The comparision among euploidy of preimplantation blastocysts in different controlled ovary stimulation (COH) protocols.

PURPOSE: To compare differences in euploidy rates for blastocysts in preimplantation genetic testing for aneuploidy (PGT-A) cycles after gonadotropin-releasing hormone agonist (GnRH-a) long and short protocols, GnRH-antagonist (GnRH-ant) protocol, progestin-primed ovarian stimulation and mild stimulation protocols, and other ovary stimulation protocols. METHODS: This was a retrospective cohort study from the Assisted Reproductive Medicine Department of Shanghai First Maternity and Infant Hospital. A total of 1657 PGT-A cycles with intracytoplasmic sperm injection after different controlled ovary hyperstimulation protocols were analyzed, and a total of 3154 embryos were biopsied. Differences in euploidy rate per embryo biopsied, embryo euploidy rate per oocyte retrieved and cycle cancellation rate were compared. RESULTS: For the PGT-A cycles, the euploidy rate per embryo biopsied was lower in the GnRH-ant protocol than in the GnRH-a long protocol (53.26 vs. 58.68%, respectively). Multiple linear regression showed that the GnRH-ant protocol was associated with a lower euploidy rate per embryo biopsied (ß = -0.079, p = 0.011). The euploidy rate per embryo biopsied was not affected by total gonadotropin dosage, duration of stimulation and number of oocytes retrieved. The embryo euploidy rate per oocyte retrieved was similar in all protocols and was negatively correlated with the total number of oocytes retrieved (ß = -0.003, p = 0.003). CONCLUSION: Compared with the GnRH-a long protocol, the GnRH-ant protocol was associated with a lower euploidy rate per embryo biopsied. The total gonadotropin dosage, duration of stimulation and number of oocytes retrieved did not appear to significantly influence euploidy rates.

Unraveling the genetic architecture of congenital vertebral malformation with reference to the developing spine.

Congenital vertebral malformation, affecting 0.13-0.50 per 1000 live births, has an immense locus heterogeneity and complex genetic architecture. In this study, we analyze exome/genome sequencing data from 873 probands with congenital vertebral malformation and 3794 control individuals. Clinical interpretation identifies Mendelian etiologies in 12.0% of the probands and reveals a muscle-related disease mechanism. Gene-based burden test of ultra-rare variants identifies risk genes with large effect sizes (ITPR2, TBX6, TPO, H6PD, and SEC24B). To further investigate the biological relevance of the genetic association signals, we perform single-nucleus RNAseq on human embryonic spines. The burden test signals are enriched in the notochord at early developmental stages and myoblast/myocytes at late stages, highlighting their critical roles in the developing spine. Our work provides insights into the developmental biology of the human spine and the pathogenesis of spine malformation.

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.

Complications and outcomes of open posterior lumbar spinal fusion surgery in obese patients: a meta-analysis.

OBJECTIVE: The aim of this study was to determine whether obesity affects the operation, complications and outcomes after open posterior lumbar spinal fusion surgery for the treatment of low back pain and leg pain. METHODS: A meta-analysis of studies that compared the outcome of posterior lumbar spinal fusion in obese and non-obese patients. A total of 16 studies were included. RESULTS: There was no difference in pain and functional outcomes. Posterior lumbar spinal fusion in obese patients resulted in a statistically significant increase in intra-operative blood loss (weighted mean difference 40.93, 95% confidence interval (CI) 15.97-65.90, n = 243, and p=.001), longer duration of surgery (weighted mean difference -1.64, 95% CI -4.12 to 0.84, n = 1460, and p=.19), more complications (odds ratio: 1.59, 95% CI 1.24-2.05, n = 339, and p<.001) and extend length of stay (weighted mean difference 0.31, 95% CI 0.07-0.55, n = 1408, and p=.01). CONCLUSIONS: Obese patients experience more blood loss, longer duration of surgery, more complications and extended length of stay, but their back and leg pain and functional outcomes are similar to non-obese patients. Based on these results, obesity is not a contraindication to open posterior lumbar spinal fusion surgery.

Bioinformatic analysis and machine learning to identify the diagnostic biomarkers and immune infiltration in adenomyosis.

Background: Adenomyosis is a hormone-dependent benign gynecological disease characterized by the invasion of the endometrium into the myometrium. Women with adenomyosis can suffer from abnormal uterine bleeding, severe pelvic pain, and subfertility or infertility, which can interfere with their quality of life. However, effective diagnostic biomarkers for adenomyosis are currently lacking. The aim of this study is to explore the mechanism of adenomyosis by identifying biomarkers and potential therapeutic targets for adenomyosis and analyzing their correlation with immune infiltration in adenomyosis. Methods: Two datasets, GSE78851 and GSE68870, were downloaded and merged for differential expression analysis and functional enrichment analysis using R software. Weighted gene co-expression network analysis (WGCNA), the least absolute shrinkage and selection operator (LASSO), and support vector machine-recursive feature elimination (SVE-RFE) were combined to explore candidate genes. Quantitative reverse transcriptase PCR (qRT-PCR) was conducted to verify the biomarkers and receiver operating characteristic curve analysis was used to assess the diagnostic value of each biomarker. Single-sample Gene Set Enrichment Analysis (ssGSEA) and CIBERSORT were used to explore immune cell infiltration in adenomyosis and the correlation between diagnostic biomarkers and immune cells. Results: A total of 318 genes were differentially expressed. Through the analysis of differentially expressed genes and WGCNA, we obtained 189 adenomyosis-related genes. After utilizing the LASSO and SVM-RFE algorithms, four hub genes, namely, six-transmembrane epithelial antigen of the prostate-1 (STEAP1), translocase of outer mitochondrial membrane 20 (TOMM20), glycosyltransferase eight domain-containing 2 (GLT8D2), and NME/NM23 family member 5 (NME5) expressed in nucleoside-diphosphate kinase, were identified and verified by qRT-PCR. Immune infiltration analysis indicated that T helper 17 cells, CD56dim natural killer cells, monocytes, and memory B-cell may be associated with the occurrence of adenomyosis. There were significant correlations between the diagnostic biomarkers and immune cells. Conclusion: STEAP1, TOMM20, GLT8D2, and NME5 were identified as potential biomarkers and therapeutic targets for adenomyosis. Immune infiltration may contribute to the onset and progression of adenomyosis.

[Study of modified subcutaneous lumbar spine index as a predictor for short-term effectiveness in transforaminal lumbar interbody fusion].

OBJECTIVE: To explore the value of modified subcutaneous lumbar spine index (MSLSI) as a predictor for short-term effectiveness of transforaminal lumbar interbody fusion (TLIF) in treatment of lumbar degenerative disease (LDD). METHODS: Between February 2014 and October 2019, 450 patients who were diagnosed as LDD and received single-segment TLIF were included in the study. Based on the MSLSI measured by preoperative lumbar MRI, the patients were sorted from small to large and divided into three groups ( n=150). The MSLSI of group A was 0.11-0.49, group B was 0.49-0.73, and group C was 0.73-1.88. There was no significance in gender, age, disease duration, diagnosis, surgical segment, and improved Charlson comorbidity index between groups ( P>0.05). There were significant differences in the subcutaneous adipose depth of the L 4 vertebral body and body mass index (BMI) between groups ( P<0.05). The operation time, intra-operative blood loss, length of incision, drainage tube placement time, drainage volume on the 1st day after operation, drainage volume on the 2nd day after operation, total drainage volume, antibiotic use time after operation, walking exercise time after operation, hospital stay, the incidences of surgical or non-surgical complications in the three groups were compared. Pearson correlation analysis was used to analyze the correlation between MSLSI and BMI, and partial correlation analysis was used to study the relationship between MSLSI, BMI, improved Charlson comorbidity index, subcutaneous adipose depth of the L 4 vertebral body and complications. The Receiver Operating Characteristic (ROC) curve was used to evaluate the value of SLSI and MSLSI in predicting the occurrence of complications after TLIF in treatment of LDD. RESULTS: There was no significant difference in operation time, length of incision, antibiotic use time after operation, walking exercise time after operation, drainage tube placement time, drainage volume on the 1st day after operation, drainage volume on the 2nd day after operation, and total drainage volume between groups ( P>0.05). The amount of intra-operative blood loss in group C was higher than that in groups A and B, and the hospital stay was longer than that in group B, with significant differences ( P<0.05). Surgical complications occurred in 22 cases (14.7%), 25 cases (16.7%), and 39 cases (26.0%) of groups A, B, and C, respectively. There was no significant difference in the incidence between groups ( χ 2=0.826, P=0.662). The incidences of nerve root injury and wound aseptic complications in group C were higher than those in groups A and B, and the incidence of nerve root injury in group B was higher than that in group A, with significant differences ( P<0.05). There were 13 cases (8.7%), 7 cases (4.7%), and 11 cases (7.3%) of non-surgical complications in groups A, B, and C, respectively, with no significant difference ( χ 2=2.128, P=0.345). There was no significant difference in the incidences of cardiovascular complications, urinary system complications, central system complications, and respiratory system complications between groups ( P>0.05). There was a correlation between MSLSI and BMI in 450 patients ( r=0.619, P=0.047). Partial correlation analysis showed that MSLSI was related to wound aseptic complications ( r=0.172, P=0.032), but not related to other surgical and non-surgical complications ( P>0.05). There was no correlation between BMI, improved Charlson comorbidity index, subcutaneous adipose depth of the L 4 vertebral body and surgical and non-surgical complications ( P>0.05). ROC curve analysis showed that the area under ROC curve (AUC) of MSLSI was 0.673 (95%CI 0.546-0.761, P=0.025), and the AUC of SLSI was 0.582 (95%CI 0.472-0.693, P=0.191). CONCLUSION: MSLSI can predict the short-term effectiveness of TLIF in treatment of LDD. Patients with high MSLSI suffer more intra-operative blood loss, longer hospital stay, and higher incidence of nerve root injury and postoperative incision complications.

Proteomic analysis of decidua in patients with recurrent pregnancy loss (RPL) reveals mitochondrial oxidative stress dysfunction.

BACKGROUND: Pregnancy is a complicated physiological process. The multifaceted regulation of maternal-fetal interface is of great importance for maintaining normal pregnancy and avoiding fetal rejection and secondary abortion. Previous studies have focused on the clinical features or pathological biomarkers of fetal rejection and abortion. However, no significant breakthrough has been made. Therefore, it is important to understand the molecular mechanisms of recurrent pregnancy loss (RPL) to identify potential therapeutic strategies. The aim of this study was to investigate the pathogenesis of RPL. METHODS: In this study, Relative and absolute quantitation (iTRAQ) technology integrated with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to identify differentially expressed proteins in decidual from RPL patients and matched normal controls. Further, Molecules NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3 (ndufb3) and cyclooxygenase-2 (COX-2) were validated by immunohistochemistry (IHC), Western blotting, CCK8 and mitochondrial red fluorescent probe (Mito-Tracker Red CMXRos). RESULTS: A total of 456 proteins reached the threshold of a 1.5-fold change were identified for further bioinformatics analysis. Upon mapping the differentially expressed proteins using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database, iTRAQ results were confirmed by assessing NDUFB3 and COX-2 protein levels in specimens of decidual tissue by Western blotting. Our study indicates that the level of COX-2 and NDUFB3 were significantly increased in decidual cell from RPL patients. Overexpression of NDUFB3 inhibited cell vitality and oxidative stress of decimal cell. Further, our found that overexpression NDUFBD3 in decidual cell decreased the mitochondrial membrane potential expression levels. These results suggest that NDUFB3 might play an important role in promote the pathological process of RPL. CONCLUSIONS: This comprehensive analysis of RPL proteomics reveals novel candidate: NDUFB3, which could be further investigated for explanation of the pathological mechanism of RPL.

The orphan nuclear receptor NUR77 promotes trophoblast invasion at early pregnancy through paracrine placental growth factor.

NR4A1 (NUR77) is an orphan nuclear receptor that has been implicated in both cell survival and apoptosis. However, the role of NUR77 in trophoblast function during early placenta development has not been fully elucidated. In this study, we showed that NUR77 expression was significantly lower in the villi of the recurrent miscarriage (RM) group compared to that in the healthy controls (HCs) group. We used immunohistochemistry and found that NUR77 was highly expressed in human placental villi during early pregnancy, especially in syncytiotrophoblast (STB), and was expressed at a much lower level in STB from the RM group than in those from HC group. Western blotting data further confirmed that NUR77 was highly expressed in primary human term placental STB and the FSK-induced BeWo cell line. Moreover, antibody array screening and ELISA revealed that NUR77 promoted significant placental growth factor (PGF) expression during trophoblast fusion. Ectopic overexpression and knockdown experiments demonstrated that PGF was a novel downstream target of NUR77, and serum PGF expression correlated positively with trophoblast NUR77 mRNA levels in HCs and RM patients. Importantly, bioinformatics analysis identified two NUR77 binding sites in the PGF promoter region, and chromatin immunoprecipitation (ChIP) coupled with Western blotting analysis further verified that NUR77 bound directly to the PGF promoter region and promoted PGF expression. Furthermore, in a BeWo/HTR-8 co-culture system, FSK-induced BeWo-secreted PGF promoted HTR-8 cell migration and invasion, and an anti-PGF antibody reversed this effect. Collectively, these results indicated that NUR77 may play a key role in regulating trophoblast invasion at early pregnancy. KEY MESSAGES: NUR77 expression was significantly decreased in the syncytiotrophoblast of the recurrent miscarriage group compared to that in the healthy control group. NUR77 promoted PGF expression during trophoblast fusion. ChIP and western blotting experiments verified that NUR77 bound directly to the PGF promoter region and activated PGF expression in trophoblast. Trophoblast-derived PGF promoted HTR-8 cell migration and invasion in a cell co-culture system.

The m6A demethylase ALKBH5 controls trophoblast invasion at the maternal-fetal interface by regulating the stability of CYR61 mRNA.

N6-Methyladenosine (m6A) is the most prevalent internal modification in mammalian mRNAs. Although m6A is important in many biological processes, its roles in the placenta are unclear. Methods: Levels of global mRNA m6A methylation and ALKBH5 expression in recurrent miscarriage (RM) patients were determined using quantitative reverse transcription-PCR (qRT-PCR), m6A RNA methylation quantification, and immunohistochemical methods. Using ALKBH5 overexpression and knockdown methods, we determined the role of ALKBH5 in trophoblast invasion at the maternal interface through trophoblasts and an extravillous explant culture experiments. Furthermore, the regulation of CYR61 by ALKBH5 was explored by RNA-sequencing coupled with methylated RNA immunoprecipitation. Results: We found that the level of global mRNA m6A methylation was significantly decreased in placental villous tissue from RM patients, while ALKBH5 expression was specifically unregulated. Furthermore, we demonstrated that ALKBH5 knockdown in human trophoblast promoted trophoblast invasion. Conversely, overexpression of ALKBH5 inhibited cell invasion. ALKBH5 knockdown promoted trophoblast invasion in villous explant culture experiments, while overexpression of ALKBH5 repressed these effects. Furthermore, we clarified that ALKBH5 inhibited trophoblast invasion by regulating CYR61 mRNA stability, and this RNA regulation is m6A dependent. Mechanistic analyses showed that decreased ALKBH5 in trophoblast increased the half-life of CYR61 mRNA and promoted steady-state CYR61 mRNA expression levels. Conclusions: We elucidated the functional roles of ALKBH5 and mRNA m6A methylation in trophoblast and identified a novel RNA regulatory mechanism, providing a basis for further exploration of broad RNA epigenetic regulatory patterns in RM diseases.

Loss of PAFR prevents neuroinflammation and brain dysfunction after traumatic brain injury.

Traumatic brain injury (TBI) is a principal cause of death and disability worldwide, which is a major public health problem. Death caused by TBI accounts for a third of all damage related illnesses, which 75% TBI occurred in low and middle income countries. With the increasing use of motor vehicles, the incidence of TBI has been at a high level. The abnormal brain functions of TBI patients often show the acute and long-term neurological dysfunction, which mainly associated with the pathological process of malignant brain edema and neuroinflammation in the brain. Owing to the neuroinflammation lasts for months or even years after TBI, which is a pivotal causative factor that give rise to neurodegenerative disease at late stage of TBI. Studies have shown that platelet activating factor (PAF) inducing inflammatory reaction after TBI could not be ignored. The morphological and behavioral abnormalities after TBI in wild type mice are rescued by general knockout of PAFR gene that neuroinflammation responses and cognitive ability are improved. Our results thus define a key inflammatory molecule PAF that participates in the neuroinflammation and helps bring about cerebral dysfunction during the TBI acute phase.

The intracellular bacterium Wolbachia uses parasitoid wasps as phoretic vectors for efficient horizontal transmission.

Facultative bacterial endosymbionts are associated with many arthropods and are primarily transmitted vertically from mother to offspring. However, phylogenetic affiliations suggest that horizontal transmission must also occur. Such horizontal transfer can have important biological and agricultural consequences when endosymbionts increase host fitness. So far horizontal transmission is considered rare and has been difficult to document. Here, we use fluorescence in situ hybridization (FISH) and multi locus sequence typing (MLST) to reveal a potentially common pathway of horizontal transmission of endosymbionts via parasitoids of insects. We illustrate that the mouthparts and ovipositors of an aphelinid parasitoid become contaminated with Wolbachia when this wasp feeds on or probes Wolbachia-infected Bemisia tabaci AsiaII7, and non-lethal probing of uninfected B. tabaci AsiaII7 nymphs by parasitoids carrying Wolbachia resulted in newly and stably infected B. tabaci matrilines. After they were exposed to infected whitefly, the parasitoids were able to transmit Wolbachia efficiently for the following 48 h. Whitefly infected with Wolbachia by parasitoids had increased survival and reduced development times. Overall, our study provides evidence for the horizontal transmission of Wolbachia between insect hosts by parasitic wasps, and the enhanced survival and reproductive abilities of insect hosts may adversely affect biological control programs.