Two-cell Rate Research Articles

Monitoring Blood Glucose in Mouse Offspring after Intracytoplasmic sperm injection - Embryo Transfer.

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life roughly equivalent to 40 human years. Intracytoplasmic sperm injection (ICSI) is a commonly used assisted reproductive technology in clinical practice. However, given its relatively recent emergence about 30 years ago, the long-term effects of this technique on human development remain unclear. In this study, we established the ICSI combined with embryo transfer (ET) method using a mouse model. The results demonstrated that normal mouse sperm, after undergoing in vitro culture and subsequent ICSI, exhibited a fertilization rate of 89.57% and a two-cell rate of 87.38%. Following ET, the birth rate of offspring was approximately 42.50%. Furthermore, as the mice aged, fluctuations in glucose metabolism levels were observed, which may be associated with the application of the ICSI technique. These findings signify that the mouse ICSI-ET technique provides a valuable platform for evaluating the impact of sperm abnormalities on embryo development and their long-term effects on offspring health, particularly concerning glucose metabolism. This study provides important insights for further research on the potential effects of the ICSI technique on human development, emphasizing the necessity for in-depth investigation into the long-term implications of this technology.

Oocyte Vitrification Reduces its Capability to Repair Sperm DNA Fragmentation and Impairs Embryonic Development.

Oocytes play a crucial role in repairing sperm DNA damage, which can affect the next generation; however, certain factors can impair this ability. This study examined whether oocyte vitrification, a widely used method for fertility preservation, negatively affects repair ability. Male DBA/2 mice (n = 28) were injected with 101.60µmol/100g body weight of tert-Butyl hydroperoxide (tBHP) for 14days to induce sperm DNA damage. Histological changes, sperm functions, and DNA fragmentation were assessed using the TUNEL assay. Cumulus-oocyte-complexes (COCs) of superovulated female DBA/2 mice (n = 28) were vitrified using the Cryotop method. Fresh and vitrified oocytes were then fertilized by tBHP-treated and untreated sperms, and subsequent embryonic development was monitored. Additionally, the expression of Mre11a, Rad51, Brca1, and Xrcc4 was assessed in resulting zygotes and blastocysts using real-time PCR. The sperm tBHP treatment reduced differentiated spermatogenic cells in the testicular tissue, sperm concentration, and motility, while increasing DNA fragmentation (P < 0.05). The fertilization rate was decreased in the tBHP-treated sperm-vitrified oocyte group (P < 0.05), and the two-cell rate diminished in tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05). The four-cell to blastocyst rate decreased in the untreated sperm-vitrified oocyte and the tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05), and the tBHP-treated sperm-vitrified oocyte groups had the lowest blastocyst rate. In zygotes, Brca1 was upregulated in the tBHP-treated sperm-vitrified oocyte group (P < 0.05). Also, in blastocysts, Rad51, Brca1, and Xrcc4 were significantly upregulated in the untreated sperm-vitrified oocytes group (P < 0.05). Damages to the oocyte due to vitrification can disrupt the repair of sperm DNA fragmentation and consequently impair the embryo development.

Iron Overload-Induced Ferroptosis Impairs Porcine Oocyte Maturation and Subsequent Embryonic Developmental Competence in vitro.

Accumulating evidence indicates that ferroptosis is an iron-dependent form of regulated cell death. This type of iron-dependent programmed cell death is different from traditional forms of regulated cell death, such as apoptosis and autophagy. However, the role of ferroptosis in porcine oocyte maturation and the associated mechanism remain unclear. In the present research, we investigated the effects of ferric ammonium citrate (FAC), a specific ferroptosis inducer, on porcine oocyte meiotic maturation and quality and subsequent embryonic developmental competence. FAC treatment caused obvious accumulation of intracellular ferrous ions in porcine oocytes. At the end of the in vitro maturation (IVM) period, there was a significant decrease in the polar body (PB) extrusion rate and an increase in the percentage of abnormal oocytes in the FAC treatment groups, indicating that iron overload-induced ferroptosis may suppress the meiotic process during porcine oocyte maturation. We also found that after FAC treatment, the subsequent two-cell rate, four-cell rate and blastocyst formation rate were significantly decreased in porcine parthenogenetic activation (PA) embryos, indicating that iron overload-induced ferroptosis decreased porcine oocyte quality. Further analysis revealed that FAC treatment not only enhanced intracellular reactive oxygen species (ROS) generation, decreased intracellular free thiol levels and induced mitochondrial dysfunction but also triggered autophagy in porcine oocytes. Taken together, these findings suggest that iron overload-induced ferroptosis impairs porcine oocyte meiosis and decreases porcine oocyte quality, possibly by increasing oxidative stress, inducing mitochondrial dysfunction and triggering autophagy.

Low cryoprotectant concentration rapid vitrification of mouse oocytes and embryos

Vitrification of mammalian oocytes and embryos is typically a two-step procedure involving two solutions of increasing concentrations of cryoprotectants. In the present study, we report a simple vitrification protocol that uses low cryoprotectant concentration and a single medium (LCSM). This medium, along with the traditional high concentration two media (HCTM) protocol, was used to vitrify mouse oocytes, zygotes, and blastocysts using silica capillary, cryotop, cryolock, and 0.25 ml straws. Survival rates, two-cell rates, and blastocyst formation rates were compared for oocytes and zygotes vitrified using both protocols. Results show that the LCSM protocol was as good as or better than the traditional HCTM protocol for vitrifying mouse MII oocytes and zygotes using silica capillary, cryotop, and cryolock. On the other hand, for blastocysts, only silica capillary using LCSM had comparable results with the traditional HCTM protocol while cryolock and cryotop had significantly lower percentages of re-expanded and hatched blastocysts. Collapsing blastocysts prior to vitrification or longer duration for better cryoprotectant distribution in multicellular embryos may improve the outcome. In conclusion, the LCSM protocol, with one medium of much lower cryoprotectant concentrations and shorter equilibration time, reduces exposure to cryoprotectant toxicity while improves efficiency, consistency and reliability for mammalian oocyte and embryo preservation.

Analysis of phytochemical constituents of zuogui wan in rat serum and its effects on early embryonic development of mice

Objective: Zuogui Wan (ZGW) has been used as a typical prescription for tonifying kidney essence in traditional Chinese medicine. The objective of this study is to elucidate the phytochemical constituents of ZGW-treated rat serum (ZGWRS) using ultra-performance liquid chromatography-electrospray ionization/quadrupole-time-of-flight high-definition mass spectrometry (UPLC-ESI-Q-TOF-MS). Methods: ZGW was administered to rats, and the phytochemical constituents in rat serum were determined using UPLC-ESI-Q-TOF-MS. MetaboLynx analysis in negative ion mode was adopted to characterize the chemical constituents of ZGWRS. Orthogonal partial least squares discriminant analysis was applied for the discovery of constituents of ZGW that entered the serum of rats. The fertilized eggs collected from the same experiment were randomly divided into four groups, including the normal, 40 mmol/L glucose, 40 mmol/L glucose 5% control rat serum, and 40 mmol/L glucose 5% ZGWRS groups. They were cultivated at 37°C and 5% CO2in a saturated humidity CO2incubator. The blastocyst rate and two-cell rate were used to evaluate the effects of ZGWRS on embryonic development. Results: Thirteen constituents were identified in the ZGWRS, among which sweroside, loganin, morroniside, loganic acid, and 8-epiloganic acid were from Fructus Corni (Shan Zhu Yu). 5-Hydroxymethyl-2-furfural-glucuronide, 2-H-5-hydroxymethyl-2-furfural-glucuronide, 3-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxylic acid, and β-D-ribofuranuronic acid methyl ester triacetate were from Radix Rehmanniae Preparata (Shu Di Huang). Coumaric acid was from Fructus Lycii (Gou Qi Zi). Kaempferol-3-beta-O-glucuronide and cuscutamine were from Semen Cuscutae (Tu Si Zi). The embryonic development was significantly inhibited using 40 mmol/L glucose. Compared with the normal group, the blastocyst rate of the glucose group was decreased. The blastocyst rate of the 40 mmol/L glucose 5% ZGWRS group was significantly higher than that of the glucose group, indicating that ZGWRS negates the effect of glucose on mouse embryonic development. Conclusion: The results verified that a rapid and robust UPLC-ESI-Q-TOF-MS-based platform had been successful for identifying multiple constituents of ZGW. ZGWRS is rich in active constituents of iridoid glycosides. The results of this study also showed that ZGWRS could negate the effect of glucose on mouse embryonic development.

Improvement of oocyte in vitro maturation from mice with polycystic ovary syndrome by human mesenchymal stromal cell-conditioned media.

The outcome of in vitro maturation (IVM) in the patients with polycystic ovary syndrome (PCOS) is poor. Abnormal intraovarian paracrine interplay alters microenvironment for oocyte development through folliculogenesis and decreases developmental competence of oocytes inpatients with PCOS. Mesenchymal stromal cells (MSCs) secrete a variety of cytokines and growth factors that could promote oocyte maturationin vitro. Thus, in the current study we aimed to evaluate the effect of human bone marrow MSC-conditioned media (hBM-MSC-CM), as a supplement, to enrich IVM medium for PCOS germinal vesicles (GVs). For this purpose, oocytes at GV and metaphase II (MII) stages were harvested from PCOS mice. The GVs were randomly divided into four groups and incubated for 24 hours in an IVM medium (TCM199, as the control group) or TCM199 supplemented by 25%, 50%, and 75% of hBM-MSC-CM (PCOS-CM25, PCOS-CM50, and PCOS-CM75 groups, respectively) so as to evaluate which dose(s) could enhance maturation rate of the GVs and their subsequent in vitro fertilization (IVF) outcome. Furthermore, MII oocytes and their subsequent IVF outcome were considered as the in vivo matured (PCOS-IVO) group. The data showed that supplementation of IVM medium with 50% hBM-MSC-CM significantly increased cytoplasmic and nuclear maturation of the GVs (P < 0.001), and also fertilization and two-cell rate (P < 0.001) and blastocyst formation (P < 0.01) of in vitro matured oocytes from mice with PCOS. Overall, higher oocyte maturation and fertilization outcome in PCOS-CM50 group proposed that enrichment of IVM medium with hBM-MSC-CM could be considered as a promising approach to improve IVM of PCOS oocytes.

Cryobiological Characteristics of L-proline in Mammalian Oocyte Cryopreservation.

Background:L-proline is a natural, nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals. The use of L-proline in mammalian oocyte cryopreservation is rare. In this study, we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation.Methods:The freezing property of L-proline was detected by Raman spectroscopy and osmometer. Mature oocytes obtained from 8-week-old B6D2F1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion of dimethyl sulfoxide (DMSO) and ethylene glycol (EG), comparing with the control group (15% DMSO and 15% EG without L-proline). The survival rate, 5-methylcytosine (5-mC) expression, fertilization rate, two-cell rate, and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization. Data were analyzed by Chi-square test.Results:L-proline can penetrate the oocyte membrane within 1 min. The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group. The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG is significantly higher than that of the control group. There is no difference of 5-mC expression between the L-proline combination groups and control. The fertilization rate, two-cell rate, and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5% DMSO and 10% EG solution are similar to that of control.Conclusions:It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG, which may be applicable to human oocyte vitrification.

Effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection in mouse mature oocytes

Better pregnancy outcomes can be obtained by human mature oocyte vitrification, but many problems remain to be resolved in human mature oocyte vitrification. Since mature oocyte development possesses its own maturity cycle, there should be the optimal timing for mature oocyte vitrification. The purpose of this study was to observe the effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection (ICSI) in vitrified mouse mature oocytes and explore its possible mechanism. Mouse oocytes were randomly divided into three groups according to different frozen timing including Groups A, B, and C in which oocytes were vitrified within 2 h after ovum pick-up, and 3-4 and 5-6 h after ovum pick-up, respectively. Spindle-related parameters were measured, ICSI was performed. The spindle occurrence rate of vitrified-thawed oocytes was 98.4% in Group A, 82.3% in Group B, and 75.8% in Group C, without statistical differences between pre-vitrification and post-thawing and among the three groups (P > 0.05). The angles between the polar body and spindle were larger after thawing than before vitrification (P < 0.01). The spindle retardance values were lower after thawing than before vitrification in Groups B and C (P < 0.05), but higher in Group A (P < 0.05). The spindle retardance values before vitrification were higher in Group B than in Groups A and C (P < 0.05), but the spindle retardance value, oocyte survival and two-cell rate after thawing were higher in Group A than in Groups B and C (P < 0.05). There were no statistical differences in ICSI fertility rate between the three groups (P > 0.05). The damage on the spindle is the slightest and embryo quality is the highest in the mouse oocytes vitrified within 2 h after ovum pick-up. The spindle retardance value is more valuable than the spindle occurrence rate in the evaluation of vitrified-thawed oocyte quality, and is positively correlated with embryo quality.

In vitro follicle growth under non-attachment conditions and decreased FSH levels reduces Lhcgr expression in cumulus cells and promotes oocyte developmental competence

The in-vitro environment influences oocyte competence and gene expression in cumulus cells and oocytes. Effects of culturing under non-attachment conditions and varying follicle exposure to FSH were investigated at the mRNA level and on oocyte developmental capacity. Quantitative PCR analysis of Gdf9, Mater, Nmp2 (in oocytes), Lhcgr and Amh (in cumulus cells), and oocyte developmental competence after in vitro follicle culture were evaluated. Follicle survival (98.7%) and polar body rate (94%) were similar for all conditions. Estradiol and progesterone production were significantly lower in non-attachment follicles (10-fold and 3-fold, respectively). Under non-attachment conditions, a higher two-cell rate (69.9%) and total blastocyst yield (48.5%) were obtained and, by decreasing FSH levels during culture, Lhcgr transcripts were significantly reduced to levels similar to in-vivo. Levels of oocyte-specific transcripts were not significantly influenced by in-vitro conditions. Non-attachment conditions influence follicle steroid secretory capacity and, together with dynamic FSH doses, positively influence cumulus cell gene expression and oocyte developmental competence.

Leukemia inhibitory factor induces cumulus expansion in immature human and mouse oocytes and improves mouse two-cell rate and delivery rates when it is present during mouse in vitro oocyte maturation

To examine the role of leukemia inhibitory factor (LIF) during in vitro maturation (IVM) on human and mice cumulus expansion and mice oocyte competence by in vitro fertilization (IVF), culture, and embryo transfer (ET). Prospective animal and human study. Serono laboratories and IVF clinic. Healthy women volunteers and 8-week-old female mice. Cumulus compacted human and mice oocytes were matured in IVM media with and without recombinant follicle-stimulating hormone (FSH) and with and without LIF. Mice IVM oocytes with and without 0.2 IU/mL of recombinant FSH; or with and without recombinant FSH + LIF (0.1, 1.0, 1000.0 ng/mL) and ovulated oocytes were in vitro fertilized and cultured. We transferred 395 blastocysts to the uterine horn of 2.5-day pseudopregnant female mice. Cumulus expansion in human and mice oocytes, and two-cell rate, blastocyst rate, and delivered rate of live pups in mice. In human and mouse oocytes, LIF induced cumulus expansion. When 1000 ng/mL of LIF was added in combination with recombinant FSH, a statistically significant increase in cleavage rate, embryo development rate, and birth rate was observed when compared with oocytes matured with FSH alone. Leukemia inhibitory factor induced cumulus expansion similarly in human and mouse cumulus-oocyte complexes, and recombinant FSH plus LIF supplementation during mouse IVM significantly improved oocyte competence as measured by cleavage rate, blastocyst development, and birth rate.

Embryonic development after follicle culture is influenced by follicle-stimulating hormone isoelectric point range.

We evaluated the effects of follicular exposure in vitro to either of two mutually exclusive isoforms of FSH (least acidic and acid) on the subsequent capacity of oocytes for embryonic development. The effects of dose and follicle culture duration were examined. At the threshold dose (that required to produce antra) and at one subthreshold dose, the major difference between the two isoform fractions was the timing and effectiveness of acquisition of two-cell embryonic developmental capacity. With the least-acidic fraction, the highest rate of two-cell development (approximately 80%) occurred after 3 days of follicle culture only at the threshold dose (2.5 ng/ml). With the acid fraction, the highest two-cell rate (approximately 60%) occurred after 5 days of culture but at equivalent rates over a range of doses between 10 ng/ml and 100 ng/ml (threshold dose was 50 ng/ml). At threshold dose or below, the capacity for two-cell embryo production appeared not to be influenced by antral status for either isoform. At above threshold doses, the least-acidic fraction induced an increasing proportion of antral follicles with increasing dose, but this increase was associated with a progressive decrease in embryo production. This relationship was more extreme after longer culture and was due to degeneration of the cumulus-oocyte complex associated with apparently increased differentiation of the mural granulosa cells. The acid fraction was by comparison less bioactive and insensitive to overdosing. The broader isoform mix of the unfractionated FSH provided a measure of protection against overdosing characteristic of the acid fraction while retaining the capacity of the least-acidic fraction to induce antral formation at a low dose.