Convenciones y tratados de protección a menores de edad

Preimplantation genetic diagnosis (PDG) can be regarded as an early form of

prenatal diagnosis. As the name implies, the process is performed before the

implantation of the embryos. This helps the couple to start a pregnancy free of the

inherited disease at risk in their family thus avoiding the problems associated with TOP

(Handyside and Delhanty, 1997). The whole process includes, after identification o f the

inherited disease at risk in the family, in vitro fertilisation (IVF), biopsy of cell(s) to be analysed and the genetic analysis.

1.7.1 IN VITRO FERTILISATION (IVF)

This process consists of ovarian stimulation, oocyte retrieval, and fertilisation

with the sperm outside the body. Ovarian stimulation is usually accomplished by

pituitary down regulation using a gonadotropin releasing hormone analogue (GnRHa)

and stimulation with an external gonadotrophic hormone such as human menopausal

gonadotropin (hMG), or synthetic follicle stimulating hormone (FSH). The woman must

be monitored for the development of ovarian follicles using an ultrasound scan and/or

blood oestradiol (E2) levels. Once there is an adequate number of suitable follicles,

ovulation is stimulated by the administration of human chorionic gonadotropin (hCG)

and the oocyte retrieval performed 34-36 hours after the administration just before the

true ovulation takes place. Oocytes are cultured and inseminated with sperm and

1.7.2 OBTAINING CELL(S) FOR ANALYSIS

After fertilisation, the oocyte completes metaphase II and the second polar body

is extruded. The zygote then undergoes mitotic divisions. From these processes, cell(s)

from several stages can be utilised for genetic analysis. Biopsy can be performed on the

polar body or cells from cleavage stage or blastocyst stage embryos. The most common

technique currently in use is cleavage stage biopsy (ESHRE PGD Consortium Steering

Committee, 1999).

1.7.2.1 Cleavage stage biopsy

The biopsy is carried out on 1-2 cells from 6- to 8-cell embryos. This is usually

on day 3 postinsemination. A small hole is made in the zona pellucida using acid

Tyrodes solution or a laser and the blastomere(s) aspirated through the hole. After the

biopsy the embryos are kept in culture and the biopsied cell(s) tested. Only unaffected

embryos are replaced into the mother. The biopsy is performed at this stage because the

cells are still totipotent. The remaining cells usually survive and can still undergo

further development (Hardy et a l, 1990). Biopsy at the 4-cell stage can retard cleavage (Tarin et a l, 1992). Soussis et al (1996) reported the obstetric outcome of 16 pregnancies following PGD at the 8- to 10-cell stage (12 singleton and 4 twins), apart

from 3 singleton pregnancies which were lost in first trimester, the remaining

pregnancies resulted in 15 healthy babies.

1.7.2.2 Polar body biopsy

Pornpim ol Ruangvutilert Chapter 1 Introduction

common aneuploidies using FISH and for some single gene disorders in 187 clinical

cycles. Three-quarters of the tested cycles resulted in embryo transfers which gave rise

to 38 clinical pregnancies and 12 births of an unaffected child (Verlinsky and Kuliev,

1996).

An important drawback of this technique is that it is prone to misdiagnosis due

to crossing over between non-sister chromatids in the first meiosis, loss of a single

chromatid or a chromosome from the polar body or the primary oocyte, or some errors

in meiosis II. In the light of this, sequential analysis of the first and second polar body

has been introdueed (Verlinsky et a l, 1997). This may reduce the chances of misdiagnosis but altogether the polar body analysis is teehnically and financially

demanding. It may not be suitable for clinical PGD in general.

1.7.2.3 Blastocyst biopsy

Blastocyst biopsy is performed on day 5 or 6 when the embryonic cells become

separated into the ICM and the TE with collection of fluid in the blastocoel. The

technique involves a slit being made in the zona pellucida opposite the ICM and the

embryo replace into culture. As the embryo expands, the TE herniates through the slit

and can be partially removed for analysis (Dokras et a l, 1990). Laser biopsy has been introduced to increase the blastocyst recovery rate (Veiga et a l, 1997). Ten to thirty cells from the TE can be biopsied thus giving more cells available for analysis than

biopsy at other stages. This could minimise the risk of error in analysis per se or error

from mosaicism. Moreover, it should not affect the future embryo/fetus which arises

human embryos arrest before the blastocyst stage, thus hampering the use of blastocyst

biopsy clinically.

1.7.3 GENETIC ANALYSIS IN PREIMPLANTATION GENETIC DIAGNOSIS

The next step in PGD is to test the biopsied cell(s). Currently, most centres

performed cleavage stage biopsy from which only 1-2 cells are available. The tests

carried out on one or two cells must be sensitive and accurate. In addition, the biopsied

cells are unlikely to be in metaphase so it is difficult to assess a full karyotype. The

development of FISH allows study of a certain chromosome or chromosomes to be

performed on interphase nuclei. FISH has been employed for embryonic sexing in

couples carrying X-linked diseases (Griffin et a l, 1994; Delhanty et a l, 1997) for the diagnosis o f some chromosomal disorders in high risk couples (Conn et a l , 1998) and for age-related aneuploidy (Gianaroli et a l, 1999). PCR, which allows DNA analysis on a small amount of DNA, can be used for the diagnosis of single gene defects and triplet

repeat disorders (Wells and Sherlock, 1998). However, some problems need to be

considered in both techniques. In FISH the major concern is mosaicism. From previous

studies, embryos with normal morphology may have sex chromosome or autosome

mosaicism or a completely chaotic pattern (Harper et a l, 1995; Munné et a l, 1995; Delhanty et a l, 1997; Magli et a l, 1998). This could also cause a problem with PCR, especially for the diagnosis of dominant single gene disorders if the biopsied cell is

haploid and carries the normal allele. Other concerns for PCR are contamination with

exogeneous DNA and allele drop out (Findlay et a l, 1995; Ray et a l, 1996). Sometimes PCR is also used for embryonic sexing but this is less informative than

Pornpim ol R uangvutilert Chapter 1 Introduction

FISH since PCR does not give the copy number of the chromosomes (Harper and

Delhanty, 1996b).