Biology of Reproduction最新文献

Human uterine spiral artery remodeling (SAR) is a tightly regulated process involving complex interactions between interstitial and endovascular extravillous trophoblasts (iEVTs and enEVTs) and diverse maternal decidual cell populations. However, the intrinsic spatiotemporal dynamics of SAR in human placentation remain poorly understood, largely due to the limited availability of high-quality maternal-fetal interface specimens. Electively terminated early pregnancies offer a valuable resource for studying SAR in situ, yet inconsistent methods for distinguishing fragmented villous and decidual tissues have hindered reproducibility and interpretation. Herein we present a standardized protocol for the classification and characterization of high-quality maternal-fetal interface specimens from elective terminations by integrating stereomicroscopic evaluation with confirmation by immunohistochemistry and immunofluorescence microscopy. Combined with multiplex immunofluorescence imaging with cell-type-specific markers, this approach enabled precise spatial mapping and quantification of key morphological and cellular events in SAR from gestational weeks 5 to 10. Our analyses reveal that SAR initiates as early as week 5 with extraluminal recruitment of natural killer (NK) cells, followed by the formation of tightly packed EVT plugs within the lumens of spiral arteries in the decidua compacta; these plugs progressively extend deeper into the vessels and gradually loosen as gestation progresses. Notably, enEVTs appear to acquire NK cell-like phenotypes that may facilitate the displacement of endothelial and smooth muscle cells, promoting progressive vessel dilation. In summary, we provide a robust and reproducible method for assessing physiological SAR in early human pregnancy, promoting the adoption of our methodology in future studies of pathological SAR and related pregnancy disorders.

The conditions imposed during in vitro embryo culture may cause modifications of the embryo epigenome and transcriptome, potentially affecting post-natal organ function. This study compared the liver and muscle transcriptome in 4-month-old male and female dairy calves conceived by artificial insemination (AI) or by the transfer of an in vitro-produced (IVP) embryo (n = 4 per sex per treatment). Biopsy tissue samples were collected and processed for RNA sequencing. Analysis of the RNAseq data revealed a distinct separation between the liver transcriptomes of female and male calves, regardless of method of production. Moreover, within the cohort of female calves, a strong separation between those derived from IVP vs AI was observed. Analysis of differentially expressed genes indicated downregulation of oxidative phosphorylation pathways and upregulation of immune system-related enriched terms, including Th17 cell differentiation and antigen processing and presentation. For the muscle transcriptome, the separation between male and female calves was less apparent compared with the liver transcriptome, but there was still a clear separation between female calves derived from IVP vs AI, with downregulated genes enriching for p53 signaling and upregulated genes enriching terms related to muscle structure development. These results suggest that female embryos derived from IVP vs AI exhibit developmental differences that manifest as differential transcriptomic profiles post-natally. In conclusion, these findings demonstrate that the IVP process induces significant alterations in the liver and muscle transcriptome of female post-natal calves. Further longitudinal studies are required to understand the potential implications for lifetime growth, health, and production characteristics.

Background: Ectopic pregnancy and tubal endometriosis directly affect the fallopian tube structure and function, while ovarian cysts and uterine fibroids may indirectly influence tubal physiology. These conditions are associated with infertility, but their impact on fallopian tube mechanical contractions remains unclear. This study aimed to assess the effects of these pathologies on fallopian tube contractility.

Method: Ampulla samples were obtained from women undergoing salpingectomy for benign causes. Based on the menstrual phases, samples were divided into two groups: proliferative (normal proliferative, tubal endometriosis, ovarian cysts, and uterine fibroids) and secretory (normal secretory and ectopic pregnancy). Normal proliferative considered control for the proliferative group, while normal secretory for the ectopic pregnancy. Contractile parameters, maximum contractile force, basal tone, frequency, and amplitude were measured using an isometric force transducer, while in another set of experiments; the oxytocin doses (1 and 10 μM) response was assessed. Smooth muscle organization and structural changes were analyzed through hematoxylin and eosin staining.

Result: Compared to the normal proliferative, the tubal endometriosis and ovarian cysts groups showed significantly lower maximum contractile force, basal tone, frequency, and amplitude, along with damaged smooth muscle layers, while uterine fibroids showed decreased frequency and amplitude, with organized muscle structure. Ectopic pregnancy showed higher maximum contractile force and basal tone than normal secretory, with increased frequency and amplitude and disorganized smooth muscle. Oxytocin increased contractility at 1 μM and reduced it at 10 μM in most groups.

Conclusion: This study demonstrated that fallopian tube contractions and tissue structure were differentially affected across groups, with increased contractility observed in the ectopic pregnancy group and reduced contractility in the uterine fibroids, ovarian cysts, and tubal endometriosis groups.

Many studies have examined the acute effects of chemotherapies and radiation on the ovary; however, few address the impact of long-term chemotherapeutic agents on future reproduction for cancer survivors. The purpose of our study was to determine the effect of long-term tamoxifen (TAM) treatment on follicle development, ovarian reserve, and embryogenesis in a murine model. Adult female mice were treated with TAM (250 mg/kg fed ad libitum in mouse chow) or control chow for three weeks, followed by 0-, 1-, or 3-weeks of TAM washout. Mice were then superovulated, mated, and their oviducts flushed to determine ovulation, fertilization, and in vitro embryo development rates. At specific timepoints, one ovary from each animal was harvested, sectioned, and stained with H&E, while the other ovary was evaluated for gene expression of targeted genes. Ovarian size was calculated based on average surface area over six slices. Follicles (primordial, primary, secondary, antral / pre-ovulatory), and corpora lutea were counted and averaged for each slice. All data were analyzed using mixed-effects analysis of variance (ANOVA), with treatment group as the fixed effect and ovary treated as a random effect nested within treatment. Ovaries of TAM treated mice were 36% smaller on average than control ovaries (p=0.01). Primordial follicle counts were 35% lower than control ovaries (p=0.007) and there were 3.4x more atretic follicles in the ovaries of TAM treated mice (p<0.001). There were no significant differences in the number of primary, secondary, antral or pre-ovulatory follicles. The number of oocytes and embryos flushed from control mice (total ovulated) was 1.65x greater than TAM treated mice. However, fertilization rates and in vitro embryo blastulation rates were not significantly different between controls and TAM groups. In summary, for this murine model, long-term TAM negatively impacted ovarian reserve (primordial follicle number) without overtly affecting the in vitro embryo development to the blastocyst.

Organophosphate esters (OPEs), widely used as flame retardants and plasticizers, are frequently detected in indoor environments and human tissues, raising concerns about their potential endocrine-disrupting effects. In this study, we examined the effects of a household dust-based mixture of OPEs, along with two structural distinct sub-mixtures, on the phenotype, function, and lipidome on MA10 Leydig cells. Using high-content imaging, we identified increase in oxidative stress levels and accumulation of lipid droplets as common phenotypic effects across mixtures. Notably, the triaryl OPE sub-mixture exhibited greater potency, suggesting that specific structural features contribute to the toxicity of OPEs. While the OPE mixture did not impair basal steroid hormone production in MA-10 cells, changes were observed in stimulated progesterone levels and transcriptional regulation of key steroidogenic transcripts. When comparing lipidomic profiles across three steroidogenic cell lines (MA-10, H295R, and KGN), we found that glycerolipids, particularly triglycerides and diglycerides, consistently appeared to be the most affected lipid species, highlighting a common disruption in the composition of lipid droplet. However, cell line specific effects were also observed, especially in the regulation of cholesterol esters, likely reflecting differences in cholesterol sourcing and steroidogenic pathways. These findings emphasize the importance of evaluating environmentally relevant chemical mixtures and demonstrate that OPEs can disrupt steroidogenic function and lipid metabolism.

Cryopreservation methods for archiving and distributing mouse strains mostly focus on freezing embryos or sperm. Although protocols for the cryopreservation of wild-type mouse oocytes are available, these methods have not been widely adopted in large biomedical research facilities. This is partly due to a lack of validation of the available methods on a large scale using a range of genetically modified oocytes. Furthermore, some of the existing methods report a relatively low rate of fertilization, requiring either the zona pellucida to be physically breached or the inclusion of cumulus cells to enable efficient fertilization, interventions which might be incompatible with maintaining hygiene barriers. Existing methods also often use cryovials rather than straws or slimline vitrification devices, which are more practical for storage and handling. Here, we present a robust vitrification protocol for large-scale oocyte cryopreservation, achieving high viability and fertilization rates comparable to fresh oocytes. We have extensively tested the protocol for in vitro fertilization of many genetically altered strains, using both genetically altered and wild-type C57BL/6J oocytes and sperm. Providing an archive of cryopreserved oocytes harboring genetically modified alleles separately from archives of cryopreserved sperm allows multiple allele combinations to be generated during the rederivation process. This reduces subsequent breeding steps and the need to maintain live stocks of mice. Furthermore, cryopreserving oocytes for later use enables them to be obtained from females at the optimal age, thereby reducing the number of mice required and providing greater scheduling flexibility for subsequent genetic modification and rederivation work.

Genome editing is a rapidly advancing technology with transformative potential in livestock, offering opportunities that range from enhanced production traits to the generation of biomedical models for human disease and xenotransplantation. The CRISPR/Cas9 system, originally identified as a bacterial defense mechanism, has become the most widely used tool for precise genome editing. In this review, we first summarize the potential applications of CRISPR/Cas9 in livestock and highlight notable successes to date. We then address the ongoing challenges associated with delivering CRISPR/Cas9 into gametes and embryos, as current methods such as microinjection and electroporation often result in high mosaicism and cellular damage. We subsequently introduce extracellular vesicles (EVs) as a promising alternative delivery system. Secreted by virtually all cell types, EVs can efficiently transport bioactive molecules and are readily internalized by gametes and embryos. Although EV-mediated delivery of CRISPR/Cas9 has shown success in somatic cells, its use in reproductive cells remains largely unexplored. We review emerging strategies for loading EVs with CRISPR/Cas components and discuss the potential advantages of combining this approach with recently developed smaller Cas variants to overcome delivery barriers. Collectively, these innovations support the promise of EVs as a biologically compatible, efficient, and minimally invasive system for targeted genome editing in livestock reproduction.

Purpose: Polycystic ovary syndrome affects 5-10% of women of reproductive age, with insulin resistance playing a central role in its pathophysiology in up to 80% of cases. This review aims to elucidate the molecular mechanisms by which insulin resistance disrupts ovarian function, contributing to menstrual irregularities and hyperandrogenism. It also evaluates current and emerging therapeutic strategies, with an emphasis on individualized management.

Methods: A comprehensive review of recent literature was conducted, focusing on molecular studies, clinical trials, and meta-analyses related to insulin signaling pathways in polycystic ovary syndrome, as well as therapeutic interventions. Special attention was paid to ethnic variations, particularly in East Asian populations, and advances in genomic and metabolomic profiling.

Results: Polycystic ovary syndrome is characterized by selective insulin resistance, wherein metabolic insulin signaling is impaired, but steroidogenic and mitogenic pathways remain responsive, promoting hyperandrogenism and anovulation. East Asian women exhibit significant insulin resistance despite lower body mass index compared with Western populations. Insulin resistance in polycystic ovary syndrome also increases cardiometabolic risks and psychological burden. While lifestyle modification, insulin sensitizers, and hormonal therapy remain first-line treatments, novel approaches such as microbiome-targeted therapies and anti-inflammatory agents show promise.

Conclusion: Understanding the complex interplay between insulin resistance and ovarian dysfunction is crucial for effective polycystic ovary syndrome management. Integrating emerging molecular insights with digital health tools can facilitate personalized, multidisciplinary approaches that address both reproductive and metabolic aspects of polycystic ovary syndrome, ultimately improving patient outcomes.

Summary sentence: This study underscores the pivotal role of insulin resistance in ovarian dysfunction and advocates for integrated, technology-enhanced strategies to personalize PCOS management, thereby improving holistic patient outcomes.

Extracellular vesicles (EVs) are promising markers for biological processes, but their role in predicting superovulatory response remains largely underexplored. We investigated blood plasma-derived EV from ewes subjected to superovulation treatment in follicular or luteal phases. 20 Santa Inês ewes underwent an estrous cycle synchronization and superovulation protocol. Based on the number of corpora lutea (CL) and after transcervical embryo collection was accomplished, 10 ewes were allocated into high- (HR; ≥ 11 CL; n = 4) or no/low-response (N/LR; CL ≤ 4; n = 6) groups. The nanoparticle tracking analysis revealed similar EV size distributions and concentrations among groups, but higher concentrations during the follicular compared to the luteal phase. Transmission electron microscopy analysis confirmed the presence of microvesicles and exosomes, while western blotting and nano-flow cytometry revealed the expression of CD63, Syntenin-1, CD9, CD81, and Alix. Tandem mass spectrometry identified a total of 357 proteins, revealing differential protein expression between phases of the estrous cycle and the superovulation response. EV proteomes differed by phase, with 17 DAP: seven up-regulated proteins in the follicular phase and 10 proteins up-regulated in the luteal phase. Comparing ewes with HR versus N/LR responses revealed only a small number of DAP across both phases. In the luteal phase, a single DAP was detected in each group, whereas in the follicular phase, four significant DAP were observed exclusively in the HR group. In conclusion, physical and molecular differences in plasma EV across the follicular and luteal phases of superovulatory treatment highlight their potential as reproductive physiology and superovulation responsiveness biomarkers.

The peri-implantation period of pregnancy in pigs is characterized by rapid morphological transitions of the conceptus necessitating a precisely regulated uterine environment to support elongation, survival, and implantation. Uterine histotroph, composed of nutrients and signaling molecules secreted by or transported by endometrial epithelia, plays a central role in mediating these events. However, dynamic changes in the metabolic composition of uterine luminal fluid (ULF) during early pregnancy are incompletely defined. In this study, we performed stage-resolved, untargeted metabolomic profiling of ULF collected from cyclic and pregnant gilts on Days 10, 12, 14, and 16 of the estrous cycle and pregnancy (n = 2-6/group). A total of 206 metabolites were identified, with amino acids, fatty acids, and carbohydrates being the dominant classes. Principal component and supervised learning analyses revealed progressive divergence in ULF composition between pregnant and cyclic gilts with the most distinct profiles observed by Day 16. Notably, pregnancy induced substantial increases in amino acids associated with mechanistic target of rapamycin (mTOR) signaling and trophectoderm proliferation, including arginine, glutamine, proline, lysine, and phenylalanine. Kyoto Encyclopedia of Genes and Genomes enrichment analyses identified gestational age-dependent activation of pathways involved in amino acid biosynthesis, nucleotide metabolism, and phospholipid turnover. Metabolites such as phosphorylcholine, succinic acid, and asymmetric dimethylarginine increased markedly in pregnancy, suggesting coordinated regulation of membrane remodeling, energy production, and nitric oxide signaling. Targeted quantification of 19 amino acids revealed both linear and quadratic trends across time and pregnancy status, with distinct differences in glycine and serine trajectories between pregnant and cyclic ULF. Collectively, these findings describe the evolving biochemical landscape of the uterine lumen during early pregnancy and highlight key metabolic pathways that likely support conceptus development and uterine receptivity to implantation.