The adverse effects of vitrification on mouse embryo development and metabolic phenotype in offspring.

Embryo vitrification is a standard procedure in assisted reproductive technology. Previous studies have shown that frozen embryo transfer is associated with an elevated risk of adverse maternal and neonatal outcomes. This study aimed to explore the effects of mouse blastocyst vitrification on the phenotype of vitrified-warmed blastocysts, their intrauterine and postnatal development, and the long-term metabolic health of the derived offspring. The vitrified-warmed blastocysts (IVF + VT group) exhibited reduced mitochondrial activity, increased apoptotic levels, and decreased cell numbers when compared to the fresh blastocysts (IVF group). Implantation rates, live pup rates, and crown-rump length at E18.5 were not different between the two groups. However, there was a significant decrease in fetal weight and fetal/placental weight ratio in the IVF + VT group. Furthermore, the offspring of the IVF + VT group at an age of 36 weeks had reduced whole energy consumption, impaired glucose and lipid metabolism when compared with the IVF group. Notably, RNA-seq results unveiled disturbed hepatic gene expression in the offspring from vitrified-warmed blastocysts. This study revealed the short-term negative impacts of vitrification on embryo and fetal development and the long-term influence on glucose and lipid metabolism that persist from the prenatal stage into adulthood in mice.

Identification of transcriptome characteristics of granulosa cells and the possible role of UBE2C in the pathogenesis of premature ovarian insufficiency.

BACKGROUND: Premature ovarian insufficiency (POI) is an important cause of infertility characterized by the functional decline of the ovary. Granulosa cells (GCs) around oocytes are critical for folliculogenesis, and GC dysfunction is one of the important etiologies of POI. The aim of this study was to explore the potential biomarkers of POI by identifying hub genes and analyze the correlation of biomarkers with immune infiltration in POI using RNA profiling and bioinformatics analysis. METHODS: RNA sequencing was performed on GCs from biochemical POI (bPOI) patients and controls. Differential expression analysis and weighted gene co-expression network analysis (WGCNA) were used to explore the candidate genes. qRT‒PCR was performed to verify the expression of hub genes. Western blot, Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine (EdU) assays, TUNEL (TdT-mediated dUTP Nick-End Labeling) and flow cytometry analysis were used to validate the possible role of ubiquitin-conjugating enzyme 2C (UBE2C) in POI. CIBERSORT was adopted to explore immune cell infiltration and the correlation between UBE2C and immune cells in bPOI. RESULTS: Through analysis of differentially expressed genes (DEGs) and WGCNA, we obtained 143 candidate genes. After construction of the protein‒protein interaction (PPI) network and analysis with Cytoscape, 10 hub genes, including UBE2C, PBK, BUB1, CDC20, NUSAP1, CENPA, CCNB2, TOP2A, AURKB, and FOXM1, were identified and verified by qRT‒PCR. Subsequently, UBE2C was chosen as a possible biomarker of POI because knockdown of UBE2C could inhibit the proliferation and promote the apoptosis of GCs. Immune infiltration analysis indicated that monocytes and M1 macrophages may be associated with the pathogenesis of POI. In addition, UBE2C was negatively correlated with monocytes and M1 macrophages in POI. CONCLUSIONS: This study identified a hub gene in GCs that might be important in the pathogenesis of POI and revealed the key role of UBE2C in driving POI. Immune infiltration may be highly related with the onset and etiology of POI.

Inhibition of Wnt activity improves peri-implantation development of somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) can reprogram differentiated somatic cells into totipotency. Although pre-implantation development of SCNT embryos has greatly improved, most SCNT blastocysts are still arrested at the peri-implantation stage, and the underlying mechanism remains elusive. Here, we develop a 3D in vitro culture system for SCNT peri-implantation embryos and discover that persistent Wnt signals block the naïve-to-primed pluripotency transition of epiblasts with aberrant H3K27me3 occupancy, which in turn leads to defects in epiblast transformation events and subsequent implantation failure. Strikingly, manipulating Wnt signals can attenuate the pluripotency transition and H3K27me3 deposition defects in epiblasts and achieve up to a 9-fold increase in cloning efficiency. Finally, single-cell RNA-seq analysis reveals that Wnt inhibition markedly enhances the lineage developmental trajectories of SCNT blastocysts during peri-implantation development. Overall, these findings reveal diminished potentials of SCNT blastocysts for lineage specification and validate a critical peri-implantation barrier for SCNT embryos.

Effects of nutritional interventions on the physical development of preschool children: a systematic review and meta-analysis.

Background: There is a significant correlation between diet and delayed growth and development in children. However, the evidence for the crucial role of dietary interventions in children's growth and development health remains inconclusive. This meta-analysis sought to comprehensively evaluate the effects of nutritional interventions on children's physical development. Methods: Articles published from January 2007 to December 2022 were retrieved from the PubMed, Embase, Cochrane Library, Wanfang, and China National Knowledge Infrastructure (CNKI) databases. Statistical analysis was conducted using Stata/SE 16.0 software, as well as Review Manager 5.4 software. Results: The meta-analysis included a total of 8 original studies. The total sample comprised 6,645 children aged <8 years. The results of meta-analysis were as follows: (I) there was no significant difference in the body mass index (BMI)-for-age z scores between the nutritional intervention group and the control group [mean difference (MD) =0.12, 95% confidence interval (CI): -0.07, 0.30]. Thus, the nutritional interventions did not significantly improve the BMI-for-age z scores; (II) when the nutritional intervention period was <6 months, there was no significant difference in the weight-for-height z scores between the nutritional intervention group and the control group (MD =0.47, 95% CI: -0.07, 1.00), but when the nutritional intervention period was ≥6 months, the nutritional interventions significantly improved the weight-for-height z scores (MD =0.36, 95% CI: 0.00, 0.72); (III) a nutritional intervention period ≥6 months cannot significantly improved children's height-for-age z scores; (4) When the nutritional intervention period was <6 months, there was no statistically significant difference in the weight-for-age z scores between the nutritional intervention group and the control group (MD =-0.20, 95% CI: -0.60, 0.20), but when the nutritional intervention period was ≥6 months, the nutritional interventions significantly increased children's weight-for-age (mean difference =2.23, 95% CI: 0.01, 4.44). Conclusions: Different nutritional interventions had a slight improvement effect on children's physical growth and development. However, the effect of the short-term nutritional interventions (<6 months) was not obvious. In clinical practice, it is recommended that nutritional intervention programs be formulated that can be implemented for longer periods. However, due to the limited literature included, further research is needed.

Bilineage embryo-like structure from EPS cells can produce live mice with tetraploid trophectoderm.

Self-organized blastoids from extended pluripotent stem (EPS) cells possess enormous potential for investigating postimplantation embryo development and related diseases. However, the limited ability of postimplantation development of EPS-blastoids hinders its further application. In this study, single-cell transcriptomic analysis indicated that the "trophectoderm (TE)-like structure" of EPS-blastoids was primarily composed of primitive endoderm (PrE)-related cells instead of TE-related cells. We further identified PrE-like cells in EPS cell culture that contribute to the blastoid formation with TE-like structure. Inhibition of PrE cell differentiation by inhibiting MEK signaling or knockout of Gata6 in EPS cells markedly suppressed EPS-blastoid formation. Furthermore, we demonstrated that blastocyst-like structures reconstituted by combining the EPS-derived bilineage embryo-like structure (BLES) with either tetraploid embryos or tetraploid TE cells could implant normally and develop into live fetuses. In summary, our study reveals that TE improvement is critical for constructing a functional embryo using stem cells in vitro.

Catalytic ozonation mechanisms of Norfloxacin using Cu-CuFe2O4.

As an easily recoverable, environmentally friendly and cost-effective catalyst, CuFe2O4 is a promising candidate for the catalytic ozonation of antibiotics in wastewater. However, its catalytic activity is restricted due to its limited active sites and low electron transfer efficiency. In this study, cetyl trimethyl ammonium bromide (CTAB) and Cu0 were doped with CuFe2O4 to introduce more OV, providing more active sites and improving electron transfer efficiency. Experimental results show that the optimum removal efficiency of the catalytic ozonation of Norfloxacin (NOR, a widely used antibiotic) using CTAB doped with Cu-CuFe2O4 as the catalyst is 81.58% with a first-order reaction kinetics constant of 0.03967 min-1. The associated O3 and catalyst dosages are 2.72 mg·L-1 and 0.1 g·L-1, respectively, which are 1.63 times and 2.22 times higher than those in an equivalent O3 system. OV can provide generation sites for surface hydroxyl groups and trigger ·O2- and 1O2 as the main active oxygen species. The synergistic redox cycles of Fe2+/Fe3+ and Cu0/Cu2+ accelerate electron transfer efficiency. The possible degradation pathways of NOR are identified as defluorination, naphthyridine ring-opening and piperazine ring-opening. In summary, this work proposes a new strategy for the modification of CuFe2O4 catalysts and provides new insights into the catalytic ozonation mechanisms for NOR removal.

The alterations of circulating mucosal-associated invariant T cells in polycystic ovary syndrome.

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder affecting reproductive age females and an important cause of infertility. Although the etiology is complex and its pathogenesis remains unclear, the pathological process of PCOS is tightly related with the immune dysfunction and gut microbial dysbiosis. Mucosal-associated invariant T (MAIT) cells are a subset of innate-like T cells which can regulate inflammation through the production of cytokines and play a role in regulating the gut microbiota. We aim to evaluate the correlation between characteristics of PCOS and MAIT cells as well as their impact on cytokine secretion. Methods: Peripheral blood samples were taken from PCOS patients (n=33) and healthy controls (n=30) during 2-5 days of the menstrual period. The frequencies of MAIT cells and T cells were measured by flow cytometry. Cytokines interleukin 17 (IL-17), interleukin 22(IL-22), interferon γ (IFN-γ) and granzyme B were determined by Enzyme-linked immunosorbent assay (ELISA). Results: The frequency of MAIT cells was significantly reduced in the blood of PCOS patients compared with the controls, and negatively correlated with Body Mass Index (BMI), Homeostatic model assessment- insulin resistance (HOMA-IR) index, and Anti Miillerian Hormone (AMH). Thus, the frequencies of MAIT cells decreased in PCOS patients with abnormal weight (BMI≥24kg/m2), higher HOMA-IR (≥1.5), and excessive AMH (≥8ng/ml). The Cytokine IL-17 was significantly higher in PCOS patients and negatively correlated with the frequency of MAIT cells. Even though the IL-22 was lower in PCOS Patients, no correlation with MAIT cells was detected. In subgroup, CD4+MAIT cells correlated with BMI, AMH, and testosterone (T) levels. Conclusion: The frequency change of MAIT cells may play a significant role in the pathogenesis of PCOS. Exploring these interactions with MAIT cells may provide a new target for PCOS treatment and prevention.

Effects of Yoga exercise on anxiety and fetus growth in pregnant women with small for gestational age fetus.

OBJECTIVE: To investigate the effect of yoga on anxiety and fetal weight of pregnant women carrying fetus small for gestational age (SGA). METHODS: In this retrospective analysis, a total of 186 pregnant women with SGA fetus in our hospital from January 2015 to December 2017 were enrolled in this study. Among them, 90 patients received routine check-up were included in the control group, and the other 96 patients who had professional yoga exercise were included in the observation group. The differences of anxiety scale scores and fetal weight between the two groups before and after intervention were compared. RESULTS: There was no significant difference in scores of anxiety scale (SAS) between the two groups before intervention. After intervention, the SAS score of pregnant women in intervention group was (46.48±3.79) was significantly lower than that in control group (60.13±4.25). There was also significant difference in fetal growth trajectory between the two groups, with a significant increase of 1021.36 g in the intervention group compared with 795.62 g in the control group (P<0.05). Furthermore, single regression analysis showed that average gestational weeks (r=0.064. P=0.011), yoga exercise (r=0.043, P<0.001), forceps use (r=0.338, P<0.001) and conversion to cesarean section (r=0.431, P<0.001) showed a significant correlation with anxiety and fetus growth in pregnant women carrying SGA fetus. Multiple regression analysis showed that yoga exercise (P<0.001) was selected as independent variables in the multiple regression model of anxiety and fetus growth in pregnant women with SGA fetus. CONCLUSION: Yoga can effectively reduce the anxiety of pregnant women with small gestational age fetus and good for the growth and development of the fetus.

The impact of different endometrial preparation protocols on obstetric and neonatal complications in frozen-thawed embryo transfer: a retrospective cohort study of 3,458 singleton deliveries.

BACKGROUND: Frozen-thawed embryo transfer (FET) is thought to be associated with obstetric and neonatal complications after in vitro fertilization/intracytoplasmic single sperm injection (IVF/ICSI) treatment. The study aimed to determine whether the endometrial preparation protocol is an influencing factor for these complications. METHODS: We conducted a retrospective cohort study of 3,458 women who had singleton deliveries after IVF/ICSI-FET treatment at the Centre for Reproductive Medicine of Shanghai First Maternity and Infant Hospital between July 2016 and April 2021. The women were divided into three groups according to the endometrial preparation protocols: 2,029 women with programmed cycles, 959 with natural cycles, and 470 with minimal ovarian stimulation cycles. The primary outcomes were the incidence rates of obstetric and neonatal complications, namely, hypertensive disorders of pregnancy (HDP), gestational diabetes mellitus (GDM), intrahepatic cholestasis of pregnancy (ICP), placenta previa, preterm rupture of membranes (PROM), preterm delivery, postpartum haemorrhage, large for gestational age (LGA), small for gestational age (SGA), and macrosomia. RESULTS: After adjustments for confounding variables by multivariate logistic regression analysis, the results showed that programmed cycles had an increased risk of HDP (aOR = 1.743; 95% CI, 1.110-2.735; P = 0.016) and LGA (aOR = 1.269; 95% CI, 1.011-1.592; P = 0.040) compared with natural cycles. Moreover, programmed cycles also increased the risk of LGA (aOR = 1.459; 95% CI, 1.083-1.965; P = 0.013) but reduced the risk of SGA (aOR = 0.529; 95% CI, 0.348-0.805; P = 0.003) compared with minimal ovarian stimulation cycles. There were no significant differences between natural cycles and minimal ovarian stimulation cycles. CONCLUSIONS: During IVF/ICSI-FET treatment, the risk of HDP and LGA was increased in women with programmed cycles. Therefore, for patients with thin endometrium, irregular menstruation or no spontaneous ovulation, minimal ovarian stimulation cycles may be a relatively safer option than programmed cycles.

Serum Cystatin C as a predictor of acute kidney injury in neonates: a meta-analysis

Abstract Objective: The objective of this meta-analysis is to evaluate the diagnostic value of serum Cystatin C in acute kidney injury (AKI) in neonates Sources: PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure (CNKI), and WanFang Database were searched to retrieve the literature related to the diagnostic value of Cystatin C for neonatal AKI from inception to May 10, 2021. Subsequently, the quality of included studies was determined using the QUADAS-2 tool. Stata 15.0 statistical software was used to calculate the combined sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). Additionally, meta-regression analysis and subgroup analysis contributed to explore the sources of heterogeneity Summary of the findings: Twelve articles were included. The pooled sensitivity was 0.84 (95%CI: 0.74-0.91), the pooled specificity was 0.81 (95%CI: 0.75-0.86), the pooled PLR was 4.39 (95%CI: 3.23-5.97), the pooled NLR was 0.19 (95%CI: 0.11-0.34), and the DOR was 22.58 (95%CI: 10.44-48.83). The area under the receiver operating characteristic curve (AUC) was 0.88 (95%CI: 0.85-0.90). No significant publication bias was identified (p > 0.05).

Aberrant H3K4me3 modification of epiblast genes of extraembryonic tissue causes placental defects and implantation failure in mouse IVF embryos.

Assisted reproductive technology has been widely applied in the treatment of human infertility. However, accumulating evidence indicates that in vitro fertilization (IVF) is associated with a low pregnancy rate, placental defects, and metabolic diseases in offspring. Here, we find that IVF manipulation notably disrupts extraembryonic tissue-specific gene expression, and 334 epiblast (Epi)-specific genes and 24 Epi-specific transcription factors are abnormally expressed in extraembryonic ectoderm (ExE) of IVF embryos at embryonic day 7.5. Combined histone modification analysis reveals that aberrant H3K4me3 modification at the Epi active promoters results in increased expression of these genes in ExE. Importantly, we demonstrate that knockdown of the H3K4me3-recruited regulator Kmt2e, which is highly expressed in IVF embryos, greatly improves the development of IVF embryos and reduces abnormal gene expression in ExE. Our study therefore identifies that abnormal H3K4me3 modification in extraembryonic tissue is a major cause of implantation failure and abnormal placental development of IVF embryos.

Serum Cystatin C as a predictor of acute kidney injury in neonates: a meta-analysis.

OBJECTIVES: The objective of this meta-analysis is to evaluate the diagnostic value of serum Cystatin C in acute kidney injury (AKI) in neonates. SOURCES: PubMed, Embase, Cochrane Library, Web of Science, China National Knowledge Infrastructure (CNKI), and WanFang Database were searched to retrieve the literature related to the diagnostic value of Cystatin C for neonatal AKI from inception to May 10, 2021. Subsequently, the quality of included studies was determined using the QUADAS-2 tool. Stata 15.0 statistical software was used to calculate the combined sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). Additionally, meta-regression analysis and subgroup analysis contributed to explore the sources of heterogeneity. SUMMARY OF THE FINDINGS: Twelve articles were included. The pooled sensitivity was 0.84 (95%CI: 0.74-0.91), the pooled specificity was 0.81 (95%CI: 0.75-0.86), the pooled PLR was 4.39 (95%CI: 3.23-5.97), the pooled NLR was 0.19 (95%CI: 0.11-0.34), and the DOR was 22.58 (95%CI: 10.44-48.83). The area under the receiver operating characteristic curve (AUC) was 0.88 (95%CI: 0.85-0.90). No significant publication bias was identified (p > 0.05). CONCLUSIONS: Serum Cystatin C has a good performance in predicting neonatal AKI; therefore, it can be used as a candidate biomarker after the optimal level is determined by large prospective studies.

Melatonin supplementation in the culture medium rescues impaired glucose metabolism in IVF mice offspring.

Increasing evidence suggests that in vitro fertilization (IVF) may be associated with an increased risk of developing obesity and metabolic diseases later in life in the offspring. Notably, the addition of melatonin to culture medium may improve embryo development and prevent cardiovascular dysfunction in IVF adult mice. This study aimed to determine if melatonin supplementation in the culture medium can reverse impaired glucose metabolism in IVF mice offspring and the underlying mechanisms. Blastocysts used for transfer were generated by natural mating (control group) or IVF with or without melatonin (10-6  M) supplementation (mIVF and IVF group, respectively) in clinical-grade culture media. Here, we first report that IVF decreased hepatic expression of Fbxl7, which was associated with impaired glucose metabolism in mice offspring. Melatonin addition reversed the phenotype by up-regulating the expression of hepatic Fbxl7. In vitro experiments showed that Fbxl7 enhanced the insulin signaling pathway by degrading RhoA through ubiquitination and was up-regulated by transcription factor Foxa2. Specific knockout of Fbxl7 in the liver of adult mice, through tail intravenous injection of recombinant adeno-associated virus, impaired glucose tolerance, while overexpression of hepatic Fbxl7 significantly improved glucose tolerance in adult IVF mice. Thus, the data suggest that Fbxl7 plays an important role in maintaining glucose metabolism of mice, and melatonin supplementation in the culture medium may rescue the long-term risk of metabolic diseases in IVF offspring.

Melatonin improves pregnancy outcomes in adenomyosis mice by restoring endometrial receptivity via NF-κB/apoptosis signaling.

Background: Adenomyosis is a common gynecological disease which seriously impacts female fertility and is increasing in incidence in women of childbearing age. Melatonin has beneficial effects on reproductive processes. However, its impact on the uterine receptivity of patients with adenomyosis remains unclear. In this study, we investigated the effect of melatonin on uterine receptivity and pregnancy outcomes in an adenomyosis mouse model. Methods: We induced an adenomyosis mouse model by oral administration of tamoxifen to neonatal female CD-1 mice, then conducted a melatonin injection experiment to investigate its effect on implantation rates (n=6 each). In a second experiment, the endometrium in the implantation state was collected to identify the local action of melatonin on adenomyosis mice (n=6 each), and in a parallel study, the pregnancy rate and number of offspring were recorded (n=6 each). Results: The number of implantation sites in the adenomyosis model mice was much less than in control group (5.0±2.10 vs. 13.3±2.38, P<0.0001), and 30 mg/kg of melatonin significantly improved this (9.0±0.63 vs. 5.0±2.10, P=0.002). Additionally, melatonin administration ameliorated the impaired endometrial receptivity [leukemia inhibitory factor (LIF), integrin ß3, homeobox A10 (HoxA10), and HoxA11], and improved the endometrium development [endometrial area (EA) and endometrial thickness index (ETI)] and pregnancy outcomes. Furthermore, the expression of implantation-related genes (Era, Pra, and P53), inflammatory factors [tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß)], oxidative stress associated genes (Gpx1 and Sod1), and apoptosis-related genes or proteins (Bax, Bcl-2, caspase-3, and cleaved caspase-3) was detected. The results showed higher local levels of reactive oxygen species (ROS) and inflammatory cytokines in the uterus of an adenomyosis model mice induced endometrial cells apoptosis and tissue damage, changed the uterine microenvironment, affected embryo implantation, and reduced the fertility of adenomyosis. Interestingly, melatonin significantly mitigated adenomyosis-induced changes by inhibiting the nuclear factor kappa B (NF-κB) signaling pathway, increasing the vascular endothelial growth factor (VEGF) expression, decreasing the endometrial cells apoptosis, and improving pregnancy outcomes. Conclusions: Melatonin treatment restored impaired uterine development and endometrial receptivity of adenomyosis mice by improving the endometrial microenvironment via the NF-κB/apoptosis signaling pathway. Our results provided new insight into melatonin-based therapy for adenomyosis-related infertility.

Relationship between bronchopulmonary dysplasia, long-term lung function, and vitamin D level at birth in preterm infants.

BACKGROUND: We aimed to investigate the relationship between the level of serum 25 hydroxyvitamin D [25-(OH)D] at birth and the complications of bronchopulmonary dysplasia (BPD), as well as the long-term lung function of preterm infants. METHODS: A total of 286 premature infants who were admitted to the neonatal ward of Haikou Maternal and Child Health Hospital from January 2018 to December 2020 and met the inclusion criteria were selected as the research objects. The level of serum 25(OH)D at birth was detected. The children were divided into a BPD group (79 cases) and a non-BPD group (207 cases). The case information and basic data of the children were recorded. The children were followed up 6 months after correcting the gestational age of 40 weeks, and their long-term lung function development was reported. Logistic regression analysis was used to evaluate the high-risk factors of BPD in preterm infants. RESULTS: The 1- and 5-minute Apgar scores of preterm infants in the BPD group were significantly lower than those in the non-BPD group. Also, the combined neonatal pneumonia, neonatal asphyxia, hospital stay, and total oxygen therapy time in the BPD group were substantially higher than those in the non-BPD group. The mean value of serum 25-(OH)D at birth in the BPD group (33.7±15.1 nmol/L) was significantly lower than that in the non-BPD group (49.5±19.6 nmol/L). Compared with the non-BPD group, the respiratory rate (RR) in the BPD group increased significantly, while the tidal volume (VT), inspiratory to expiratory ratio (TI/TE), ratio of time to peak tidal expiratory flow to total expiratory time (TPEF/TE), and 25% tidal expiratory flow rate (TEF25%) decreased markedly (P<0.05). Total oxygen therapy time, neonatal pneumonia, neonatal asphyxia, and serum 25-(OH)D level at birth were identified as independent risk factors for BPD in preterm infants. CONCLUSIONS: The level of serum 25-(OH)D in preterm infants at birth is closely related to the occurrence of BPD and long-term lung function damage, and is affected by multiple high-risk factors. This study provides a theoretical basis for the individualized treatment of preterm infants and the early prevention of BPD.

Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation.

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.

Genome transfer for the prevention of female infertility caused by maternal gene mutation.

Poor oocyte quality is associated with early embryo developmental arrest and infertility. Maternal gene plays crucial roles in the regulation of oocyte maturation, and its mutation is a common cause of female infertility. However, how to improve oocyte quality and develop effective therapy for maternal gene mutation remains elusive. Here, we use Zar1 as an example to assess the feasibility of genome transfer to cure maternal gene mutation-caused female infertility. We first discover that cytoplasmic deficiency primarily leads to Zar1-null embryo developmental arrest by disturbing maternal transcript degradation and minor zygotic genome activation (ZGA) during the maternal-zygotic transition. We next perform genome transfer at the oocyte (spindle transfer or polar body transfer) and zygote (early pronuclear transfer or late pronuclear transfer) stages to validate the feasibility of preventing Zar1 mutation-caused infertility. We finally demonstrate that genome transfer either at the oocyte or at the early pronuclear stage can support normal preimplantation embryo development and produce live offspring. Moreover, those pups grow to adulthood and show normal fertility. Therefore, our findings provide an effective basis of therapies for the treatment of female infertility caused by maternal gene mutation.

Identification and rescue of a novel TUBB8 mutation that causes the first mitotic division defects and infertility.

PURPOSE: Tubulin beta eight class VIII (TUBB8) is essential for oogenesis, fertilization, and pre-implantation embryo development in human. Although TUBB8 mutations were recently discovered in meiosis-arrested oocytes of infertile females, there is no effective therapy for this gene mutation caused infertility. Our study aims to further reveal the infertility-causing gene mutations in the patient's family and to explore whether the infertility could be rescued by optimizing the conditions of embryo culture and finally achieve the purpose of making the patient pregnant. METHODS: Whole-exome sequence analysis and Sanger sequencing were performed on patients' family members to screen and identify candidate mutant genes. Construction of plasmids, in vitro transcription, microinjection of disease-causing gene cRNA, and immunofluorescence staining were used to recapitulate the infertility phenotype observed in patients and to understand the pathogenic principles. Simultaneously, overexpression of mutant and wild-type cRNA of the candidate gene in mouse oocytes at either germinal vesicle (GV) or metaphase II (MII) stage was performed in the rescue experiment. RESULTS: We first identified a novel heritable TUBB8 mutation (c.1041C>A: p.N347K) in the coding region which specifically affects the first mitosis and causes the developmental arrest of early embryos in a three-generation family. We further demonstrated that TUBB8 mutation could lead to abnormal spindle assemble. And moreover, additional expression of wild-type TUBB8 cRNA in the mouse oocytes in which the mutant TUBB8 were expressed can successfully rescue the developmental defects of resulting embryo and produce full-term offspring. CONCLUSIONS: Our study not only defines a novel mutation of TUBB8 causing the early cleavage arrest of embryos, but also provides an important basis for treating such female infertility in the future.

Participation of the inositol 1,4,5-trisphosphate-gated calcium channel in the zona pellucida- and progesterone-induced acrosome reaction and calcium influx in human spermatozoa.

The acrosome reaction is a prerequisite for fertilization, and its signaling pathway has been investigated for decades. Regardless of the type of inducers present, the acrosome reaction is ultimately mediated by the elevation of cytosolic calcium. Inositol 1,4,5-trisphosphate-gated calcium channels are important components of the acrosome reaction signaling pathway and have been confirmed by several researchers. In this study, we used a novel permeabilization tool BioPORTER® and first demonstrated its effectiveness in spermatozoa. The inositol 1,4,5-trisphosphate type-1 receptor antibody was introduced into spermatozoa by BioPORTER® and significantly reduced the calcium influx and acrosome reaction induced by progesterone, solubilized zona pellucida, and the calcium ionophore A23187. This finding indicates that the inositol 1,4,5-trisphosphate type-1 receptor antibody is a valid inositol 1,4,5-trisphosphate receptor inhibitor and provides evidence of inositol 1,4,5-trisphosphate-gated calcium channel involvement in the acrosome reaction in human spermatozoa. Moreover, we demonstrated that the transfer of 1,4,5-trisphosphate into spermatozoa induced acrosome reactions, which provides more reliable evidence for this process. In addition, by treating the spermatozoa with inositol 1,4,5-trisphosphate/BioPORTER® in the presence or absence of calcium in the culture medium, we showed that the opening of inositol 1,4,5-trisphosphate-gated calcium channels led to extracellular calcium influx. This particular extracellular calcium influx may be the major process of the final step of the acrosome reaction signaling pathway.

[Effect of Ca2+ on the Nitrification Activity and the Flocculation and Sedimentation Performances of the Activated Sludge].

Ca2+ is an important microbial growth factor that can affect the activity, flocculation, and sedimentation of activated sludge. In order to study the roles of Ca2+ in the activated sludge system, the activity changes of ammonium oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) were analyzed using the specific oxygen uptake rates (SOURAOB and SOURNOB). The changes in composition and structure of extracellular polymeric substances (EPS) were analyzed using Fourier transform infrared spectroscopy (FTIR) and three-dimensional excitation emission fluorescence spectroscopy (3D-EEM). The effects of Ca2+on the nitrification activity and microbial metabolites were investigated. The results showed that when the Ca2+concentration increased from 0.45 mmol·L-1 to 3 mmol·L-1, SOURAOB and SOURNOB increased from 6.3 mg·(g·h)-1 to 10.4 mg·(g·h)-1 and from 2.3 mg·(g·h)-1 to 3.7 mg·(g·h)-1, respectively. The EPS concentrations increased from 68 mg·g-1 to 93 mg·g-1, and the flocculation ability (FA) of the sludge was improved. When the Ca2+ concentration was higher than 3 mmol·L-1, SOURAOB and SOURNOBboth decreased. The FA was maintained at about 30%, and the particle size of the sludge continued to increase. Based on FTIR analysis, the main components of EPS were always amino, amide Ⅰ, and carboxyl with an increase in Ca2+ concentration. Based on EEM analysis, the composition of loosely-bound (LB)-EPS did not change, and humic acid substances appeared in the tightly-bound (TB)-EPS at low nitrification rates. Low concentrations of Ca2+ promoted nitrification activity and flocculation of the sludge. However, high concentrations of Ca2+ led to a decline in the sludge nitrification activity.