Bioluminescent Imaging Reveals Divergent Viral Pathogenesis in Two Strains of Stat1-Deficient Mice, and in αßγ Interferon Receptor-Deficient Mice

Pivotal components of the IFN response to virus infection include the IFN receptors (IFNR), and the downstream factor signal transducer and activator of transcription 1 (Stat1). Mice deficient for Stat1 and IFNR (Stat1−/− and IFNαßγR−/− mice) lack responsiveness to IFN and exhibit high sensitivity to various pathogens. Here we examined herpes simplex virus type 1 (HSV-1) pathogenesis in Stat1−/− mice and in IFNαßγR−/− mice following corneal infection and bioluminescent imaging. Two divergent and paradoxical patterns of infection were observed. Mice with an N-terminal deletion in Stat1 (129Stat1−/− (N-term)) had transient infection of the liver and spleen, but succumbed to encephalitis by day 10 post-infection. In stark contrast, infection of IFNαßγR−/− mice was rapidly fatal, with associated viremia and fulminant infection of the liver and spleen, with infected infiltrating cells being primarily of the monocyte/macrophage lineage. To resolve the surprising difference between Stat1−/− and IFNαßγR−/− mice, we infected an additional Stat1−/− strain deleted in the DNA-binding domain (129Stat1−/− (DBD)). These 129Stat1−/− (DBD) mice recapitulated the lethal pattern of liver and spleen infection seen following infection of IFNαßγR−/− mice. This lethal pattern was also observed when 129Stat1−/− (N-term) mice were infected and treated with a Type I IFN-blocking antibody, and immune cells derived from 129Stat1−/− (N-term) mice were shown to be responsive to Type I IFN. These data therefore show significant differences in viral pathogenesis between two commonly-used Stat1−/− mouse strains. The data are consistent with the hypothesis that Stat1−/− (N-term) mice have residual Type I IFN receptor-dependent IFN responses. Complete loss of IFN signaling pathways allows viremia and rapid viral spread with a fatal infection of the liver. This study underscores the importance of careful comparisons between knockout mouse strains in viral pathogenesis, and may also be relevant to the causation of HSV hepatitis in humans, a rare but frequently fatal infection

Bioinformatics and Functional Analysis of an Entamoeba histolytica Mannosyltransferase Necessary for Parasite Complement Resistance and Hepatical Infection

The glycosylphosphatidylinositol (GPI) moiety is one of the ways by which many cell surface proteins, such as Gal/GalNAc lectin and proteophosphoglycans (PPGs) attach to the surface of Entamoeba histolytica, the agent of human amoebiasis. It is believed that these GPI-anchored molecules are involved in parasite adhesion to cells, mucus and the extracellular matrix. We identified an E. histolytica homolog of PIG-M, which is a mannosyltransferase required for synthesis of GPI. The sequence and structural analysis led to the conclusion that EhPIG-M1 is composed of one signal peptide and 11 transmembrane domains with two large intra luminal loops, one of which contains the DXD motif, involved in the enzymatic catalysis and conserved in most glycosyltransferases. Expressing a fragment of the EhPIG-M1 encoding gene in antisense orientation generated parasite lines diminished in EhPIG-M1 levels; these lines displayed reduced GPI production, were highly sensitive to complement and were dramatically inhibited for amoebic abscess formation. The data suggest a role for GPI surface anchored molecules in the survival of E. histolytica during pathogenesis

Molecular genetic approaches for the determination of embryo viability

Since its first application in the 1990s, genetic testing of preimplantation embryos has undergone a major transformation and its utilisation continues to grow. While in preimplantation genetic screening, euploid embryos are identified and prioritised for uterine transfer, preimplantation genetic diagnosis identifies embryos free of genetic disease to avoid transmission of hereditary genetic disorders from parents to offspring. This study aimed to deliver a novel preimplantation genetic screening protocol, which is capable of high accuracy aneuploidy detection utilising array-based, nano-scale quantitative real-time polymerase chain reaction. Furthermore, it focused on the development of a preimplantation genetic diagnosis protocol, which is capable of accurate detection of maternally inherited mitochondrial DNA mutations and which is fully compatible with comprehensive chromosome screening. After extensive validation, the novel preimplantation genetic screening technique was successfully implemented clinically for testing of blastocyst-stage embryos and now offers a valuable option for couples undergoing in vitro fertilisation combined with preimplantation genetic screening. It is a unique method in that it is streamlined, rapid, versatile and less labour intensive compared to other conventional methods for aneuploidy screening. Furthermore, it has the potential to become an in-house component of an in vitro fertilisation laboratory, thereby reducing the processing time and eliminating the need for embryo cryopreservation. This in turn will help reduce the cost of in vitro fertilisation cycles with preimplantation genetic screening. In addition, a new preimplantation genetic diagnosis protocol for detection of mitochondrial DNA diseases in conjunction with comprehensive chromosome screening using a next-generation sequencing technology was developed and delivered. The new method was successfully applied in four in vitro fertilisation patients transmitting different mutations causing mitochondrial DNA disease. In one patient, the transfer of an unaffected embryo resulted in the birth of a healthy child. In summary, two novel methods were successfully developed, validated and clinically implemented for preimplantation genetic screening and preimplantation genetic diagnosis of mitochondrial DNA diseases.</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

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 implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study

Study Question Can quantification of mitochondrial DNA (mtDNA) in trophectoderm (TE) biopsy samples provide information concerning the viability of a blastocyst, potentially enhancing embryo selection and improving IVF treatment outcomes? Summary Answer This study demonstrated that euploid blastocysts of good morphology, but with high mtDNA levels had a greatly reduced implantation potential. What is Known Already Better methods of embryo selection leading to IVF outcome improvement are necessary, as the transfer of chromosomally normal embryos of high morphological grade cannot guarantee the establishment of an ongoing pregnancy. The quantity of mtDNA in embryonic cells has been proposed as a new biomarker of viability—higher levels of mtDNA associated with reduced implantation potential. Study Design, Size, Duration mtDNA was quantified in 199 blastocysts, previously biopsied and shown to be chromosomally normal using preimplantation genetic testing for aneuploidy (PGT-A). These were generated by 174 couples (average female age 37.06 years). All patients underwent IVF in a single clinic. The study took place in a blinded, non-selection manner—i.e. mtDNA quantity was not known at the time of single embryo transfer. The fate of the embryos transferred was subsequently compared to the mtDNA levels measured. Participants/Materials, Settings, Methods Embryos were biopsied at the blastocyst stage. The TE samples obtained were subjected to whole genome amplification followed by comprehensive chromosome analysis via next generation sequencing. The same biopsy specimens were also tested using quantitative PCR, allowing highly accurate mtDNA quantification. After blastocyst transfer, the code used for blinding was broken and analysis undertaken to reveal whether the amount of mtDNA had any association with embryo implantation. Main Results and the Role of Chance mtDNA analysis of the 199 blastocysts revealed that 9 (5%) contained unusually high levels of mtDNA. All embryo transfers involved a single chromosomally normal blastocyst of good morphology. Of these, 121 (60%) led to ongoing pregnancies, 11(6%) led to biochemical pregnancies, and 10 (5%) spontaneously miscarried. All (100%) of these blastocysts had mtDNA levels considered to be normal/low. The remaining 57 (29%) blastocysts failed to implant. Among these non-viable embryos there were 9 (16%) with unusually high levels of mtDNA. This meant that the ongoing pregnancy rate for morphologically good, euploid blastocysts, with normal/low levels of mtDNA was 64% (121/190). In contrast, the ongoing pregnancy rate for the same type of embryos, but with elevated mtDNA levels, was 0/9 (0%). This difference was highly statistically significant (P Limitations Reasons for Caution To determine the true extent of any clinical benefits a randomized clinical trial will be necessary. Research is needed to improve understanding of the biology of mtDNA expansion. Wider Implications of the Findings This is the first investigation to evaluate the clinical impact of increased mtDNA in a prospective blinded manner. Results confirm that embryos with elevated mtDNA rarely implant, supporting its use as a viability biomarker. A total of 64% of euploid blastocysts with normal/low mtDNA implanted versus 60% for the cohort as a whole.</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

Clinical implications of mitochondrial DNA quantification on pregnancy outcomes: a blinded prospective non-selection study

Study Question Can quantification of mitochondrial DNA (mtDNA) in trophectoderm (TE) biopsy samples provide information concerning the viability of a blastocyst, potentially enhancing embryo selection and improving IVF treatment outcomes? Summary Answer This study demonstrated that euploid blastocysts of good morphology, but with high mtDNA levels had a greatly reduced implantation potential. What is Known Already Better methods of embryo selection leading to IVF outcome improvement are necessary, as the transfer of chromosomally normal embryos of high morphological grade cannot guarantee the establishment of an ongoing pregnancy. The quantity of mtDNA in embryonic cells has been proposed as a new biomarker of viability—higher levels of mtDNA associated with reduced implantation potential. Study Design, Size, Duration mtDNA was quantified in 199 blastocysts, previously biopsied and shown to be chromosomally normal using preimplantation genetic testing for aneuploidy (PGT-A). These were generated by 174 couples (average female age 37.06 years). All patients underwent IVF in a single clinic. The study took place in a blinded, non-selection manner—i.e. mtDNA quantity was not known at the time of single embryo transfer. The fate of the embryos transferred was subsequently compared to the mtDNA levels measured. Participants/Materials, Settings, Methods Embryos were biopsied at the blastocyst stage. The TE samples obtained were subjected to whole genome amplification followed by comprehensive chromosome analysis via next generation sequencing. The same biopsy specimens were also tested using quantitative PCR, allowing highly accurate mtDNA quantification. After blastocyst transfer, the code used for blinding was broken and analysis undertaken to reveal whether the amount of mtDNA had any association with embryo implantation. Main Results and the Role of Chance mtDNA analysis of the 199 blastocysts revealed that 9 (5%) contained unusually high levels of mtDNA. All embryo transfers involved a single chromosomally normal blastocyst of good morphology. Of these, 121 (60%) led to ongoing pregnancies, 11(6%) led to biochemical pregnancies, and 10 (5%) spontaneously miscarried. All (100%) of these blastocysts had mtDNA levels considered to be normal/low. The remaining 57 (29%) blastocysts failed to implant. Among these non-viable embryos there were 9 (16%) with unusually high levels of mtDNA. This meant that the ongoing pregnancy rate for morphologically good, euploid blastocysts, with normal/low levels of mtDNA was 64% (121/190). In contrast, the ongoing pregnancy rate for the same type of embryos, but with elevated mtDNA levels, was 0/9 (0%). This difference was highly statistically significant (P &lt; 0.0001). Limitations Reasons for Caution To determine the true extent of any clinical benefits a randomized clinical trial will be necessary. Research is needed to improve understanding of the biology of mtDNA expansion. Wider Implications of the Findings This is the first investigation to evaluate the clinical impact of increased mtDNA in a prospective blinded manner. Results confirm that embryos with elevated mtDNA rarely implant, supporting its use as a viability biomarker. A total of 64% of euploid blastocysts with normal/low mtDNA implanted versus 60% for the cohort as a whole.</p