, mosaicism with normal
ties seen in individuals with an abnormal number of and abnormal cell lines is often observed.
X chromosomes.178 The mechanism whereby specific
genes escape X-inactivation remains enigmatic and is an Turner Syndrome and Variants.  Turner syndrome is
area of intense research. widely known as 45,X, although approximately 50% of
Numerous aberrations of the X chromosome have been individuals with Turner syndrome have a variation of
identified and used to define regions of the chromosome. this karyotype (see Chapters 16 and 17). Approximately
Just distal to XIC is the “critical region” that appears to 15% of patients have one normal X chromosome and one
be required on both X chromosomes for normal ovarian structurally aberrant X chromosome. These structural
function.179 Although many balanced rearrangements of abnormalities of the X chromosome include deletions of
the X chromosome have no phenotype, this critical region portions of the short and long arms as well as isochromo-
must remain intact for normal female sexual development. somes. Approximately 25% to 30% of patients are mosaic,
In addition to this critical region, specific ovarian mainte- with one 45,X cell line and a second cell line that might
nance determinants may exist on both arms of the X chro- contain, among others, either two normal X chromosomes
mosome because a variety of deletions in either the short (i.e., 45,X/46,XX), one normal and one abnormal X chro-
or the long arm are associated with ovarian dysgenesis in mosome (i.e., 45,X/46,X,i[Xq]), or one X and one Y chro-
Turner syndrome. However, it is also possible that the ovar- mosome (i.e., 45,X/46,XY).
ian dysgenesis seen with deletions or translocations of the In apparent nonmosaic 45,X individuals, the single X
X chromosome is caused, not by deletion of specific criti- chromosome is maternal in origin 80% of the time. In other
cal genes, but by an alteration of chromosome structure words, the meiotic error is usually paternal.183,184 The ba-
or dynamics, perhaps affecting the ability of the X chro- sis for the unusually high frequency of X or Y chromosome
mosomes to pair completely during meiosis.180,181 Sev- nondisjunction in paternal meiosis is unknown, but may
eral genes along the X chromosome contribute to ovarian relate to limited meiotic recombination between the X and Y
796 PART III  Reproductive Technologies

chromosomes. Of note, the X–Y bivalent is estimated to material. As such, molecular studies for the detection of Y
have the highest rate of nondisjunction in paternal meiosis chromosomal DNA should be considered. In addition, rare
I among all chromosome pairs.185 Whether paternal age is a patients with features of Turner syndrome are determined
risk factor for Turner syndrome is unclear. Advanced mat­ to have a 46,XY karyotype missing a portion of the Y chro-
ernal age, however, is not correlated with this disorder. An mosome.187 These individuals also have an increased risk
alternative mechanism is postfertilization loss of either the of gonadoblastoma.
X or the Y chromosome, based on the high frequency of Mosaicism for 45,X and a second cell line is not uncom-
45,X (1% to 2% of clinically recognized pregnancies) com- mon. In addition, 45,X/46,XX mosaicism is the most com-
pared with the reciprocal products of 47,XXY, 47,XYY, and mon sex chromosome mosaicism diagnosed from genetic
47,XXX. Random loss of an X or a Y chromosome in a nor- amniocentesis. Of 114 cases reviewed,89 approximately
mal XX or XY embryo would result in an excess of 45,X of 10% had some features of Turner syndrome and approxi-
paternal origin and the 45,Y embryo would not be viable.185 mately 4% had other anomalies. Although the majority
A number of phenotypic abnormalities are character- of these cases were phenotypically normal at either birth
istic of Turner syndrome. The most common findings are or termination, many of the features of Turner syndrome
short stature (<5 feet or 150 cm) and gonadal dysgenesis might not be recognized until puberty, and prenatal coun-
(usually streak gonads). Fetal cystic hygroma, resulting seling remains difficult. For 45,X/46,XY individuals, the
from lymphedema and leading to postnatal webbed neck, phenotype ranges from normal fertile males to individu-
is also common. Other associated anomalies include low als with ambiguous genitalia to females with Turner syn-
posterior hairline, shield chest with widely spaced nipples, drome. Presumably, different phenotypes reflect different
cubitus valgus, cardiac anomalies (frequently coarctation tissue distributions of the various cell lines. In a compila-
of the aorta), and renal anomalies. Although mental re- tion of prenatally diagnosed cases,89 90% to 95% of cases of
tardation is not more common in individuals with Turner 45,X/46,XY resulted in a normal male phenotype. When a
syndrome than in females with two X chromosomes, karyotype of 45,X/46,XY is detected at prenatal diagnosis,
­deficiencies in spatial perception, perceptual motor orga­ detailed ultrasonography of the genitalia is recommended.
nization, and fine motor skills are more frequent. Turner If the genitalia appear to be normal male, the prognosis
syndrome is fully compatible with life. It is therefore puz- is fairly reliable for a phenotypically normal male. If the
zling why 45,X is usually lethal in utero. Furthermore, genitalia appear to be female, Turner syndrome or ambigu-
45,X is the most common chromosomal abnormality in ous genitalia are more likely to be present at birth.
spontaneous abortions, with more than 99% of 45,X con- In normal females and males, loss of a sex chromosome
ceptuses being spontaneously aborted.74 in peripheral blood cells can occur, leading to apparent
Another phenotype of Turner syndrome is infertility, re- mosaicism for 45,X. Although this type of X chromo-
sulting from increased germ cell attrition. In the absence some aneuploidy was once thought to be more frequent
of a Y chromosome, the gonad develops into an ovary in in women with multiple pregnancy losses, it is now recog-
which germ cells are initially present. However, germ cells nized that 45,X aneuploidy in peripheral blood increases
rapidly degenerate during the fetal period and the result- with age and that this age-related phenomenon is not as-
ing lack of oocytes leads to streak gonads and amenorrhea. sociated with reproductive loss.188,189
Most patients carrying deletions of either the short or the
long arm of the X chromosome have ovarian dysgenesis.179 47,XXX.  Individuals with three X chromosomes, or tri-
Thus, two complete X chromosomes are necessary for nor- ple X syndrome, appear physically normal, although by
mal ovarian development and function. adolescence, many are taller than average. Despite the ex-
Obtaining the karyotype of individuals with Turner pectation that 50% of offspring would carry an extra X
syndrome is clinically important, because although many chromosome, the actual empirical risk to offspring is low.
of the distinct symptoms of Turner syndrome seem to be Prospective studies with long-term follow-up of 47,XXX
randomly distributed with respect to different deletions individuals indicate that although mental retardation is
throughout the X chromosome, some correlations with not present, the IQ of many of these patients is 10 to 15
phenotype can be made.180 Most individuals with break- points below that of their siblings. Language delay, learn-
points distal to Xq25 have few abnormalities except oc- ing disabilities, and impaired gross motor skills are also
casional secondary amenorrhea or premature menopause. common, as are psychosocial disorders, particularly de-
Short stature is almost always associated with deletions pression.190-193
of the distal portion of the short arm, and less often with In females with three X chromosomes, approximately
long arm deletions. Determination of the presence of Y 90% have two maternal X chromosomes and 10% have
chromosomal material is of critical medical importance two paternal X chromosomes. When there are two mater-
because it leads to an increased risk of gonadoblastoma in nal X chromosomes, 66% of cases are due to meiosis I er-
sex-reversed individuals, as mentioned earlier. Routine cy- rors, 18% are due to meiosis II errors, and 16% are due to
togenetic testing for individuals with a suspected sex chro- postzygotic errors. In two of the informative cases describ-
mosome disorder often includes a count of 30 metaphase ing two paternally derived X chromosomes, both were due
preparations, which rules out greater than 10% mosaicism to postzygotic errors.183 Increased maternal age is a risk
at a 95% confidence level.186 factor for 47,XXX.
Even with this stringent level of classic cytogenetic
workup, some individuals with a nonmosaic 45,X karyo- Klinefelter Syndrome/47,XXY.  Males with the 47,XXY
type may have an undetected cell line with Y chromosomal karyotype have a fairly well-defined phenotype known
 CHAPTER 31  Cytogenetics in Reproduction 797

as Klinefelter syndrome. They are tall and thin, with long in a temporal and tissue-specific manner, indicating
legs (see Chapters 16 and 17). Physical appearance is fairly interaction of upstream regulatory genes, such as GATA4,
normal until puberty, when a characteristic eunuchoid FOG2, and WT1.131,194 Although no direct target gene for
habitus presentation develops. Secondary sexual charac- SRY has been conclusively identified, indirect observa-
teristics are underdeveloped and testes remain small, with tions suggest that SOX9 (SRY HMG box–related gene 9)
azoospermia and subsequent ­ infertility. Gynecomastia is one molecular target. The SRY gene product binds
may occur. IQ is reduced in this patient population, and DNA sequences in vitro, ­ indicating that the SRY pro-
two thirds of patients have educational problems, particu- tein exerts its effect in vivo by regulating transcription
larly dyslexia. of other genes.131 Loss-of-function mutations in SRY ac-
Approximately 50% of 47,XXY individuals have two count for approximately 10% to 15% of XY sex reversal
paternally derived sex chromosomes, and approximately through gonadal dysgenesis after birth.195
50% have two maternally inherited X chromosomes.77,183 The existence of several chromosome abnormalities as-
In all cases, when there are two paternally derived sex sociated with sex reversal has played an integral role in the
chromosomes (i.e., one X and one Y chromosome), the identification of additional genes involved in sex determi-
error must have occurred in meiosis I. When there are two nation. Both deletions of 9p24 and duplications of Xp21
maternal sex chromosomes (i.e., two X chromosomes), have been associated with XY sex reversal.196,197 DMRT1
approximately 70% of cases are due to a meiosis I error, has emerged as a likely gene within 9p24 to be involved
25% to a meiosis II error, and 5% to postzygotic nondis- in male sex determination.198,199 DMRT1 contains a zinc
junction.183 Increased maternal age appears to be associ- finger DNA-binding domain (termed “DM”) that is also
ated only with maternal meiosis I errors. present in two genes involved in invertebrate sex deter-
mination, the Drosophila doublesex (dsx) gene and the
47,XYY.  The most consistent phenotype in 47,XYY indi- Caenorhabditis elegans mab-3 gene. In the mouse, DMRT1
viduals is increased height. Increased risk of behavioral was also shown to be required for testis formation.200,201
problems and perhaps some decrease in intelligence may Within Xp21, NROB1 was identified as encoding another
be associated with this karyotype. Fertility is normal, and critical component of the male sex determination pathway.
these individuals are not found to be at increased risk NROB1, an orphan nuclear hormone receptor, exerts an
for having a chromosomally abnormal child. All 47,XYY antagonistic effect in the male sex determination pathway.
cases result from either a paternal meiosis II error or a Overexpression of the gene prevents testes development,
postzygotic ­error. despite the presence of an intact SRY gene.197,202 Because
duplications of NROB1 lead to XY sex reversal, whereas
Sex Chromosome Tetrasomy and Pentasomy.  Although supernumerary X chromosomes do not, NROB1 is presum-
studies of the phenotype of sex chromosome tetrasomy ably subject to X-inactivation.
and pentasomy do not exist from unbiased ascertainment Other transcription factors implicated in male sex de-
(i.e., all information is based on case reports of postna- termination include SOX9, SF1 (steroidogenic factor-1),
tally identified individuals), it is generally assumed that WT1 (Wilms tumor 1), and GATA4 (a zinc finger tran-
with an increasing number of supernumerary sex chro- scription factor). SOX9 was first mapped to 17q24-q25
mosomes comes an increasingly severe phenotype. Super- in patients with apparently balanced translocations and
numerary X chromosomes are clinically more severe than campomelic dysplasia.203 A portion of the SOX9 gene has
supernumerary Y chromosomes, presumably because the significant homology to the DNA-binding domain (high-
Y chromosome encodes so few genes. Even though su- mobility group box) of SRY and has been shown to bind to
pernumerary X chromosomes are inactivated essentially, the same DNA sequences in vitro.204 SOX9 is likely to be a
some genes escape X-inactivation, and increased dosage critical component of sex determination in all vertebrates.
of these gene products presumably leads to the clinical In mammals, SRY may act directly through SOX9.205
phenotype. Mental capacity is increasingly diminished Any phenotypic female with the SRY gene is suscep-
with each supernumerary X chromosome, with an es- tible to gonadoblastoma. At risk are not only patients with
timated decrease of 15 IQ points for each additional X Turner syndrome, but also XY individuals with gonadal
chromosome. In males, supernumerary X chromosomes dysgenesis or an androgen insensitivity syndrome. As
lead not only to infertility but also to malformed genitalia. more of the genes involved in sex determination are iden-
The effect on fertility in females with supernumerary X tified, it will remain important to identify all phenotypic
chromosomes is unclear. Fewer cases of supernumerary Y females carrying the SRY gene.
chromosomes have been reported, but additional Y chro-
mosomes also appear to cause decreased mental capacity,
although the effect is less pronounced than with supernu- Preimplantation Genetic
merary X chromosomes (reviewed by Linden et al.192). ­Diagnosis
Over the last decade, multicolor FISH has contributed to
Chromosome Abnormalities and Sex Reversal
the field of preimplantation genetic diagnosis ([PGD] see
Although the testis-determining factor on the Y chromo- Chapter 30). By combining assisted reproductive tech-
some has been identified for almost two decades, eluci- nologies with genetic analysis of single cells, PGD allows
dation of the human sex determination pathway is far the screening of pre-embryos before their transfer, with
from complete (see Chapter 16). SRY itself is expressed the goal of transferring only those embryos that would not
798 PART III  Reproductive Technologies

develop into an individual with a specific genetic disease these patient populations, it is controversial whether this
or condition. However, because the genetic analyses of type of aneuploidy screen actually increases the likelihood
PGD are performed on only one or two cells per embryo, of a successful pregnancy (reviewed by Handyside and
it is critical to view these tests as screening tests only. Ogilvie218). Recent guidelines from the British Fertility So-
To obtain material for genetic analysis, most centers ciety show no robust evidence that the use of preimplanta-
performing PGD employ embryo biopsy.206 Blastomeres tion genetic screening for advanced maternal age improves
removed from pre-embryos are tested for specific gene de- the live birth rate.219
fects using single-cell PCR, followed by a variety of molec-
ular diagnostic techniques or FISH screening for specific The complete reference list can be found on the companion Expert
chromosomal aberrations. PGD with FISH has been used Consult Web site at www.expertconsultbook.com.
in the following three ways: sex identification for carriers
of X-linked recessive disorders207,208; chromosome imbal- Cytogenetics Web Resources
ance screening for couples in which one member is a car-
rier of an apparently balanced chromosome rearrangement www.biologia.uniba.it/rmc/
This site from the Cytogenetics Unit at the University of Bari, Italy, provides
(e.g., chromosome translocation carriers)209-211; and aneu- information on probes, protocols, and chromosome idiograms.
ploidy screening for patients with advanced maternal age,
www.bwhpathology.org/dgap
a history of multiple spontaneous abortions, or repeated in This site represents the Developmental Genome Anatomy Project (DGAP)
vitro fertilization failures.212-215 at Harvard University. The DGAP posts balanced chromosome rearrange-
Among these three applications of FISH, identification ments in individuals with congenital anomalies with the goal of identifying
of presumptive fetal sex is the most straightforward because genes critical to human development that are disrupted or dysregulated at
it requires analysis of only two chromosomal regions, one ­ the breakpoints.
Y-specific region and one X-specific region. Commercial www.hcforum.net
probes producing bright hybridization signals are available The Human Cytogenetics Forum provides a database of chromosome rear-
rangements and their references. Chromosomal rearrangements can be
for both the X and Y chromosomes in a variety of colors. With queried for their previous description and for their potential to result in
just two probes labeled in different colors, hybridization pat- liveborn infants, based on their genetic content, as assessed by R-banding.
terns for normal male, normal female, and sex chromosome This site provides a useful resource for genetic counseling for couples with
aneuploidy can be distinguished. FISH analysis is the method chromosomal rearrangements.
of choice for sex identification because 45,X and 47,XXY em- www.mcndb.org
bryos have been misdiagnosed as normal by PCR.216 This site represents a network of cytogenetic laboratories, Mendelian
When the indication for PGD is the presence of a bal- Cytogenetics Network (MCN), for the systematic identification and mapping
of disease-­associated balanced chromosome rearrangements (DBCRs) that
anced chromosomal rearrangement in one member of a truncate or inactivate specific genes. The online database, MCNdb, serves as
couple, testing is complicated by the need to distinguish a resource for genotype–phenotype delineation in humans.
the two balanced chromosomal configurations (balanced
www.molgen.mpg.de/~cytogen
carrier and normal noncarrier) from the numerous pos- This site at the Max-Planck-Institute for Molecular Genetics includes the
sible unbalanced segregation products. Probe strategies YAC/BAC FISH mapping resource for the Mendelian Cytogenetics Network
that detect all possible unbalanced products may not be as (MCN Reference Center).
good as strategies that miss some of the unbalanced prod-
ucts, especially if the undetected products are unlikely to Suggested Readings
occur or unlikely to be viable. The addition of extra probes
required to detect all possible segregation products will re- Anderson RA, Pickering S. The current status of preimplantation genetic
screening: British Fertility Society Policy and Practice Guidelines.
duce the overall efficiency of hybridization and increase Hum Fertil (Camb) 11(2):71-75, 2008.
the likelihood of visualizing overlap of the probe signals, Blaschke RJ, Rappold G. The pseudoautosomal regions, SHOX and dis-
thus reducing the diagnostic accuracy of the test.217 For ease. Curr Opin Genet Dev 16(3):233-239, 2006.
this reason, when devising the detection strategy, it is im- Camerino G, Parma P, Radi O, Valentini S. Sex determination and sex
reversal. Curr Opin Genet Dev 16(3):289-292, 2006.
portant to balance the risks of occurrence and potential Disteche CM. Escape from X inactivation in human and mouse. Trends
viability for any specific abnormal segregation product Genet 11:17-22, 1995.
against the decreased accuracy created by the addition of Hall H, Hunt P, Hassold T. Meiosis and sex chromosome aneuploidy:
any probe. Similarly, the use of each additional color can how meiotic errors cause aneuploidy; how aneuploidy causes meiotic
increase the information provided (e.g., discriminate be- errors. Curr Opin Genet Dev 16(3):323-329, 2006.
Higgins AW, Alkuraya FS, Bosco AF, et al. Characterization of appar-
tween a balanced carrier and a normal noncarrier), but si- ently balanced chromosomal rearrangements from the developmental
multaneously can increase the difficulty of interpretation. genome anatomy project. Am J Hum Genet 82(3):712-722, 2008.
A limited number of centers have offered preimplan- Iafrate AJ, Feuk L, Rivera MN, et al. Detection of large-scale variation in
tation genetic screening for aneuploidy for indications of the human genome. Nat Genet 36(9):949-951, 2004.
advanced maternal age, history of multiple in vitro fertil- Kallioniemi A, Kallioniemi OP, Sudar D, et al. Comparative genomic
hybridization for molecular cytogenetic analysis of solid tumors.
ization failures, and repeat spontaneous abortions.212-215 Science 258(5083):818-821, 1992.
With the commercial availability of probe sets designed to Lee C, Iafrate AJ, Brothman AR. Copy number variations and clinical
enumerate five chromosomes simultaneously (either 13, cytogenetic diagnosis of constitutional disorders. Nat Genet 39(7
18, 21, X, and Y or 13, 16, 18, 21, and 22), additional Suppl):S48-S54, 2007.
Ogawa Y, Sun BK, Lee JT. Intersection of the RNA interference and
laboratories may begin to offer preimplantation genetic X-inactivation pathways. Science 320(5881):1336-1341, 2008.
diagnosis–based aneuploidy screening. Although the data Ross MT, Bentley DR, Tyler-Smith C. The sequences of the human sex
support a high rate of aneuploidy in the embryos from chromosomes. Curr Opin Genet Dev 16(3):213-218, 2006.
 CHAPTER 31  Cytogenetics in Reproduction 799

Schreck RR, Disteche CM. Chromosome banding techniques. Curr Shaffer LG, Agan N, Goldberg JD, et al. American College of Medical
Protoc Hum Genet Chapter 4:Unit 4.2, 2001. Genetics statement of diagnostic testing for uniparental disomy. Genet
Schreck RR, Warburton D, Miller OJ, et al. Chromosome structure as Med 3(3):206-211, 2001.
revealed by a combined chemical and immunochemical procedure. Shaffer LG, Tommerup N (eds). ISCN 2005: An International System for
Proc Natl Acad Sci U S A 70(3):804-807, 1973. Human Cytogenetic Nomenclature. Basel, S. Karger, 2005.