Preimplantation genetic diagnosis: Polar body biopsy
Markus Montag, Department of Gynecological Endocrinology & Reproductive Medicine, University Clinics of Bonn, Germany
Abstract
One technique used in preimplantation genetic diagnosis (PGD) is polar body (PB) biopsy. The first and second
polar body of the oocyte are extruded at the conclusion of the meiotic division; normally the first polar body is
noted after ovulation, and the second 2 - 3 h after sperm entry into the oocyte. The extraction of the first and second
polar body is done 6 - 12 hours after intracytoplasmic sperm injection (ICSI) has been carried out. The biopsy
of the polar bodies is followed by a detection of certain chromosomes using fluorescence in-situ hybridisation
(FISH) or detection of all chromosomes by comparative genomic hybridization. Polar body biopsy has been used
for diagnosing translocations and monogenic disorders of maternal origin.
Introduction
Over the past few decades the mean age of women conceiving
their first child has steadily increased. However,
advanced maternal age lowers the chance for pregnancy
and increases the risk of miscarriage once a woman is
pregnant. One major problem strongly correlated to maternal
age is the occurrence of numerical chromosomal abnormalities
in human oocytes. In women who are 40 years and
older, up to 70% of their oocytes can be chromosomally
abnormal [1]. In the context of assisted reproduction treatment,
it is possible to identify and exclude such oocytes
thereby increasing the success rates. The underlying
technique is the biopsy of the first and second polar body
(Figure 1), followed by a detection of certain chromosomes
using fluorescence in-situ hybridization (FISH) or detection
of all chromosomes by comparative genomic hybridization
(CGH). To understand these techniques it is important
to know some aspects of the fertilization and subsequent
early embryo development. The cell division that results
in sperm and eggs containing 23 chromosomes each is
called meiosis. In their resting state, eggs exist in a state
of arrested meiosis and still contain all 23 paired chromosomes.
During ovulation, meiosis resumes, and the
egg extrudes one set of its 23 chromosomes in a small
structure called the first polar body. Soon after fertilization
occurs, a second polar body, containing 23 maternal
chromatides, is expelled. On the day after oocyte retrieval,
both polar bodies can be seen in the normally fertilized
egg under the microscope. Polar bodies with missing chromosomal
material is indicative of an oocyte that contains
an excess chromosome, which, after fertilization, results
in an embryo with a trisomy. Vice versa, excess chromosomal
material in the polar bodies indicates that an oocyte
is missing chromosomes which, after fertilization, results
in an embryo with a monosomy. Thus, polar body biopsy
provides an indirect diagnosis of the oocyte for aneuploidy
testing of up to 10 chromosomes. This method can also
be applied to couples with a balanced translocation of the
mother and to couples who are aware of maternal predisposition
for a genetic disease that may manifest itself in the
child.
Polar body biopsy was first presented in 1990 [2], and
several aspects of this technique have been technically
refined including the use of lasers to facilitate the biopsy
procedure [3].
Materials and methods
Compared to ICSI, polar body biopsy requires additional
manipulation steps; thus it greatly benefits from the proper
instrumentation (Figure 2) which makes the procedure as
economically as possible. This helps reducing exposure
time of the oocytes outside the incubator, and it minimizes
the risk of loosing the material of interest.
Devices for polar body biopsy
• Inverted microscope with a heated plate and Hoffmann
contrast objectives
• Laser system (Octax, MTG)
• 2 TransferMan NK2 micromanipulators (Eppendorf)
• CellTram Air microinjector for holding the embryo (Eppendorf)
• CellTram vario microinjector for removal and transfer of
the polar body (Eppendorf)
• CustomTip capillary especially designed for polar body
biopsy (Polar body tip MM or MML, Eppendorf)
Polar body biopsy
The most important feature of TransferMan NK2 for polar
body biopsy is its ability to store several free definable capillary
positions. Polar body biopsy is done in a culture dish,
then a biopsy capillary is used to transfer the polar bodies
directly into a drop of water on a glass slide. This technique
requires three user-defined capillary positions (Figure 3, 6):
position 1B for biopsy, position 2B for easily replacing the
culture dish with a glass slide, and position 3B for releasing
the polar bodies into the water droplets on the slide.
Additionally, two positions are required for the holding
capillary: position 1H for holding the oocyte for biopsy and
position 2H for the changing of dishes. Once stored, these
positions can be automatically activated by pressing the
relevant position button on the device.
This set-up enables a fast and economic change from one
capillary working position to another, but most importantly,
it controls the capillary that holds the aspirated polar bodies,
helping to reduce the rate of lost polar bodies to less
than 0.5 %. An overview of the different positions required
during the biopsy procedure is shown in Figure 3.
For polar body biopsy we recommend setting up a dish with
a single droplet that contains PVP and two rows of droplets,
3 - 5 ìl each, that contain buffered culture medium. The
buffered culture medium is used to maintain the proper pH
during manipulation outside the incubator. The PVP is used
for rinsing the biopsy capillary, which helps to avoid polar
bodies sticking to the inner glass wall. The left row of droplets
is used for the oocytes (one per droplet) and the right
row for sampling of the polar bodies after biopsy. For biopsy,
an oocyte is gently aspirated by the holding capillary and
affixed as closely as possible to the bottom of the dish. This
position is stored as position 1H. To get a proper alignment
of the first and second polar body in one focal plane rotating
the oocyte may be necessary (Figure 4).
This focal plane defines position 1B for the biopsy capillary,
and, once adjusted, this position should also be stored.
Following laser-assisted opening of the zona pellucida, the
biopsy capillary is pushed through the opening of the zona
towards the polar body. While doing this, a slight suction is
usually helpful. By rotating the knob of CellTram vario both
polar bodies are slowly aspirated into the capillary (Figure 5).
Once both polar bodies are completely aspirated, the capillary
is removed from the zona and the oocyte is released
from the holding capillary. If several oocytes need to be
biopsied, the first and second polar body can be temporarily
stored in the neighbouring medium droplet while
the biopsy of the next oocyte is performed as previously
described. Once all polar bodies are biopsied, it is advisable
to first place the oocytes back into the incubator. To
do so, both capillaries are brought into a position above the
culture dish (positions 2H/B), and the dish can be removed
so that the oocytes may be transferred into another culture
dish.
Transfer of polar bodies
The dish still holding the polar bodies is placed back on
the microscope stage, and the biopsy capillary is lowered
automatically into position 1B by pressing Pos1. The polar
bodies corresponding to oocyte 1 are then aspirated into
the biopsy capillary. Next, the capillary is moved to position
2B (by pressing Position 2), the culture dish is removed,
and a glass slide holding a 0.2 µl droplet of pure water is
placed under the capillary. The biopsy capillary still holding
the polar bodies is lowered into the water droplet so that
it just touches the glass surface. This position is stored as
position 3B (Figure 6).
The first and second polar body are carefully released into
the droplet, and the capillary is first drawn back and then
brought into position 2B by pressing Pos 2. The small volume
ensures that the polar body will attach to a small area
on the slide and the fluid will dry fast, thereby reducing
the risk of a dislocation on the slide. Even so, the drying
process must be observed under a stereo microscope, and
the final location of the polar body after air-drying must be
circled on top of the slide by using a diamond marker. This
procedure can be repeated until all polar bodies have been
transferred to the slide. With some experience, 4 to 6 polar
bodies can be placed within an approx. 10 mm area, each
encircled using a diamond marker [4]. Because all relevant
capillary positions have been stored during the first round,
further manipulation of polar bodies is less time consuming.
Fluoresence in-situ hybridisation (FISH)
For FISH analysis, the dried polar bodies are fixed by adding
2 x 10 µl ice-cold methanol: acetic acid (3:1), followed
by incubation in methanol at room temperature for another
5 min. The slides are dried, and the FISH probe for chromosomal
detection is directly applied to the slide, which
is covered by a cover slip and sealed with rubber cement.
The slide is placed into an Eppendorf Thermomixer comfort
with exchangeable thermoblock for slides. Co-denaturation
of the probe and the genomic DNA, as well as subsequent
hybridization, is performed with the Thermomixer at the
time and temperature indicated by the manufacturer of the
chromosome probe. Following hybridization, unbound probe
is washed off, and the FISH signals can be evaluated using
a fluorescence light microscope equipped with appropriate
filter sets (Figure 7).
Results and discussion
Each chromosome should show two signals in the 1st polar
body and one signal in the 2nd polar body.
By analysing the 1st polar body chromosomal disorders
which happened during meiosis I can be detected. Approx.
70 % chromosomal disorders are found in the 1st polar
body; however, a disorder may also occur in the formation
of the 2nd polar body during meiosis II. Therefore 30% of
the chromosomal disorders can only be detected by analyzing
both polar bodies.
A frequent problem when judging FISH results is the occurrence
of chromatin degeneration which can be detected
by speckled signals. Interestingly, this phenomenon
occurs most often when using LSI (locus-specific identifier)
probes. Nevertheless, it is still possible to draw conclusions
about the respective chromosomes, since early
segregation of chromatides means that the regions with
speckled signals are also separated.
The main problem with polar body biopsy is the fragmentation
of polar bodies, particularly the first polar body.
Because each fragment can contain chromosomes, it is
crucial that all fragments be removed during biopsy. In this
case identifying the number and location of the fragments
is critical, because they can disconnect while drying and
move to different areas of the slide. Therefore it is absolutely
necessary to compile a drawing. Otherwise the risk
is quite high that signals in small fragments may be disregarded
which can cause a false diagnosis.
Depending on the results of the FISH analysis, chromosomally
normal oocytes can be selected for further culture
and transfer.
Corresponding author
Markus Montag, Ph.D.
Department of Gynecological Endocrinology &
Reproductive Medicine
University Clinics of Bonn, Germany,
Sigmund-Freud-Str. 25, D-53105 Bonn
Phone: +49 228 287 15449; Fax: +49 228 287 14651
e-mail: markus.montag@ukb.uni-bonn.de
References
[1] Hassold T, Chiu D. Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Hum
Genet 1985;70(1):11-7.
[2] Verlinsky Y, Ginsberg N, Lifchez A, Valle J, Moise J, Strom CM. Analysis of the first polar body: preconception genetic diagno
sis. Hum Reprod 1990 Oct;5(7):826-9.
[3] Montag M, van d, V, Delacretaz G, Rink K, van d, V. Laser-assisted microdissection of the zona pellucida facilitates polar body
biopsy. Fertil Steril 1998 Mar;69(3):539-42.
[4] Montag M, van der Ven K, van der Ven H. Polar body biopsy. In: Gardner, Weissmann, Howles, Shoham, editors. Textbook of
Assisted Reproductive Techniques: Laboratory and Clinical Perspectives.Lancaster: Taylor & Francis Medical Text Books; 2004.
p. 391-404.
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