Machine learning in time-lapse imaging to differentiate embryos from young vs old mice†.

Time-lapse microscopy for embryos is a non-invasive technology used to characterize early embryo development. This study employs time-lapse microscopy and machine learning to elucidate changes in embryonic growth kinetics with maternal aging. We analyzed morphokinetic parameters of embryos from young and aged C57BL6/NJ mice via continuous imaging. Our findings show that aged embryos accelerated through cleavage stages (from 5-cells) to morula compared to younger counterparts, with no significant differences observed in later stages of blastulation. Unsupervised machine learning identified two distinct clusters comprising of embryos from aged or young donors. Moreover, in supervised learning, the extreme gradient boosting algorithm successfully predicted the age-related phenotype with 0.78 accuracy, 0.81 precision, and 0.83 recall following hyperparameter tuning. These results highlight two main scientific insights: maternal aging affects embryonic development pace, and artificial intelligence can differentiate between embryos from aged and young maternal mice by a non-invasive approach. Thus, machine learning can be used to identify morphokinetics phenotypes for further studies. This study has potential for future applications in selecting human embryos for embryo transfer, without or in complement with preimplantation genetic testing.

Stability of Blueberry Extracellular Vesicles and Their Gene Regulation Effects in Intestinal Caco-2 Cells.

Plant extracellular vesicles (P-EVs) are considered promising functional food ingredients due to their various health benefits. In this study, blueberry extracellular vesicles (B-EVs) were collected and purified by size exclusion chromatography (SEC). The chemical compounds in B-EV extracts were analyzed by LC-MS/MS. In addition, the stability of B-EVs was evaluated during short- and long-term storage, heating, and in vitro digestion. The results showed that the B-EVs had a desirable particle size (88.2 ± 7.7 nm). Protein and total RNA concentrations were 582 ± 11.2 µg/mL and 15.4 µg/mL, respectively. The optimal storage temperatures for B-EVs were 4 °C and -80 °C for short- and long-term storage, respectively. Fluorescent labeling and qRT-PCR tests showed that B-EVs could be specifically internalized by Caco-2 cells, whereas virtually no cytotoxic or growth-inhibitory effects were observed. B-EVs down-regulated the expression levels of IL-1ß and IL-8 and up-regulated the expression levels of NF-κß and TLR5 in Caco-2 cells. Overall, the results proved that the intact structure of B-EVs could be preserved during food storage and processing conditions. B-EVs had the ability to reach the human intestine through oral delivery. Moreover, they could be absorbed by intestinal cells and affect human intestinal function.

Comparison of Female Ovarian Reserve Before vs After COVID-19 Vaccination.

This cohort study examines the association of COVID-19 vaccination with levels of anti-Mullerian hormone and antral follicle count in women seeking fertility treatment.

Small Bowel Obstruction in Postpartum Vaginal Delivery due to Prior Abdominal Adhesions Case Report.

Intestinal obstruction rarely occurs after uncomplicated vaginal deliveries. Here, we present a case of a multiparous woman with a history of prior appendectomy presenting with generalized, nonspecific abdominal pain that was out of proportion to exam findings. Initial abdominal X-ray was nonspecific, and subsequent computed tomography (CT) abdomen showed closed small bowel obstruction requiring surgical repair. We present a case of intestinal obstruction occurring within 24 hours of uncomplicated vaginal delivery with a risk factor of a prior appendectomy surgery and the use of CT abdomen and pelvis to expedite diagnose.

Transcriptome identification of genes associated with uterus-vagina junction epithelial folds formation in chicken hens.

The development regulation of the uterine-vaginal junction (UVJ) epithelial folds during the sexual maturation of female birds played crucial roles in the adults' sperm storage duration and fertilization capability. However, there is a lack of studies on it in the breeding field of laying hens. In this study, White Leghorn was used for the morphological and developmental studies. According to the morphological characteristics, the development of the UVJ epithelial folds was classified into 4 stages (morphological stage T1-T4). Significant individual differences were observed simultaneously, which is one of the factors leading to the adults' UVJ morphological differences. Bulk RNA-seq suggested the different regulations of UVJ epithelial folds were classified into 3 stages (developmental stage S1-S3). Genes enriched in cell proliferation, differentiation, polarity, migration, adhesion and junction were supposed to regulate UVJ epithelial fold formation. Single-cell RNA-sequencing (scRNA-seq) showed significant differences between different types of cells within UVJ at the developmental stage S2. Immunohistochemical studies confirmed that the different proliferation rates between the epithelium and nonepithelium were one of the key factors leading to the formation of UVJ epithelial folds. Genes in the TGF-beta and WNT pathways may play roles in regulating the proliferation and differentiation of epithelium. Some factors, such as CHD2, CDC42, and carbonic anhydrases, were important participants in forming UVJ epithelial folds. This study will provide new thoughts on the differential regulation of fertilization traits from the developmental biology perspective.

Transcriptomic and Metabolomic Profile Analysis of Muscles Reveals Pathways and Biomarkers Involved in Flavor Differences between Caged and Cage-Free Chickens.

The cage-free system has gained a lot of interest in recent years because it can offer chickens more freedom and is easier to manage compared with free-range rearing systems, but few studies have focused on the effect of the cage-free rearing system on meat quality and flavor. In this study, 44 Jianghan chickens were reared in caged or cage-free systems to explore the effect of different rearing systems on meat-eating quality. Sensory evaluation of cooked muscles showed that the leg muscle aroma, juiciness, and flavor intensity significantly improved by the cage-free rearing. The cage-free hens had significantly lower body weight, abdominal fat percentage, and meat fat content, but higher meat moisture content. The cage-free group had brighter breast muscle and redder leg muscle color 24 h after slaughter. Transcriptomic and metabolomic profile analysis of the leg muscle samples showed that the cage-free rearing changed biosynthesis pathways associated with glycogen metabolism, lipid and fatty acid biosynthesis and transport, muscle cellular type, and cellular components, which were related to raw meat quality. Different rearing systems also resulted in differences in glycolipid metabolism, lipid metabolism, and altered levels of intramuscular fat content and other flavor precursors. Pathways such as glycerolipid metabolism, adipocytokine signaling, and metabonomic pathways such as linoleic acid, glycerophospholipid, arginine, proline, and ß-alanine metabolism may be responsible for the meat quality and flavor change.

Development of a dynamic machine learning algorithm to predict clinical pregnancy and live birth rate with embryo morphokinetics.

Objective: To evaluate the feasibility of generating a center-specific embryo morphokinetic algorithm by time-lapse microscopy to predict clinical pregnancy rates. Design: A retrospective cohort analysis. Setting: Academic fertility clinic in a tertiary hospital setting. Patients: Patients who underwent in vitro fertilization with embryos that underwent EmbryoScope time-lapse microscopy and subsequent transfer between 2014 and 2018. Interventions: None. Main Outcome Measures: Clinical pregnancy. Results: A supervised, random forest learning algorithm from 367 embryos successfully predicted clinical pregnancy from a training set with overall 65% sensitivity and 74% positive predictive value, with an area under the curve of 0.7 for the test set. Similar results were achieved for live birth outcomes. For the secondary analysis, embryo growth morphokinetics were grouped into five clusters using unsupervised clustering. The clusters that had the fastest morphokinetics (time to blastocyst = 97 hours) had pregnancy rates of 54%, whereas a cluster that had the slowest morphokinetics (time to blastocyst = 122 hours) had a pregnancy rate of 71%, although the differences were not statistically significant (P=.356). Other clusters had pregnancy rates of 51%-60%. Conclusions: This study shows the feasibility of a clinic-specific, noninvasive embryo morphokinetic simple machine learning model to predict clinical pregnancy rates.

De novo assembly of the complete mitochondrial genome of sweet potato (Ipomoea batatas [L.] Lam) revealed the existence of homologous conformations generated by the repeat-mediated recombination.

Sweet potato (Ipomoea batatas [L.] Lam) is an important food crop, an excellent fodder crop, and a new type of industrial raw material crop. The lack of genomic resources could affect the process of industrialization of sweet potato. Few detailed reports have been completed on the mitochondrial genome of sweet potato. In this research, we sequenced and assembled the mitochondrial genome of sweet potato and investigated its substructure. The mitochondrial genome of sweet potato is 270,304 bp with 23 unique core genes and 12 variable genes. We detected 279 pairs of repeat sequences and found that three pairs of direct repeats could mediate the homologous recombination into four independent circular molecules. We identified 70 SSRs in the whole mitochondrial genome of sweet potato. The longest dispersed repeat in mitochondrial genome was a palindromic repeat with a length of 915 bp. The homologous fragments between the chloroplast and mitochondrial genome account for 7.35% of the mitochondrial genome. We also predicted 597 RNA editing sites and found that the rps3 gene was edited 54 times, which occurred most frequently. This study further demonstrates the existence of multiple conformations in sweet potato mitochondrial genomes and provides a theoretical basis for the evolution of higher plants and cytoplasmic male sterility breeding.

Identification of genes associated with feather color in Liancheng white duck using FST analysis.

Liancheng white duck has two phenotypic traits: white feather and black beak-black foot, but the genes controlling these phenotypic traits are unknown. The objective of this study is to identify various candidate genes related to the plumage of Liancheng white duck. This study used F2 population construction generated between white Kaiya duck and Liancheng white duck and FST analysis between the dominant and recessive loci associated with the Liancheng white duck white feather in order to identify specific gene regions. As per the feather color statistics of the F2 population, it is estimated that there are about three or four genes controlling the white feather of Liancheng white ducks, and the FST results showed that four significant signals were found on chromosomes 4, 12, 13, and 21. Further annotation of these regions led to the identification of five genes involved in the melanin pathway, namely, KIT, CLOCK, MITF, CEBPA, and DOK5. Among them, CEBPA and DOK5 might be affecting the white feather traits of Liancheng white duck by regulating the melanin production and its transfer to the feather. The results provide insightful understanding into the genetic mechanisms of white feather in Liancheng white duck.

Fetal programming of polycystic ovary syndrome: Effects of androgen exposure on prenatal ovarian development.

Polycystic ovary syndrome (PCOS) is a common form of anovulatory infertility with a strong hereditary component but no candidate genes have been found. The inheritance pattern may be due to in utero androgen programming on gene expression and mitochondria. Mitochondria are maternally inherited and alterations to mitochondria after fetal androgen exposure may explain one of the mechanisms of fetal programming in PCOS. Our aim was to investigate the role of excessive prenatal androgens in ovarian development by identifying how hyperandrogenemia affects gene expression and mitochondria in neonatal ovary. Pregnant dams were injected with dihydrotestosterone on days 16-18 of pregnancy. Day 0 ovaries were collected for gene expression and mitochondrial studies. RNAseq showed differential gene expressions which were related to mitochondrial dysfunction, fetal gonadal development, oocyte maturation, metabolism, angiogenesis, and PCOS. Top 20 up and downregulated genes were validated with qPCR and Western Blot. Transcriptional pathways involved in folliculogenesis and genes involved in ovarian and mitochondrial function were dysregulated. Further, DHT exposure altered mitochondrial ultrastructure and function by increasing mitochondrial oxygen consumption and decreasing mitochondrial efficiency with increased proton leak within the first day of life. Our data indicates that one path that leads to PCOS begins at birth and is programmed in utero by androgens.

Transcriptome analysis and identification of age-associated fertility decreased genes in hen uterovaginal junction.

Avian sperm storage tubules (SSTs), which are located in the uterovaginal junction (UVJ) of the oviduct, are primary sperm storage sites after mating or artificial insemination. The mechanism underlying reduced sperm storage efficiency of SSTs which is highly correlated with decreased fertility rates in aged laying breeders remains largely unclear. Here, comparative transcriptomic analysis between the aged and young White Leghorn hens (120 vs. 30 wk) was applied to identify gene expression changes of UVJs containing SSTs. Bioinformatics analysis revealed 567 upregulated and 1998 downregulated differentially expressed genes. Gene ontology analysis was highly enriched in terms of immune system, cell adhesion, and cytoskeleton proteins. Kyoto Encyclopedia of Genes and Genomes analysis revealed 5 significant (P < 0.05) pathways including inositol phosphate and glycerophospholipid metabolism. ß-Galactosidase staining of chicken UVJ sections suggested increased cell senescence via aging. Oil Red O staining and immunohistochemistry detection of ADFP both confirmed distribution of lipid droplets in SST cells with increased intensity in aged breeders. The lipid synthesis and metabolism-related genes represented by TFAP2 and PLD1 were differentially expressed in aged laying breeders. The upregulation of IL15 and downregulation of a large number of immune-related genes in aged breeders indicate altered immune homeostasis in UVJs and SSTs. The increased accumulation of lipids, and altered immunity homeostasis, combined with other factors (TJP1, MYL9, AFDN, and RPL13, etc.) are potentially dominant effectors to decrease the sperm storage efficiency and egg fertility in aged laying breeders.

Transcriptome analysis and identification of genes associated with chicken sperm storage duration.

The sperm storage tubules located in the mucosal folds of the uterovaginal junction (UVJ) are the primary site of sperm storage in chicken hens after natural mating or artificial insemination (AI). The short-term sperm storage (24 h after mating or AI) in hens was highly associated with immunity and pH-related pathway genes. However, the underlying mechanism of longer duration of sperm storage in female birds remains largely unclear. In the present study, transcriptome analysis was applied to uncover the dynamic gene expression changes in chicken UVJ tissues at two time points (day 3 and day 9) after AI. A total of 574 differentially expressed genes (DEG) were enriched, including 266 upregulated and 308 downregulated DEG. The validation of 5 DEG using quantitative PCR showed a similar expression tendency with RNA sequencing results. The gene ontology terms of DEG were highly enriched in heparin binding (9 genes including COMP, CTGF, and IMPG2), glycosaminoglycan binding (10 genes including PCOLCE, POSTN, and RSPO3), and response to estradiol and ion transport (AREG, RAMP3, SFRP1, and SSTR1). Kyoto encyclopedia of genes and genomes pathway-enrichment analyses of DEG revealed 10 significant pathways (P < 0.05) represented by calcium signaling pathway (7 genes including CACNA1G, PDE1C, PDGFRB, and SLC8A1) and glycosaminoglycan biosynthesis (B3GNT7, CSGALNACT1, GLCE, and ST3GAL1). Protein-protein interaction network of DEG established the connection-regulating epithelial cell or cell-matrix adhesion and migration. The enriched pathways and genes were highly correlated with temporary sperm storage in and possibly sequential sperm release from chicken UVJ overtime after AI. Of these, HIP1, PDE1C, and calcium-related genes were the most interesting candidates associated with sperm storage duration. This report provided a global gene expression profile of the chicken UVJ regarding the capacity of sperm storage overtime after AI. The outcome of this study will contribute to further understanding of the long-term sperm maintenance in avian females and eventually improving the duration of fertile egg performance by selected chicken breeding.

RAI14 in the blood feather regulates chicken pigmentation.

A genome-wide association study (GWAS) was performed on a resource family consisting of white and colored chickens for identification of genes related to plumage coloration using the Fixed and random model Circulating Probability Unification (FarmCPU) package. GWAS identified three chromosomal single-nucleotide polymorphisms (SNPs), demonstrating the polygenic basis of plumage phenotypes. Herein, retinoic acid-induced protein 14 (RAI14), a developmentally regulated gene that encodes a protein containing many ankyrin repeats, was identified as a candidate gene involved in plumage color. In this study, mRNA expression profiles of chicken RAI14 were determined, indel (insertion-deletion) variants were identified, and their association was analyzed in white and colored chickens. RA114 mRNA was expressed in all tissues tested (brain, spleen, liver, heart, oviduct, kidney, lung, pituitary gland, ovary, muscle, feather bulb, and skin). A relatively high RAI14 expression in white feather bulb compared to colored feather bulb ( P < 0.01 ) indicated a potential association with plumage color. Additionally, statistical analysis revealed that a 4 bp indel genetic variation in RAI14 was associated with plumage phenotypes ( P < 0.01 ). Together, our analysis of the identification of the RAI14 gene will enable us to understand the genetic mechanisms behind chicken pigmentation.

Embryos from polycystic ovary syndrome patients with hyperandrogenemia reach morula stage faster than controls.

OBJECTIVE: To investigate if patients with polycystic ovary syndrome (PCOS) have altered embryo morphokinetics when compared with controls. DESIGN: Retrospective cohort analysis. SETTING: Single academic fertility clinic in a tertiary hospital setting. PATIENTS: Age- and body mass index-matched patients who underwent in vitro fertilization diagnosed with PCOS using the Rotterdam criteria. A subanalysis was performed on patients with PCOS with hyperandrogenemia. Sixty-four patients with PCOS were identified with 990 embryos that were matched with 64 control patients with 628 embryos. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Time to blastulation. RESULTS: Embryos from women with PCOS displayed faster growth rate at t7, t8, and t9; all other morphokinetic points were similar. Patients with PCOS also had a higher number of oocytes retrieved. No differences were seen in the fertilization rate or blastulation rate. Patients with PCOS had a higher miscarriage rate (38.1% in PCOS vs. 18.8% in controls). Patients with hyperandrogenic PCOS showed a faster growth rate at t5, t6, t7, t8, t9, and morula. CONCLUSIONS: Embryos from women with PCOS grew faster until 9-cell stage and women with hyperandrogenic PCOS until morula. Patients with PCOS also showed a higher miscarriage rate. The alterations in early embryo development are consistent with altered fertility and obstetric outcomes in the population with PCOS and may be due to the hyperandrogenic microenvironment in the ovarian follicle.

The capability of L-carnitine-mediated antioxidant on cock during aging: evidence for the improved semen quality and enhanced testicular expressions of GnRH1, GnRHR, and melatonin receptors MT 1/2.

Precise natural anti-oxidative compounds have facilitated the research of infertile gametes and the development of novel bio-therapeutics, especially the molecules that are based on the reduction of oxidative stress, such as L-carnitine (LC). In addition to, the defect in the functioning of sperm mitochondrial and the decreasing seminal antioxidant ability due to aging, its essential role in permitting the mitochondrial import and oxidation of long chain fatty acids is worthy. Therefore, current study was designed to investigate the effects of dietary LC on semen quality, seminal antioxidant activity, and their implications for the fertility in aged cocks for 12 wk. Supplementation of the feed with two different doses of LC (50 and 150 mg/kg body weight/day) for 12 wk showed significantly increased in the reproductive activity of cock, in comparison to the control group. Seminal analysis showed that supplementation of LC significantly increased (P < 0.05) the sperm motility, concentration, livability, semen quality factor, seminal malondialdehyde concentration, catalase, and glutathione peroxidase activities. In addition, addition of LC significantly increased (P < 0.05) the plasma concentration of testosterone and prostaglandin E2 but posed no significant effect on the concentration of follicle-stimulating hormone. Furthermore, the findings of artificial insemination showed significant increased (P < 0.05) in the percentage of fertility in LC groups, while the percentage hatchability and mortality remained unchanged. Immunohistochemistry analysis revealed that LC significantly increased (P < 0.05) the testicular immunopositivity of MT1 and MT2. Moreover, the administration of LC to the aged cocks enhanced (P < 0.05) GnRH1 and GnRHR mRNA levels when compared with untreated cocks. The results of the present study suggest that LC treatment of aged cocks increases the seminal antioxidant enzymes and sexual hormones levels, which may improve the semen quality by increasing the expression of GnRH1 and melatonin receptors (MT1 and MT2) activities. Collectively, LC could be a suitable feed supplementation to increase reproductive activities through enhancing semen quality in aging cocks.

Identifying artificial selection signals in the chicken genome.

Identifying the signals of artificial selection can contribute to further shaping economically important traits. Here, a chicken 600k SNP-array was employed to detect the signals of artificial selection using 331 individuals from 9 breeds, including Jingfen (JF), Jinghong (JH), Araucanas (AR), White Leghorn (WL), Pekin-Bantam (PB), Shamo (SH), Gallus-Gallus-Spadiceus (GA), Rheinlander (RH) and Vorwerkhuhn (VO). Per the population genetic structure, 9 breeds were combined into 5 breed-pools, and a 'two-step' strategy was used to reveal the signals of artificial selection. GA, which has little artificial selection, was defined as the reference population, and a total of 204, 155, 305 and 323 potential artificial selection signals were identified in AR_VO, PB, RH_WL and JH_JF, respectively. We also found signals derived from standing and de-novo genetic variations have contributed to adaptive evolution during artificial selection. Further enrichment analysis suggests that the genomic regions of artificial selection signals harbour genes, including THSR, PTHLH and PMCH, responsible for economic traits, such as fertility, growth and immunization. Overall, this study found a series of genes that contribute to the improvement of chicken breeds and revealed the genetic mechanisms of adaptive evolution, which can be used as fundamental information in future chicken functional genomics study.

DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias.

DNMT3A, the gene encoding the de novo DNA methyltransferase 3A, is among the most frequently mutated genes in hematologic malignancies. However, the mechanisms through which DNMT3A normally suppresses malignancy development are unknown. Here, we show that DNMT3A loss synergizes with the FLT3 internal tandem duplication in a dose-influenced fashion to generate rapid lethal lymphoid or myeloid leukemias similar to their human counterparts. Loss of DNMT3A leads to reduced DNA methylation, predominantly at hematopoietic enhancer regions in both mouse and human samples. Myeloid and lymphoid diseases arise from transformed murine hematopoietic stem cells. Broadly, our findings support a role for DNMT3A as a guardian of the epigenetic state at enhancer regions, critical for inhibition of leukemic transformation.

Long non-coding RNAs control hematopoietic stem cell function.

Hematopoietic stem cells (HSCs) possess unique gene expression programs that enforce their identity and regulate lineage commitment. Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression and cell fate decisions, although their functions in HSCs are unclear. Here we profiled the transcriptome of purified HSCs by deep sequencing and identified 323 unannotated lncRNAs. Comparing their expression in differentiated lineages revealed 159 lncRNAs enriched in HSCs, some of which are likely HSC specific (LncHSCs). These lncRNA genes share epigenetic features with protein-coding genes, including regulated expression via DNA methylation, and knocking down two LncHSCs revealed distinct effects on HSC self-renewal and lineage commitment. We mapped the genomic binding sites of one of these candidates and found enrichment for key hematopoietic transcription factor binding sites, especially E2A. Together, these results demonstrate that lncRNAs play important roles in regulating HSCs, providing an additional layer to the genetic circuitry controlling HSC function.

DNMT3A in haematological malignancies.

DNA methylation patterns are disrupted in various malignancies, suggesting a role in the development of cancer, but genetic aberrations directly linking the DNA methylation machinery to malignancies were rarely observed, so this association remained largely correlative. Recently, however, mutations in the gene encoding DNA methyltransferase 3A (DNMT3A) were reported in patients with acute myeloid leukaemia (AML), and subsequently in patients with various other haematological malignancies, pointing to DNMT3A as a critically important new tumour suppressor. Here, we review the clinical findings related to DNMT3A, tie these data to insights from basic science studies conducted over the past 20 years and present a roadmap for future research that should advance the agenda for new therapeutic strategies.