Characterization of wheat varieties (Triticum spp.) through seed morphology

An experiment was carried out at the Department of Seed Science and Technology, Junagadh Agricultural University, Junagadh, to characterize 28 wheat varieties of different species [17 varieties (MP 4010, HI 1500, HI 1531, HI 1544, GW 1, GW 503, DL 788-2, HD 2932, GW 11, GW 173, GW 190, GW 273, LOK 1, GW 322, MP 3288, GW 366 and GW 496) of Triticum aestivum, 9 varieties (HI 8381, HI 8498, HI 8627, HI 8713, A 28, A 206, GDW 1255, GW 1139 and RAJ 1555) of Triticum durum and 2 varieties (DDK 1025, DDK 1029) of Triticum dicoccum)] released for general cultivation in Gujarat at state level as well as at the National level in Central India based on the seed morphological characters. Based on the seed colour, the varieties were grouped into amber (25), white (1) and red (2). On the basis of seed shape, wheat varieties were separated in to round (2) ovate (7), oblong (12) and elliptical (7). On the basis of seed size, varieties were grouped into medium seed size (5), bold seed size (15) and very bold seed size (8). On the basis of seed hardness, varieties were grouped into soft (1), semi-hard (7) and hard (20). Based on the seed germ width, varieties were grouped into medium (15), wide (9) and narrow (4) seed germ width types. The varieties were grouped based on the seed crease into three groups, as medium (11),shallow (12) and deep (5) grain crease types. Based on the brush hair length, wheat varieties were grouped as short (17), medium (7) and long (4) brush hair length types

The first ongoing pregnancy following comprehensive aneuploidy assessment using a combined blastocenetesis, cell free DNA and trophectoderm biopsy strategy

Background: The exact origin of cell-free DNA found in spent culture media or blastocoel fluid is currently unknown but with the potential to become an improved source of DNA for chromosomal analysis than trophectoderm biopsy samples, it provides a superior representation of the fetal genetic status. However, the genetic material contained within the blastocoel cavity may be more reliable to assessment of embryo euploidy in a clinical context than trophectoderm of cell-free DNA. Case Presentation: This is the first UK case report where all three sources of DNA were analyzed in a clinical setting on 29 th January 2018 at the Centre for Reproductive and Genetic Health, London, leading to an ongoing clinical pregnancy. Conclusion: The experience from this case report suggests that removal of blasto-coel fluid, sampling of spent culture media and trophectoderm biopsy can be carried out in parallel. Gathering genetic information from two to three independent samples of embryo DNA may provide enhanced diagnostic accuracy and may clarify cytogenetic status of mosaic embryos

The incidence and origin of segmental aneuploidy in human oocytes and preimplantation embryos

Study Questions What is the incidence, origin and clinical significance of segmental aneuploidy in human oocytes and preimplantation embryos? Summary Answer Segmental aneuploidy occurs at a considerable frequency in preimplantation embryos with a majority being mitotic in origin. What is Known Already In recent years, accurate techniques for the detection of aneuploidy in single cells have been developed. Research using such methods has confirmed that aneuploidy is a common feature of human oocytes and preimplantation embryos. However, thus far research has mainly focused on loss or gain of whole chromosomes. We utilized sensitive molecular methods to study another important form of cytogenetic abnormality at the earliest stages of human development, namely segmental aneuploidy. Study Design, Size, Duration Chromosomal copy number data was obtained from oocytes and embryos of 635 IVF patients, who requested chromosome screening for various reasons, most commonly for advanced maternal age or previously unsuccessful IVF treatments. A total of 3541 samples comprising of 452 human oocytes, 1762 cleavage stage and 1327 blastocyst stage embryos were investigated in the present study. Participants/Materials, Setting, Methods Whole genome amplification (Sureplex, Illumina) was performed on cells biopsied from oocytes and embryos of IVF patients who requested chromosome screening. The samples were subsequently processed and analyzed for their chromosome complement using microarray comparative genomic hybridization (aCGH), (Illumina, Cambridge, UK). Main Results and the Role of Chance Segmental abnormalities, involving loss or gain of chromosomal fragments in excess of 15 Mb, were found to occur at a high frequency. The incidence of such abnormalities was 10.4% in oocytes, but this increased dramatically during the first 3 days of embryonic development (24.3%), before starting to decline as embryos reached the final (blastocyst) stage of preimplantation development (15.6%). While some segmental errors were clearly of meiotic origin, most appear to arise during the first few mitoses following fertilization. The reduction in frequency at the blastocyst stage suggests that many cells/embryos affected by segmental abnormalities are eliminated (e.g. via arrest of the affected embryos or apoptosis of abnormal cells). Interestingly, sites of chromosome breakage associated with segmental aneuploidy were not entirely random but tended to occur within distinct chromosomal regions. Some of the identified hotspots correspond to known fragile sites while others may be considered novel and may be specific to gametogenesis and/or embryogenesis. Limitations Reasons for Caution The cytogenetic analysis was performed on biopsies of embryos, which might not be representative of the true incidence of mosaic segmental aneuploidy of the entire embryo. Wider Implications of the Findings The findings of this study are valuable for understanding the origin of subchromosomal duplications and deletions, a clinically important class of abnormalities that are a common cause of congenital abnormalities and miscarriage. Furthermore, the results provide additional evidence that control of the cell cycle is more relaxed during the first few mitotic divisions following fertilization, permitting DNA double-strand breaks to occur and persist through cell division. The data are also of great relevance for preimplantation genetic testing, where the detection of segmental aneuploidy is currently considered problematic for embryo diagnosis and patient counseling.</p

Clinical application of a protocol based on universal next-generation sequencing for the diagnosis of beta-thalassaemia and sickle cell anaemia in preimplantation embryos

Research question Mutations of the beta-globin gene (HBB) cause beta-thalassaemia and sickle cell anaemia and are the most common cause of severe inherited disease in humans. Traditional PGD protocols for the detection of HBB mutations frequently involve labour intensive, patient-specific test design due to the wide diversity of disease associated HBB mutations. For this reason, we aimed to develop and clinically apply a universal PGD method to test for mutations in the HBB gene. Design A multiplex PCR 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 (SNPs). Amplicons were then analyzed using a next generation sequencing (NGS) method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified (WGA) DNA derived from embryos of three couples carrying different combinations of β-thalassemia mutations. Results The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Interestingly, analysis of 141 heterozygous sites showed no instances of allele dropout (ADO) and the test displayed 100% concordance when compared to the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive NGS may provide superior accuracy than typically achieved using traditional PGD 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 PGD, especially in less affluent parts of the world.</p

The incidence and origin of segmental aneuploidy in human oocytes and preimplantation embryos

Study Questions What is the incidence, origin and clinical significance of segmental aneuploidy in human oocytes and preimplantation embryos? Summary Answer Segmental aneuploidy occurs at a considerable frequency in preimplantation embryos with a majority being mitotic in origin. What is Known Already In recent years, accurate techniques for the detection of aneuploidy in single cells have been developed. Research using such methods has confirmed that aneuploidy is a common feature of human oocytes and preimplantation embryos. However, thus far research has mainly focused on loss or gain of whole chromosomes. We utilized sensitive molecular methods to study another important form of cytogenetic abnormality at the earliest stages of human development, namely segmental aneuploidy. Study Design, Size, Duration Chromosomal copy number data was obtained from oocytes and embryos of 635 IVF patients, who requested chromosome screening for various reasons, most commonly for advanced maternal age or previously unsuccessful IVF treatments. A total of 3541 samples comprising of 452 human oocytes, 1762 cleavage stage and 1327 blastocyst stage embryos were investigated in the present study. Participants/Materials, Setting, Methods Whole genome amplification (Sureplex, Illumina) was performed on cells biopsied from oocytes and embryos of IVF patients who requested chromosome screening. The samples were subsequently processed and analyzed for their chromosome complement using microarray comparative genomic hybridization (aCGH), (Illumina, Cambridge, UK). Main Results and the Role of Chance Segmental abnormalities, involving loss or gain of chromosomal fragments in excess of 15 Mb, were found to occur at a high frequency. The incidence of such abnormalities was 10.4% in oocytes, but this increased dramatically during the first 3 days of embryonic development (24.3%), before starting to decline as embryos reached the final (blastocyst) stage of preimplantation development (15.6%). While some segmental errors were clearly of meiotic origin, most appear to arise during the first few mitoses following fertilization. The reduction in frequency at the blastocyst stage suggests that many cells/embryos affected by segmental abnormalities are eliminated (e.g. via arrest of the affected embryos or apoptosis of abnormal cells). Interestingly, sites of chromosome breakage associated with segmental aneuploidy were not entirely random but tended to occur within distinct chromosomal regions. Some of the identified hotspots correspond to known fragile sites while others may be considered novel and may be specific to gametogenesis and/or embryogenesis. Limitations Reasons for Caution The cytogenetic analysis was performed on biopsies of embryos, which might not be representative of the true incidence of mosaic segmental aneuploidy of the entire embryo. Wider Implications of the Findings The findings of this study are valuable for understanding the origin of subchromosomal duplications and deletions, a clinically important class of abnormalities that are a common cause of congenital abnormalities and miscarriage. Furthermore, the results provide additional evidence that control of the cell cycle is more relaxed during the first few mitotic divisions following fertilization, permitting DNA double-strand breaks to occur and persist through cell division. The data are also of great relevance for preimplantation genetic testing, where the detection of segmental aneuploidy is currently considered problematic for embryo diagnosis and patient counseling.</p

Clinical application of a protocol based on universal next-generation sequencing for the diagnosis of beta-thalassaemia and sickle cell anaemia in preimplantation embryos

Research question Mutations of the beta-globin gene (HBB) cause beta-thalassaemia and sickle cell anaemia and are the most common cause of severe inherited disease in humans. Traditional PGD protocols for the detection of HBB mutations frequently involve labour intensive, patient-specific test design due to the wide diversity of disease associated HBB mutations. For this reason, we aimed to develop and clinically apply a universal PGD method to test for mutations in the HBB gene. Design A multiplex PCR 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 (SNPs). Amplicons were then analyzed using a next generation sequencing (NGS) method, revealing mutations and SNP genotypes. The protocol was extensively validated, optimized and eventually clinically applied on whole-genome amplified (WGA) DNA derived from embryos of three couples carrying different combinations of β-thalassemia mutations. Results The HBB mutation status and associated SNP haplotypes were successfully determined in all 21 embryos. Interestingly, analysis of 141 heterozygous sites showed no instances of allele dropout (ADO) and the test displayed 100% concordance when compared to the results obtained from karyomapping. This suggests that the combination of trophectoderm biopsy and highly sensitive NGS may provide superior accuracy than typically achieved using traditional PGD 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 PGD, especially in less affluent parts of the world.</p

Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts

Preimplantation genetic testing for aneuploidy (PGT-A) is widely used in IVF and aims to improve outcomes by avoiding aneuploid embryo transfers. Chromosomal mosaicism is extremely common in early development and could affect the efficacy of PGT-A by causing incorrect embryo classification. Recent innovations have allowed accurate mosaicism detection in trophectoderm samples taken from blastocysts. However, there is little data concerning the impact of mosaicism on viability, and the optimal clinical pathway for such embryos is unclear. This study provides new information concerning the extent to which mosaic preimplantation embryos are capable of producing pregnancies and births. Archived trophectoderm biopsy specimens from transferred blastocysts were analyzed using next generation sequencing (NGS). Unlike other PGT-A methods, NGS accurately detects mosaicism in embryo biopsies. 44 mosaic blastocysts were identified. Their clinical outcomes were compared to 51 euploid blastocysts, derived from a well-matched, contemporary control group. Mosaic embryos were associated with outcomes that were significantly poorer than those of the control group: implantation 30.1 versus 55.8% (P = 0.038); miscarriage rate 55.6 versus 17.2% (P = 0.036); and ongoing pregnancy 15.4 versus 46.2% (P = 0.003). 61% of the mosaic errors affected whole chromosomes and 39% were segmental aneuploidies. Embryo viability is compromised by the presence of aneuploid cells. However, a minority of affected embryos can produce successful pregnancies. Hence, such embryos should not necessarily be excluded, but given a lower priority for transfer than those that are fully euploid. It is recommended that pregnancies established after mosaic embryo transfers be subjected to prenatal testing, with appropriate patient counselling

Analysis of implantation and ongoing pregnancy rates following the transfer of mosaic diploid-aneuploid blastocysts

Preimplantation genetic testing for aneuploidy (PGT-A) is widely used in IVF and aims to improve outcomes by avoiding aneuploid embryo transfers. Chromosomal mosaicism is extremely common in early development and could affect the efficacy of PGT-A by causing incorrect embryo classification. Recent innovations have allowed accurate mosaicism detection in trophectoderm samples taken from blastocysts. However, there is little data concerning the impact of mosaicism on viability, and the optimal clinical pathway for such embryos is unclear. This study provides new information concerning the extent to which mosaic preimplantation embryos are capable of producing pregnancies and births. Archived trophectoderm biopsy specimens from transferred blastocysts were analyzed using next generation sequencing (NGS). Unlike other PGT-A methods, NGS accurately detects mosaicism in embryo biopsies. 44 mosaic blastocysts were identified. Their clinical outcomes were compared to 51 euploid blastocysts, derived from a well-matched, contemporary control group. Mosaic embryos were associated with outcomes that were significantly poorer than those of the control group: implantation 30.1 versus 55.8% (P = 0.038); miscarriage rate 55.6 versus 17.2% (P = 0.036); and ongoing pregnancy 15.4 versus 46.2% (P = 0.003). 61% of the mosaic errors affected whole chromosomes and 39% were segmental aneuploidies. Embryo viability is compromised by the presence of aneuploid cells. However, a minority of affected embryos can produce successful pregnancies. Hence, such embryos should not necessarily be excluded, but given a lower priority for transfer than those that are fully euploid. It is recommended that pregnancies established after mosaic embryo transfers be subjected to prenatal testing, with appropriate patient counselling