The synergistic effects of anoikis-related genes and EMT-related genes in the prognostic prediction of Wilms tumor.

Wilms tumor (WT) is the most common type of malignant abdominal tumor in children; it exhibits a high degree of malignancy, grow rapidly, and is prone to metastasis. This study aimed to construct a prognosis model based on anoikis-related genes (ARGs) and epithelial-mesenchymal transition (EMT)-related genes (ERGs) for WT patients; we assessed the characteristics of the tumor microenvironment and treatment efficacy, as well as identifying potential therapeutic targets. To this end, we downloaded transcriptome sequencing data and clinical data for WT and normal renal cortices and used R to construct and validate the prognostic model based on ARGs and ERGs. Additionally, we performed clinical feature analysis, nomogram construction, mutation analysis, drug sensitivity analysis, Connectivity Map (cMAP) analysis, functional enrichment analysis, and immune infiltration analysis. Finally, we screened the hub gene using the STRING database and validated it via experiments. In this way, we constructed a model with good accuracy and robustness, which was composed of seven anoikis- and EMT-related genes. Paclitaxel and mesna were selected as potential chemotherapeutic drugs and adjuvant chemotherapeutic drugs for the WT high-risk group by using the Genomics of Drug Sensitivity in Cancer (GDSC) and cMAP compound libraries, respectively. We proved the existence of a strong correlation between invasive immune cells and prognostic genes and risk scores. Next, we selected NTRK2 as the hub gene, and in vitro experiments confirmed that its inhibition can significantly inhibit the proliferation and migration of tumor cells and promote late apoptosis. In summary, we screened out the potential biomarkers and chemotherapeutic drugs that can improve the prognosis of patients with WT.

NET1 is a critical regulator of spindle assembly and actin dynamics in mouse oocytes.

BACKGROUND: Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily guanine nucleotide exchange factor that governs a wide array of biological processes. However, its roles in meiotic oocyte remain unclear. We herein demonstrated that the NET1-HACE1-RAC1 pathway mediates meiotic defects in the progression of oocyte maturation. METHODS: NET1 was reduced using a specific small interfering RNA in mouse oocytes. Spindle assembly, chromosomal alignment, the actin cap, and chromosomal spreads were visualized by immunostaining and analyzed under confocal microscopy. We also applied mass spectroscopy, and western blot analysis for this investigation. RESULTS: Our results revealed that NET1 was localized to the nucleus at the GV stage, and that after GVBD, NET1 was localized to the cytoplasm and predominantly distributed around the chromosomes, commensurate with meiotic progression. NET1 resided in the cytoplasm and significantly accumulated on the spindle at the MI and MII stages. Mouse oocytes depleted of Net1 exhibited aberrant first polar body extrusion and asymmetric division defects. We also determined that Net1 depletion resulted in reduced RAC1 protein expression in mouse oocytes, and that NET1 protected RAC1 from degradation by HACE1, and it was essential for actin dynamics and meiotic spindle formation. Importantly, exogenous RAC1 expression in Net1-depleted oocytes significantly rescued these defects. CONCLUSIONS: Our results suggest that NET1 exhibits multiple roles in spindle stability and actin dynamics during mouse oocyte meiosis.

Individualized Use of 6-Mercaptopurine in Chinese Children with ALL: A Multicenter Randomized Controlled Trial.

Continuous 6-mercaptopurine (6-MP) dose titration is necessary because of its narrow therapeutic index and frequently encountered dose-limiting hematopoietic toxicity. However, evidence-based guidelines for gene-based 6-MP dosing have not been established for Chinese children with acute lymphoblastic leukemia (ALL). This multicenter, randomized, open-label, active-controlled clinical trial randomly assigned Chinese children with low- or intermediate-risk ALL in a 1:1 ratio to receive TPMT-NUDT15 gene-based dosing of 6-MP (N = 44, 10 to 50 mg/m2 /day) or standard dosing (N = 44, 50 mg/m2 /day) during maintenance therapy. The primary end point was the incidence of 6-MP myelosuppression in both groups. Secondary end points included frequencies of 6-MP hepatotoxicity, duration of myelosuppression and leukopenia, event-free survival, and steady-state concentrations of active metabolites (6-thioguaninenucleotides and 6-methylmercaptopurine nucleotides) in erythrocytes. A 2.2-fold decrease in myelosuppression, the primary end point, was observed in the gene-based-dose group using ~ 50% of the standard initial 6-MP dose (odds ratio, 0.26, 95% confidence interval, 0.11 to 0.64, P = 0.003). Patients in the gene-based-dose group had a significantly lower risk of developing thiopurine-induced myelosuppression and leukopenia (P = 0.015 and P = 0.022, respectively). No significant differences were observed in the secondary end points of the incidence of hepatotoxicity and steady-state concentrations of active metabolites in erythrocytes between the two groups. TPMT- and NUDT15-based dosing of 6-MP will significantly contribute toward further reducing the incidence of leukopenia in Chinese children with ALL. This trial is registered at www.clinicaltrial.gov as #NCT04228393.

An Integrative ATAC-Seq and RNA-Seq Analysis of the Endometrial Tissues of Meishan and Duroc Pigs.

Meishan pigs are a well-known indigenous pig breed in China characterized by a high fertility. Notably, the number of endometrial grands is significantly higher in Meishan pigs than Duroc pigs. The characteristics of the endometrial tissue are related to litter size. Therefore, we used the assay for transposase-accessible chromatin with sequencing (ATAC-seq) and RNA-sequencing (RNA-seq) to analyze the mechanisms underlying the differences in fecundity between the breeds. We detected the key transcription factors, including Double homeobox (Dux), Ladybird-like homeobox gene 2 (LBX2), and LIM homeobox 8 (Lhx8), with potentially pivotal roles in the regulation of the genes related to endometrial development. We identified the differentially expressed genes between the breeds, including SOX17, ANXA4, DLX3, DMRT1, FLNB, IRF6, CBFA2T2, TFCP2L1, EFNA5, SLIT2, and CYFIP2, with roles in epithelial cell differentiation, fertility, and ovulation. Interestingly, ANXA4, CBFA2T2, and TFCP2L1, which were upregulated in the Meishan pigs in the RNA-seq analysis, were identified again by the integration of the ATAC-seq and RNA-seq data. Moreover, we identified genes in the cancer or immune pathways, FoxO signaling, Wnt signaling, and phospholipase D signaling pathways. These ATAC-seq and RNA-seq analyses revealed the accessible chromatin and potential mechanisms underlying the differences in the endometrial tissues between the two types of pigs.

Effects of Selenium Yeast on Egg Quality, Plasma Antioxidants, Selenium Deposition and Eggshell Formation in Aged Laying Hens.

Internal egg and eggshell quality are often deteriorated in aging laying hens, which causes huge economic losses in the poultry industry. Selenium yeast (SY), as an organic food additive, is utilized to enhance laying performance and egg quality. To extend the egg production cycle, effects of selenium yeast supplementation on egg quality, plasma antioxidants and selenium deposition in aged laying hens were evaluated. In this study, five hundred and twenty-five 76-week-old Jing Hong laying hens were fed a selenium-deficient (SD) diet for 6 weeks. After Se depletion, the hens were randomly divided into seven treatments, which included an SD diet, and dietary supplementation of SY and sodium selenite (SS) at 0.15, 0.30, and 0.45 mg/kg to investigate the effect on egg quality, plasma antioxidant capacity, and selenium content in reproductive organs. After 12 weeks of feeding, dietary SY supplementation resulted in higher eggshell strength (SY0.45) (p < 0.05) and lower shell translucence. Moreover, organs Se levels and plasma antioxidant capacity (T-AOC, T-SOD, and GSH-Px activity) were significantly higher with Se supplementation (p < 0.05). Transcriptomic analysis identified some key candidate genes including cell migration inducing hyaluronidase 1 (CEMIP), ovalbumin (OVAL), solute carrier family 6 member 17 (SLC6A17), proopiomelanocortin (POMC), and proenkephalin (PENK), and potential molecular processes (eggshell mineralization, ion transport, and eggshell formation) involved in selenium yeast's effects on eggshell formation. In conclusion, SY has beneficial functions for eggshell and we recommend the supplementation of 0.45 mg/kg SY to alleviate the decrease in eggshell quality in aged laying hens.

A unique 15-bp InDel in the first intron of BMPR1B regulates its expression in Taihu pigs.

BACKGROUND: BMPR1B (Bone morphogenetic protein receptor type-1B) is a receptor in the bone morphogenetic protein (BMP) family and has been identified as a candidate gene for reproductive traits in pigs. Our previous study in Taihu pigs found a specific estrogen response element (ERE) in the first intron of the BMPR1B gene that is associated with the number born alive trait. However, little is known about the mechanism by which the ERE regulates the expression of BMPR1B in the endometrium. RESULTS: Here, a 15-bp InDel (insertion/deletion) (AGCCAGAAAGGAGGA) was identified as a unique variation in Taihu pigs, and was shown to be responsible for the binding of the type I receptor of estrogen (ESR1) to the ERE using dual-luciferase assays. Four BMPR1B transcripts (T1, T2, T3, and T4) were identified by 5' RACE in endometrial tissue. Expression of T3 and T4 in the endometrium of Meishan pigs was significantly higher than in Duroc pigs during pregnancy. Luciferase assays showed that three distinct BMPR1B promoters may drive expression of T1, T3, and T4. Interestingly, ERE-mediated enhancement of T4 promoter activity significantly increased expression of Transcript T4 in the endometrium of Taihu pigs (P < 0.05). In contrast, the ERE inhibited activity of the T3 promoter and decreased expression of the T3 transcript in the Duroc background (P < 0.05). In summary, we identified a 15-bp InDel in the Taihu ERE that can be used as a molecular marker for the number born alive trait, characterized the 5' untranslated regions (UTRs) of BMPR1B transcripts in the endometrium, and determined how the transcripts are processed by alternative splicing events. CONCLUSIONS: Our results provide a foundation for understanding the transcriptional regulation of BMPR1B and its contributions to the unique breeding prolificacy characteristics of Taihu pigs.

Comparison of clinical outcomes of frozen-thawed D5 and D6 blastocysts undergoing preimplantation genetic testing.

BACKGROUND: This study aimed to analyze the clinical outcomes of blastocyst which undergo the preimplantation genetic testing (PGT) transplantation from frozen-thawed D5 and D6. In addition, the effect of blastocyst grade on clinical and neonatal outcomes was also investigated in this study. METHODS: The pregnancy and miscarriage rates of 1130 cycles of frozen embryo transfer, including 784 D5 frozen embryos and 346 D6 frozen embryos in the Reproductive Hospital of Shandong University from January to December 2020 were analyzed. Gardner blastocyst scoring was used for blastocyst evaluation. RESULTS: The pregnancy rate of D5 blastocyst was significantly higher, whereas the miscarriage rate of D5 blastocyst was lower, than that of D6 blastocyst tissue biopsy. No significant difference was observed in birth weight and low birth weight of D5 blastocyst and D6 blastocyst, preterm birth, gestational age, and neonatal sex. Frozen-thawed D5 blastocysts have higher pregnancy success rates and lower miscarriage rates compared to D6 blastocysts. CONCLUSION: Therefore, both blastocyst grade and embryo biopsy date must be considered when transferring frozen embryos.

Microbiome and ileum transcriptome revealed the boosting effects of selenium yeast on egg production in aged laying hens.

The declines in laying performance during the late production period have adverse effects on the length of the production cycle. Improving the nutrition of laying hens is a crucial measure to reverse this declination. This study investigated the effect of selenium yeast (SY) on egg production, ileal gene expression and microbiota, as well as elucidating their associations in aged laying hens. A total of 375 Jinghong laying hens at 76 weeks old were randomly assigned into 5 dietary treatments, which included a selenium-deficient basal diet based on corn-soybean meal, and dietary supplementation of SY at 0.15, 0.30 and 0.45 mg/kg, and sodium selenite at 0.45 mg/kg. The results showed that SY ameliorated the depression in aged laying performance in the 0.30 mg/kg group (P < 0.01). Selenium yeast significantly increased ileum selenium concentration (P < 0.05), and SY groups had higher selenium deposition efficiency than the sodium selenite group. Functional enrichment and Short Time-series Expression Miner (STEM) analysis indicated that SY activated metabolic progress (e.g., glycerolipid metabolism, glycerophospholipid metabolism, and fatty acid metabolism), immune response and oxidative stress response. Four hub genes including thioredoxin reductase 1 (TXNRD1), dihydrolipoamide dehydrogenase (DLD), integrin linked kinase (ILK) and leucine zipper tumor suppressor 2 (LZTS2) were involved in intestinal metabolism which was closely associated with selenium deposition/status. Moreover, the relative abundance of Veillonella, Turicibacter and Lactobacillus was significantly increased, but the relative abundance of Stenotrophomonas was significantly decreased by SY supplementation. Multi-omics data integration and Canonical correspondence analysis (CCA) showed that both the ileum selenium content and the laying rate were highly correlated with pathways and bacteria enriched in metabolism and immune response. Meanwhile, the "switched on" gene prostate stem cell antigen (PSCA) had a positive relationship with Veillonella and a negative relationship with the opportunistic pathogens Stenotrophomonas. Overall, our study offered insight for the further exploration of the role of SY on boosting egg production and balancing ileum intestinal flora in aged laying hens.

Effect of the amount of correction on posterior tibial slope and patellar height in open-wedge high tibial osteotomy.

Objectives: To evaluate the effect of amount of correction on postoperative changes in PTS (posterior tibial slope), PH (patellar height), and clinical outcomes following biplanar OWHTO (open-wedge high tibial osteotomy). Method: This study included 79 knees (32 left and 47 right) of 79 patients (mean age 60.28 ± 4.2 years, 24 males, 55 females) with varus malalignment and symptomatic isolated medial joint osteoarthritis who underwent OWHTO. According to the amount of correction angles, all patients were divided into three groups: LCA (large correction angle) group (>14°), MCA (medium correction angle) group (10°-14°), and SCA (small correction angle) group (<10°). All patients were clinically assessed according to the Lysholm score, HSS (hospital for special surgery knee score), and KSS (knee society score) prior to and after surgery. For radiographic analysis, we measured the PTS, PH [ISI (Insall-Salvati index), and BPI (Blackburne-Peel index)]. The pre-post difference of PTS, ISI, and BPI was calculated by subtracting the post-OWHTO value to the pre-OWHTO value in three groups, respectively. The preoperative, postoperative, and difference of PTS, ISI, and BPI values were analyzed according to the correction angle. The mean follow-up period was 28.5 months (SD, 4.9; range 18-52 months). Results: Radiologically, PTS increased and PH decreased after surgery on the whole (p < .05). The relationship between amount of correction and slope increase is significant (p < .001). Furthermore, the pairwise difference between the LCA group and SCA group and MCA group is significant respectively (p < .05). In terms of PH, the LCA group yielded ISI and BPI that were significantly different from baseline for the SCA group and MCA group. In addition, the pairwise difference between the SCA group and LCA group in ISI and BPI is significant (p = .031). Clinically, significant improvements were observed in postoperative clinical scores of the Lysholm score, HSS, and KSS (p < .05). Seventy-four patients (93.67%) reported satisfaction with surgery. However, no correlation was found between changes in PTS and PH with postoperative knee score. No severe adverse complications were observed. Conclusions: The amount of correction angle is a significant factor affecting the PTS and PH in OWHTO. With increased correction angle, the likelihood of increasing the PTS and decreasing the PH increases. Special attention should be paid to keep PTS and PH unchanged in cases where large corrections are required. Otherwise, closing wedge osteotomy or other intraoperative effective measures are supposed to be adopted.

Human embryo polarization requires PLC signaling to mediate trophectoderm specification.

Apico-basal polarization of cells within the embryo is critical for the segregation of distinct lineages during mammalian development. Polarized cells become the trophectoderm (TE), which forms the placenta, and apolar cells become the inner cell mass (ICM), the founding population of the fetus. The cellular and molecular mechanisms leading to polarization of the human embryo and its timing during embryogenesis have remained unknown. Here, we show that human embryo polarization occurs in two steps: it begins with the apical enrichment of F-actin and is followed by the apical accumulation of the PAR complex. This two-step polarization process leads to the formation of an apical domain at the 8-16 cell stage. Using RNA interference, we show that apical domain formation requires Phospholipase C (PLC) signaling, specifically the enzymes PLCB1 and PLCE1, from the eight-cell stage onwards. Finally, we show that although expression of the critical TE differentiation marker GATA3 can be initiated independently of embryo polarization, downregulation of PLCB1 and PLCE1 decreases GATA3 expression through a reduction in the number of polarized cells. Therefore, apical domain formation reinforces a TE fate. The results we present here demonstrate how polarization is triggered to regulate the first lineage segregation in human embryos.

Long-term survival and clinical outcomes of non-vascularized autologous and allogeneic fibular grafts are comparable for treating osteonecrosis of the femoral head.

BACKGROUND: Osteonecrosis of the femoral head (ONFH) is a disabling disease, which often involves young patients. Recently, various hip-preserving surgeries were recommended to delay total hip arthroplasty (THA). QUESTIONS/PURPOSES: This study aimed to compare clinical outcomes and survival rate in the long-term follow-up between core decompression combined with a non-vascularized autologous fibular graft (group A) and an allogeneic fibular graft (group B) for the treatment of ONFH. PATIENTS AND METHODS: We retrospectively evaluated 117 patients (153 hips) with ONFH (Association Research Circulation Osseous [ARCO] stages IIa to IIIc) who underwent the abovementioned hip-preserving surgeries between January 2003 and June 2012. The mean (range) follow-up times (years) were 12.9 (7-16) and 9.3 (6-16) in groups A and B, respectively. Clinical outcomes were assessed using the Harris Hip Score (HHS), visual analog scale (VAS) score, and forgotten joint score (FJS). A survival analysis was performed using the Kaplan-Meier method. The end point was THA. RESULTS: Groups A and B showed postoperative improvements, respectively, in HHS from 65 ± 7.2 to 80.3 ± 14.5 and from 66 ± 5.9 to 82.4 ± 13.6 (p < 0.05), and in VAS score from 6.3 ± 1.1 to 2.3 ± 1.6 and from 6.1 ± 1 to 2.2 ± 2.2 (p < 0.05). However, no significant differences in the HHS, VAS score, and hip FJS at the last follow-up (p > 0.05) and 15-year survival rate (84.1% and 86%, respectively, p > 0.05) were found between groups A and B. CONCLUSIONS: Autologous and allogeneic fibular grafts can attain equally good clinical outcomes and high survival rates in long-term follow-up, and thus can greatly delay THA owing to good bone osseointegration and sufficient mechanical support. Notably, the ratio of failure will increase when patients were more than 37 years old. LEVEL OF EVIDENCE: Level III, therapeutic study.

Total Calcanectomy and Bilateral Iliac Bone Autograft Reconstruction for the Treatment of Calcaneal Chondroblastoma Involving a Secondary Aneurysmal Bone Cyst: A Case Report and Literature Review.

Chondroblastoma is a rare, benign, cartilaginous-derived tumor accounting for ∼1% to 2% of all primary bone tumors and almost 9% of all benign bone tumors. In this case report, we describe a patient with chondroblastoma and a secondary aneurysmal bone cyst, with the adjacent talus being mildly affected. The initial diagnosis was giant cell tumor and was then confirmed after computed tomography-assisted biopsy. We performed a total calcanectomy via bilateral structural iliac bone autografting to relieve pain and reconstruct the loadbearing function because of the presence of extensive lesions. The patient was pain free and expressed satisfaction with postsurgical dorsiflexion and plantarflexion function at the 60-month follow-up visit. Radiographic images showed that the autografted iliac bone was completely healed, with no evidence of local recurrence.

Molecular cloning of SLC35D3 and analysis of its role during porcine intramuscular preadipocyte differentiation.

BACKGROUND: Solute carrier family 35 (SLC35) is one of a large number of membrane transporter protein families. Member D3 of this family is thought to be involved in adipose deposition and metabolic control. RESULTS: We obtained 2238 bp cDNA of porcine SLC35D3, it contains a 1272 bp ORF, encoding a 423 amino acid polypeptide, and a 966 bp 3' UTR. BLAST results revealed that the amino acid sequence of porcine SLC35D3 had the closest phylogenetic relationship with members of the genus Ovis aries. Further bioinformatics analysis showed that the SLC35D3 protein contains 8 transmembrane domains, and that there is no signal peptide structure. The secondary structure of the protein mainly contains 37.12% α-helixes, 7.8% in ß-folds, and 33.57% random coils. mRNA expression analysis showed that SLC35D3 is expressed in lung, liver, heart, spleen, kidney, longissimus dorsi muscle (LDM), leaf fat (LF), and subcutaneous adipose tissue (SAT). To examine the effects of SLC35D3 expression on fat synthesis and catabolism, SLC35D3-siRNA was transfected into cultured intramuscular adipocytes. SLC35D3 silenced cells showed increased expression of genes related to fat synthesis, and increased deposition of intramuscular fat (IMF), abundance of lipid droplets, and the level of free fatty acid (FFA) in the culture medium. In contrast, the siRNA decreased the expression genes involved in fat catabolism. CONCLUSIONS: Our results demonstrate that silenced SLC35D3 results in increased adipogenic processes in pig intramuscular adipocytes. These data represent the first exploration of SLC35D3 expression in swine, and provide valuable insights into the functions of SLC35D3 in adipocyte differentiation.

Protein expression profiles in Meishan and Duroc sows during mid-gestation reveal differences affecting uterine capacity, endometrial receptivity, and the maternal-fetal Interface.

BACKGROUND: Embryonic mortality is a major concern in the commercial swine industry and primarily occurs early in gestation, but also during mid-gestation (~ days 50-70). Previous reports demonstrated that the embryonic loss rate was significant lower in Meishan than in commercial breeds (including Duroc). Most studies have focused on embryonic mortality in early gestation, but little is known about embryonic loss during mid-gestation. RESULTS: In this study, protein expression patterns in endometrial tissue from Meishan and Duroc sows were examined during mid-gestation. A total of 2170 proteins were identified in both breeds. After statistical analysis, 70 and 114 differentially expressed proteins (DEPs) were identified in Meishan and Duroc sows, respectively. Between Meishan and Duroc sows, 114 DEPs were detected at day 49, and 98 DEPs were detected at day 72. Functional enrichment analysis revealed differences in protein expression patterns in the two breeds. Around half of DEPs were more highly expressed in Duroc at day 49 (DUD49), relative to DUD72 and Meishan at day 49 (MSD49). Many DEPs appear to be involved in metabolic process such as arginine metabolism. Our results suggest that the differences in expression affect uterine capacity, endometrial matrix remodeling, and maternal-embryo cross-talk, and may be major factors influencing the differences in embryonic loss between Meishan and Duroc sows during mid-gestation. CONCLUSIONS: Our data showed differential protein expression pattern in endometrium between Meishan and Duroc sows and provides insight into the development process of endometrium. These findings could help us further uncover the molecular mechanism involved in prolificacy.

Melatonin promotes human oocyte maturation and early embryo development by enhancing clathrin-mediated endocytosis.

Embryo development potential and reproductive clinical outcomes are all deeply rooted in oocyte maturation. Melatonin has been reported to promote oocyte maturation as an antioxidant in nonprimate species. Its antioxidative functions also help reduce plasma membrane rigidity, which facilitates clathrin-mediated endocytosis (CME). Whether melatonin has effects on human oocyte maturation by regulating CME is worthy of exploration. In this study, we found that the optimal melatonin concentration for human oocyte maturation was 10-11  M, and the maturation rate of this group was 71.9% (P = .03). The metaphase II (MII) stage oocytes obtained by in vitro maturation with 10-11  M melatonin had a significantly higher fertilization rate (81.4% vs 61.4%, respectively, P = .017) and blastocyst rate (32.2% vs 15.8%, respectively, P = .039) compared to controls. During maturation, antioxidative melatonin greatly enhanced CME and decreased intra-oocyte cAMP level. The former was evidenced by the increasing numbers of coated pits and vesicles, and the upregulated expression of two major CME markers-clathrin and adaptor protein-2 (AP2). CME inhibitor dynasore increased intra-oocyte cAMP level and blocked oocyte maturation, and melatonin could partly rescue oocyte maturation and significantly elevate the expression of clathrin and AP2 in the presence of dynasore. Therefore, we conclude that melatonin could promote human oocyte maturation and early embryo development through enhancing CME.

Effects of Acupoint Application Therapy with TianGui Powder on Osteoporosis in Ovariectomized Rats through TGF-ß1 and Smad2/3 Signaling Pathway.

OBJECTIVES: To explore the effects of acupoint application therapy (AAT) with TianGui Powder (TGP) on the expressions of the transforming growth factor ß1 (TGF-ß1) and Smad-2/3 signaling pathway in ovariectomized osteoporosis rats. METHODS: Sixty rats were randomly divided into four groups: normal group (group A), model group (group B), TGP group (group C), and Western medicine group (group D). Group A had only the corresponding amount of adipose tissue around the ovary removed; rats in the other groups had bilateral ovariectomies. After 1 week, groups A and B were given 1 mL/100 mg normal saline solution by gavage, group C was treated with AAT with TGP on ShenQue acupoint (0.2 piece/rat, 6 h/time, 1 time/d) and group D was given calcium carbonate vitamin D3 (36 mg/kg/d) and alfacalcidol (0.05 µg/kg/d) tablet suspension. In this study, the bone mineral density (BMD) , the levels of BALP, TRAP-5b, and BGP in serum and the changes in bone histomorphology was detected. For acquiring lumbar experimental data, the expression of TGF-ß1, Smad-2/3 proteins and mRNA of TGF-ß1and Smad-2/3 were assessed. After 12 weeks, the data were collected for analysis. RESULTS: Compared with group A, the bone trabecula was thinner and significantly reduced in other groups. The result of BMD improved significantly in both groups C and D compared to group B after intervention (P < 0.05). In contrast, compared to group B, the levels of BALP, TRAP-5b, and BGP significantly declined in both groups C and D. In group C, the results of protein expressions in TGF-ß1, Smad-2/3 were 2.870 ± 0.270, 1.552 ± 0.111, and 1.420 ± 0.079, respectively. In groups C and D, those indications significantly declined compared to group B (P < 0.01). In group C, the level of mRNA expressions of TGF-ß1, Smad-2/3 were 1.872 ± 0.177, 1.672 ± 0.086, and 1.790 ± 0.136, respectively. Compared with group B, those indications had significant difference in groups C and D (P < 0.05). CONCLUSION: Acupoint application therapy with TGP could significantly improve the BMD. The TGF-ß1 and Smad-2/3 signaling pathway could be a therapeutic target of TGP in postmenopausal osteoporosis rats.

Comparison of two protocols of blastocyst biopsy submitted to preimplantation genetic testing for aneuploidies: a randomized controlled trial.

PURPOSE: To compare the effectiveness of two protocols of blastocyst biopsy submitted to preimplantation genetic testing for aneuploidies (PGT-A). METHODS: This is a randomized controlled trial of a cohort of 221 patients undergoing PGT-A. 106 female patients aged ≤ 40 years with no less than 8 mature oocytes retrieved and ≥ 3 good-quality embryos on day 3 were randomly assigned to the day-3 hatching-based TE biopsy. The remaining 115 females aged ≤ 40 years with ≥ 8 MII oocytes obtained and no less than 3 high-quality embryos on day 3 were assigned to the TE biopsy without hatching group (also called the new biopsy group). The primary outcome was measured by a live birth after the first embryo transfer. RESULTS: The live birth rate did not differ significantly between the two groups (50.00% vs. 59.26%, P > 0.05, OR 1.46; 95% CI 0.78-2.70). There was no significant between-group difference in the rates of implantation, clinical pregnancy, and miscarriage. However, the frozen blastocyst rate was significantly lower in the day-3 hatching-based TE biopsy compared with the new biopsy group (47.54% vs. 53.96%, P < 0.05, OR 1.29; 95% CI 1.08-1.56). CONCLUSIONS: Our study provides strong evidence that the new blastocyst biopsy method exhibits advantages over day-3 hatching-based TE biopsy method. Using this method, we were able to obtain more blastocysts to perform trophectoderm biopsy in patients subjected to PGT-A.

Morphokinetic parameters from a time-lapse monitoring system cannot accurately predict the ploidy of embryos.

PURPOSE: This study aimed to test whether there is an association between embryo morphokinetic parameters and ploidy status. METHODS: Patients with high risk of aneuploidy were analyzed by time-lapse microscopy combined with preimplantation genetic screening (PGS). Accordingly, 256 blastocysts from 75 patients were subjected to trophectoderm biopsy and microarray comparative genomic hybridization (array-CGH). Blastocyst development process was analyzed using time-lapse images. RESULTS: Morphokinetic parameters: tPNf, t2, t3, t4, t5, t8, t9, tcom, tM, tSB, tB, tEB, CC1, CC2, CC3, S2, S3, t5-t2, and tB-tSB showed no significant difference in euploid embryos compared to aneuploid counterparts. In addition, two risk models based on previously published morphokinetic parameters failed to segregate euploid from aneuploid embryos. CONCLUSIONS: Morphokinetic parameters subjected to investigation in the present study failed to improve the chance of selecting euploid embryos.

Reproductive management through integration of PGD and MPS-based noninvasive prenatal screening/diagnosis for a family with GJB2-associated hearing impairment.

A couple with a proband child of GJB2 (encoding the gap junction protein connexin 26)-associated hearing impairment and a previous pregnancy miscarriage sought for a reproductive solution to bear a healthy child. Our study aimed to develop a customized preconception-to-neonate care trajectory to fulfill this clinical demand by integrating preimplantation genetic diagnosis (PGD), noninvasive prenatal testing (NIPT), and noninvasive prenatal diagnosis (NIPD) into the strategy. Auditory and genetic diagnosis of the proband child was carried out to identify the disease causative mutations. The couple then received in-vitro-fertilization treatment, and eight embryos were obtained for day 5 biopsy. PGD was performed by short-tandem-repeat linkage analysis and Sanger sequencing of GJB2 gene. Transfer of a GJB2c.235delC heterozygous embryo resulted in a singleton pregnancy. At the 13th week of gestation, genomic DNA (gDNA) from the trio family and cell-free DNA (cfDNA) from maternal plasma were obtained for assessment of fetal chromosomal aneuploidy and GJB2 mutations. NIPT and NIPD showed the absence of chromosomal aneuploidy and GJB2-associated disease in the fetus, which was later confirmed by invasive procedures and postnatal genetic/auditory diagnosis. This strategy successfully prevented the transmission of hearing impairment in the newborn, thus providing a valuable experience in reproductive management of similar cases and potentially other monogenic disorders.