Glucose metabolism disorder related to follicular fluid exosomal miR-122-5p in cumulus cells of endometriosis patients.

In Brief: Elevated expression of miR-122-5p in exosomes in the follicular fluid of patients with endometriosis impairs glucose metabolism in cumulus cells and may further impair oocyte quality. Abstract: Endometriosis (EMs) affects fertility in women of childbearing age in many ways. The underlying mechanisms, including the decrease in oocyte quality, require further investigation. Exosomes, small vesicles responsible for intercellular information exchange, have been found to be involved in many biological events, including follicle development and oocyte meiosis recovery. From the perspective of follicular fluid exosomes, this study aimed to elucidate the mechanisms involved in EMs-related oocyte quality decline. Follicular fluid was collected from three groups of women: the untreated EMs group (EMs_UT), the satisfactorily treated EMs group (EMs_ST), and the control group (Ctrl). Mouse cumulus-oocyte complexes (COCs) were co-cultured with exosomes extracted from follicular fluid during in vitro maturation. Oocyte quality and cumulus cell function were assessed. High-throughput sequencing of miRNA in exosomes was conducted. The function of differentially expressed miRNAs was studied by using SVOG human ovarian granulosa cells transfected with an miRNA mimic and inhibitor. It was found that the follicular fluid exosomes from patients with untreated EMs reduced both the rate of maturation and the quality of mouse oocytes. Overexpression of miR-122-5p in untreated EMs inhibited the translation of key aldolase enzymes related to glucose metabolism and partly impaired glucose metabolism in the cumulus cells of patients with endometriosis. miR-122-5p was also observed to reduce proliferation and increase apoptosis after cell transfection with an miR-122-5p mimic and inhibitor. Further experiments are needed to determine whether there are additional small molecules in the follicular fluid of patients with endometriosis that could be involved in damaging oocyte quality and to identify where harmful substances in follicular fluid exosomes are loaded.

Single-Cell Quantitative Proteomic Analysis of Human Oocyte Maturation Revealed High Heterogeneity in In Vitro-Matured Oocytes.

Oocyte maturation is pertinent to the success of in vitro maturation (IVM), which is used to overcome female infertility, and produced over 5000 live births worldwide. However, the quality of human IVM oocytes has not been investigated at single-cell proteome level. Here, we quantified 2094 proteins in human oocytes during in vitro and in vivo maturation (IVO) by single-cell proteomic analysis and identified 176 differential proteins between IVO and germinal vesicle oocytes and 45 between IVM and IVO oocytes including maternal effect proteins, with potential contribution to the clinically observed decreased fertilization, implantation, and birth rates using human IVM oocytes. IVM and IVO oocytes showed separate clusters in principal component analysis, with higher inter-cell variability among IVM oocytes, and have little correlation between mRNA and protein changes during maturation. The patients with the most aberrantly expressed proteins in IVM oocytes had the lowest level of estradiol per mature follicle on trigger day. Our data provide a rich resource to evaluate effect of IVM on oocyte quality and study mechanism of oocyte maturation.

A comprehensive characterization of cell-free RNA in spent blastocyst medium and quality prediction for blastocyst.

The rate of pregnancy can be affected by many factors in assisted reproductive technology (ART), and one of which is the quality of embryos. Therefore, selecting the embryos with high potential is crucial for the outcome. Fifteen spent blastocyst medium (SBM) samples were collected from 14 patients who received in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), seven from high-grade embryos and eight from low-grade embryos. Cell-free RNA (cf-RNA) profile of SBM samples were analyzed by RNA sequencing in the present study. It was found that a large amount of cf-RNA were released into SBM, including protein-coding genes (68.9%) and long noncoding RNAs (lncRNAs) (17.26%). Furthermore, a high correlation was observed between blastocyst genes and SBM genes. And the cf-mRNAs of SBM were highly fragmented, and coding sequence (CDS) and untranslated (UTR) regions were released equally. Two hundred and thirty-two differentially expressed genes were identified in high-grade SBM (hSBM) and low-grade SBM (lSBM), which could be potential biomarker in distinguishing the embryos with different quality as an alternative or supplementary approach for subjective morphology criteria. Hence, cf-RNAs sequencing revealed the characterization of circulating transcriptomes of embryos with different quality. Based on the results, the genes related to blastocyst quality were screened, including the genes closely related to translation, immune-signaling pathway, and amino acid metabolism. Overall, the present study showed the types of SBM cf-RNAs, and the integrated analysis of cf-RNAs profiling with morphology grading displayed its potential in predicting blastocyst quality. The present study provided valuable scientific basis for noninvasive embryo selection in ART by RNA-profiling analysis.

[Effect of complete Y chromosome AZFc microdeletion on embryo euploidy].

OBJECTIVE: Preimplantation genetic testing (PGT) was performed to analyze the embryo euploidy in patients with complete Y chromosome AZFc microdeletion. METHODS: The clinical data of complete AZFc microdeletion underwent PGT from January 2013 to December 2021 in Reproductive Medicine Center of the First Affiliated Hospital of Nanjing Medical University were retrospectively analyzed. The patients with monogenic disease who underwent PGT during the same period were set as the control group. The basic characteristics, fertilization rate, Day 3 high quality embryo rate, blastocyst formation rate, embryo euploid rate, 45, X embryo ratio was compared between the two groups. RESULTS: A total of 220 patients were included, including 91 patients with complete AZFc microdeletion and 129 patients with monogenic disease. There was no significant difference in age between the two groups. In semen parameters, the sperm concentration, total sperm count and progressive motility in AZFc microdeletion group were lower than those in monogenic disease group, and the differences were statistically significant (P=0.001). The fertilization rate of AZFc microdeletion group was lower than that in monogenic disease group (P=0.012), and there was no significant difference in the number of MII oocytes, Day 3 high-quality embryo rate and blastocyst formation rate. A total of 933 blastocysts were successfully tested, including 496 blastocysts in AZFc deletion group and 437 blastocysts in monogenic disease group. The euploid, aneuploid and mosaic rates of the AZFc microdeletion group were 57.26%, 24.60% and 18.14%, respectively, while those of the monogenic disease group were 66.13%, 23.80% and 10.07%, with statistically significant differences between the two groups (P=0.001). Further analysis of the two groups of aneuploid embryos showed that aberrations occurred most commonly in chromosome16 (3.87%), X (3.46%), 13 (2.44%), 22 (2.24%) and 19 (2.03%) in AZFc microdeletion group, respectively, while the monogenic disease group was 22 (4.35%), 16 (2.97%), 7 (2.74%), 15(1.60%) and 2(1.60%), respectively. The proportion of sex chromosome abnormality in AZFc microdeletion group was higher than that in monogenic disease group (P=0.039), and there was no significant difference in the proportion of 45,X between the two groups. CONCLUSION: Compared with monogenic disease group, The embryo euploid rate in AZFc microdeletion patients decreased and the proportion of 45, X embryos did not increase significantly. It is recommended to select euploid female embryos by PGT, which not only avoids vertical transmission of AZFc microdeletion, but also reduces the risk of miscarriage due to aneuploid embryos.

Elevated prohibitin 1 expression mitigates glucose metabolism defects in granulosa cells of infertile patients with endometriosis.

Endometriosis is a common disease in women of childbearing age and is closely associated with female infertility. However, the pathogenesis of endometriosis-related infertility is still not fully understood. Prohibitin 1 (PHB1), a highly conserved protein related to mitochondrial function, is differentially expressed in the endometrium of patients with endometriosis. However, the role of PHB1 in glucose metabolism in granulosa cells remains unclear. In this study, we investigated whether PHB1 expression and glucose metabolism patterns differ in the granulosa cells of patients with endometriosis and those of patients serving as controls. We then evaluated these changes after PHB1 was upregulated or downregulated in the human granulosa cell line (KGN) using a lentivirus construct. In the granulosa cells of patients with endometriosis, significantly elevated PHB1 expression, increased glucose consumption and lactic acid production, as well as aberrant expression of glycolysis-related enzymes were found compared to those without endometriosis (P < 0.05). After PHB1 expression was upregulated in KGN cells, and the expression of enzymes related to glucose metabolism, glucose consumption and lactic acid production was strikingly increased compared to controls (P < 0.05). The opposite results were found when PHB1 expression was downregulated in KGN cells. Additionally, the cell proliferation and apoptosis rates, ATP synthesis, reactive oxygen species (ROS) levels and mitochondrial membrane potential (MMP) were significantly altered after down-regulation of PHB1 expression in KGN cells (P < 0.05). This study suggested that PHB1 plays a pivotal role in mitigating the loss of energy caused by impaired mitochondrial function in granulosa cells of patients with endometriosis, which may explain, at least in part, why the quality of oocytes in these patients is compromised.

Nucleoporin37 may play a role in early embryo development in human and mice.

Maternal-effect genes (MEGs) play an important role in maintaining the survival and development of mammalian embryos at the cleavage stage after fertilization. Despite long-term efforts, the MEGs that regulate preimplantation embryo development remain largely unknown. Here, using whole-exome sequencing and homozygosity mapping, we identified a potential candidate gene associated with early embryo development: nucleoporin37 (NUP37), a nucleoporin gene that encodes a member of the nuclear pore complexes and regulates nuclear pore permeability and nucleocytoplasmic transport. Moreover, we determined the temporal and spatial expression patterns of Nup37 in mouse oocytes and early embryos, and explored the role of NUP37 in oocyte maturation and preimplantation embryo development. Immunoprecipitation assays confirmed that yes-associated protein-1 (YAP1) binds to TEA domain transcription factor 4 (TEAD4) and NUP37. Furthermore, Nup37 gene knockdown reduced the nuclear import of YAP1 and down-regulated the expression of YAP1-TEAD pathway downstream genes Rrm2 and Rpl13 in early embryos. Our study provides evidence that maternal NUP37 contributes to the nuclear import of YAP1 and then activates the YAP1-TEAD pathway, a signalling pathway essential for zygotic genome activation. Nup37 may be a key gene involved in preimplantation embryo development in mammals.

Majority of transferred mosaic embryos developed healthy live births revealed by a preclinical study using embryonic morphology assessment and noninvasive PGT-A on cell-free DNA in blastocoel fluid.

PURPOSE: This preclinical study aimed to evaluate whether using transferred mosaic embryos (primarily selected by embryonic morphology assessment (EMA) and compared by the noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) on cell-free DNA in blastocoel fluid (BF)) increases the rates of clinical pregnancies (CPs) and healthy live births (HLBs) and to investigate whether niPGT-A could provide valuable genetic information for the EMA-selected transferred mosaic embryos. METHODS: This study collected 215 blastocyst culture samples and 182 BF samples. Cell-free DNA from the BF was amplified and examined by next-generation sequencing-based niPGT-A. All 182 patients underwent EMA. However, only 147 underwent in vitro fertilization and embryo transfer, and only 113 clinical outcomes were followed up. Comprehensive chromosome screening for the chorionic villus sampling of spontaneous miscarriages and noninvasive prenatal testing for ongoing pregnancies were also performed. RESULTS: The implantation rate was 77.55% in 147 transferred high-quality embryos selected by EMA. Among 113 CPs, 16 led to spontaneous miscarriage (14.16%), and 97 resulted in HLBs (85.84%). According to the niPGT-A results for 113 patients with clinical outcomes, 80.4% had CP (euploid, 20.54%; single aneuploid, 1.79%; mosaic chromosome aneuploid and/or segmental aneuploid, 58.04%). Of all the mosaic aneuploids, 90.76% were false positive, transforming to euploid. CONCLUSIONS: Transferred EMA-selected embryos showed higher implantation rates. The niPGT-A of BF provided valuable genetic status ("-ploid") information, which helped reduce aneuploid-induced implantation failure and miscarriage, thereby increasing the CP and HLB rates. Additionally, majority of the transferred embryos with complex/chaotic mosaic aneuploid would likely develop HLBs.

Novel bi-allelic variants in ACTL7A are associated with male infertility and total fertilization failure.

STUDY QUESTION: What are the genetic causes of total fertilization failure (TFF) in a proband suffering from male infertility? SUMMARY ANSWER: Novel compound heterozygous variants (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) in actin-like protein 7A (ACTL7A) were identified as a causative genetic factor for human TFF. WHAT IS KNOWN ALREADY: ACTL7A, an actin-related protein, is essential for spermatogenesis. ACTL7A variants have been reported to cause early embryonic arrest in humans but have not been studied in human TFF. STUDY DESIGN, SIZE, DURATION: We recruited a non-consanguineous family whose son was affected by infertility characterized by TFF after ICSI. Whole-exome sequencing was used to identify the potential pathogenic variants. Artificial oocyte activation (AOA) after ICSI was performed to overcome TFF and any resulting pregnancy was followed up. PARTICIPANTS/MATERIALS, SETTING, METHODS: Sanger sequencing was performed to validate the variants. Pathogenicity of the identified variants was predicted by in silico tools. The ultrastructure of spermatozoa was studied by transmission electron microscopy (TEM). Immunofluorescence staining and western blotting were used to investigate the mechanism of the variants on the affected spermatozoa. MAIN RESULTS AND THE ROLE OF CHANCE: Novel compound heterozygous variants in ACTL7A (c.[463C>T];[1084G>A], p.[(Arg155Ter)];[(Gly362Arg)]) were identified in a family with TFF after ICSI. In silico analysis predicted that the variants lead to a disease-causing protein. TEM showed that the ACTL7A variants caused ultrastructural defects in the acrosome and perinuclear theca. Protein expression of ACTL7A and phospholipase C zeta, a key sperm-borne oocyte activation factor, was significantly reduced in the affected sperm compared to healthy controls, suggesting that the ACLT7A variants lead to an oocyte activation deficiency and TFF. AOA by calcium ionophore (A23187) after ICSI successfully rescued the TFF and achieved a live birth for the patient with ACTL7A variants. LIMITATIONS, REASONS FOR CAUTION: Given the rarity of sperm-associated TFF, only one family with an only child carrying the ACTL7A variants was found. In addition, the TFF phenotype was not assessed in two or more ICSI cycles, due to the intervention in ICSI with AOA after one failed ICSI cycle. Further studies should validate the ACTL7A variants and its effect on male infertility in larger independent cohorts. WIDER IMPLICATIONS OF THE FINDINGS: : Our findings revealed a critical role of ACTL7A in male fertility and identified bi-allelic variants in ACTL7A associated with human TFF, which expands the genetic spectrum of TFF and supports the genetic diagnosis of TFF patients. We also rescued TFF by AOA and obtained a healthy live birth, which provides a potentially effective intervention for patients with ACTL7A pathogenic variants. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81971374 and 81401267). No conflicts of interest were declared. TRIAL REGISTRATION NUMBER: N/A.

The integrity of cfDNA in follicular fluid and spent medium from embryo culture is associated with embryo grade in patients undergoing in vitro fertilization.

PURPOSE: This study was conducted to verify if the cfDNA integrity (cfDI) in follicular fluid and subsequent spent embryo medium (SEM) could serve as potential non-invasive biomarker for high-grade embryo selection during IVF/ICSI. METHODS: Thirty-two follicular fluids, 32 subsequent corresponding cleavage embryo SEM, and 23 subsequent blastocyst SEM were collected from 11 patients undergoing IVF/ICSI. CfDI was measured by ALU gene amplicons with different sizes by qPCR, as the ratio of long to short fragments. RESULTS: CfDI in follicular fluid corresponding to subsequent high-grade cleavage embryos and blastocysts was significantly lower than that related to low-grade embryos (p = 0.018). Conversely, cfDI in SEM was significantly and positively correlated with high-grade embryos at both stages (p = 0.009). ROC curves of the analysis of cfDI in follicular fluid showed great potential in predicting subsequent embryogenesis and embryo grade (AUC > 0.927). Regardless of the cleavage embryo grade by morphology, cfDI in day 3 SEM could predict if the cleavage embryo could develop to a high-grade blastocyst (AUC = 0.820). A concordant shift pattern of cfDI from follicular fluid to subsequent day 3 SEM and day 5 SEM was found in 81.82% participants featured by various clinical characteristics. CONCLUSION: CfDI in follicular fluid and SEM was significantly correlated with embryogenesis and embryo grade and could serve as a potential non-invasive biomarker in high-grade embryo selection. Direct qPCR was proved as a labor-saving and sensitive method for the analysis of cfDI in low volume of SEM.

A novel homozygous nonsense mutation in zona pellucida 1 (ZP1) causes human female empty follicle syndrome.

PURPOSE: To identify a pathogenic gene mutation in a female infertility proband characterized by empty follicle syndrome (EFS) and explore the genetic cause of EFS. METHODS: Whole exome sequencing (WES) was performed to identify the candidate pathogenic mutation. Sanger sequencing was used to validate the mutation in family members. The pathogenicity of the identified variant and its possible effects on the protein were evaluated with in silico tools. Immunofluorescence staining was used to study the possible mechanism of the mutation on affected oocyte. RESULTS: We identified a family with a novel homozygous nonsense mutation in zona pellucida 1 (ZP1) (c.199G > T [p.Glu67Ter]). Based on bioinformatics analysis, the mutation was predicted to be pathogenic. This variant generates a premature stop codon in exon 2 at the 199th nucleotide, and was inferred to result in a truncated ZP1 protein of 67 amino acids at the ZP-N1 domain. An in vitro study showed that the oocyte of the EFS proband was degenerated and the zona pellucida was absent. Additionally, the mutant ZP1 proteins were localized in the cytoplasm of the degenerated oocyte but not at the surface. CONCLUSIONS: The novel mutation in ZP1 is a genetic cause of female infertility characterized by EFS. Our finding expands the genetic spectrum for EFS and will help justify the EFS diagnosis in patients.

Genetic programs in human and mouse early embryos revealed by single-cell RNA sequencing.

Mammalian pre-implantation development is a complex process involving dramatic changes in the transcriptional architecture. We report here a comprehensive analysis of transcriptome dynamics from oocyte to morula in both human and mouse embryos, using single-cell RNA sequencing. Based on single-nucleotide variants in human blastomere messenger RNAs and paternal-specific single-nucleotide polymorphisms, we identify novel stage-specific monoallelic expression patterns for a significant portion of polymorphic gene transcripts (25 to 53%). By weighted gene co-expression network analysis, we find that each developmental stage can be delineated concisely by a small number of functional modules of co-expressed genes. This result indicates a sequential order of transcriptional changes in pathways of cell cycle, gene regulation, translation and metabolism, acting in a step-wise fashion from cleavage to morula. Cross-species comparisons with mouse pre-implantation embryos reveal that the majority of human stage-specific modules (7 out of 9) are notably preserved, but developmental specificity and timing differ between human and mouse. Furthermore, we identify conserved key members (or hub genes) of the human and mouse networks. These genes represent novel candidates that are likely to be key in driving mammalian pre-implantation development. Together, the results provide a valuable resource to dissect gene regulatory mechanisms underlying progressive development of early mammalian embryos.

Homozygous missense mutation Arg207Cys in the WEE2 gene causes female infertility and fertilization failure.

PURPOSE: To investigate a novel mutation in the WEE2 gene in a female patient with primary infertility and fertilization failure. METHODS: Sanger sequencing was used to detect mutations in WEE2. The pathogenicity of the identified variant and its possible effects on the WEE2 protein were evaluated with in silico tools and molecular modeling. We used the calcium ionophore A23187 as a chemical activator of oocytes after intracytoplasmic sperm injection (ICSI). RESULTS: We identified a consanguineous family with a novel homozygous missense mutation in WEE2 (c.619C>T [p.R207C]). Based on preliminary bioinformatics analysis, we speculate that the novel homozygous missense mutation is pathogenic. ICSI combined with assisted oocyte activation (ICSI-AOA) did not overcome fertilization failure in this patient with WEE2 mutation. CONCLUSIONS: We identified a novel mutation in WEE2 (c.619C>T [p.R207C]) in a female patient with fertilization failure after ICSI, and we provide evidence that this novel homozygous missense mutation can cause fertilization failure.

Preimplantation genetic testing for complex chromosomal rearrangement carriers by next-generation sequencing.

RESEARCH QUESTION: Can preimplantation genetic testing (PGT) with next-generation sequencing (NGS) increase the chance of achieving a balanced euploid pregnancy in complex chromosome rearrangement (CCR) carriers? DESIGN: Six couples underwent PGT at the Clinical Centre of Reproductive Medicine, First Affiliated Hospital, Nanjing Medical University. The CCR carriers in the six couples were: Case A: 46,XY,t(1;4;11)(p31;p16;q22); Case B: 46,XY,t(3;13;5)(p14;q21;p14); Case C: 46,XX,t(6;11;21)(q21;q21;q13); Case D: 46,XX,inv(9)(p12; q13),t(13;15)(q14;q24); Case E: 46,XX,inv(9)(p12;q13),t(7;9)(q22;p22); and Case F: 46,XX,t(2;7)(q21;q36),t(2;4)(p10;q10),t(2;4)(q15;q10). After ovarian stimulation followed by oocyte retrieval and embryo culture, PGT was performed on day 5 or 6 blastocyst biopsies using NGS to identify normal/balanced euploid embryos. Vitrified-warmed single embryo transfers were performed using normal/balanced euploid embryos. RESULTS: After seven cycles, 84 oocytes were retrieved. Whole genome sequencing by NGS was performed on 25 trophectoderm biosies. Six (24%) embryos were identified as normal/balanced euploid, four were transferred resulting in four live births. Case A, C, D and E each gave birth to a healthy baby after their first cycle. There was no transferable embryo after two cycles for Case B and one cycle for Case F. The implantation rate per transfer was 4/4 and the live birth rate was 4/4. CONCLUSION: These results strongly support the use of NGS for CCR carriers.

[Outcomes of micro-TESE combined with ICSI for non-obstructive azoospermia].

OBJECTIVE: To investigate the clinicaleffects of micro-dissection of testicular sperm extraction (micro-TESE) and its combination with intracytoplasmic sperm injection (ICSI) in the treatment of non-obstructive azoospermia (NOA). METHODS: We retrospectively analyzed the clinical data on 130 NOA patients treated between December 2015 and December 2017, including36 cases of idiopathic NOA, 22 cases of idiopathic NOA with small testis, 18 cases of Klinefelter syndrome, 46 cases of surgically treated cryptorchidism and 8 cases of AZFc microdeletion.All the patients underwent micro-TESE and 29 of them with sperm received micro-TESE + ICSI. We observed the changes in the postoperative serum T level, analyzed the influences of different types of NOA and testicular pathology on the sperm retrieval rate (SRR) and the outcomes of ICSI. RESULTS: All the micro-TESE operations were successfully completed. The SRR was significantly higher in those surgically treated for cryptorchidism (60.9%) than in the other NOA groups (P<0.05), withstatistically significant differencesnot among the latter groups (P>0.05) but among differenttypes of testicular pathology (P<0.05), the highest in the hypospermatogenesis group (100%), very low in those with Sertoli-cell-only syndrome (11.8%), and the lowest in the cases of spermatogenesis arrest (0). The serum T level was remarkably decreased at 1 and 6 months after operation (P<0.01), but markedly higher at 6 than at 1 month (P<0.01). Of the 52 patients with sperm retrieved at micro-TESE, 29 of their spouses received ICSI, resulting in 17 pregnancies, 6 live births, and 6 spontaneous abortions. CONCLUSIONS: Micro-TESE is an effective method for the treatment of NOA, and its combination with ICSI can help the NOA patients obtain their genetic offspring.

[Differentiation of embryonic stem cells from the embryos of the couples with male asthenozoospermia and Robertsonian translocation into germ cells].

OBJECTIVE: To assess the risk of male infertility in the offspring conceived through assisted reproductive technology (ART) byin vitroinductionof the differentiation of embryonic stem cells (ESCs) derived from the embryos of the couples with male asthenozoospermia and Robertsonian translocation (RT) into germ cells. METHODS: We established a CCRM16ESC line with the karyotype of 46, XY, +14, rob(13; 14) (q10; q10) from the embryo donated by a patientwithasthenozoospermiaand RT and his wife by isolation of the inner cell mass of blastula, culturing, passaging, and amplification,followed by in vitro induction and differentiationof the ESCs into germ cells with ratinoic acid(RA) at 2 mol/L. Then, we analyzed the process of differentiation and the expressions of its related genes and compared them with those in the normal CCRM23ESCs. RESULTS: CCRM16 showed the typical characteristics of ESCs, expressing the pluripotency makers of NANOG, OCT4, TRA-1-181 and SSEA4, forming embryoid bodies, and differentiating into three germlayer tissues in vitro and in vivo. Intervention with 2 mol/LRAinduced direct differentiation of the ESCs into germ cells. The expressions of the primordial germ cell marker geneDAZLand the meiosis marker geneSCP3were markedly decreased in the CCRM16 as compared with those in the normal CCRM23 ESCs. CONCLUSIONS: The CCRM16ESC linewith the karyotype of46, XY, +14, rob(13; 14) (q10; q10) has thetypical characteristics of ESCs but an abnormal process of differentiation into germ cells in the early stage. In vitroinductionof the differentiation of ESCs into germ cells can be used for assessing the risk of male infertility in the offspring conceived through ART for asthenozoospermia patients.

Sparse-View Photoacoustic Reconstruction Method for Diabetic Retinopathy Using Feature Fusion Network.

Diabetic retinopathy is one of the most prevalent microvascular complications of diabetes mellitus, and photoacoustic imaging is an effective method for imaging diabetic retinal vessels. Photoacoustic imaging is an emerging noninvasive imaging method based on the photoacoustic effect, which offers advantages of contrast, resolution, and depth imaging. Appropriate photoacoustic reconstruction methods are essential for obtaining high-quality photoacoustic images. In this study, a multi-input self-attention multiscale feature fusion network (SAMF-Net) is proposed for photoacoustic reconstruction. The algorithm accepts two inputs, namely the original photoacoustic signal and the traditional reconstructed image. Furthermore, a global feature extraction module based on the self-attention mechanism is employed to focus on the global information. The results demonstrate that the proposed method exhibits superior reconstruction capability under different sparse detection views. The method has instructive value for photoacoustic image reconstruction and has the potential for further application in the diagnosis of diabetic retinopathy.

The biological characteristics of long cell-free DNA in spent embryos culture medium as noninvasive biomarker in in-vitro embryo selection.

An improved understanding of the cfDNA fragmentomics has proved it as a promising biomarker in clinical applications. However, biological characteristics of cfDNA in spent embryos culture medium (SECM) remain unsolved obstacles before the application in non-invasive in-vitro embryo selection. In this study, we developed a Tn5 transposase and ligase integrated dual-library construction sequencing strategy (TDual-Seq) and revealed the fragmentomic profile of cfDNA of all sizes in early embryonic development. The detected ratio of long cfDNA (>500 bp) was improved from 4.23 % by traditional NGS to 12.80 % by TDual-Seq. End motif analysis showed long cfDNA molecules have a more dominance of fragmentation intracellularly in apoptotic cells with higher predominance of G-end, while shorter cfDNA undergo fragmentation process both intracellularly and extracellularly. Moreover, the mutational pattern of cfDNA and the correlated GO biological process were well differentiated in cleavage and blastocyst embryos. Finally, we developed a multiparametric index (TQI) that employs the fragmentomic profiles of cfDNA, and achieved an area under the ROC curve of 0.927 in screening top quality embryos. TDual-Seq strategy has facilitated characterizing the fragmentomic profile of cfDNA of all sizes in SECM, which are served as a class of non-invasive biomarkers in the evaluation of embryo quality in in-vitro fertilization. And this improved strategy has opened up potential clinical utilities of long cfDNA analysis.

Agarose amplification based sequencing characterization cell-free RNA in preimplantation spent embryo medium.

BACKGROUND: The cell-free RNA (cf-RNA) of spent embryo medium (SEM) has aroused a concern of academic and clinical researchers for its potential use in non-invasive embryo screening. However, comprehensive characterization of cf-RNA from SEM still presents significant technical challenges, primarily due to the limited volume of SEM. Hence, there is urgently need to a small input liquid volume and ultralow amount of cf-RNA library preparation method to unbiased cf-RNA sequencing from SEM. (75) RESULT: Here, we report a high sensitivity agarose amplification-based cf-RNA sequencing method (SEM-Acf) for human preimplantation SEM cf-RNA analysis. It is a cf-RNA sequencing library preparation method by adding agarose amplification. The agarose amplification sensitivity (0.005 pg) and efficiency (105.35 %) were increased than that of without agarose addition (0.45 pg and 96.06 %) by âˆ¼ 90 fold and 9.29 %, respectively. Compared with SMART sequencing (SMART-seq), the correlation of gene expression was stronger in different SEM samples by using SEM-Acf. The cf-RNA number of detected and coverage uniformity of 3' end were significantly increased. The proportion of 5' end adenine, alternative splicing events and short fragments (<400 bp) were increased. It is also found that 4-mer end motifs of cf-RNA fragments was significantly differences between different embryonic stage by day3 spent cleavage medium and day5/6 spent blastocyst medium. (141) SIGNIFICANCE: This study established an efficient SEM amplification and library preparation method. Additionally, we successfully described the characterizations of SEM cf-RNA in preimplantation embryo using SEM-Acf, including expression features and fragment lengths. SEM-Acf facilitates the exploration of cf-RNA as a noninvasive embryo screening biomarker, and opens up potential clinical utilities of small input liquid volume and ultralow amount cf-RNA sequencing. (59).

The synergy of morphokinetic parameters and sHLA-G in cleavage embryo enhancing implantation rates.

Objective: This study aimed to assess the relationship between implantation and soluble HLA-G (sHLA-G) expression in cleavage embryo culture medium (ECM) in conjunction with early developmental kinetics determined by time-lapse imaging (TLI). Methods: A retrospective, single-center study was conducted involving 238 embryos from 165 patients who underwent Frozen-thawed embryo transfer (FET) using autologous oocytes, with either single or double embryo transfer. TLI morphokinetic parameters (t2, t3, t4, t5, t6, t7, t8, cc2, s2, cc3, s3) of embryos were analyzed, and sHLA-G levels in D3 ECM were measured using an enzyme-linked immunosorbent assay (ELISA). A hierarchical classification model was developed to categorize embryos into five groups (A, B, C, D, E). The correlation between sHLA-G levels, TLI classification of embryos, and embryo implantation was investigated to establish a non-invasive method for evaluating implantation potential. Multivariate logistic regression analysis was performed to identify potential influencing factors, and receiver operating characteristic (ROC) curves were used to evaluate the predictive value for implantation. Results: Multivariate unconditional logistic regression analysis indicated that TLI parameters t5 and s3 and sHLA-G level in ECM were independent risk factors affecting embryo implantation. The implantation rate decreased from TLI classification A to E. The proposed classification model effectively assessed the implantation potential of embryos. The implantation rate was higher in the sHLA-G positive group compared to the sHLA-G negative group (p < 0.001). The expression of sHLA-G in D3 ECM, combined with the TLI classification model, accurately evaluated the implantation potential of embryos with an AUC of 0.876. Conclusion: The integration of cleavage kinetics and embryonic sHLA-G expression could reliably identify embryos with a high likelihood of successful implantation.

Normal human embryonic stem cell lines were derived from microsurgical enucleated tripronuclear zygotes.

A normal fertilized human zygote contains two pronuclei, but zygotes may also display one, three, or even more pronuclei resulting from irregular insemination or meiotic division. Today diploid and triploid human embryonic stem cell (hESC) lines have been derived from tripronuclear (3PN) triploid zygotes, and an in-vitro fertilization (IVF) baby was born from a rescued diploid zygote by removing the extra male pronucleus of the 3PN zygote. However, whether hESCs can be derived from a rescued 3PN zygote is still unknown. Here, by microsurgical pronuclear removal, we restored 61 diploid zygotes from 3PN zygotes donated by 35 couples, and 11 blastocysts developed with a blastocyst rate of 18.0%, which seems higher than that of nonrescued 3PN zygotes according to previous reports. After the whole zona pellucida free embryos were plated onto feeder cells to grow and passage, 2 hESC lines (CCRM-hESC-22 and CCRM-hESC-23) were generated and both carried normal karyotype (46, XY). The hESC lines were then characterized by morphology, expansion in vitro, and expression of specific markers of alkaline phosphatase, OCT4, SSEA4, TRA-1-60 and TRA-1-81. Furthermore, the pluripotency of these 2 hESC lines was confirmed by in vitro embryoid body formation and in vivo teratoma production. Our study indicates that depronucleared 3PN zygotes can improve the blastocysts formation rate, and normal hESC lines can be derived from those corrected 2PN embryos. Based on their multi-directional differentiation potential in vitro, the established hESC lines could be applied to the developmental risk assessment for IVF babies born from restored zygotes.