With pleasure we present the first part of the unique cycle of expert articles developed by INVICTA Fertility Clinics in cooperation with the web-portal ginekolog.pl. Every month, on our website and on our web-portal, we will publish substantive materials dedicated to broadly understood issues of infertility diagnosis and treatment.
The assumption which underpins this initiative is the presentation of the professional approach to these issues and the provision of information about the most recent achievements of medicine in the area of assisted reproductive technologies. The articles are prepared by the experienced practitioner and scientist, Professor Krzysztof Łukaszuk, PhD, MD, Chief of INVICTA Fertility Clinics, and a team of experts.
Part one of the cycle “Preimplantation diagnosis” discusses the possibilities of using the innovative method of PGD in case of couples carrying or affected by genetic diseases who try for a child. In subsequent parts, the Readers will have the opportunity to read, among others, the description of the methods used to diagnose infertility, characteristics of the modern techniques of assisted reproduction and publications pertaining to the role of tests in this process.
We hope that these articles will prove to be a valuable assistance in daily work. Enjoy the reading!
PGD Preimplantation Diagnosis
Preimplantation Genetic Diagnosis (PGD) is a method which allows to assess the genetic material of egg cells before their fertilization or of embryos before their transfer to the uterus . It is considered that the pioneers of the idea of PGD are R.G. Edwards and R.L. Gardner, who, in 1968, performed the first blastocyst biopsy in a rabbit in order to confirm its sex . The application of preimplantation diagnosis with human reproductive cells in 1989 initiated the stage of development of this area regarding its diagnostic possibilities. In 1990, Handyside et al. described the first case of pregnancy after PGD in patients with a genetic disease linked to the Y chromosome (adrenoleukodystrophy) . In the result of tests using the technique of polymerase chain reaction – PCR, female embryos without the risk of occurrence of this disease were transferred to the uterus. In the same year, a team led by Verlinsky carried out successfully preimplantation diagnosis for another disease – cystic fibrosis . The simultaneous development of diagnostic techniques allowed to expand the diagnostic possibilities of PGD. Application of FISH – fluorescent in situ hybridization enabled fast analysis of aneuploidies and chromosomal aberrations in egg cells and embryos .
PGD is an early form of prenatal diagnosis related to assisted reproductive technologies . Indications for PGD can be divided into 2 groups:
- The first group, characterized by a high risk of passing genetic disorders to progeny:
– one of partners carries a recessive or dominant allele or a balanced reciprocal translocation 
– couples who several times had to take the decision to terminate pregnancy due to abnormal results of prenatal tests
– recurrent miscarriages of unexplained etiology [4, 6]
- The second group, with a low risk of passing genetic disorders to progeny, in which PGD is performed for the purpose of increasing the chances for successful IVF (In Vitro Fertilization) procedure. This type of diagnosis is erroneously referred to as PGS Preimplantation Genetic Screening):
– advanced age of a mother (>37 years at the expected date of delivery), where the reason can be increased risk of aneuploidies in embryos 
– lack of positive effects of treatment in the course of ART (Assisted Reproductive Technologies) procedures [4, 6].
The material for the genetic analysis can be collected with the use of one of the currently available 3 methods:
1) Blastomere biopsy – this method is used most often. It is carried out on day 3 following fertilization. Only 6-8 cell embryos are eligible for the biopsy. After opening the embryo’s zona pellucida (mechanically, using enzymes or laser light), 1, or less commonly 2, blastomeres are collected . The transfer of a genetically unaffected embryo is carried out on day 5 from the egg fertilization.
2) Biopsy of I or II polar body. Polar bodies are fragments of cytoplasm within the egg cell, containing only the genetic material of a mother. They form during the first and the second meiotic division of an oocyte. They do not have any physiological function in further embryo development. The procedure of their collection does not differ from that applied in the case of blastomere biopsy. The first polar body is collected from the oocyte before fertilization, while the second polar body – after fertilization. This method allows avoiding intervention into the embryo’s genetic material. The limitation in this case is the inability to examine the genetic material of a father or to detect genetic anomalies occurring during the embryo’s development .
3) Biopsy of embryonic trophectoderm cells (TF). It is performed on day 5 after the egg fertilization. Cells for analysis are collected from trophectoderm. The embryo has to be in the blastocyst stage. For the analysis, several cells from the trophectoderm area are collected. This test limits the scope of selected chromosomes in case the embryo transfer is planned for day 5.
The advantage of the TF biopsy is a large amount of material for testing, although the short time for the analysis and the lack of certainty that TF cells reflect the actual DNA of the inner cell mass limit its application .
PCR (polymerase chain reaction) and FISH have been used for genetic analysis in preimplantation diagnosis for many years now.
PCR allows the duplication of a selected DNA fragment; with this, we can obtain multiple copies of this fragment. PCR is used in the diagnosis of single-gene diseases. The limitation of PCR is the risk of contamination of the foreign DNA (e.g. originating from a sperm when the procedure applied to fertilize the egg cell was the classical fertilization technique and not ICSI). Another disadvantage is the allele drop-out (ADO) effect where one of heterozygous alleles is amplified improperly, which can lead both to false positive and false negative effects. The application of the modified PCR methods such as multiplex PCR, fluorescent PCR and real-time PCR allow to avoid the aforementioned problems [5, 9]. Further method applied in PGD using PCR is the duplication of the whole genome of the analyzed cell – WGA (whole genome amplification). This procedure is recommended when the amount of material for testing is small . False results of the genetic evaluation of embryos for autosomal recessive disorders are on the level of 2% and for dominant traits – on the level of 11% .
PGD with the use of FISH technique uses fluorescent probes to assess autosomal aneuploidies, detect sex chromosomes and chromosomal aberrations. False results of the aneuploidy assessment based on 1 blastomere amount to 7%, of which 6% are mosaics, therefore a biopsy of 2 blastomeres is recommended in order to increase the sensitivity of this method .
The results of the correct PCR diagnosis for the biopsy of 1 cell as compared to the biopsy of 2 cells are 88.6% and 96.4%, respectively. In the case of FISH, differences are not statistically significant. The differences in the live birth rate after PCR or FISH have not been statistically significant .
Comparative genomic hybridization (CGH) and array comparative genomic hybridization (aCGH) have been used for only several years to select embryos with higher implantation rate, diagnosis of aneuploidies or mosaicism. CGH allows the analysis of differences in the number of DNA copies of whole genome, thus giving a chance to carry out combined diagnosis of various point mutations or point mutations and aneuploidies. As for now, insufficient efficacy and complexity of the developed methods and their high costs limit their large-scale application. Their definite deficiency is a long time of waiting for test results (CGH up to 72 hours and aCGH up to 30 hours). For the time of test performance, embryos or oocytes have to be subjected to cryopreservation [3, 16]. Vitrification is a new method of oocyte and embryo freezing developed by M. Kuwayama. It has been proved that it increases the birth rate of healthy children  and survival rate of embryos after blastocyst biopsy as compared to the standard methods of cryopreservation . Zhang et al. studied survival rates of vitrified embryos in various stages of development, subjected to biopsy on day 3 after fertilization as compared to embryos without biopsy. The results of the study permit to formulate the thesis that embryos after biopsy, cryopreserved at the higher stage of development can be used after thawing with satisfactory effect .
At the moment, in theory, PGD can be carried out for all single-gene diseases: autosomal dominant (e.g. Huntington’s disease, Marfan syndrome, myotonic dystrophy), autosomal recessive (e.g. cystic fibrosis, beta thalassemia, spinal muscular atrophy), sex-linked diseases (e.g. Duchenne muscular dystrophy, fragile X syndrome, hemophilia A) and chromosomal aberrations: aneuploidies, balanced and Robertsonian translocations .
Aneuploidies are the known causal factor of the failures of assisted reproduction and recurrent miscarriages. In approximately 70% of embryos created in the result of IVF procedures, at least one of five chromosome disorders is present (X, Y, 13, 18, 21) . With maternal age, the risk of trisomy 21 increases. High risk of miscarriage, stillbirth of birth of fetus with congenital disorders has been confirmed in cases when one parent is a carrier of a balanced reciprocal translocation . Carriers of balanced reciprocal translocations are at higher risk of miscarriage than carriers of Robertsonian translocations. In the group of patients with a history of genetic disorders, recurrent miscarriages and lack of live births, PGD reduces the risk of miscarriages and increases the rate of live births .
Although the efficacy of PGD in the group with high risk of passing genetic disorders to their progeny is proven, the application of PGD creates a lot of controversy. Referring to randomized studies, Geraedts et al. (2010) state that PGS does not rise the rate of live births in couples with advanced maternal age, failures of IVF/ICSI . Moreover, PGD in this group can decrease the rate of pregnancies and pregnancies carried to term .
In conclusion, preimplantation diagnosis is the alternative for prenatal diagnosis in couples who choose ARTs. With its extensive options of genetic diagnosis, it gives not only the possibility to have healthy children, but also the chance of having a child for couples with recurrent miscarriages or ART failures. It also provides the option of somewhat controversial choice of siblings for compatible HLA, for the purposes of umbilical cord blood stem cell acquisition e.g. for stem cell transplantation needed by siblings .
Authors of the article: A. Litwin, dr n. med. J. Liss, dr hab. n. med. K. Łukaszuk, prof GUM-ed
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