BACKGROUND: Monozygotic (MZ) twins are believed to arise from the fission of a single fertilized embryo at different stages. Monochorionic MZ twins, who share one chorion, originate from the splitting of the inner cell mass (ICM) within a single blastocyst. In the classic model for dichorionic MZ twins, the embryo splits before compaction, developing into two blastocysts. However, there are a growing number of ART cases where a single blastocyst transfer results in dichorionic MZ twins, indicating that embryo splitting may occur even after blastocyst formation.
OBJECTIVE: For monochorionic MZ twins, we conducted a comprehensive analysis of the cellular mechanisms involved in ICM splitting, drawing from both ART cases and animal experiments. In addition, we critically re-examine the classic early splitting model for dichorionic MZ twins. We explore cellular mechanisms leading to two separated blastocysts in ART, potentially causing dichorionic MZ twins.
METHODS: Relevant studies including research articles, reviews, and conference papers were searched in the PubMed database. Cases of MZ twins from IVF clinics were found by using combinations of terms including \'monozygotic twins\' with \'IVF case report\', \'ART\', \'single embryo transfer\', or \'dichorionic\'. The papers retrieved were categorized based on the implicated mechanisms or as those with unexplained mechanisms. Animal experiments relating to MZ twins were found using \'mouse embryo monozygotic twins\', \'mouse 8-shaped hatching\', \'zebrafish janus mutant\', and \'nine-banded armadillo embryo\', along with literature collected through day-to-day reading. The search was limited to articles in English, with no restrictions on publication date or species.
RESULTS: For monochorionic MZ twins, ART cases and mouse experiments demonstrate evidence that a looser ICM in blastocysts has an increased chance of ICM separation. Physical forces facilitated by blastocoel formation or 8-shaped hatching are exerted on the ICM, resulting in monochorionic MZ twins. For dichorionic MZ twins, the classic model resembles artificial cloning of mouse embryos in vitro, requiring strictly controlled splitting forces, re-joining prevention, and proper aggregation, which allows the formation of two separate human blastocysts under physiological circumstances. In contrast, ART procedures involving the transfer of a single blastocysts after atypical hatching or vitrified-warmed cycles might lead to blastocyst separation. Differences in morphology, molecular mechanisms, and timing across various animal model systems for MZ twinning can impede this research field. As discussed in future directions, recent developments of innovative in vitro models of human embryos may offer promising avenues for providing fundamental novel insights into the cellular mechanisms of MZ twinning during human embryogenesis.
CONCLUSIONS: Twin pregnancies pose high risks to both the fetuses and the mother. While single embryo transfer is commonly employed to prevent dizygotic twin pregnancies in ART, it cannot prevent the occurrence of MZ twins. Drawing from our understanding of the cellular mechanisms underlying monochorionic and dichorionic MZ twinning, along with insights into the genetic mechanisms, could enable improved prediction, prevention, and even intervention strategies during ART procedures.
UNASSIGNED: N/A.

In the last decades, to enhance success rates in assisted reproductive technology (ART) cycles, scientists have continually tried to optimize embryo culture and selection to increase clinical outcomes. In this scenario, the application of laser technology has increased considerably worldwide and is currently applied across ART in several ways: for assisted hatching (AH) or thinning of the zona pellucida (ZP), embryo biopsy, to immobilize and select the sperm during intracytoplasmic sperm injection, as well as to induce artificial blastocyst shrinkage before cryopreservation. Laser-AH has been suggested as a procedure to improve embryo implantation: the concept is that drilling holes through or thinning of the ZP could improve the hatching process and implantation. The artificial disruption of the ZP can be performed by different approaches: mechanically, chemically and with the laser, which is one of the most favourable and easy methods to remove part of the ZP and to augment the possibilities of implantation in patients defined as having a poor prognosis of success, or when the ZP is too thick. However, in the current literature, there is not sufficient evidence about the potential risk or impairment that laser utilization might induce on embryo development; therefore, the main aim of the current review is to provide an overview of the existing knowledge on the ZP and the mechanisms of manipulating it to improve the effectiveness of ART. Also, it emphasizes the positive aspect of laser application as a powerful tool that might increase the chance of pregnancy for infertile couples undergoing ART cycles.

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.

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.

The present study is set in the broad field of assisted reproductive technologies (ARTs) and examines various procedures under assisted hatching (AH). It also reviews their effects on implantation success rates. The primary emphasis is on explaining who has benefited and how many have benefited from these interventions. The most important factor determining the success rate of ART is implantation. To increase these rates, we use AH in our clinics to enhance each embryo\'s chances at life and substantially improve overall results. This comprehensive review includes various approaches, such as chemical-based measures (such as applying Tyrode\'s solution) and mechanical techniques (such as zona drilling and partial zona dissection). The individual techniques are carefully scrutinized, considering their mechanical detailing, methods of applying therapeutic effects, and the appropriateness of matching present social circumstances. The review begins by analyzing the basic nature of AH as a medium for embryo implantation and then focuses on how this detailed view reveals the advantages and drawbacks of various methods. Moreover, the articles discuss improvements in AH technology and many of the most modern technological developments that can help fine-tune ART issues. A major problem with these methodologies is that they involve serious risks and legal complications. However, a broad assessment of these topics allows us to understand their impact on fertility treatments. This review is written as a guidebook for physicians and researchers working in reproductive medicine. It compiles all current knowledge, providing literature to build on successes that will make breakthroughs possible in ART. Indeed, this is a valuable reference for guiding us in navigating complex AH procedures. It advances ARTs step by step toward perfection.

BACKGROUND: 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).
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.

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.

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.

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.

暂无摘要。