Search

ABSTRACT Empty follicle syndrome (EFS) is a disorder characterised by the unsuccessful retrieval of oocytes from matured follicles following ovarian stimulation for in vitro fertilisation (IVF). Genetic factors significantly contribute to this pathology. To date, an increasing number of genetic mutations associated with GEFS have been documented, however, some cases still remain unexplained by these previously reported mutations. Here, we identified a novel homozygous missense ZP1 variant (c.1096 C > T, p.Arg366Trp) in a female patient with GEFS from a consanguineous family who failed to retrieve any oocytes during two cycles of IVF treatment. We conducted a molecular dynamics simulation analysis on the mutant ZP1 model, revealing that the mutant ZP1 protein has an altered 3D structure, lower fluctuation, higher compactness and higher instability than wild‐type ZP1. Immunostaining, immunoblotting and co‐immunoprecipitation results showed that the homozygous missense mutation in ZP1 impaired protein secretion and weakened interactions between ZP1 and other ZP proteins, which may affect the ZP assembly. This study contributes to a more comprehensive understanding of the genetic aetiopathogenesis of GEFS. [ABSTRACT FROM AUTHOR]

Nanotechnology and its applications have advanced significantly in recent decades, contributing to various fields, including reproduction. This study introduces a novel method to label porcine oocytes with nanoparticles (NPs) bound to oviductin (OVGP1, Ov) for use in Assisted Reproductive Technologies (ARTs). Despite promising developments, concerns about NP toxicity in gametes necessitate thorough investigation. This research aims to assess the impact of functionalized NPs (NPOv) on sperm functionality. Boar sperm were co‐incubated with NPOv for 0, 0.5 and 1 h in two media: BTS (semen dilution and conservation) and TALP (sperm capacitation and in vitro fertilization‐IVF). Sperm quality parameters (viability, motility and kinematics) showed no significant differences in TALP medium (p >.05). In BTS, although some kinetic parameters were altered, motility, progressive motility and viability remained unaffected (p >.05). Additionally, NPs presence on the zona pellucida (ZP) of oocytes did not affect sperm attachment (p >.05). In conclusion, in vitro exposure of boar sperm to OVGP1‐functionalized NPs in IVF medium or attached to the ZP surface of matured oocytes does not impair sperm functionality, including their binding ability to the ZP. [ABSTRACT FROM AUTHOR]

Although monotremes diverged from the therian mammal lineage approximately 187 million years ago, they retain various plesiomorphic and/or reptilian‐like anatomical and physiological characteristics. This study examined the morphology of juvenile and adult female reproductive tracts across various stages of the presumptive oestrous cycle, collected opportunistically from cadaver specimens submitted to wildlife hospitals during the breeding season. In adult females, ovaries had a convoluted cortex with follicles protruding from the ovarian surface. While protruding antral follicles were absent from the ovaries of juvenile echidnas, histological analysis identified early developing primordial and primary follicles embedded into the ovarian cortex. The infundibulum epithelial cells of the oviducts were secretory during the follicular phase but not at other stages, the ampulla region was secretory at all stages and is likely responsible for the mucoid layer deposited around the zona pellucida, and the isthmus region of the oviduct appeared to be responsible for initial deposition of the shell coat, as in marsupials. Female echidnas have two separate uteri, which never merge and enter separately into the urogenital sinus (UGS). This study confirmed that both uteri are functional and increase in glandular activity during the luteal phase. In the juvenile uteri, the endometrium was immature with minimal, small uterine glands. A muscular cervical region at the caudal extremity of each uterus, just before the cranial region of the UGS was defined by the absence of glandular tissue in all female echidnas, including the juveniles. There was no evidence of a definitive vaginal region. A clitoris was also detected that possessed a less developed but similar structural (homologous) anatomy to the male penis; urethral ducts while present did not appear to be patent. [ABSTRACT FROM AUTHOR]

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO’s role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5’-TAACNGT-3’ OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/ germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development. [ABSTRACT FROM AUTHOR]

Although under appropriate laboratory conditions, sperm from different mammalian species can be capacitated in vitro , the optimal conditions for sperm capacitation in the stallion have been elusive. This study evaluated the effect of different capacitating inducers in Whitten and Tyrode media and assessed their impact on capacitation-related factors. Stallion sperm were incubated with different combinations of capacitating inducers at 38.5 °C in an air atmosphere. Sperm quality variables such as motility, mitochondrial membrane potential, and lipid peroxidation were assessed. Membrane fluidity and intracellular calcium levels were evaluated as early markers of capacitation, while tyrosine phosphorylation events and the sperm's ability to perform acrosomal exocytosis were used as late capacitation markers. Finally, these sperm were evaluated using a heterologous zona pellucida binding assay. The findings confirm that capacitating conditions evaluated increase intracellular calcium levels and membrane fluidity in both media. Similarly, including 2 or 3 inducers in both media increased tyrosine phosphorylation levels and acrosomal exocytosis after exposure to progesterone, confirming that stallion sperm incubated in these conditions shows cellular and molecular changes consistent with sperm capacitation. Furthermore, the zona pellucida binding assay confirmed the binding capacity of sperm incubated in capacitation conditions, a key step for stallion in vitro fertilization success. Further studies are needed to evaluate the effect of these conditions on in vitro fertilization in the horse. • Different treatments for equine sperm capacitation were evaluated in two media. • Combination of capacitating inducers boosts phosphorylation and acrosomal exocytosis. • Capacitating conditions improve sperm's zona pellucida binding capacity. [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) fish hepatocyte cultures are promising alternative models for replicating in vivo data. Few studies have attempted to characterise the structure and function of fish 3D liver models and illustrate their applicability. This study aimed to further characterise a previously established spheroid model obtained from juvenile brown trout (Salmo trutta) primary hepatocytes under estrogenic stimulation. The spheroids were exposed for six days to environmentally relevant concentrations of 17α-ethinylestradiol—EE2 (1–100 ng/L). The mRNA levels of peroxisome (catalase—Cat and urate oxidase—Uox), lipid metabolism (acyl-CoA long chain synthetase 1—Acsl1, apolipoprotein AI—ApoAI, and fatty acid binding protein 1—Fabp1), and estrogen-related (estrogen receptor α—ERα, estrogen receptor β—ERβ, vitellogenin A—VtgA, zona pellucida glycoprotein 2.5—ZP2.5, and zona pellucida glycoprotein 3a.2—ZP3a.2) target genes were evaluated by quantitative real-time polymerase chain reaction. Immunohistochemistry was used to assess Vtg and ZP protein expressions. At the highest EE2 concentration, VtgA and ZP2.5 genes were significantly upregulated. The remaining target genes were not significantly altered by EE2. Vtg and ZP immunostaining was consistently increased in spheroids exposed to 50 and 100 ng/L of EE2, whereas lower EE2 levels resulted in a weaker signal. EE2 did not induce significant changes in the spheroids' viability and morphological parameters. This study identified EE2 effects at environmentally relevant doses in trout liver spheroids, indicating its usefulness as a proxy for in vivo impacts of xenoestrogens. [ABSTRACT FROM AUTHOR]

Handmade cloning (HMC) has a higher yield and is relatively less difficult to operate compared to traditional micromanipulation cloning. Yet, there are few reports on handmade cloning in sheep. Therefore, this study investigates the key nodes such as AC and DC voltage, denucleation method and fusion method in sheep handmade cloning. In addition, it compares the effects of fibroblasts (FC) and umbilical cord mesenchymal stem cells (UC‐MSCs) of different states as donors on the development of HMC embryos. Furthermore, the effect of different freezing solutions on the survival rate of frozen blastocysts without zona pellucida was also investigated. The results indicate that an AC voltage of 150 V/cm and a DC voltage of 1800 V/cm significantly enhanced the fusion and blastocyst rates (p <.01). The blastocyst rate achieved with umbilical cord MSCs as nucleus donors was significantly higher (40.3%) than that achieved with fibroblasts and differentiated umbilical cord MSCs (21.5%, 22.5%) (p <.01). The highest survival rate was achieved using 20% DMSO + 20% EG for freezing without zona pellucida. In conclusion, the most efficient and pregnant ovine HMC cloning method using 150 V/cm AC, 1800 V/cm DC, knife‐cut denucleation, two‐step fusion and the use of UC‐MSCs as nucleus donors resulted in the highest overall efficiency and pregnancy after transplantation. [ABSTRACT FROM AUTHOR]

Background: Zona pellucida is one of the main selective barriers for the spermatozoa before reaching the oocyte. Using native zona in the sperm selection prior to Intracytoplasmic Sperm Injection (ICSI) has been proven effective but inconvenient approach in In Vitro Fertilisation (IVF) laboratory. The application of autologous solubilised zonae pellucidae in the sperm selection prior to ICSI has not been studied yet. Objectives: To compare the assisted reproductive treatment (ART) outcomes (implantation, pregnancy, live birth, and miscarriage rates) after ICSI performed with spermatozoa selected on their ability to adhere to immobilised solubilised zonae pellucidae and conventionally selected spermatozoa. Materials and methods: In total, 500 couples fulfilled the inclusion criteria and 368 of them were included in the study. After random allocation, 192 couples had spermatozoa selected by sperm–zona adhesion for ICSI (study group) and 176 patients underwent standard ICSI (control group). In the study group, patients' own zonae were acid solubilised and immobilised on petri dishes. The partner's motile spermatozoa were placed in the dishes and the adhered spermatozoa were used for ICSI. For the control group, the conventional sperm selection by morphological criteria was applied prior ICSI. All women underwent frozen ET with euploid embryos. Chi square test was used to compare the data. Results: The sperm selection by zona adhesion resulted in significantly higher implantation rate (50.4% vs. 37.0%, p = 0.003), clinical pregnancy rate (43.8% vs. 33.3%, p = 0.018) and live birth rate (38.0% vs. 25.9%, p = 0.004) and significantly lower incidence of miscarriage (11.3% vs. 22.2%, p = 0.044) in comparison to the conventional method of the sperm selection. Discussion and conclusion: The application of solubilised zonae pellucidae in the sperm selection for ICSI benefits ART outcomes in couples with unexplained infertility. Moreover, sperm–zona selection significantly reduces the risk of miscarriages. [ABSTRACT FROM AUTHOR]

A priority to facilitate the application of lipofection to generate genetically modified porcine embryos and animals will be the use of zona pellucida (ZP)-intact oocytes and zygotes. Recently, our group produced genetically modified embryos by lipofection of ZP-intact oocytes during in vitro fertilization (IVF). This study investigates the effect of two commercial lipofection reagents, Lipofectamine 3000 and Lipofectamine CRISPRMAX, on embryo development and mutation efficiency in ZP-intact porcine oocytes. We compared these reagents with the electroporation method and a control group using two sgRNAs targeting the CAPN3 and CD163 genes. The detrimental effects on cleavage rates were observed in both lipofection treatments compared to the control and electroporated groups. However, blastocyst rates were higher in the Lipofectamine 3000 group than in the electroporated group for both genes. Mutation parameters varied by target gene, with Lipofectamine 3000 achieving higher mutation rates for CD163, while all groups were similar for the CAPN3 gene. Overall efficiency was similar for both lipofectamines, confirming their feasibility for use. In addition, we evaluated the effect of coincubation time (4, 8, and 24 h) on IVF outcomes, embryo development, and mutation parameters. Results indicated that an 8-h coincubation period optimized fertilization and mutation efficiency without significant toxic effects. This study demonstrates that lipofection with either Lipofectamine 3000 or CRISPRMAX during IVF is an effective method for generating genetically modified porcine embryos without the need for specialized equipment or trained personnel, with efficiencies similar to or greater than electroporation. This study also highlights the importance of optimizing reagent selection and coincubation times. There is no difference between Lipofectamine 3000 and CRISPRMAXTM in terms of embryo development and mutation efficiency, and under our experimental conditions, the optimal coincubation time with lipofectamine is 8 h. • Lipofection during IVF is an effective method to generate mutant porcine embryos. • Lipofectamine 3000 and CRISPRMAX are similar in efficiency. • Lipofection maintains high blastocyst rates. • Optimal coincubation time with Lipofectamine is 8 h. [ABSTRACT FROM AUTHOR]

Highlights • LAH might effectively increase pregnancy outcomes for frozen-thawed embryo transfer. • Zona pellucida drilling might help increase pregnancy outcomes in FET. Abstract Background: Zona Pellucida (ZP) thickness of less than 16 mm is better for embryo implantation inside the endometrium. Laser Assisted Hatching (LAH) is commonly used, especially in non-contact mode, using a 1.48-um infrared diode laser beam because of its short exposure time, accurate positioning, simple operation, indirect contact, safety, and effectiveness. Objective: This paper describes the potential of laser-assisted hatching in biochemical pregnancy outcomes in Frozen Embryo Thawed transfers. Material and Method: The total number of patients enrolled in this study was 141. Patients were prospectively treated during embryo transfers at Pusat Fertilitas Bocah Indonesia, Primaya Hospital at Tangerang, Indonesia, from December 2020 until December 2021. Result: There were no significant differences between the LAH and no-LAH groups regarding average age, infertility duration, infertility type, and etiology of infertility (p>0.05). In the same line, the blastocyst (0.76 ± 0.87; 1.25 ± 1.08) compared with cleavage (0.72 ± 0.84; 0.67 ± 0.98) (p

High quality label-free imaging of oocytes and early embryos is essential for accurate assessment of their developmental potential, a key element of assisted reproduction procedures. To achieve this goal, we propose full-field optical coherence microscopy (FF-OCM), constructed as a compact module fully integrated with a commercial wide-field fluorescence microscope. Our system achieves optical sectioning in wide-field, high in-plane resolution of 0.5 µm, and high sensitivity to backscattered light. To demonstrate its imaging capabilities, we study live mouse oocytes and embryos at all important stages of meiotic maturation and early embryogenesis. Our system enables visualization of intracellular structures, which are not visible in common bright-field microscopy, i.e., internal structure of nuclear apparatus, cytoskeletal filaments, cellular cortex, cytoplasmic protrusions, or zona pellucida features. Additionally, we visualize and quantify intracellular dynamics like cytoplasmic stirring motion, nuclear envelope fluctuations and nucleolus mobility. Altogether, we demonstrate that FF-OCM is a powerful tool for research in developmental biology that also holds great potential for non-invasive time-lapse monitoring of oocyte and embryo quality in assisted reproduction. Full-field optical coherence microscopy enables high-contrast and high-resolution label-free imaging of the intracellular structure and the dynamics of live mouse oocytes and early embryos for non-invasive quality assessment in assisted reproduction. [ABSTRACT FROM AUTHOR]

The Drosophila corneal lens is entirely composed of chitin and other apical extracellular matrix components, and it is not known how it acquires the biconvex shape that enables it to focus light onto the retina. We show here that the zona pellucida domain-containing protein Dusky-like is essential for normal corneal lens morphogenesis. Dusky-like transiently localizes to the expanded apical surfaces of the corneal lens-secreting cells and prevents them from undergoing apical constriction and apicobasal contraction. Dusky-like also controls the arrangement of two other zona pellucida domain proteins, Dumpy and Piopio, external to the developing corneal lens. Loss of either dusky-like or dumpy delays chitin accumulation and disrupts the outer surface of the corneal lens. We find that artificially inducing apical constriction by activating myosin contraction is sufficient to similarly alter chitin deposition and corneal lens morphology. These results demonstrate the importance of cell shape in controlling the morphogenesis of overlying apical extracellular matrix structures such as the corneal lens. [ABSTRACT FROM AUTHOR]

Apical extracellular matrices (aECMs) coat the exposed surfaces of animal bodies to shape tissues, influence social interactions, and protect against pathogens and other environmental challenges. In the nematode Caenorhabditis elegans , collagenous cuticle and zona pellucida protein-rich precuticle aECMs alternately coat external epithelia across the molt cycle and play many important roles in the worm's development, behavior, and physiology. Both these types of aECMs contain many matrix proteins related to those in vertebrates, as well as some that are nematode-specific. Extensive differences observed among tissues and life stages demonstrate that aECMs are a major feature of epithelial cell identity. In addition to forming discrete layers, some cuticle components assemble into complex substructures such as ridges, furrows, and nanoscale pillars. The epidermis and cuticle are mechanically linked, allowing the epidermis to sense cuticle damage and induce protective innate immune and stress responses. The C. elegans model, with its optical transparency, facilitates the study of aECM cell biology and structure/function relationships and all the myriad ways by which aECM can influence an organism. [ABSTRACT FROM AUTHOR]

Autoimmune factors play an important role in premature ovarian insufficiency (POI). Human amniotic epithelial stem cells (hAESCs) have recently shown promising treatment effects on chemotherapy-induced POI. However, the therapeutic efficacy and underlying mechanisms of hAESCs in autoimmune POI remain to be investigated. In this study, we showed for the first time that intravenous transplantation of hAESCs could reside in the ovary of zona pellucida 3 peptide (pZP3) induced autoimmune POI mice model for at least 4 weeks. hAESCs could improve ovarian function and fertility, alleviate inflammation and reduce apoptosis of granulosa cells (GCs) in autoimmune POI mice. The transcriptome analysis of mice ovaries and in vitro co-cultivation experiments suggest that activation of the AKT and ERK pathways may be the key mechanism in the therapeutic effect of hAESCs. Our work provides the theoretical and experimental foundation for optimizing the administration of hAESCs, as well as the clinical application of hAESCs in autoimmune POI patients. [ABSTRACT FROM AUTHOR]

Oogenesis has universal characteristics among fishes, in which structures of vital importance for embryos are assembled. However, these universal structures may have additional functions depending on the evolutionary ecology of each species. The aim of this research was to describe histologically the structure of the ovary and the oogenesis of the pupfishes Cyprinodon artifrons and Garmanella pulchra, with emphasis on the follicular structures linked to the resistance of embryos to desiccation. The embryos of pupfishes experience periodic deleterious environmental changes caused by estuarine tides. They can survive periods out of water protected by accessory egg layers assembled in the ovaries during oogenesis. This research found that both species have a single ovary with asynchronous follicle development, associated with a frequent breeding activity. Also, multiple generations of cortical alveoli were described, larger in G. pulchra, related with the formation of perivitelline space, a structure that can buffer the effects of desiccation when embryos remain out of water. In both species, the accumulation of oil droplets in the oocytes was simultaneous with the synthesis of cortical alveoli. Oil droplets may be related to energy reserves during extended incubation periods. The zona pellucida of both species presented an intermediate thickness with respect to other species, and hair-like filaments arranged on the zona pellucida of G. pulchra were clearly detected. These structures as a whole, could be related to the survival of the embryos of the pupfishes of Yucatan in the tidal fluctuations in the estuarine environments in which they inhabit. [ABSTRACT FROM AUTHOR]

Submucous cleft palate (SMCP) is a congenital anomaly characterized by the presence of Calnan's triad. However, in clinical practice, it is common for individuals to exhibit one or two anatomical abnormalities within the triad. Furthermore, the definition of SMCP has been diverse and ambiguous in literature. Therefore, this study aimed to analyze the correlation between anatomical abnormalities and development of velopharyngeal insufficiency (VPI). We conducted a retrospective analysis of 99 patients referred to our clinic for speech issues or anatomical abnormalities identified during routine oral examinations from January 2012 to June 2023. A single surgeon performed all physical examinations. We evaluated the presence of bony notch, zona pellucida, and bifid uvula, assigned a score to each abnormality, and analyzed their correlation with VPI. The correlation of each of the abnormalities with VPI development was examined, along with the relationship between the number of abnormalities and VPI. Among the 99 patients, 27 were diagnosed with VPI. Only the bony notch had a significant correlation with VPI development. The incidence of VPI tended to increase with the presence of more anatomical abnormalities. VPI occurred in approximately 40% of patients exhibiting all three anatomical abnormalities. The study findings highlight the importance of meticulous intraoral examinations in patients with SMCP and careful monitoring of patients with a bony notch or two or more anatomical abnormalities. [ABSTRACT FROM AUTHOR]

Genetic mosaicism, characterized by multiple genotypes within an individual, is considered an obstacle to CRISPR/Cas9 genome editing in animal models. Despite the various strategies for minimizing mosaic mutations, no definitive methods exist to eliminate them. This study aimed to enhance gene editing efficiency in porcine zygotes using CRISPR/Cas9, which targets specific genes through centrifugation and zona pellucida removal before electroporation. Centrifugation at 2000 × g did not adversely affect blastocyst formation rates in zygotes electroporated with gRNA targeting the GGTA1 gene; instead, it led to increased total and monoallelic mutation rates compared with control zygotes without centrifugation. However, the groups had no significant differences in biallelic mutation rates. In zygotes electroporated with gRNA targeting the CMAH gene, centrifugation treatments exceeding 1000 × g significantly increased both biallelic mutation rates and mutation efficiency. The combination of centrifugation and zona pellucida removal did not have a detrimental effect on blastocyst formation rates. It led to a higher rate of double biallelic mutations in embryos targeting both GGTA1 and CMAH compared to embryos without centrifugation treatment. In summary, our results demonstrate that pre-electroporation treatments, including centrifugation and zona pellucida removal, positively influenced the reduction of mosaic mutations, with the effectiveness of centrifugation depending on the specific gRNA used. [ABSTRACT FROM AUTHOR]

Cryopreservation is crucial for conserving genetic diversity in endangered species including the critically endangered group of sturgeons (Acipenseridae), but it can compromise sperm quality and protein profiles. Although cryopreservation with dimethyl sulfoxide (DMSO) and methanol (MeOH) results in the recovery of good post-thaw motility, DMSO-preserved sperm show reduced fertilization ability. This study was conducted in Siberian sturgeon as a model for Acipenserid fishes to explore the effects of DMSO and MeOH on the proteome of semen using advanced proteomics methods—liquid chromatography‒mass spectrometry and two-dimensional difference gel electrophoresis. We analyzed the proteomic profiles of fresh and cryopreserved spermatozoa and their extracellular medium and showed that cryopreservation decreases motility and viability and increases reactive oxygen species levels, membrane fluidity, and acrosome damage. Despite having similar post-thaw semen motility, sperm treated with DMSO had significantly lower fertilization success (6.2%) than those treated with MeOH (51.2%). A total of 224 and 118 differentially abundant proteins were identified in spermatozoa preserved with MeOH and DMSO, respectively. MeOH-related proteins were linked to chromosomal structure and mitochondrial functionality, while DMSO-related proteins impacted fertilization by altering the acrosome reaction and binding of sperm to the zona pellucida and nuclear organization. Additionally, cryopreservation led to alterations in the proacrosin/acrosin system in both cryoprotectants. This study provides the first comprehensive proteomic characterization of Siberian sturgeon sperm after cryopreservation, offering insights into how cryoprotectants impact fertilization ability. [ABSTRACT FROM AUTHOR]

Infertility affects a significant proportion of the reproductive‐aged population, with male‐associated factors contributing to over half of the cases. However, current diagnostic tools have limitations, leading to an underestimation of the true prevalence of male infertility. While traditional semen parameters provide some insights, they fail to determine the true fertility potential in a substantial number of instances. Therefore, it is crucial to investigate additional molecular targets responsible for male infertility to improve understanding and identification of such cases. Seminal plasma, the main carrier of molecules derived from male reproductive glands, plays a crucial role in reproduction. Amongst its multifarious functions, it regulates processes such as sperm capacitation, sperm protection and maturation, and even interaction with the egg's zona pellucida. Seminal plasma offers a non‐invasive sample for urogenital diagnostics and has shown promise in identifying biomarkers associated with male reproductive disorders. This review aims to provide an updated and comprehensive overview of seminal plasma in the diagnosis of male infertility, exploring its composition, function, methods used for analysis, and the application of emerging markers. Apart from the application, the potential challenges of seminal plasma analysis such as standardisation, marker interpretation and confounding factors have also been addressed. Moreover, we have also explored future avenues for enhancing its utility and its role in improving diagnostic strategies. Through comprehensive exploration of seminal plasma's diagnostic potential, the present analysis seeks to advance the understanding of male infertility and its effective management. [ABSTRACT FROM AUTHOR]

Background: Spermatozoa become mature and competent for fertilization during transit from the caput epididymis to the cauda epididymis. However, detailed molecular mechanisms of epididymal sperm maturation are still unclear. Here, we focused on multiple epididymis‐enriched genes: lipocalin family genes (Lcn5, Lcn6, Lcn8, Lcn9, and Lcn10) and Ly6 family genes (Ly6g5b and Ly6g5c). These genes are evolutionarily conserved in mammals and form clusters on chromosomes 2 and 17 in the mouse, respectively. Objective: To clarify whether these genes are required for epididymal sperm maturation and acquisition of fertilizing ability, we generated knockout (KO) mice using the CRISPR/Cas9 system and analyzed their phenotype. Materials and methods: We generated four lines of KO mice: Lcn9 single KO, the lipocalin family quadruple KO (Lcn5, Lcn6, Lcn8, and Lcn10), quintuple KO (Lcn5, Lcn6, Lcn8, Lcn10, and Lcn9), and double KO of Ly6 family genes (Ly6g5b and Ly6g5c). Results: Although the Lcn9 single KO did not affect male fertility, the quadruple KO and quintuple KO male mice were subfertile and mostly infertile, respectively, with a reduced amount of ADAM3, an essential protein for sperm binding to the zona pellucida. Further analysis revealed that the quintuple KO spermatozoa lack the CMTM2A/B that are required for ADAM3 maturation. Intriguingly, Ly6g5b and Ly6g5c double KO male mice also showed subfertility with reduced sperm ADAM3. Conclusion: These results suggest epididymal secretory proteins are involved in ADAM3 maturation and acquisition of sperm fertilizing ability. [ABSTRACT FROM AUTHOR]

To evaluate whether laser-mediated assisted hatching (AH) performed on vitrified/warmed blastocysts before embryo transfer can improve live birth rate. The "pArtiaL zonA pelluciDa removal by assisteD hatchINg of blastocysts (ALADDIN)" is a 2-center comparative study with a parallel randomized controlled design. University hospital. Participants were recruited between September 2018 and November 2021. They were aged 18–39 years, underwent nondonor in vitro fertilization cycles, and were scheduled for elective single embryo transfer with vitrified/warmed blastocysts. Those with uterine abnormalities, body mass index of >35 kg/m2, severe male factor infertility, or performing preimplantation genetic testing were excluded. Assisted hatching was performed using a 1,480 nm diode laser, removing approximately one-third of the zona pellucida with continuous 0.2 ms pulses applied from the 1–5 o'clock positions. The primary outcome was the live birth rate. Secondary end points included clinical pregnancy, miscarriage, multiple pregnancies, preterm births, obstetric and neonatal complications, and congenital anomalies. Overall, 698 participants met the inclusion criteria and were randomized: 352 patients were assigned to the AH arm and 346 to the control arm. Of the participants, 105 (29.8%) and 101 (29.2%), respectively, achieved a live birth after treatment. The relative risk of live birth in patients with vitrified/warmed blastocysts treated with AH was 1.02 (95% confidence interval, 0.86–1.19). Exploratory subgroup analyses for women's age, recruiting centers, indications for in vitro fertilization, method of insemination, blastocyst quality, and days of blastocyst development failed to highlight any clinical situation that could benefit from AH in thawed blastocysts. In patients undergoing frozen embryo transfer with vitrified/warmed blastocysts, laser AH does not improve the live birth rate. Further studies are required to rule out milder but potentially interesting benefits in specific subgroups of patients. ClinicalTrials.gov: NCT03623659. [ABSTRACT FROM AUTHOR]

Defective oocyte maturation is a common cause of female infertility. The loss of the zona pellucida (ZP) represents a specific condition of impaired oocyte maturation. The extracellular matrix known as the ZP envelops mammalian oocytes and preimplantation embryos, exerting significant influence on oogenesis, fertilization, and embryo implantation. However, the genetic factors leading to the loss of the ZP in oocytes are not well understood. This study focused on patients who underwent oocyte retrieval surgery after ovarian stimulation and were found to have abnormal oocyte maturation without the presence of the ZP. Ultrasonography was performed during the surgical procedure to evaluate follicle development. Peripheral blood samples from the patient were subjected to exome sequencing. Here, a novel, previously unreported heterozygous mutation in the ZP1 gene was identified. Within the ZP1 gene, we discovered a novel heterozygous mutation (ZP1 NM_207341.4: c.785A>G (p.Y262C)), specifically located in the trefoil domain. Bioinformatics comparisons further revealed conservation of the ZP1-Y262C mutation across different species. Model predictions of amino acid mutations on protein structure and cell immunofluorescence/western blot experiments collectively confirmed the detrimental effects of the ZP1-Y262C mutation on the function and expression of the ZP1 protein. The ZP1-Y262C mutation represents the novel mutation in the trefoil domain of the ZP1 protein, which is associated with defective oocyte maturation in humans. Our report enhances comprehension regarding the involvement of ZP-associated genes in female infertility and offers enriched understanding for the genetic diagnosis of this condition. [ABSTRACT FROM AUTHOR]

The process of growing and maturing into a Graafian follicle, which has the capacity to either ovulates its eggs into the oviduct for fertilisation or undergo atresia, is known as folliculogenesis. Follicle growth in sheep and cattle begins prior to the final formation of primordial follicles and persists during the foetal, neonatal, and adult stages of life. The inhibitory factors may be important for controlled exit from the pool of primordial follicles and there is evidence that anti-Mullerian hormone (AMH) inhibits follicle activation and early follicular growth in bovine ovaries. A number of genes present in primordial follicles are active during the growth stage; these genes include those related to the formation of the zona pellucida, cell signalling and communication and mitochondrial activity. The capacity of a mature oocyte to support the very earliest stages of life, fertilization, preimplantation of embryo development and implantation is termed oocyte developmental competence. In this article, we elaborate the development of oocyte from embryonic stage to ovulatory stage. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate whether it is possible to prevent oolemma lysis after Piezo-assisted intracytoplasmic sperm injection (Piezo-ICSI) caused by abnormal membrane rupture. A total of 489 mature oocytes were obtained from 85 patients who underwent Piezo-ICSI in an infertility clinic. Inseminated oocytes were classified into the following two groups: normal rupture and abnormal rupture, and a portion of abnormally ruptured oocytes was randomly exposed to 7% PVP solution. Normal fertilization rate, degeneration rate, proportion of high-quality embryos on day 3, blastocyst formation, and morphologically high-quality blastocysts were compared. Abnormal rupture was defined as cytoplasmic membrane rupture before piezo pulse driving. Among the abnormal rupture groups, the normal fertilization and degeneration rates were compared between the high-stretched (ruptured at ≥ 50% during oocyte membrane stretching) and low-stretched (< 50% position) oocytes. The degeneration rate was significantly higher in abnormally ruptured oocytes than in normally ruptures oocytes (14.3% vs 1.3%, p < 0.001), and there was no significant difference in embryonic development after fertilization. PVP treatment immediately after oolemma rupture significantly decreased the degeneration rate (6.0% vs 19.7%, p = 0.031) and increased the normal fertilization rate. Similarly, there were no significant differences in the developmental potential. Furthermore, pregnancy outcome data showed no significant differences in pregnancy and live birth rates. The degeneration rate was reduced by treating low-stretched oocytes with PVP. Exposure to polyvinylpyrrolidone (PVP) immediately after abnormal membrane rupture represents an effective strategy to prevent oocyte degeneration. This is the first study to propose a strategy for the prevention of oocyte degeneration in Piezo-ICSI. These findings are expected to reduce the oocyte degeneration rate and increase normal fertilization rate as well as assist patients who can only acquire oocytes with weak plasma membranes. [ABSTRACT FROM AUTHOR]

Background: Agar-like zona pellucida (ZP) is the most common type of abnormal ZP, and is one of the causes of low fertility or infertility. However, the molecular mechanism of agar-like ZP is unclear. Single-cell RNA-sequencing (scRNA-seq) analysis was used to assess the cellular and molecular landscape of oocytes with agar-like ZP. Methods: Human metaphase I (MI) oocytes were collected from four patients with agar-like ZP and four healthy donors. Total RNA was isolated, cDNA was synthesized, and libraries were generated and subsequently sequenced on a HiSeq 2500 instrument. The scRNA-seq data were analyzed with R software. Results: We identified 1320 genes that were differentially expressed between agar-like ZP oocytes and healthy donor oocytes. Gene Ontology term enrichment results showed that the genes downregulated in agar-like ZP oocytes were significantly related to extracellular matrix organization, while the genes upregulated in agar-like ZP oocytes were significantly related to the regulation of response to DNA damage stimulus. The Kyoto Encyclopedia of Genes and Genomes enrichment results showed that genes were enriched in the ECM-receptor interaction pathway and focal adhesion pathway. Other signaling pathways important in oocyte development were also enriched, such as PI3K-Akt. Differential expression analysis identified UBC, TLR4, RELA, ANXA5, CAV1, KPNA2, CCNA2, ACTA2, FYN and ITGB3 as genetic markers of oocytes with agar-like ZP. Conclusions: Our findings suggest that agar-like ZP oocytes exhibit significant downregulation of genes involved in the ECM-receptor interaction signaling pathway and focal adhesion pathway, which could lead to aberrant ZP formation, while the upregulated genes were significantly related to regulation of the response to DNA damage stimulus. Agar-like ZP formation may interfere with the normal exchange of signals between oocytes and perivitelline granulosa cells, thereby preventing cumulus cells from participating in oocyte DNA damage repair and causing MI arrest. [ABSTRACT FROM AUTHOR]

According to previous studies, the quality and fertilization rate of fresh sperm from boars of different ages were significantly different. However, the difference of freeze-thaw sperm quality and fertility in boars of different ages is unclear. In this study, boars of a Chinese native breed were assigned into two groups. Each group consisted of five boars aged aged either 2-3 years (young boars = YB) or 5-6 years (aging boars = AB) A total of 60 ejaculates for each group were collected and cryopreserved. Semen quality and in vitro fertility of post-thaw sperm was evaluated. The results showed that the concentration and motility of fresh sperm collected from AB were similar to YB, but their semen volume was higher than that in YB (p < 0.05). Frozen-thawed sperm of AB had lower viability than YB, and higher abnormal rate and reactive oxygen species (ROS) levels of YB (p < 0.05). There was no effect of the age on post-thaw sperm motility and time survival. Functional assessments indicated that increasing age markedly compromises the integrity of the sperm plasma membrane and acrosome, as well as mitochondrial functionality post-thaw, albeit without affecting DNA integrity. Furthermore, increasing age of boars reduces the ability of sperm to bind to the oocyte zona pellucida after thawing, delaying the time of the first embryo cleavage after fertilization. Finally, the early developmental efficiency of in vitro fertilized embryos progressing from 4-cell to blastocyst derived from post-thaw sperm in AB significantly decreased compared to those from YB (p < 0.05). Taken together, these results suggest that increasing age in boars impairs the quality and in vitro fertility of frozen thawed sperm. [ABSTRACT FROM AUTHOR]

Worldwide meat consumption and production have nearly quintupled in the last 60 years. In this context, research and the application of new technologies related to animal reproduction have evolved in an accelerated way. The objective of the present study was to apply nanoemulsions (NEs) as carriers of lipids to feed bovine embryos in culture media and verify their impact on the development of embryos produced in vitro. The NEs were characterized by particle size, polydispersity, size distribution, physical stability, morphology using atomic force microscopy (AFM), surface tension, density, pH, and rheological behavior. The NEs were prepared by the emulsification/evaporation technique. A central composite rotatable design (CCRD) was used to optimize the NE fabrication parameters. The three optimized formulations used in the embryo application showed an emulsion stability index (ESI) between 0.046 and 0.086, which reflects high stability. The mean droplet diameter analyzed by laser diffraction was approximately 70–80 nm, suggesting a possible transit across the embryonic zona pellucida with pores of an average 90 nm in diameter. AFM images clearly confirm the morphology of spherical droplets with a mean droplet diameter of less than 100 nm. The optimized formulations added during the higher embryonic genome activation phase in bovine embryos enhanced early embryonic development. [ABSTRACT FROM AUTHOR]

Abstract Background Laser-assisted hatching (LAH) stands as the predominant technique for removing the zona pellucida (ZP) in embryos, primarily consisting of two methods: drilling laser-assisted hatching (D-LAH) and thinning laser-assisted hatching (T-LAH). Presently, both methods have limitations, and their comparative efficacy for embryo implantation and clinical pregnancy remains uncertain. Aim Evaluate the impact of D-LAH and T-LAH on clinical pregnancy rates within assisted reproductive technology (ART). Methods We systematically searched electronic databases including PubMed, Web of Science, and Cochrane Library until July 20, 2022. This study encompassed observational studies and randomized controlled trials (RCTs). A 95% confidence interval (CI) was utilized for assessing the risk ratio (RR) of pregnancy outcomes. The level of heterogeneity was measured using I2 statistics, considering a value exceeding 50% as indicative of substantial heterogeneity. Results The meta-analysis scrutinized 9 studies involving 2405 clinical pregnancies from D-LAH and 2239 from T-LAH. Findings suggested no considerable variation in the clinical pregnancy rates between the two techniques (RR = 0.93, 95% CI: 0.79–1.10, I2 = 71%, P = 0.41). Subgroup analyses also revealed no substantial differences. However, D-LAH exhibited a notably higher occurrence of singleton pregnancies compared to T-LAH (RR = 2.28, 95% CI: 1.08–4.82, I2 = 89%, P = 0.03). There were no noteworthy distinctions observed in other secondary outcomes encompassing implantation rate, multiple pregnancies, ongoing pregnancy, miscarriage, premature birth, and live birth. Conclusion Both the primary findings and subgroup analyses showed no marked variance in clinical pregnancy rates between D-LAH and T-LAH. Therefore, patients with varying conditions should select their preferred LAH technique after assessing their individual situation. However, due to the restricted number of studies involved, accurately gauging the influence of these laser techniques on clinical outcomes is challenging, necessitating further RCTs and high-quality studies to enhance the success rate of ART. Trial registration PROSPERO: CRD42022347066.

Thoroughbred stallions that carry a double-homozygous genotype A/A-A/A for SNPs rs397316122 and rs69101140 in exon 5 of the FKBP6 gene (chr13; EquCab3.0) are uniquely subfertile due to impaired acrosomal exocytosis (IAE). In this study, the sperm proteome in frozen/thawed semen from subfertile Thoroughbred stallions was studied and compared to that of frozen/thawed sperm from fertile Thoroughbred stallions. A total of 2,220 proteins was identified, of which 140 proteins were found to be differentially abundant in sperm from the subfertile stallions compared to that of fertile stallions (83 less and 57 more abundant). Proteins of differential abundance in sperm from the subfertile stallions were mainly overrepresented in the "metabolism" and the "metabolism of lipids" pathways. One of these proteins, arylsulfatase F (ARSF), was studied by immunofluorescence. A lower proportion of sperm displaying ARSF signal at the acrosome region was observed in sperm from subfertile Thoroughbred stallions. In addition, heterologous zona pellucida binding assays revealed that sperm from subfertile Thoroughbred stallions bound at a lower proportion to zonae pellucidae than sperm from fertile Thoroughbred stallions. In conclusion, a group of differential abundance proteins, including some of acrosome origin, were identified in sperm from subfertile stallions with acrosome dysfunction. [ABSTRACT FROM AUTHOR]

The yellow fever mosquito Aedes aegypti is a major disease vector and an increasingly popular emerging model research organism. We present here an improved protocol for the collection, fixation, and preparation of A. aegypti embryos for immunohistochemical and in situ hybridization studies. The processing of A. aegypti embryos for such studies is complicated by the inability to easily remove the vitelline membrane, which prevents the reagents needed for staining from reaching their targets, and which furthermore obscures visualization of the embryo since the membrane is highly sclerotized. Previously described protocols for removal of the vitelline membrane are very low throughput, limiting the capacity of work that can be accomplished in a reasonable timeframe. Our adapted protocol increases the throughput capacity of embryos by an individual user, with experienced users able to prepare an average of 100–150 embryos per hour. The protocol provides high-quality intact embryos that can be used for morphological, immunohistochemical, and in situ hybridization studies. The protocol has been successfully tested on embryos of ages ranging from 14h after egg laying (AEL) at 27°C through to 55h AEL. Critical to the success of the optimized protocol is the selection, fabrication, and description of the tools required. To this end, a video-demonstrated protocol has been placed at protocols.io to clarify the protocol and provide easy access and training to anyone interested in the preparation of A. aegypti embryos for biological studies. [ABSTRACT FROM AUTHOR]

Eggs and embryo manipulation is an important biotechnological challenge to enable positioning, entrapment, and selection of reproductive cells to advance into a new era of nature‐like assisted reproductive technologies. Oviductin (OVGP1) is an abundant protein in the oviduct that binds reversibly to the zona pellucida, an extracellular matrix that surrounds eggs and embryos. Here, the study reports a new method coupling OVGP1 to magnetic nanoparticles (NP) forming a complex (NPOv). NPOv specifically surrounds eggs and embryos in a reversible manner. Eggs/embryos bound to NPOv can be moved or retained when subjected to a magnetic force, and interestingly only mature‐competent eggs are attracted. This procedure is compatible with normal development following gametes function, in vitro fertilization, embryo development and resulting in the birth of healthy offspring. The results provide in vitro proof‐of‐concept that eggs and embryos can be precisely guided in the absence of physical contact by the use of magnets. [ABSTRACT FROM AUTHOR]

Background: In poultry, the smooth transition of follicles from the preovulatory-to-postovulatory phase impacts egg production in hens and can benefit the poultry industry. However, the regulatory mechanism underlying follicular ovulation in avians is a complex biological process that remains unclear. Results: Critical biochemical events involved in ovulation in domestic chickens (Gallus gallus) were evaluated by transcriptomics, proteomics, and in vitro assays. Comparative transcriptome analyses of the largest preovulatory follicle (F1) and postovulatory follicle (POF1) in continuous laying (CL) and intermittent laying (IL) chickens indicated the greatest difference between CL_F1 and IL_F1, with 950 differentially expressed genes (DEGs), and the smallest difference between CL_POF1 and IL_POF1, with 14 DEGs. Additionally, data-independent acquisition proteomics revealed 252 differentially abundant proteins between CL_F1 and IL_F1. Perivitelline membrane synthesis, steroid biosynthesis, lysosomes, and oxidative phosphorylation were identified as pivotal pathways contributing to ovulation regulation. In particular, the regulation of zona pellucida sperm-binding protein 3, plasminogen activator, cathepsin A, and lactate dehydrogenase A (LDHA) was shown to be essential for ovulation. Furthermore, the inhibition of LDHA decreased cell viability and promoted apoptosis of ovarian follicles in vitro. Conclusions: This study reveals several important biochemical events involved in the process of ovulation, as well as crucial role of LDHA. These findings improve our understanding of ovulation and its regulatory mechanisms in avian species. [ABSTRACT FROM AUTHOR]

Background: Laser-assisted hatching (LAH) stands as the predominant technique for removing the zona pellucida (ZP) in embryos, primarily consisting of two methods: drilling laser-assisted hatching (D-LAH) and thinning laser-assisted hatching (T-LAH). Presently, both methods have limitations, and their comparative efficacy for embryo implantation and clinical pregnancy remains uncertain. Aim: Evaluate the impact of D-LAH and T-LAH on clinical pregnancy rates within assisted reproductive technology (ART). Methods: We systematically searched electronic databases including PubMed, Web of Science, and Cochrane Library until July 20, 2022. This study encompassed observational studies and randomized controlled trials (RCTs). A 95% confidence interval (CI) was utilized for assessing the risk ratio (RR) of pregnancy outcomes. The level of heterogeneity was measured using I2 statistics, considering a value exceeding 50% as indicative of substantial heterogeneity. Results: The meta-analysis scrutinized 9 studies involving 2405 clinical pregnancies from D-LAH and 2239 from T-LAH. Findings suggested no considerable variation in the clinical pregnancy rates between the two techniques (RR = 0.93, 95% CI: 0.79–1.10, I2 = 71%, P = 0.41). Subgroup analyses also revealed no substantial differences. However, D-LAH exhibited a notably higher occurrence of singleton pregnancies compared to T-LAH (RR = 2.28, 95% CI: 1.08–4.82, I2 = 89%, P = 0.03). There were no noteworthy distinctions observed in other secondary outcomes encompassing implantation rate, multiple pregnancies, ongoing pregnancy, miscarriage, premature birth, and live birth. Conclusion: Both the primary findings and subgroup analyses showed no marked variance in clinical pregnancy rates between D-LAH and T-LAH. Therefore, patients with varying conditions should select their preferred LAH technique after assessing their individual situation. However, due to the restricted number of studies involved, accurately gauging the influence of these laser techniques on clinical outcomes is challenging, necessitating further RCTs and high-quality studies to enhance the success rate of ART. Trial registration: PROSPERO: CRD42022347066. [ABSTRACT FROM AUTHOR]

Porcine seminal plasma (SP) is loaded with a heterogeneous population of extracellular vesicles (sEVs) that modulate several reproductive-related processes. This study investigated the effect of two sEV subsets, small (S-sEVs) and large (L-sEVs), on porcine in vitro fertilization (IVF). The sEVs were isolated from nine SP pools (five ejaculates/pool) using a size-exclusion chromatography-based procedure and characterized for quantity (total protein), morphology (cryogenic electron microscopy), size distribution (dynamic light scattering), purity and EV-protein markers (flow cytometry; albumin, CD81, HSP90β). The characterization confirmed the existence of two subsets of high purity (low albumin content) sEVs that differed in size (S- and L-sEVs). In vitro fertilization was performed with in vitro matured oocytes and frozen-thawed spermatozoa and the IVF medium was supplemented during gamete coincubation (1 h at 38.5 °C, 5 % CO 2 in a humidified atmosphere) with three different concentrations of each sEV subset: 0 (control, without sEVs), 0.1, and 0.2 mg/mL. The first experiment showed that sEVs, regardless of subset and concentration, decreased penetration rates and total IVF efficiency (P < 0.0001). In a subsequent experiment, it was shown that sEVs, regardless of subset and concentration, impaired the ability of spermatozoa to bind to the zona pellucida of oocytes (P < 0.0001). The following experiment showed that sEVs, regardless of the subset, bound to frozen-thawed sperm but not to in vitro matured oocytes, indicating that sEVs would affect sperm functionality but not oocyte functionality. The lack of effect on oocytes was confirmed by incubating sEVs with oocytes prior to IVF, achieving sperm-zona pellucida binding results similar to those of control. In the last experiment, conducted under IVF conditions, sperm functionality was analyzed in terms of tyrosine phosphorylation, acrosome integrity and metabolism. The sEVs, regardless of the subset, did not affect sperm tyrosine phosphorylation or acrosome integrity, but did influence sperm metabolism by decreasing sperm ATP production under capacitating conditions. In conclusion, this study demonstrated that the presence of sEVs on IVF medium impairs IVF outcomes, most likely by altering sperm metabolism. • Seminal extracellular vesicles decrease sperm-zona pellucida binding during porcine IVF. • Seminal extracellular vesicles impair IVF process during gamete co incubation. • Seminal extracellular vesicles can interact with pig frozen thawed spermatozoa. • Sperm metabolism can be modulated by seminal extracellular vesicles. [ABSTRACT FROM AUTHOR]

Domestic cat blastocysts cultured without the zona pellucida exhibit reduced implantation capacity. However, the protein expression profile has not been evaluated in these embryos. The objective of this study was to evaluate the protein expression profile of domestic cat blastocysts cultured without the zona pellucida. Two experimental groups were generated: (1) domestic cat embryos generated by IVF and cultured in vitro (zona intact, (ZI)) and (2) domestic cat embryos cultured in vitro without the zona pellucida (zona-free (ZF group)). The cleavage, morula, and blastocyst rates were estimated at days 2, 5 and 7, respectively. Day 7 blastocysts and their culture media were subjected to liquid chromatography–tandem mass spectrometry (LC–MS/MS). The UniProt Felis catus database was used to identify the standard proteome. No significant differences were found in the cleavage, morula, or blastocyst rates between the ZI and ZF groups (p > 0.05). Proteomic analysis revealed 22 upregulated and 20 downregulated proteins in the ZF blastocysts. Furthermore, 14 proteins involved in embryo development and implantation were present exclusively in the culture medium of the ZI blastocysts. In conclusion, embryo culture without the zona pellucida did not affect in vitro development, but altered the protein expression profile and release of domestic cat blastocysts. [ABSTRACT FROM AUTHOR]

The evaluation of oocytes and zygotes, based on their size, shape and morphology, is a valuable tool for predicting subsequent embryo development. While this assessment is non-invasive and made possible with time-lapse monitoring systems, not all the assessment criteria used for zygotes with pale cytoplasm can be used for domestic cat zygotes, which have dark cytoplasm. In this study, feline presumptive zygotes were evaluated for shape, size, and morphology. Measurements were also made of the diameter of the entire zygote, its cytoplasm, and the zona pellucida. Differences in the dataset were assessed using the generalized linear model (GLM) procedure. While there was no relationship between a combination of the tested parameters with the potential for cleavage, blastocyst development, and hatching, the parameters of the shape and size of the entire oocyte, and of the zona pellucida, were related to the development potential. The results presented in this study indicate that the assessment procedure for human zygotes has to be adjusted to be used in the cat model, however the relationship between measurements of the diameter of presumptive feline zygotes and the thickness of zona pellucida with their developmental potential deserves further investigation to optimize assessment of cat presumptive zygotes. [ABSTRACT FROM AUTHOR]

Avian embryos develop in an egg composition which reflects both maternal condition and the recent environment of their mother. In birds, yolk corticosterone (CORT) influences development by impacting pre‐ and postnatal growth, as well as nestling stress responses and development. One possible mechanism through which maternal CORT may affect offspring development is via changes to offspring DNA methylation. We sought to investigate this, for the first time in birds, by quantifying the impact of manipulations to maternal CORT on offspring DNA methylation. We non‐invasively manipulated plasma CORT concentrations of egg‐laying female zebra finches (Taeniopygia castanotis) with an acute dose of CORT administered around the time of ovulation and collected their eggs. We then assessed DNA methylation in the resulting embryonic tissue and in their associated vitelline membrane blood vessels, during early development (5 days after lay), using two established methods – liquid chromatography–mass spectrometry (LC–MS) and methylation‐sensitive amplification fragment length polymorphism (MS‐AFLP). LC–MS analysis showed that global DNA methylation was lower in embryos from CORT‐treated mothers, compared to control embryos. In contrast, blood vessel DNA from eggs from CORT‐treated mothers showed global methylation increases, compared to control samples. There was a higher proportion of global DNA methylation in the embryonic DNA of second clutches, compared to first clutches. Locus‐specific analyses using MS‐AFLP did not reveal a treatment effect. Our results indicate that an acute elevation of maternal CORT around ovulation impacts DNA methylation patterns in their offspring. This could provide a mechanistic understanding of how a mother's experience can affect her offspring's phenotype. [ABSTRACT FROM AUTHOR]

Simple Summary: The objective was to identify candidate miRNAs and their integrated genes regulating acrosome function in capacitated sperm of high- versus low-fertility dairy bulls and to elucidate functional biological pathways using a systems biology approach featuring miRNA–mRNA cluster analyses. Based on categorized bovine miRNAs (n = 84), 19 were differentially expressed in high- compared to low-fertility capacitated sperm (p ≤ 0.05, fold regulation ≥ 2 magnitudes). mRNA expression of highly scored integrated genes of differentially expressed miRNAs was greater, ranging from 2.0 to 9.1-fold (p < 0.05) in high- compared to low-fertility sperm, with predicted pathways regulating acrosome vesicle exocytosis, acrosome reaction, and binding of sperm to zona pellucida. In conclusion, highly differentially expressed miRNAs in high-fertility bovine sperm regulating acrosome function have potential for predicting bull fertility. Bioinformatics envisage experimental data as illustrated biological networks, exploring roles of individual proteins and their interactions with other proteins in regulation of biological functions. The objective was to identify differentially expressed miRNAs and their associated genes regulating the acrosome reaction in capacitated sperm of high- compared to low-fertility dairy bulls and to elucidate biological functional pathways using a systems biology approach, featuring miRNA–mRNA cluster analysis. Categorized bovine-specific miRNAs (n = 84) were analyzed by RT-PCR; 19 were differentially expressed in high- compared to low-fertility sperm (p ≤ 0.05, fold regulation ≥ 2 magnitudes). Six miRNAs (bta-miR-129-5p, bta-miR-193a-3p, bta-miR-217, bta-mir-296-5p, bta-miR-27a, and bta-miR-320a) were highly upregulated (p < 0.05; fold regulation ≥ 5 magnitudes) in high- compared to low-fertility sperm. Highly scored integrated genes of differentially expressed miRNAs predicted associations with pathways regulating acrosome vesicle exocytosis, acrosome reaction, and sperm-oocyte binding. The mRNA expressions of genes associated with the acrosome reaction (including hub genes) were greater, ranging from 2.0 to 9.1-fold (p < 0.05) in high- compared to low-fertility capacitated bull sperm. In conclusion, differentially expressed miRNAs in high-fertility bovine sperm regulating acrosome functions have potential for predicting bull fertility. [ABSTRACT FROM AUTHOR]

Egg activation, representing the critical oocyte-to-embryo transition, provokes meiosis completion, modification of the vitelline membrane to prevent polyspermy, and translation of maternally provided mRNAs. This transition is triggered by a calcium signal induced by spermatozoon fertilization in most animal species, but not in insects. In Drosophila melanogaster, mature oocytes remain arrested at metaphase-I of meiosis and the calcium-dependent activation occurs while the oocyte moves through the genital tract. Here, we discovered that the oenocytes of fruitfly females are required for egg activation. Oenocytes, cells specialized in lipid-metabolism, are located beneath the abdominal cuticle. In adult flies, they synthesize the fatty acids (FAs) that are the precursors of cuticular hydrocarbons (CHCs), including pheromones. The oenocyte-targeted knockdown of a set of FA-anabolic enzymes, involved in very-long-chain fatty acid (VLCFA) synthesis, leads to a defect in egg activation. Given that some but not all of the identified enzymes are required for CHC/pheromone biogenesis, this putative VLCFA-dependent remote control may rely on an as-yet unidentified CHC or may function in parallel to CHC biogenesis. Additionally, we discovered that the most posterior ventral oenocyte cluster is in close proximity to the uterus. Since oocytes dissected from females deficient in this FA-anabolic pathway can be activated in vitro, this regulatory loop likely operates upstream of the calcium trigger. To our knowledge, our findings provide the first evidence that a physiological extra-genital signal remotely controls egg activation. Moreover, our study highlights a potential metabolic link between pheromone-mediated partner recognition and egg activation. Author summary: Efficient sexual reproduction requires gamete formation and maturation, partner mating, fecundation and the triggering of embryonic development. Deciphering the underlying physiological regulatory processes represents a major scientific challenge, crucial both for medical issues and agricultural pest control. A pivotal step in the reproductive process, known as egg activation, is the oocyte-to-embryo transition. In a wide range of metazoan species, this activation is provoked by the entry of the spermatozoon into the oocyte. Conversely, in insects, egg activation is initiated as the oocyte moves through the female genital tract, operating independently of spermatozoon entry. Here, we discovered that egg activation in Drosophila depends on a set of anabolic enzymes required for very-long-chain fatty acid (VLCFA) synthesis in the oenocytes. The oenocytes, abdominal cells specialized in lipid metabolism, are necessary for the biogenesis of cuticular hydrocarbons and pheromones. To our knowledge, our findings provide the first evidence that a non-genital physiological signal remotely governs egg activation. Given that pheromone-deficient Drosophila females attract males regardless of their species, we propose that pheromone biogenesis and this VLCFA-dependent remote signal might have co-evolved to prevent the development of hybrid individuals. [ABSTRACT FROM AUTHOR]

Simple Summary: Insulin-like growth factors improve oocyte quality and are necessary for oocyte maturation. However, their effectiveness is contingent upon regulated availability, influenced by binding proteins. Specifically, IGFBP-4 has been identified as a factor blocking the positive effects of insulin-like growth factors, with higher expression observed in degenerating cattle follicles. Therefore, we studied the effect of IGFBP-4 on oocyte developmental potential using an in vitro embryo production system. We determined the ability of the oocyte to mature and cleave; proper embryo development; as well as the total cell number and some relevant genes for embryo quality. We found that IGFBP-4 did not affect the early stages of embryo development or growth. However, high IGFBP-4 concentrations decreased the number of embryos able to break and leave the Zona Pellucida, an important event before elongation and pregnancy establishment. We concluded that IGFBP-4 impairs embryo quality by decreasing the capacity of an embryo to hatch, and therefore the further development and pregnancy establishment could be impaired. These results provide new insights about the impact of IGFBP-4 on embryo quality and may have implications for cattle fertility. Insulin-like growth factors (IGFs) are essential for oocyte maturation. Their bioavailability is regulated by their respective binding proteins (IGFBPs) and proteases. IGFBP-4 blocks the biological effects of IGFs. High IGFBP-4 expression has been associated with follicle atresia. We hypothesized that IGFBP-4 affects oocyte developmental competence during maturation. Therefore, the aim of this study was to examine the effect of IGFBP-4 on the developmental rate of bovine cumulus–oocyte complexes (COCs) during in vitro embryo production. Abattoir-derived COCs were matured with rbIGFBP-4 (2000, 540, and 54 ng/mL) compared to a control. Cumulus expansion, oocyte maturation, cleavage, blastocyst, and hatching rates were evaluated. Furthermore, blastocyst gene expression of SOCS2, STAT3, SLC2A1, SLCA3, BAX, and POU5F1 transcripts were quantified using RT-qPCR. No statistical differences were detected among the groups for cumulus expansion, maturation, cleavage, blastocyst rates, or all gene transcripts analyzed. However, at day 8 and 9, the number of total hatching and successfully hatched blastocysts was lower in 2000 ng/mL rbIGFBP-4 compared to the control (day 8: total hatching: 17.1 ± 0.21 vs. 31.2 ± 0.11%, p = 0.02 and hatched blastocyst 6.7 ± 0.31 vs. 21.5 ± 0.14%, p = 0.004; day 9 total hatching 36.4 ± 0.18 vs. 57.7 ± 0.10%, p = 0.009 and hatched blastocyst 18.2 ± 0.21 vs. 38.1 ± 0.11%, p = 0.004). We concluded that high concentrations of rbIGFBP-4 might negatively affect the subsequent ability of the embryo to hatch and possibly compromise further elongation. [ABSTRACT FROM AUTHOR]

The pathogenesis of empty follicle syndrome has not been clarified. More and more research has focused on the genetic factors of empty follicle syndrome, especially the genes involved in regulating oocyte development. Zona pellucida glycoprotein 1 (ZP1) is a key component of the structural integrity of the zona pellucida matrix. Structural and functional defects of zona pellucida due to the mutations of ZP1 gene often lead to the impaired oocyte maturation and the increased fragility, which may lead to the repeated oocyte retrieval failures. Various mutations of ZP1 gene that cause empty follicle syndrome phenotypes have been identified. In this paper, we reviewed the action and mechanism of ZP1 gene mutations at different sites in causing the empty follicle syndrome phenotype. [ABSTRACT FROM AUTHOR]

The present study investigated the effects of ellagic acid, a type of polyphenol that does not have a glycan and is composed of four hydroxyl groups and two lactone functional groups, on porcine in vitro fertilization (IVF) by focusing on its anti-hyaluronidase activity. A comparative analysis of ellagic acid and apigenin, which is commonly used as a hyaluronidase inhibitor, was performed. It compared the effects of ellagic acid and apigenin on hyaluronidase activity at different concentrations. The results showed that 10, 20, and 40 μM ellagic acid strongly reduced hyaluronidase activity (P < 0.05). The addition of 20 μM ellagic acid, but not apigenin, to porcine IVF medium effectively reduced polyspermy without decreasing sperm penetration or the formation rates of male pronuclei in cumulus-free oocytes. However, neither ellagic acid nor apigenin affected the number of sperm that bound to zona pellucida (ZP) or the induction of zona hardening and protease resistance. The percentage of acrosome-reacting sperm that bound to the ZP was markedly lower in the presence of 20 μM ellagic acid than in the untreated and apigenin-treated groups, even though the antioxidant capacity of ellagic acid was weaker than that of apigenin. Furthermore, a markedly higher percentage of embryos developed to the blastocyst stage in the ellagic acid-treated group, and the apoptotic indexes of expanded blastocysts produced by the ellagic acid treatment during IVF were significantly low. Therefore, the anti-hyaluronidase effect of ellagic acid markedly suppressed the induction of the acrosome reaction in sperm that bound to the ZP, resulting in a marked decrease in polyspermy under conditions that maintained high sperm penetrability during IVF and sustainment of the developmental potency in porcine oocytes. [ABSTRACT FROM AUTHOR]

Key clinical message: In in vitro fertilization (IVF), laser offers several advantages. In this study, we employed laser to eliminate the zona pellucida of a contaminated embryo. This approach helps to rescue embryo with bacterial contamination, and improve embryo‐endometrium interaction. To present a case report on the removal of a contaminated zona pellucida from an embryo of patient with a history of recurrent implantation failure (RIF), which was followed by a successful live birth. We present the case of a 34‐year‐old patient with a history of 3 years of infertility who underwent in vitro fertilization. During the culture process, the embryos became contaminated, leading to three failed implantations. Despite the aneuploidy of the embryo and the implementation of a washing technique, the contamination persisted. In the final attempt, the contaminated zona pellucida was successfully removed using laser, followed by embryo transfer, resulting in a live birth. We provided detailed clinical information, including patient demographics, infertility history, ovarian response, evidence of bacterial contamination, embryo development, treatment protocols, and outcomes. Laser excision of the zona pellucida is a safe and effective method for addressing bacterial infection in embryos. [ABSTRACT FROM AUTHOR]

Context: In pigs, in vitro fertilisation (IVF) is associated with high polyspermy rates, and for this reason, in vitro embryo production (IVP) is still an inefficient biotechnology. Coculture with somatic cells is an alternative to improve suboptimal in vitro maturation (IVM) conditions. Aim: This study was conducted to test a coculture system of porcine luteal cells (PLC) and cumulus–oocyte complexes (COC) to improve oocyte metabolism. Methods: COC were matured in vitro with PLC. Oocyte lipid content, mitochondrial activity, zona pellucida (ZP) digestibility and pore size, cortical reaction and in vitro embryo development were assessed. Key results: Coculture reduced cytoplasmic lipid content in the oocyte cytoplasm without increasing mitochondrial activity. Although ZP digestibility and ZP pore number were not different between culture systems, ZP pores were smaller in the coculture. Coculture impacted the distribution of cortical granules as they were found immediately under the oolemma, and more of them had released their content in the ZP. Coculture with porcine luteal cells during IVM increased monospermic penetration and embryo development after IVF. Conclusions: The coculture of COC with PLC affects the metabolism of the oocyte and benefits monospermic penetration and embryo development. Implications: The coculture system with PLC could be an alternative for the conventional maturation medium in pigs. In vitro -produced embryos are essential for employing assisted reproductive technologies to establish porcine models. However, polyspermy remains a major obstacle to the production of porcine embryos in vitro. This study was conducted to test a coculture system of porcine luteal cells during in vitro maturation to improve suboptimal conditions. Our model could be an alternative to replace the conventional maturation medium, affecting the metabolism of the oocytes and leading to lower rates of polyspermy and higher rates of embryo development. Image by Gabriela Teplitz and Alexandre Bastien. [ABSTRACT FROM AUTHOR]

Abnormalities in the zona pellucida (ZP) adversely affect oocyte maturation, embryo development and pregnancy outcomes. However, the assessment of severity is challenging. To evaluate the effects of different degrees of ZP abnormalities on embryo development and clinical outcomes, in total, 590 retrieval cycles were scored and divided into four categories (control, mild, moderate and severe) based on three parameters: perivitelline space, percentage of immature oocytes and percentage of oocytes with abnormal morphology. As the severity of abnormal ZP increased, both the number of retrieved oocytes and mature oocytes decreased. The fertilization rate did not differ significantly among groups. The rates of embryo cleavage and day-3 high-quality embryos in the mild group and the moderate group did not vary significantly between the two groups but were significantly higher than those in the severe group. The blastulation rates of the abnormal ZP groups were similar; however, they were lower than those of the control group. Moreover, the cycle cancellation rate of the severe abnormal ZP group was as high as 66.20%, which was significantly higher than that of the other three groups. Although the rates of cumulative clinical pregnancy and live births were lower than those in the control group, they were comparable among the abnormal ZP groups. There were no differences in the neonatal outcomes of the different groups. Together, ZP abnormalities show various degrees of severity, and in all patients regardless of the degree of ZP abnormalities who achieve available embryos, there will be an opportunity to eventually give birth. [ABSTRACT FROM AUTHOR]

Laser-assisted hatching (LAH) is a widely used and unavoidable technique in assisted reproductive technology after frozen embryo transfer (FET). We aimed to investigate the safety and efficacy of completely ZP (zona pellucida) removed by an assisted laser group compared to an intact group after FET. This retrospective study involved 320 SVBT single vitrified-warmed blastocyst transfers of 213 patients who underwent clomiphene citrate (CC) based minimal stimulation protocol. This study compared 160 SVBT with ZP that were completely removed using laser-assisted (laser beams of 10–15, a wavelength of 1.48 μm, and duration of 1.8–2.2 ms) to 160 cases of intact SVBT. Fisher's exact test, chi-squared test, and logistic regression are used for statistical analysis. The women's age, cause of infertility, BMI, transferred embryo day, insemination methods, and blastocyst grade were not significantly different between two groups. The implantation rate (66.2% vs. 51.2%, P < 0.01), the clinical pregnancy rate (CPR, 52.5% vs. 39.3%, P = 0.01), and the live birth rate (LBR, 43.7% vs. 29.3%, P < 0.01) were significantly higher in the completely ZP removed than the intact group. In logistic regression analysis, the laser-assisted hatching group showed about 2 times higher implantation rate and CPR and LBR than the control group. But miscarriage, gestational weeks, gender, birth weight, and twin births were insignificant between two groups. Our study suggests ZP free embryo transfer by assisted laser is more effective and safer than intact embryo transfer. [ABSTRACT FROM AUTHOR]

The metalloproteinase ovastacin is released by the mammalian egg upon fertilization and cleaves a distinct peptide bond in zona pellucida protein 2 (ZP2), a component of the enveloping extracellular matrix. This limited proteolysis causes zona pellucida hardening, abolishes sperm binding, and thereby regulates fertility. Accordingly, this process is tightly controlled by the plasma protein fetuin‐B, an endogenous competitive inhibitor. At present, little is known about how the cleavage characteristics of ovastacin differ from closely related proteases. Physiological implications of ovastacin beyond ZP2 cleavage are still obscure. In this study, we employed N‐terminal amine isotopic labeling of substrates (N‐TAILS) contained in the secretome of mouse embryonic fibroblasts to elucidate the substrate specificity and the precise cleavage site specificity. Furthermore, we were able to unravel the physicochemical properties governing ovastacin–substrate interactions as well as the individual characteristics that distinguish ovastacin from similar proteases, such as meprins and tolloid. Eventually, we identified several substrates whose cleavage could affect mammalian fertilization. Consequently, these substrates indicate newly identified functions of ovastacin in mammalian fertilization beyond zona pellucida hardening. [ABSTRACT FROM AUTHOR]