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Research question Can next-generation sequencing (NGS) based on copy number variation sequencing (CNV-Seq) identify normal/balanced embryos in balanced reciprocal translocation carriers and what are their reproductive outcomes? Design One hundred couples with balanced reciprocal translocation who underwent a total of 134 preimplantation genetic testing (PGT) cycles between January 2015 and October 2017 were evaluated. Trophectoderm cells of blastocysts were biopsied for CNV-Seq-based NGS. All the balanced/normal blastocysts were vitrified and cryopreserved. Single balanced/normal blastocysts were warmed and transferred in the subsequent frozen embryo transfer (FET) cycle. Results During the study period, 400 blastocysts were analysed by NGS-PGT, of which 109 (27.25%) were balanced and euploid. A total of 52 blastocysts were transferred in the FET cycle. Clinical pregnancy was confirmed in 34 women (65.38%), with a miscarriage rate of 2.94%; 26 healthy term babies were born, including 24 singletons and one set of twins, while eight couples had ongoing pregnancies. Amniocentesis revealed a fetal chromosome status that was consistent with the NGS-PGT results. Female carriers had a significantly higher blastocyst rate than did the male carriers (37.01% versus 31.27%, P = 0.04). The transferable blastocyst rate was higher in couples treated with gonadotrophin-releasing hormone (GnRH) antagonist than in those treated with GnRH agonist (38.20% versus 24.37%, P = 0.01). However, neither carrier sex nor ovarian stimulation protocol influenced the clinical pregnancy rate. Conclusions CNV-Seq-based NGS is an efficient and reliable PGT method for balanced reciprocal translocation.

Research question: Does newborn gender affect placental histopathology pattern and perinatal outcome in singleton live births following IVF treatment? Design: Retrospective cohort study evaluating data of all live births from one academic tertiary hospital following IVF treatment during 2009–2017. All patients had placentas sent for pathological evaluation irrelevant of maternal and fetal complications status. Exclusion criteria were abnormal uterine cavity findings, previous uterine surgery, in-vitro maturation cycles, gestational carrier cycles, oocyte recipient cycles, preimplantation genetic diagnosis cycles and multiple pregnancies. The primary outcomes included anatomical, inflammation, vascular malperfusion and villous maturation placental features. The secondary outcomes included fetal, maternal, perinatal and delivery complications. A multivariate analysis was conducted to adjust the results for factors potentially associated with placental pathology features. Results: A total of 1057 live births were included in the final analysis and were allocated to the study groups according to fetal gender: males (n = 527) and females (n = 530). After adjustment for potential confounding factors, male gender was significantly associated with villous agglutination (odds ratio [OR] 9.8; 95% confidence interval [CI] 1.4–78.2), avascular villi (OR 4.1; 95% CI 1.3–12.6) and maternal vascular malperfusion (OR 1.8; 95% CI 1.2–2.7). Female gender was significantly associated with bilobed placenta (OR 0.2; 95% CI 0.06–0.8) and subchorionic thrombi (OR 0.5; 95% CI 0.3–0.9). The prevalence of adverse fetal, maternal and delivery outcomes was similar between the groups. Conclusions: Newborn gender has a significant impact on the placental histopathology pattern, which can contribute to the development of adverse perinatal outcomes.

Recurrent pregnancy loss (RPL) is a common, yet elusive, complication of pregnancy. Among couples at high risk of RPL, such as those carrying a structural chromosomal rearrangement, preimplantation genetic diagnosis (PGD) has been proposed as a tool to improve live birth rates and reduce the incidence of miscarriage; however, no clear consensus has been reached on its benefits in this population. This systematic review summarizes existing published research on the effect of PGD on pregnancy outcomes among carriers of chromosomal abnormalities with RPL. A comprehensive search of common databases was conducted, which yielded 20 studies. Meta-analysis was precluded owing to significant heterogeneity between studies. The primary outcome of interest was live birth rate (LBR), and a pooled total of 847 couples who conceived naturally had a LBR ranging from 25–71% compared with 26.7–87% among 562 couples who underwent IVF and PGD. Limitations of the study include lack of large comparative or randomized control studies. Patients experiencing RPL with structural chromosomal rearrangement should be counselled that good reproductive outcomes can be achieved through natural conception, and that IVF–PGD should not be offered first-line, given the unproven benefits, additional cost and potential complications associated with assisted reproductive technology.

The aim of this study was to determine whether patients with transthyretin-related hereditary amyloidosis (V30M), after transplantation or under tafamidis treatment, have normal gamete reproductive capacity. A retrospective analysis was carried out of all preimplantation genetic diagnosis (PGD) cycles performed in patients with the V30M mutation. The groups analysed were: total cases with V30M, female cases with V30M and male cases with V30M. Detailed demographic, stimulation, embryological, clinical and newborn outcomes were evaluated. Comparisons revealed that patients have a high likelihood of achieving a live birth per PGD treatment cycle (48%). This is the first large report on patients with the V30M mutation treated with PGD. The high rate of live birth obtained should represent a strong stimulus for patients to use PGD as it proved to be effective and safe. As a neurodegenerative disease that leads to death, it is of maximum importance that it could be eradicated using PGD in order to definitively avoid the transmission of the disease.

Research question What are the incidence and patterns of meiotic trisomies and recombination separately and in relation to each other at the blastocyst stage via single nucleotide polymorphism genotyping combined with array comparative genomic hybridization. Design Single nucleotide polymorphism microarrays were carried out on a total of 1442 blastocyst stage embryos derived from 268 fertile couples undergoing preimplantation genetic diagnosis for the purposes of avoiding transmittance of known single gene disorders to their offspring; 24-chromosome aneuploidy screening via array comparative genomic hybridization was carried out in parallel. Results One hundred per cent of meiotic trisomies identified in these embryos were of maternal origin and their incidence increased significantly with advancing maternal age (P < 0.0001). A total of 55.8% of meiotic trisomies were meiosis I-type and 44.2% were meiosis II-type. Certain chromosomes were affected more by meiosis I-type errors, whereas others experienced more meiosis II-type errors. A detailed recombination analysis was carried out for 11,476 chromosomes and 17,763 recombination events were recorded. The average number of recombination sites was 24.0 ± 0.3 for male meiosis and 41.2 ± 0.6 for female meiosis (autosomes only). Sex-specific differences were observed in the locations of recombination sites. Comparative analysis conducted between 190 euploid embryos and 69 embryos presenting maternal meiotic trisomies showed similar recombination rates (P = 0.425) and non-recombinant chromatid rates (P = 0.435) between the two categories; differences, however, were observed when analysing embryos affected with specific maternal meiotic trisomies. Conclusions This study yielded unique data concerning recombination and the origin of aneuploidies observed during the first few days of life and provides a novel insight into these important biological processes.

Preimplantation genetic diagnosis (PGD) aims to test the embryo for specific conditions before implantation in couples at risk of transmitting genetic abnormality to their offspring. The couple must undergo IVF procedures to generate embryos in vitro. The embryos can be biopsied at either the zygote, cleavage or blastocyst stage. Preimplantation genetic screening uses the same technology to screen for chromosome abnormalities in embryos from patients undergoing IVF procedures as a method of embryo selection. Fluorescence in-situ hybridization was originally used for chromosome analysis, but has now been replaced by array comparative genomic hybridization or next generation sequencing. For the diagnosis of single gene defects, polymerase chain reaction is used and has become highly developed; however, single nucleotide polymorphism arrays for karyomapping have recently been introduced. A partnership between IVF laboratories and diagnostic centres is required to carry out PGD and preimplantation genetic screening. Accreditation of PGD diagnostic laboratories is important. Accreditation gives IVF centres an assurance that the diagnostic tests conform to specified standards. ISO 15189 is an international laboratory standard specific for medical laboratories. A requirement for accreditation is to participate in external quality assessment schemes.

Preimplantation genetic diagnosis (PGD) was carried out for a couple carrying a de-novo deletion in the TSC2 gene, responsible for tuberous sclerosis. Karyomapping, a method employing genome-wide analysis of single nucleotide polymorphisms (SNP), was used as PGD protocol. Analysis of DNA from the affected parent using karyomapping confirmed the region covered by the deletion and revealed more than 30 SNP located within the affected region. These SNP were subsequently used for embryo diagnosis (deletion revealed by hemizygosity and/or reduced probe intensity). Seven blastocyst embryos underwent trophectoderm biopsy followed by vitrification. Biopsied cells were subjected to comprehensive aneuploidy screening using microarray comparative genomic hybridization (aCGH), with karyomapping for the detection of embryos carrying the mutant TSC2 gene carried out in tandem. Two embryo transfers were performed, the second of which resulted in the birth of a child. This study highlights that karyomapping may be applicable to a subset of de-novo mutations undetectable using standard PGD strategies. Additionally, karyomapping results were in complete concordance with aCGH, both methods revealing the same aneuploidies in the embryos tested. It was concluded that karyomapping may represent a valuable advance in cases of PGD for monogenic diseases.

Massively parallel genome sequencing, also known as next-generation sequencing (NGS), is the latest approach for preimplantation genetic diagnosis. The purpose of this study was to determine whether NGS can accurately detect aneuploidy in human embryos. Low coverage genome sequencing was applied to trophectoderm biopsies of embryos at the blastocyst stage of development. Sensitivity and specificity of NGS was determined by comparison of results with a previously validated platform, array-comparative genomic hybridization (aCGH). In total, 156 samples (116 were blindly assessed) were tested: 40 samples were re-biopsies of blastocysts where the original biopsy specimen was previously tested for aCGH; four samples were re-biopsies of single blastomeres from embryos previously biopsied at the cleavage stage and tested using aCGH; 18 samples were single cells derived from well-characterized cell lines; 94 samples were whole-genome amplification products from embryo biopsies taken from previous preimplantation genetic screening cycles analysed using aCGH. Per embryo, NGS sensitivity was 100% (no false negatives), and 100% specificity (no false positives). Per chromosome, NGS concordance was 99.20%. With more improvement, NGS will allow the simultaneous diagnosis of single gene disorders and aneuploidy, and may have the potential to provide more detailed insight into other aspects of embryo viability.

Research question Mutations of the beta-globin gene ( HBB ) cause beta-thalassaemia and sickle cell anaemia. These are the most common cause of severe inherited disease in humans. Traditional preimplantation genetic testing protocols for detecting HBB mutations frequently involve labour intensive, patient-specific test designs owing to the wide diversity of disease-associated HBB mutations. We, therefore, asked the question whether a universally applicable preimplantation genetic testing method can be developed to test for HBB gene mutations. Design A multiplex polymerase chain reaction protocol was designed, allowing simultaneous amplification of multiple overlapping DNA fragments encompassing the entire HBB gene sequence in addition to 17 characterized, closely linked single nucleotide polymorphisms (SNP). Amplicons were then analysed using a next-generation sequencing method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified DNA derived from embryos of three couples carrying different combinations of beta-thalassaemia mutations. Results The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Analysis of 141 heterozygous sites showed no instances of allele dropout and the test displayed 100% concordance compared with the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive next-generation sequencing may provide superior accuracy than typically achieved using traditional preimplantation genetic testing methods. Importantly, no patient-specific test design or optimization was needed. Conclusions It is hoped that protocols that deliver almost universally applicable low-cost tests, without compromising diagnostic accuracy, will improve patient access to preimplantation genetic testing, especially in less affluent parts of the world.

Introduction Despite the use of more precise techniques that indicates greater incidence, meiotic segregation of interchromosomal insertion has rarely been studied. Theoretically, the risk to have a child at term with malformation or mental retardation is high but there are no tools to determine this risk. We have investigated the Preimplantation genetic diagnosis (PGT) management of patients with interchromosomal insertion (IT) by fluorescent in situ hybridization (FISH) to obtain a better understanding and improve the genetic counseling. Material & methods Two couples were enrolled in our PGT unit. The male of the first couple carries the IT 46,XY,ins(4;11)(q22;p12p13) and the female karyotype of the second couple is 46,XX,ins(2;10)(q21;q25.2q22). We have investigated, by FISH technique, the meiotic segregation analysis of the sperm of the IT male carrier and embryos providing from the two carriers. One FISH analysis round was performed for the male IT carrier. Five specific probes of chromosomes 4 and 11 were applied to nuclei from spermatozoa and blastomeres from embryo biopsy. An independent X2 test was used to compare abnormal spermatozoa from the patient and the control. Two FISH analysis rounds were performed for the female IT carrier. Six specific probes of chromosomes 2 and 10 were applied to blastomeres of PGT embryos. Result(s) The sperm-FISH segregation analysis showed a significantly increased percentage of unbalanced gametes (52.7%) compared with the control sperm (3.4%, p Conclusion(s) These results permit to reassure the physicians in the management of IT carriers and better guide the genetic counseling. PGT is a suitable diagnostic tool for IT carriers. Sperm FISH analysis contributes to a better IT behavior and segregation understanding. The reanalysis of not transferred whole embryos is essential to avoid misdiagnosis and therefore to contribute to a better IT behavior and segregation understanding. Several studies are needed to specify the risk to ITs carriers and to better assess the risk incurred by the couples.

Introduction Preimplantation genetic diagnosis (PGD) has become a crucial approach to help carriers of inherited disorders give birth to healthy offspring. Here, we review the PGD methodologies and explore the use of polymerase chain reaction (PCR)-based technologies for PGD in neurodegenerative diseases that are clinically relevant and have typical features such as late-onset and severely debilitating. Material and Methods Trophectoderm biopsies were performed at Day5/6 blastocyst stage. PGD using amplification refractory mutation system quantitative PCR (ARMS-qPCR) and/or linkage analysis was performed for 10 cases of various neurodegenerative diseases, underwent 13 oocyte retrieval cycles for in vitro fertilization (IVF) with PGD at the core laboratory of Dr. Ming Chen, a major PGD laboratory in Taiwan, during 2013-2016. Results A total of 13 oocyte retrieval cycles were conducted. Among the 59 embryos analyzed, 49.2% (29/59) were unaffected and 50.8% (30/59) were affected. There were 12 embryo transfer cycles, of which 3 became pregnant 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, respectively. 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 of them were correctly diagnosed. Conclusions PGD by PCR-based methods (e.g., ARMS-qPCR and linkage analysis) is a feasible strategy whereas ADO is always a concern if ARMS-qPCR is used as the sole technology in PGD, especially in autosomal dominant diseases. Robust methodologies, proper genetic counseling that covers technical and ethical aspects of genetic testing, and confirmatory invasive prenatal diagnosis are important in PGD of neurodegenerative diseases.

Technological developments in gene editing raise high expectations for clinical applications, first of all somatic gene editing but in theory also germline gene editing. The latter is currently not allowed in many countries. This makes clinical applications in these countries impossible now, even if germline gene editing would become safe and effective. What were the arguments behind this legislation, and are they still convincing? If a technique can help to avoid serious genetic disorders, in a safe and effective way, would this be a reason to reconsider earlier standpoints? The European Society of Human Reproduction and Embryology (ESHRE) and European Society of Human Genetics (ESHG) together developed a Background Document and Recommendations to inform and stimulate ongoing societal debates. After consulting its membership and experts, this final version of the Recommendations was endorsed by the Board and Executive Committee of the Societies in April/May 2017. Taking account of ethical arguments, it is argued that both basic and preclinical research regarding human germline gene editing can be justified on conditions. Furthermore, while clinical germline gene editing would be totally premature, it might become a responsible intervention in the future, but only after adequate preclinical research. Safety of the child and future generations is a major concern. Future discussions must also address priorities among reproductive and potential non-reproductive alternatives, such as preimplantation genetic diagnosis and somatic editing if that would be safe and successful. The prohibition of human germline modification, however, needs renewed discussion among relevant stakeholders, including the general public and legislators.

Introduction Embryonic mosaicism is defined as the presence of multiple distinct cell lines within an embryo. Mosaicism is relatively common in preimplantation embryos derived from IVF and can be detected from trophectoderm (TE) biopsy provided we use a high resolution diagnostics platform (Ion Torrent). It is well known that advanced maternal age is linked to higher incidence of embryo aneuploidy. However, the relationship between mosaicism rate and maternal age is still unclear. Therefore, the purpose of this retrospective study is to investigate the relationship between mosaicism rate and maternal age in Alpha Fertility Centre, Malaysia from August 2017 to February 2018. Materials & Methods Four hundred and ten (410) patients (age range 18 – 47, mean age 30.7) underwent IVF cycles and their blastocysts were cultured to blastocyst stage. These patients were divided into 2 age groups, Group A (≤35 years, mean age 27.4) and Group B (>35 years, mean age 38.6). One thousand and two (1002) blastocysts from Group A and four hundred and twenty-nine (429) blastocysts from Group B with at least fair grade (Gardner, 1999) were biopsied respectively. Number of biopsy cells ranged from 2 to 10. All biopsied samples were subjected to preimplantation genetic screening (PGS) with next-generation sequencing (NGS). All samples were amplified and DNA libraries were constructed for sequencing using Ion TorrentTM PGM benchtop sequencer according to manufacturer's specifications (Ion Torrent, USA). The results were analysed by using ReproSeq Mosaic PGS w1.1 workflow (Ion Reporter Version 5.4). By following guidelines provided by Preimplantation Genetic Diagnosis International Society (PGDIS) 2016 , embryos with less than 20% aneuploidy in the TE sample were reported as euploid, over 80% aneuploidy were considered as aneuploidy and the remaining 20% to 80% aneuploidy were mosaic. Results Of the 1431 blastocysts assessed for ploidy status, 181 blastocysts were mosaic in Group A and 40 blastocysts were mosaic in Group B. Mosaicism rate for Group A was 18.1% (181/1002) and 9.3% (40/429) for Group B. The mosaicism rate for patients in Group B were significantly lower compared to patients in Group A (p=0.002). Conclusions Escudero et al, 2016 reported that mosaicism is independent of maternal age. However, our study shows that mosaicism rate is significantly higher in patients aged ≤35 years as compared to patient aged >35 years. Therefore, mosaicism rate might show correlation with maternal age. Nevertheless, further studies should be carried out to ascertain the relationship between maternal age and mosaicism rate.

Large scale whole genome and exome sequencing is uncovering a plethora of novel mutations that cause highly penetrant, early-onset, severe, or later-onset life-threatening dominant and recessive disorders. For couples who are known carriers of mutant alleles, preimplantation genetic diagnosis enables the detection of genetic disorders in embryos that have been fertilized in vitro, thereby avoiding their transmission to offspring. Traditional PGD methods require a mutation and family specific work-up. We and others have developed generic methods that can be readily applied for all transmitted genetic disorders. They are termed karyomapping and haplarithmisis. The methods reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes byemploying phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions. I will present the principles, the use and the results of its implementation since clinical implementation. The introduction of genome wide screening of embryo's raised novel ethical questions. The principles guiding embryo selection and prioritization that are applied at our center according to the chromosomal content and mutational load of the embryos, will be presented. I will present novel technical developments broadening the scope of use. In addition, I will present new insights in the origins and mechanisms leading to early meiotic and mitotic errors as well as on the origin of mixoploid and chimaeric embryos.

Introduction Thalassaemia affects 4.5% of the population in Malaysia. Complications of β- thalassaemia major in children pose a heavy load on transfusion and paediatric services. Preimplantation Genetic Diagnosis (PGD) in conjunction with IVF to avoid transferring affected blastocyst is one strategy to eradicate this disease. Allele drop out in PGD however may lead to misdiagnosis. Single nucleotide polymorphism (SNP) is a useful marker to confirm the inheritance of the parental wild-type or mutant allele thus increasing the accuracy of the diagnosis. The objective of this study is to evaluate the efficiency of an in-house developed SNP assay for PGD used in conjunction with aneuploidy screening. Methodology Beta-globin gene common mutation at CD41/42 and IVS2-654 were studied. Haplotyping was carried out to determine the SNPs that present in the wild-type and mutant allele of the parent. Five pairs of primers were designed specifically to amplify the region containing potential SNPs (3 pairs) and the mutations stated earlier (2 pairs). A total of 27 blastocysts were biopsied from 3 couples. Biopsies were amplified using Sureplex whole genome amplification kit. Aliquots of amplified DNA were subjected to second round of amplification to determine the presence of SNP and the mutation. Gel electrophoresis and sequencing were carried out and the resulting sequences were aligned. The genotype of the blastocysts was compared and confirmed with the SNP that present either in the wild-type or the mutant allele. The unaffected and heterozygous carrier blastocysts will subsequently be subjected to aneuploidy screening using Veriseq PGS Protocol (Illumina). Only blastocysts that were not affected or aneuploidy were suitable for blastocyst transfer. Results Of the 27 blastocysts tested for beta globin gene mutation at CD41/42 and IVS2-654, 7 was shown to be unaffected, 11 heterozygous carriers for thalassaemia and 9 were affected. For the first couple, the SNP is present in the paternal wild-type allele while for second couple, the SNP is present in the maternal mutant allele. For third couple the SNP is present in the paternal mutant allele and maternal wild-type allele. The results from SNP analysis were then compared with the genotype results obtained from direct sequencing on the targeted mutation, and the comparison showed 100% concordance. Aneuploidy screening on unaffected and heterozygous carrier blastocyst revealed 6 euploid, 7 mosaic and 5 aneuploid blastocysts. Five blastocysts (3 unaffected euploid, 1 heterozygous carrier euploid and 1 heterozygous carrier mosaic) were transferred with three successful implantations (2 unaffected euploid and 1 heterozygous carrier mosaic). Conclusion The presence of wild-type allele means that the blastocyst is either unaffected or is a heterozygous carrier and vice-versa. On the other hand, the presence of mutant allele means that the blastocyst is either affected or is a heterozygous carrier and vice-versa. This in-house developed SNP assay shows concordance results with the established mutation screening assay. The combination of SNP and mutation screening assay improves the accuracy of PGD by minimizing the risk of misdiagnosis caused by allele drop out.

Introduction Numerous publications have demonstrated that preimplantation genetic diagnosis (PGD) increases the implantation rate and percentage of healthy newborns or at least decreases themiscarriage rate for translocation carriers with recurrent pregnancy losses (Fisher et al. 2010, Ikumaet al. 2018). The aim of this study was to evaluate whether VeriSeq PGS, a next generation sequencingbasedmethod for aneuploidy screening, is also suitable for the detection of structural aberrations inpolar bodies and trophectoderm cells. Since the product provider didn´t validate the method forunbalanced structural changes, our goal was to determine its sensitivity. Material & methods Array-based preimplantation genetic diagnostic was performed on polar bodies obtained from 12 women as well as on trophectoderm samples from five couples being translocation carriers. Furthermore, polar bodies from four women carrying other structural aberrations (i.e.versions, insertions, duplications) were included in the study. Whole genome amplification (WGA)was performed using the SurePlex DNA Amplification System. Cy3-labelled sample DNA and Cy5-labelled male reference DNA were hybridized for 4-16 hours on 24sure+ arrays. The slides were washedand scanned according to the manufacturer´s protocol. NGS libraries (VeriSeq PGS, Illumina Inc.) wereprepared from the same WGA products and sequenced on a MiSeq as described by the manufacturer. Data obtained with both methods were analyzed using the BlueFuse Multi Software. Fisher's exact testwas applied for statistical analyses. Results Reciprocal translocations present on a total of 18 chromosomes were included in this study. The results obtained with next generation sequencing in general delivered an excellent signal-to- noiseratio and a high dynamic range. Although, no statistically significant differences in the detection ofstructural variants between aCGH and NGS were observed, the comparison of both methods revealedseveral discordant results. In three cases, only one out of two translocated segments was detectableby NGS, whereas the aCGH method identified both fragments. By contrast, in the cohort with otherstructural aberrations, a 17 Mb insertion of chromosome 3 was detected only by NGS. Conclusions Our data indicate that although it is possible to analyze structural aberrations in singlecells with VeriSeq PGS, this method has some limitations in the detection of small telomericimbalances. The detection of structural aberrations is not only determined by their size but moreimportantly, it is locus-dependent. Hence, unbalanced reciprocal translocations with both translocatedfragments smaller than 8 Mb containing repetitive or paralogous sequences, might not be detectablewith VeriSeq PGS resulting in false negative results. Therefore, PGD for such translocations requires aprevalidation by e.g. sequencing of single sperm cells or unbalanced products of conception in orderto determine the locus-specific detection sensitivity.

Preimplantation genetic diagnosis (PGD) for human leukocyte antigen (HLA) typing is an established procedure for conceiving a child who may donate cord blood or haematopoietic stem cells for transplantation to save an ill sibling. Haematopoietic stem cell transplantation (HSCT) from related matched donors improves overall survival compared with unrelated or non-matched donors. Since HSCT from related matched-donors is unavailable for 70% of patients, IVF for PGD-HLA is a relevant clinical option. Recent success of HSCT after PGD-HLA, and excellent health and family support of the children born, suggests that debate over this kind of 'designer baby' and 'gift of life' should subside. Discussions about IVF for PGD-HLA should be held with families when a related matched-donor is unavailable, when HSCT can wait at least 9-12 months, within weeks of diagnosis irrespective of prognosis, and when the mother is of reproductive age. Related half-matched egg donors may also be considered. National and international collaborations should be established, and couples choosing this modality should be referred to experienced IVF and PGD centres. Clinical guidelines will improve physician and patient awareness of IVF for PGD-HLA and its role in advancing the clinical care of children in need of HSCT.

Preimplantation genetic diagnosis (PGD) for monogenic disorders has the drawback of time and cost associated with tailoring a specific test for each couple, disorder, or both. The inability of any single assay to detect the monogenic disorder in question and simultaneously the chromosomal complement of the embryo also limits its application as separate tests may need to be carried out on the amplified material. The first clinical use of a novel approach ('karyomapping') was designed to circumvent this problem. In this example, karyomapping was used to confirm the results of an existing PGD case detecting both chromosomal abnormalities and a monogenic disorder (Smith-Lemli-Opitz [SLO] syndrome) simultaneously. The family underwent IVF, ICSI and PGD, and both polar body and cleavage stage biopsy were carried out. Following whole genome amplification, array comparative genomic hybridisation of the polar bodies and minisequencing and STR analysis of single blastomeres were used to diagnose maternal aneuploidies and SLO status, respectively. This was confirmed, by karyomapping. Unlike standard PGD, karyomapping required no a-priori test development. A singleton pregnancy and live birth, unaffected with SLO syndrome and with no chromosome abnormality, ensued. Karyomapping is potentially capable of detecting a wide spectrum of monogenic and chromosome disorders and, in this context, can be considered a comprehensive approach to PGD.

Myotonic dystrophy (DM) is the most common form of muscular dystrophy in adults. There are conflicting reports about its effect on female fertility. This study investigated ovarian reserve and IVF-preimplantation genetic diagnosis (PGD) outcome in women with DM1. A total of 21 women undergoing PGD for DM1 were compared with 21 age- and body mass index-matched women undergoing PGD for other diseases. Ovarian reserve markers, response to stimulation, embryo quality and clinical pregnancy and live birth rates were compared. Day-3 FSH concentration was higher, while anti-Mullerian hormone concentration and antral follicle count were lower in the DM1 group (median, range: 6.9 (1.8-11.3) versus 5.7 (1.5-10.7)IU/l; 0.9 (0.17-5.96) versus 2.68 (0.5-9.1)ng/ml; and 13 (0-63) versus 23 (8-40) follicles, respectively, all P < 0.05). Total FSH dose was higher (5200 versus 2250 IU, P = 0.004), while the numbers of oocytes retrieved (10 versus 16, P < 0.04) and metaphase-II oocytes (9 versus 12, P < 0.03) were lower in the DM1 group. The number of cycles with top-quality embryos and the clinical pregnancy rate were lower in the DM1 group. In conclusion, there is evidence of diminished ovarian reserve and less favourable IVF-PGD outcome in women with DM1. Myotonic Dystrophy (DM) is the most common form of muscular dystrophy in adults. There is evidence of subfertility in males affected with the disease but conflicting reports about the effect of the disease on female fertility. The aim of our study was to investigate ovarian reserve and IVF-PGD results in women with DM. Twenty-one women undergoing preimplantation genetic diagnosis (PGD) treatment for DM were compared to 21 age- and BMI matched women undergoing PGD treatment for other diseases. The two groups were compared for antral follicle count (AFC) and serum anti-Mullerian hormone (AMH) levels (the best known markers of ovarian reserve and fertility potential), ovarian response, embryo quality and pregnancy and live birth rates. AFC and the AMH levels were statistically significant lower in the DM group. Total medication dose needed for ovarian stimulation was higher, the number of oocytes and mature oocytes retrieved, and the number of cycles with top quality embryos were lower in the DM group compared to the controls. In conclusion, there is evidence of diminished ovarian reserve, and less favorable IVF-PGD outcome in women with DM. Therefore, we recommend advising these women about the possibility of early decreasing ovarian function in order to prevent any delay in reproductive planning.

The objective of this retrospective study was to investigate the incidence and clinical implications of multinucleation in blastomeres biopsied from cleavage-stage embryos obtained from patients undergoing preimplantation genetic screening (PGS) for aneuploidies or preimplantation genetic diagnosis (PGD) for translocations or single-gene defects (SGD). A total of 3515 embryos were obtained from 306 couples in 380 PGD or PGS cycles. Incidence of multinucleation, chromosomal complement in multinucleated (MN) and sibling embryos and the characteristics of MN embryos resulting in healthy births were investigated. Of all cycles, 41.3% involved at least one MN embryo. There were more uniformly diploid than uniformly haploid nuclei (22.0% versus 7.9%, P0.01). The most common form of abnormality was chaotic chromosomal complement (39.9%, 147/368). Transfer of embryos that had MN blastomeres free of the genetic abnormalities tested resulted in three healthy deliveries. It is concluded that, although the majority of MN blastomeres are chromosomally abnormal, healthy births are possible after transfer of embryos containing these blastomeres subjected to genetic analysis. As far as is known, this is the first report of healthy births after transfer of embryos with MN blastomeres tested for translocations or SGD in PGD cycles. Preimplantation genetic diagnosis (PGD) is an established method for selecting genetically healthy embryos for transfer. A blastomere sampled from the developing embryo is subjected to genetic analysis. Some of these blastomeres may contain multiple nuclei, complicating the genetic diagnosis. We investigated clinical implications of multinucleation in PGD cycles. Our results indicate that majority of the multinucleated blastomeres, and consequently embryos, are genetically abnormal. However, healthy births are possible after transfer of multinucleated embryos that are free of the genetic abnormalities screened.

Introduction Robertsonian translocation (RT) results from centric fusion of two acrocentric chromosomes. RT carriers are phenotypically normal, but they are known to be at increased risk of repeated miscarriages or of pregnancies resulting in the birth of a child with congenital anomalies, mental retardation. Preimplantation Genetic Diagnosis (PGD) is therefore a solution for RT carriers. A suitable probe strategy on cleavage stage allows us to differentiate balanced embryos, unbalanced embryos, mosaic and chaotic embryos. According to ESHRE recommendations, only balanced embryos are considered for transfer. However, transfer of some mosaic embryos has resulted in live births. We performed the first comparative analysis between a two or a three probes FISH strategies, in order to enhance the safety and security of embryos transferred and to establish the best reliable and low-cost strategy for RT PGD management. Material and methods Retrospective analysis of 253 preimplantation genetic diagnosis (PGD) cycles were performed on 91 men and 42 women carriers of different Robertsonian translocations t(13;14), t(14;21), t(14;15), t(15;21), t(13;15), t(13;21), t(14;22), t(15;22), t(21;22), t(13;13). The first strategy involved two relevant probes use. Each probe is located within the terminal long arm. The second strategy required a third probe addition on one of the two translocated chromosomes. The additional probe is located close to centromeric regions. Result(s) Regardless of probe strategies, 253 PGD cycles were performed in the aim to exclude imbalances in both carriers. On a total of 1,549 biopsied cells, 1,458 were successfully analyzed. Among these, 232 were transferred. Clinical pregnancy was achieved in 108 patients, and 85 healthy babies were delivered. No significant difference was observed in the balanced embryo rates (40.5% and 40.4% in a two probes and a three probe strategies respectively). Among these balanced embryos, diagnostic established on single or two mononucleate cells, wasn't affected by the probe strategies. However, a significant difference (p Conclusion(s) According to the present findings, concerning balanced embryo rates, the two probes strategy seems more suitable for Robertsonian translocation PGD analysis, firstly because it led to similar results as the three probes strategy. Secondly, this study also highlighted an increase mosaic embryo rate observed with the two probes strategy. Recent data indicated that mosaic embryos may represent a second category to transfer after balanced embryos. Obviously, balanced embryos must be transferred in priority. However in absence of balanced embryos, transfer of a mosaic embryo should be considered. In this case, genetic counseling about risks and potential benefits is of utmost importance to ensure informed decision-making by patients.

Introduction Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy with incidence of 1 in 8,000 worldwide. The relationship between DM1 and female infertility remains controversial. This study investigated ovarian reserve, ovarian response to stimulation and IVF-PGD outcome in women with DM1 Materials & Methods This study included female patients with DM1 who performed IVF-PGD cycles in Cheil General Hospital & Women's Healthcare Center from January 2013 to December 2017. During this period, a total of 12 women undergoing PGD for DM1 were compared with 10 age matched women undergoing PGD for X-linked disorders (XLD). Ovarian reserve markers, response to controlled ovarian stimulation, embryo quality, and clinical pregnancy rate were compared. Results Day 3 FSH, E2 and anti-mullerian hormone concentration (median, range: 7.5 (4.6-14.8) verse 7.0 (5.7-10.0) mIU/ml; 22.3 (15.9-53.2) verse 27.5 (9.4-54.3) pg/ml; 1.8 (0.7-7.3) verse 3.6 (1.0-7.5) ng/ml, respectively), the mean number of oocytes retrieved and metaphase II oocytes (15.7±12.7 verse 15.6±6.2; 11.9±10.3 verse 12.4±4.9 respectively), fertilization rate (77.3% verse 83.9%) and prevalence of top quality embryo (39.3% vs 33.4%) showed no significant difference between DM1 and XLD groups. Clinical pregnancy rates (17% vs 21%) were similar in both groups Conclusions The results showed that DM1 does not influence ovarian reserve and reproductive outcomes in female patients. Further studies with sufficient number of cases are required to elucidate the impact of DM1 on female infertility.

Introduction Preimplantation Genetic Diagnosis- Aneuploidy Screening (PGD-A) is a technique commonly used in conjunction with IVF treatments. PGD-A is intended to identify those embryos with greater likelihood to implant. It is offered to patients at increased risk of producing chromosomally abnormal embryos (increased maternal age, Recurrent miscarriage, IVF failure and severe male factor). Technical related Error rates of PGD-A have fallen from 7% when using FISH to less than 1% when using modern techniques such as Next Generation Sequencing (NGS). Another source of error that is related to the physiology of the embryos is mosaicism described as the presence of two or more cells lines within an embryo. The advantage of NGS made the detection of mosaicism easier due to the i) higher sensitivity in aneuploidy detection and ii) the reliability of the technique making non-mosaicism related error minimal. The objective of our study is to assess the mosaicism rate and the misdiagnosis rate due to euploid/aneuploid mosaic embryos on day 3 biopsy. Material and Methods Embryos from patients undergoing PGD-A were biopsied on day 3 and sent to our laboratory for Aneuploidy Screening by NGS. Blind Reanalysis ofembryonic cells from embryos diagnosed as aneuploid but with good morphology and development was performed. Rebiopsy was carried out either on day 4, day 5 or day 6. Both, original analysis and reanalysis were performed by NGS (MySeq, Illumina platform). Result(s) PGD-A was performed in our laboratory on 29309 embryos at cleavage stage from 2016 to date. Rebiopsy for 173 embryos, originally diagnosed as aneuploidy, were blindly reanalyzed. There was 118 out of 173 showed aneuploidy (91.3%) while 15 out of 173 were found euploids (8.7%). The aneuploid embryos were divided into 3 categories: first where 118 embryos out of 173 (68.2%) showed the same initial result; second, 37 of the 173 (21.4%) showed at least one aneuploidy that was present in the initial result and third 3 out 173 (1.7%) showed a different an inverted figure of deletion/duplication for the same chromosome. Conclusions This study shows a very high confirmation rate (more than 90%) for day 3 biopsies. Our misdiagnosis rate after day 3 biopsy is 8.7%, due to mosaicism. Our data support the usefulness of day 3 biopsy PGD-A by NGS in places where day 5 biopsy is not possible. The mosaic rate on a relatively high number of embryos observed in this study is in line with other similar reported data. Our reanalysis was based on rebiopsies on embryos showing a good division after the first biopsy which might explain the low mosaic rate. Despite the high accuracy in day 3 biopsy diagnosis, implantation/pregnancy rate is still in the range of 50-60% implicating the necessity of finding more embryonic viability markers allowing a better outcome after IVF/PGD-A.

Aneuploidy is the most common genetic abnormality in human embryos. Preimplantation genetic diagnosis for aneuploidy (PGT-A) can increase pregnancy rates in infertile couples requiring assisted reproductive technology (ART). Large datasets from testing preimplantation embryos demonstrate that over half of embryos produced by in vitro fertilization (IVF) are aneuploid. Conventional embryo morphology evaluation as well as morphokinetics can not properly discriminate euploid from aneuploid embryos. Particularly, trisomic embryos that behave very similarly to euploid embryos. For these reasons, PGT-A started to be applied for several indications. In the present talk the results of two randomized trials (RCTs) in advanced maternal age (AMA) and severe male factor (SMF) will be discussed. In both studies, livebirths/ongoing pregnancy rates were compared among patients with blastocyst transfer as a control group and a study group in which day-3 biopsy was performed, and array CGH was applied for 24-chromosome analysis, with the transfer of euploid blastocysts. The final results of the AMA study conducted in women between 38 and 41 years of age included a total of 205 cycles. The PGT-A group exhibited significantly fewer embryo transfers (68.0%, vs. 90.5% for control; p=0.0001), and lower miscarriage rates (2.7%, vs. 39.0% for control; p=0.0007). Delivery rate after the first transfer attempt was significantly higher in the PGT-A group per transfer (52.9% vs. 24.2%; p=0.0002), and per patient (36.0% vs. 21.9%; p=0.0309). No significant differences were observed in the cumulative delivery rates per patient six months after closing the study. However, the mean number of embryo transfers needed per live birth was lower in the PGD-A group compared to control group (1.8 vs. 3.7), as was the time to pregnancy (7.7 vs. 14.9 weeks). The interim analysis of SMF study was performed when 101 cycles were completed and included women We can conclude that the benefits of testing embryos for common chromosomal abnormalities, include: increased ongoing implantation and pregnancy rates per transfer, decreased miscarriage rates per patient, and faster time to pregnancy when compared to conventional embryo scoring by morphology alone. Also, we concluded that PGT-A with the new approaches with throphectoderm biopsy and Next Generation Sequencing (NGS) the cost per treatment can decrease, being like a conventional cycle if we consider the endpoint of a healthy livebirth per cycle.

Introduction Achondroplasia a type of dwarfisim is an autosomal dominant condition. usually caused by a heterozygous mutation in the fibroblast growth factor receptor 3 gene (FGFR3).Majority of these are sporadic and rare familial cases are 10%. We report our first PGD case of achondroplasia for a couple where male aged 39 years was affected with a mutation (FGFR3:c.1620C>G). The couple never conceived before undergoing treatment. Couple KT was normal. The male partner parents were normal without the mutation and this represents a sporadic case. Materials and Methods Couple opted for IVF and PGD. Primers specific to the mutation was designed and nested PCR was performed with the inner and outer primers. As a standardization method male partner's mutation was checked if the designed primers have picked up the mutation.After controlled ovarian stimulation 5 good quality blastocysts were developed. The blastocysts were subjected to biopsy and PGD was carried out. Results Out of 5 blasts tested. 3 embryos were normal without the mutation , one embryo was affected and gave inconclusive result . Two embryos were transferred which resulted in a successful pregnancy with twins. And now the babies are 21 days old post delivery. Conclusion Achondroplasia is a rare condition , PGD was attempted which resulted in a successful delivery of healthy twins. This is an addition to one of the few cases reported on achondoplasia so far . Hence it proves that PGD is a powerful technology to avoid the birth of a dwarf child and new NGS panels can be developed so that the rarest mutations can be diagnosed. References: Successful birth with preimplantation genetic diagnosis using single-cell allele-specific PCR and sequencing in a woman with hypochondroplasia due to FGFR3 mutation (c.1620C>A, p. N540K) Kyung,Sung, Moo et al. 2013

As we enter the fifth decade of human IVF, this technique presents a paradox. On the one hand, IVF has become more regular and ordinary, even a new norm of social life. On the other hand, it has arguably become, as Alice might have said, ‘curiouser and curiouser', with the development of its applications such as intracytoplasmic sperm injection, preimplantation genetic diagnosis and gestational surrogacy, as well as human embryonic stem cell derivation. Five million miracle babies later, in the midst of ‘the age of biological control', IVF can be seen as the source of important changes in how reproductive biology is understood – socially, ethically, medically and in terms of basic science. This article reviews three decades of social scientific research into IVF and suggests that, while the passage of time may have allowed IVF to become more ‘routine', the opposite is also true. With hindsight, some of the more radical changes to the understandings of parenthood, kinship, fertility and technology to which IVF has contributed can be appreciated. Learning from this paradox must be part of the legacy of IVF's first half-century if its future evolution is to be directed wisely, safely and conscientiously. As we enter the fifth decade of human IVF, this technique is starting to present a paradox. On the one hand, IVF has become more regular and ordinary, even having become a new norm of social life. On the other hand, it has arguably become, as Alice might have said, ‘curiouser and curiouser', with the development of its applications such as intracytoplasmic sperm injection, preimplantation genetic diagnosis and gestational surrogacy, as well as human embryonic stem cell derivation. Five million miracle babies later, in the midst of ‘the age of biological control', IVF can be seen as the source of important changes in how reproductive biology is understood – not only socially and ethically, but medically and in terms of basic science. This article reviews three decades of social scientific research into IVF and suggests that, while the passage of time may have allowed IVF to become more ‘routine', the opposite is also true: with the benefit of hindsight, some of the more radical changes to the understandings of parenthood, kinship, fertility and technology to which IVF has contributed can also be appreciated. Learning from this paradox must be part of the legacy of IVF's first half century if its future evolution is to be directed wisely, safely and conscientiously. VIDEO LINK: http://sms.cam.ac.uk/media/1401097

Mitochondrial disorders represent the most common group of inborn errors of metabolism. Clinical manifestations can be extremely variable, ranging from single affected tissues to multisystemic syndromes. Maternally inherited mitochondrial DNA (mtDNA) mutations are a frequent cause, affecting about one in 5000 individuals. The expression of mtDNA mutations differs from nuclear gene defects. Mutations are either homoplasmic or heteroplasmic, and in the latter case disease becomes manifest when the mutation load exceeds a tissue-specific threshold. Mutation load can vary between tissues and in time, and often an exact correlation between mutation load and clinical manifestations is lacking. Because of the possible clinical severity, the lack of treatment and the high recurrence risk of affected offspring for female carriers, couples request prevention of transmission of mtDNA mutations. Previously, choices have been limited due to a segregational bottleneck, which makes the mtDNA mutation load in embryos highly variable and the consequences largely unpredictable. However, recently it was shown that preimplantation genetic diagnosis offers a fair chance of unaffected offspring to carriers of heteroplasmic mtDNA mutations. Technically and ethically challenging possibilities, such maternal spindle transfer and pronuclear transfer, are emerging and providing carriers additional prospects of giving birth to a healthy child.

Preimplantation genetic diagnosis (PGD) for inherited disorders is presently applied for more than 300 different conditions. The most frequent PGD indication is cystic fibrosis (CF), the largest series of which is reviewed here, totalling 404 PGD cycles. This involved testing for 52 different CFTR mutations with almost half of the cases (195/404 cycles) performed for ΔF508 mutation, one-quarter (103/404 cycles) for six other frequent mutations and only a few for the remaining 45 CFTR mutations. There were 44 PGD cycles performed for 25 CF-affected homozygous or double-heterozygous CF patients (18 male and seven female partners), which involved testing simultaneously for three mutations, resulting in birth of 13 healthy CF-free children and no misdiagnosis. PGD was also performed for six couples at a combined risk of producing offspring with CF and another genetic disorder. Concomitant testing for CFTR and other mutations resulted in birth of six healthy children, free of both CF and another genetic disorder in all but one cycle. A total of 96 PGD cycles for CF were performed with simultaneous aneuploidy testing, including microarray-based 24-chromosome analysis, as a comprehensive PGD for two or more conditions in the same biopsy material.

Preimplantation genetic diagnosis (PGD) is increasingly available, but how physicians view it is unclear. Internists are gatekeepers and sources of information, often treating disorders for which PGD is possible. This quantitative study surveyed 220 US internists, who were found to be divided. Many would recommend PGD for cystic fibrosis (CF; 33.7%), breast cancer (BRCA; 23.4%), familial adenomatous polyposis (FAP; 20.6%) and familial hypertrophic cardiomyopathy (19.9%), but few for social sex selection (5.2%); however, in each case,50% were unsure. Of those surveyed, 4.9% have suggested PGD to patients. Only 7.1% felt qualified to answer patient questions about it. Internists who would refer for PGD had completed medical training less recently and, for CF, were more likely to have privately insured patients (P0.033) and patients who reported genetic discrimination (P0.013). Physicians more likely to refer for BRCA and FAP were less likely to have patients ask about genetic testing. This study suggests that internists often feel they have insufficient knowledge about it and may refer for PGD based on limited understanding. They view possible uses of PGD differently, partly reflecting varying ages of onset and disease treatability. These data have critical implications for training, research and practice. Preimplantation genetic diagnosis (PGD) allows embryos to be screened prior to transfer to a woman's womb for various genetic markers. This procedure raises complex medical, social, psychological and ethical issues, but how physicians view it is unclear. Internists are gatekeepers and sources of information, often treating disorders for which PGD use is possible. We surveyed 220 US internists, who were found to be divided: many would recommend PGD for cystic fibrosis (CF; 33.7%), breast cancer (BRCA; 23.4%), familial adenomatous polyposis (FAP; 20.6%), and familial hypertrophic cardiomyopathy (FHC; 19.9%) and a few for sex selection (5.2%); but in each case,50% were unsure. Of those surveyed, 4.9% have suggested PGD to patients. Only 7.1% felt qualified to answer patient questions. Internists who would refer for PGD completed medical training less recently and, for CF, were more likely to have privately insured patients and patients who reported genetic discrimination. Physicians more likely to refer for BRCA and FAP were less likely to have patients ask about genetic testing. This quantitative study suggests that internists often feel they have insufficient knowledge and may refer for PGD based on limited understanding. They view possible uses of PGD differently, partly reflecting varying ages of onset and disease treatability. Internists should be made aware of the potential benefit of PGD, but also be taught to refer patients, when appropriate, to clinical geneticists who could then refer the patient to an IVF/PGD team. These data thus have critical implications for training, research and practice.

Genome editing based on site-directed nucleases facilitated efficient and versatile genetic modifications in human cells. However, recent reports, demonstrating CRISPR/Cas9-mediated genome editing in human embryos have raised profound concerns worldwide. This commentary explores the clinical justification and feasibility of reproductive medicine using germline genome editing. Despite the perceived utility of reproductive medicine for treating intractable infertility, it is difficult to justify germline genome editing from the perspective of the prospective child. As suggested by the UK legalization regarding mitochondrial donation, the prevention of genetic disease in offspring by genome editing might be acceptable in limited cases of serious or life-threatening conditions, where no alternative medicine is available. Nonetheless, the mosaicism underlying human embryos as well as the off-target effect by artificial nucleases will likely hamper preimplantation genetic diagnosis prior to embryo transfer. Such considerations suggest that this type of reproductive medicine should not be developed toward a clinical application. However, the clinical uncertainties underscore the need for embryology that can address fundamental questions regarding germline aneuploidy and mosaicism using genome editing.

This case study reports the first successful birth outcome following preimplantation genetic diagnosis (PGD) for a chromosome translocation in embryos generated by serial vitrification of oocytes. A couple presented to the fertility clinic with 2 years of primary infertility. The woman was diagnosed with poor ovarian reserve and her partner was diagnosed with severe oligoteratozoospermia and the reciprocal translocation 46,XY,t(1;7)(p36.1;q11.23). Following counselling, the couple opted for serial vitrification of oocytes followed by PGD. A total of 31 oocytes were obtained in five egg collection cycles over a period of 12 months and 27 metaphase-II oocytes were vitrified. Nineteen of the 27 vitrified oocytes survived warming: 14 oocytes from the vitrified group and three oocytes from the fresh cycle were fertilized by intracytoplasmic sperm injection. Eleven embryos, including three from the fresh cycle, were biopsied on day 3 post insemination. Fluorescence in-situ hybridization was performed for the specific chromosomes involved in translocation. Only two embryos from the cryopreservation cycles were diagnosed as normal/balanced, one of which was transferred on day 5 post insemination. A normal healthy female infant was born at week 42 of gestation.

Preimplantation genetic diagnosis (PGD) is an increasingly common adjunct to IVF. The information gained from PGD may be used to reduce the incidence of chromosomally abnormal pregnancies and augment the current selection process of embryos. As such, patients may choose to utilize PGD in either fresh or cryopreserved IVF cycles. It is a common practice to cryopreserve excess embryos at the blastocyst stage. In these cases, trophectoderm biopsy is the only technique available for PGD. This articles reports this study centre's experience with trophectoderm biopsies of cryopreserved blastocysts in 12 patients who underwent 13 cycles of PGD. The implantation rate per embryo transferred was 46% and the ongoing pregnancy rate per embryo transfer was 63%. The results from this case series demonstrate that trophectoderm biopsy on cryopreserved blastocysts to perform PGD is logistically feasible. In addition, the rate of implantation and ongoing pregnancy were maintained within a reasonable range to justify the procedure. Preimplantation genetic diagnosis (PGD) is an increasingly common adjunct to IVF and is used to evaluate the genetic makeup of the embryo prior to transfer of the embryo into the uterus. The information gained from PGD may be used to identify single-gene disorders that result in genetic disease, reduce the incidence of chromosomally abnormal pregnancies and/or augment the selection process of embryos to be transferred. In order to perform PGD, a biopsy of the embryo is the performed and cells are removed for testing. PGD may be performed in either fresh or frozen (cryopreserved) IVF cycles. Patients who have cryopreserved embryos remaining in storage from a previous fresh cycle may wish to have these embryos tested with PGD. Many embryos are frozen on day 5 of development, referred to as the blastocyst stage. At this stage of development, embryo biopsy is performed via a technique known as 'trophectoderm biopsy', in which 1-3 of the cells destined to become the placenta are removed from the embryo for chromosomal testing. We report our experience with trophectoderm biopsy of frozen blastocysts in 12 patients who underwent 13 cycles of PGD. The implantation rate per embryo transferred was 46% and the ongoing pregnancy rate per embryo transfer was 63%. The results from this case series demonstrate that trophectoderm biopsy on cryopreserved blastocysts to perform PGD is logistically feasible. In addition, the rate of implantation and ongoing pregnancy were maintained within a reasonable range to justify the procedure.

The aim of this study was to develop and perform a preimplantation genetic diagnosis (PGD) assay discriminating between wild-type and mutant alleles in two families with germline mosaicism. Family 1 had two children affected with severe myoclonic epilepsy (SCNA1A del exons 1-22). Family 2 had two children with tuberous sclerosis (TSC2 C1327T) and two healthy children. Neither mutation was detected in genomic DNA derived from the parents in either family. Informative microsatellite markers flanking SCNA1A and TSC2 along with the identified mutations were used to construct haplotypes. For tuberous sclerosis, single spermatozoa were analysed using a multiplex assay that included six informative markers and the TSC2 mutation. In family 1, deletion in the maternal allele was detected in the affected child. In family 2, both affected children and one healthy child shared the same paternal allele. To confirm mutant paternal transmission, single spermatozoa were analysed for the mutation along with six markers. Of 44 single spermatozoa, four showed the mutant T allele, allowing linkage between the mutation and the genetic markers. Both families delivered healthy children following IVF/PGD. In conclusion, germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm analysis is a useful tool for verifying allelic linkage.

Fluorescent in-situ hybridization (FISH) for preimplantation genetic diagnosis (PGD) of structural chromosome abnormalities has limitations, including carrier testing, inconclusive results and limited aneuploidy screening. Array comparative genome hybridization (CGH) was used in PGD cases for translocations. Unbalances could be identified if three fragments were detectable. Smallest detectable fragments were ∼6Mbp and ∼5Mbp for blastomeres and trophectoderm, respectively. Cases in which three or more fragments were detectable by array CGH underwent PGD by FISH and concordance was obtained in 53/54 (98.1%). The error rate for array CGH was 1.9% (1/54). Of 402 embryos analysed, 81 were normal or balanced, 92 unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. FISH with additional probes to detect other aneuploidies would have missed 28 abnormal embryos in the reciprocal group and 10 in the Robertsonian group. PGD cases (926) were retrospectively reviewed for reciprocal translocations performed by FISH to identify which could have been analysed by array CGH. This study validates array CGH in PGD for translocations and shows that it can identify all embryos with unbalanced reciprocal and Robertsonian translocations. Array CGH is a better approach than FISH since it allows simultaneous screening of all chromosomes for aneuploidy. Fluorescent in-situ hybridization (FISH) has been used in preimplantation genetic diagnosis (PGD) for structural chromosome abnormalities. This approach has limitations such as the need for testing of carriers, poor fixation and no possibility for aneuploidy screening of all chromosomes. The use of array comparative genome hybridization (CGH) can solve these limitations. Array CGH was used in 47 PGD cases for Robertsonian or reciprocal translocations. The smallest detectable fragment size was ∼6Mbp for single blastomeres (day-3) and ∼5Mbp for trophectoderm (day-5) biopsies. Translocation cases in which three or more translocated fragments were detectable by array CGH underwent PGD. Of those, a total of 54 non-transferred embryos analysed by array CGH were reanalysed by FISH. The error rate for array CGH was 1.9% (1/54). Of the 402 embryos analysed by array CGH, 81 embryos were normal or balanced, 92 were unbalanced but euploid, 123 unbalanced and aneuploid and 106 balanced but aneuploid. We retrospectively reviewed 926 PGD cases for reciprocal translocations performed by FISH in order to identify those in which fewer than two fragments were less than ∼6Mbp. In conclusion, this study validates the use of array CGH in PGD for translocations showing a low error rate and shows that array CGH can identify all embryos with unbalanced reciprocal and Robertsonian translocations. Array CGH is a better approach than FISH or single-nucleotide polymorphism arrays because it allows simultaneous screening of all chromosomes for aneuploidy and does not require previous genetic testing of the parents.

Preimplantation genetic diagnosis and aneuploidy testing (PGD/PGS) use is constantly growing in IVF, and embryo/biopsy traceability during the additional laboratory procedures needed is pivotal. An electronic witnessing system (EWS), which showed a significant value in decreasing mismatch occurrence and increasing detection possibilities during standard care IVF, still does not guarantee the same level of efficiency during PGD/PGS cycles. Specifically, EWS cannot follow single embryos throughout the procedure. This is however critical when an unambiguous diagnosis corresponds to each embryo. Failure Mode and Effects Analysis (FMEA) is a proactive method generally adopted to define tools ensuring safety along a procedure. Due to the implementation of a large quantitative PCR (qPCR)-based blastocyst stage PGD/PGS programme in our centre, and to evaluate the potential procedural risks, a FMEA was performed in September 2014. Forty-four failure modes were identified, among which six were given a moderate risk priority number (>15) (RPN; product of estimated occurrence, severity and detection). Specific corrective measures were then introduced and implemented, and a second evaluation performed six months later. The meticulous and careful application of such measures allowed the risks to be decreased along the whole protocol, by reducing their estimated occurrence and/or increasing detection possibilities.

Regulatory differences between countries are an important driver of the cross-border trade in assisted reproduction as people move to seek services unavailable in their home countries. The development of a lucrative global trade in non-medical sex selection needs to be considered in ethical debates over its availability. I suggest that depictions of non-medical sex selection as a means of 'family balancing' or supportive of reproductive autonomy serve to distance the technologies rhetorically from the gender stereotyping inherent in their use and the commodification upon which they depend. They construct new social categories such as the 'unbalanced' family, the pathologization of 'gender disappointment' and a limited and highly individualized definition of reproductive freedom that permits medical interventions on healthy bodies. Orientalism pervades ethical debate depicting non-medical sex selection in the West as more acceptable to practices in 'Asia'. A case study of the interconnections between Australia and Thailand highlights the global economy sustaining the practice.

The early influences on Robert Edwards’ approach to the scientific research that led to human IVF are described. His interest as a graduate student in the genetics of early mammalian development stimulated him later to investigate whether the origins of human genetic diseases such as Down, Klinefelter and Turner syndromes might be explained by events during egg maturation. This clinical problem provided the most powerful stimulus to achieve both oocyte maturation and fertilization in vitro in humans. Indeed, preimplantation genetic diagnosis was his main goal until he met Patrick Steptoe in 1968. A re-evaluation of his meeting with Steptoe suggests that initially Steptoe’s laparoscopic skill was of interest for its potential to solve the sperm capacitation problem. Steptoe’s impact on Edwards was twofold. First, Steptoe’s long-held interest in infertility raised this application of IVF higher in Edwards’ priorities. Second, Steptoe offered a long-term partnership, in which oocyte collection without in-vitro maturation was a possibility. The professional criticism generated by their work together encouraged Edwards to pursue a deliberate programme of public education about the issues raised and to challenge and develop professional bioethical thought and discourse about reproduction.The early life and career of Robert Edwards are described and re-evaluated in the light of documentary evidence. His early interest in the genetics of development provided the major motivation behind his goal of achieving IVF in humans. Through this work, he aimed to understand and hopefully to reduce the transmission of genetic disease in humans. His meeting with Patrick Steptoe, the details of which are re-examined, increased the significance for Edwards of infertility as an outcome of IVF. It also led to a creative long-term research partnership, initiated a long-term programme of public education in the UK about reproductive science and stimulated the development of bioethical thinking.

This article reports a historical study of factors influencing the achievement of clinical preimplantation genetic diagnosis (PGD) in 1990, 22 years after its first demonstration in animals. During the 1970s, research on PGD continued in large farm animals, but serious interest in human PGD was not evident until 1986. First, interest in PGD during the 1970s waned with the advent of prenatal testing, which for gynaecologists was clinically more familiar, technically simpler and ethically less challenging than IVF. Indeed, IVF was viewed with widespread suspicion until the first IVF births in 1978. Second, interest in clinical PGD was stimulated by the UK Parliamentary reaction against human embryo research that greeted the Warnock Report in 1984. This hostility led scientists to initiate a pro-research campaign, further galvanized in 1985 by MP Enoch Powell’s bid to ban such research. However, while Powell abhorred embryo research, he approved of PGD, a stance that divided the anti-research lobby. Accordingly, the campaigners for research emphasized that it was needed to achieve PGD. Powell demanded evidence of such projects and PGD research increased from 1986. It is concluded that UK political debates on embryo research played a critical role in stimulating the achievement of clinical PGD.Human pregnancies following preimplantation genetic diagnosis (PGD) for embryo sex were announced in 1990, 22 years after the technique was pioneered in animals. PGD in humans required not only technological advances, such as IVF and sensitive diagnostic tests, but also the motivation to develop and apply them. Our historical analysis shows that, although research on PGD continued in large farm animals during the 1970s, and techniques of the required sensitivity were developed on mouse embryo models, interest in clinical PGD was not evident until 1986. Two factors stimulated this sudden change in motivation. First, interest in PGD was depressed during the 1970s by the advent of prenatal diagnostic techniques, which for gynaecologists were clinically, technically and ethically less challenging than IVF. IVF was then regarded with a suspicion that only started to wane in the early 1980s following the first IVF births. Second, the UK Parliamentary reaction against human embryo research that greeted the Warnock Report in 1984 provided a positive stimulus to clinical PGD by prompting scientists to form a pro-research lobby, which was further galvanized in early 1985 by MP Enoch Powell’s almost-successful bid to ban human embryo research. We show that while Powell abhorred embryo research, he approved of PGD, a stance that fractured the unity of the anti-research lobby. Accordingly, the pro-research lobby emphasized that embryo research was needed to achieve PGD. Powell demanded evidence of such projects, thereby, we argue, stimulating PGD research from 1986. Our evidence shows that UK political debates about PGD played a critical role in stimulating the achievement of PGD clinically.

Congenital hyperinsulinism is the most common cause of persistent hypoglycaemia in infancy. Early surgical intervention is usually required to prevent brain damage. The prevention of the transmission to the offspring is important in families carrying the mutated gene. Preimplantation genetic diagnosis (PGD) is an early genetic testing procedure for couples at risk of transmitting inherited diseases. A 36-year-old Saudi woman married to her first cousin with four affected children was referred for PGD. The hyperinsulinism disease was caused by a novel homozygous mutation in the KCNJ11 gene, an arginine 301 to proline (R301P) substitution.PGD was achieved by whole genome amplification followed by mutation detection combined with short tandem repeat identifier analysis in the first cycle and with haplotyping in the second cycle. The first and second cycles resulted in the births of healthy twin girls and a boy, respectively. As far as is known, this is the first application of PGD to hyperinsulinism. A feasible strategy including whole genome amplification followed by direct mutation detection combined with haplotyping is described.Utilizing haplotyping increases the efficiency of PGD diagnosis as well as confirming the genetic diagnosis. It reveals the parental origin of each inherited chromosome.

This report describes an unusual case of ovarian torsion during an IVF cycle prior to vaginal oocyte retrieval and the subsequent embryo development. A 27-year-old, whose husband carries a balanced translocation, presented on stimulation day 11 (day after human chorionic gonadotrophin administration) with signs of right ovarian torsion. Transvaginal ultrasound identified decreased right ovarian venous flow but preservation of right ovarian arterial flow. She underwent emergency laparoscopic right ovarian detorsion followed by vaginal oocyte retrieval on postoperative day 1. Ten oocytes were retrieved from the right detorted ovary, 4/10 (40%) were fertilized and 3/4 (75%) became blastocysts. Fifteen oocytes were retrieved from the left ovary, 14/15 (93%) were fertilized and 9/14 (64%) became blastocysts. All 18 embryos biopsied for preimplantation genetic diagnosis carried unbalanced translocations and none were transferred. The markedly reduced fertilization rate of the oocytes from the previously torted ovary is similar to the rate described in a prior report and likely related to decreased but maintained ovarian arterial flow. This report is unique because not only was the patient's ovarian torsion surgically corrected prior to oocyte retrieval but also the embryos originating from the previously torted ovary had excellent development with 75% progressing to the blastocyst stage.

Chromosome abnormalities are common among human oocytes and are usually lethal to any embryos they produce. It therefore seems logical that a reliable technique for distinguishing between normal and aneuploid embryos would be a useful tool for physicians and embryologists, assisting the choice of which embryo(s) to prioritize for uterine transfer. This concept has led to the development of a variety of methods for the detection of chromosome abnormalities in oocytes and embryos, most often referred to as preimplantation genetic screening (PGS). However, several well-controlled studies have been unable to show an advantage of chromosome screening in terms of pregnancy and birth rates. Some investigators have suggested that damage to embryos, sustained during cleavage-stage biopsy, might explain why PGS has not always provided the anticipated benefits. This paper asks whether there is evidence that a non-invasive, morphological analysis could allow chromosomally normal embryos to be accurately identified and reviews data from the most recent publication concerning IVF outcome following PGS.

Over 1000 children affected with cystic fibrosis (CF) are born annually in the USA. Since IVF with preimplantation genetic diagnosis (PGD) is an alternative to raising a sick child or to aborting an affected fetus, a cost-benefit analysis was performed for a national IVF-PGD program for preventing CF. The amount spent to deliver healthy children for all CF carrier-couples by IVF-PGD was compared with the average annual and lifetime direct medical costs per CF patient avoided. Treating annually about 4000 CF carrier-couples with IVF-PGD would result in 3715 deliveries of non-affected children at a cost of $57,467 per baby. Because the average annual direct medical cost per CF patient was $63,127 and life expectancy is 37 years, savings would be $2.3 million per patient and $2.2 billion for all new CF patients annually in lifetime treatment costs. Cumulated net saving of an IVF-PGD program for all carrier-couples for 37 years would be $33.3 billion. A total of 618,714 cumulative years of patients suffering because of CF and thousands of abortions could be prevented. A national IVF-PGD program is a highly cost-effective novel modality of preventive medicine and would avoid most births of individuals affected with debilitating genetic disease.

Herein is reported efficient erythropoietic differentiation of a human embryonic stem cell (ESC) line derived from a preimplantation genetic diagnosis (PGD)-screened embryo that harbours the homozygous sickle cell disease (SCD) haemoglobinopathy mutation. This human ESC line possesses typical pluripotency characteristics and forms multilineage teratomas in vivo. SCD-human ESC efficiently differentiated to the haematopoietic lineage under serum-free and stromal co-culture conditions and gave rise to robust primitive and definitive erythrocytes. Expression of embryonic, fetal and adult sickle globin genes in SCD PGD-derived human ESC-derived erythrocytes was confirmed by quantitative real-time PCR, intracytoplasmic fluorescence-activated cell sorting and in-situ immunostaining of PGD-derived human ESC teratoma sections. These data introduce important methodologies and paradigms for using patient-specific human ESC to generate normal and haemoglobinopathic erythroid progenitors for biomedical research.

This study aimed at evaluating parameters and results of ovarian stimulation for myotonic dystrophy type 1 (DM1) female patients undergoing preimplantation genetic diagnosis (PGD) and to assess an eventual association between genotype and ovarian reserve. A retrospective study involved all 17 DM1 patients treated in the study centre's PGD programme. The control group consisted of 22 patients treated for X-linked disorders in the same period. Comparative analysis of ovarian stimulation parameters and results was performed with bivariate and multivariate analysis. Then, among DM1 patients, a correlation between genotype (number of CTG repeats) and ovarian reserve, assessed by antral follicle count, was investigated. Comparative study showed no difference concerning the number of oocytes, embryos and pregnancy rate between the two groups. Multivariate analysis demonstrated that DM1 patients needed a significantly higher dose of gonadotrophins (+544IU, P0.001) than X-linked disorders patients and suggests a decreased ovarian sensitivity. However, with higher dose of gonadotrophins, PGD for DM1 offers good reproductive outcomes with a clinical pregnancy rate of 35.7%. Genotype was not correlated to ovarian reserve and appeared not to be helpful for the choice of the dose of gonadotrophins.

The aim of the present study was to evaluate the usefulness of pronuclear patterns, according to the classifications of Tesarik and Scott, as predictors of embryo chromosome constitution. Up to 73 preimplantation genetic diagnosis/preimplantation genetic screening (PGD/PGS) cycles were analysed in this retrospective study including 17 cycles of translocation carriers and 56 PGS cycles. A total of 331 biopsied embryos were studied assessing pronuclear (PN) pattern, embryo quality and chromosome constitution. As regards to the relationship between PN pattern and embryo quality, the data obtained in this study show no correlation between both parameters. Although there were no significant differences when comparing the distribution of chromosomally normal and abnormal embryos with respect to embryo quality, such differences were observed when distinguishing between normal, aneuploid and polyploid embryos. The results show that the PN pattern using Tesarik's and Scott's classification systems is not related to the embryo developmental potential or its chromosome constitution. Therefore, in the context of a PGD/PGS programme, the PN pattern cannot be used as a tool to predict embryo quality or chromosome status.

Preimplantation genetic diagnosis using whole genome amplification and a haplotyping approach (PGH) was first described in 2006 and suggested as an efficient alternative to single-cell PCR for monogenic disorders. DNA from single cells was amplified using multiple displacement amplification; the resulting products were then tested using disease-specific PCR multiplexes applied under standard laboratory conditions to determine the haplotypes in the embryo. This study reports on a total of 127 completed biopsy cycles for 101 couples at risk of: autosomal recessive disease (71 cycles, 53 couples including one germ-line mosaic carrier), autosomal dominant disease (31 cycles, 26 couples including one germ-line mosaic carrier), X-linked recessive disease (18 cycles, 16 couples including one germ-line mosaic carrier), X-linked dominant disease (six cycles, five couples) and a double inheritance of both autosomal and X-linked recessive diseases (one cycle, one couple). Of these, 107 cycles reached embryo transfer. Overall success rates were: fetal heart beat-positive pregnancies (FHB+)/biopsy cycle = 28%; FHB+/embryo transfer = 34%; FHB+/couple = 36%; 26 babies born, 13 ongoing pregnancies. These data demonstrate that PGH provides a robust, efficient and successful alternative to single-cell PCR for monogenic diseases.