The gut microbial composition in polycystic ovary syndrome with hyperandrogenemia and its association with steroid hormones.

Background: Polycystic ovary syndrome (PCOS) is characterized by excess androgens, ovulatory dysfunction, and polycystic ovaries. The mechanisms underlying ovulatory and metabolic disorders in PCOS remain elusive, hampering therapeutic development. Enhanced metabolic health correlates with increased microbiota gene content and microbial diversity. We aimed to explore the impact of gut microbiota and serum steroids on PCOS regulation associated with androgen excess. Methods: The fecal samples of patients with hyperandrogenic PCOS (n = 14) and control group with PCOS (n = 14) were analyzed by 16S rRNA gene sequencing. The peripheral venous blood of all subjects was collected to detect serum hormones. The association between gut microbiota and serum hormones was analyzed with the R language. Results: Our findings reveal that the hyperandrogenic PCOS group exhibits lower richness and diversity of gut microbiota compared to the control group. Characteristic genera in PCOS patients with hyperandrogenism include Bifidobacterium, Enterobacteriaceae_unclassified, Streptococcus, Saccharimonadaceae, Enterococcus, and Eubacterium_nodatum_group. Five hormones, including 5ß-androsterone, deoxycorticosterone, corticosterone, 11-dehydrocorticosterone, and cortexolone, emerge as potential serum biomarkers for identifying patients with hyperandrogenic-PCOS (HA-PCOS). Furthermore, a lower vitamin D3 level may act as a susceptibility factor, suggesting that vitamin D3 supplementation could serve as a potential intervention for PCOS with hyperandrogenism. Conclusion: Specific fecal microbiota and serum steroids may be used as characteristic markers for clinical diagnosis of hyperandrogenic-PCOS. This research enhances our understanding of the intricate interplay among hormones, gut microbiota, and hyperandrogenemia in patients with PCOS.

Relationship of the levels of reactive oxygen species in the fertilization medium with the outcome of in vitro fertilization following brief incubation.

Embryo selection in in vitro fertilization-embryo transfer (IVF-ET) mostly relies on morphological assessment using a conventional microscope or the time-lapse monitoring system, which is not comprehensive. Inappropriate levels of reactive oxygen species (ROS) in the fertilization medium may cause damage to gametes, eventually leading to adverse IVF outcomes. The present study aimed to identify the optimal oxidation-reduction level in the fertilization medium for IVF outcomes by measuring the static oxidation-reduction potential (sORP) using a highly accurate and sensitive MiOXSYS system. A total of 136 patients undergoing IVF following brief incubation were divided equally into 4 groups in this prospective cohort study. The sORP value in the fertilization medium was detected using the MiOXSYS system, and its relationship with IVF outcomes was analyzed. The primary outcome was pregnancy outcomes, including live birth rate (LBR), clinical pregnancy rate (CPR), biochemical pregnancy rate (BPR), and implantation rate (IR). The secondary outcome was embryo quality, including fertilization rate (FR), cleavage rate (CR), available embryo rate (AER), and good-quality embryo rate (GQER). Group II (sORP: 228.7-235.3 mV) showed a higher LBR, CPR, BPR, and IR compared with Group III (sORP: 235.4-242.7 mV), presented as follows: LBR (32.0% for Group II vs 3.6% for Group III, P = 0.033), CPR (32.0% for Group II vs 3.6% for Group III, P = 0.033), BPR (36.0% for Group II vs 3.6% for Group III, P = 0.019), and IR (31.3% for Group II vs 2.7% for Group III, P = 0.003). The FR in Groups I and II had lower significant differences compared with that in Groups III and IV (71.7% and 70.3% for Groups I and II vs 83.5% and 80.4% for Groups III and IV, P = 0.000). The GQER in Group I to Group IV was 32.7%, 37.4%, 26.5%, and 33.3%, respectively (P = 0.056). This study indicated that the sORP value in the fertilization medium might be a potential indicator of embryo quality and pregnancy outcome.

[Clinical and genetic analysis of a Chinese pedigree affected with Dyggve-Melchior-Clausen syndrome due to a novel frameshift variant of DYM gene].

OBJECTIVE: To explore the genetic basis of a Chinese pedigree affected with Dyggve-Melchior-Clausen syndrome. METHODS: Whole exome sequencing and Sanger sequencing were carried out to detect potential pathogenic variants associated with the syndrome. The function of candidate variant was verified by Western blotting. RESULTS: A novel homozygous variant, c.1222delG of the DYM gene was detected in the two affected siblings, for which both parents were heterozygous carriers. The variant has caused replacement of Asp by Met at amino acid 408 and generate a premature stop codon p.Asp408Metfs*10. Western blotting confirmed that the variant can result in degradation of the mutant DYM protein, suggesting that it is a loss of function variant. CONCLUSION: The homozygous c.1222delG frameshift variant of the DYM probably underlay the Dyggve-Melchior-Clausen syndrome in the two affected siblings. Above findings has enabled clinical diagnosis and genetic counseling for the family.

A new perspective on semen quality of aged male: The characteristics of metabolomics and proteomics.

Background: Semen quality is negatively correlated with male age and is mainly quantified by a routine semen analysis, which is descriptive and inconclusive. Sperm proteins or semen metabolites are used as the intermediate or end-products, reflecting changes in semen quality, and hold much promise as a new biomarker to predict fertility in advanced-aged males. Objectives: In this study, we sought to assess whether the semen metabolome and proteome of aged males can affect semen quality and serve as biomarkers for predicting semen quality. Materials and methods: We retrospectively analyzed 12825 males that underwent semen routine analysis to understand the age-dependent changes in sperm quality. To identify the difference between aged and young adults, metabolomics (n=60) analyses of semen and proteomics (n=12) analyses of sperm were conducted. Finally, integrated machine learning of metabolomics was conducted to screen biomarkers to identify aging semen. Results: We discovered that male age was positively correlated with sperm concentration as well as DNA fragmentation index(DFI), and negatively with progressive motile sperm count, total sperm count, sperm volume and progressive sperm motility. The differential metabolites were significantly enriched in various metabolic pathways, and four of these differential metabolites (Pipamperone, 2,2-Bis(hydroxymethyl)-2,2',2''-nitrilotriethanol, Arg-Pro and Triethyl phosphate) were utilized to establish a biomarker panel to identify aging semen. Proteomic analysis showed that differential proteins were significantly enriched in protein digestion and absorption and some energy-related pathways. An integrated analysis of the metabolome and proteome identified differential energy metabolism and oxidative stress-related proteins, which could explain the decreased motility and the increased DFI of aging sperm. Discussion and conclusion: We provide compelling evidence that the changes in semen metabolome and sperm proteome are related to the decline of semen quality in aged males. Moreover, a biomarker panel based on four metabolites was established to identify aging semen.

Production of offspring by intracytoplasmic sperm injection using sperm from deceased transgenic mice at different postmortem intervals.

Successful fertilization by intracytoplasmic sperm injection (ICSI) is possible as long as the sperm genome is intact, even in the context of defective sperm or sustained adverse treatment. However, there are few reports on rescuing gene-modified mouse lines after accidental death. To investigate whether sperm from a dead transgenic mouse can fertilize an oocyte and enable embryo development into a pup, Nestin-GFP transgenic male mice were sacrificed, and sperm was collected 14 h, 24 h, and 48 h after death. The collected sperm was injected into oocytes from hybrid B6D2F1 or inbred C57BL/6 N mice. The results showed that the sperm in the three groups activated oocytes from B6D2F1 and supported embryo development to the blastocyst stage. For ICSI embryos derived from B6D2F1 mice, the cleavage and blastocyst rates were significantly lower in the three experimental groups than in the control group (0 h) (P < 0.05), and the birth rate in the 24 h and 48 h groups was significantly lower than that in the 14 h and control groups (0 h). For C57BL/6N-derived ICSI embryos, the cleavage rates were significantly lower at 24 h and 48 h than at 14 h and 0 h (control group), and the birth rate in the three experimental groups was significantly lower than that in the control group (0 h). The F0 mice derived from B6D2F1 and C57BL/6 N oocytes had normal reproductive ability, and F1 mice were successfully obtained. The characteristics of the GFP gene were preserved and inherited. The histone H2AX phosphorylation assay showed that the proportion of focus-negative embryos was markedly and significantly lower in the 14 h, 24 h, and 48 h groups than in the control group (0 h). The proportion of focus-negative embryos was significantly lower at 48 h than at 14 h or 24 h. The number of foci was significantly higher in the three experimental groups than in the control group (0 h), indicating that sperm DNA sustained more damage after death and that few sperm had an intact genome. In summary, sperm obtained from mice 14 h, 24 h, and 48 h after death is capable of activating an oocyte and supporting complete embryo development into a pup. This study provides an effective way to rescue accidently died mouse strains.

In Vivo Exon Replacement in the Mouse Atp7b Gene by the Cas9 System.

The application of CRISPR/Cas9 has opened a new era in gene therapy, making it possible to correct mutated genomes in vivo. Exon replacement can correct many mutations and has potential clinical value. In this study, we used a lentivirus-delivered transgene to obtain transgenic mice in which Cas9 and green fluorescent protein (GFP) were driven by the hTBG promoter and were specifically expressed in the liver. In Cas9-positive mice, only ∼11.6% of hepatocytes were GFP positive. The newborn Cas9-positive F1 mice were injected via the temporal vein with rAAV carrying a modified homologous replacement sequence for exon 8 of Atp7b and a pair of single-strand guide RNAs targeting the introns surrounding exon 8. When the Cas9-positive hepatocytes were sorted and analyzed by PCR and next-generation deep sequencing with different labels, ∼16.34 ± 4.02% to 19.37 ± 6.50% of the analyzed copies of exon 8 were replaced by the donor template in the genome of GFP-positive hepatocytes, that is, 1.81 ± 0.29% to 2.09 ± 0.54% replacement occurred in all liver genomes. However, when rAAV carrying a modified homologous replacement sequence was injected into the adult spCas9 mice, a double-cut deletion ratio of up to 99%, only about 1.10-1.13% of the exon 8 replacement rate was detected in Cas9-positive hepatocytes. This study is the first to achieve exon replacement via CRISPR/Cas9, which will benefit research on CRISPR/Cas9 technology for gene therapy.

Production of Wilson Disease Model Rabbits with Homology-Directed Precision Point Mutations in the ATP7B Gene Using the CRISPR/Cas9 System.

CRISPR/Cas9 has recently been developed as an efficient genome engineering tool. The rabbit is a suitable animal model for studies of metabolic diseases. In this study, we generated ATP7B site-directed point mutation rabbits to simulate a major mutation type in Asians (p. Arg778Leu) with Wilson disease (WD) by using the CRISPR/Cas9 system combined with single-strand DNA oligonucleotides (ssODNs). The efficiency of the precision point mutation was 52.94% when zygotes were injected 14 hours after HCG treatment and was significantly higher than that of zygotes injected 19 hours after HCG treatment (14.29%). The rabbits carrying the allele with mutant ATP7B died at approximately three months of age. Additionally, the copper content in the livers of rabbits at the onset of WD increased nine-fold, a level similar to the five-fold increase observed in humans with WD. Thus, the efficiency of precision point mutations increases when RNAs are injected into zygotes at earlier stages, and the ATP7B mutant rabbits are a potential model for human WD disease with applications in pathological analysis, clinical treatment and gene therapy research.