Preimplantation genetic diagnosis (PGD) was introduced in the late 1980s and represents an option for couples at risk of transmitting an inherited, debilitating or neurological disorder to their children. From a cleavage or blastocyst stage embryo, cell(s) are collected and then genetically analyzed for disease; enabling an unaffected embryo to be transferred into the uterus cavity. Nowadays, PGD has been carried out for several hundreds of heritable conditions including myotonic dystrophy, and for susceptibility genes involved in cancers of the nervous system. Currently, advanced molecular technologies with better resolution, such as array comparative genomic hybridisation, quantitative polymerase chain reaction, and next generation sequencing, are on the verge of becoming the gold standard in embryo preimplantation screening. Given this, it may be time for neurological societies to consider the published evidence to develop new guidelines for the integration of PGD into modern preventative neurology. Therefore, the main aim of this review is to illustrate the option of PGD to enable conception of an unaffected baby, and to assist clinicians and neurologists in the counseling of the patient at risk of transmitting an inherited disease, to explore the genetic journey throughout in vitro fertilization IVF with PGD.

Preimplantation genetic diagnosis (PGD) has become a crucial approach in helping carriers of inherited disorders to give birth to healthy offspring. In this study, we review PGD methodologies and explore the use of amplification refractory mutation system quantitative polymerase chain reaction (ARMS-qPCR) and/or linkage analysis for PGD in neurodegenerative diseases that are clinically relevant with typical features, such as late onset, and which are severely debilitating. A total of 13 oocyte retrieval cycles were conducted in 10 cases with various neurodegenerative diseases. Among the 59 embryos analyzed, 49.2% (29/59) were unaffected and 50.8% (30/59) were affected. Of the 12 embryo transfer cycles, three resulted in pregnancy, and all pregnancies were delivered. The implantation rate and livebirth rate were 23.1% (3/13) per oocyte retrieval cycle and 25.0% (3/12) per embryo transfer cycle. Allele dropout (ADO) was noted in two embryos that were classified as unaffected by ARMS-qPCR but were evidenced as affected after prenatal diagnosis, rendering the false negative rate as 6.3% (2/32). Four among the 13 cycles underwent PGD by ARMS-qPCR coupled with linkage analysis, and all were correctly diagnosed. We conclude that PGD by ARMS-qPCR and/or linkage analysis is a feasible strategy, whereas ADO is a concern when ARMS-qPCR is used as the sole technology in PGD, especially in autosomal dominant diseases.

Background: Preimplantation genetic testing for monogenic disease (PGT-M) has become an effective method for providing couples with the opportunity of a pregnancy with a baby free of spinal muscular atrophy (SMA). Multiple displacement amplification (MDA) overcomes the innate dilemma of very limited genetic material available for PGT-M. Objective: To evaluate the use of MDA, combined with haplotype analysis and mutation amplification, in PGT-M for families with SMA. Methods: MDA was used to amplify the whole genome from single blastomeres or trophectoderm (TE) cells. Exon 7 of the survival motor neuron gene 1 (SMN1) and eleven STRs markers flanking the SMN1 gene were incorporated into singleplex polymerase chain reaction (PCR) assays on MDA products. Results: Sixteen cycles (19 ovum pick-up cycles) of PGT-M were initiated in 12 couples. A total of 141 embryos were tested: 90 embryos were biopsied at the cleavage stage and 51 embryos were biopsied at the blastocyst stage. MDA was successful on 94.44% (85/90) of the single blastomeres and on 92.16% (47/51) of the TE cells. And the PCR efficiency were 98.4% (561/570) and 100% (182/182), respectively. In addition, the average allele drop-out (ADO) rates were 13.3% (60/392) and 9.8% (11/112), respectively. The results for SMN1 exon 7 were all matched with haplotype analysis, which allowed an accurate diagnosis of 93.62% (132/141) embryos. Twelve families had unaffected embryos available for transfer and a total of 38 embryos were transferred in 20 embryo transfer cycles. Eight transfers were successful, resulting in a clinical pregnancy rate of 40% (8/20) and an implantation rate of 28.95% (11/38). Finally, 11 healthy babies were born. Among them, 5 SMA carriers were singleton live births and 3 SMA carriers had twin births. Conclusion: Careful handling during the MDA procedure can improve subsequent PCR efficiency and reduce the ADO rate. We suggest that this protocol is reliable for increasing the accuracy of the PGT-M for SMA.

Spinal muscular atrophy is an autosomal recessive neurodegenerative disease characterised by degeneration of spinal cord motor neurons, atrophy of skeletal muscles, and generalised weakness. It is caused by homozygous disruption of the survival motor neuron 1 (SMN1) gene by deletion, conversion, or mutation. Although no medical treatment is available, investigations have elucidated possible mechanisms underlying the molecular pathogenesis of the disease. Treatment strategies have been developed to use the unique genomic structure of the SMN1 gene region. Several candidate treatment agents have been identified and are in various stages of development. These and other advances in medical technology have changed the standard of care for patients with spinal muscular atrophy. In this Seminar, we provide a comprehensive review that integrates clinical manifestations, molecular pathogenesis, diagnostic strategy, therapeutic development, and evidence from clinical trials.

To develop and apply efficient multiplex preimplantation genetic diagnosis (PGD) protocols for spinal muscular atrophy (SMA).

Two multiplex PGD protocols were developed allowing the detection of the common homozygous deletion of the telomeric spinal muscular atrophy gene (SMN1), together with two microsatellites located on each side of SMN1.

The molecular genetics laboratory of the university hospital in Montpellier.

A couple who had already given birth to a child affected with SMA.

In vitro fertilization using intracytoplasmic sperm injection (ICSI) and blastomere biopsy.

Improvement of PGD for SMA.

Two different multiplex protocols were set up on 81 (multiplex A) and 64 single cells (multiplex B) from normal controls, affected patients, and individuals with homozygous SMN2 deletion. In one PGD cycle that used one of these protocols, two embryos were transferred, which resulted in the birth of a healthy baby.

Analysis of microsatellite markers in addition to the SMN1 deletion allows the detection of contamination, the study of ploidy of the biopsied blastomeres, and the performance of an indirect genetic diagnosis, thereby increasing the reliability of the results. This PGD assay may be applied to all families with the common deletion of SMN1 and also to couples in whom one of the partners carries a small intragenic mutation in SMN1, identified in about 6% of affected individuals who do not lack both copies of SMN1.

Detection of genetic diseases before implantation for couples at risk helps ensure healthy children, but testing for aneuploidy does not improve the chances of live birth in normal infertile women, say Peter Braude and Frances Flinter

To report on our experience with preimplantation genetic diagnosis (PGD) cycles performed for serious genetic disease in relation to the clinical factors affecting outcome.

Retrospective review of data from a single centre.

Tertiary referral PGD centre in a London teaching hospital.

The PGD cycles included 172 cycles for chromosome rearrangements, 96 cycles for single-gene disorders and 62 cycles for X-linked disorders. In vitro fertilisation was the preferred method in chromosome rearrangement and X-linked cases, while intra cytoplasmic sperm injection was used in all single-gene disorders. Appropriate in situ hybridisation fluorescence probes were used in chromosome rearrangement and X-linked cases and polymerase chain reaction was used in single-gene disorders. All pregnancies were followed till delivery.

Live birth rate per PGD cycle started.

Eighty-six percent of cycles started (283) reached oocyte retrieval and 3743 eggs were collected, of which 2086 fertilised normally (55.7%). Two hundred and fifty cycles (76%) had embryos sutiable for biopsy on day 3 of in vitro culture, 1714 embryos were biopsied, and in 205 cycles (62%), there was at least one unaffected embryo available for transfer, resulting in 90 pregnancies, 68 clinical pregnancies and 58 live birth. The live birth rate was 18% per cycle started, 21% per egg retrieval and 28% per embryo transfer which significantly affected the live birth outcome. Woman age, number of eggs collected and achieving cryopreservation of surplus embryos had no statistically significant effect on treatment outcome.

The live birth outcome of PGD cycles for serious genetic disorder is modest and is affected by the number of embryos genetically suitable for transfer.

The molecular basis of spinal muscular atrophy (SMA), an autosomal recessive neuromuscular disorder, is the homozygous loss of the survival motor neuron gene 1 (SMN1). A nearly identical copy of the SMN1 gene, called SMN2, modulates the disease severity. The functional difference between both genes is a translationally silent mutation that, however, disrupts an exonic splicing enhancer causing exon 7 skipping in most SMN2 transcripts. Only 10% of SMN2 transcripts encode functional full-length protein identical to SMN1. Transcriptional activation, facilitation of correct SMN2 splicing, or stabilization of the protein are considered as strategies for SMA therapy. Among various drugs, histone deacetylase inhibitors such as valproic acid (VPA) or 4-phenylbutyrate (PBA) have been shown to increase SMN2-derived RNA and protein levels. Recently, in vivo activation of the SMN gene was shown in VPA-treated SMA patients and carriers. Clinical trials are underway to investigate the effect of VPA and PBA on motor function in SMA patients.

To develop and validate a simple and reliable single-cell analysis protocol for the preimplantation genetic diagnosis (PGD) of spinal muscular atrophy (SMA).

Molecular tests based on specific enzymatic digestion have already been described for SMA diagnosis. We modified the amplified DNA fragments so as to introduce a novel restriction site that provides an internal control for the completeness of the digestion.

The genetics and reproduction departments of two teaching hospitals.

Six informed couples at risk of transmitting SMA.

All patients underwent standard procedures associated with intracytoplasmic sperm injection.

Improvement of SMA diagnostic efficiency and accuracy on single cell.

One hundred fifty lymphocytes were analyzed with our protocol. One hundred percent diagnostic accuracy was achieved from both homozygous normal and SMN1-deleted leukocytes. Successful molecular analysis was achieved for 36 of 42 biopsied embryos (86%). Twenty-five normal embryos were transferred, but no pregnancy was achieved.

We developed an improved protocol for PGD of SMA that is simple, robust, and accurate; unfortunately, no pregnancies were achieved for any of the six patients who have undergone PGD in the program thus far.

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.

Spinal muscular atrophy (SMA) type I is an autosomal recessive disorder characterized by loss of lower motor neurons in the spinal cord. This severe hereditary neurodegenerative disorder is an important cause of morbidity in the neonate and the leading hereditary cause of infant mortality. The characteristic degeneration of anterior horn cells in the spinal cord leads to progressive muscular weakness and atrophy of the skeletal muscles. In SMA type I, the most severe form of SMA, death usually ensues by 2 years of age from respiratory failure or infection. Accurate diagnosis is now available through genetic testing, and progress is being made toward the development of therapy based on understanding of the disease mechanism. The neonatal nurse plays a pivotal role in identifying and caring for these medically fragile infants and in providing support and education for parents and families.

To establish strategies for the implementation of a successful preimplantation genetic diagnosis (PGD) service.

Retrospective review of data from a single center.

A United Kingdom National Health Service hospital.

Patients (60 couples) were referred for PGD from UK genetic centers.

We followed the protocol of ovarian stimulation, oocyte retrieval, fertilization, single cell biopsy on day 3, and embryo transfer on day 4. Pregnancies unaffected by the familial genetic condition.

A total of 60 couples was treated for 20 different conditions. Early cycles using nonsequential embryo culture media were less successful (13% pregnancy rate/embryo transfer) than later cycles using sequential media (33.5%). Ninety-four percent of embryos (n = 473) had a single cell removed at biopsy. The overall pregnancy rate was 24% per cycle started, 29% per egg collection, 38% per transfer, and 40% per couple treated. In one cycle, an affected pregnancy followed PGD for spinal muscular atrophy (SMA).

The use of sequential media and single cell biopsy results in a successful PGD program with encouraging pregnancy rates.

Preimplantation genetic diagnosis (PGD) has become an established procedure for avoiding the birth of affected children with single gene disorders. PGD is performed through polar body or blastomere biopsy, which has no deleterious effect on pre- and post-implantation development. This review describes the most recent developments and current changes in the spectrum of conditions for which PGD has been applied. The most recent applications of PGD include congenital malformations, blood group incompatibility and an increasing number of late onset disorders with genetic predisposition, all of which have not previously been diagnosed using PGD. Despite ethical concerns, PGD has also been used for preselection of unaffected and HLA matched embryos, and recently for preimplantation HLA matching without testing for the causative gene. This extends the practical value of PGD, with its utility being no longer limited to prevention of single gene disorders, by expanding it to treatment of siblings requiring stem cell transplantation.

Ongoing trends in prenatal diagnosis aim at early, rapid, and ideally noninvasive diagnosis as well as at the improvement of risk-screening for aneuploidy. Interphase-fluorescence in situ hybridization and quantitative fluorescence polymerase chain reaction are efficient tools for the rapid exclusion of selected aneuploidies in addition to the established direct preparation of chromosomes from chorionic villi. Interphase fluorescence in situ hybridization has also made possible the diagnosis of selected chromosome abnormalities in single cells (e.g. in preimplantation genetic diagnosis) or noninvasive diagnosis. More complex multicolor fluorescence in situ hybridization approaches are currently being evaluated. Single cell polymerase chain reaction is the key technique for the molecular diagnosis of a growing number of monogenic conditions before implantation or, still more experimental, in fetal cells retrieved from the maternal circulation. New sources for noninvasive diagnosis came into play such as fetal DNA or cell nuclei in maternal plasma. The combination of biochemical parameters in the maternal serum, namely free beta-human chorionic gonadotropin with pregnancy associated plasma protein A and sonographic markers, has already dramatically increased the sensitivity of risk screening in the first trimester of pregnancy.