Background: In males with extremely severe oligospermia (MESO), single density gradient centrifugation (SDGC) has low sperm enrichment efficiency, making intracytoplasmic sperm injection (ICSI) challenging. This study aimed to determine whether double density gradient centrifugation (DDGC) can efficiently enrich sperm from MESO samples and whether these sperm are safe for clinical use. Methods: MESO was defined as having ≤2000 motile sperm/mL of semen, whereas males with severe oligospermia (MSO) were defined as having 2000–10,000 motile sperm/mL. We compared sperm recovery between SDGC and DDGC in MESO samples and retrospectively analyzed in vitro fertilization (IVF) data from 39 MESO cases (sperm prepared using DDGC) and 78 MSO cases (sperm prepared using SDGC) collected from 2017 to 2023. The SDGC group served as the control group. Results: The results showed that the sperm recovery rate of DDGC was approximately three-fold higher than that of SDGC in MESO samples. We hypothesized that in normal semen samples, sperm aggregate into a pellet during centrifugation, enabling efficient enrichment by SDGC. In MESO, where sperm count is extremely low, sperm fail to form a pellet, leading to slower sedimentation and lower recovery rates with SDGC, thereby necessitating additional centrifugation. Importantly, sperm prepared by DDGC from MESO semen samples showed comparable in vitro and in vivo embryo developmental parameters to sperm prepared by SDGC. Interestingly, the DDGC group showed a significantly higher usable blastocyst formation rate compared to SDGC group (73.48% vs. 62.63%, p = 0.009). Conclusions: In conclusion, DDGC can effectively enrich sperm from MESO samples, and no obvious adverse clinical outcomes were observed. The method is simple, requires no additional equipment, and may be considered as a routine sperm preparation technique for MESO in clinical use. However, the long-term safety of using DDGC for sperm preparation from MESO for ICSI still requires further confirmation, and more effective methods for sperm enrichment from MESO are needed.

The heterogeneity in health effects of circulating proteins remained unclear. Previous studies have identified that glycoprotein acetyls (GlycA), a stable biomarker of inflammation, were associated with risks of mortality and chronic diseases. However, it remained unclear whether the health risks of proteins may differ across people with varied GlycA levels. Based on the multi‐omics profiling of the UK Biobank prospective cohort, we evaluated whether GlycA statistically modifies the protein–mortality associations. In the discovery dataset ( n = 24,134), we observed that GlycA significantly modified the associations of ANG, CRHBP, CXCL16, and PRRT3 with all‐cause mortality, and these findings were replicated in the validation dataset ( n = 6081). Subgroup analyses further indicated that associations between these proteins and mortality can be modulated by GlycA levels. Through exploratory analyses focused on chronic diseases and cause‐specific mortality, we identified that cross‐products of GlycA with these proteins can be associated with cancer mortality and heart failure. Together, the findings will expand our understanding of heterogeneity in protein‐health associations and highlight several potential therapeutic targets for interventions across people with varied inflammation status.

Tris-EDTA buffer (pH 9.0) is widely used for heat-induced antigen retrieval (HIAR) in immunohistochemistry due to its high efficiency. However, it remains unclear whether Tris-EDTA is equally suitable for all tissues and for the detection of both nuclear and non-nuclear antigens. In this study, we systematically evaluated the effects of Tris-EDTA HIAR on mouse and human tissues. Nuclear morphology was assessed by DAPI and hematoxylin staining, while immunohistochemistry and immunofluorescence were used to detect nuclear and non-nuclear antigens. We found that Tris-EDTA HIAR caused selective nuclear disruption (nuclear content leakage) in thyroid, lung, and kidney tissues, manifested as weak or abnormal staining. Consequently, nuclear antigens in these tissues showed poor detection, including weak signals, mislocalization, or complete loss. In contrast, nuclear antigen detection was preserved when citrate buffer (pH 6.0) was used. For non-nuclear antigens, however, Tris-EDTA HIAR generally produced stronger staining than citrate buffer. Although nuclear content leakage may lead to slightly higher background, Tris-EDTA HIAR did not appear to damage the antigens themselves. In addition, both the pH and the antigen retrieval method influence the nuclear morphology in Tris-EDTA–treated sensitive tissues. In conclusion, Tris-EDTA HIAR should be used with caution for nuclear antigen detection in thyroid, lung, and kidney tissues but remains effective for non-nuclear antigens. These findings provide practical guidance for optimizing antigen retrieval strategies in both clinical diagnostics and research.

Background: This study aimed to compare the effects of different gamete co-incubation times on fertilization, embryo development potential, along with clinical outcomes. Methods: The study included 530 cycles with short co-incubation times (4–6 hours), referred to as the S group, and 1653 cycles with long co-incubation times (16–24 hours), referred to as the L group, all undergoing in vitro fertilization (IVF) without rescue intracytoplasmic sperm injection (R-ICSI). The study analyzed the basic characteristics and clinical outcomes of these two groups. Additionally, the study stratified the cycles based on the number of oocytes retrieved and performed multivariate logistic regression analysis with the normal fertilization (two pronuclei, 2PN) rate as the dependent variable and co-incubation time as the main independent variable. The study also examined 79 partial short co-incubation cycles, dividing them into a short co-incubation part (10 oocytes) and a long co-incubation part (the remaining oocytes), to analyze embryo developmental parameters. Results: There were no significant differences in the rates of top-quality blastocyst formation, clinical pregnancy, and implantation between the two groups (p > 0.05). 2PN rate in S group was slightly lower than in L group (68.06% vs. 71.08%, p < 0.01). After stratifying by the number of oocytes obtained, multiple logistic regression analysis demonstrated no significant correlation between co-incubation time and 2PN rate (p > 0.05). In the 79 partial short co incubation cycles, there were no significant differences in the rates of 2PN oocytes, ≥3PN (3 pronuclei) oocytes, top-quality cleavage embryos, and top-quality blastocysts on Day 5/Day 6 between the two parts (p > 0.05). Conclusions: Short oocyte-sperm co-incubation is an effective strategy in preventing fertilization failure. Short and long oocyte-sperm co-incubation times have similar outcomes on in vitro development.