Maternal inheritance of glucose intolerance via oocyte TET3 insufficiency.

Diabetes mellitus is prevalent among women of reproductive age, and many women are left undiagnosed or untreated1. Gestational diabetes has profound and enduring effects on the long-term health of the offspring2,3. However, the link between pregestational diabetes and disease risk into adulthood in the next generation has not been sufficiently investigated. Here we show that pregestational hyperglycaemia renders the offspring more vulnerable to glucose intolerance. The expression of TET3 dioxygenase, responsible for 5-methylcytosine oxidation and DNA demethylation in the zygote4, is reduced in oocytes from a mouse model of hyperglycaemia (HG mice) and humans with diabetes. Insufficient demethylation by oocyte TET3 contributes to hypermethylation at the paternal alleles of several insulin secretion genes, including the glucokinase gene (Gck), that persists from zygote to adult, promoting impaired glucose homeostasis largely owing to the defect in glucose-stimulated insulin secretion. Consistent with these findings, mouse progenies derived from the oocytes of maternal heterozygous and homozygous Tet3 deletion display glucose intolerance and epigenetic abnormalities similar to those from the oocytes of HG mice. Moreover, the expression of exogenous Tet3 mRNA in oocytes from HG mice ameliorates the maternal effect in offspring. Thus, our observations suggest an environment-sensitive window in oocyte development that confers predisposition to glucose intolerance in the next generation through TET3 insufficiency rather than through a direct perturbation of the oocyte epigenome. This finding suggests a potential benefit of pre-conception interventions in mothers to protect the health of offspring.

Overweight and obesity determined by body mass index criteria for Asian populations adversely affect assisted reproductive outcomes among Chinese women with polycystic ovary syndrome.

BACKGROUND: Obesity is a common feature in women with polycystic ovary syndrome (PCOS) and is associated with multiple adverse reproductive outcomes. However, the impact of overweight and obesity on reproductive outcomes of women with PCOS who underwent in vitro fertilization-embryo transfer (IVF-ET) is currently controversial and appropriate body mass index (BMI) levels differ across ethnic groups. METHODS: This was a retrospective study including 1066 women with PCOS receiving IVF treatment at our institution between January 2018 and June 2021, among whom 960 underwent their first fresh or frozen embryo transfer. Participants were categorized according to BMI cut-off values proposed by the World Health Organization for Asian populations: normal weight (BMI < 23 kg/m2), overweight (BMI: 23-24.9 kg/m2), and obesity (BMI ≥ 25 kg/m2). The effect of BMI on clinical and embryological outcomes was evaluated by descriptive statistics and logistic regression models with confounders adjusted. The dose-response relationship between BMI as a continuous variable and IVF outcomes is also explored. INTERVENTIONS: no RESULTS: Increasing BMI was associated with significantly lower numbers of total oocytes retrieved, metaphase II oocytes, two pronuclear (2PN) zygotes, and good-quality embryos among women with PCOS. Patients with PCOS with a BMI ≥ 23 kg/m2 had significantly lower live birth rates (41.9% vs. 49.1%; adjusted odds ratio [aOR], 0.75; 95% confidence interval [CI], 0.57-0.97) and implantation rates (35.8% vs. 43.9%; aOR, 0.76; 95% CI, 0.61-0.93) than those with normal BMI. Moreover, BMI showed a non-linear relationship (p for nonlinearity <0.001) with the number of 2PN zygotes with the curve becoming steeper as BMI surpassed 22.4 kg/m2. CONCLUSIONS: Patients with PCOS with a BMI ≥ 23 kg/m2 have lower live birth rates than those with a BMI < 23 kg/m2. Defining obesity and overweight with ethnicity-specific BMI cut-offs may help to improve IVF outcomes among PCOS patients.

Sperm-borne microRNA-34c regulates maternal mRNA degradation and preimplantation embryonic development in mice.

BACKGROUND: Studies have shown that sperm-borne microRNAs (miRNAs) are involved in mammalian preimplantation embryonic development. In humans, spermatozoan miR-34c levels are correlated with in vitro fertilization outcomes, such as embryo quality and the clinical pregnancy and live birth rates. In rabbits and cows, miR-34c improves the developmental competence of embryos generated by somatic cell nuclear transfer. However, the mechanisms underlying the regulation of embryonic development by miR-34c remain unknown. METHODS: Female C57BL/6 mice (6-8 weeks old) were superovulated, and pronucleated zygotes were collected and microinjected with an miR-34c inhibitor or a negative-control RNA. The embryonic development of the microinjected zygotes was evaluated, and the messenger RNA (mRNA) expression profiles of the embryos at the two-cell, four-cell and blastocyst stages (five embryos per group) were determined by RNA sequencing analysis. Gene expression levels were verified by reverse transcription-quantitative polymerase chain reaction. Cluster analysis and heat map visualization were performed to detect differentially expressed mRNAs. Pathway and process enrichment analyses were performed using ontology resources. Differentially expressed mRNAs were systematically analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins database to determine their biological functions. RESULTS: Embryonic developmental potential was significantly reduced in zygotes microinjected with the miR-34c inhibitor compared with those microinjected with a negative-control RNA. Two-cell stage embryos microinjected with an miR-34c inhibitor presented altered transcriptomic profiles, with upregulated expression of maternal miR-34c target mRNAs and classical maternal mRNAs. Differentially expressed transcripts were mainly of genes associated with lipid metabolism and cellular membrane function at the two-cell stage, with cell-cycle phase transition and energy metabolism at the four-cell stage; and with vesicle organization, lipid biosynthetic process and endomembrane system organization at the blastocyst stage. We also showed that genes related to preimplantation embryonic development, including Alkbh4, Sp1, Mapk14, Sin3a, Sdc1 and Laptm4b, were significantly downregulated after microinjection of an miR-34c inhibitor. CONCLUSIONS: Sperm-borne miR-34c may regulate preimplantation embryonic development by affecting multiple biological processes, such as maternal mRNA degradation, cellular metabolism, cell proliferation and blastocyst implantation. Our data demonstrate the importance of sperm-derived miRNAs in the development of preimplantation embryos.

Inhibition of USP10 induces myeloma cell apoptosis by promoting cyclin D3 degradation.

The cell cycle regulator cyclin D3 (CCND3) is highly expressed in multiple myeloma (MM) and it promotes MM cell proliferation. After a certain phase of cell cycle, CCND3 is rapidly degraded, which is essential for the strict control of MM cell cycle progress and proliferation. In the present study, we investigated the molecular mechanisms regulating CCND3 degradation in MM cells. By utilizing affinity purification-coupled tandem mass spectrometry, we identified the deubiquitinase USP10 interacting with CCND3 in human MM OPM2 and KMS11 cell lines. Furthermore, USP10 specifically prevented CCND3 from K48-linked polyubiquitination and proteasomal degradation, therefore enhancing its activity. We demonstrated that the N-terminal domain (aa. 1-205) of USP10 was dispensable for binding to and deubiquitinating CCND3. Although Thr283 was important for CCND3 activity, it was dispensable for CCND3 ubiquitination and stability modulated by USP10. By stabilizing CCND3, USP10 activated the CCND3/CDK4/6 signaling pathway, phosphorylated Rb, and upregulated CDK4, CDK6 and E2F-1 in OPM2 and KMS11 cells. Consistent with these findings, inhibition of USP10 by Spautin-1 resulted in accumulation of CCND3 with K48-linked polyubiquitination and degradation that synergized with Palbociclib, a CDK4/6 inhibitor, to induce MM cell apoptosis. In nude mice bearing myeloma xenografts with OPM2 and KMS11 cells, combined administration of Spautin-l and Palbociclib almost suppressed tumor growth within 30 days. This study thus identifies USP10 as the first deubiquitinase of CCND3 and also finds that targeting the USP10/CCND3/CDK4/6 axis may be a novel modality for the treatment of myeloma.

A New Hemoglobin Variant: Hb Tangshan [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)] Detected by MALDI-TOF MS.

In this report we decribed a new α-chain variant found during the measurement of hemoglobin A1c (Hb A1c) using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). MALDI-TOF MS analysis detected an α-chain variant with a mass of 15,155 Da. However, this Hb variant was not detected during Hb A1c measurement by cation-exchange high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) methods. Sanger sequencing validated the presence of a heterozygous missense mutation [HBA1: c.239C > T, CD79(GCG > GTG)(Ala > Val)]. The observed 28 Da mass difference exactly matches the theoretical mass difference (28 Da) resulting from the substitution of alanine (89.079) with valine (117.133). As this represents the initial documentation of the mutation, we named it Hb Tangshan after the proband's residence.

Long-term cryopreservation and frozen embryo transfer do not impact clinical and neonatal outcomes: a retrospective cohort study of slow-frozen early-cleavage human embryos.

This study aimed to evaluate the effect of the cryopreservation duration (up to 160 months) on the clinical and neonatal outcomes of slow-frozen early-cleavage human embryos. Clinical data collected between February 2013 and August 2017 were included in this retrospective study. Cases were classified into five groups by the duration of cryopreservation: Group 1, 6-12 months; Group 2, 13-36 months; Group 3, 37-60 months; Group 4, 61-84 months; and Group 5, >84 months. The embryo survival rate, implantation rate, clinical pregnancy rate, live-birth rate, newborn sex ratio, singleton gestational age, singleton birth weight and malformation rate were compared between the groups. The cryopreservation duration did not significantly affect the rates of clinical pregnancy (P = 0.119) and live birth (P = 0.354), the newborn sex ratio (P = 0.614) or the singleton gestational age (P = 0.212) and birthweight (P = 0.212). Although decreases in the embryo survival and implantation rates were observed in groups 4 and 5 compared with those in groups 1-3, these differences were not statistically significant (P = 0.329, P = 0.279, respectively). Long-term cryopreservation does not appear to adversely affect the clinical and neonatal outcomes of slow-frozen early-cleavage human embryos.

Preimplantation genetic diagnosis and screening (PGD/S) using a semiconductor sequencing platform.

BACKGROUND: Recent advances in semiconductor sequencing platform (SSP) have provided new methods for preimplantation genetic diagnosis/screening (PGD/S). The present study aimed to evaluate the applicability and efficiency of SSP in PGD/S. METHODS: The artificial positive single-cell-like DNAs and normal single-cell samples were chosen to test our semiconductor sequencing platform for preimplantation genetic diagnosis/screening (SSP-PGD/S) method with two widely used whole-genome amplification (WGA) kits. A total of 557 single blastomeres were collected from in vitro fertilization (IVF) couples, and their WGA products were processed and analyzed by our SSP-PGD/S method in comparison with array comparative genomic hybridization (array-CGH). RESULTS: Our SSP-PGD/S method indicated high compatibilities with two commercial WGA kits. For 557 single blastomeres, our method with four million reads in average could detect 24-chromosome aneuploidies as well as microdeletion/microduplication of the size over 4 Mb, providing 100% consistent conclusion with array-CGH method in the classification of whether it was transplantable. CONCLUSIONS: Our studies suggested that SSP-PGD/S represents a valuable alternative to array-CGH and brought PGD/S into a new era of more rapid, accurate, and economic.

Serum and follicular fluid fetuin-B levels are correlated with fertilization rates in conventional IVF cycles.

PURPOSE: To investigate the relationship between serum/follicular fluid fetuin-B levels and fertilization outcomes in conventional IVF cycles. METHODS: A prospective cohort study of conventional IVF treatments including 78 cycles with low fertilization rates (two pronuclei [2PN] rate < 30%; LF group) and 104 cycles performed during the same period with 2PN rate > 70% (high fertilization group, HF). To calculate the required sample size, a two-sample t test power analysis was applied to data from our pilot study, using PASS 11.0 software. Fetuin-B was measured using a commercial sandwich enzyme-linked immunosorbent assay. RESULTS: Serum fetuin-B and follicular fluid fetuin-B were positively correlated (r = 0.703, P < 0.001). Compared to the HF group, the LF group had significantly lower levels of fetuin-B, both in serum (5.81 ± 1.53 vs. 7.19 ± 1.42, P < 0.001) and follicular fluid (5.06 ± 1.29 vs. 6.16 ± 1.52, P < 0.001). The serum fetuin-B level from cycles with polypronuclear (PPN) zygotes was significantly lower when compared to cycles without PPN zygotes (6.82 ± 1.65 vs. 6.10 ± 1.43, P = 0.006). However, serum fetuin-B level was not correlated with preimplantation embryo development or clinical pregnancy. CONCLUSION: Serum fetuin-B level is correlated with fertilization rate in conventional IVF and it may be used as a predictive marker of fertilization in IVF treatment.

Endothelin-1 promotes human germinal vesicle-stage oocyte maturation by downregulating connexin-26 expression in cumulus cells.

STUDY QUESTION: Does endothelin-1 (ET-1) promote human oocyte maturation and by what mechanism? SUMMARY ANSWER: Addition of ET-1 to the medium in which human germinal vesicle (GV)-stage immature oocytes are cultured enhances the GV breakdown (GVBD) rate; the resumption of meiosis may be initiated by ET-1 downregulating the expression of connexin-26 (Cx26) in cumulus cells via endothelin receptor type B (ETRB), leading to decreased cAMP levels in the oocyte. WHAT IS KNOWN ALREADY: The paracrine factor ET-1 is secreted by ovarian somatic cells in pre-ovulatory follicles and regulates oocyte maturation in mice. Connexins, or gap junction proteins, form intercellular membrane channels that play important roles in the resumption of meiosis. STUDY DESIGN, SIZE, DURATION: This laboratory study was conducted over a 1-year period. The effects of ET-1 on meiotic resumption were evaluated in human GV-stage cumulus-oocyte complexes (COCs; 70 oocytes/group). The transcriptome profiles of ET-1-treated or untreated cumulus cells were compared to explore the possible mechanisms by which ET-1 may regulate oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ET-1, ETRA and ETRB expression levels in human cumulus cells from oocytes at different stages of maturation were evaluated using real-time quantitative PCR. Human GV-stage COCs collected from patients undergoing IVF at a university-affiliated infertility centre were cultured with or without ET-1, and cumulus cells were subsequently denuded using hyaluronidase and cultured in α-MEM. A GeneChip® Human Transcriptome Array was applied to explore differences in the whole-genome transcriptome profiles of cumulus cells treated with or without ET-1. Real-time quantitative PCR and Western blotting were used respectively to examine Cx26 mRNA and protein levels in cumulus cells. Changes in cAMP levels in both oocytes and cumulus cells after ET-1 treatment were measured using an enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: Cumulus cells from MII-stage oocytes exhibited upregulated ET-1 expression, compared to those from GV-stage oocytes. The addition of ET-1 to the culture medium enhanced the GVBD rate of cumulus cell-enclosed human oocytes. Whole-genome transcriptome microarray analyses revealed significantly downregulated Cx26 expression in cumulus cells after ET-1 treatment, and this action was blocked by an ETRB antagonist. The involvement of Cx26 was further supported by the finding that ET-1 treatment led to decreased cAMP levels in oocytes but increased cAMP levels in cumulus cells. LARGE SCALE DATA: Microarray data are published in the GEO database (GSE97684). LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of human COCs collected from patients undergoing IVF might affect the maturation results in vitro. Although we focused on the effects of ET-1 on human oocyte maturation in the present study, mammalian oocyte maturation is a complicated process involving many endocrine and paracrine factors. WIDER IMPLICATIONS OF THE FINDINGS: Our present study suggests that in vitro, human GV-stage oocyte maturation could be enhanced by adding ET-1 to the culture medium. In the present study, we explored the molecular mechanisms by which ET-1 initiates the resumption of meiosis and demonstrated that ET-1 promotes oocyte maturation by downregulating the expression of the gap junction protein Cx26 in cumulus cells. These results expand our understanding of the molecular mechanisms underlying mammalian oocyte maturation and provide a basis for better in-vitro maturation strategies. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by grants from the China Natural Science Foundation (Grant Nos. 81170567 and 81370761). The authors declare that they have no conflicts of interest associated with this manuscript.

[Effect of oxygen concentration on outcome of in-vitro fertilization-embryo transfer].

OBJECTIVE: To investigate the effect of low and high oxygen concentration on embryo development, pregnancy outcome and birth defects of in vitro fertilization-embryo transfer (IVF-ET). METHODS: According to the oxygen concentration of in vitro culture environment, the IVF-ET performed in the Women's Hospital, Zhejiang University School of Medicine during 2013 and 2015 were divided into low oxygen concentration group (n=2036, 5% O2) and high oxygen concentration group (n=4617, 20% O2). The rate of fertilization, good quality embryo, clinical pregnancy, ectopic pregnancy, abortion and birth defect were compared between two groups. RESULTS: The good quality embryo rate was significantly higher in the low oxygen concentration group (P<0.05). However, no significant differences were found between two groups in the rate of fertilization, clinical pregnancy, ectopic pregnancy, abortion and birth defect (all P>0.05). CONCLUSIONS: Low oxygen environment may improve the potential of embryonic development, but its impact on pregnancy outcome and birth defect is not significant.

[Effects of embryo cryopreservation and thawing on clinical outcomes of transplantable embryos after cleavage-stage preimplantation genetic diagnosis or screening].

OBJECTIVE: To investigate the effects of embryo cryopreservation and thawing on clinical outcomes of transplantable embryos after preimplantation genetic diagnosis (PGD) or preimplantation genetic screening (PGS) in cleavage-stage. METHODS: The clinical data of 302 cases (including 118 cases using frozen/thawing embryos and 184 cases using fresh embryos) undergoing PGD/PGS in Women's Hospital, Zhejiang University School of Medicine during January 2011 and December 2016 were retrospectively analyzed. The pregnancy rate, implantation rate, live birth rate and abortion rate of fresh and frozen-thawed embryo transfer (FET) cycles were compared. And the influencing factors for pregnancy outcome was analyzed by multivariate logistic regression. RESULTS: The rate of normal or balanced translocation embryos in fresh cycle was higher than that in FET cycle (23.52% vs 16.67%, P<0.05), and the average number of transplanted embryos was more than that in FET cycle (1.54±0.56 vs 1.33±0.51, P<0.05). But there were no significant differences in pregnancy rate (36.42% vs 40.00%, P>0.05), implantation rate (26.62% vs 32.91%, P>0.05), abortion rate (19.44% vs 8.33%, P>0.05) and live birth rate (25.96% vs 28.33%, P>0.05) between fresh cycle and FET cycle. Multivariate logistic regression showed that, parent ages, embryo status (fresh or frozen), the mode of PGD/PGS and the findings of PGD/PGS had no impact on pregnancy outcome (all P>0.05). CONCLUSIONS: Cryopreservation do not have significant effects on the clinical outcomes of transplantable embryos after PGD/PGS in cleavage-stage.

[Correlation of fetuin-B concentrations in serum and follicular fluid with outcomes of in vitro fertilization].

OBJECTIVE: To investigate the relationship between serum/follicular fluid fetuin B levels and outcome of in vitro fertilization (IVF). METHODS: Infertility women (28 with low fertilization rates, 44 with normal fertilization rates) receiving IVF in Women's Hospital of Zhejiang University School of Medicine during June and December 2016 were enrolled in the study. Serum/follicular fluid fetuin B levels were measured with ELISA method. Correlations of serum and follicular fetuin B level with fertilization outcome of IVF were analyzed with Pearson correlation coefficient and receiver operating characteristic (ROC) curve. RESULTS: A positive correlation between serum fetuin B and follicular fluid fetuin B levels was observed (r=0.675, P<0.01). Both serum and follicular fluid fetuin B levels in women with low fertilization rates of IVF were lower than those in women with normal fertilization rates[(6.09±1.31) µg/mL vs. (7.13±1.47) µg/mL, t=3.050, P<0.05; (5.13±0.96)µg/mL vs. (6.22±1.33) µg/mL, t=3.755, P<0.01]. ROC analysis showed that the area under curve (AUC) of serum fetuin B level in predicting fertilization rate was 0.832 (95% CI:0.729-0.934, P<0.01), and 6.08 µg/mL could be used as cut-off value. CONCLUSIONS: Serum fetuin B level is correlated with follicular fluid fetuin B level, and it may be used for predicting the fertilization outcome of IVF.

Clinical evaluation of two formulations of slow-freezing solutions for cleavage stage embryos.

PURPOSE: The aim was to investigate if improved survival rates could be achieved using a new formulation of solutions for slow freezing of human cleavage stage embryos. METHODS: The evaluation was divided into two parts. The first part was a retrospective analysis of results obtained after freezing and thawing of day 3 embryos from 400 women using an old formulation of cryopreservation solutions compared to results from 108 women for which cryopreservation had been performed using new compositions of solutions. The second part was prospective, adding cycles until similar numbers of patients had been included in both groups. In total, 2274 embryos from 897 patients were thawed using the old formulation of solutions while 1273 embryos from 542 patients were frozen and thawed using the new solutions. The primary endpoint was survival rate. RESULTS: With the new solutions, the survival rate increased from 82.1 to 94.4 % and the complete embryo survival rate increased from 54.9 to 81.3 %. The implantation rate, clinical pregnancy rate per embryo transfer, and per cycle were 28.2, 45.2, and 43.7 %, respectively, using the old formulations of cryosolutions. With the new solutions, the results reached 33.7, 54.1, and 54.1 %, respectively. All differences in results were statistically significant. The number of cancelled embryo transfers due to no survived embryos was 18 with the old solutions and 0 using the new solutions. CONCLUSION: With the new composition of solutions for slow freezing and thawing of embryos, significantly improved results were obtained. Additionally, the number of cancelled embryo transfers was reduced.

Preimplantational genetic diagnosis and mutation detection in a family with duplication mutation of DMD gene.

Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disease caused by mutation in the DMD gene. A 38-year-old woman was referred to our hospital with her son who was diagnosed with DMD. Multiplex PCR failed to detect DMD mutations in the affected child. The female carrier underwent preimplantation genetic diagnosis by linkage analysis and gender determination. Eight embryos were biopsied after in vitro fertilization. Two healthy embryos determined both as females (E1 and E3) were transferred. Although the paternal allele was absent in E3, it was considered to be a result of allele dropout for the STR-49 marker. Surprisingly, a female and a male baby were delivered at 38 gestational weeks, suggesting that E3 was a male embryo with the allele dropout occurring at the SRY gene. Exon 2 duplication was detected in the DMD patient and the carrier mother using next-generation sequencing and multiple ligation-dependent probe amplification. Next, we verified the duplication of exon 2 by real-time PCR, using a special primer at 3' of intron 1, very close to exon 2. Finally, we confirmed that both newborns inherited the normal allele, using quantitative real-time PCR and linkage analysis.

Identification of Hb Lepore, Hb anti-Lepore, and α-globin gene triplications by long-read single-molecule real-time sequencing.

OBJECTIVES: Hemoglobin (Hb) Lepore and Hb anti-Lepore are infrequent fusion gene variants that result from nonhomologous crossovers during meiosis. Conventional molecular testing methods may face challenges in identifying these variants. During Hb analysis using capillary electrophoresis, we encountered 6 cases with unusual Hb variants. Our aim was to identify the alterations in their globin genes. METHODS: Gap-polymerase chain reaction (PCR), reverse dot-blot assay (RDB), Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and long-read single-molecule real-time (SMRT) sequencing were used to confirm the presence of globin gene alterations. RESULTS: The routine thalassemia gene test kit using the gap-PCR and RDB techniques did not detect common gene variations. Direct sequencing failed to identify any known or unknown globin gene alterations. The MLPA analysis, however, revealed the possible presence of α-globin gene triplications as well as 2 types of fusion gene alterations. Further analysis using long-read SMRT sequencing accurately identified 3 rare gene variations: αααanti-3.7, Hb Lepore-Boston-Washington, and Hb anti-Lepore P-India. CONCLUSIONS: Conventional methods may overlook rare thalassemias or Hb variants. Long-read SMRT sequencing has the potential to identify breakpoints in fusion genes, demonstrating that it is a promising technique for detecting rare thalassemias.

Prevalence of monoclonal gammopathy of undetermined significance in Shenzhen, China.

BACKGROUND: Data on the prevalence of monoclonal gammopathy of undetermined significance (MGUS) in China are very limited. Our aim was to determine the prevalence and clinical characteristics of MGUS in a large Chinese population. METHODS: This study included 49,220 healthy people who received serum immunofixation electrophoresis (sIFE) and serum protein electrophoresis (SPE) tests. Serum free light chain ratio, immunoglobulin quantification, and other clinically correlates of MGUS were performed for all patients with M-protein. RESULTS: A total of 576 MGUS patients were identified by sIFE, with a median age of 58 years and an overall prevalence of 1.17% (95% CI, 1.08-1.27). Among those aged 50 years and older, the prevalence of MGUS was 2.26% (95% CI, 2.04-2.50). The prevalence of MGUS was significantly higher in males than in females (P < 0.05). The median concentration of M-protein was 3.1 g/L, ranging from 0.5 g/L to 25.1 g/L. The M-protein type was IgG in 55.4% of MGUS patients, followed by IgA (31.1%), IgM (9.5%), IgD (0.5%), biclonal (2.3%), and light chain (1.2%). Abnormalities in SPE, FLC ratios, and immunoglobulin levels were observed in 78.3%, 31.1%, and 38.4% of MGUS patients, respectively. CONCLUSIONS: The prevalence of MGUS is substantially lower in southern China than in whites and blacks.

Identification of tumor heterogeneity associated with KRAS/TP53 co-mutation status in lung adenocarcinoma based on single-cell RNA sequencing.

Lung cancer stands as the predominant cause of cancer-related mortality globally. Lung adenocarcinoma (LUAD), being the most prevalent subtype, garners extensive attention due to its notable heterogeneity, which significantly influences tumor development and treatment approaches. This research leverages single-cell RNA sequencing (scRNA-seq) datasets to delve into the impact of KRAS/TP53 co-mutation status on LUAD. Moreover, utilizing the TCGA-LUAD dataset, we formulated a novel predictive risk model, comprising seven prognostic genes, through LASSO regression, and subjected it to both internal and external validation sets. The study underscores the profound impact of KRAS/TP53 co-mutational status on the tumor microenvironment (TME) of LUAD. Crucially, KRAS/TP53 co-mutation markedly influences the extent of B cell infiltration and various immune-related pathways within the TME. The newly developed predictive risk model exhibited robust performance across both internal and external validation sets, establishing itself as a viable independent prognostic factor. Additionally, in vitro experiments indicate that MELTF and PLEK2 can modulate the invasion and proliferation of human non-small cell lung cancer cells. In conclusion, we elucidated that KRAS/TP53 co-mutations may modulate TME and patient prognosis by orchestrating B cells and affiliated pathways. Furthermore, we spotlight that MELTF and PLEK2 not only function as prognostic indicators for LUAD, but also lay the foundation for the exploration of innovative therapeutic approaches.

Expression of aquaporins in human embryos and potential role of AQP3 and AQP7 in preimplantation mouse embryo development.

BACKGROUND/AIMS: Water channels, also named aquaporins (AQPs), play crucial roles in cellular water homeostasis. METHODS: RT-PCR indicated the mRNA expression of AQPs 1-5, 7, 9, and 11-12, but not AQPs 0, 6, 8, and 10 in the 2∼8-cell stage human embryos. AQP3 and AQP7 were further analyzed for their mRNA expression and protein expression in the oocyte, zygote, 2-cell embryo, 4-cell embryo, 8-cell embryo, morula, and blastocyst from both human and mouse using RT-PCR and immunofluorescence, respectively. RESULTS: AQP3 and AQP7 were detected in all these stages. Knockdown of either AQP3 or AQP7 by targeted siRNA injection into 2-cell mouse embryos significantly inhibited preimplantation embryo development. However, knockdown of AQP3 in JAr spheroid did not affect its attachment to Ishikawa cells. CONCLUSION: These data demonstrate that multiple aquaporins are expressed in the early stage human embryos and that AQP3 and AQP7 may play a role in preimplantation mouse embryo development.

[Analysis on nutritional component of Cervus elaphus products].

OBJECTIVE: To investigate nutrient values in the Cervus elaphus, so as to provide a scientific basis for choosing products by businesses and consumers. METHODS: According to the national standards required for various nutritional elements, the amount of protein, energy, fat, water, ash, amino acids, minerals, vitamin in different parts of the Cervus elaphus (venison, tail, penis cervi, sinew) were determined, carbohydrate and energy were calculated, and were compared with Cervus nippon. RESULTS: Nutritive characteristics of Cervus elaphus and Cervus nippon were similar. The respective portion of protion in Cervus elaphus venison, Cervus elaphus tail, Cervus elaphus penis cervi, Cervus elaphus sinew were 22.6, 70.7, 85.8 and 89.5 g/100 g; The respective Amino acid score portion in Cervus elaphus venison, Cervus elaphus tail, Cervus elaphus penis cervi, Cervus elaphus sinew were 97, 78, 45, 37. In all Cervus elaphus products, the highest contents of fat, cholesterol, calcium, potassium, iron, vitamin B2 and vitamin B12 were found in Cervus elaphus tail. The highest contents of protion and the lowest contents of potassium, iron and zinc were found in Cervus elaphus sinew. The INQ of protein, cholesterol, potassium, iron, zinc, vitamin B2 and vitamin B12 in Cervus elaphus venison were all above 2, and fat INQ was under 1. CONCLUSION: Cervus elaphus is a kind of high quality animal food which is rich in protein, potassium, iron, zinc, vitamin B2, vitamin B12 and essential amino acids, and low in fat.

Case report: A reciprocal translocation-free and pathogenic DUOX2 mutation-free embryo selected by complicated preimplantation genetic testing resulted in a healthy live birth.

Preimplantation genetic testing (PGT) is an effective approach to improve clinical outcomes and prevent transmission of genetic imbalances by selecting embryos free of disease-causing genes and chromosome abnormalities. In this study, PGT was performed for a challenging case in which a couple simultaneously carried a maternal subchromosomal reciprocal translocation (RecT) revealed by fluorescence in situ hybridization involving the chromosome X (ChrX) and heterozygous mutations in dual oxidase 2 (DUOX2). Carriers of RecT are at increased risk for infertility, recurrent miscarriages, or having affected children due to the unbalanced gametes produced. DUOX2 mutation results in congenital hypothyroidism. Pedigree haplotypes for DUOX2 was constructed after the mutations were verified by Sanger sequencing. Since male carriers of X-autosome translocations may exhibit infertility or other abnormalities, pedigree haplotype for chromosomal translocation was also constructed to identify embryo with RecT. Three blastocysts were obtained by in vitro fertilization and underwent trophectoderm biopsy, whole genomic amplification, and next-generation sequencing (NGS). A blastocyst lacking copy number variants and RecT but carrying the paternal gene mutation in DUOX2, c.2654G>T (p.R885L) was used for embryo transfer, resulting in a healthy female infant whose genetic properties were confirmed by amniocentesis. Cases containing RecT and single gene disorder are rare. And the situation is more complicated when the subchromosomal RecT involving ChrX cannot be identified with routine karyotype analysis. This case report contributes significantly to the literature and the results have shown that the NGS-based PGT strategy may be broadly useful for complex pedigrees.