Clinical applications of MARSALA for preimplantation genetic diagnosis of spinal muscular atrophy

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.

Sperm retrieval rates and clinical outcomes for patients with different causes of azoospermia who undergo microdissection testicular sperm extraction-intracytoplasmic sperm injection

The aim of our study was to compare the sperm retrieval rates(SRRs)and clinical outcomes of patients with different causes of azoospermia who underwent microdissection testicular sperm extraction-intracytoplasmic sperm injection(micro-TESE-ICSI).We conducted a retrospective study at the Reproductive Medicine Center of Peking University Third Hospital in Beijing,China,from January 2014 to December 2017.This study examined 769 patients with nonobstructive azoospermia who underwent 347 cycles of micro-TESE-ICSI.Patients with azoospermia were classified into Group A(Klinefelter syndrome,n=284,125 cycles),Group B(azoospermia Y chromosome factor c[AZFc]microdeletion,n=91,64 cycles),Group C(cryptorchidism,n=52,39 cycles),Group D(previous mumps and bilateral orchitis,n=23,23 cycles),and Group E(idiopathic azoospermia,n=319,96 cycles).Clinical characteristics,SRR,embryonic development,and pregnancy outcomes of the patients were compared between all groups.Patients in Group D had the highest and most successful SRR.The average SRR for all patients was 46.0%.The rates of clinical pregnancy,implantation,and live birth in Group D were 78.3%,65.0%,and 74.0%,respectively,which were higher than those in all other groups(P<0.05).Group B patients had the lowest clinical pregnancy,implantation,and live birth rates of all groups(P<0.05).No differences were found in the miscarriage rate or birth defects among the groups(P>0.05).Patients with orchitis had the highest SRR and best clinical outcomes.Although AZFc microdeletion patients had a higher SRR,their clinical outcomes were worse.

Genome wide abnormal DNA methylome of human blastocyst in assisted reproductive technology

Proper reprogramming of parental DNA methylomes is essential for mammalian embryonic development. However, it is unknown whether abnormal methylome reprogramming occurs and is associated with the failure of embryonic development. Here we analyzed the DNA methylomes of 57 blastocysts and 29 trophectoderm samples with different morphological grades during assisted reproductive technology （ART） practices. Our data reveal that the global methylation levels of high-quality blastocysts are similar （0.30 ± 0.02, mean ± SD）, while the methylation levels of low-quality blastocysts are divergent and away from those of high-quality blastocysts. The proportion of blastocysts with a methylation level falling within the range of 0.30± 0.02 in different grades correlates with the live birth rate for that grade. Moreover, abnormal methylated regions are associated with the failure of embryonic development. Furthermore, we can use the methylation data of cells biopsied from trophectoderm to predict the blastocyst methylation level as well as to detect the aneuploidy of the blastocysts. Our data indicate that global abnormal methylome reprogramming often occurs in human embryos, and suggest that DNA methylome is a potential biomarker in blastocyst selection in ART.

Fargeted elimination of mutant mitochondria DNA in MELAS-iPSCs by mitoTALENs

Mitochondrial diseases are maternally inherited hetero- geneous disorders that are primarily caused by mitochondrial DNA （mtDNA） mutations. Depending on the ratio of mutant to wild-type mtDNA, known as heteroplasmy, mitochondrial defects can result in a wide spectrum of clinical manifestations. Mitochondria-targeted endonucleases provide an alternative avenue for treating mitochondrial disorders via targeted destruc- tion of the mutant mtDNA and induction of heteroplasmic shifting. Here, we generated mitochondrial disease patient-specific induced pluripotent stem cells （MiPSCs） that harbored a high proportion of m.3243A〉G mtDNA mutations and caused mitochondrial encephalomyopathy and stroke-like episodes （MELAS）. We engineered mitochondrial-targeted transcription activator-like effector nucleases （mitoTALENs） and successfully eliminated the m.3243A〉G mutation in MiPSCs. Off-target mutagenesis was not detected in the targeted MiPSC clones. Utilizing a dual fluorescence iPSC reporter cell line expressing a 3243G mutant mtDNA sequence in the nuclear genome, mitoTALENs displayed a significantly limited ability to target the nuclear genome compared with nuclear-localized TALENs. Moreover, genetically rescued MiPSCs displayed normal mitochondrial respiration and energy production. Moreover, neuronal progenitor cells differentiated from the rescued MiPSCs also demonstrated normal metabolic profiles. Further- more, we successfully achieved reduction in the human m.3243A〉G mtDNA mutation in porcine oocytes via injection of mitoTALEN mRNA. Our study shows the great potential for using mitoTALENs for specific targeting of mutant mtDNA both in iPSCs and mammalian oocytes, which not only provides a new avenue for studying mitochondrial biology and disease but also suggests a potential therapeutic approach for the treatment of mitochondrial disease, as well as the prevention of germline transmission of mutant mtDNA.

Dynamic changes in soil chemical properties and microbial community structure in response to different nitrogen fertilizers in an acidified celery soil

To determine the effects of different kinds of nitrogen fertilizer,especially high-efficiency slowrelease fertilizers,on soil pH,nitrogen(N)and microbial community structures in an acidic celery soil,four treatments(CK,no N fertilizer;NR,urea;PE,calcium cyanamide fertilizer;and SK,controlled-release N fertilizer)were applied,and soil pH,total soil N,inorganic N,and soil microbial biomass C were analyzed.Phospholipid fatty acids(PLFAs)were extracted and detected using the MIDI Sherlock microbial identification system.The PE treatment significantly improved soil pH,from 4.80 to>6.00,during the whole growth period of the celery,and resulted in the highest celery yield among the four treatments.After 14 d application of calcium cyanamide,the soil nitrate content significantly decreased,but the ammonium content significantly increased.The PE treatment also significantly increased soil microbial biomass C during the whole celery growth period.Canonical variate analysis of the PLFA data indicated that the soil microbial community structure in the CK treatment was significantly different from those in the N applied treatments after 49 d fertilization.However,there was a significant difference(P<0.05)in soil microbial community structure between the PE treatment and the other three treatments at the end of the experiment.Calcium cyanamide is a good choice for farmers to use on acidic celery land because it supplies sufficient N,and increases soil pH,microbial biomass and the yield of celery.

Clinical outcomes of microdissection testicular sperm extraction-intracytoplasmic sperm injection with fresh or cryopreserved sperm in patients with nonobstructive azoospermia

We performed this study to evaluate the clinical outcomes of microdissection testicular sperm extraction-intracytoplasmic sperm injection(micro-TESE-ICSI)treatment that used fresh or cryopreserved sperm in patients with nonobstructive azoospermia(NOA).A total of 338 NOA patients with 344 consecutive cycles received treatment in the reproductive medicine center of Peking University Third Hospital in Beijing,China,from January 2014 to December 2017.Fresh oocytes and fresh sperm were used in 222 patients with 234 cycles(Group A).Fresh oocytes and cryopreserved sperm were used in 116 patients with 110 cycles(Group B).We compared patient characteristics,embryonic development,and pregnancy outcomes between Groups A and B.There was no statistical difference in the patient characteristics,and no differences were observed with fertilization or quality embryo rates between Groups A and B.The rates of clinical pregnancy and live birth were both higher for Group A than those for Group B(both P<0.05).In conclusion,fresh testicular sperm appears to produce better ICSI outcomes than cryopreserved testicular sperm in patients with NOA.

Sperm origins and concentration do not impact the clinical outcomes in intracytoplasmic sperm injection cycles

In the present study, we evaluated the impact of sperm origins and concentration on the clinical outcomes of intracytoplasmic sperm injection （ICSI） cycles. A total of 1201 IC51 cycles were retrospectively analyzed for male azoospermia or oligozoospermia between January 2015 and December 2015 in the Peking University Third Hospital. Patients were divided into three groups （Group 1 vs Group 2/3; surgically extracted sperm vs ejaculated sperms）： Group 1 included 343 ICSI cycles and Group 2 analyzed 388 cycles on semen with sperm concentration 〈5 × 10^6 m1-1 （severe oligozoospermia group）. Group 3 included 470 cycles with sperm concentration between 5×10^6 m1-1 and 15 × 10^6 m1-1 （mild oligozoospermia group）. Fertilization rates, clinical pregnancy rates, and live birth rates were analyzed and compared among groups of different semen origins and concentrations on the oocyte retrieval day. Group 2 showed a lower fertilization rate than Group 3 （62.9%± 21.6% vs 66.8% ± 22.1%, P 〈 0.05）. There were no statistically significant differences in clinical pregnancy rate per transfer （51.3%, 46.7%, and 50.0%, respectively）, live birth rate per transfer （44.4%, 40.9%, and 41.4%, respectively）, accumulative live birth rate （58.3%, 51.0%, and 52.1%, respectively）, twin birth rate （18.4%, 10.6%, and 12.6%, respectively）, and birth defects rate （0, 0.3%, and 0.2%, respectively） among three groups. The results of this study indicated that sperm origins and concentration do not imDact the clinical outcomes in ICSI cycles.

A novel pathogenic mutation in FBN2 associated with congenital contractural arachnodactyly for preimplantation genetic diagnosis

Congenital contractural arachnodactyly(CCA,OMIM:121050),also known as Beals syndrome,belongs to a group of rare autosomal dominant(AD)diseases of connective tissue(Maslen et al.,1997).People with CCA share many distinguishing features,such as arachnodactyly,camp tod actyly,multiple joint contractures(especially finger,elbow,and knee joints),crumpled ears,scoliosis,pectus deformities,and muscular hypoplasia(Jurko et al.,2013).It exhibits no specific geographic or ethnic predilection(Frederic et al.,2009).

Effect of vitrification on clinical outcomes of cleavage-stage embryos with poor quality in human embryo cryopreservation

Objective: To evaluate the effect of morphologic factors on survival rate (SR), pregnancy rate (PR), and implantation rate (IR) of human embryo vitrification following frozen embryo transfer (FET) on day 3 post-ovulation.Methods: Women undergoing FET (n = 921) with embryos cryopreserved by vitrification between 2012 and 2013 were enrolled in this retrospective cohort study.Results: Embryos with >9 blastomeres yielded the highest SR of 100%. Lower SR was observed in embryos with 5 to 6 (57.5%) and 4 blastomeres (41.4%). In terms of blastomere symmetry, the SR of embryos with equally sized blastomeres was significantly higher than that of embryos with unequally sized cells (82.5%vs. 64.6%,P < 0.05). As fragmentation increased, SR decreased from 92.1% to 20.6% (P < 0.05). Significant differences were observed among groups when analyzing PR and IR according to the 3 embryonic parameters before vitrification. Embryos with 13 to 16 blastomeres yielded the highest PR (39.5%) and IR (24.1 %). The PR and IR of embryos with blastomeres of equal size were significantly higher than those with unequally sized blastomeres (36.5%vs. 21.7%, 23.7%vs. 12.4%,P < 0.05). After warming, embryos with 13 to 16 blastomeres yielded the highest PR and IR (40.9% and 24.2%, respectively). The PR and IR were observed to grow with an increase in the percentage of intact blastomeres (23.2%-38.2%, 14.2%-23.2%).Conclusions: These results show that vitrification methods do not effectively improve survival outcomes for embryos of poor quality and it is needed to develop a comprehensive vitrification protocol that considers all the practical aspects, including the current limitation regarding cleavage-stage embryos of poor quality.

THE APPLICATION OF COBB-DOUGLAS FUNCTION IN FORECASTING THE DURATION OF INTERNET PUBLIC OPINIONS CAUSED BY THE FAILURE OF PUBLIC POLICIES

Gushes of Internet public opinions may trigger unexpected incidents that significantly affectsocial security and stability, especially for ones caused by the failure of public policies. Therefore,forecasting this kind of Interact public opinions is of great significance. The duration could be citedas one of the most direct indicators that can reflect the severity of a specific Internet public opinioncase. Based on this background, this paper aims to find the factors that may affect the duration of Internet public opinions, and accordingly proposes a model that can accurately predict the durationbefore the release of public policies. Specifically, an index system including 8 factors by consideringfour dimensions, namely, object, environment, reality （offline）, and the network （online）, isestablished. In addition, based on the dataset containing 23 typical Internet public opinion casescaused by the failure of public policies, 9 prediction models are gained by applying the multivariatelinear regression model, multivariate nonlinear regression model, and the Cobb-Douglas function.