Download Analysis of the first polar body: preconception genetic

Human Reproduction vol.5 no.7 pp.826-829, 1990
Analysis of the first polar body: preconception genetic
diagnosis
Yury Verlinsky1, Norman Ginsberg2,
Aaron Lifchez2, Jorge Valle2, Jacob Moise2 and
Charles M.Strom2
'To whom correspondence should be addressed
In women who are heterozygous for a genetic disease, genetic
analysis of the first polar body allows the identification of
oocytes that contain the maternal unaffected gene. These
oocytes can be fertilized and transferred to the mother without
risk of establishing a pregnancy with a genetically abnormal
embryo. We have demonstrated that removal of the first polar
body has no effect on subsequent fertilization rates or
embryonic growth to the blastocyst stage. We have developed
a PCR technique to successfully analyze the PI type Z and
PI type M genotypes of alpha-1-antitrypsin deficiency and
applied this technique for a couple at risk for PI type ZZ
alpha-1-antitrypsin deficiency. After standard FVF treatment
to stimulate multiple follicle development, eight oocytes were
aspirated transvaginally. Polar bodies were removed by
micromanipulation from seven oocytes and fertilization
occurred in six cases. PCR analysis was successful in five
oocytes. One was PI type M, two were PI type Z and two
were heterozygous MZ due to crossing over. Embryos from
the two oocytes containing the unaffected gene (polar body
PI type Z) were transferred in the same cycle 48 h after
insemination. No pregnancy was established. The accuracy
of the polar body diagnosis was confirmed by polymerase
chain reaction (PCR) analysis of an oocyte that failed to
fertilize.
Key words: embryo/genetic/IVF/PCR/preimplantation
Introduction
The diagnosis of human genetic diseases prior to implantation
has been proposed for high risk families to eliminate the
possibility of initiating a genetically abnormal pregnancy
(Handyside etai, 1989; Coutell etal., 1989). Methods have
been developed to biopsy the preimplantation-embryo and to
determine its genotype. One or more blastomeres can be removed
between the 2-cell and 8-cell stages (Handyside etal., 1989;
Coutell et al., 1989) or trophectoderm tissue herniating through
an incision in the zona pellucida of the blastocyst can be excised.
We have developed a new approach, preconception analysis of
826
Materials and methods
Polar body analysis using PCR
Each polar body was lysed by freezing at -70°C for 20 min
followed by incubation at 95°C for 20 min. PCR was performed
using primers 5'-TCAGCCTTACAACGTGTCTCT-3' and
5'-GTTTGTTGAACTTGACCTCG-3' designed to span the
a-l-AT PI type Z mutation from published DNA sequence data
(Dermer and Johnson, 1988). All buffers, oligonucleotide primers
and reagents, including Taq polymerase, were first digested with
© Oxford University Press
Downloaded from http://humrep.oxfordjournals.org/ at Pennsylvania State University on September 11, 2016
Reproductive Genetics Institute and 2 Departments of Obstetrics and
Gynecology, Illinois Masonic Medical Center, Chicago, IL 60657,
USA
the genotype of human oocytes by removal and genetic analysis
of the first polar body (PB) using the polymerase chain reaction
(PCR). We have applied this approach to a patient at risk for
alpha-1-antitrypsin (a-l-AT) deficiency and have transferred only
diose embryos derived from oocytes possessing the normal PIM allele. Theoretically, this technique can be applied to any
genetic disorder amenable to genetic analysis using PCR.
We have genetically analysed the first polar body of oocytes
aspirated from a woman at risk for a-l-AT. These studies may
serve as a model for the preconception diagnosis of human genetic
disorders in high-risk families. Such couples currently have
limited options to prevent the birth of affected children, namely
fetal diagnosis in the first or second trimesters by chorionic villus
sampling or amniocentesis, respectively, followed by elective
termination of affected fetuses. The possibility of diagnosis prior
to conception provides an alternative for these high-risk families.
In a woman who is a carrier for a single gene mutation such
as a-l-AT, the first polar body, in the absence of crossing over,
will contain a genotype opposite to mat of its corresponding
oocyte. If crossing over does occur, the first polar body will
contain both the normal and the mutant genes. Only embryos
developing from oocytes whose polar body analysis revealed them
to have the normal gene would be transferred in the same cycle.
When polar body analysis determines that an oocyte contains the
mutant gene, reveals crossing over (heterozygous polar bodies),
or fails because of technical difficulties, these oocytes can be
fertilized and the resulting pre-embryo biopsied for the
determination of their diploid genotype.
The first polar body is extruded from the developing oocyte
during meiosis I and is not required for successful fertilization
or normal embryonic development. Normal newborns have
resulted following destruction of the first polar body during 'zona
pellucida dissection' (PZD) as a treatment of male factor infertility
(J.Cohen, personal communication). Therefore removal of the
first polar body for the purposes of genetic diagnosis should have
no deleterious effect on the developing embryo.
Analysis of first polar body
HaeUi, in order to eliminate contaminating DNA sequences. This
was followed by incubation at 95°C for 5 min to inactivate the
restriction enzyme. Seventy-five cycles of PCR were performed.
The PCR cycles consisted of 1 min at 95°C, 30 s at 50°C and
1 min at 72 °C. Duplicate filters were prepared of slot blotted
PCR products and were hybridized with allele-specific oligonucleotides to the PI type Z and PI type M (normal) a-l-AT
alleles (Dermer and Johnson, 1988).
Results
Fig. 1. Illustration of polar body removal. A Nikon inverted microscope was used with Hoffman optics at 400x magnification. Upper left,
sampling pipette is placed through the zona pellucida. Upper right, sampling pipette reaches first polar body. Lower left, polar body being
aspirated into sampling pipette. Lower right, pipette leaving zona pellucida after polar body removal.
827
Downloaded from http://humrep.oxfordjournals.org/ at Pennsylvania State University on September 11, 2016
In preliminary experiments, the first polar body was removed
from 12 human oocytes from patients participating in a clinical
research protocol involving PZD for male factor infertility.
Successful fertilization occurred in five oocytes (42%), a rate
not statistically different (P = 0.9) from that observed in oocytes
not subjected to polar body aspiration (48%).
A technique for the PCR analysis of single cells was developed
for the a-l-AT mutation and applied to the analysis of the first
polar body. Since the a-l-AT locus is located in the telomeric
region of chromosome 14 (q24.3-q32.2), (Cox elal., 1982;
Darlington et al., 1982; Lai et al., 1983) it was predicted that
one-half of the first polar bodies would be heterozygous due to
crossing over between the centromere and the a-l-AT gene.
Preconception diagnosis was undertaken in a couple whose first
child succumbed to pulmonary emphysema at 3 years of age as
a consequence of homozygosity for the a-l-AT mutant allele,
PI type Z. Genetic analysis by PCR confirmed that both parents
were heterozygous for the PI type Z mutation. The mother
underwent standard IVF treatment to stimulate maturation of
multiple follicles and eight oocytes were aspirated transvaginally
under ultrasound guidance. The first polar body was removed
from seven oocytes by micromanipulation without visible damage
(Figure 1). Fertilization occurred in six of the seven oocytes,
as shown by the formation of two pronuclei with 15 h of
insemination with spermatozoa from the husband.
Genetic analysis was successful for five of seven polar bodies
(Figures 2 and 3); one was PI type M (Figure 3, slot 2B), two
were PI type Z (Figure 2, upper polar body, and Figure 3, slot
2C) and two were heterozygous due to crossing over (Figure 2,
lower polar body; Figure 3, slot IB). The analysis of two polar
bodies revealed no hybridization with either probe (Figure 3, slots
3B and 4B), indicating failed analyses. The light hybridization
band of one of the heterozygous parents with the M probe
(Figure 3, slot 4A) was probably due to poor membrane transfer.
Slot blots from single cells are difficult to interpret due to the
small amount of amplified material. Often, hybridization to both
probes is seen in cells known to be homozygous. The relative
intensity of hybridization was therefore used to determine the
genotype. Controls containing the PCR products of individuals
known to be heterozygous for the mutation were placed on each
filter. By examining the relative hybridization intensities of the
two probes with the heterozygous DNA the expected intensities
of hybridization for the specific experiment could be assessed.
Y.Veriinsky et al.
Fig. 2. Slot blot analysis of PCR reactions from polar bodies. Left
panel, hybridization with allele-specific oligonucleotide to PI
type M allele: 5'-TCCCTTTCTCGTCGATGGT-3'. Right panel,
hybridization with allele-specific oligonucleotide to PI type Z allele:
5'-TCCCTTTCTTGTCGATGGT-3'. C, control reaction with no
DNA added; MZ, parents (PI type MZ); PB, two polar bodies.
B
C
1
2
A
B
1
3
4
5
6
7
8
M-Probe
Z-probe
Fig. 3. Slot blot analysis of seven polar bodies and one oocyte.
Methods as described in Figure 2. Left panel, ASO to PI type M.
Right panel, ASO to PI type Z. Slot Al, ink marker; Slot A2,
control with no added DNA; Slot A3, 0.1 jtg normal control DNA
(PI type MM); Slot A4, 0.1 ng heterozygote paternal DNA (PI
type MZ); Slot A5, 0.1 /tg heterozygote maternal DNA (PI type
MZ); Slot A 6 - A 8 , no DNA; Slot Bl, polar body #1; Slot B2,
polar body #2; Slot B3, polar body #3; Slot B4, polar body #4;
Slot B5—B8, polar bodies from other experiments; Slot C2, polar
body #5; Slot C3, oocyte corresponding to polar body #2.
For example, in Figure 2, the slots labelled MZ represent
amplification of the DNA from both parents. The M probe
hybridizations were clearly slightly stronger in intensity than the
Z probe hybridizations. In the lower polar body in Figure 2, there
was hybridization with both probes. The intensity of the Z probe
hybridization was slightly less than that of the M probe. This
828
Discussion
This study has established the feasibility of performing preconception genetic analysis by removal and analysis of the first polar
body. The accuracy of this diagnosis was confirmed by the
analysis of an oocyte predicted to be PI type Z by virtue of the
polar body having the genotype of homozygous PI type M.
Theremovaland genetic analysis of the first polar body is likely
to have several advantages over preimplantation diagnosis based
on the biopsy of pre-embryonic or extra-embryonic cells.
Manipulation of the oocyte prior to fertilization is minimal and
no manipulation of the pre-embryo is necessary. Genetic analysis
of the first polar body allows embryo transfer in the same cycle
eliminating the need for cryopreservation, as in blastomere biopsy
at the 8-cell stage and trophectoderm biopsy at the blastocyst
stage. Finally, aspiration of the first polar body should have no
detrimental effects on fertilization or embryonic development,
since the first polar body is not involved in these processes.
Preconception diagnosis by the removal and genetic analysis
of the first polar body can be applied to any gene or restriction
fragment length polymorphism (RFLP) amenable to PCR
analysis, including sickle cell anaemia (Saikai et al., 1985), athalassaemia (Saikai etal., 1985; Chehab etal, 1987), /3thalassaemia (Saikai et al., 1988), cystic fibrosis (Kerem et al.,
1989), DuChenne's muscular dystrophy (Speer et al., 1989) and
Tay Sach's disease (Myerowitz, 1988; Myerowitz and Costigan,
1988).
The possibility of crossing over between homologous
chromosomes must be considered when performing preconception genetic diagnosis by analysis of the first polar body. For
any single meiosis, the probability of a cross over occurring
Downloaded from http://humrep.oxfordjournals.org/ at Pennsylvania State University on September 11, 2016
M
PROBE
is what would be predicted for a heterozygous MZ cell. In the
upper polar body, the Z probe hybridization was quite intense
and the M probe hybridization barely visible, indicating that the
genotype of this polar body was ZZ. Similar reasoning was used
in the interpretation of Figure 3. The heterozygous control in
lane 5C and an MM control in lane 3A were used as hybridization standards. The hybridizations of the amplified DNA from
the control (Figure 3, slot 5A) to both probes is similar in
magnitude. The filter with the Z probe was exposed for a longer
period of time to achieve this equivalence. As expected, the slot
3A (MM control) there is intense hybridization with the M probe
and little or no hybridization with the Z probe. The intense
hybridization in lane 2B with the M probe is in contrast to the
minimal hybridization with the Z probe indicating the genotype
of MM.
Embryos from the two oocytes with the normal PI type M allele
(polar body PI type Z) were transferred at the 6-cell stage, ~ 52 h
after removal of the first polar body and 48 h after insemination.
Pregnancy did not occur.
Confirmation of the accuracy of the genetic analysis of the first
polar body was accomplished by analysis of an oocyte that failed
to fertilize following removal of the polar body. Analysis revealed
that the polar body of this oocyte was PI type M (Figure 3, slot
2B), predicting that the oocyte was PI type Z. Analysis of the
oocyte revealed that expected PI type Z genotype (Figure 2, slot
3C).
Analysis of first polar body
Kerem,B.-E., Rommens.J.M., Buchanan.J.A., Markiewicz.D.,
Cox.T.K., Chakravarti.A., Buchwald.M. and Tsiu,L.-C. (1989)
Identification of the cystic fibrosis gene: genetic analysis. Science,
245, 1073-1080.
Lai.E.C, Kao.F.T., Law.M.L. and Woo.S.L.C. (1983) Assignment
of the alpha- 1-antitrypsin gene and a sequence-related gene to
chromosome 14 by molecular hybridization. Am. J. Hum. Genet.,
35, 385-392.
Myerowitz,R. (1988) Splice junction mutation in some Ashkenazi Jews
with Tay—Sachs disease; evidence against a single defect within this
ethnic group. Proc. Natl. Acad. Sci. USA, 85, 3955-3995.
Myerowitz.R. and Cosugan.F.C. (1988) The major defect in Ashkenazi
Jews with Tay—Sachs disease is an insertion in the gene for the alphachain of beta-hexosaminidase. J. Biol. Chem., 263, 18587-18589.
Saikai,R.K., Scharf.S., Faloona.F., Mullis.K.B., Horn.G.T.,
Erlich.H.A. and Arnheim,N. (1985) Enzymatic amplification of betaglobin genomic sequences and restriction site analysis of diagnosis
of sickle cell anemia. Science, 230, 1350-1354.
Saikai,R.K., Chang,C.-A., Levensen.C.H., Warren,T.C., Boehm.C.D.,
Kazazian,H.H. and Erlich,H.A. (1988) Diagnosis of sickle cell anemia
and beta-thalassemia with enzymatically amplified DNA and
nonradioactive allele-specific oligonucleotide probes. New Engl. J.
Med., 319, 537-541.
Speer.A., Rosenthal.A., Billwitz.H. and Hanke,R. (1989) DNA
amplification of a further exon of DuChenne muscular dystrophy locus
increase possibilities for deletion screening. Nucleic Acids Res., 17,
6775-6784.
Received on March 29, 1990; accepted on May 22, 1990
References
Chehab.F.F., Doherty,M., Cai,S., Kan,Y.W., Cooper,S. and
Rubin,E.M. (1987) Detection of sickle cell anaemia and thalassaemias.
Science, 329, 293-294.
Coutell.C, Williams.C, Handyside,A., Hardy,K., Winston,R. and
Williamson,R. (1989) Genetic analysis of DNA from single human
oocytes: a model for preimplantation diagnosis of cystic fibrosis. Br.
Med. J., 299, 22-24.
Cos.D.W., Markovic.V.D. and Teshima.I.E. (1982) Genes for
immunoglobulin heavy chains and for alpha- 1-antitrypsin are localized
to specific regions of chromosome 14q. Nature, 297, 428-430.
Darlington.G.J., Astrin,K.H., Muirhead,S.P., Desnick.R.J. and
Smith,M. (1982) Assignment of human alpha-1-antitrypsin to
chromosome 14 by somatic cell hybrid analysis. Proc. Natl. Acad.
Sci. USA, 79, 870-873.
Dermer.S.J. and Johnson,E.M. (1988) Rapid DNA analysis of
alpha-1-antitrypsin deficiency: application of an improved method for
amplifying mutated gene sequences. Lab. Invest., 59, 403—408.
Handyside.A.H., Penketh.R.J.A., Winston,R.M.L., Pattinson.J.K.,
Delhanty,J.D.A. and Tudenham.E.G.D. (1989) Biopsy of human
preimplantation embryos and sexing by DNA amplification. Lancet,
I 347-349.
829
Downloaded from http://humrep.oxfordjournals.org/ at Pennsylvania State University on September 11, 2016
between any gene and the centromere is a function of the distance
of the gene from the centromere. For telomeric genes, such as
the ar-l-AT locus, crossing over occurs with sufficient frequency
that the gene segregates independently from the centromere. In
polar body analysis of oocytes aspirated from a female
heterozygous for a genetic disease, meioses that have no crossing
over or an even number of cross overs between the centromere
and the a-l-AT gene will result in homozygous polar bodies.
In this situation, the genotype of the oocyte can be accurately
determined by polar body analysis. If an odd number of cross
overs has occurred, the polar body will be heterozygous. In this
situation it is impossible to predict the eventual haploid genotype
of the mature oocyte by analysis of the first polar body. In this
situation, either aspiration and analysis of the second polar body
or pre-embryo biopsy and analysis would be necessary for
accurate genetic diagnosis.
For telomeric genes, therefore, the prediction is that one half
of aspirated polar bodies will be heterozygous as a result of
crossing over, that one quarter will be homozygous for the mutant
gene and that one quarter will be homozygous for the normal
gene. Thus only one quarter of the embryos will be suitable for
transfer without further analysis. For genes situated close to the
telomere, the percentage of meiosis with crossing over between
the locus and the centromere will be lower. Since preconception
genetic diagnosis is designed for couples without underlying
fertility problems, IVF stimulation protocols result in the
development of multiple follicles which should provide ample
material for transfer. In addition, pregnancy rates following IVF
may be higher for these couples than in couples using FVF for
fertility problems.
Preconception genetic diagnosis by polar body aspiration and
PCR analysis can provide an alternative to prenatal diagnosis and
pregnancy termination for couples at high risk for genetic
diseases. This procedure can be used in conjunction with embryo
biopsy to enable couples to conceive children known to be
unaffected by the genetic disease.