BACKGROUND Dyschromatosis universalis hereditaria(DUH)is a rare type of autosomal dominant inheritance disease.It has varying gene mutation sites among different ethnicities.SASH1 and ABCB6 have been identified as the...BACKGROUND Dyschromatosis universalis hereditaria(DUH)is a rare type of autosomal dominant inheritance disease.It has varying gene mutation sites among different ethnicities.SASH1 and ABCB6 have been identified as the causative genes of this disorder.CASE SUMMARY A 30-year-old woman presented with irregular black pigmentation spots to our department.Upon examination,the pigmentations were found to be especially dense on the extremities and the face.She had no family history of inbreeding,nor any previous chemical exposure.Genetic testing confirmed that the disease occu-rred because the patient has a SASH1 gene mutation.Following the use of assisted reproductive technology and preimplantation genetic testing for monogenic disorders,the patient give birth to a health baby.CONCLUSION Using assisted reproductive technology/preimplantation genetic testing for monogenic disorders is an option for DUH patients to reduce the risk of trans-mitting the pathogenic variant to their offspring.展开更多
The cattle different stage embryos obtained from in vitro was studied using the technology of single preimplantation embryo mRNA different display:single 8-cell and blastocyst stage embryos were studied using technolo...The cattle different stage embryos obtained from in vitro was studied using the technology of single preimplantation embryo mRNA different display:single 8-cell and blastocyst stage embryos were studied using technology of mRNA different display and one different fragment was found. The result suggested that this fragment displayed high homology (99%) to cattle mRNA for ribosomal protein L31. Then to detect the expression of RPL31mRNA in 8 cell and blastocyst stage embryos by real-time quantitative PCR,the result showed the relative amount of 8 cells was 3.2 times of blastocyst's.展开更多
Conventional PCR methods combined with linkage analysis based on short tandem repeats （STRs） or Karyomapping with single nucleotide polymorphism （SNP） arrays, have been applied to preimplantation genetic diagnosis...Conventional PCR methods combined with linkage analysis based on short tandem repeats （STRs） or Karyomapping with single nucleotide polymorphism （SNP） arrays, have been applied to preimplantation genetic diagnosis （PGD） for spinal muscular atrophy （SMA）, an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wild- type embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses （MARSALA） is a new method allowing embryo selection by a one-step next-generation sequencing （NGS） procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion （carriers） from the wild-type （normal） ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos （homozygous deletion）, which can be regarded as probands for linkage analysis, in case that the affected family member is absent, In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation.展开更多
Oocyte quality has long been considered as a main limiting factor for in vitro fertilization （IVF）. In the past decade, extensive observations demonstrated that the mitochondrion plays a vital role in the oocyte cyt...Oocyte quality has long been considered as a main limiting factor for in vitro fertilization （IVF）. In the past decade, extensive observations demonstrated that the mitochondrion plays a vital role in the oocyte cytoplasm, for it can provide adenosine triphosphate （ATP） for fertilization and preimplantation embryo development and also act as stores of intracellular calcium and proapoptotic factors. During the oocyte maturation, mitochondria are characterized by distinct changes of their distribution pattern from being homogeneous to heterogeneous, which is correlated with the cumulus apoptosis. Oocyte quality decreases with the increasing maternal age. Recent studies have shown that low quality oocytes have some age-related dysfunctions, which include the decrease in mitochondrial membrane potential, increase of mitochondrial DNA （mtDNA） damages, chromosomal aneuploidies, the incidence of apoptosis, and changes in mitochoncLrial gene expression. All these dysfunctions may cause a high level of de- velopmental retardation and arrest of preimplantation embryos. It has been suggested that these mitochondrial changes may arise from excessive reactive oxygen species （ROS） that is closely associated with the oxidative energy production or calcium overload, which may trigger permeability transition pore opening and subsequent apoptosis. Therefore, mitochondria can be seen as signs for oocyte quality evaluation, and it is possible that the oocyte quality can be improved by enhancing the physical function of mitochondria. Here we reviewed recent advances in mitochondrial functions on oocytes.展开更多
Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited dis...Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.展开更多
Evidence has shown in mouse that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis. In the current paper, attempts were made to clone and characterize a gene encoding L...Evidence has shown in mouse that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis. In the current paper, attempts were made to clone and characterize a gene encoding Lhx8 from pig. Rapid amplification of cDNA ends （RACE） gave rise to a full-length of Lhx8 which contained 1 681 bp nucleotides, with a complete open reading frame of 885 bp, encoding a 295 amino acid polypeptide. Homology search and sequence multialignment demonstrated that the deduced pig Lhx8 protein sequence shared a high identity with Lhx8 from other mammals, including several highly conservative motifs and amino acids. The phylogenetic tree of the LIM superfamily proteins has been constructed to reveal the evolutionary relationship of various species. RT-PCR analysis showed that the Lhx8 gene was expressed in gonad and immunity tissues. In preimplantation embryos, Lhx8 mRNA expression profiling using realtime PCR revealed that its mRNA levels were highest in 4-cell stage embryos and gradually decreased until the blastocyst stage.展开更多
Objective: To evaluate the effect of preimplantation genetic diagnosis (PGD) conducted for women who had Down syndrome pregnancy previously. Methods: Trisomy 21 was diagnosed by using fluorescence in site hybridizatio...Objective: To evaluate the effect of preimplantation genetic diagnosis (PGD) conducted for women who had Down syndrome pregnancy previously. Methods: Trisomy 21 was diagnosed by using fluorescence in site hybridization (FISH) before embryo transfer in two women who had Down syndrome pregnancies. Each received one or two PGD cycles respectively. Results: Case 1: one PGD cycle was conducted, two oocytes were fertilized and biopsied. One embryo is of trisomy 21 and the other of monosomy 21. No embryo was transferred. Case 2: two PGD cycles were conducted, in total, sixteen oocytes were fertilized and biopsied. Four embryos were tested to be normal, six of trisomy 21, and one of monosomy 21. Five had no signal. Four normal embryos were transferred but no pregnancy resulted. Conclusion: For couples who had pregnancies with Down syndrome pre-viously, PGD can be considered, and has been shown to be an effective strategy.展开更多
Charcot-Marie-Tooth （CMT） disease is the most common hereditary neuropathy, with a population prevalence of 1 in 2500. CMT disease type 1A （CMT1A）, accounting for ~70% of CMT1 cases and ~ 50% of all CMT cases, is ...Charcot-Marie-Tooth （CMT） disease is the most common hereditary neuropathy, with a population prevalence of 1 in 2500. CMT disease type 1A （CMT1A）, accounting for ~70% of CMT1 cases and ~ 50% of all CMT cases, is transmitted in an autosomal dominant manner. CMT1A maps to chromo- some 17pl 1.2 and is caused, in the majority of cases, by a 1.4- Mb tandem duplication that includes the peripheral myelin protein22 （PMP22） gene （Li et al., 2013）. The disease usually presents in the first 20 years of age, causing difficulty in walking or running, distal symmetrical muscle weakness and wasting, and sensory loss （van Paassen et al., 2014）.展开更多
In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of bl...In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further de- velopment capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%) (P>0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P<0.05). There was no significant difference in cyto- plasm remains between methanol/acetic acid method and Tween/HCL method (P>0.05). The hy- bridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P>0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chro- mosomal diseases.展开更多
Objective:To observe the effects of vitamin E on post-vitrification preimplantation development,gross morphology as well as mitochondrial distribution and ultrastructure.Methods:Twenty-four female C57BL/6NTac mice,age...Objective:To observe the effects of vitamin E on post-vitrification preimplantation development,gross morphology as well as mitochondrial distribution and ultrastructure.Methods:Twenty-four female C57BL/6NTac mice,aged 12-16 weeks,were randomly divided into four groups.Group A did not receive any treatment and served as the control group.Group B was treated with corn oil stripped of tocopherols and served as the vehicle group.Group C was treated with 60 mg/kg body weight of tocotrienol-rich-fraction with corn oil stripped of tocopherols.Group D was treated with 60 mg/kg body weight of alpha-tocopherol with corn oil stripped of tocopherols.All treatments were administered orally for 7 consecutive days.After superovulation and mating with fertile males,2-cell stage embryos were harvested for vitrification.Post vitrification development in vitro,gross morphology and ultrastructure were compared between groups.Results:The number of 2 and 8-cell embryo,and blastocysts in the treatment and control groups were not significantly different(P>0.05).Following vitrification,all 2-cell embryos had equal-sized blastomeres and intact zona pellucida.Mitochondrial aggregation toward the perinuclear region was seen in all of the treatment groups.Both groups C and D had vacuolated mitochondria,which was reflected in the trend of preimplantation development reduction.Conclusions:Vitamin E supplementation of 60 mg/kg body weight does not improve the viability of healthy embryos according to this study.As a result,the most effective dose of vitamin E supplementation may be determined by the initial quality of the embryos.展开更多
Objective:To detect common chromosomal aneuploidy variations in embryos from couples undergoing assisted reproductive technology and preimplantation genetic screening and their possible associations with embryo qualit...Objective:To detect common chromosomal aneuploidy variations in embryos from couples undergoing assisted reproductive technology and preimplantation genetic screening and their possible associations with embryo quality.Methods:In this study,359 embryos from 62 couples were screened for chromosomes 13,21,18,X,and Y by fluorescence insitu hybridization.For biopsy of blastomere,a laser was used to remove a significantly smaller portion of the zona pellucida.One blastomere was gently biopsied by an aspiration pipette through the hole.After biopsy,the embryo was immediately returned to the embryo scope until transfer.Embryo integrity and blastocyst formation were assessed on day 5.Results:Totally,282 embryos from 62 couples were evaluated.The chromosomes were normal in 199(70.57%)embryos and abnormal in 83(29.43%)embryos.There was no significant association between the quality of embryos and numerical chromosomal abnormality(P=0.67).Conclusions:Embryo quality is not significantly correlated with its genetic status.Hence,the quality of embryos determined by morphological parameters is not an appropriate method for choosing embryos without these abnormalities.展开更多
Objective To explore the effect of hepatocyte growth factor （HGF） and fibroblast growth factor （FGF） on preimplantation embryo development of mouse.Methods Mated mice were killed by cervical dislocation to collect...Objective To explore the effect of hepatocyte growth factor （HGF） and fibroblast growth factor （FGF） on preimplantation embryo development of mouse.Methods Mated mice were killed by cervical dislocation to collect two pronucleous （2 PN） zygotes from oviduct of pregnant 1 d NMRI mice and were cultured to the hatching blastocyst stage and the number of embryo in different stages was recorded under an invert microscope. The cleavage rates of formed 2 PN zygotes were compared with blastocyst and hatching blastocyst stage in drops of T6 medium with or without HGF or FGF （0 ng/ml, 10 ng/ml, 20 ng/ml, 50 ng/ml, 1 000 ng/ml）. Results HGF and FGF enriched embryo culture media promotea the aevetopment from 2 PN stage embryos to blastocyst. Adding 20 ng/ml of FGF or HGF to the culture medium significantly increased the percentage of 2PN embryos that developed into blastocysts （P〈0.05）, but culture of embryos in drops of T6 medium with HGF （20 ng/ml） had no improvement in in-vitro hatching.Conclusion Exogenous HGF and FGF at low concentrations promote in-vitro mouse blastocyst formation.展开更多
BACKGROUND Identifying a potential single monogenetic disorder in healthy couples is costly due to the Assisted Reproduction facilities'current methodology for screening,which focuses on the detecting multiple gen...BACKGROUND Identifying a potential single monogenetic disorder in healthy couples is costly due to the Assisted Reproduction facilities'current methodology for screening,which focuses on the detecting multiple genetic disorders at once.Here,we report the successful application of a low-cost and fast preimplantation genetic testing for monogenic/single gene defects(PGT-M)approach for detecting propionic acidemia(PA)in embryos obtained from a confirmed heterozygous propionyl-CoA carboxylase alpha subunit(PCCA)couple.CASE SUMMARY A fertile 32-years old Mexican couple with denied consanguinity sought antenatal genetic counseling.They were suspected obligate PA carriers due to a previous deceased PA male newborn with an unknown PCCA/propionyl-CoA carboxylase beta subunit(PCCB)genotype.Next-Generation Sequencing revealed a heterozygous genotype for a pathogenic PCCA variant(c.2041-1G>T,ClinVar:RCV-000802701.1;dbSNP:rs1367867218)in both parents.The couple requested in vitro fertilization(IVF)and PGT-M for PA.From IVF,12 oocytes were collected and fertilized,of which two resulted in high-quality embryos.Trophectoderm biopsies and Whole Genome Amplification by a fragmentation/amplification-based method were performed and revealed that the two embryos were euploid.Endpoint polymerase chain reaction and further Sanger sequencing of the exon-intron borders revealed a wild-type PCCA male embryo and a heterozygous c.2041-1G>T female embryo.Both embryos were transferred,resulting in a clinical pregnancy and the delivery of a healthy male newborn(38 wk,weight:4080 g,length:49 cm,APGAR 9/9).The absence of PA was confirmed by expanded newborn screening.CONCLUSION We show that using PGT-M with Whole Genome Amplification templates,coupled with IVF,can reduce the transmission of a pathogenic variant of the PCCA gene.展开更多
Objective To investigate whether CD146, a cell adhesion molecule mouse and human preimplantation blastocysts and to localize CD146 trophectoderm（TE） and/or inner cell mass（ICM）. is expressed in in the layer of Met...Objective To investigate whether CD146, a cell adhesion molecule mouse and human preimplantation blastocysts and to localize CD146 trophectoderm（TE） and/or inner cell mass（ICM）. is expressed in in the layer of Methods Human and mouse embryos were collected. Using reverse transcriptionpolymerase chain reaction（RT-PCR）, the expression of CD146 mRNA in blastocyst was evaluated in human and mouse embryos. Single embryo immunohistochemical staining was applicated in the examination of the expression of CD146 in protein level. The statistical significance of the data was analyzed using t-test. Results CD146 transcript was detected in all human and mouse preimplantation morula and blastocyst. The expression of CD146 was found to localize in human and mouse compacted morula stage embryos and the TE and ICM of the expanded blastocysts. Conclusion mRNA and protein of CD1 46 was expressed in preimplantation embryos, which may have a profound influence on early preimplantation development for the differentiation of the trophectoderm and the morphogenesis of the blastocyst. Furthermore, the expression of CD146 in blastocyst stage may be implicated in the assistance of embryo implantation.展开更多
Preimplantation genetic testing refers to the procedure to determine the genetic status of embryos formed by in vitro fertilization(IVF) prior to initiating a pregnancy.Traditional genetic methods for preimplantation ...Preimplantation genetic testing refers to the procedure to determine the genetic status of embryos formed by in vitro fertilization(IVF) prior to initiating a pregnancy.Traditional genetic methods for preimplantation genetic diagnosis(PGD) examine distinct parts of an individua genome, require the development of a custom assay for every patient family, and are time consuming and inefficient. In the last decade technologies for wholegenome amplification(WGA) from single cells have led to innovative strategies for preimplantation testing.Applications of WGA technology can lead to a universa approach that uses single-nucleotide polymorphisms(SNPs) and mutations across the entire genome for the analysis. Single-cell WGA by multiple displacement amplification has enabled a linkage approach to PGD known as "preimplantation genetic haplotyping", as well as microarray-based techniques for preimplantation diagnosis. The use of microarrays in preimplantation diagnosis has provided genome-wide testing for gains or losses of single chromosomes(aneuploidies)or chromosomal segments. Properly designed randomized controlled trials are, however, needed to determine whether these new technologies improve IVF outcomes by increasing implantation rates and decreasing mis-carriage rates. In genotype analysis of single cells, allele dropout occurs frequently at heterozygous loci. Preimplantation testing of multiple cells biopsied from blastocysts, however, can reduce allele dropout rates and increase the accuracy of genotyping, but it allows less time for PGD. Future development of fast SNP microarrays will enable a universal preimplantation testing for aneuploidies, single-gene disorders and unbalanced translocations within the time frame of an IVF cycle.展开更多
It is well known that to achieve an acceptable engraftment and survival in stem cell therapy, an human leukocyte antigens(HLA) identical stem cell transplant is strongly required. However, the availability of the HLA ...It is well known that to achieve an acceptable engraftment and survival in stem cell therapy, an human leukocyte antigens(HLA) identical stem cell transplant is strongly required. However, the availability of the HLA matched donors even among family members is extremely limited, so preimplantation HLA typing provides an attractive practical tool of stem cell therapy for children requiring HLA matched stem cell transplantation. The present experience of preimplantation genetic diagnosis(PGD) for HLA typing of over one thousand cases shows that PGD provides the at-risk couples with the option to establish an unaffected pregnancy, which may benefit the affected member of the family with hemoglobinopathies, immunodeficiencies and other congenital or acquired bone marrow failures. Despite ethical issues involved in preimplantation HLA typing, the data presented below show an extremely high attractiveness of this option for the couples with affected children requiring HLA compatible stem cell transplantation.展开更多
Preimplantation genetic diagnosis allows to test the genetic status of embryos prior to implantation. In order to obtain genetic material, on which carry out a genetic diagnosis, a procedure named embryo biopsy is req...Preimplantation genetic diagnosis allows to test the genetic status of embryos prior to implantation. In order to obtain genetic material, on which carry out a genetic diagnosis, a procedure named embryo biopsy is required. In the last two decades, embryo biopsy at the cleavage stage has been the mostly performed procedure. However, recently, alternative methods allowing the retrieval of a larger number of cells (blastocyst stage biopsy), or representing a valid alternative to overcome ethical issues (polar body biopsy) have obtained increasing consensus. This article reviews different methods of embryo biopsy and points out their positive and negative aspects.展开更多
Atelosteogenesis type II (AO2) and diastrophic dysplasia (DTD) are two recessively inherited, severe skeletal dysplasias caused by mutations in the SLC26A2 gene. AO2 is an invariably lethal condition, while DTD patien...Atelosteogenesis type II (AO2) and diastrophic dysplasia (DTD) are two recessively inherited, severe skeletal dysplasias caused by mutations in the SLC26A2 gene. AO2 is an invariably lethal condition, while DTD patients may reach adult life, although both diseases have overlapping diagnostic features. Here we report a patient with an intermediate phenotype between AO2 and DTD and present the successful application of preimplantation genetic diagnosis (PGD) in this situation. Sequencing of SLC26A2 alleles in the infant identified two compound heterozygous mutations, p.Arg178Ter and p.Arg279Trp, of paternal and maternal origin, respectively. At request from the parents, PGD was developed by haplotype mapping of parental SLC26A2 alleles in eleven five-day embryos. Transference to the mother was attempted twice, finally resulting in pregnancy and delivery of a healthy baby. This exemplifies the utility of PGD for inherited lethal conditions with a significant risk of recurrence, and highlights the importance of accurate diagnosis of skeletal dysplasias with prenatal manifestation.展开更多
Mus musculus tyrosine 3-monooxygenase/trytophan 5-monooxygenase activation protein,zeta polypeptide(Ywhaz)is related to communication among blastomeres and con-tributes to buildup of celiular communication system,and ...Mus musculus tyrosine 3-monooxygenase/trytophan 5-monooxygenase activation protein,zeta polypeptide(Ywhaz)is related to communication among blastomeres and con-tributes to buildup of celiular communication system,and ATPase 6 is essential for one-cell to two-cell transition and play an important role in establishment of oxidative phosphorylation.We report the expression analysis of mouse two-celi stage specific genes using modified mR-NA differential display.The results of sequence analysisand reverse northern blot indicate that the genes for ATPase 6and Ywhaz are expressed just in 2-cell embryo.展开更多
文摘BACKGROUND Dyschromatosis universalis hereditaria(DUH)is a rare type of autosomal dominant inheritance disease.It has varying gene mutation sites among different ethnicities.SASH1 and ABCB6 have been identified as the causative genes of this disorder.CASE SUMMARY A 30-year-old woman presented with irregular black pigmentation spots to our department.Upon examination,the pigmentations were found to be especially dense on the extremities and the face.She had no family history of inbreeding,nor any previous chemical exposure.Genetic testing confirmed that the disease occu-rred because the patient has a SASH1 gene mutation.Following the use of assisted reproductive technology and preimplantation genetic testing for monogenic disorders,the patient give birth to a health baby.CONCLUSION Using assisted reproductive technology/preimplantation genetic testing for monogenic disorders is an option for DUH patients to reduce the risk of trans-mitting the pathogenic variant to their offspring.
基金Supported by National "863" Project (2008AA101007)~~
文摘The cattle different stage embryos obtained from in vitro was studied using the technology of single preimplantation embryo mRNA different display:single 8-cell and blastocyst stage embryos were studied using technology of mRNA different display and one different fragment was found. The result suggested that this fragment displayed high homology (99%) to cattle mRNA for ribosomal protein L31. Then to detect the expression of RPL31mRNA in 8 cell and blastocyst stage embryos by real-time quantitative PCR,the result showed the relative amount of 8 cells was 3.2 times of blastocyst's.
基金supported by the National Natural Science Foundation of China (Nos. 31522034, 31571544 and 31230047)the National High Technology Research and Development Program (No. 2015AA020407)+1 种基金Beijing Municipal Science and Technology Commission (No. D151100002415004)Research Fund of National Health and Family Planning Commission of China (No. 201402004)
文摘Conventional PCR methods combined with linkage analysis based on short tandem repeats （STRs） or Karyomapping with single nucleotide polymorphism （SNP） arrays, have been applied to preimplantation genetic diagnosis （PGD） for spinal muscular atrophy （SMA）, an autosome recessive disorder. However, it has limitations in SMA diagnosis by Karyomapping, and these methods are unable to distinguish wild- type embryos with carriers effectively. Mutated allele revealed by sequencing with aneuploidy and linkage analyses （MARSALA） is a new method allowing embryo selection by a one-step next-generation sequencing （NGS） procedure, which has been applied in PGD for both autosome dominant and X-linked diseases in our group previously. In this study, we carried out PGD based on MARSALA for two carrier families with SMA affected children. As a result, one of the couples has given birth to a healthy baby free of mutations in SMA-causing gene. It is the first time that MARSALA was applied to PGD for SMA, and we can distinguish the embryos with heterozygous deletion （carriers） from the wild-type （normal） ones accurately through this NGS-based method. In addition, direct mutation detection allows us to identify the affected embryos （homozygous deletion）, which can be regarded as probands for linkage analysis, in case that the affected family member is absent, In the future, the NGS-based MARSALA method is expected to be used in PGD for all monogenetic disorders with known pathogenic gene mutation.
基金supported by the National Natural Science Foundation of China (No. 30772345)the Research Program of the Science and Technology Bureau of Zhejiang Province (No. 2006C33016)+1 种基金the Natural Science Foundation of Zhejiang Province (No. Y204202)the Chinese Medicine Research Program of Zhejiang Province (No. 2007CA071), China
文摘Oocyte quality has long been considered as a main limiting factor for in vitro fertilization （IVF）. In the past decade, extensive observations demonstrated that the mitochondrion plays a vital role in the oocyte cytoplasm, for it can provide adenosine triphosphate （ATP） for fertilization and preimplantation embryo development and also act as stores of intracellular calcium and proapoptotic factors. During the oocyte maturation, mitochondria are characterized by distinct changes of their distribution pattern from being homogeneous to heterogeneous, which is correlated with the cumulus apoptosis. Oocyte quality decreases with the increasing maternal age. Recent studies have shown that low quality oocytes have some age-related dysfunctions, which include the decrease in mitochondrial membrane potential, increase of mitochondrial DNA （mtDNA） damages, chromosomal aneuploidies, the incidence of apoptosis, and changes in mitochoncLrial gene expression. All these dysfunctions may cause a high level of de- velopmental retardation and arrest of preimplantation embryos. It has been suggested that these mitochondrial changes may arise from excessive reactive oxygen species （ROS） that is closely associated with the oxidative energy production or calcium overload, which may trigger permeability transition pore opening and subsequent apoptosis. Therefore, mitochondria can be seen as signs for oocyte quality evaluation, and it is possible that the oocyte quality can be improved by enhancing the physical function of mitochondria. Here we reviewed recent advances in mitochondrial functions on oocytes.
基金Project supported by the National Basic Research Program (973) of China (No.2007CB948104)the Natural Science Foundation of Zhejiang Province,China (No.Z207021)
文摘Preimplantation genetic diagnosis (PGD) refers to a procedure for genetically analyzing embryos prior to implantation,improving the chance of conception for patients at high risk of transmitting specific inherited disorders.This method has been widely used for a large number of genetic disorders since the first successful application in the early 1990s.Polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) are the two main methods in PGD,but there are some inevitable shortcomings limiting the scope of genetic diagnosis.Fortunately,different whole genome amplification (WGA) techniques have been developed to overcome these problems.Sufficient DNA can be amplified and multiple tasks which need abundant DNA can be performed.Moreover,WGA products can be analyzed as a template for multi-loci and multi-gene during the subsequent DNA analysis.In this review,we will focus on the currently available WGA techniques and their applications,as well as the new technical trends from WGA products.
基金supported by the High Technology Research and Development Program of China(2006AA10Z136)
文摘Evidence has shown in mouse that Lhx8 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis. In the current paper, attempts were made to clone and characterize a gene encoding Lhx8 from pig. Rapid amplification of cDNA ends （RACE） gave rise to a full-length of Lhx8 which contained 1 681 bp nucleotides, with a complete open reading frame of 885 bp, encoding a 295 amino acid polypeptide. Homology search and sequence multialignment demonstrated that the deduced pig Lhx8 protein sequence shared a high identity with Lhx8 from other mammals, including several highly conservative motifs and amino acids. The phylogenetic tree of the LIM superfamily proteins has been constructed to reveal the evolutionary relationship of various species. RT-PCR analysis showed that the Lhx8 gene was expressed in gonad and immunity tissues. In preimplantation embryos, Lhx8 mRNA expression profiling using realtime PCR revealed that its mRNA levels were highest in 4-cell stage embryos and gradually decreased until the blastocyst stage.
基金Project supported by the National Basic Research Program of China(Nos. 2006CB944006 and 2006CB504004)the Key Research Pro-gram of Zhejiang Province, China (No. 2006C13078)the Bureau of Science and Technology of Hangzhou, China (No. 20061123B03)
文摘Objective: To evaluate the effect of preimplantation genetic diagnosis (PGD) conducted for women who had Down syndrome pregnancy previously. Methods: Trisomy 21 was diagnosed by using fluorescence in site hybridization (FISH) before embryo transfer in two women who had Down syndrome pregnancies. Each received one or two PGD cycles respectively. Results: Case 1: one PGD cycle was conducted, two oocytes were fertilized and biopsied. One embryo is of trisomy 21 and the other of monosomy 21. No embryo was transferred. Case 2: two PGD cycles were conducted, in total, sixteen oocytes were fertilized and biopsied. Four embryos were tested to be normal, six of trisomy 21, and one of monosomy 21. Five had no signal. Four normal embryos were transferred but no pregnancy resulted. Conclusion: For couples who had pregnancies with Down syndrome pre-viously, PGD can be considered, and has been shown to be an effective strategy.
文摘Charcot-Marie-Tooth （CMT） disease is the most common hereditary neuropathy, with a population prevalence of 1 in 2500. CMT disease type 1A （CMT1A）, accounting for ~70% of CMT1 cases and ~ 50% of all CMT cases, is transmitted in an autosomal dominant manner. CMT1A maps to chromo- some 17pl 1.2 and is caused, in the majority of cases, by a 1.4- Mb tandem duplication that includes the peripheral myelin protein22 （PMP22） gene （Li et al., 2013）. The disease usually presents in the first 20 years of age, causing difficulty in walking or running, distal symmetrical muscle weakness and wasting, and sensory loss （van Paassen et al., 2014）.
文摘In order to establish a simple and useful way for preimplantation genetic diagnosis (PGD) of chromosomal diseases in general IVF laboratory, the methods that are most commonly used in the embryo biopsy, fixation of blastomere and fluorescence in situ hybridization were compared. The three aspects of PGD were analyzed respectively. There was no significant difference in further de- velopment capacity of embryos between mechanical (79.7%) and chemical biopsy group (78.6%) (P>0.05). In this study, more cells were successfully fixed with the Tween/HCL method (93.8%) than with the methanol/acetic acid method (80.5%, P<0.05). There was no significant difference in cyto- plasm remains between methanol/acetic acid method and Tween/HCL method (P>0.05). The hy- bridization efficiency of fluorescence in situ hybridization was 89.5% in successive denaturation method and 90.9% in codenaturation method with the difference being not significant (P>0.05). In conclusion, the mechanical or chemical method, Tween/HCL fixation method and codenaturation fluorescence in situ hybridization method can constitute a simple and useful way for PGD of chro- mosomal diseases.
基金This research was supported financially by Universiti Teknologi MARA Grants[600-IRMI/REI/5/3(022/2019)]and[600-IRMI/MYRA 5/3/MITRA(008/2017)-2].
文摘Objective:To observe the effects of vitamin E on post-vitrification preimplantation development,gross morphology as well as mitochondrial distribution and ultrastructure.Methods:Twenty-four female C57BL/6NTac mice,aged 12-16 weeks,were randomly divided into four groups.Group A did not receive any treatment and served as the control group.Group B was treated with corn oil stripped of tocopherols and served as the vehicle group.Group C was treated with 60 mg/kg body weight of tocotrienol-rich-fraction with corn oil stripped of tocopherols.Group D was treated with 60 mg/kg body weight of alpha-tocopherol with corn oil stripped of tocopherols.All treatments were administered orally for 7 consecutive days.After superovulation and mating with fertile males,2-cell stage embryos were harvested for vitrification.Post vitrification development in vitro,gross morphology and ultrastructure were compared between groups.Results:The number of 2 and 8-cell embryo,and blastocysts in the treatment and control groups were not significantly different(P>0.05).Following vitrification,all 2-cell embryos had equal-sized blastomeres and intact zona pellucida.Mitochondrial aggregation toward the perinuclear region was seen in all of the treatment groups.Both groups C and D had vacuolated mitochondria,which was reflected in the trend of preimplantation development reduction.Conclusions:Vitamin E supplementation of 60 mg/kg body weight does not improve the viability of healthy embryos according to this study.As a result,the most effective dose of vitamin E supplementation may be determined by the initial quality of the embryos.
文摘Objective:To detect common chromosomal aneuploidy variations in embryos from couples undergoing assisted reproductive technology and preimplantation genetic screening and their possible associations with embryo quality.Methods:In this study,359 embryos from 62 couples were screened for chromosomes 13,21,18,X,and Y by fluorescence insitu hybridization.For biopsy of blastomere,a laser was used to remove a significantly smaller portion of the zona pellucida.One blastomere was gently biopsied by an aspiration pipette through the hole.After biopsy,the embryo was immediately returned to the embryo scope until transfer.Embryo integrity and blastocyst formation were assessed on day 5.Results:Totally,282 embryos from 62 couples were evaluated.The chromosomes were normal in 199(70.57%)embryos and abnormal in 83(29.43%)embryos.There was no significant association between the quality of embryos and numerical chromosomal abnormality(P=0.67).Conclusions:Embryo quality is not significantly correlated with its genetic status.Hence,the quality of embryos determined by morphological parameters is not an appropriate method for choosing embryos without these abnormalities.
文摘Objective To explore the effect of hepatocyte growth factor （HGF） and fibroblast growth factor （FGF） on preimplantation embryo development of mouse.Methods Mated mice were killed by cervical dislocation to collect two pronucleous （2 PN） zygotes from oviduct of pregnant 1 d NMRI mice and were cultured to the hatching blastocyst stage and the number of embryo in different stages was recorded under an invert microscope. The cleavage rates of formed 2 PN zygotes were compared with blastocyst and hatching blastocyst stage in drops of T6 medium with or without HGF or FGF （0 ng/ml, 10 ng/ml, 20 ng/ml, 50 ng/ml, 1 000 ng/ml）. Results HGF and FGF enriched embryo culture media promotea the aevetopment from 2 PN stage embryos to blastocyst. Adding 20 ng/ml of FGF or HGF to the culture medium significantly increased the percentage of 2PN embryos that developed into blastocysts （P〈0.05）, but culture of embryos in drops of T6 medium with HGF （20 ng/ml） had no improvement in in-vitro hatching.Conclusion Exogenous HGF and FGF at low concentrations promote in-vitro mouse blastocyst formation.
文摘BACKGROUND Identifying a potential single monogenetic disorder in healthy couples is costly due to the Assisted Reproduction facilities'current methodology for screening,which focuses on the detecting multiple genetic disorders at once.Here,we report the successful application of a low-cost and fast preimplantation genetic testing for monogenic/single gene defects(PGT-M)approach for detecting propionic acidemia(PA)in embryos obtained from a confirmed heterozygous propionyl-CoA carboxylase alpha subunit(PCCA)couple.CASE SUMMARY A fertile 32-years old Mexican couple with denied consanguinity sought antenatal genetic counseling.They were suspected obligate PA carriers due to a previous deceased PA male newborn with an unknown PCCA/propionyl-CoA carboxylase beta subunit(PCCB)genotype.Next-Generation Sequencing revealed a heterozygous genotype for a pathogenic PCCA variant(c.2041-1G>T,ClinVar:RCV-000802701.1;dbSNP:rs1367867218)in both parents.The couple requested in vitro fertilization(IVF)and PGT-M for PA.From IVF,12 oocytes were collected and fertilized,of which two resulted in high-quality embryos.Trophectoderm biopsies and Whole Genome Amplification by a fragmentation/amplification-based method were performed and revealed that the two embryos were euploid.Endpoint polymerase chain reaction and further Sanger sequencing of the exon-intron borders revealed a wild-type PCCA male embryo and a heterozygous c.2041-1G>T female embryo.Both embryos were transferred,resulting in a clinical pregnancy and the delivery of a healthy male newborn(38 wk,weight:4080 g,length:49 cm,APGAR 9/9).The absence of PA was confirmed by expanded newborn screening.CONCLUSION We show that using PGT-M with Whole Genome Amplification templates,coupled with IVF,can reduce the transmission of a pathogenic variant of the PCCA gene.
文摘Objective To investigate whether CD146, a cell adhesion molecule mouse and human preimplantation blastocysts and to localize CD146 trophectoderm（TE） and/or inner cell mass（ICM）. is expressed in in the layer of Methods Human and mouse embryos were collected. Using reverse transcriptionpolymerase chain reaction（RT-PCR）, the expression of CD146 mRNA in blastocyst was evaluated in human and mouse embryos. Single embryo immunohistochemical staining was applicated in the examination of the expression of CD146 in protein level. The statistical significance of the data was analyzed using t-test. Results CD146 transcript was detected in all human and mouse preimplantation morula and blastocyst. The expression of CD146 was found to localize in human and mouse compacted morula stage embryos and the TE and ICM of the expanded blastocysts. Conclusion mRNA and protein of CD1 46 was expressed in preimplantation embryos, which may have a profound influence on early preimplantation development for the differentiation of the trophectoderm and the morphogenesis of the blastocyst. Furthermore, the expression of CD146 in blastocyst stage may be implicated in the assistance of embryo implantation.
基金Supported by Department of Pediatrics,Medical College of Wisconsin,Milwaukee,WI,United States
文摘Preimplantation genetic testing refers to the procedure to determine the genetic status of embryos formed by in vitro fertilization(IVF) prior to initiating a pregnancy.Traditional genetic methods for preimplantation genetic diagnosis(PGD) examine distinct parts of an individua genome, require the development of a custom assay for every patient family, and are time consuming and inefficient. In the last decade technologies for wholegenome amplification(WGA) from single cells have led to innovative strategies for preimplantation testing.Applications of WGA technology can lead to a universa approach that uses single-nucleotide polymorphisms(SNPs) and mutations across the entire genome for the analysis. Single-cell WGA by multiple displacement amplification has enabled a linkage approach to PGD known as "preimplantation genetic haplotyping", as well as microarray-based techniques for preimplantation diagnosis. The use of microarrays in preimplantation diagnosis has provided genome-wide testing for gains or losses of single chromosomes(aneuploidies)or chromosomal segments. Properly designed randomized controlled trials are, however, needed to determine whether these new technologies improve IVF outcomes by increasing implantation rates and decreasing mis-carriage rates. In genotype analysis of single cells, allele dropout occurs frequently at heterozygous loci. Preimplantation testing of multiple cells biopsied from blastocysts, however, can reduce allele dropout rates and increase the accuracy of genotyping, but it allows less time for PGD. Future development of fast SNP microarrays will enable a universal preimplantation testing for aneuploidies, single-gene disorders and unbalanced translocations within the time frame of an IVF cycle.
文摘It is well known that to achieve an acceptable engraftment and survival in stem cell therapy, an human leukocyte antigens(HLA) identical stem cell transplant is strongly required. However, the availability of the HLA matched donors even among family members is extremely limited, so preimplantation HLA typing provides an attractive practical tool of stem cell therapy for children requiring HLA matched stem cell transplantation. The present experience of preimplantation genetic diagnosis(PGD) for HLA typing of over one thousand cases shows that PGD provides the at-risk couples with the option to establish an unaffected pregnancy, which may benefit the affected member of the family with hemoglobinopathies, immunodeficiencies and other congenital or acquired bone marrow failures. Despite ethical issues involved in preimplantation HLA typing, the data presented below show an extremely high attractiveness of this option for the couples with affected children requiring HLA compatible stem cell transplantation.
文摘Preimplantation genetic diagnosis allows to test the genetic status of embryos prior to implantation. In order to obtain genetic material, on which carry out a genetic diagnosis, a procedure named embryo biopsy is required. In the last two decades, embryo biopsy at the cleavage stage has been the mostly performed procedure. However, recently, alternative methods allowing the retrieval of a larger number of cells (blastocyst stage biopsy), or representing a valid alternative to overcome ethical issues (polar body biopsy) have obtained increasing consensus. This article reviews different methods of embryo biopsy and points out their positive and negative aspects.
基金The authors would like to thank the subjects reported here and Hospital de Clínicas de Porto Alegre Research Fund 12-0467 for supporting this publication.
文摘Atelosteogenesis type II (AO2) and diastrophic dysplasia (DTD) are two recessively inherited, severe skeletal dysplasias caused by mutations in the SLC26A2 gene. AO2 is an invariably lethal condition, while DTD patients may reach adult life, although both diseases have overlapping diagnostic features. Here we report a patient with an intermediate phenotype between AO2 and DTD and present the successful application of preimplantation genetic diagnosis (PGD) in this situation. Sequencing of SLC26A2 alleles in the infant identified two compound heterozygous mutations, p.Arg178Ter and p.Arg279Trp, of paternal and maternal origin, respectively. At request from the parents, PGD was developed by haplotype mapping of parental SLC26A2 alleles in eleven five-day embryos. Transference to the mother was attempted twice, finally resulting in pregnancy and delivery of a healthy baby. This exemplifies the utility of PGD for inherited lethal conditions with a significant risk of recurrence, and highlights the importance of accurate diagnosis of skeletal dysplasias with prenatal manifestation.
基金The project was supported by the State Basic Research Development Program of“973”(No.G200016107)
文摘Mus musculus tyrosine 3-monooxygenase/trytophan 5-monooxygenase activation protein,zeta polypeptide(Ywhaz)is related to communication among blastomeres and con-tributes to buildup of celiular communication system,and ATPase 6 is essential for one-cell to two-cell transition and play an important role in establishment of oxidative phosphorylation.We report the expression analysis of mouse two-celi stage specific genes using modified mR-NA differential display.The results of sequence analysisand reverse northern blot indicate that the genes for ATPase 6and Ywhaz are expressed just in 2-cell embryo.
