Improved clinical utility of preimplantation genetic testing through the integration of ploidy and common pathogenic microdeletions analyses.

STUDY QUESTION: Can chromosomal abnormalities beyond copy-number aneuploidies (i.e. ploidy level and microdeletions (MDs)) be detected using a preimplantation genetic testing (PGT) platform? SUMMARY ANSWER: The proposed integrated approach accurately assesses ploidy level and the most common pathogenic microdeletions causative of genomic disorders, expanding the clinical utility of PGT. WHAT IS KNOWN ALREADY: Standard methodologies employed in preimplantation genetic testing for aneuploidy (PGT-A) identify chromosomal aneuploidies but cannot determine ploidy level nor the presence of recurrent pathogenic MDs responsible for genomic disorders. Transferring embryos carrying these abnormalities can result in miscarriage, molar pregnancy, and intellectual disabilities and developmental delay in offspring. The development of a testing strategy that integrates their assessment can resolve current limitations and add valuable information regarding the genetic constitution of embryos, which is not evaluated in PGT providing new level of clinical utility and valuable knowledge for further understanding of the genomic causes of implantation failure and early pregnancy loss. To the best of our knowledge, MDs have never been studied in preimplantation human embryos up to date. STUDY DESIGN, SIZE, DURATION: This is a retrospective cohort analysis including blastocyst biopsies collected between February 2018 and November 2021 at multiple collaborating IVF clinics from prospective parents of European ancestry below the age of 45, using autologous gametes and undergoing ICSI for all oocytes. Ploidy level determination was validated using 164 embryonic samples of known ploidy status (147 diploids, 9 triploids, and 8 haploids). Detection of nine common MD syndromes (-4p=Wolf-Hirschhorn, -8q=Langer-Giedion, -1p=1p36 deletion, -22q=DiGeorge, -5p=Cri-du-Chat, -15q=Prader-Willi/Angelman, -11q=Jacobsen, -17p=Smith-Magenis) was developed and tested using 28 positive controls and 97 negative controls. Later, the methodology was blindly applied in the analysis of: (i) 100 two pronuclei (2PN)-derived blastocysts that were previously defined as uniformly euploid by standard PGT-A; (ii) 99 euploid embryos whose transfer resulted in pregnancy loss. PARTICIPANTS/MATERIALS, SETTING, METHODS: The methodology is based on targeted next-generation sequencing of selected polymorphisms across the genome and enriched within critical regions of included MD syndromes. Sequencing data (i.e. allelic frequencies) were analyzed by a probabilistic model which estimated the likelihood of ploidy level and MD presence, accounting for both sequencing noise and population genetics patterns (i.e. linkage disequilibrium, LD, correlations) observed in 2504 whole-genome sequencing data from the 1000 Genome Project database. Analysis of phased parental haplotypes obtained by single-nucleotide polymorphism (SNP)-array genotyping was performed to confirm the presence of MD. MAIN RESULTS AND THE ROLE OF CHANCE: In the analytical validation phase, this strategy showed extremely high accuracy both in ploidy classification (100%, CI: 98.1-100%) and in the identification of six out of eight MDs (99.2%, CI: 98.5-99.8%). To improve MD detection based on loss of heterozygosity (LOH), common haploblocks were analyzed based on haplotype frequency and LOH occurrence in a reference population, thus developing two further mathematical models. As a result, chr1p36 and chr4p16.3 regions were excluded from MD identification due to their poor reliability, whilst a clinical workflow which incorporated parental DNA information was developed to enhance the identification of MDs. During the clinical application phase, one case of triploidy was detected among 2PN-derived blastocysts (i) and one pathogenic MD (-22q11.21) was retrospectively identified among the biopsy specimens of transferred embryos that resulted in miscarriage (ii). For the latter case, family-based analysis revealed the same MD in different sibling embryos (n = 2/5) from non-carrier parents, suggesting the presence of germline mosaicism in the female partner. When embryos are selected for transfer based on their genetic constitution, this strategy can identify embryos with ploidy abnormalities and/or MDs beyond aneuploidies, with an estimated incidence of 1.5% (n = 3/202, 95% CI: 0.5-4.5%) among euploid embryos. LIMITATIONS, REASONS FOR CAUTION: Epidemiological studies will be required to accurately assess the incidence of ploidy alterations and MDs in preimplantation embryos and particularly in euploid miscarriages. Despite the high accuracy of the assay developed, the use of parental DNA to support diagnostic calling can further increase the precision of the assay. WIDER IMPLICATIONS OF THE FINDINGS: This novel assay significantly expands the clinical utility of PGT-A by integrating the most common pathogenic MDs (both de novo and inherited ones) responsible for genomic disorders, which are usually evaluated at a later stage through invasive prenatal testing. From a basic research standpoint, this approach will help to elucidate fundamental biological and clinical questions related to the genetics of implantation failure and pregnancy loss of otherwise euploid embryos. STUDY FUNDING/COMPETING INTEREST(S): No external funding was used for this study. S.C., M.F., F.C., P.Z., I.P., L.G., C.P., M.P., D.B., J.J.-A., D.B.-J., J.M.-V., and C.R. are employees of Igenomix and C.S. is the head of the scientific board of Igenomix. A.C. and L.P. are employees of JUNO GENETICS. Igenomix and JUNO GENETICS are companies providing reproductive genetic services. TRIAL REGISTRATION NUMBER: N/A.

Number of embryos biopsied as a predictive indicator for the outcome of preimplantation genetic diagnosis by fluorescence in situ hybridisation in translocation cases.

This study aimed to investigate the optimum number of embryos to be biopsied in order to increase the likelihood of obtaining a balanced/normal embryo following preimplantation genetic diagnosis (PGD) by fluorescence in situ hybridisation (FISH) for translocation carriers. Patients with low number of fertilised oocytes (≤5) or low number of embryos available for PGD (<7) underwent multiple hormonal stimulation cycles and their embryos from each cycle were vitrified and accumulated to obtain at least three embryos for PGD. Fifty-seven PGD cycles were performed for translocation carriers by FISH on day 3 of embryo development. PGD and pregnancy outcomes were examined according to the number of embryos biopsied. The cancellation rates of embryo transfer for the reciprocal translocation carriers were 40% when more than eight embryos were biopsied and it was as high as 78% when low number of embryos (less than nine) were biopsied. For Robertsonian translocation carriers, when more than eight embryos were biopsied, there were no embryo transfer cancellations. This study showed that when there are more than nine embryos biopsied for PGD, the likelihood of obtaining a balanced embryo and positive pregnancy outcome is significantly higher (P < 0.05) in such the overall pregnancy rate was 63% for reciprocal and 86% for Robertsonian carriers. This was reduced to only 7% for reciprocal and 14% for Robertsonian translocation carriers when less than nine embryos were biopsied. One of the limitations of this study was that the analysis was performed by FISH and more studies should investigate the outcomes of embryo accumulation following comprehensive chromosome analysis.

Poor embryo development and preimplantation genetic diagnosis outcomes of translocations involving chromosome 10: Do we blame genetics?

Balanced reciprocal translocation carriers are usually phenotypically normal. Although the reproductive risk of these carriers varies, they generally have a lower chance to produce normal or balanced gametes. Preimplantation genetic diagnosis (PGD) is offered to these patients to increase their chances of becoming pregnant by selecting a balanced embryo for transfer. This study aimed to analyse the development and the PGD outcome of the embryos obtained from reciprocal translocation carriers focusing on ones with chromosome 10 rearrangements. In total, 27 reciprocal translocation carriers underwent 31 cycles of PGD. PGD was performed using multicolour fluorescence in situ hybridisation for 298 embryos and of these 136 were obtained from couples carrying translocations involving chromosome 10 rearrangements. Carriers of translocations involving chromosome 10 rearrangements have a lower chance of producing normal or balanced embryos compared with the carriers with other rearrangements. The development of embryos obtained from the patients with chromosome 10 rearrangements was impaired and only a limited number of embryos developed to the blastocyst stage.

Human embryonic stem cell culture: current limitations and novel strategies.

Embryonic stem cells (ESC) are multipotent cells isolated from blastocyst-stage preimplantation embryos. Since their first culture in 1998, human ESC have revolutionized reproductive and regenerative medicine by allowing the establishment of detailed molecular and therapeutic models for certain metabolic pathways and life-threatening disorders. They also offer significant contributions to genetics and pharmacology in designing and analysing disease models that can be closer to in vivo than any other procedures available. However, the procedures by which they are obtained and manipulated also create intense ethical and social debates worldwide. This article discusses the current limitations and recent advances in isolation, culture and differentiation of human ESC from the laboratory perspective.

Embryo aneuploidy screening for repeated implantation failure and unexplained recurrent miscarriage.

Among other factors, chromosomal abnormalities that originate from gametogenesis and preimplantation embryonic development are thought to be one of the major contributing factors for early embryonic death and failure of pregnancy. However, so far, no non-invasive technique exists that allows the detection of the chromosomal complement of an oocyte or a developing embryo as a whole. Rather, by removing polar bodies/blastomeres, recent developments on preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) have paved the way to detect and possibly eliminate the majority of chromosomally abnormal embryos, thereby increasing the chance of a healthy pregnancy. This article summarizes the origin and impact of chromosomal abnormalities on human reproduction in cases with repeated implantation failure (RIF) and unexplained recurrent miscarriage. It also discusses recent advances regarding the possible benefits of PGD-AS in such cases.

Preliminary FISH studies on spermatozoa and embryos in patients with variable degrees of teratozoospermia and a history of poor prognosis.

The aim of this study was to analyse to what extent sperm aneuploidy is associated with sperm morphology and subsequently with embryo aneuploidy. Fifty-nine men with variable degrees of teratozoospermia and previously poor assisted reproduction prognosis were included in the study. Samples from 10 normozoospermic men with proven fertility were used as controls. Individual spermatozoa were scored for chromosomes 13, 21 and for 18, X, Y separately. Compared with controls, 23 out of 59 cases (39.0%) were found to have increased sperm aneuploidy for at least one of the chromosomes analysed in a treatment cycle. Fifty-two patients underwent a treatment cycle and were documented according to the pregnancy and spermatozoa fluorescence in-situ hybridization results. A total of 121 previous unsuccessful assisted reproduction cycles of the cases were then retrospectively reviewed. In 23 of the latest cycles, preimplantation genetic diagnosis was applied to 106 cleavage stage embryos and 47 of 94 embryos analysed (50.0%) were found to be chromosomally abnormal. Furthermore, 16 of 47 (34.0%) embryos with chromosomal abnormality were carrying complex chromosomal defects. The results imply that increased aneuploidy is present in both spermatozoa and embryos in couples with severe male infertility with a history of repeated unsuccessful attempts. Therefore, proper genetic counselling should be considered in these cases.

Clinical aspects of preimplantation genetic diagnosis for single gene disorders combined with HLA typing.

Preimplantation genetic diagnosis (PGD) for single gene disorders combined with human leukocyte antigen (HLA) matching has recently emerged as a therapeutic tool for stem cell transplantation in couples bearing an affected offspring. There may exist, however, several patient- or cycle-specific limitations for certain couples. This article documents data regarding experience of single gene disorders combined with HLA matching obtained at Istanbul Memorial Hospital, Turkey. The data were obtained from 20 couples undergoing 26 PGD-HLA cycles for thalassaemia (n = 23), Wiscott-Aldrich syndrome (n = 1) and acute lymphoblastic leukaemia (n = 2). A total of 206 embryos was biopsied on day 3 of embryo development and subsequently analysed. After the analysis, 26 (12.6%) of them were found to be both healthy and HLA compatible. In 16 embryo transfers performed, seven (43.7%) clinical pregnancies were obtained, one of which resulted in miscarriage. Ten of the 26 cycles started (38.4%) were cancelled due to a lack of suitable (mutation-free and/or HLA-compatible) embryos. The data suggest that application of PGD in combination with HLA typing is a promising therapeutic tool for an affected sibling.

Effect of PGD on implantation and ongoing pregnancy rates in cases with predominantly macrocephalic spermatozoa.

Although its occurrence is rare, the presence of large headed or macrocephalic spermatozoa and increased chromosomal abnormality has recently been reported by several groups. Moreover, when intracytoplasmic sperm injection (ICSI) was performed with samples containing macrocephalic spermatozoa, lower fertilization and implantation rates result in poor clinical outcome. In order to evaluate the impact of preimplantation genetic diagnosis (PGD) on implantation and ongoing pregnancy rates in these couples, the results of 23 PGD cycles were compared with non-PGD cycles (n = 60) as well as cycles with absolute teratozoospermia (having zero normal morphology) with (n = 14) or without PGD (n = 66). Out of 82 embryos biopsied in the macrocephalic sperm group, abnormalities were detected in 46.4% of the embryos analysed. Most of the abnormalities were trisomies (37.0%) and complex aneuploidies (51.9%). A 33.3% pregnancy rate was achieved by selectively transferring euploid embryos after PGD with the statistically higher implantation rate of 25.0% compared with non-PGD cycles (IR: 12.3%, P < 0.01). Moreover, only one missed abortion (14.3%) was observed in the PGD group, whereas seven of the 15 pregnancies resulted in abortion in the non-PGD group (46.7%). Preliminary results indicate that patients should be counselled for increased chromosomal abnormality and a possible beneficial effect of eliminating chromosomally abnormal embryos with PGD on a bortion rates.

The results of aneuploidy screening in 276 couples undergoing assisted reproductive techniques.

Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) using sequential in situ hybridization was applied for aneuploidy testing in 276 couples with 282 ART cycles. Patients with advanced maternal age (AMA, n = 147), recurrent implantation failure (RIF, n = 48), repeated early spontaneous abortion (RSA, n = 32) and abnormal gamete cell morphology (AGCM, n = 55) including macrocephal sperm forms or cytoplasmic granular oocytes were included. Embryo biopsy was performed on day 3 in a calcium-magnesium-free medium by using a noncontact diode laser system. After fixation and enzymatic treatment, fluorescent in situ hybridization (FISH) was carried out on 1147 blastomeres with specific probes for chromosomes 13, 16, 18, 21 and 22 for AMA group, 13, 18, 21, X and Y for AGCM group and 13, 16, 18, 21, 22, X and Y for RIF and RSA groups respectively. The overall chromosomal abnormality rate in analyzed embryos was 40.9%, with no significant difference between AMA, RIF and RSA groups (p > 0.05). However, AGCM group presented a higher rate of chromosomal aneuploidies (57.4%) than the other three groups (p < 0.01). A total of 84% biopsied embryos presented cleavage in 24 h and embryo transfer was realized in 278 cycles. In four cycles, no chromosomally normal embryo was found for embryo transfer. A total of 88 pregnancies (31.6%) were achieved, 19.3% resulted in abortion and 63 healthy births were obtained, with a total of 93 babies born. Aneuploidy testing in couples with poor prognosis undergoing ART cycles is a useful tool to increase the chance of ART success. Furthermore, abnormal gamete cell morphology should be considered one of the major indications for PGD in ART programs as high aneuploidy rates were observed in this group.

Assessment of DNA fragmentation and aneuploidy on poor quality human embryos.

In human assisted reproduction, low embryo quality due to retarded growth and abnormal cellular morphology results in fewer embryos suitable for transfer. This study aimed to assess the extent of DNA fragmentation and aneuploidy in spare slow growing or arrested human embryos. In 19 assisted reproduction cycles, a total of 57 embryos unsuitable for embryo transfer were used for simultaneous apoptosis and aneuploidy assessment. Among them, 31 (54.3%) showed DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) analysis. Among 26 embryos that were negative for TUNEL, interpretable fluorescence in-situ hybridization (FISH) results were obtained for 25 embryos (96.2%). Sixteen embryos were detected to be chromosomally abnormal (64.0%); three were found to be chaotic, six had complex aneuploidy, six had complete monosomy and one was polyploid. The results show that a high level of DNA fragmentation and aneuploidy are common in embryos with slow growth and/or low quality. More detailed studies are needed to assess the effect of factors such as ovarian stimulation regimens and in-vitro culture conditions. Moreover, application of simultaneous TUNEL and FISH techniques can be informative regarding DNA integrity and aneuploidy.

Embryo development characteristics in Robertsonian and reciprocal translocations: a comparison of results with non-translocation cases.

The effect of translocations on embryo development was evaluated and results were compared in terms of embryo development with those of embryos obtained from standard intracytoplasmic sperm injection (ICSI) cycles. In 23 translocation carriers with 34 cycles, fertilization, pronuclear morphology scoring (PMS), developmental arrest, cleavage and blastocyst formation were evaluated and compared with embryos obtained from non-translocation cases undergoing ICSI (n = 98 cycles). In 28 cycles, preimplantation genetic diagnosis (PGD) was performed on prezygotes (first and second polar body biopsy for female carriers; n = 3) or on embryos having seven or more blastomeres (blastomere biopsy; n = 25). In six cycles for four couples, probes for translocated chromosomes were not available, so PGD could not be performed. Overall, in translocation cases, a lower fertilization rate, a higher rate of retarded embryo development, and a lower rate of blastocyst formation were observed compared with embryos of non-translocation cases. Fluorescence in-situ hybridization (FISH) analysis showed a 70.9% abnormality rate for reciprocal translocations and 55.0% for Robertsonian translocations respectively. In cases with Robertsonian and reciprocal translocation carriers, the probability of poor embryo development, which may be a result of high segregation abnormalities, may negatively affect the outcome of assisted reproductive techniques. This poor prognosis should also be considered when genetic counselling for translocation is given.

Results of preimplantation genetic diagnosis in patients with Klinefelter's syndrome.

With the application of preimplantation genetic diagnosis (PGD), a possible genetic contribution of spermatozoa obtained from 47,XXY non-mosaic Klinefelter patients on preimplantation embryos was analysed in eight couples. Interpretable fluorescence in-situ hybridization results were obtained for 28 out of 33 embryos biopsied (84.8%) and 23 blastomeres were analysed for chromosomes 13, 18, 21, X and Y. Nine out of 23 embryos were diagnosed as abnormal (39.1%). Five out of nine contained sex chromosome abnormalities (55.5%). Two were diagnosed as 47,XXY and three were found to have monosomy X. Besides sex chromosomal abnormalities, other abnormalities detected were haploidy, triploidy, monosomy 13, monosomy 18 and trisomy 13. Five blastomeres were analysed for sex chromosomes only and all of them were found to be normal. Overall, the rate of sex chromosome abnormality in biopsied embryos was found to be 17.8% (5/28). Moreover, among 33 embryos biopsied, five of the eight zygotes, which were classified as a poor prognosis group according to pronuclear morphology scoring, showed an impaired growth profile after biopsy and were found to be chromosomally abnormal. Elimination of abnormal embyos and transfer of normal ones resulted in four pregnancies in eight cycles (50%). Two pregnancies, one singleton and one twins resulted in healthy births. Two pregnancies, one singleton and one twins are continuing beyond the second trimester. These results show that there is in fact elevated chromosomal abnormality for both sex chromosomes and autosomes in embryos developed from Klinefelter males. Furthermore together with PGD, embryo scoring according to pronuclear morphology can give additional benefit for selecting chromosomally abnormal embryos. Therefore, PGD should be recommended in cases with Klinefelter's syndrome and this information should be discussed with the couple when genetic counselling is given.

Pronuclear morphology scoring and chromosomal status of embryos in severe male infertility.

BACKGROUND: The study aim was to evaluate the relationship between pronuclei morphology scoring (PNMS) and the chromosomal complement of embryos in couples with severe male infertility undergoing ICSI. A total of 3116 pre-embryos was scored according to PNMS in 452 cycles. METHODS: Pre-embryos were classified into eight categories based on the alignment, size, linear or irregular distribution of pronuclear bodies (PNB), position and clarity of cytoplasmic halo and abutting of the pronucleus. These categories were subdivided into groups I and II according to the similarity and distribution of PNB. RESULTS: In total, 2574 pre-embryos formed by using ejaculated sperm, while 542 pre-embryos developed by injection of testicular sperm or round spermatids. More group II pre-embryos with markedly different morphology from group I were formed after ICSI with testicular sperm than with fresh ejaculated sperm (32.1 versus 22.7%, P < 0.01). Of 490 pre-embryos in which pronuclear morphology was evaluated, 263 were biopsied for preimplantation genetic diagnosis. The rate of chromosomal abnormality was higher in embryos developed from group II pre-embryos (52.2%) than in embryos developed from group I prezygotes (37.6%, P < 0.05). CONCLUSIONS: Group II pre-embryos had markedly different morphology from group I, and had a low rate of blastocyst formation and high risk of chromosomally abnormal embryos. When testicular sperm and round spermatids were used for ICSI, more group II pre-embryos and chromosomally abnormal embryos were produced than with ejaculated sperm. Pronuclear morphology was correlated with chromosomal complement, and impacted upon by the sperm source.

Acquired expression of transcriptionally active p73 in hepatocellular carcinoma cells.

p53 and p73 proteins activate similar target genes and induce apoptosis and cell cycle arrest. However, p53, but not p73 is considered a tumour-suppressor gene. Unlike p53, p73 deficiency in mice does not lead to a cancer-prone phenotype, and p73 gene is not mutated in human cancers, including hepatocellular carcinoma. Here we report that normal liver cells express only DeltaN-p73 transcript forms giving rise to the synthesis of N-terminally truncated, transcriptionally inactive and dominant negative p73 proteins. In contrast, most hepatocellular carcinoma cells express TA-p73 transcript forms encoding full-length and transcriptionally active p73 proteins, in addition to DeltaN-p73. We also show that together with the acquired expression of TA-p73, the 'retinoblastoma pathway' is inactivated, and E2F1-target genes including cyclin E and p14(ARF) are activated in hepatocellular carcinoma. However, there was no full correlation between 'retinoblastoma pathway' inactivation and TA-p73 expression. Most TA-p73-expressing hepatocellular carcinoma cells have also lost p53 function either by lack of expression or missense mutations. The p73 gene, encoding only DeltaN-p73 protein, may function as a tumour promoter rather than a tumour suppressor in liver tissue. This may be one reason why p73 is not a mutation target in hepatocellular carcinoma.