F&S science最新文献

Objective: To study the protective effects of bioactive peptides derived from whiteleg shrimp (Litopenaeus vannamei) by-products on testicular function in a rat model of nonalcoholic fatty liver disease (NAFLD).

Design: Experimental study.

Setting: Urmia University, Urmia, Iran.

Animals: Twenty-four adults male Wistar rats (8 weeks old, average weight 230.2 ± 23 g).

Exposure: Rats were divided into four groups (n=6): control (standard chow), high-fat diet (HFD), HFD + 20 mg peptide/kg body weight (BW), and HFD + 300 mg peptide/kg BW. Peptides were enzymatically hydrolyzed at 40-60°C, yielding 60-70% low molecular weight peptides (<500 Da), and administered via oral gavage for 10 weeks.

Main outcome measures: Spermatogenic indices (tubule diameter, Sertoli/Leydig cell counts, tubular differentiation index [TDI], spermiogenesis index [SPI]), oxidative stress markers (malondialdehyde [MDA], reduced glutathione [GSH], oxidized glutathione [GSSG], total antioxidant capacity [TAC]), autophagy-related gene expression (Beclin-1, Atg7, LC3-I, p62), and glucose/lactate transporter expression (GLUT1/3, MCT1/4) assessed via quantitative PCR and immunohistochemistry.

Results: HFD significantly reduced tubule diameter, Sertoli/Leydig cell numbers, TDI, and SPI while increasing MDA (11.7 ± 2.9 vs. 8.3 ± 1.0 nmol/mL, P < 0.05) and disrupting GSH/GSSG ratio (78.6 ± 8.7 vs. 48.2 ± 9.3, P < 0.05). Autophagy genes were upregulated, and GLUT/MCT expression decreased at mRNA and protein levels. Peptide supplementation, particularly at 300 mg/kg BW, dose-dependently reversed these effects by preserving tubular structure, normalizing oxidative markers (MDA: 8.8 ± 0.55 nmol/mL; GSH/GSSG: 51.5 ± 3.0), reducing autophagy gene expression, and enhancing GLUT/MCT expression in germ and Sertoli cells (P < 0.05), with superior efficacy at the higher dose.

Conclusions: Shrimp-derived bioactive peptides mitigate NAFLD-induced testicular dysfunction by modulating oxidative stress, autophagy pathways, and energy metabolism. They are promising natural therapeutics for preserving male fertility under metabolic stress and warrant clinical investigation.

Objective: To characterize the redistribution patterns of zinc and calcium ions during human sperm capacitation and to investigate their roles in sperm maturation and fertilization.

Design: In vitro experimental study using synchrotron-based X-ray Fluorescence (XRF) microscopy and complementary immunohistochemistry to assess elemental changes during different capacitation stages at nanometric spatial resolution.

Setting: Synchrotron imaging conducted at the NanoMAX beamline at MAX IV Laboratory (Lund, Sweden); sperm samples obtained and processed at Institute for Maternal and Child Health IRCCS Burlo Garofolo (Trieste, Italy).

Subjects: Semen samples collected from healthy donors, evaluated according to WHO guidelines.

Exposure(s): None MAIN OUTCOME MEASURE(S): Nanometric distribution and quantification of zinc and calcium in different sperm region (head, midpiece, tail, centriole) during different stages of capacitation.

Result(s): XRF mapping allowed to evaluate the nanometric redistribution of zinc and calcium in human sperm during capacitation. Distinct "zinc signatures" were observed during different capacitation stages, with zinc initially abundant throughout the cell, later concentrating in the midpiece after capacitation, and further decreasing during acrosomal exocytosis. A persistent presence of zinc-rich areas at the centriole was also observed, which likely helps maintain the integrity of the head and midpiece. Concurrently, increased calcium levels in the flagellum during capacitation suggest potentially linked dynamics between zinc efflux and calcium influx. These findings provide new insight into elemental dynamics underlying sperm maturation and fertilization potential.

Conclusion(s): A deeper understanding of male fertility may be achieved by elucidating the multifaceted role of zinc in sperm function, particularly its interaction with calcium signaling pathways. By considering both the biochemical and ionic mechanisms alongside the physical aspects of sperm activity, a more precise assessment of sperm functionality becomes possible.

Objective: To investigate the effect of angiotensin II (Ang II) on proliferation and extracellular matrix (ECM) deposition in human uterine leiomyoma cells and normal myometrial cells.

Design: Experimental in vitro study using immortalized human leiomyoma (HuLM) cells, immortalized human uterine smooth muscle (UTSM) cells, and patient-derived primary fibroid and myometrial cells.

Subjects: Women with uterine fibroids who underwent hysterectomy.

Exposure(s): Administration of physiological and supra-physiological levels of Ang II (to mimic essential hypertension) to cultured HuLM, UTSM, primary fibroid, and myometrial cells to assess effects on cellular proliferation and ECM deposition.

Main outcome measure(s): We evaluated HuLM, UTSM, primary fibroid, and uterine smooth muscle cells for the presence of the angiotensin II type 1 receptor (AT1R). Ang II-induced proliferation and ECM deposition was assessed through MTS assay, Western blot analysis, immunofluorescence, and real time PCR.

Result: (s): Immortalized and primary leiomyoma and myometrial cells expressed AT1R. Leiomyoma cells responded to Ang II with increased cellular proliferation measured by MTS assay, PCNA protein levels, and Ki67 staining. Ang II treatment of fibroid cells showed increased expression of COL1A1, the predominant fibroid and myometrial collagen. Integrin β1, an upstream regulator of fibrosis, also showed an increase in protein and mRNA expression in fibroid cells treated with Ang II. No difference in proliferation or ECM production was observed in myometrial cell controls.

Conclusion: (s): Ang II promoted growth and matrix accumulation in fibroid cells, highlighting a potential link between fibroids and cardiovascular disease.

Objective: To study the levels of oxidant and antioxidant parameters, including total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI), in the follicular fluid of patients with diminished ovarian reserve (DOR) or unexplained infertility (UEI), to compare these groups, and to analyze the relationship between these parameters and anti-Mullerian hormone (AMH).

Design: The study was planned and completed as a prospective cohort study.

Subjects: A total of 90 patients (n(DOR)=45, n(UEI)=45) aged 23-38 years, who applied to underwent in vitro fertilization between February 2024 and August 2024 and were diagnosed with DOR or UEI, were included in the study. TOS and TAS levels were determined in follicle fluid obtained during oocyte retrieval via double-antibody sandwich enzyme-linked immunosorbent assay.

Exposure: Patients categorized as having diminished ovarian reserve (DOR) or unexplained infertility (UEI).

Main outcome measures: Oocyte retrieval was performed on all patients and follicular fluid was obtained simultaneously. TAS and TOS values were measured in the follicular fluid and the OSI value was calculated. TAS, TOS and OSI values were compared between the groups. The relationship between the three parameters and AMH was analyzed separately in each group. To analyze the relationship between low OSI values and AMH, mean OSI values within the group were determined. The AMH values of the populations above and below the mean OSI value within the same group were compared.

Results: No significant differences in TAS, TOS and OSI were observed between groups (p>0.05). A significant positive correlation was found between TOS and AMH in DOR group (p<0.05). Statistically significant difference was found between AMH values of the populations with OSI values above or below the mean in the UEI group (p<0.05). AMH values of the population with lower OSI values were higher. A high TOS value may not indicate that AMH is negatively affected. AMH levels may be negatively affected at high OSI values indicating increased oxidative stress levels.

Conclusion: In evaluating the relationship between the oxidant-antioxidant system and infertility, it may not be appropriate to comment with the TOS or TAS value alone. The OSI level may be taking an effective role in the AMH level.

Objective: To study the maternal contribution to early human embryogenesis by describing the transcriptional dynamics and regulatory roles of maternal effect genes (MEGs) and transcription factors (TFs) throughout the first four cell cycles. This will be achieved using pathenogenotes, such as the human uniparental bioconstruct model.

Design: Descriptive observational study based on single-cell transcriptomic analysis.

Subjects: Nineteen single human parthenocytes were derived from six parthenogenotes at the first (n=2), third (n=2), and fourth (n=2) cell cycles.

Exposure: Transcriptomic changes occurring during early embryonic development in the absence of paternal genomic input.

Main outcome measures: Transcript abundance of MEGs, expression levels of key TFs, number and identity of differentially expressed genes (DEGs), pathway enrichment associated with DEGs, transcriptional complexity, temporal patterns of MEG transcript decay and persistence and timing of embryonic genome activation (EGA).

Results: Transcriptomic analysis revealed progressive increases in transcriptional complexity, with major shifts between the third and fourth cell cycles coinciding with EGA. A total of 212 and 1,515 DEGs were identified in the third and fourth cycles, respectively (fold change ≥|2|, p<0.05 vs. first cycle), predominantly involved in RNA biosynthesis and cell proliferation pathways. Principal component and hierarchical clustering analyses showed distinct transcriptomic profiles by cell cycle and oocyte origin. Key TFs (DUXA, DUX4, Elk-1, E2F-1, Sp1) were implicated in cell cycle regulation. MEG analysis revealed decay of transcripts associated with mRNA clearance, alongside sustained expression of MEGs linked to cell cycle progression and spindle assembly, suggesting a non-random, structured maternal regulatory program.

Conclusion: This study provides the first comprehensive single-cell transcriptomic characterisation of early human parthenogenotes, suggesting a structured, genome-driven maternal programme that governs early embryonic development in the absence of paternal input. The identification of key TFs and MEG signature highlights the pivotal regulatory role of the maternal genome prior to EGA and may inform strategies to improve outcomes in assisted reproductive technologies.

Objective: To evaluate the feasibility of using endometrial stromal cells (EmSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs) as alternative sources of donor nuclei for somatic cell haploidization.

Design: To perform somatic cell haploidization, mouse MII oocytes were enucleated and injected with the nucleus of cumulus cells (CCs), EMSCs or BM-MSCs. Intact MII oocytes served as controls. Oocytes following haploidization and control were inseminated and cultured up to 96 hours in a time-lapse incubator to assessed embryo development and morphokinesis. Blastocysts were either cryopreserved for future genetic analysis or transferred to surrogate pseudo-pregnant mice.

Subjects: Female B2D2F1 mice (ooplasm donor), Male B6-EGFP mice (spermatozoa donor), CD-1 female mice (surrogate) EXPOSURE: Enucleated oocytes underwent somatic cell nuclear transfer using nucleus of CCs, EMSCs or BM-MSCs to generate functional oocytes. These oocytes are fertilized to generate conceptuses.

Main outcome measures: The primary outcome compared embryo development in the two experimental groups versus control. The secondary outcome was embryo morphokinetics evaluated with time-lapse microscopy.

Results: A total of 811 oocytes were enucleated, with a survival rate of 97.5%. SCNT was performed using CCs (n=90), EmSCs (n=394), or BM-MSCs (n=327) resulting in comparable somatic cell fusion rates of 95.2%, 96.0%, and 96.5%, respectively (P=0.53). Comparing the different SCNT groups, the CC cohort had a higher fertilization rate (45.6%) than the EmSC (30.8%, P<0.05) and BM-MSC (26.5%, P<.01) cohorts. However, subsequent embryo development showed significant attrition in the CC cohort where only 14.4% of CC embryos developed into blastocysts compared to 25.6% of EmSC embryos (P=0.04) and 19.6% of BM-MSC embryos (P=0.29). In terms of embryo morphokinetics, all SCNT groups had slower embryo progression (P<0.01) than the control group. However, EmSC and BM-MSC embryo development was faster than CC embryos from syngamy to the 8-cell stage (48.8 vs. 48.5 vs. 58.5 h, respectively, P<0.01). All EmSC embryos and eight out of nine BM-MSC embryos displayed heterozygosity, confirming biparental contribution from the somatic cell and sperm genome. In the EmSC cohort, 42 blastocysts were transferred, yielding 3 healthy mouse pups (2 male, 1 female). All 3 pups displayed biparental inheritance, grew to adulthood and produced 3 healthy first-generation litters.

Conclusion: Somatic cell haploidization of EmSCs and BM-MSCs generated oocytes capable of full pre-implantation development and yielded healthy offspring. The utilization of genotyped donor nuclei for neogametogenesis may represent a feasible fertility treatment option for individuals with complete absence of oocytes.

Objective: To characterize the dynamic secretion patterns of extracellular cytokines in three in vitro human endometrial culture models subjected to a hormonally simulated 28-day menstrual cycle, and to assess the influence of cellular composition on paracrine signaling relevant to endometrial receptivity.

Design: Experimental in vitro study.

Setting: Academic research laboratory.

Subjects: Three endometrial culture models were developed using combinations of endometrial epithelial cells (RL95-2), stromal cells (T0533) and primary myometrial smooth muscle cells.

Exposure: The three multicellular models were subjected to a sequential hormonal treatment protocol simulating the proliferative, ovulatory and secretory phases of the human menstrual cycle.

Main outcome measures: Quantitative profiling of 105 extracellular cytokines across four hormonal phases (control, proliferative, ovulatory, and secretory) using a cytokine array platform. Comparative analysis of cytokine expression patterns, correlation matrices, and hierarchical clustering to identify model-specific and hormone-dependent regulatory networks.

Results: We identified 12 highly expressed cytokines with distinct phase and model-specific expression profiles. EMMPRIN, MIF, and IL-8 increased consistently across all phases, peaking during the window of implantation. VEGF exhibited biphasic expression patterns, EGF upregulated by estradiol and downregulated progesterone. Dkk-1 was upregulated in progesterone-dominant phases. Serpin E1 and Dkk-1 showed phase-specific regulation and also influenced by cellular composition. IGFBP-3 downregulated proliferative and ovulatory phases. Clustering analyses revealed co-regulatory modules (e.g., EMMPRIN/MIF, GRO-α/Lipocalin-2) and distinct cytokine networks modulated by stromal and myometrial components. Notably, Model C (EEC+ESC+MSMC) demonstrated profiles more similar to Model A (EEC alone) than to Model B (EEC+ESC), suggesting MSMCs may attenuate certain epithelial-stromal paracrine interactions.

Conclusions: The phase-specific and model-dependent cytokine secretion patterns highlight the complexity of endometrial paracrine signaling and underscore the importance of multicellular in vitro models. These findings advance our understanding of cytokine dynamics during the menstrual cycle and provide a platform for future studies on endometrial receptivity and implantation.

Objective: To assess the impact of different recombinant follicle-stimulating hormone (recFSH) dosages in ovarian stimulation (OS) on luteinizing hormone (LH)-receptor and FSH-receptor expression in granulosa cells (GCs) and progesterone level.

Design: Prospective interventional study (07/2023-02/2024).

Subjects: Six volunteers with regular cycles and normal ovarian reserve undergoing two OS cycles for oocyte vitrification as a means of elective fertility preservation.

Exposure: Different recFSH dosages (150 IU vs. 300 IU) in two OS cycles, performed in a gonadotropin-releasing hormone-antagonist protocol.

Main outcome measures: The LH receptor expression on GCs.

Results: Volunteers had a median age of 34 years (range: 28-36), a median antimüllerian hormone-level of 2.28 ng/mL (interquartile range [IQR]: 2.41-3.35) and a median antral follicle count of 13.5 (IQR:12-14) in the 150 IU and 13 (12-15) in the 300 IU OS cycle. Median total recFSH dosages were 1650 IU (IQR: 1350-1800IU)/3150 IU (IQR: 3000-3300) in the 150 IU/300 IU OS cycle, respectively. The number of retrieved cumulus-oocyte-complexes was (median and IQR) 11.5 (10-14)/12.5 (7-15), the number of mature oocytes (median and IQR) of 10.5 (7-12)/10 (6-12), respectively, without a statistically significant difference. Besides the stimulation start, serum FSH levels of the 300 IU-cycle were significantly higher compared with the 150 IU-cycle. Estradiol levels were significantly higher in the 300 IU-group on day 5 and at follicle size of 14-16 mm. Progesterone (P4) levels were not statistically significantly different between the stimulation cycles. The LH receptor expression in the GCs at oocyte-pick up was significantly higher and FSH-receptor expression was significantly lower in the 300 IU-group compared with the 150 IU-group. The P4 levels on oocyte-pick up day were significantly associated with LH receptor levels after adjusting for the recFSH dose. Furthermore, a significant and independent association of the recFSH dose between the P4 levels and the LH receptor levels was seen on the day of oocyte retrieval.

Conclusion: Higher recFSH dosages during OS are associated with increased LH receptor expression, and LH receptor count had a significant association with serum P4 levels.

Objective: To evaluate the effect of Shikonin, a potent direct reactive oxygen species (ROS) scavenger, and Indole propionic acid (IPA), a microbiome-derived metabolite, on epididymis and sperm morphology and function in diabetic rats.

Design: A rat model of Type 2 diabetes mellitus (T2DM) was induced in male Wistar rats using a high-fat diet and streptozotocin (50 mg/kg). The animals were subsequently divided into seven groups: Control, T2DM, T2DM + IPA (50 mg/kg), T2DM + Shikonin (0.5 mg/kg), T2DM + Metformin (50 mg/kg), IPA-only, and Shikonin-only. Treatments were administered orally for 4 weeks. We assessed histological integrity, sperm motility, and viability (via computer-assisted sperm analysis), serum lipid profiles, epididymal antioxidant activity (superoxide dismutase, malondialdehyde, and catalase), and apoptotic marker (cytochrome C, caspase-9, and caspase-3) and metabolic signature (fibroblast growth factor 21 [FGF21]) expression via quantitative reverse transcription polymerase chain reaction.

Subjects: Male Wistar albino rats (n = 42, Age: 8-12 weeks old, 200 ± 2.4 g).

Intervention: Streptozotocin, Shikonin, Indole propionic acid.

Main outcome measures: Type 2 diabetes mellitus profoundly impairs male fertility, with oxidative stress in the epididymis being a key driver of sperm damage.

Results: Streptozotocin-induced diabetic rats exhibited increased oxidative damage, characterized by an impaired antioxidant system, high expression of apoptotic markers, and reduced metabolic gene expression. These effects ultimately damaged epididymis tissue and resulted in poor sperm quality. Shikonin and IPA treatments significantly attenuated oxidative stress and apoptosis, restored antioxidant activity and lipid profiles, and improved sperm parameters. Both treatments enhanced metabolic balance, as indicated by improved FGF21 expression, and restored epididymal histoarchitecture. Shikonin was superior in improving the epididymal somatic index and sperm motility, whereas IPA was more effective in normalizing FGF21 expression and lipid profiles, highlighting its primary metabolic role.

Conclusion: Our findings reveal that Shikonin and IPA protect against diabetic reproductive damage through complementary pathways. This work not only introduces promising natural therapeutic strategies but also provides a mechanistic framework for their combined or targeted use in managing diabetic infertility.