呂英仁醫師

Introduction:

Chromosomal anomalies account for a large percentage of early pregnancy losses, congenital malformations and postnatal functional impairments. Conventional prenatal genetic diagnosis involves obtaining fetal/trophoblastic samples during late first trimester (chorionic villi sampling, between 8-10 wks GA) or early second trimester (amniocentesis, between 15-17 wks GA). The diagnostic sensitivity and specificity are high, although with the inconvenience of having to terminate pregnancy relatively late and the associated risks of late abortion.

However, with the wide spread use and mature procedures of artificial reproductive technology (ART), new approaches to have genetic diagnosis before embryonic implantation are possible, or even before conception takes place, thus saving the patient from the traumatic experience of therapeutic abortion.

In this brief discussion, attempts are made to describe available methods of preimplantation genetic diagnosis (PGD), advantages and disadvantages.

Obtaining samples for PGD:

The sources for PGD come from 3 mayor procedures:

1.     Polar body biopsy:

The so called preconception diagnosis. The first polar body extrude from primary oocyte after meiosis I, which contains a haploid set of chromosomes but double genetic materials, as each chromosome is composed of 2 chromatids. If the genetic disease in question is recessive, the genotype of oocyte can be inferred indirectly by knowing the karyotype of the first polar body. However the situation might be complicated by the fact that crossing over occurs during prophase of meiosis I, specially if the gene is located far from centromere or within telomeric loci. In such cases, biopsy of second polar body becomes necessary.The advantage of polar body biopsy is that diagnosis is made before conception, with ample time to implantation. However, the procedure is technically difficult and only maternal genetic material is assessed.

2.     Trophectoderm biopsy:

Human blastocyst is obtained usually after 5 days of in vitro culture, which is composed of inner cell mass and trophectoderm. More cells, with less fragmentation and degeneration, are disposable for further manipulation. Nevertheless, the source of blastocyst is scarce, since fertilized eggs reach blastocyst stage in only 20-25% under the best circumstances and trophectoderm biopsy has not been applied clinically.

3.     Cleavage stage biopsy:

In human 8 cells pre-embryo, up to 3 blastomeres can be excised without compromising its developmental competence to blastocyst stage. Usually 1 or 2 blastomeres are extracted by "drilling" a hole in zona pellucida and then remove blastomeres by aspiration or extrusion. It is the clinically most used method.

Preimplantation diagnosis methods:

1.    DNA methods:             Polymerase chain reaction (PCR)

                                     Restriction fragment length polymorphism

                                     Primer extension pre-amplification

                                     Southern blotting

                                     Fluorescence in situ hybridization (FISH)

                                     Short tandem repeat (STR)

2.    Enzymatic methods:     unreliable ( due to persistent maternal enzymes)

3.    Histological methods:   sex chromatin and F body

4.    Growth in vitro:            male embryo grows faster than female embryo.

Polymerase chain reaction (PCR):

Few cells obtained in PGD require highly sensitive and specific DNA diagnostic methods. PCR use a thermo-stable polymerase (Taq) to synthesize copies of target DNA sequences. Millions of copies can be made within several hours, which are then analyzed with electrophoresis or other methods. Variations of the original PCR were developed recently: nested primers, heteroduplexing, restriction fragment length polymorphism (RFLPs), etc. 

Fluorescence in situ hybridization (FISH):

Fluorochrome-labeled DNA probes were designed to identify centromeres or other structures of the chromosome arms in a histological specimen. Fluorochromes of different colors can be used simultaneously to give a mapping of target chromosomes. Result can be available within 6-8 hs. 

Conclusion:

PGD is accomplished through several steps that require highly trained procedures, like ART, micromanipulation, blastomere biopsies, and DNA analyses. Not every infertility center will be capable of such facilities, but its value in early diagnosis of sex-chromosome linked or autosomal diseases is promising. Since aneuploidy is suspected to be a mayor cause of implantation failure of morphologically normal embryos, the implantation rate could be improved by selecting only those embryos with normal karyotyping for implantation, with the additional benefit of reducing the number of embryos transferred and the risk of multiple pregnancy.