Pgd Activities In The Different Countries

All countries with centres practising PGD have well-developed IVF programmes that offer a full range of treatment procedures including intracytoplasmic sperm injection (ICSI). Therefore, the different procedures of assisted reproduction were fully represented and considered standard medical treatments for infertility. This, however, was not true for PGD, which is still considered an experimental procedure. The experimental status of PGD and related ethical concerns are reflected in the...

Metabolic Requirements Of The Early Mammalian Embryo In Vitro

The metabolic requirements of the preimplantation embryo vary through the preimplantation period. Prior to genome activation pyruvate and lactate are the primary energy sources. After activation of the embryonic genome, metabolism shifts to glucose-dependent pathways. Sequential media for use in IVF culture has been developed which complements this shift in metabolic requirements, with low glucose concentrations for the initial stages of culture, followed by medium containing glucose for...

Microdeletion Syndromes

Chromosome microdeletion carriers have a 50 risk of transmitting the defect to their offspring. If the phenotypic effect of the deletion is variable then prenatal diagnosis can create the dilemma of deciding whether to continue an affected pregnancy when the fetus may be severely impaired or only wildly so. PGD has been used in such a situation for an asymptomatic female carrier of. a microdeletion of chromosome 22 associated with DiGeorge syndrome this was only diagnosed after the birth of a...

Spermatid Injection

In some cases of severe testicular dysfunction, no spermatozoa can be found either in the ejaculate or in testicular tissue, but precursor cells (round, elongating or elongated spermatids) may be identified. Although pregnancies and live births have been achieved following injection of these cells (Sousa et al., 1998 Vanderzwalmen et al., 1997, 1998), fertilization and pregnancy rates are dramatically lower than following the use of mature spermatozoa, partially due to the technical difficulty...

Oogenesis

With the advent of IVF technology, human oocytes that failed to fertilize after in vitro insemination became available for chromosome analysis (Figures 7.2, p. 106 and 7.3 see Plate I). Initial estimates of the incidence of hyperhaploidy in unfertilized oocytes ranged from 2 to 14.5 (reviewed by Zenzes & Casper, 1992). Based on the 1120 oocytes karyotyped in the 11 studies, the weighted mean percentage of aneuploidy was estimated to be approximately 13 (Zenzes & Casper, 1992). More recent...

Alan H Handyside

School of Biology, University of Leeds, UK Chichester New York Weinheim Brisbane Singapore Toronto Copyright 2001 by John Wiley & Sons, Ltd Baffins Lane, Chichester, West Sussex P019 1UD, England National 01243 779777 International (+44) 1243 779777 e-mail (for orders and customer service enquiries) cs-books wiley.co.uk Visit our Home Page on http www.wiley.co.uk or http www.wiley.com All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system, or...

Blastocyst Biopsy

The human blastocyst consists of an outer layer of trophectoderm cells (TE) which goes on to make the placenta and an inner cluster of cells, the inner cell mass (ICM) from which the embryo proper is derived (see Chapter 6 p. 92). The use of differential labelling, whereby the ICM and the TE nuclei are stained with different coloured polynucleotide-specific fluorochromes, has shown that during blastocyst formation, cell death occurs in both the ICM and the TE. The mitotic index for both cell...

Fragments

Most IVF embryologists would agree that fragmentation is the norm in routine IVF, but it is not clear whether this is an effect of culture conditions and follicular stimulation, or a characteristic of human development (see Chapter 6 p. 97). The degree of fragmentation varies from 5 or 10 to 100 ), and the fragments may be either localized or scattered. Alikani et al. (1995) used an analysis of patterns of cell fragmentation in the human embryo as a means of determining the relationship between...

Locus Or Unique Sequence Probes

Locus or unique sequence probes are homologous to particular genes or loci and so hybridize to a specific location on the chromosome (Figure 11.1c and d see Plate IV). It is hoped that in the near future there will be unique sequence probes available for most of the unique sequence regions of all of the chromosomes. Because they bind to a unique sequence, these probes are made from larger pieces of DNA. Those originally used for PGD were cloned in yeast artificial chromosomes (YACs) (Conn et...

Whole Genome Amplification

Multiplex PCR is not the only method that allows more than one fragment to be amplified from a single cell. A variety of methods aimed at non-specific amplification of the entire genome (whole genome amplification WGA) have also been applied to single cell analysis (Zhang et al., 1992 Wells et al., 1996, 1999). Using these techniques a single genome can be amplified numerous times, thus providing sufficient DNA templates for many independent PCR amplifications. The WGA method that has most...

Foreword

Few developments are anticipated to affect human beings more profoundly in the coming years than the knowledge gained from the complete sequencing of the human genome. This knowledge combined with the power and sensitivity of recombinant DNA technologies promises to revolutionize the diagnostic, predictive and life-enhancing capabilities of medicine. Nowhere will these medical developments become more apparent than in the field of preimplantation genetics, particularly as the functional nature...

Polygenic Inheritance

Disorders such as diabetes and schizophrenia are caused by the interaction of many different genes together with environmental influences. Technically it is not possible to offer PGD for this type of condition at present since the genetic basis is not fully understood and the exact environmental component is unknown.

The Legal Status Of

It is difficult to generalize the current legal status of PGD because each country has its own distinct legal traditions and cultural and or religious beliefs that influence perceptions of IVF, prenatal diagnosis and embryo manipulation. Four distinct legal frameworks affecting the performance of PGD were revealed by the ESHRE survey 1. Countries with legislation regulating research on human embryos, including PGD. 2. Countries with legislation that specifically address PGD. 3. Countries where...

Xlinked Inheritance

X-linked disorders (caused by mutation in genes that are carried on the X-chromosome) can be either recessively or dominantly inherited. In practice, almost all severe examples are recessives and are carried by females who are themselves unaffected or only mildly so, because of the normal copy of the gene on their second X chromosome. Half their sons (who get their single X chromosome from their mother) will be normal however, the others will be affected with the disease as their Y chromosome...

Follicle Tracking

Follicular tracking is undertaken by serial ultrasound scans (Figure 5.1) and measurement of serum oestradiol (see Chapter 8 p. 130). When there are three follicles over 18 mm, a single dose of HCG is administered. This mimics the luteinizing hormone (LH) surge (see Chapter 6) and allows the final stages of oocyte maturation to occur. The oocyte retrieval takes place approximately 36 hours later. Since the pituitary is downregulated, endogenous progesterone is administered as luteal support.

Chromosome Paints

Chromosome painting probes contain a cocktail of DNA sequences resulting in targeted chromosomes being completely covered by the hybridization signal (Figure 11.1a see Plate IV). Specific chromosome paints are available for every chromosome but they can only be used on metaphase chromosomes. If used in interphase they form a non-identifiable signal usually in the middle of the interphase nucleus. Chromosome paints have been used for PGD on polar bodies for patients carrying chromosome...

Spermatogenesis

The process of spermatogenesis can be divided into three phases proliferation, meiosis and differentiation. These are associated with specific germ cell types spermatogonia, spermatocytes and spermatids, respectively. In the male, interphase germ cells start to proliferate by mitosis at puberty. This is followed by meiosis and a gradual reorganization of cellular components, characterized by a loss of cytoplasm. The stem cells, or AO spermatogonia, are located in the intratubular compartment,...

Info

Figure 8.4 Receiver operating characteristic curves of predictive tests for ovarian reserve. The number of follicles produced through ovarian stimulation best correlates with rise in oestradiol and day 3 oestradiol compared to FSH and FSH LH ratio. Rise in oestradiol from day 2 to day 3 of the G-test * FSH O day 3 oestradiol of the G-test FSH LH ratio. Reprinted by permission from the American Socicty for Reproductive Medicine (Ranieri et al. Fertility and Sterility, 1998, 70(2), 227-33) Figure...

Fertile Patients

The analysis of chromosomes in human embryos has been based almost entirely on embryos generated from IVF, which may not be representative of in vivo development. However, the classic studies of Hertig et al. (1954) detected high levels of nuclear abnormalities in embryos from natural cycles. Until recently, PGD was performed primarily on fertile patients who were at significant reproductive risk for a Mendelian mutation. For example, the majority of patients who have requested embryo sex...

Semen Assessment And Sperm Preparation

Prior to the oocyte retrieval procedure, the male partner provides a semen sample. After a period in the laboratory of approximately 30 minutes to allow liquefaction, the sample is thoroughly mixed and carefully assessed. By the time of oocyte retrieval, the laboratory should already be familiar with the male partner's semen profile, and can refer to features that might influence the choice of sperm preparation method used. The choice of sperm preparation method or combination of methods...

Technical Considerations And Methods

Whether the fertilization has been achieved by conventional IVF or by ICSI the possibility of contamination by maternal cumulus cells is potentially disastrous to the diagnosis (particularly in a case involving sex-linked disease). Before transferring the embryo to the biopsy dish, it is essential to render the zona completely free from cumulus cells. Cumulus cell contamination may have been the cause of some of the recent misdiagnoses reported (see Chapter 14 p. 242). A number of methods are...

Blastocyst Expansion And Hatching

The early blastocyst (day 4 5) initially shows no increase in size, but it subsequently expands over the next one or two days (day 5 6) by active accumulation of fluid in the central blastocoelic cavity. Throughout these early stages the embryo is enclosed in the ZP, which keeps the cells together prior to compaction and prevents two embryos fusing and forming a chimaera. If the ICM divides at this early stage monozygotic (identical) twins may develop. During the transition from morula to...

First Trimester Ultrasonography

The Diagnosis of Structural Anomalies in the First Trimester First trimester sonographic assessment of fetal anatomy has become an important component of prenatal diagnosis. It requires a good understanding of embryological development, because findings the sonographer regards as abnormal in the second trimester may be considered normal in the first. The optimal gestational age for early fetal anomaly scanning will depend on the time that the majority of fetal structures can be visualized, at...

The Potential For Misdiagnosis And The Implication Of Mosaicism

The possible causes of misdiagnosis in PGD are summarized in Table 14.1. The first misdiagnosis reported was in the first series of PGD cases where embryos were sexed amplifying only a Y chromosome sequence (see Chapter 11). This was due to either amplification failure or an XO blastomere in a male embryo (Handyside et al., 1991). With the use of dual FISH and improved PCR methods there have been no further reported misdiagnoses of sex. The two instances of CF misdiagnosis were both cases where...

Embryo Sexing

Sexing for X-linked disease is one of the major indications for PGD (Verlinsky et al., 1994 ESHRE PGD Consortium, 1999). X-linked recessive diseases account for 6-7 of single gene defects and include conditions such as Duchenne muscular dystrophy (DMD), haemophilia, and various mental retardation syndromes. Table 11.1 lists some of the common X-linked diseases for which PGD sexing has been offered. The mother carries the mutation on one of her X chromosomes and so transmits the defective gene...

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Figure 12.6 PGD of ring chromosomes. (a) Diagrams show two possible approaches to detecting a ring chromosome 16 depending on position of breakpoints (chromosome 16 centromere orange, 16p green, 16q red). (i) Simplest scheme whereby the ring is deleted for the sub-telomeric probe region. (ii) Actual location of sub-telomeric probes on the ring chromosome in a prospective PGD patient 46, XX, r (16) (p13.3q24). (b) Probe combination on patient lymphocyte metaphase chromosomes (orange shown where...

Centrosomes

The centrosome represents a fundamental paternal contribution to embryogenesis, providing a 'division centre' for the zygote. Although it is generally accepted that in humans the centrosome is of paternal origin, in the mouse and hamster the centrosome is apparently of maternal origin, lending support to the observation that these rodents are poor model systems for human fertilization. In somatic cells the centrosome is composed of two structures called centrioles, placed at right angles to...

Coverage Of The Cost Of Ivf And

In eight countries (Australia, Belgium, France, Germany, Italy, the Netherlands, Spain and Sweden), the IVF component of the cost of PGD, usually up to four to six IVF cycles, is covered by the social security system. The couple, however, pay the diagnostic component. In the United Kingdom, the availability of coverage is dependent on the regional funding scheme providing coverage to individual couples. Therefore the National Health Service does not always cover PGD (J. Harper, personal...

Reciprocal Translocations

Balanced reciprocal translocations are by far the most frequently encountered chromosome rearrangement found in couples requesting PGD. As breakpoints can occur theoretically at any point on any chromosome, each translocation case represents a unique event with its own risk of chromosome imbalance at conception. As discussed in Chapter 2, reciprocal translocations associate in a quadrivalent during meiosis to allow the homologous translocated and centric segments to pair. This arrangement can...

Interphase Chromosome Conversion

The ideal way to examine chromosomes from any tissue is to arrest the nuclei in metaphase and examine the chromosomes individually. It has been possible to induce metaphase nuclei in sperm by injecting sperm directly into hamster oocytes and recently this technique has been modified to fuse polar bodies or blastomeres into oocytes or zygotes to induce metaphase formation (Evsikov & Verlinsky, 1999 Willadsen et al., 1999). This technique has been termed nuclear conversion. In one method human...

Chorionic Villus Sampling

The indications for CVS are similar to those of amniocentesis, although it has not been used as frequently for karyotyping. It can be performed transcervically or transabdominally. The preferred route and technique is determined by the placental localization, gestation, safety of the procedure and operator experience. The transcervical (TC) approach to the placenta is limited to 10-13 weeks' gestation, by the distance to, and localization of, the placenta. The transabdominal (TA) approach can...

Embryo Transfer

For standard IVF embryo transfer is normally performed on day 2 after the egg collection. For PGD, embryos are usually biopsied on day 3 (at the six- to eight-cell stage). Diagnosis usually takes two to seven hours depending on the disease and unaffected embryos can be transferred to the mother during the afternoon or evening of day 3. However, some diagnoses take 24 hours and it may be day 4 before it is known which embryos are suitable for transfer. Embryos are transferred using a Frydman...

Embryo Quality And Selection For Transfer

On day 2-3 (approximately 48-72 hours post oocyte retrieval), oocytes with normal fertilization and cleavage may contain from two to eight blastomeres. The embryos should then be carefully evaluated in order to select those with the highest implantation potential. The limitations of evaluating embryos based on morphological criteria alone are well recognized. Correlation between gross morphology and implantation are weak and inaccurate, unless the embryos are clearly fragmenting. Many studies...

Prospective Parents

It is reasonable to assume that the impetus for parents seeking PGD at the present time is the desire to have a healthy baby free from a predictable and debilitating genetic disease, and without the need for a possible termination in the course of the pregnancy. In seeking PGD for these reasons, parents are, in effect, exercising both their liberty to procreate and their discretionary judgement as parents (UN Universal Declaration of Human Rights, 1948). Indeed, unless met with resistance from...

Programmed Superovulation Protocols

These protocols provide a convenient and effective means of scheduling and organizing a clinical IVF programme, allowing oocyte retrievals to be done on specific days of the week, or in 'batches'. A standard protocol which can be used to maximize the number of oocytes recovered uses pituitary downregulation with a gonadotrophin-releasing hormone (GnRH) agonist preparation, commencing in the luteal phase of the previous cycle, and ovarian stimulation with purified follicle-stimulating hormone...

References

Abdel-Rahman B, Fiddler M, Rappolee D & Pergament E (1995) Expression of transcription regulating genes in human preimplantation embryos. Mo Hum Reprod 10 2787-2792. Adjaye J, Daniels R & Monk M (1998) The construction of cDNA libraries from single human preimplantation embryos and their use in the study of gene expression during development. J Assist Reprod Genet 15 344-348. Almeida PA & Bolton VN (1994) The relationship between chromosomal abnormalities in the human ooctye and...

Chromosomal Translocations

The most common type of chromosome rearrangement is a translocation, which is the movement of a segment of chromosome from its normal position to a new site. Reciprocal translocations involve breaks along the arms of two chromosomes and Figure 2.4 (a) Reciprocal translocation reciprocal exchange of material between two nonhomologous chromosomes, (b) Cross-shaped arrangement (quadrivalent) adopted by reciprocal translocations during early meiosis allows pairing of homologous chromosomes. A, B...

Dagan Wells And Jon Sherlock

The polymerase chain reaction (PCR) is designed to enrich a DNA sample for one specific fragment, amplifying it to a level at which it can be visualized and subjected to further genetic analysis. The success of PCR in achieving this objective has enabled it to become one of the most important methods in genetic testing, and has led to a proliferation of PCR-based techniques for mutation detection. Application of PCR protocols to single cell analyses has proven highly successful, and remains the...

Oocyte Identification

Follicle Ultrasound After Clomid

Follicular aspirates are examined under a stereo dissecting microscope with transmitted illumination base and heated stage. Contents of each test tube are aliquoted into two or three Petri dishes, forming a thin layer of fluid that can be quickly, carefully and easily scanned for the presence of an oocyte in the follicular tissue. Low power magnification (6x to 12x) can be used for scanning the fluid, and oocyte identification verified using higher magnification (25* to 50x). The oocyte usually...

Polar Bodies

Genetic analysis of the first and second polar bodies formed during oogenesis has been applied for PGD of both single gene mutations and chromosome aberrations (Chapters 10 and 12). The use of the first polar body was initially premised on two advantages first, the genotype of the oocyte is presumably complementary to that of the first polar body following the first meiotic division and, second, as no manipulation is performed on the embryo itself, further development of the oocyte would not be...

Amplification Refractory Mutation System

The annealing of allele-specific oligonucleotides is the basis of the amplification refractory mutation system ARMS . This employs three oligonucleotide primers one which anneals upstream of the mutation site and two which have sequences that anneal at their 3' terminus to only one of the possible alleles i.e. either mutant or normal . With this technique allele-specific oligonucleotides are not detected directly, but serve as primers for PCR. Amplification of the DNA indicates annealing of the...

Sperm Activation And The Acrosome Reaction

Prior activation of the spermatozoon is a prerequisite for successful fertilization. Activation of the male gamete involves several behavioural, physiological and structural changes, some of which are induced by exposure to environmental signals, and others are induced whilst the spermatozoon is interacting with the oocyte and its extracellular investments. All of these changes are essential for successful fertilization, including changes in motility, capacitation, acrosome reaction,...

Diagnostic And Strategic Considerations

The number of cells present at the blastocyst stage makes blastocyst biopsy more akin to early CVS and therefore, to some, more ethically acceptable as it does not remove any cells that would make up the embryo proper. TE cells can be removed Figure 9.6 Blastocyst biopsy. A hole is made in the zona pellucida and the blastocyst is left until some of the trophectoderm cells herniate through the hole. These can be removed and used for diagnosis. Courtesy of Anna Veiga, Institute Dexeus Figure 9.6...