PGDIS position statement on the transfer of mosaic embryos 2021.

Chromosome testing strategies, such as preimplantation genetic testing for aneuploidy (PGT-A), improve initial IVF outcomes by avoiding unwitting transfer of aneuploid embryos in morphology-based selection practices. Newer technologies have revealed that some embryos may appear to have intermediate whole chromosome (or parts of a chromosome termed segmental) copy number results suggesting trophectoderm mosaicism. An embryo with a trophectoderm mosaic-range result may be the only option for transfer for some patients. Recent data suggest that such mosaic embryos can be transferred without added risk of abnormal birth outcomes but may be associated with increased implantation failure and miscarriage rates, with higher values of mosaicism appearing to be less favourable for producing good outcomes. In this Position Statement, we provide guidance to laboratories, clinics, clinicians and counsellors to assist in discussions on the utility and transfer of mosaic embryos.

Transcriptome dissection of candidate genes associated with lentil seed quality traits.

Lentils provide a rich plant-based protein source and staple food in many parts of the world. Despite numerous nutritional benefits, lentil seeds also possess undesirable elements, such as anti-nutritional factors. Understanding the genetic networks of seed metabolism is of great importance for improving the seed nutritional profile. We applied RNA sequencing analysis to survey the transcriptome of developing lentil seeds and compared this with that of the pod shells and leaves. In total, we identified 2622 genes differentially expressed among the tissues examined. Genes preferentially expressed in seeds were enriched in the Gene Ontology (GO) terms associated with development, nitrogen and carbon (N/C) metabolism and lipid synthesis. We further categorized seed preferentially expressed genes based on their involvement in storage protein production, starch accumulation, lipid and suberin metabolism, phytate, saponin and phenylpropanoid biosynthesis. The availability of transcript profile datasets on lentil seed metabolism and a roadmap of candidate genes presented here will be of great value for breeding strategies towards further improvement of lentil seed quality traits.

Noninvasive prenatal diagnosis for pregnancies at risk for ß-thalassaemia: a retrospective study.

OBJECTIVE: To evaluate the clinical feasibility of noninvasive prenatal diagnosis (NIPD) for ß-thalassaemia using circulating single molecule amplification and re-sequencing technology (cSMART). DESIGN: Through carrier screening, 102 pregnant Chinese couples carrying pathogenic HBB gene variants were recruited to the study. Pregnancies were managed using traditional invasive prenatal diagnosis (IPD). Retrospectively, we evaluated the archived pregnancy plasma DNA by NIPD to evaluate the performance of our cSMART assay for fetal genotyping. SETTING: Chinese prenatal diagnostic centres specialising in thalassaemia testing. POPULATION: Chinese carrier couples at high genetic risk for ß-thalassaemia. METHODS: Fetal cell sampling was performed by amniocentesis and HBB genotypes were determined by reverse dot blot. NIPD was performed by a newly designed HBB cSMART assay and fetal genotypes were called by measuring the allelic ratios in the maternal cell-free DNA. MAIN OUTCOME MEASURES: Concordance of HBB fetal genotyping between IPD and NIPD and the sensitivity and specificity of NIPD. RESULTS: Invasive prenatal diagnosis identified 29 affected homozygotes or compound heterozygotes, 54 heterozygotes and 19 normal homozygotes. Compared with IPD results, 99 of 102 fetuses (97%) were correctly genotyped by our NIPD assay. Two of three discordant samples were false positives and the other sample involved an incorrect call of a heterozygote carrier as a homozygote normal. Overall, the sensitivity and specificity of our NIPD assay was 100% (95% CI 88.06-100.00%) and 97.26% (95% CI 90.45-99.67%), respectively. CONCLUSIONS: This study demonstrates that our cSMART-based NIPD assay for ß-thalassaemia has potential clinical utility as an alternative to IPD for pregnant HBB carrier couples. TWEETABLE ABSTRACT: A new noninvasive test for pregnancies at risk for ß-thalassaemia.

Noninvasive fetal genotyping in pregnancies at risk for PKU using a comprehensive quantitative cSMART assay for PAH gene mutations: a clinical feasibility study.

OBJECTIVE: To assess the diagnostic performance of a novel circulating single molecule amplification and re-sequencing technology (cSMART) method for noninvasive prenatal testing (NIPT) of Phenylketonuria (PKU). DESIGN: Blinded NIPT analysis of pregnancies at high risk for PKU. SETTING: Shanghai Xinhua Hospital and Hunan Jiahui Genetics Hospital, China. POPULATION: Couples (n = 33) with a child diagnosed with PKU. METHODS: Trio testing for pathogenic PAH mutations was performed by Sanger sequencing. In second pregnancies, invasive prenatal diagnosis (IPD) was used to determine fetal genotypes. NIPT was performed using a PAH gene-specific cSMART assay. Based on the plasma DNA mutation ratio relative to the fetal DNA fraction, fetal genotypes were assigned using a maximum-likelihood algorithm. MAIN OUTCOME MEASURES: Concordance of fetal genotyping results between IPD and NIPT, and the sensitivity and specificity of the NIPT assay. RESULTS: Compared with gold standard IPD results, 32 of 33 fetuses (96.97%) were accurately genotyped by NIPT. The sensitivity and specificity of the NIPT assay was 100.00% (95% CI 59.04-100.00%) and 96.15% (95% CI 80.36-99.90%), respectively. CONCLUSIONS: The novel cSMART assay demonstrated high accuracy for correctly calling fetal genotypes. We propose that this test has useful clinical utility for the rapid screening of high-risk and low-risk pregnancies with a known history of PKU on one or both sides of the family. TWEETABLE ABSTRACT: NIPT of couples at high risk for PKU using a full-coverage cSMART PAH gene test.

The time to and determinants of first fractures in boys with Duchenne muscular dystrophy.

Boys with vertebral fractures (VF) identified through routine spine radiographs had milder, less symptomatic, and fewer VF compared to those diagnosed with VF following consultation for back pain. Spontaneous (i.e., medication-unassisted) reshaping of fractured vertebral bodies was absent. Long bone fractures were present even before Duchenne muscular dystrophy (DMD) diagnosis in some boys. INTRODUCTION: The objective of the study was to determine the time to and characteristics of first fractures in Duchenne muscular dystrophy. METHODS: This study was a retrospective longitudinal study of 30 boys with DMD <18 years. Boys were classified into four groups according to their first fracture: those with VF identified on routine lateral spine radiographs, those with VF diagnosed following consultation for back pain, those with long bone fractures, and those without fractures. RESULTS: Compared to boys diagnosed with VF as their initial fracture following consultation for back pain, those with VF surveillance radiographs had shorter durations of glucocorticoid (GC) therapy at the time of VF diagnosis (median 1.6 versus 5.3 years, p < 0.01), higher areal (mean ± standard deviation -1.4 ± 0.7 versus -3.1 ± 0.8, p = 0.01), and volumetric (-0.3 ± 0.5 versus -2.6 ± 0.8, p < 0.01) lumbar spine bone mineral density Z-scores, as well as fewer VF (median 1.4 versus 5.2 per person, p < 0.01) and a lower median spinal deformity index (median 1.5 versus 9.5, p < 0.01). Vertebral body reshaping following VF was not observed. Ten boys sustained a long bone fracture as their first fracture at a mean age of 8.9 ± 4.0 years; four of these boys later sustained a total of 27 incident VF. CONCLUSIONS: Routine lateral spine radiographs led to detection of VF in their earlier stages, vertebral body reshaping following VF was absent, and VF were frequent after the first long bone fracture. These results support the inclusion of a lateral spine radiograph starting at the time of GC initiation as part of routine bone health monitoring in DMD.

Three-dimensional imaging of human stem cells using soft X-ray tomography.

Three-dimensional imaging of human stem cells using transmission soft X-ray tomography (SXT) is presented for the first time. Major organelle types--nuclei, nucleoli, mitochondria, lysosomes and vesicles--were discriminated at approximately 50 nm spatial resolution without the use of contrast agents, on the basis of measured linear X-ray absorption coefficients and comparison of the size and shape of structures to transmission electron microscopy (TEM) images. In addition, SXT was used to visualize the distribution of a cell surface protein using gold-labelled antibody staining. We present the strengths of SXT, which include excellent spatial resolution (intermediate between that of TEM and light microscopy), the lack of the requirement for fixative or contrast agent that might perturb cellular morphology or produce imaging artefacts, and the ability to produce three-dimensional images of cells without microtome sectioning. Possible applications to studying the differentiation of human stem cells are discussed.

Traditional karyotyping vs copy number variation sequencing for detection of chromosomal abnormalities associated with spontaneous miscarriage.

OBJECTIVES: To compare the performance of traditional G-banding karyotyping with that of copy number variation sequencing (CNV-Seq) for detection of chromosomal abnormalities associated with miscarriage. METHODS: Products of conception (POC) were collected from spontaneous miscarriages. Chromosomal abnormalities were detected using high-resolution G-banding karyotyping and CNV sequencing. Quantitative fluorescent polymerase chain reaction analysis of maternal and POC DNA for short tandem repeat (STR) markers was used to both monitor maternal cell contamination and confirm the chromosomal status and sex of the miscarriage tissue. RESULTS: A total of 64 samples of POC, comprising 16 with an abnormal and 48 with a normal karyotype, were selected and coded for analysis by CNV-Seq. CNV-Seq results were concordant for 14 (87.5%) of the 16 gross chromosomal abnormalities identified by karyotyping, including 11 autosomal trisomies and three sex chromosomal aneuploidies (45,X). Of the two discordant results, a 69,XXX polyploidy was missed by CNV-Seq, although supporting STR marker analysis confirmed the triploidy. In contrast, CNV-Seq identified a sample with 45,X karyotype as a 45,X/46,XY mosaic. In the remaining 48 samples of POC with a normal karyotype, CNV-Seq detected a 2.58-Mb 22q deletion associated with DiGeorge syndrome and nine different smaller CNVs of no apparent clinical significance. CONCLUSIONS: CNV-Seq used in parallel with STR profiling is a reliable and accurate alternative to karyotyping for identifying chromosome copy number abnormalities associated with spontaneous miscarriage.

Non-invasive prenatal testing for fetal aneuploidies in the first trimester of pregnancy.

OBJECTIVES: To evaluate the feasibility of non-invasive prenatal testing (NIPT) of maternal plasma samples collected from pregnant Chinese women in early gestation, between 8 + 0 and 12 + 6 weeks' gestation. METHODS: In this pilot study, 212 women with high-risk pregnancies were recruited at a single Chinese Hospital. Fetal aneuploidies associated with chromosomes 21, 18, 13, X and Y were detected by massively parallel sequencing of maternal plasma DNA samples. Invasive prenatal diagnosis by either chorionic villus sampling or amniocentesis and then karyotyping was offered to all women to confirm both positive and negative NIPT results. Fetal DNA fraction was also determined in male pregnancies, by the relative percentage of Y-chromosome sequences. All confirmed NIPT-negative pregnancies were followed up to birth and neonates were clinically evaluated for any symptoms of chromosomal disease. RESULTS: Autosomal aneuploidies trisomy 21 (n = 2), 18 (n = 1) and 13 (n = 1) were detected by NIPT and confirmed by amniocentesis and karyotyping. There were one false-positive 45,X sample and two false-negative samples associated with fetal karyotypes 47,XXY and 45,X[16]/47,XXX[14]. In the 100 male pregnancies, the median fetal DNA fraction was 8.54% and there was a trend towards an increasing fetal fraction from 8 + 0 to 12 + 6 weeks' gestation. The majority (95%) of pregnancies had a fetal DNA fraction > 4%, which is generally the limit for accurate aneuploidy detection by NIPT. Across this early gestational time period, there was a weak inverse relationship (R(2) = 0.186) between fetal DNA fraction and maternal weight. CONCLUSIONS: NIPT is highly reliable and accurate when applied to maternal DNA samples collected from pregnant women in the first trimester between 8 + 0 and 12 + 6 weeks.

Extra-group mating increases inbreeding risk in a cooperatively breeding bird.

In many cooperatively breeding species, females mate extra-group, the adaptive value of which remains poorly understood. One hypothesis posits that females employ extra-group mating to access mates whose genotypes are more dissimilar to their own than their social mates, so as to increase offspring heterozygosity. We test this hypothesis using life history and genetic data from 36 cooperatively breeding white-browed sparrow weaver (Plocepasser mahali) groups. Contrary to prediction, a dominant female's relatedness to her social mate did not drive extra-group mating decisions and, moreover, extra-group mating females were significantly more related to their extra-group sires than their social mates. Instead, dominant females were substantially more likely to mate extra-group when paired to a dominant male of low heterozygosity, and their extra-group mates (typically dominants themselves) were significantly more heterozygous than the males they cuckolded. The combined effects of mating with extra-group males of closer relatedness, but higher heterozygosity resulted in extra-group-sired offspring that were no more heterozygous than their within-group-sired half-siblings. Our findings are consistent with a role for male-male competition in driving extra-group mating and suggest that the local kin structure typical of cooperative breeders could counter potential benefits to females of mating extra-group by exposing them to a risk of inbreeding.

Microarray detection of Y chromosome deletions associated with male infertility.

Array-comparative genomic hybridization (CGH) has emerged as a powerful new molecular tool for the high-resolution analysis of copy-number variation and breakpoint analysis. In this study, array-CGH was used to analyse known Yq deletions associated with male infertility. A microarray platform encompassing probes for chromosomes 13, 14, 21, X and Y was developed in-house and was used to detect different Yq deletion types. The successful application of this array for the detection of Yq deletions involving either the AZFb or AZFc region was demonstrated. Partial and complete AZF deletions were correctly detected in 13 patients with Yq deletions previously identified by multiplex polymerase chain reaction (PCR). This study demonstrates that array-CGH may be an alternative approach to multiplex PCR for the diagnosis of known Yq deletions and potentially a useful tool for the discovery of other Y chromosome deletions/polymorphisms associated with defective spermatogenesis.

Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study.

BACKGROUND: Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in PGD cycles for beta-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts. METHODS: This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis. RESULTS: In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born. CONCLUSIONS: This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.

Preimplantation genetic diagnosis of familial adenomatous polyposis.

Familial adenomatous polyposis is a severe autosomal dominant cancer predisposition syndrome. It is characterized by the development of hundreds to thousands of polyps in the gastrointestinal tract, primarily in the colon, at a mean age of 16 years. Without a colectomy, colon cancer is inevitable. FAP is caused by mutations in the adenomatous polyposis coli (APC) gene. A couple was referred to Monash IVF following a request to undertake preimplantation genetic diagnosis for FAP. The female proband had an AGTT deletion mutation in exon 15 of the APC gene. Analysis of the APC-linked marker D5S346 showed that the parental alleles were fully informative. A multiplex polymerase chain reaction (PCR) test was developed involving direct mutation detection and the co-analysis of D5S346 to identify allele drop out and avoid a misdiagnosis. The delAGTT mutation and D5S346 alleles were diagnosed by fluorescent PCR and allele sizing. Following standard hormone stimulation and IVF procedures, 14 oocytes were collected, 11 inseminated and nine embryos were biopsied on day 3. Of the nine embryos that were analysed, five embryos were affected and four were unaffected. Two unaffected embryos were transferred on day 4 resulting in a triplet pregnancy and the birth of three healthy babies.

The Y chromosome gr/gr subdeletion is associated with male infertility.

Men with Y chromosome (Yq) AZFc deletions lack all copies of the DAZ gene and have severe spermatogenic failure. A recently described gr/gr subdeletion of AZFc removes two of four copies of DAZ. To better understand the relative frequencies of AZFc and gr/gr deletions and their associated phenotypes, we analysed two large groups of infertile men. A total of 788 men from the Monash Male Infertility (MMI) database with a range of fertility disorders showed similar overall prevalences of AZFc (2.5%) and gr/gr deletions (3.4%). There was no association of gr/gr deletions with sperm density. In 234 control men of known or presumed fertility, only one gr/gr deletion was found. In a further 599 consecutive men presenting for assisted reproductive technologies, we detected 13 (2.2%) AZFc deletions and 28 (4.7%) gr/gr deletions. All AZFc deletions were seen with sperm densities <5 million/ml but again the gr/gr deletion occurred with similar frequency across all sperm density categories. These data show that gr/gr deletions are significantly associated with infertility in the Australian population (P = 0.0015) but not exclusively with reduced sperm density suggesting a complex interaction with other factors important for male fertility. Vertical transmission of gr/gr deletions from father to son by ICSI was demonstrated in four cases. Analysis of 130 ICSI-conceived sons revealed no de novo gr/gr deletions indicating that ICSI is not a risk factor. The data suggest that testing for gr/gr deletions should be considered in the routine genetic assessment of men with idiopathic infertility.

Birth of a healthy infant following trophectoderm biopsy from blastocysts for PGD of beta-thalassaemia major.

PGD is a well accepted reproductive choice for couples at genetic risk and involves the diagnosis and transfer of unaffected IVF embryos. PGD for monogenetic diseases is most commonly accomplished by the biopsy of one or two blastomeres from cleavage stage embryos, followed by PCR-based protocols. However, PCR-based DNA analysis of one or two cells is subject to several problems, including total PCR failure, or failure of one allele to amplify. Trophectoderm biopsy at the blastocyst stage enables the removal of more than two cells for diagnosis while being non-invasive to the inner cell mass which is destined for fetal development. The aim of this study was to develop a safe, reliable technique for the biopsy of trophectoderm cells from human blastocysts. This case report demonstrates that removal of trophectoderm cells prior to blastocyst transfer is compatible with implantation and development to term. Here we report successful PGD for beta-thalassaemia following trophectoderm cell biopsy from blastocysts and the birth of a healthy infant.

A QF-PCR system to detect chromosome 13 aneuploidy from as few as ten cells.

Complete and partial trisomies of chromosome 13 are characterized by abnormal fetal development and birth defects. Despite the severe abnormalities associated with trisomy 13, some couples elect not to undergo invasive prenatal diagnosis (PND) due to the 0.5%-1.0% risk of pregnancy loss. As a result, current studies are focusing on refining non-invasive prenatal diagnostic techniques, such as screening fetal cells isolated from maternal blood or cervical smears. As these techniques only provide a limited number of cells for analysis, any progress in this field depends on the development of a highly sensitive genetic screening strategy. We have developed a quantitative fluorescent PCR (QF-PCR) system capable of detecting chromosome 13 aneuploidy from as few as 10 cells. The system was further validated by screening 13 amniocyte samples, three of which had been diagnosed by FISH as having chromosomal abnormalities involving chromosome 13. In all cases, the QF-PCR results were concordant with those obtained using FISH. The high reliability (99%) and accuracy (96%) of the QF-PCR system at the 10 cell level makes this technique ideal for use in non-invasive PND.

Towards preimplantation diagnosis of cystic fibrosis using microarrays.

Cystic fibrosis (CF) is a common indication for preimplantation genetic diagnosis (PGD). A 3-bp deletion (DeltaF508) in the cftr gene, which accounts for approximately 80% of all CF mutations in the Caucasian population, is normally diagnosed in IVF embryos using fluorescent PCR (FL-PCR) and allelic sizing. In PGD, the possibility of using microarrays for genetic diagnosis is largely unexplored. Therefore, the aim of this study was to prove the diagnostic capability of microarrays for PGD, using DeltaF508 as a model mutation. To this end, oligonucleotide probes representing both the normal and DeltaF508 disease alleles were used to construct a single microarray platform. Target DNA, which was generated by PCR and labelled with the fluorescent dye Cy3, was hybridized to the array and the DeltaF508 genotypes assigned from the fluorescence bound to each allelic probe. The performance of the array was evaluated by its ability to detect DeltaF508 mutations in target DNA. Strong binding of the target to the probes was observed, allowing the expected DeltaF508 genotypes to be assigned. The reliability and accuracy of the microarray diagnosis for DeltaF508 was blindly assessed on 10 samples with either a homozygous normal, homozygous affected or heterozygous genotype. All samples were correctly genotyped. In addition, PCR products from a previous PGD case involving DeltaF508 were re-evaluated on the array, with results in complete concordance with allelic sizing methods used to make the original diagnosis. Together, these findings prove the concept that the DeltaF508 mutation of CF can be reliably and accurately diagnosed at the single cell level using microarray analysis. The availability of more cost-effective array platforms comprising mutation probes for common single-gene disorders and a reliable method of whole genome amplification (WGA) would allow PGD to be offered to the majority of PGD patients with minimal or no change to methodology.

Mitotic errors in chromosome 21 of human preimplantation embryos are associated with non-viability.

Fluorescent in situ hybridization (FISH) studies of human preimplantation embryos have demonstrated a high proportion of chromosomal mosaicism. To investigate the different timings and nature of chromosomal mosaicism, we developed single cell multiplex fluorescent (FL)-PCR to distinguish meiotic and mitotic cell division errors. Chromosome 21 was investigated as the model chromosome as trisomy 21 (Down's syndrome) represents the most common chromosomal aneuploidy that reaches live birth. Sister blastomeres from a total of 25 chromosome 21 aneuploid embryos were analysed. Of these, 13 (52%) comprised cells with concordant DNA fingerprints indicative of meiotic non-disjunction errors. The remaining 12 (48%) aneuploid embryos comprised discordant sister blastomere allelic profiles and thus were mosaic. Errors at all stages including metaphase (MI) (12%) and first (38%), second (31%) and third (19%) mitotic cleavage divisions were identified from the types and proportion of different allelic profiles. In addition, three embryos showed combined meiotic and mitotic cell division errors including non-disjunction and anaphase lag, suggesting that diploid cells had resulted from an aneuploid zygote. However, the majority of the mosaic aneuploid embryos showed mitotic gains and losses from a diploid zygote occurring prior to the activation of the embryonic genome. Allelic profiling of amniocytes from 15 prenatal diagnosis samples displayed only meiotic errors. There appears to be a large difference between the proportion of mosaic mitotic-derived trisomy 21 embryos and fetuses. These findings indicate that mosaic mitotic error of chromosome 21 is associated with non-viability.

Preimplantation diagnosis of Lesch-Nyhan using mini-sequencing primer extension.

Lesch-Nyhan syndrome (LN) is a severe X-linked disorder of males characterized by hyperuricaemia, choreoathetosis, spasticity, mental retardation and self-mutilation. The disorder is caused by a wide spectrum of mutations distributed throughout the hypoxanthine phosphoribosyltransferase (HPRT) gene. Female carriers of LN display no clinical symptoms but are at 50% risk of passing on the affected gene to their male offspring. A couple who had a boy with LN were referred to Monash IVF for preimplantation genetic diagnosis (PGD) because the woman had undergone tubal ligation and the couple wanted to have another child. A test was developed for the causative mutation IVS8+6 T-->G mutation based on minisequencing primer extension that also incorporated the co-analysis of an informative tetranucleotide marker in intron 3 of the HPRT gene to identify allelic dropout. All four biopsied embryos from their first IVF cycle were diagnosed as unaffected, and transfer of two embryos in the cohort with the highest morphological quality resulted in a singleton pregnancy and the birth of a healthy girl. Direct mutation detection by mini-sequencing and parallel analysis of an informative linked marker provides an alternative strategy for molecular diagnosis of point mutations that will have useful application in PGD for other single gene disorders.

Diagnosis of trisomy 21 in preimplantation embryos by single-cell DNA fingerprinting.

Many couples presenting for preimplantation genetic diagnosis (PGD) for a single gene disorder are of advanced reproductive age (>35 years) and have a greater chance of producing embryos with chromosomal aneuploidies. The most common chromosomal aneuploidy observed in newborns is trisomy 21, or Down's syndrome. Consequently, the availability of a highly reliable system that simultaneously detects the heritable gene disorder and trisomy 21 would be beneficial to couples at specific risk. A pentaplex chromosome 21 (Ch 21) single-cell DNA fingerprinting system was developed in a multiplex fluorescence polymerase chain reaction (FL-PCR) on single cells. High reliability and accuracy rates were observed, together with low allele dropout (ADO) and preferential amplification rates on diploid buccal cells, trisomy 21 buccal cells and blastomeres derived from Ch 21 aneuploid embryos. A combined multiplex FL-PCR format was optimized with the common cystic fibrosis delta F508 mutation and validated on single buccal cells from a carrier of the cystic fibrosis delta F508 mutation. This new test is a very powerful technique, which also allows confirmation of the embryo parentage and the identification of extraneous DNA contamination that could cause a misdiagnosis in PGD cases.